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Structural and functional properties of pectin and lignin–carbohydrate complexes de-esterases: a review

Structural and functional properties of pectin and lignin–carbohydrate complexes de-esterases: a... Biological conversion of plant biomass into commercially valuable products is one of the highly studied subjects in the last two decades. Studies were continuously being conducted to understand and develop efficient enzymes for the breakdown and conversion of plant cell-wall components into valuable commercial products. Naturally, plant cell-wall components are differentially esterified to protect from the invading microorganisms. However, during the process of evolution, microorganisms have developed special set of enzymes to de-esterify the plant cell-wall com- ponents. Among the carbohydrate-active enzymes (CAZy), carbohydrate esterases stand first during the process of enzymatic conversion of plant biomass, as these enzymes de-esterify the plant biomass and make it accessible for the hydrolytic enzymes such as cellulases, hemicellulases, ligninolytic and pectinases. In this article, we have extensively discussed about the structural and functional properties of pectin methyl esterases, feruloyl, cinnamoyl and glucu- ronoyl esterases which are required for the de-esterification of pectin and lignin–carbohydrate complexes. Pectin esterases are classified among CE8, CE12, CE13 and CE15 carbohydrate esterase class of CAZy database. Whereas, lignin–carbohydrate complex de-esterifying enzymes are classified among CE1 (feruloyl esterase) and CE15 (glu- curonoyl esterase) classes. Understanding the structural and functional abilities of pectin and lignin–carbohydrate esterases will significantly aid in developing efficient class of de-esterases for reducing the recalcitrant nature of plant biomass. These efficient de-esterases will have various applications including pretreatment of plant biomass, food, beverage, pulp and paper, textile, pharmaceutical and biofuel industries. Keywords: Pectin, Lignin–carbohydrate complexes, Pectin methyl esterase, Feruloyl esterase, Cinnamoyl esterase, Glucuronoyl esterase Background 35% of pectin, grasses contain 2–10% and in woody tis- Pectin is a natural heterogenous polysaccharide present sue 5% (Voragen et al. 2009). The exact molecular weight in plant cell walls especially between the middle lamella, of pectic substances is not well defined compared to occurring as calcium and magnesium salts. Plant physio- other plant cell-wall components. However, studies have logical studies based on the uptake of ruthenium red and reported that the relative molecular weight of pectic sub- alkaline hydroxylamine stains (Albersheim and Killias stances ranges between 25 and 300  kDa, e.g., apple and 1963; Gee et  al. 1959; McCready and Reeve 1955; Ster- lemon—200–360  kDa, pear and prune—25–35  kDa, ling 1970) have confirmed that pectic substances majorly orange—40–50  kDa and sugar beet pulp—40–50  kDa occur in the middle lamella of the plant cell wall. The (Jayani et al. 2005). Naturally, pectic substances occur in composition of pectin in plant cell walls varies based on galacturonans and rhamnogalacturonans-I and -II. These the type of plants, e.g., dicotyledonous plant cells contain pectic substances contain anhydrogalacturonic acid backbone, where carboxyl groups are partially esterified by O-acetyl and methyl groups with the hydroxyl groups *Correspondence: wqin@lakeheadu.ca present on the C-2 and -3 positions (Table 1). Pectin con Department of Biology, Lakehead University, 955 Oliver Road, Thunder tains long chains of α-d-galacturonate units joined by Bay, ON P7B 5E1, Canada © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 2 of 16 Table 1 Structural properties and  occurrence of  different O-acetylated pectin structures reported below  were retrieved from (Pawar et al. 2013) Acetylated forms of polysaccharides Properties and occurrence Rhamnogalacturonan I Rhamnogalacturonan I, a group of structurally complex pectic polysaccharides. With a repeat- ing backbone composed of diglycosyl [→ 2)-α-l -Rhap-(1 → 4)-α-d -GalpA-(1 →], branched at O-4/O-3 positions by four different side chain types: (1 → 5)-α-l -arabinan, (1 → 4)-β-d -galactan, arabinogalactan-I, and sometimes with arabinogalactan-II (Pawar et al. 2013; Yapo 2011). It is present in primary cell walls of soft and hardwoods (Pawar et al. 2013) Rhamnogalacturonan II Rhamnogalacturonan II is a low-molecular weight pectic polysaccharide with 5–10 kDa released upon treatment with endo-α-1,4-polygalacturonases. Structurally, rhamnogalacturonan II is a homogalacturonan backbone containing at least 7–9 residues containing five oligosaccharide side chains such as A–E [as described in Pérez et al. (2003)]. Naturally, rhamnogalacturonan II was found to occur in primary cell walls of soft and hard wood, also in cell walls of growing plant cell walls (Pérez et al. 2003) Homogalacturonan Homogalacturonan is one of the major constituents of the pectic polysaccharides. Structurally, it contains long chains of d -galacturonic acid units linked through α-(1 → 4) bonds, which are methyl or acetyl esterified at C-6 position with acetyl/methyl group on O-2 or O-3 positions. It is synthesized from the nucleotide sugars in the Golgi apparatus and are then transported to cell wall in fully methylesterified forms (Sénéchal et al. 2014) α (1 → 4) linkages and 2–3% of l-rhamnose units were and B-type (outer site/polysaccharide chains con- found in association with the galacturonate units joined nected to polygalacturonic acid chain). A-type proto- through β (1 → 2) and (1 → 4) linkages forming the pri- pectinases were majorly reported to be secreted in the mary chain of pectic substances. The American chemi - cultures of yeast and yeast like fungi, whereas B-type cal society has classified the pectic substances into four proto-pectinases were majorly reported in the cultures different types as (a) protopectin, (b) pectic acid, (c) of Bacillus strains and especially in Bacillus subtilis pectinic acid and (d) pectin. Thus, degradation of these cultures (Sakai and Okushima 1982; Sakai et  al. 1993; pectin structural variants requires different pectinolytic Sakamoto et  al. 1994). Polygalacturonases are class of enzymes, which can be broadly classified into three main pectinolytic enzymes which performs the hydrolytic classes (a) protopectinases (b) esterases and (c) depoly- cleavage of polygalacturonic acid by introducing water merases (Table 2). across the oxygen bridge. Based on its reactivity, polyg- Naturally, most of the fungi, bacteria and yeast secrete alacturonases are divided into endo- (widely reported wide range of pectin methyl esterases and pectin- among fungi, bacteria and yeast, and were also reported depolymerizing enzymes for the degradation of pectin. in higher plants and parasitic nematodes) (De Lorenzo Previous studies have extensively reported about vari- et  al. 1987; Luh and Phaff 1951; Manachini et  al. 1987; ous endogenous pectinases secreted by plants (Sakai Marcus et al. 1986; Maria de Lourdes et al. 1991; Sakai and Okushima 1982; Sakai et  al. 1993; Sakamoto et  al. et al. 1984) and exo-polygalacturonases (well studied in 1994; Whitaker 1990). Based on their specific location Erwinia carotovora, Agrobacterium tumefaciens, Bac- of activity, protopectinases were classified as A-type teroides thetaiotaomicron, E. chrysanthemi, Alternaria (inner site/reacts at the polygalacturonic acid region) mali, Fusarium oxysporum, Ralstonia solanacearum, Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 3 of 16 Table 2 Different types of pectic substances and pectinolytic enzymes responsible for its degradation S. no Different types of pectic substances 1. Protopectin: Protopectin is present in the inner tissues of plant cell walls which is insoluble in water. Upon restricted hydrolysis yields pectin or pectic acids 2. Pectic acid: Pectic acids are soluble pectic substances (galacturonans) with lesser number of methoxyl groups. Normal and pectic acid salts are called as pectates 3. Pectinic acids: Are long polygalacturonans with < 75% methylated galacturonate units, salts of pectinic acids are called pectinates 4. Pectin: (or) Polymethyl galacturonate is a polymeric material with 75% of the carboxyl groups are esterified with methanol. Pectin provides rigid- ity to the plant cell walls Pectinolytic enzymes 1. Pectin methyl esterases: These esterases catalyze the de-esterification pectin by releasing methoxy esters, resulting in pectic acids and methanol 2. Pectin-depolymerizing enzymes (a) Protopectins are enzymatically hydrolyzed by set of enzymes called as protopectinases (PPase). PPase are classified into two types (a) A-type PPase, which reacts with polygalacturonic acid regions and (b) B-type PPase reacts with the polysaccharide chains on outer region. (Protopectin + H O–(PPase)– → Pectin ) (insoluble) 2 (soluble) (b) The pectin-depolymerizing enzymes can be majorly classified as hydrolases divided into: Endo and Exo polygalacturonases such as (Exo- polygalacturonan-digalacturono hydrolase, Oligo galacturonate hydrolase, Delta 4:5 Unsaturated oligo galacturonate hydrolases, Endo-poly- methyl-galacturonases, Endo-polymethyl-galacturonases). Lyases which majorly contains enzymes such as Endo and Exo polygalacturonase lyases Plant cell wall Pectin Protopectin Pectic acid Pectic acid Pectin Proto pectinase Polygalacturonases Lyases Pectin Esterase A-type B-type Endo Exo Endo Exo EndoPMGL ExoPMGL Assay method Assay method Assay method Assay method 3,5- Increase in absorbance Gel diffusion Carbozole- dinitrosalicylate at 235 nm to detect assay sulphuric acid Arsenomolybdate formation delta 4:5 Binding of method –copper reagents double bonds produced Ruthenium red Occurrence at the non-reducing to pectin Occurrence Yeast and ends of the unsaturated Occurrence Bacteria, Fungi, yeast-like fungi products Plants and Yeast, higher Occurrence pathogenic plants and Bacteria and pathogenic bacteria and parasitic plants fungi fungi Fig. 1 Brief illustration of different pectin-degrading enzymes, its occurrence and assay methods used for its characterization Bacillus sp) (Garcıća Maceira et  al. 1997; Kobayashi Polygalacturonate lyases were majorly reported to be et  al. 2001; Nozaki et  al. 1997; Reymond et  al. 1994; secreted by bacteria and some pathogenic fungi, espe- Rodriguez-Palenzuela et  al. 1991; Tierny et  al. 1994). cially soft rot fungi (Fig.  1). Pectin esterases are a class Pectin lyases catalyze non-hydrolytic cleavage of pec- of carbohydrate esterases which are involved in dees- tates or pectinates, lyases cleaves the glycosidic link- terification of methyl ester linkages present on the ages at C-4 by simultaneously eliminating the H at C-5 galacturonan chains of pectic substances present in by producing 4:5 double-bonded unsaturated products. the plant cell wall (Cosgrove 1997; Micheli 2001; Prade Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 4 of 16 et  al. 1999; Whitaker 1984). The de-esterified pectin is 2011). Pectinolytic enzymes are widely observed among further degraded by the polygalacturonases and lyases plants, bacterial and fungal species and most of the pec- (Fig.  1) (Prade et al. 1999; Sakai et al. 1993). The mode tin methyl esterases can be divided based on their opti- of action of the pectin esterases differs significantly mum pH; bacteria and plant PME exhibit an optimum pH based on its origin; pectin esterases secreted by fungi range between 6 and 8 and PME secreted by fungi exhibit act through a multichain mechanism to cleave methyl a pH 4–6 (Gonzalez and Rosso 2011; Jayani et  al. 2005). groups randomly. Whereas, pectin methyl esterases Pectin-degrading enzymes have attained high commer- originated from plant act either on the non-reducing cial importance since early 1930s in wine and fruit juice ends or on the groups next to free carboxyl groups by industries; pectinolytic enzymes secreted by Aspergillus a single chain mechanism (Förster 1988; Micheli 2001). species are highly used in industries (Alkorta et al. 1998). Lignin, the heterophenolic polymer present in the plant Pectin present in vegetable tissues and majorly in fruits cell wall, is considered as the major drawback in the pro- contains complex hetero polysaccharides at a molecular duction of bioethanol. Naturally, lignin is found to occur weight ranging between 25 and 360  kDa. Calcium and in intricate networks with plant cell-wall carbohydrates. magnesium pectate forms the major constituent of the Lignin forms ester linkages through its alcohols with hemi- plant cell walls especially in middle lamella (Jayani et  al. cellulose through 4-O-methyl-d-glucuronic acid side resi- 2005). The gelling property of pectin majorly employed in dues of glucuronoxylans (Pokkuluri et al. 2011). According the food industries is directly dependent on its degree of to Del Río et  al. (2007) similar to pectin and hemicellu- esterification; pectins with higher degree of esterification lose, lignin present in plant cell walls, was also found to gel around pH 3.0 in the presence of sugar, whereas pec- be acetylated on the aliphatic side chains (gamma carbon) tins with low degree of esterification gel in the presence of of lignin S and G monomers (Del Río et  al. 2007). Studies calcium ions under wide pH ranges and with or without have reported that extraaxillary fibers in jute, abaca and sugar (Fu and Rao 1999, 2001; Gonzalez and Rosso 2011). kenaf exhibit highest degree of acetylation with degree of Pectin methyl esterases or alkaline reagents were acetylation up to 0.8 and lignin acetylation in hardwood majorly used to demethoxylate large galacturonic chain xylem varies between 1 and 50% (w/w). However, studies for reducing the overall pectin methoxylation con- need to be conducted to determine the degree of acetyla- tent (Gonzalez and Rosso 2011). Majorly, pectinolytic tion in softwood xylem and the reasons behind the vari- enzymes were highly applied in fruit juice and wine indus- ation of lignin acetylation in plants (Del Río et  al. 2007; tries for the clarification of the fruit juices, modification of Pawar et  al. 2013). Ferulic acid is a most abundant phy- fruits and vegetables (Kohli et al. 2015). Apart from these tochemical phenolic compound derived from cinnamic applications, pectinolytic enzymes were also used for acid 3-(4-hydroxy-3-methoxyphenyl-2-proponoic acid), extracting oils from germ, palm, coconut, sunflower seed 4-hydroxy-3-methoxycinnamic acid, or coniferic acid and kernel rape seeds, by replacing the conventionally (Fazary and JU 2007). Ferulic  acid is commercially an used carcinogenic solvents like hexane. These pectinolytic important compound and it is widely distributed in the enzymes extract oil from different crops by liquefying the whole plant kingdom, it is highly studied for its anti-oxidant structural components of the cell walls. Commercial pec- properties (Fazary and JU 2007). However, in the nature, tinase preparations called Olivex were applied in olive ferulic acid occurs in its esterified form which is covalently oil industries for the extraction of oil and to increase the connected to the lignin, glycoproteins, and to the insoluble quality (Kashyap et al. 2001; Vierhuis et al. 2003). Rham- carbohydrate components mono- and di-saccharides of the nogalacturonan, a complex polysaccharide unit present in plant cell-wall components (Fazary and JU 2007; Kroon the primary cell walls and middle lamella of higher plants, et  al. 1997). The feruloyl esterases attack and convert fer - with alternating rhamnose and galacturonic acid residues ulic acid and cinnamic acid present in the plant biomass; acetylated majorly at C-2 and C-3 positions (Ishii 1997). because of this ability, feruloyl or cinnamoyl esterases have As the acetylation of these residues sterically hinders the gained higher industrial importance (Benoit et al. 2008). catalytic function of the corresponding lyases and hydro- lases on the glycosidic linkages, deacetylation facilitates Carbohydrate esterases de‑acetylating pectin the action of the lyases and hydrolases. Thus, rhamnoga - Enzymes required for the breakdown of pectin can be lacturonan acetyl esterase belonging to CE-12 family has majorly classified into three categories as protopectinases gained significance in deacetylation of these residues and (involved in breaking insoluble protopectin and results also been used industrially for the production of β-lactam in soluble polymerized pectin), depolymerizing enzymes antibiotics and paper bleaching purposes (Navarro- (required for breaking down α(1 → 4) glycosidic linkages Fernández et al. 2008). We have specifically reviewed the of pectin) and esterases (required for the de-esterifica - structural and functional properties of pectin methyl and tion and de-acetylation of pectin) (Gonzalez and Rosso acetyl esterases below (Table 3). Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 5 of 16 Table 3 List of  the  structural and  functional properties of  pectin-deacetylating esterases belonging to  CE-8 and  CE-12 of carbohydrate esterases Strain, PDB ID, CE class Catalytic residues CATH and Pfam Enzyme and fold and reaction type Substrates and Refs Erwinia chrysanthemi, [1QJV ], CE-8 Asp178, Asp199, Arg267 3-Solenoid Pectin methyl esterase, single stranded right- Aspartic esterase Pectinase lyase C-like pectinesterase handed β-helix, pectin present in cell wall polysaccharides is hydrolyzed to pectate and methanol (Jenkins et al. 2001) Aspergillus aculeatus, [1DEX], CE-12 Ser9, His195, Asp192 3-Layer α/β sandwich Rhamnogalacturonan acetylesterase flavodoxin- Serine esterases Rossmann fold like (SGNH hydrolase). Rhamnogalacturonan GDSL-like lipase/acylhydrolase present in the cell walls. RGAE functions in coordination with rhamnogalacturonan lyases and hydrolases (Mølgaard et al. 2000) The structural and functional properties such as SCOPE and CATH related information was retrieved from RCSB PDB data repository, appropriate references and the corresponding PDB ID’s were reported in the table loops and T1 loops are arranged near C-terminal end of Several studies were conducted in the past to under- parallel β-helix. The occurrence of α-helix allows the exist stand the structural and functional properties of pec- - tinolytic enzymes. Present day, National Center for ing structures of the pectinolytic enzymes to align struc- Biotechnological Information (NCBI) repositories turally, so that half of the α-carbon atoms are arranged resides about 20,933 reports on pectin esterases which approximately at 2 Å. Loop conformations extended from can be majorly classified as 1758 (NCBI Literature), 15 the pemA parallel β-helix were not comparable to that (NCBI Health), 3503 (NCBI Genomes), 15,230 (NCBI of other pectinolytic enzymes even at the position of T3 Genes), 395 (NCBI Proteins) and 42 (NCBI Chemicals). loops in space, as they might come from different coils. Till date, there are 189 protein sequences, 160 protein The β-structure formed from two long T1 loops are inter clusters (expressing seven conserved domains related nally hydrogen bonded, and the β-hairpins and the hydro- to pectin esterases) and 30 experimentally-determined gen bond present in between the hairpins give a suggestion biomolecular structures of pectin methyl esterases avail - of the anti-parallel four strand sheet. Aminoacid residues able in NCBI and PDB (protein data bank) repositories from 342 to 359 of parallel β-helix are involved in the respectively. The protein data bank (PDB) contains struc - formation of one distorted α-helix (342–359) or two dis- tures of 3-pectin methyl esterases of bacteria (Dickeya torted α-helix (342–352 and 353–359) containing Tyr242 dadantii, Yersinia enterocolitica, Dickeya chrysanthemi) and Thr353 residues, where the side chains of these resi - and one fungal pectin methyl esterase of Aspergillus dues interact with 351-O and 356-N residues through the niger and four pectin methyl esterases of plants (Arabi- hydrogen bonds, forming an antiparallel structure against dopsis thaliana, Solanum lycopersicum, Daucus carota, parallel β-helix as the C-terminal of α-helix of lyases. Sitophilus oryzae, Actinidia chinensis). In this article, we Unlike the conserved C-terminal extension and α-helix of rhamnogalacturonase A (RGase A) arranged against have extensively focused on understanding the structural PB3, aminoacid residues from 345 to 359 are packed and functional properties of pectin methyl esterases of microorganisms. against PB2, which further interact with equivalent residues in pectate lyases forming an extended chain reaching to the conserved α-helix against PB3. Struc Erwinia chrysanthemi (CE class‑8) - Jenkins et  al. (2001), for the first time, have studied and turally, several lipases and esterases exhibited a com- revealed the 3D structure of pectin methyl esterase iso- mon α/β hydrolase fold with a catalytic Ser–His–Asp lated from Erwinia chrysanthemi refined at 2.4  Å, where triad. But pectin methylesterases showed some variation it contains two identical molecules with 342 amino acid with respect to the protein structure and catalytic triad residues in the crystallographic asymmetric unit (Jen- location. Interestingly, sequence alignments of pectin methylesterases does not show any conserved histidine kins et al. 2001). Pectin methyl esterase (pemA) is a right- and serine residues but show various other conserved handed parallel β-helix structure resembling other pectin aminoacid residues. The conserved aminoacid residues and pectate lyase, by having same number of total coils. mapped against the structure of the pemA shows a differ Each parallel β-helix turn further contains three β-strands - PB1, PB2 and PB3 which are in turn connected through ent cluster with a deep cleft on the surface of the enzyme. loops called as T1 (connecting PB1 and PB2), T2 (PB2 to The cleft present on the surface of the enzyme is involved PB3) and T3 (PB3 to PB1) of another coil. It also contains a in substrate binding and active site formation, by con- α-helix at the end of N-terminal of parallel β-helix; long T3 taining several aromatic residues. Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 6 of 16 The active site region was found to contain three con - through simple activation of methyl ester by Asp178 by served aminoacid residues Asp178, Asp199 and Arg267. avoiding the nucleophilic assistance of Asp199, which According to Jenkins et  al. (2001), these two conserved might question the conservation of Asp199 residue and aspartate residues and their adjacent arginine residue are its stereochemical chemistry at the catalytic site across considered as the catalytic residues, as there is no other the pectin methyl ester family. Though Arg267 and substantial candidate to be determined as catalytic sites Trp269 residues are not directly involved in catalytic (Jenkins et  al. 2001). The aspartate residues are equally reaction, they are required for the substrate binding. The arranged on the neighboring coils, thus having same left- hydrolytic water molecule which is involved in hydroly- handed α-helices. The Asp199 was found to be in close sis of the acyl enzyme is observed during the low pH and contact with Arg267 which partially blocks Asp199 from time course adjacent to the Asp178 (general base). This the solvent and forms hydrogen bonds with NE and NH1 protein structure also suggests that Gln177 is involved in of the side chain oxygen atoms (Jenkins et al. 2001). The the formation of oxyanion hole and the kinetic analysis second aspartate residue Asp178 interacts with two water proves the role of Gln177 in stabilization of transition molecules which shields it form interacting with the sol- state; similarly, Gln153 is required for substrate binding vent molecule from glutamine residues (Gln153 and (Fries et al. 2007; Jenkins et al. 2001) (Fig. 2). Gln177) and with the β-carbon atom of Asp199 residue. The aromatic residues Tyr158, Tyr181 and Phe202 on the Aspergillus aculeatus (Rhamnogalacturonan acetylesterase exposed surface of PB1 forms a floor of valley at Tyr269 CE class‑12) of a T1 loop. With the Tyr181, Phe202 and Trp269 resi- The three-dimensional structure of the rhamnogalactu - dues are found to be conserved in most pectin methyl- ronan acetylesterase (RGAE) from Aspergillus aculeatus esterases sequences. Fries et  al. (2007) have performed was revealed by (Mølgaard et  al. 2000). Protein struc- site-directed mutagenesis of the pectin methylesterase ture of RGAE was determined by multiple isomorphous (E. chrysanthemi) to understand the functional prop- replacement and refined at 1.55  Å resolution (Mølgaard erties of aminoacid residues in catalytic site (Fries et  al. et  al. 2000). The protein structure of RGAE exhibited a 2007). This study has also reported that following resi - α/β/α fold containing a central five-stranded parallel dues Asp199, Asp178, Gln177 are involved in reaction β-sheet surrounded by α-helices, with a structural topol- mechanism, though Arg267 was found to be conserved ogy of central sheet to be found as—1X 2X1X1X with among all the pectin methyl esterases and is not directly two stranded antiparallel β-sheet insertion after the involved in enzyme catalysis (Fries et  al. 2007). The E. third β-strand. There are total of 11 helices in the protein chrysanthemi pemA was found to be active between pH structure containing four 3 helices (A, B, E and K). Due 5 and 9; however, at pH 5 pemA showed highest activity to the lack of backbone hydrogen bonds between Glu25- (Pitkänen et al. 1992). The catalytic Asp178 residue might Ala28 and Tyr26-Ser29, A and B are assigned as two sep- mostly be found in its protonated state due to its position arate helices. There are two disulfide bridges formed by in a hydrophobic environment, while the Asp199 occurs four cysteine residues, which are involved in linking the in deprotonated at neutral pH as it is accessible by the aminoacid residues 88–96, 214–232 with other disulfide solvents. The negative charge and the position of Asp199 bridges anchoring the C-terminal loop and α-helix. are favored by hydrophobic residues Arg267, Ala233, Though, electron density maps do not show any indica - Tyr230 and Val198 and direct Asp199 towards incoming tion of O-glycosylation sites, but it showed two N-glyco- substrate and solvent. The presence of hydrogen bonds sylation sites (Asn104 and Asn182) which allows a total between the Arg267 and Asp199 oxygen atom assists in of seven carbohydrate residues in the electron density maintaining the Asp199 residues deprotonated state. The map (Mølgaard et al. 2000). two glutamine residues Gln153 and Gln177 present near The active site inferred from the 3D structure of RAGE the Asp199 and Asp178 residues might contribute to the shows that Ser9 residue present on the topological point oxyanion hole (Fries et al. 2007; Johansson et al. 2002). at the end of β-strand is involved in forming the charac- These crystallographic studies have provided clear teristic hydrogen bonds in the catalytic triad with His195 insights on the reaction mechanism of pectin methyl and Asp192. The electron density maps showed that the esterase at atomic resolution (Fries et  al. 2007; Jenkins sulfite ion is bound to the active site of the RGAE protein et al. 2001). The Asp199 acts as nucleophile, as it directly with oxygen atom occupying the oxyanion hole through attacks the carbonyl carbon of methyl ester, with Asp178 making hydrogen bonds with main chain NH groups of acting as general acid–base in the reaction. Lack of water Ser9, Gly 42 and side chain amide of Asn74, which are molecule between Asp199 and substrate rules out the located in the loop regions next to carboxy ends of first earlier proposed mechanism (reaction is initiated by three strands in central β-sheet (Mølgaard et  al. 2000). activating the water molecule). The reaction proceeds The second sulfate oxygen atom is hydrogen bonded with Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 7 of 16 Erwinia chrysanthemi(PDBID-1QJV) (PMEA) Fig. 2 The protein secondary structure of Erwinia chrysanthemi pectin methyl esterase (carbohydrate esterase class-8) (PDB ID:1QJV ) (Jenkins et al. 2001), where alpha-helices (red), beta strands (blue) and coils (gray). (Image was developed using Discovery Studio Visualizer ) the imidazole group of His195, thus connecting four dif- the protein structure of RGAE violates this consensus ferent regions of the protein structure. These structural sequence as large side chains of methionine (Met11) 2+ studies have extensively revealed the structural localiza- located at N u position making it sterically impossible tion of catalytic triad residues. The Ser9 residue which to form a sharp turn which is mandatory for nucleo- acts as nucleophile is present in the type-I β turn, Asp192 phile elbow motif. Secondly, the most conserved α-helix and His195 are arranged in loop region which connects C present in α/β hydrolases is not conserved in the pro- the two helices near the C-terminal end. The position tein structure of RGAE (Schrag and Cygler 1997). The 3D of the active site makes it accessible for a wide range structure of RGAE also revealed that nucleophile back- of large substrates, as it is situated in the bottom of the bone conformation is unstrained, as it is situated on type- open cleft. Most of the arginine residues are situated in I β turn following a loop region. Although RGAE follows suitable positions for its interactions with the negatively similar sequential order of catalytic residues (Ser–Asp– charged carboxylate groups of the substrates (Mølgaard His) as α/β hydrolase fold, they differ in their location. et al. 2000). In α/β hydrolases, the Asp and His are located quite far The α/β hydrolase fold adopted by most of the neutral from each other in sequence; whereas, in RGAE they are lipases and esterases consists of a central parallel β-sheet only separated by two residues. RGAE differs from α/β (eight stranded) surrounded by α-helices (Ollis et  al. hydrolases with respect to the orientation of the catalytic 1992). Majorly, the catalytic triad residues from the α/β residues and the remaining protein structure. The cata - hydrolases follow an order of nucleophile–acid–histidine lytic triad residues in α/β hydrolases are arranged parallel and in most of the protein structures, the topology and to the central β-sheet; whereas in RGAE, these residues the positions of these residues are similar. The nucleo - are arranged perpendicularly to the central β-sheet. phile is situated at the end of 5th β-strand, acid is located However, these differences do not severely affect the cat - at the end of 7th β-strand and the histidine follows the alytic functions of the nucleophile on carbonyl carbon or loop region of 8th strand; thus, α/β hydrolase fold char- the enzyme (Mølgaard et al. 2000) (Fig. 3; Table 3). acteristic motif is also called as nucleophilic elbow (Derewenda and Derewenda 1991; Ollis et al. 1992). This Carbohydrate esterases for de‑esterification of lignin– nucleophile backbone attains a strained ε conformation carbohydrate complexes due to situation of nucleophile in a sharp turn between a Plants contain a range of hydroxycinnamic acids like β-strand and α-helix, the small helix (Sm) any residue (X) caffeic, p-coumaric, ferulic and sinapic acids which and nucleophile (Nu) residues are arranged specifically can be broadly classified as phenolic compounds and as Sm-X-Nu-X-Sm–Sm for forming a motif. However, highly abundant among foods (Guglielmetti et  al. 2008). Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 8 of 16 Aspergillus aculeatus (PDBID-1DEX) (Rhamnogalacturonan acetylesterase ) Fig. 3 The protein secondary structure of Aspergillus aculeatus rhamnogalacturonan acetylesterase (carbohydrate esterase class-12) (PDB ID:1DEX) (Mølgaard et al. 2000), where alpha-helices (red), beta strands (blue) and coils (gray). (Image was developed using Discovery Studio Visualizer ) Feruloyl esterases (or ferulic acid esterases) or cinnamoyl the fungal glucuronoyl esterase using a series of uronic esterases are carbohydrate esterase class of enzymes acids and their glycoside derivatives (Duranová et  al. which hydrolyze the ester linkages between hydroxycin- 2009). These studies have revealed the specificity of GE namic acids and plant cell-wall carbohydrates by releas- towards 4-O-methyl-d-glucuronic acid, its methyl esters ing ferulic and cinnamic acid (Topakas et  al. 2007). and d-glucuronic acid containing 4-nitrophenyl agly - Combinatorial usage of feruloyl esterase or cinnamoyl con, showing that GE attack the ester bonds between esterases with glycoside hydrolases for the liberation of 4-O-methyl-d-glucuronic acid of glucuronoxylan and free carbohydrate residues and phenolic acids can signifi - alcohols of lignin (Ďuranová et  al. 2009; Topakas et  al. cantly aid in different preprocessing steps of biofuel and 2010). Glucuronoyl esterase finds its applications in biorefining industries (Faulds 2010). The activity of FAE growing biofuel and biorefinery industries as it breaks and CAE are chiefly limited to the position and confor - down and separates the hemicellulose and lignin. mations of the feruloyl groups present in the feruloylated Pretreatment step is currently being used in bioethanol polysaccharides and other surrounding cell-wall compo- industries for releasing free carbohydrate residues from nents. Recent studies conducted by Faulds et  al. (2003, the other aromatic components of the cell wall. The het - 2006) have revealed the preferential partnership between erophenolic lignin compounds interacting with the poly- glycoside hydrolase class-11 (GH-11) xylanases and FAE saccharide units increase the recalcitrant nature of the for liberating ferulic acid from the insoluble biomass, plant cell wall and the percentage of lignin in plant tis- while partnership between GH-10 xylanases and FAE will sues is directly proportional to its digestibility. It has been liberate 5,5′dimers (Faulds et al. 2003, 2006). Glucuronoyl assumed that FAEs and CAEs are required for breaking esterases are the class of carbohydrate esterases involved the lignin and carbohydrate linkages. According to Benoit in hydrolysis of the ester linkages present between et  al. (2006), type-C and type-B FAEs isolated from 4-O-methyl-d-glucuronic acid residues of glucuronoxy - A. niger release higher proportions of ferulic acid and lans and aromatic alcohols of lignin (Špániková and Biely p-coumaric acid from the steam-exploded wheat straw 2006). Glucuronoyl esterase which is involved in plant (Benoit et  al. 2006). The type-A FAE from A. niger was cell-wall degradation was discovered in Schizophyllum found to be effective against the steam-exploded wheat commune for the first time (Špániková and Biely 2006). straw in the presence of cellulases and xylanases, and at Duranová et  al. (2009) have purified and characterized 50  °C the rate hydrolysis increased significantly (Tabka Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 9 of 16 et al. 2006). Similarly, Selig et al. (2008) have used a com- involved in forming the dimer interface where a total of bination of cellobiohydrolase Cel7A, xylanase, feruloyl 2373 Å is buried between these chains (Lai et al. 2011). esterase and acetyl xylan esterase and reported enhanced The 3PF8 is considered as a dimer interface protein due breakdown of the hot water-treated corn stover cellulose to (a) the primary hydrophobic interface formed by the (Selig et al. 2008). Apart from its long list of applications, 18/37 residues of chain A and chain B (b) the presence FAEs were also used for the utilization of straws in paper of six salt bridges. Total of 54 aminoacid residues Pro131 industries (Record et al. 2003; Tapin et al. 2006), detoxifi - to Gln184 are involved in formation of inserted α/β sub- cation of animal feed (Laszlo et al. 2006), for the removal domain which is arranged in between the β6 and β11 of cinnamic acids and p-coumaric acids from coffee pulp strands. (Benoit et  al. 2006). Thus, wide applications of FAE and The catalytic site of the 3PF8 resembles an open canal CAE in food, paper and pulp, biofuel industries make in the shape of boomerang ending with a hydrophobic these enzymes economically and commercially impor- pocket buried in between α5 and α6 of the inserted α/β tant enzymes (Table 4). subdomain (Lai et al. 2011). The Ser106 residue situated in the center of boomerang with two clefts present in Lactobacillus johnsonii (Cinnamoyl esterase CE class I) the catalytic site is of approximately 13 Å and 7 Å which The three-dimensional structures of native and mutant is large enough for accommodating the acyl groups of cinnamoyl esterase secreted by Lactobacillus johnsonii the aromatic substrates. The roof of the catalytic site is was revealed by (Lai et  al. 2011). The native apo cin - formed by two protruding hairpins of the inserted α/β namoyl esterase (3PF8) structure consists of central subdomain, with another cleft of 12  Å accommodating β-sheet containing seven parallel β-strands (β1, β3, β4, the alkoxyl group of other atoms present on the larger β5, β6, β11, β12) and one anti-parallel β-strand (β2). substrates. The catalytic triad of the catalytic site con - The central β-sheet exhibits a superhelical twist with sists of Ser106, His225 and Asp197, where the Ser106 120  °C angle from β1 to β2 strands, central β-strand is is situated at the nucleophile elbow formed by the β5 edged by five α-helices where two α-helices are located and α4. The backbone nitrogen atoms of Phe34 and externally (α1 and α9) and three α-helices are internally Gln107 are buried near the base of inserted α/β subdo- located (α3, α4 and α8) near the dimer interface. Two main, which constitutes for the oxyanion hole. The site- protein molecules of the 3PF8 are involved in exten- directed mutagenesis experiments conducted on the sive interface formation which consists of α4, α6 and catalytic triad by Lai et  al. (2011) revealed that Ser106, β1 strand. Lai et  al. (2011) have also reported the pro- Ala and Asp197, Ala mutants failed to exhibit the cata- tein interactions and surface assemblies analysis (PISA lytic activity; thus, Ser106, His225 and Asp197 consti- server); based on these results, it was revealed that 34 tute for the catalytic function of 3PF8 (Lai et  al. 2011). residues from chain A and 37 residues from chain B are The catalytic site analysis of 3PF8 has revealed that α/β Table 4 List of  the  structural and  functional properties of  lignin–carbohydrate complex deacetylating esterases belonging to carbohydrate esterases CE1, CE15 classes Strain, PDB ID, CE class Enzyme and fold Catalytic residues CATH and Pfam Substrates and Refs and reaction type Lactobacillus johnsonii, [3PF8], CE-1 Cinnamoyl esterase Ser106, His225, Asp197 3-Layer α/β sandwich Ferulic acid, Caffeic acid and ethyl α/β hydrolases Serine esterase Rossmann fold ferulate. Cinnamoyl esterase is α/β hydrolase family active against short acyl chain aliphatic esters and phenolic esters (Lai et al. 2011) Aspergillus niger, [1UWC], CE-1 Feruloyl esterase Ser133, His247, Asp194 3-Layer α/β sandwich Arabinoxylans (hydrolysis of feruloyl- α/β hydrolases Serine esterase Rossmann fold arabinose ester bond) pectin Lipase class 3 (feruloyl-galactose ester). High substrate specificity towards esters where feruloyl group is attached to C-5 of arabinose and inactive against C-2 esters (McAuley et al. 2004) Sporotrichum thermophile, [4G4J ], Glucuronoyl ester- Ser213, Glu236, His346 3-Layer α/β sandwich It has high specificity for hydrolyzing CE-15 ase, α/β hydrolases Serine hydrolase Rossmann fold the ester bonds of 4-O-methyl- d -glucuronic acid units of glucu- ronoxylan and alcohols of lignin (Charavgi et al. 2013) Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 10 of 16 Aspergillus niger (Feruloyl esterase CE class‑I) subdomain residues from Pro131 to Gln184 might play The three-dimensional structure of feruloyl esterase iso - a crucial role in substrate binding. Lai et al. (2011) have lated from A. niger was revealed by (McAuley et al. 2004). hypothesized that inserted α/β subdomain plays a criti- The protein structure of feruloyl esterase (FAE) contains cal role in holding the phenolic ring of phenolic esters two FAE-III molecules, two sulfate ions and 399 water in the appropriate position for the catalytic reaction; molecules. This protein contains the standard α/β hydro - however, its role diminishes when aliphatic esters were lase fold with five α-helices and two β-sheets surrounding used as the enzyme substrates. Trp160 residue situ- the central core (nine β-sheets). FAE-III is glycosylated at ated on the second hairpin formed between the β9 and Asp79 residue and at every glycosylation site, a N-acetyl β10 plays an important role in fixing the substrate as glucosamine was observed. The clear and organized elec - the rotation of Trp160 forms a tunnel for burying the tron density maps of FAE-III prove that NAG residues ferulic acid in the catalytic site. In addition, the side were not attached to any other carbohydrate residues. chains of Asp138 and Trp169 are involved in forming The catalytic site of the FAE-III contains Ser133, Asp194 hydrogen bonds with the hydroxyl groups of aromatic and His247; His247 is well organized to form hydrogen rings of ferulic acid and caffeic acid in the catalytic bonds with other two residues (Ser133 and Asp194); N pocket of 3PF8. It was reported that substrate orienta- atom forms hydrogen bonds with O atom of Ser133; and tion in the 3PF8 catalytic site plays a critical role in its similarly, N connects with the Asp194 through hydro- catalysis; thus, appropriate orientation by the substrate gen bonding. This characteristic arrangement of catalytic is acquired through its interactions with the enzyme triad residues denotes that enzymes involved breakdown (aromatic substituents on one end of ligand and ester of amide or ester bonds through nucleophile attack. The groups on another end). These interactions cease the serine residue (Ser133) performs nucleophilic attack on carbonyl group at the oxyanion hole, thus allowing the the carbonyl atom of the substrate molecule and His247 molecule to bend. Contrastingly, the functional groups residue deprotonates Ser133 residue by acting as general other than ester groups such as alkoxy groups of feru- base and finally, the positive charge on the His247 is sta - lic acid/ethyl ferulate, quinic acid group of chlorogenic bilized by the negatively charged Asp194 residue through acid do not exhibit any role in substrate binding (Lai electrostatic interaction. This catalytic reaction results in et al. 2011) (Fig. 4). Lactobacillus johnsonii (PDBID-3PF8) Cinnamoyl esterase Fig. 4 The protein secondary structure of Lactobacillus johnsonii cinnamoyl esterase (carbohydrate esterase class-I) (PDB ID:3PF8) (Lai et al. 2011), where alpha-helices (red), beta strands (blue) and coils (gray). (Image was developed using Discovery Studio Visualizer ) Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 11 of 16 a tetrahedral transition state which is further collapsed helix in two different conformations in FAE-III is ques - to give an acyl-enzyme intermediate. This intermedi - tionable as it lacks the lipase activity (Aliwan et al. 1999). ate is attacked by the nucleophilic water molecule which Although FAE-III and lipases are similar in their over- was in turn activated by the histidine residue. The second all structure, difference in their active site regions was tetrahedral transition intermediate is formed due to the expected as they accommodate different substrates. FAE- release of acid; the oxyanion formed by the backbone N III contains large number of polar amino acids exposed atoms of Thr68 and Leu134 residues stabilizes the nega - around the catalytic site, but it is expected that lipases tive charge on O atom of carbonyl group present in the contain hydrophobic residues on its surface. The interac - tetrahedral transition state. tion between ferulic acid and FAE-III happens through The two molecules of FAE-III contain the well-organ - OH, CH3 side chains and Tyr80 hydroxyl groups. The ized active site residues having single conformation to carboxylate group is situated near the oxyanion hole the catalytic triad and with the known hydrogen bond- near the catalytic site serine residue, main chain N atom ing patterns between residues (Hedstrom 2002; McAuley (Leu134) and backbone N and OH atoms of Thr68. The et al. 2004). The tertiary structure of FAE-III is similar to presence of Leu199 and Ile196 residues provides a hydro- other fungal lipases, as the rmsd values between the FAE- phobic environment. McAuley et al. (2004) have reported III and RmL (Rhizomucor miehei), FAE-III and TIL (Ther - that residues involved in binding to the ferulic acid were momyces lanuginosa) were 1.0  Å (over 217 Cα atoms) found to be similar in both native and complexed struc- and 1.3 Å (over 205 Cα atoms). Based on the conforma- tures of FAE-III. This study has also reported that feru - tion states, the lipases are classified as active and inac - lic acid binds to the proteins in an unproductive way as tive forms; the catalytic residues are exposed in active it contains third molecule of ferulic acid present in the form and in inactive forms, these residues are closed by FAE-III complex at approximately 20 Å distance from the a helical lid. FAE-III residues ranging from 71 to 77 are catalytic site. Ferulic acid interacts with AsnA223 of mol- involved in formation of the helical lid but it does not ecule A and AspB217 of molecule B of other asymmet- cover the active site and functions as an active form. ric unit through a water molecule (McAuley et  al. 2004) According to Aliwan et  al. (1999), the occurrence of the (Fig. 5). Aspergillus niger (PDBID: 1UWC) Feruloyl esterase Fig. 5 The protein secondary structure of Aspergillus niger feruloyl esterase (carbohydrate esterase class-I) (PDB ID:1UWC) (McAuley et al. 2004), where alpha-helices (red), beta strands (blue) and coils (gray). (Image was developed using Discovery Studio Visualizer ) Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 12 of 16 Myceliophthora thermophile (Glucuronoyl esterase) sequence (Ser–Asp–His). The Ser213 situated between Three-dimensional structure of glucuronoyl esterase the S6 strand and H6 helix (forming a characteristic isolated from Sporotrichum thermophile (StGE2) was nucleophilic elbow) performs nucleophilic attack on the explained by (Charavgi et  al. 2013). The StGE2 is a ser - substrate, His346 acts as acid/base suggesting the nature ine-type hydrolase family containing a three-layer αβα- of the substrate and Glu236 stabilizes the positive charge sandwich hydrolase fold with a Rossmann-fold topology of His346. The structural organization and conforma - (Charavgi et al. 2013). Though StGE2 structure belongs to tion of the catalytic triad are stabilized by the internal α/β hydrolase superfamily, it deviates from the standard hydrogen bonds formed between Ser213 hydroxyl group α/β hydrolase fold. The β-sheet of StGE2 is expanded due and NE2 of His346, OE1 of Glu236 and ND1 of His346 to the insertion of two antiparallel β-strands at the N-ter- (Charavgi et al. 2013). The catalytic triad is also stabilized minus of StGE2 forming a twisted β-sheet. This twisted by the direct and water-mediated polar linkages formed β-sheet is sandwiched between 18 helices arranged in by Arg214, Gly216, Lys217, Gln235, Phe304 and Asn306 two layers, eight α-helices and 3 helices are arranged on residues. Along with these interactions, the disulfide one side of the β-sheet and on the opposite side, another bridges formed by the Cys347 and Cys212 also enhance set of eight α-helices and 3 helices are equally divided. the rigidity of the catalytic site by bridging Ser213 and However, the standard α/β hydrolase fold exhibits eight His346 residues. Active site of StGE2 is located on the strands of β-sheets in the central core of the structure surface of the molecule and exposed to the solvent mol- sandwiched between two clusters of α-helices (Charavgi ecules and unaffected by other interactions. The site et  al. 2013; Ollis et  al. 1992) (Fig.  6). The StGE2 struc - directed mutagenesis experiments of Ser213 residue to ture is stabilized by three disulfide bonds (Cys31–Cys65, understand the structural and functional properties of Cys212–Cys347 and Cys244–Cys319). The catalytic triad StGE2 (Charavgi et al. 2013). However, the superposition of StGE2 is represented by Ser213, Glu236 and His346, of StGE2 with the S213A mutant structures on C atoms where it deviates from the standard catalytic triad with a rmsd value of 0.4  Å shows that both StGE2 and Sporotrichum thermophile (PDBID: 4G4G) glucuronoyl esterase Fig. 6 The protein secondary structure of Sporotrichum thermophile glucuronoyl esterase (carbohydrate esterase class-I) (PDB ID:4G4G) (Charavgi et al. 2013), where alpha-helices (red), beta strands (blue) and coils (gray). (Image was developed using Discovery Studio Visualizer ) Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 13 of 16 S213A mutants differ slightly. Similarly, the superposition dehydration, sterilization and pasteurization lead to of S213A mutant complexed with methyl 4-O-methyl- severe physical damage of plant cellular tissues including β-d-glucopyranuronate (MCU) with StGE2 and S213A primary cell wall and middle lamella. However, recently, mutant crystals showed that substrate analog of MCU these traditional methods were replaced with vacuum binds to the active site without disturbing the struc- infusion methods using pectin methyl esterases and cal- ture of the protein. The RMSD values obtained from the cium chloride mixture to overcome the negative effects superposition of S213A mutant bound to MCU with the of fruits- and vegetables-processing methods (Suutar- S213A mutant and StGE2 on C atoms was 0.40  Å and inen et  al. 2000, 2002). Based on its degree of esterifica - 0.12  Å. The residues present around and in the catalytic tion, pectin is divided into low-methoxylated (LM) and 2 2 triad with surface areas 27.1 Å (Ser213), 2.7 Å (Glu236) high-methoxylated (HM) (Kohli et  al. 2015). The HM and 75.7 Å (His346) are exposed to the solvent. The site- pectin is generally used for the production of high-sugar directed mutagenesis experiment on Ser213 with Ala jams and jellies. As the HM pectin follows a hydropho- residue has confirmed that lack of the hydroxyl group did bic interaction and dehydration at low pH for its gelling not affect the catalytic site conformation. and to achieve proper gelling of the HM pectin, higher The structure analysis of S213A mutant bound with sugar concentrations are essential (Kohli et  al. 2015). MCU has revealed the catalytic function of the StGE2 Contrastingly, LM pectin follows different gelling mecha - protein. The substrate MCU is bound to the catalytic nisms through ionic interactions as it contains calcium site residues through seven direct, four indirect and 59 (divalent cations) which interacts with the free carboxylic van der Waals interactions (Charavgi et  al. 2013). The acid groups resulting in a successful cross linkage; there- MCU analog binds to the cavity through its O atom of fore, LM pectin does not require higher concentrations the hydroxyl group present on the glucopyranose ring; of sugar for gelling. Thus, industrially LM pectin is used the side chains of Gln259 and Glu267 residues of S213A for the production of low-sugar jellies and jams suit- mutant interact with O1, O2 and O3 of sugar by hydro- able for the consumption of diabetic patients (Kohli et al. gen bonding. When compared to the structure of StGE2, 2015). The apple- and orange-peel wastes are rich in pec - the Glu267 side chain residue conformation in S213A tin; thus, it is industrially used as gelling and thickening mutant differs by dihedral angle of ~ 74° supporting the agents. However, the degree of esterification significantly strong binding of the substrate analog, whereas StGE2 affects the thickening and gelling property of the pec - Glu267 is arranged at dihedral angle of ~ 127°. The oxy - tin. Pectin methyl esterases are used for retrieving pec- gen groups present on the substrate interact with resi- tin with lower degree of esterification (Kohli et  al. 2015; dues Trp310 NE1 (O2) and Lys217 NZ (O3 and O4) of Morris et al. 2000). the protein (Charavgi et  al. 2013). Similarly, O6a which Industrially, feruloyl esterases are applied in various relates to the ester bond interacts with NE2 atom present biotechnological processes such as production of plat- on the imidazole ring of His 346. The interactions among form chemicals, fuel, animal feed, textile, paper and pulp, the N atom of Arg214 and its side chains with the meth- food processing, agriculture and pharmaceutical indus- oxy group (O4 atom) and ester group (O6 atom) which tries (Fazary and Ju 2013). In the last few decades, several further forms hydrogen bonds with Ser213 OG suggests studies were being conducted to understand and increase that Arg214 is involved in the oxyanion hole formation the activity of enzymes used in paper and pulp indus- (Charavgi et al. 2013) (Fig. 6). tries. Enzyme mixtures containing feruloyl esterases and acetyl xylan esterases enhance the enzymatic activity of Commercial significance of pectin and lignin de‑esterases hemicellulases and cellulases, by breaking down the sub- Commercially, pectin methyl esterases are majorly stitutions and linkages between lignin and carbohydrate applied in association with pectinases in food and bev- complexes and solubilize the complex (Fazary and Ju erage processing industries. The presence of pectin leads 2013; Mathew and Abraham 2004; Topakas et  al. 2004). to cloudiness of the fruit juices; pectinase preparations The lignocellulolytic enzyme mixtures including feruloyl are applied industrially for clarifying and reducing the esterases, secreted by fungi, were being applied for the viscosity of fruit juices and eases the process of concen- pretreatment of animal feedstock. Pretreatment of ani- tration and filtration (Demir et  al. 2001). Pectinases are mal feedstock with the lignocellulolytic enzyme mixtures applied in combinations with cellulases; hemicellulases were significantly found to increase the activity of rumen were applied for the disruption of plant cell walls and microbiota by 80%, thus increasing the rate of digestion commercially used to produce juices from tropical fruits (Mathew and Abraham 2004; Tarbouriech et  al. 2005; (Alkorta et  al. 1998; Massiot et  al. 1997; Wicker et  al. Topakas et al. 2004). Major applications of feruloyl ester- 2002). Industrial processes used for preservation of fruits ases were reported in food and pharmaceutical indus- and vegetables such as blanching, washing, freezing, tries by converting cinnamic acid/ferulic acid and other Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 14 of 16 phenolic compounds into commercially valuable prod- these de-esterifying enzymes are highly significant as ucts like vanillin (Fazary and Ju 2013). Feruloyl esterases they are involved in the production of commercially are also applied for improving the quality of bread, prepa- valuable products ranging from food, beverage, tex- ration and clarification of juices, synthesis of oligosaccha - tile, pulp and paper, biofuel and pharmaceutical indus- rides and production of medicinal compounds (Fazary tries. Continuous studies were being conducted to and Ju 2013). Glucuronoyl esterases are active against a understand and reveal the catalytic mechanisms and to wide range of natural and synthetic lignin–carbohydrate develop an efficient set of de-esterifying enzymes with complexes (Hüttner et al. 2017). Specifically, glucuronoyl high rate of substrate hydrolysis. In this article, we have esterases cleave ester linkages between the lignin and discussed specifically about the structural and func - glucuronoxylan complexes (Arnling Bååth et  al. 2016). tional aspects of pectin (pectin methyl esterase) and uTh s, glucuronoyl esterases share various industrial lignin–carbohydrate complex (feruloyl/cinnamoyl and applications in common with feruloyl esterases. Glucu- glucuronoyl esterases) de-esterifying enzymes. We have ronoyl esterases are primarily used in biofuel industries extensively conversed by comparing the structures of E. in combinations with other lignocellulolytic enzymes chrysanthemi and A. aculeatus for pectin esterases, L. for conversion of plant biomass into fuel. Studies have johnsonii, A. niger for feruloyl/cinnamoyl esterases and reported that the presence of glucuronoyl esterases in S. thermophile for glucuronoyl esterase. Understand- the enzyme mixture has significantly improved the rate ing the active catalytic residues of these de-esterifying of substrate hydrolysis (d’Errico et al. 2016). Glucuronoyl enzymes will significantly help in developing recom - esterases are also applied in food and beverage industries binantly active enzymes with higher substrate hydrol- for the clarification and concentration of fruit juices and ysis rates. It is highly necessary to understand the wine. Glucuronoyl esterases are also applied for syntheti- structural and functional properties of plant biomass cally altering glucuronic acid derivatives for developing de-esterifying enzymes, as they stand on first place in nonionic surfactants and other bioactive substances with bioconversion of plant biomass into commercially valu- great applications in the pharmaceutical (anti tumorous able products. prodrugs) and cosmetic preparations (De Graaf et  al. 2004). Studies were continuously being conducted for Abbreviations developing recombinant plants-vulnerable plant cell-wall CE: carbohydrate esterases; CAZy: carbohydrate active enzymes; PDB: protein components (Lionetti et  al. 2010; Sticklen 2006); paral- data bank; PME: pectin methyl esterase; FAE: ferulic acid esterases (or) feruloyl esterases; CAE: cinnamic acid esterase (or) cinnamoyl esterase; StGE2: Sporotri- lelly, studies were also reported to develop the recombi- chum thermophile glucuronoyl esterase 2; HM: highly methoxylated; LM: low nant enzymes with higher rate of hydrolysis and greater methoxylated. substrate-binding abilities (Himmel et  al. 2007; Mar- Authors’ contributions tinez et  al. 2009). In this article, we have listed some of AKS is a Ph.D. research fellow wrote the manuscript. WQ was the principal the state-of-the-art articles based on enzyme engineering supervisor who supervised in writing the manuscript and provided comments and developing efficient recombinant enzymes (Bloom and revisions to the manuscript. Both authors read and approved the final manuscript. et  al. 2005; Chen et  al. 2018; Dalby 2007; Damborsky and Brezovsky 2014; Gaj et  al. 2013; Jørgensen et  al. 2007; Martinez et al. 2009; Taylor et al. 2001; Tischer and Acknowledgements Not applicable. Wedekind 1999). Competing interests The authors declare that they have no competing interests. Conclusions In nature, plant cell-wall components occur in esterified Availability of data and materials The data presented and supporting the conclusion of our manuscript were form a type defensive mechanism adapted for inhibit- mostly presented in the form of text; structural images and functional proper- ing the activity of enzymes secreted by invading micro- ties were previously reported and published. The PDB IDs and structural and organisms. However, these invading microorganisms functional properties reported in our manuscript were cited in the manuscript. have evolved over the course of time and developed an Consent for publication efficient enzyme system for the de-esterification of the Not applicable. plant cell-wall components. Pectin methyl esterases, Ethics approval and consent to participate feruloyl/cinnamoyl esterases and glucuronoyl ester- Not applicable. ases play a significant role in plant biomass conversion by de-esterifying pectin and lignin–carbohydrate com- Funding This work was supported by Natural Sciences and Engineering Research plexes and provide an accessible substrate for the act- Council of Canada Funding (RGPIN-2017-05366) to Wensheng Qin and Ontario ing enzymes secreted by microorganisms. Industrially, Trillium Scholarship (OTS) to Ayyappa Kumar Sista Kameshwar. Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 15 of 16 and family 11 and a feruloyl esterase in the release of phenolic acids from Publisher’s Note cereal arabinoxylan. Appl Microbiol Biotechnol 71:622–629 Springer Nature remains neutral with regard to jurisdictional claims in pub- Fazary AE, JU YH (2007) Feruloyl esterases as biotechnological tools: current lished maps and institutional affiliations. and future perspectives. Acta Biochim Biophys Sin 39:811–828 Fazary AE, Ju Y-H (2013) The large-scale use of feruloyl esterases in industry. Received: 7 August 2018 Accepted: 3 October 2018 Biotechnol Mol Biol Rev 3:95–110 Förster H (1988) Pectinesterases from Phytophthora infestans. In: Wood WA, Kellogg ST (eds) Methods in enzymology, vol 161. Academic Press, pp 355–361 Fries M, Ihrig J, Brocklehurst K, Shevchik VE, Pickersgill RW (2007) Molecular References basis of the activity of the phytopathogen pectin methylesterase. 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Structural and functional properties of pectin and lignin–carbohydrate complexes de-esterases: a review

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2018 The Author(s)
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10.1186/s40643-018-0230-8
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Abstract

Biological conversion of plant biomass into commercially valuable products is one of the highly studied subjects in the last two decades. Studies were continuously being conducted to understand and develop efficient enzymes for the breakdown and conversion of plant cell-wall components into valuable commercial products. Naturally, plant cell-wall components are differentially esterified to protect from the invading microorganisms. However, during the process of evolution, microorganisms have developed special set of enzymes to de-esterify the plant cell-wall com- ponents. Among the carbohydrate-active enzymes (CAZy), carbohydrate esterases stand first during the process of enzymatic conversion of plant biomass, as these enzymes de-esterify the plant biomass and make it accessible for the hydrolytic enzymes such as cellulases, hemicellulases, ligninolytic and pectinases. In this article, we have extensively discussed about the structural and functional properties of pectin methyl esterases, feruloyl, cinnamoyl and glucu- ronoyl esterases which are required for the de-esterification of pectin and lignin–carbohydrate complexes. Pectin esterases are classified among CE8, CE12, CE13 and CE15 carbohydrate esterase class of CAZy database. Whereas, lignin–carbohydrate complex de-esterifying enzymes are classified among CE1 (feruloyl esterase) and CE15 (glu- curonoyl esterase) classes. Understanding the structural and functional abilities of pectin and lignin–carbohydrate esterases will significantly aid in developing efficient class of de-esterases for reducing the recalcitrant nature of plant biomass. These efficient de-esterases will have various applications including pretreatment of plant biomass, food, beverage, pulp and paper, textile, pharmaceutical and biofuel industries. Keywords: Pectin, Lignin–carbohydrate complexes, Pectin methyl esterase, Feruloyl esterase, Cinnamoyl esterase, Glucuronoyl esterase Background 35% of pectin, grasses contain 2–10% and in woody tis- Pectin is a natural heterogenous polysaccharide present sue 5% (Voragen et al. 2009). The exact molecular weight in plant cell walls especially between the middle lamella, of pectic substances is not well defined compared to occurring as calcium and magnesium salts. Plant physio- other plant cell-wall components. However, studies have logical studies based on the uptake of ruthenium red and reported that the relative molecular weight of pectic sub- alkaline hydroxylamine stains (Albersheim and Killias stances ranges between 25 and 300  kDa, e.g., apple and 1963; Gee et  al. 1959; McCready and Reeve 1955; Ster- lemon—200–360  kDa, pear and prune—25–35  kDa, ling 1970) have confirmed that pectic substances majorly orange—40–50  kDa and sugar beet pulp—40–50  kDa occur in the middle lamella of the plant cell wall. The (Jayani et al. 2005). Naturally, pectic substances occur in composition of pectin in plant cell walls varies based on galacturonans and rhamnogalacturonans-I and -II. These the type of plants, e.g., dicotyledonous plant cells contain pectic substances contain anhydrogalacturonic acid backbone, where carboxyl groups are partially esterified by O-acetyl and methyl groups with the hydroxyl groups *Correspondence: wqin@lakeheadu.ca present on the C-2 and -3 positions (Table 1). Pectin con Department of Biology, Lakehead University, 955 Oliver Road, Thunder tains long chains of α-d-galacturonate units joined by Bay, ON P7B 5E1, Canada © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 2 of 16 Table 1 Structural properties and  occurrence of  different O-acetylated pectin structures reported below  were retrieved from (Pawar et al. 2013) Acetylated forms of polysaccharides Properties and occurrence Rhamnogalacturonan I Rhamnogalacturonan I, a group of structurally complex pectic polysaccharides. With a repeat- ing backbone composed of diglycosyl [→ 2)-α-l -Rhap-(1 → 4)-α-d -GalpA-(1 →], branched at O-4/O-3 positions by four different side chain types: (1 → 5)-α-l -arabinan, (1 → 4)-β-d -galactan, arabinogalactan-I, and sometimes with arabinogalactan-II (Pawar et al. 2013; Yapo 2011). It is present in primary cell walls of soft and hardwoods (Pawar et al. 2013) Rhamnogalacturonan II Rhamnogalacturonan II is a low-molecular weight pectic polysaccharide with 5–10 kDa released upon treatment with endo-α-1,4-polygalacturonases. Structurally, rhamnogalacturonan II is a homogalacturonan backbone containing at least 7–9 residues containing five oligosaccharide side chains such as A–E [as described in Pérez et al. (2003)]. Naturally, rhamnogalacturonan II was found to occur in primary cell walls of soft and hard wood, also in cell walls of growing plant cell walls (Pérez et al. 2003) Homogalacturonan Homogalacturonan is one of the major constituents of the pectic polysaccharides. Structurally, it contains long chains of d -galacturonic acid units linked through α-(1 → 4) bonds, which are methyl or acetyl esterified at C-6 position with acetyl/methyl group on O-2 or O-3 positions. It is synthesized from the nucleotide sugars in the Golgi apparatus and are then transported to cell wall in fully methylesterified forms (Sénéchal et al. 2014) α (1 → 4) linkages and 2–3% of l-rhamnose units were and B-type (outer site/polysaccharide chains con- found in association with the galacturonate units joined nected to polygalacturonic acid chain). A-type proto- through β (1 → 2) and (1 → 4) linkages forming the pri- pectinases were majorly reported to be secreted in the mary chain of pectic substances. The American chemi - cultures of yeast and yeast like fungi, whereas B-type cal society has classified the pectic substances into four proto-pectinases were majorly reported in the cultures different types as (a) protopectin, (b) pectic acid, (c) of Bacillus strains and especially in Bacillus subtilis pectinic acid and (d) pectin. Thus, degradation of these cultures (Sakai and Okushima 1982; Sakai et  al. 1993; pectin structural variants requires different pectinolytic Sakamoto et  al. 1994). Polygalacturonases are class of enzymes, which can be broadly classified into three main pectinolytic enzymes which performs the hydrolytic classes (a) protopectinases (b) esterases and (c) depoly- cleavage of polygalacturonic acid by introducing water merases (Table 2). across the oxygen bridge. Based on its reactivity, polyg- Naturally, most of the fungi, bacteria and yeast secrete alacturonases are divided into endo- (widely reported wide range of pectin methyl esterases and pectin- among fungi, bacteria and yeast, and were also reported depolymerizing enzymes for the degradation of pectin. in higher plants and parasitic nematodes) (De Lorenzo Previous studies have extensively reported about vari- et  al. 1987; Luh and Phaff 1951; Manachini et  al. 1987; ous endogenous pectinases secreted by plants (Sakai Marcus et al. 1986; Maria de Lourdes et al. 1991; Sakai and Okushima 1982; Sakai et  al. 1993; Sakamoto et  al. et al. 1984) and exo-polygalacturonases (well studied in 1994; Whitaker 1990). Based on their specific location Erwinia carotovora, Agrobacterium tumefaciens, Bac- of activity, protopectinases were classified as A-type teroides thetaiotaomicron, E. chrysanthemi, Alternaria (inner site/reacts at the polygalacturonic acid region) mali, Fusarium oxysporum, Ralstonia solanacearum, Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 3 of 16 Table 2 Different types of pectic substances and pectinolytic enzymes responsible for its degradation S. no Different types of pectic substances 1. Protopectin: Protopectin is present in the inner tissues of plant cell walls which is insoluble in water. Upon restricted hydrolysis yields pectin or pectic acids 2. Pectic acid: Pectic acids are soluble pectic substances (galacturonans) with lesser number of methoxyl groups. Normal and pectic acid salts are called as pectates 3. Pectinic acids: Are long polygalacturonans with < 75% methylated galacturonate units, salts of pectinic acids are called pectinates 4. Pectin: (or) Polymethyl galacturonate is a polymeric material with 75% of the carboxyl groups are esterified with methanol. Pectin provides rigid- ity to the plant cell walls Pectinolytic enzymes 1. Pectin methyl esterases: These esterases catalyze the de-esterification pectin by releasing methoxy esters, resulting in pectic acids and methanol 2. Pectin-depolymerizing enzymes (a) Protopectins are enzymatically hydrolyzed by set of enzymes called as protopectinases (PPase). PPase are classified into two types (a) A-type PPase, which reacts with polygalacturonic acid regions and (b) B-type PPase reacts with the polysaccharide chains on outer region. (Protopectin + H O–(PPase)– → Pectin ) (insoluble) 2 (soluble) (b) The pectin-depolymerizing enzymes can be majorly classified as hydrolases divided into: Endo and Exo polygalacturonases such as (Exo- polygalacturonan-digalacturono hydrolase, Oligo galacturonate hydrolase, Delta 4:5 Unsaturated oligo galacturonate hydrolases, Endo-poly- methyl-galacturonases, Endo-polymethyl-galacturonases). Lyases which majorly contains enzymes such as Endo and Exo polygalacturonase lyases Plant cell wall Pectin Protopectin Pectic acid Pectic acid Pectin Proto pectinase Polygalacturonases Lyases Pectin Esterase A-type B-type Endo Exo Endo Exo EndoPMGL ExoPMGL Assay method Assay method Assay method Assay method 3,5- Increase in absorbance Gel diffusion Carbozole- dinitrosalicylate at 235 nm to detect assay sulphuric acid Arsenomolybdate formation delta 4:5 Binding of method –copper reagents double bonds produced Ruthenium red Occurrence at the non-reducing to pectin Occurrence Yeast and ends of the unsaturated Occurrence Bacteria, Fungi, yeast-like fungi products Plants and Yeast, higher Occurrence pathogenic plants and Bacteria and pathogenic bacteria and parasitic plants fungi fungi Fig. 1 Brief illustration of different pectin-degrading enzymes, its occurrence and assay methods used for its characterization Bacillus sp) (Garcıća Maceira et  al. 1997; Kobayashi Polygalacturonate lyases were majorly reported to be et  al. 2001; Nozaki et  al. 1997; Reymond et  al. 1994; secreted by bacteria and some pathogenic fungi, espe- Rodriguez-Palenzuela et  al. 1991; Tierny et  al. 1994). cially soft rot fungi (Fig.  1). Pectin esterases are a class Pectin lyases catalyze non-hydrolytic cleavage of pec- of carbohydrate esterases which are involved in dees- tates or pectinates, lyases cleaves the glycosidic link- terification of methyl ester linkages present on the ages at C-4 by simultaneously eliminating the H at C-5 galacturonan chains of pectic substances present in by producing 4:5 double-bonded unsaturated products. the plant cell wall (Cosgrove 1997; Micheli 2001; Prade Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 4 of 16 et  al. 1999; Whitaker 1984). The de-esterified pectin is 2011). Pectinolytic enzymes are widely observed among further degraded by the polygalacturonases and lyases plants, bacterial and fungal species and most of the pec- (Fig.  1) (Prade et al. 1999; Sakai et al. 1993). The mode tin methyl esterases can be divided based on their opti- of action of the pectin esterases differs significantly mum pH; bacteria and plant PME exhibit an optimum pH based on its origin; pectin esterases secreted by fungi range between 6 and 8 and PME secreted by fungi exhibit act through a multichain mechanism to cleave methyl a pH 4–6 (Gonzalez and Rosso 2011; Jayani et  al. 2005). groups randomly. Whereas, pectin methyl esterases Pectin-degrading enzymes have attained high commer- originated from plant act either on the non-reducing cial importance since early 1930s in wine and fruit juice ends or on the groups next to free carboxyl groups by industries; pectinolytic enzymes secreted by Aspergillus a single chain mechanism (Förster 1988; Micheli 2001). species are highly used in industries (Alkorta et al. 1998). Lignin, the heterophenolic polymer present in the plant Pectin present in vegetable tissues and majorly in fruits cell wall, is considered as the major drawback in the pro- contains complex hetero polysaccharides at a molecular duction of bioethanol. Naturally, lignin is found to occur weight ranging between 25 and 360  kDa. Calcium and in intricate networks with plant cell-wall carbohydrates. magnesium pectate forms the major constituent of the Lignin forms ester linkages through its alcohols with hemi- plant cell walls especially in middle lamella (Jayani et  al. cellulose through 4-O-methyl-d-glucuronic acid side resi- 2005). The gelling property of pectin majorly employed in dues of glucuronoxylans (Pokkuluri et al. 2011). According the food industries is directly dependent on its degree of to Del Río et  al. (2007) similar to pectin and hemicellu- esterification; pectins with higher degree of esterification lose, lignin present in plant cell walls, was also found to gel around pH 3.0 in the presence of sugar, whereas pec- be acetylated on the aliphatic side chains (gamma carbon) tins with low degree of esterification gel in the presence of of lignin S and G monomers (Del Río et  al. 2007). Studies calcium ions under wide pH ranges and with or without have reported that extraaxillary fibers in jute, abaca and sugar (Fu and Rao 1999, 2001; Gonzalez and Rosso 2011). kenaf exhibit highest degree of acetylation with degree of Pectin methyl esterases or alkaline reagents were acetylation up to 0.8 and lignin acetylation in hardwood majorly used to demethoxylate large galacturonic chain xylem varies between 1 and 50% (w/w). However, studies for reducing the overall pectin methoxylation con- need to be conducted to determine the degree of acetyla- tent (Gonzalez and Rosso 2011). Majorly, pectinolytic tion in softwood xylem and the reasons behind the vari- enzymes were highly applied in fruit juice and wine indus- ation of lignin acetylation in plants (Del Río et  al. 2007; tries for the clarification of the fruit juices, modification of Pawar et  al. 2013). Ferulic acid is a most abundant phy- fruits and vegetables (Kohli et al. 2015). Apart from these tochemical phenolic compound derived from cinnamic applications, pectinolytic enzymes were also used for acid 3-(4-hydroxy-3-methoxyphenyl-2-proponoic acid), extracting oils from germ, palm, coconut, sunflower seed 4-hydroxy-3-methoxycinnamic acid, or coniferic acid and kernel rape seeds, by replacing the conventionally (Fazary and JU 2007). Ferulic  acid is commercially an used carcinogenic solvents like hexane. These pectinolytic important compound and it is widely distributed in the enzymes extract oil from different crops by liquefying the whole plant kingdom, it is highly studied for its anti-oxidant structural components of the cell walls. Commercial pec- properties (Fazary and JU 2007). However, in the nature, tinase preparations called Olivex were applied in olive ferulic acid occurs in its esterified form which is covalently oil industries for the extraction of oil and to increase the connected to the lignin, glycoproteins, and to the insoluble quality (Kashyap et al. 2001; Vierhuis et al. 2003). Rham- carbohydrate components mono- and di-saccharides of the nogalacturonan, a complex polysaccharide unit present in plant cell-wall components (Fazary and JU 2007; Kroon the primary cell walls and middle lamella of higher plants, et  al. 1997). The feruloyl esterases attack and convert fer - with alternating rhamnose and galacturonic acid residues ulic acid and cinnamic acid present in the plant biomass; acetylated majorly at C-2 and C-3 positions (Ishii 1997). because of this ability, feruloyl or cinnamoyl esterases have As the acetylation of these residues sterically hinders the gained higher industrial importance (Benoit et al. 2008). catalytic function of the corresponding lyases and hydro- lases on the glycosidic linkages, deacetylation facilitates Carbohydrate esterases de‑acetylating pectin the action of the lyases and hydrolases. Thus, rhamnoga - Enzymes required for the breakdown of pectin can be lacturonan acetyl esterase belonging to CE-12 family has majorly classified into three categories as protopectinases gained significance in deacetylation of these residues and (involved in breaking insoluble protopectin and results also been used industrially for the production of β-lactam in soluble polymerized pectin), depolymerizing enzymes antibiotics and paper bleaching purposes (Navarro- (required for breaking down α(1 → 4) glycosidic linkages Fernández et al. 2008). We have specifically reviewed the of pectin) and esterases (required for the de-esterifica - structural and functional properties of pectin methyl and tion and de-acetylation of pectin) (Gonzalez and Rosso acetyl esterases below (Table 3). Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 5 of 16 Table 3 List of  the  structural and  functional properties of  pectin-deacetylating esterases belonging to  CE-8 and  CE-12 of carbohydrate esterases Strain, PDB ID, CE class Catalytic residues CATH and Pfam Enzyme and fold and reaction type Substrates and Refs Erwinia chrysanthemi, [1QJV ], CE-8 Asp178, Asp199, Arg267 3-Solenoid Pectin methyl esterase, single stranded right- Aspartic esterase Pectinase lyase C-like pectinesterase handed β-helix, pectin present in cell wall polysaccharides is hydrolyzed to pectate and methanol (Jenkins et al. 2001) Aspergillus aculeatus, [1DEX], CE-12 Ser9, His195, Asp192 3-Layer α/β sandwich Rhamnogalacturonan acetylesterase flavodoxin- Serine esterases Rossmann fold like (SGNH hydrolase). Rhamnogalacturonan GDSL-like lipase/acylhydrolase present in the cell walls. RGAE functions in coordination with rhamnogalacturonan lyases and hydrolases (Mølgaard et al. 2000) The structural and functional properties such as SCOPE and CATH related information was retrieved from RCSB PDB data repository, appropriate references and the corresponding PDB ID’s were reported in the table loops and T1 loops are arranged near C-terminal end of Several studies were conducted in the past to under- parallel β-helix. The occurrence of α-helix allows the exist stand the structural and functional properties of pec- - tinolytic enzymes. Present day, National Center for ing structures of the pectinolytic enzymes to align struc- Biotechnological Information (NCBI) repositories turally, so that half of the α-carbon atoms are arranged resides about 20,933 reports on pectin esterases which approximately at 2 Å. Loop conformations extended from can be majorly classified as 1758 (NCBI Literature), 15 the pemA parallel β-helix were not comparable to that (NCBI Health), 3503 (NCBI Genomes), 15,230 (NCBI of other pectinolytic enzymes even at the position of T3 Genes), 395 (NCBI Proteins) and 42 (NCBI Chemicals). loops in space, as they might come from different coils. Till date, there are 189 protein sequences, 160 protein The β-structure formed from two long T1 loops are inter clusters (expressing seven conserved domains related nally hydrogen bonded, and the β-hairpins and the hydro- to pectin esterases) and 30 experimentally-determined gen bond present in between the hairpins give a suggestion biomolecular structures of pectin methyl esterases avail - of the anti-parallel four strand sheet. Aminoacid residues able in NCBI and PDB (protein data bank) repositories from 342 to 359 of parallel β-helix are involved in the respectively. The protein data bank (PDB) contains struc - formation of one distorted α-helix (342–359) or two dis- tures of 3-pectin methyl esterases of bacteria (Dickeya torted α-helix (342–352 and 353–359) containing Tyr242 dadantii, Yersinia enterocolitica, Dickeya chrysanthemi) and Thr353 residues, where the side chains of these resi - and one fungal pectin methyl esterase of Aspergillus dues interact with 351-O and 356-N residues through the niger and four pectin methyl esterases of plants (Arabi- hydrogen bonds, forming an antiparallel structure against dopsis thaliana, Solanum lycopersicum, Daucus carota, parallel β-helix as the C-terminal of α-helix of lyases. Sitophilus oryzae, Actinidia chinensis). In this article, we Unlike the conserved C-terminal extension and α-helix of rhamnogalacturonase A (RGase A) arranged against have extensively focused on understanding the structural PB3, aminoacid residues from 345 to 359 are packed and functional properties of pectin methyl esterases of microorganisms. against PB2, which further interact with equivalent residues in pectate lyases forming an extended chain reaching to the conserved α-helix against PB3. Struc Erwinia chrysanthemi (CE class‑8) - Jenkins et  al. (2001), for the first time, have studied and turally, several lipases and esterases exhibited a com- revealed the 3D structure of pectin methyl esterase iso- mon α/β hydrolase fold with a catalytic Ser–His–Asp lated from Erwinia chrysanthemi refined at 2.4  Å, where triad. But pectin methylesterases showed some variation it contains two identical molecules with 342 amino acid with respect to the protein structure and catalytic triad residues in the crystallographic asymmetric unit (Jen- location. Interestingly, sequence alignments of pectin methylesterases does not show any conserved histidine kins et al. 2001). Pectin methyl esterase (pemA) is a right- and serine residues but show various other conserved handed parallel β-helix structure resembling other pectin aminoacid residues. The conserved aminoacid residues and pectate lyase, by having same number of total coils. mapped against the structure of the pemA shows a differ Each parallel β-helix turn further contains three β-strands - PB1, PB2 and PB3 which are in turn connected through ent cluster with a deep cleft on the surface of the enzyme. loops called as T1 (connecting PB1 and PB2), T2 (PB2 to The cleft present on the surface of the enzyme is involved PB3) and T3 (PB3 to PB1) of another coil. It also contains a in substrate binding and active site formation, by con- α-helix at the end of N-terminal of parallel β-helix; long T3 taining several aromatic residues. Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 6 of 16 The active site region was found to contain three con - through simple activation of methyl ester by Asp178 by served aminoacid residues Asp178, Asp199 and Arg267. avoiding the nucleophilic assistance of Asp199, which According to Jenkins et  al. (2001), these two conserved might question the conservation of Asp199 residue and aspartate residues and their adjacent arginine residue are its stereochemical chemistry at the catalytic site across considered as the catalytic residues, as there is no other the pectin methyl ester family. Though Arg267 and substantial candidate to be determined as catalytic sites Trp269 residues are not directly involved in catalytic (Jenkins et  al. 2001). The aspartate residues are equally reaction, they are required for the substrate binding. The arranged on the neighboring coils, thus having same left- hydrolytic water molecule which is involved in hydroly- handed α-helices. The Asp199 was found to be in close sis of the acyl enzyme is observed during the low pH and contact with Arg267 which partially blocks Asp199 from time course adjacent to the Asp178 (general base). This the solvent and forms hydrogen bonds with NE and NH1 protein structure also suggests that Gln177 is involved in of the side chain oxygen atoms (Jenkins et al. 2001). The the formation of oxyanion hole and the kinetic analysis second aspartate residue Asp178 interacts with two water proves the role of Gln177 in stabilization of transition molecules which shields it form interacting with the sol- state; similarly, Gln153 is required for substrate binding vent molecule from glutamine residues (Gln153 and (Fries et al. 2007; Jenkins et al. 2001) (Fig. 2). Gln177) and with the β-carbon atom of Asp199 residue. The aromatic residues Tyr158, Tyr181 and Phe202 on the Aspergillus aculeatus (Rhamnogalacturonan acetylesterase exposed surface of PB1 forms a floor of valley at Tyr269 CE class‑12) of a T1 loop. With the Tyr181, Phe202 and Trp269 resi- The three-dimensional structure of the rhamnogalactu - dues are found to be conserved in most pectin methyl- ronan acetylesterase (RGAE) from Aspergillus aculeatus esterases sequences. Fries et  al. (2007) have performed was revealed by (Mølgaard et  al. 2000). Protein struc- site-directed mutagenesis of the pectin methylesterase ture of RGAE was determined by multiple isomorphous (E. chrysanthemi) to understand the functional prop- replacement and refined at 1.55  Å resolution (Mølgaard erties of aminoacid residues in catalytic site (Fries et  al. et  al. 2000). The protein structure of RGAE exhibited a 2007). This study has also reported that following resi - α/β/α fold containing a central five-stranded parallel dues Asp199, Asp178, Gln177 are involved in reaction β-sheet surrounded by α-helices, with a structural topol- mechanism, though Arg267 was found to be conserved ogy of central sheet to be found as—1X 2X1X1X with among all the pectin methyl esterases and is not directly two stranded antiparallel β-sheet insertion after the involved in enzyme catalysis (Fries et  al. 2007). The E. third β-strand. There are total of 11 helices in the protein chrysanthemi pemA was found to be active between pH structure containing four 3 helices (A, B, E and K). Due 5 and 9; however, at pH 5 pemA showed highest activity to the lack of backbone hydrogen bonds between Glu25- (Pitkänen et al. 1992). The catalytic Asp178 residue might Ala28 and Tyr26-Ser29, A and B are assigned as two sep- mostly be found in its protonated state due to its position arate helices. There are two disulfide bridges formed by in a hydrophobic environment, while the Asp199 occurs four cysteine residues, which are involved in linking the in deprotonated at neutral pH as it is accessible by the aminoacid residues 88–96, 214–232 with other disulfide solvents. The negative charge and the position of Asp199 bridges anchoring the C-terminal loop and α-helix. are favored by hydrophobic residues Arg267, Ala233, Though, electron density maps do not show any indica - Tyr230 and Val198 and direct Asp199 towards incoming tion of O-glycosylation sites, but it showed two N-glyco- substrate and solvent. The presence of hydrogen bonds sylation sites (Asn104 and Asn182) which allows a total between the Arg267 and Asp199 oxygen atom assists in of seven carbohydrate residues in the electron density maintaining the Asp199 residues deprotonated state. The map (Mølgaard et al. 2000). two glutamine residues Gln153 and Gln177 present near The active site inferred from the 3D structure of RAGE the Asp199 and Asp178 residues might contribute to the shows that Ser9 residue present on the topological point oxyanion hole (Fries et al. 2007; Johansson et al. 2002). at the end of β-strand is involved in forming the charac- These crystallographic studies have provided clear teristic hydrogen bonds in the catalytic triad with His195 insights on the reaction mechanism of pectin methyl and Asp192. The electron density maps showed that the esterase at atomic resolution (Fries et  al. 2007; Jenkins sulfite ion is bound to the active site of the RGAE protein et al. 2001). The Asp199 acts as nucleophile, as it directly with oxygen atom occupying the oxyanion hole through attacks the carbonyl carbon of methyl ester, with Asp178 making hydrogen bonds with main chain NH groups of acting as general acid–base in the reaction. Lack of water Ser9, Gly 42 and side chain amide of Asn74, which are molecule between Asp199 and substrate rules out the located in the loop regions next to carboxy ends of first earlier proposed mechanism (reaction is initiated by three strands in central β-sheet (Mølgaard et  al. 2000). activating the water molecule). The reaction proceeds The second sulfate oxygen atom is hydrogen bonded with Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 7 of 16 Erwinia chrysanthemi(PDBID-1QJV) (PMEA) Fig. 2 The protein secondary structure of Erwinia chrysanthemi pectin methyl esterase (carbohydrate esterase class-8) (PDB ID:1QJV ) (Jenkins et al. 2001), where alpha-helices (red), beta strands (blue) and coils (gray). (Image was developed using Discovery Studio Visualizer ) the imidazole group of His195, thus connecting four dif- the protein structure of RGAE violates this consensus ferent regions of the protein structure. These structural sequence as large side chains of methionine (Met11) 2+ studies have extensively revealed the structural localiza- located at N u position making it sterically impossible tion of catalytic triad residues. The Ser9 residue which to form a sharp turn which is mandatory for nucleo- acts as nucleophile is present in the type-I β turn, Asp192 phile elbow motif. Secondly, the most conserved α-helix and His195 are arranged in loop region which connects C present in α/β hydrolases is not conserved in the pro- the two helices near the C-terminal end. The position tein structure of RGAE (Schrag and Cygler 1997). The 3D of the active site makes it accessible for a wide range structure of RGAE also revealed that nucleophile back- of large substrates, as it is situated in the bottom of the bone conformation is unstrained, as it is situated on type- open cleft. Most of the arginine residues are situated in I β turn following a loop region. Although RGAE follows suitable positions for its interactions with the negatively similar sequential order of catalytic residues (Ser–Asp– charged carboxylate groups of the substrates (Mølgaard His) as α/β hydrolase fold, they differ in their location. et al. 2000). In α/β hydrolases, the Asp and His are located quite far The α/β hydrolase fold adopted by most of the neutral from each other in sequence; whereas, in RGAE they are lipases and esterases consists of a central parallel β-sheet only separated by two residues. RGAE differs from α/β (eight stranded) surrounded by α-helices (Ollis et  al. hydrolases with respect to the orientation of the catalytic 1992). Majorly, the catalytic triad residues from the α/β residues and the remaining protein structure. The cata - hydrolases follow an order of nucleophile–acid–histidine lytic triad residues in α/β hydrolases are arranged parallel and in most of the protein structures, the topology and to the central β-sheet; whereas in RGAE, these residues the positions of these residues are similar. The nucleo - are arranged perpendicularly to the central β-sheet. phile is situated at the end of 5th β-strand, acid is located However, these differences do not severely affect the cat - at the end of 7th β-strand and the histidine follows the alytic functions of the nucleophile on carbonyl carbon or loop region of 8th strand; thus, α/β hydrolase fold char- the enzyme (Mølgaard et al. 2000) (Fig. 3; Table 3). acteristic motif is also called as nucleophilic elbow (Derewenda and Derewenda 1991; Ollis et al. 1992). This Carbohydrate esterases for de‑esterification of lignin– nucleophile backbone attains a strained ε conformation carbohydrate complexes due to situation of nucleophile in a sharp turn between a Plants contain a range of hydroxycinnamic acids like β-strand and α-helix, the small helix (Sm) any residue (X) caffeic, p-coumaric, ferulic and sinapic acids which and nucleophile (Nu) residues are arranged specifically can be broadly classified as phenolic compounds and as Sm-X-Nu-X-Sm–Sm for forming a motif. However, highly abundant among foods (Guglielmetti et  al. 2008). Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 8 of 16 Aspergillus aculeatus (PDBID-1DEX) (Rhamnogalacturonan acetylesterase ) Fig. 3 The protein secondary structure of Aspergillus aculeatus rhamnogalacturonan acetylesterase (carbohydrate esterase class-12) (PDB ID:1DEX) (Mølgaard et al. 2000), where alpha-helices (red), beta strands (blue) and coils (gray). (Image was developed using Discovery Studio Visualizer ) Feruloyl esterases (or ferulic acid esterases) or cinnamoyl the fungal glucuronoyl esterase using a series of uronic esterases are carbohydrate esterase class of enzymes acids and their glycoside derivatives (Duranová et  al. which hydrolyze the ester linkages between hydroxycin- 2009). These studies have revealed the specificity of GE namic acids and plant cell-wall carbohydrates by releas- towards 4-O-methyl-d-glucuronic acid, its methyl esters ing ferulic and cinnamic acid (Topakas et  al. 2007). and d-glucuronic acid containing 4-nitrophenyl agly - Combinatorial usage of feruloyl esterase or cinnamoyl con, showing that GE attack the ester bonds between esterases with glycoside hydrolases for the liberation of 4-O-methyl-d-glucuronic acid of glucuronoxylan and free carbohydrate residues and phenolic acids can signifi - alcohols of lignin (Ďuranová et  al. 2009; Topakas et  al. cantly aid in different preprocessing steps of biofuel and 2010). Glucuronoyl esterase finds its applications in biorefining industries (Faulds 2010). The activity of FAE growing biofuel and biorefinery industries as it breaks and CAE are chiefly limited to the position and confor - down and separates the hemicellulose and lignin. mations of the feruloyl groups present in the feruloylated Pretreatment step is currently being used in bioethanol polysaccharides and other surrounding cell-wall compo- industries for releasing free carbohydrate residues from nents. Recent studies conducted by Faulds et  al. (2003, the other aromatic components of the cell wall. The het - 2006) have revealed the preferential partnership between erophenolic lignin compounds interacting with the poly- glycoside hydrolase class-11 (GH-11) xylanases and FAE saccharide units increase the recalcitrant nature of the for liberating ferulic acid from the insoluble biomass, plant cell wall and the percentage of lignin in plant tis- while partnership between GH-10 xylanases and FAE will sues is directly proportional to its digestibility. It has been liberate 5,5′dimers (Faulds et al. 2003, 2006). Glucuronoyl assumed that FAEs and CAEs are required for breaking esterases are the class of carbohydrate esterases involved the lignin and carbohydrate linkages. According to Benoit in hydrolysis of the ester linkages present between et  al. (2006), type-C and type-B FAEs isolated from 4-O-methyl-d-glucuronic acid residues of glucuronoxy - A. niger release higher proportions of ferulic acid and lans and aromatic alcohols of lignin (Špániková and Biely p-coumaric acid from the steam-exploded wheat straw 2006). Glucuronoyl esterase which is involved in plant (Benoit et  al. 2006). The type-A FAE from A. niger was cell-wall degradation was discovered in Schizophyllum found to be effective against the steam-exploded wheat commune for the first time (Špániková and Biely 2006). straw in the presence of cellulases and xylanases, and at Duranová et  al. (2009) have purified and characterized 50  °C the rate hydrolysis increased significantly (Tabka Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 9 of 16 et al. 2006). Similarly, Selig et al. (2008) have used a com- involved in forming the dimer interface where a total of bination of cellobiohydrolase Cel7A, xylanase, feruloyl 2373 Å is buried between these chains (Lai et al. 2011). esterase and acetyl xylan esterase and reported enhanced The 3PF8 is considered as a dimer interface protein due breakdown of the hot water-treated corn stover cellulose to (a) the primary hydrophobic interface formed by the (Selig et al. 2008). Apart from its long list of applications, 18/37 residues of chain A and chain B (b) the presence FAEs were also used for the utilization of straws in paper of six salt bridges. Total of 54 aminoacid residues Pro131 industries (Record et al. 2003; Tapin et al. 2006), detoxifi - to Gln184 are involved in formation of inserted α/β sub- cation of animal feed (Laszlo et al. 2006), for the removal domain which is arranged in between the β6 and β11 of cinnamic acids and p-coumaric acids from coffee pulp strands. (Benoit et  al. 2006). Thus, wide applications of FAE and The catalytic site of the 3PF8 resembles an open canal CAE in food, paper and pulp, biofuel industries make in the shape of boomerang ending with a hydrophobic these enzymes economically and commercially impor- pocket buried in between α5 and α6 of the inserted α/β tant enzymes (Table 4). subdomain (Lai et al. 2011). The Ser106 residue situated in the center of boomerang with two clefts present in Lactobacillus johnsonii (Cinnamoyl esterase CE class I) the catalytic site is of approximately 13 Å and 7 Å which The three-dimensional structures of native and mutant is large enough for accommodating the acyl groups of cinnamoyl esterase secreted by Lactobacillus johnsonii the aromatic substrates. The roof of the catalytic site is was revealed by (Lai et  al. 2011). The native apo cin - formed by two protruding hairpins of the inserted α/β namoyl esterase (3PF8) structure consists of central subdomain, with another cleft of 12  Å accommodating β-sheet containing seven parallel β-strands (β1, β3, β4, the alkoxyl group of other atoms present on the larger β5, β6, β11, β12) and one anti-parallel β-strand (β2). substrates. The catalytic triad of the catalytic site con - The central β-sheet exhibits a superhelical twist with sists of Ser106, His225 and Asp197, where the Ser106 120  °C angle from β1 to β2 strands, central β-strand is is situated at the nucleophile elbow formed by the β5 edged by five α-helices where two α-helices are located and α4. The backbone nitrogen atoms of Phe34 and externally (α1 and α9) and three α-helices are internally Gln107 are buried near the base of inserted α/β subdo- located (α3, α4 and α8) near the dimer interface. Two main, which constitutes for the oxyanion hole. The site- protein molecules of the 3PF8 are involved in exten- directed mutagenesis experiments conducted on the sive interface formation which consists of α4, α6 and catalytic triad by Lai et  al. (2011) revealed that Ser106, β1 strand. Lai et  al. (2011) have also reported the pro- Ala and Asp197, Ala mutants failed to exhibit the cata- tein interactions and surface assemblies analysis (PISA lytic activity; thus, Ser106, His225 and Asp197 consti- server); based on these results, it was revealed that 34 tute for the catalytic function of 3PF8 (Lai et  al. 2011). residues from chain A and 37 residues from chain B are The catalytic site analysis of 3PF8 has revealed that α/β Table 4 List of  the  structural and  functional properties of  lignin–carbohydrate complex deacetylating esterases belonging to carbohydrate esterases CE1, CE15 classes Strain, PDB ID, CE class Enzyme and fold Catalytic residues CATH and Pfam Substrates and Refs and reaction type Lactobacillus johnsonii, [3PF8], CE-1 Cinnamoyl esterase Ser106, His225, Asp197 3-Layer α/β sandwich Ferulic acid, Caffeic acid and ethyl α/β hydrolases Serine esterase Rossmann fold ferulate. Cinnamoyl esterase is α/β hydrolase family active against short acyl chain aliphatic esters and phenolic esters (Lai et al. 2011) Aspergillus niger, [1UWC], CE-1 Feruloyl esterase Ser133, His247, Asp194 3-Layer α/β sandwich Arabinoxylans (hydrolysis of feruloyl- α/β hydrolases Serine esterase Rossmann fold arabinose ester bond) pectin Lipase class 3 (feruloyl-galactose ester). High substrate specificity towards esters where feruloyl group is attached to C-5 of arabinose and inactive against C-2 esters (McAuley et al. 2004) Sporotrichum thermophile, [4G4J ], Glucuronoyl ester- Ser213, Glu236, His346 3-Layer α/β sandwich It has high specificity for hydrolyzing CE-15 ase, α/β hydrolases Serine hydrolase Rossmann fold the ester bonds of 4-O-methyl- d -glucuronic acid units of glucu- ronoxylan and alcohols of lignin (Charavgi et al. 2013) Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 10 of 16 Aspergillus niger (Feruloyl esterase CE class‑I) subdomain residues from Pro131 to Gln184 might play The three-dimensional structure of feruloyl esterase iso - a crucial role in substrate binding. Lai et al. (2011) have lated from A. niger was revealed by (McAuley et al. 2004). hypothesized that inserted α/β subdomain plays a criti- The protein structure of feruloyl esterase (FAE) contains cal role in holding the phenolic ring of phenolic esters two FAE-III molecules, two sulfate ions and 399 water in the appropriate position for the catalytic reaction; molecules. This protein contains the standard α/β hydro - however, its role diminishes when aliphatic esters were lase fold with five α-helices and two β-sheets surrounding used as the enzyme substrates. Trp160 residue situ- the central core (nine β-sheets). FAE-III is glycosylated at ated on the second hairpin formed between the β9 and Asp79 residue and at every glycosylation site, a N-acetyl β10 plays an important role in fixing the substrate as glucosamine was observed. The clear and organized elec - the rotation of Trp160 forms a tunnel for burying the tron density maps of FAE-III prove that NAG residues ferulic acid in the catalytic site. In addition, the side were not attached to any other carbohydrate residues. chains of Asp138 and Trp169 are involved in forming The catalytic site of the FAE-III contains Ser133, Asp194 hydrogen bonds with the hydroxyl groups of aromatic and His247; His247 is well organized to form hydrogen rings of ferulic acid and caffeic acid in the catalytic bonds with other two residues (Ser133 and Asp194); N pocket of 3PF8. It was reported that substrate orienta- atom forms hydrogen bonds with O atom of Ser133; and tion in the 3PF8 catalytic site plays a critical role in its similarly, N connects with the Asp194 through hydro- catalysis; thus, appropriate orientation by the substrate gen bonding. This characteristic arrangement of catalytic is acquired through its interactions with the enzyme triad residues denotes that enzymes involved breakdown (aromatic substituents on one end of ligand and ester of amide or ester bonds through nucleophile attack. The groups on another end). These interactions cease the serine residue (Ser133) performs nucleophilic attack on carbonyl group at the oxyanion hole, thus allowing the the carbonyl atom of the substrate molecule and His247 molecule to bend. Contrastingly, the functional groups residue deprotonates Ser133 residue by acting as general other than ester groups such as alkoxy groups of feru- base and finally, the positive charge on the His247 is sta - lic acid/ethyl ferulate, quinic acid group of chlorogenic bilized by the negatively charged Asp194 residue through acid do not exhibit any role in substrate binding (Lai electrostatic interaction. This catalytic reaction results in et al. 2011) (Fig. 4). Lactobacillus johnsonii (PDBID-3PF8) Cinnamoyl esterase Fig. 4 The protein secondary structure of Lactobacillus johnsonii cinnamoyl esterase (carbohydrate esterase class-I) (PDB ID:3PF8) (Lai et al. 2011), where alpha-helices (red), beta strands (blue) and coils (gray). (Image was developed using Discovery Studio Visualizer ) Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 11 of 16 a tetrahedral transition state which is further collapsed helix in two different conformations in FAE-III is ques - to give an acyl-enzyme intermediate. This intermedi - tionable as it lacks the lipase activity (Aliwan et al. 1999). ate is attacked by the nucleophilic water molecule which Although FAE-III and lipases are similar in their over- was in turn activated by the histidine residue. The second all structure, difference in their active site regions was tetrahedral transition intermediate is formed due to the expected as they accommodate different substrates. FAE- release of acid; the oxyanion formed by the backbone N III contains large number of polar amino acids exposed atoms of Thr68 and Leu134 residues stabilizes the nega - around the catalytic site, but it is expected that lipases tive charge on O atom of carbonyl group present in the contain hydrophobic residues on its surface. The interac - tetrahedral transition state. tion between ferulic acid and FAE-III happens through The two molecules of FAE-III contain the well-organ - OH, CH3 side chains and Tyr80 hydroxyl groups. The ized active site residues having single conformation to carboxylate group is situated near the oxyanion hole the catalytic triad and with the known hydrogen bond- near the catalytic site serine residue, main chain N atom ing patterns between residues (Hedstrom 2002; McAuley (Leu134) and backbone N and OH atoms of Thr68. The et al. 2004). The tertiary structure of FAE-III is similar to presence of Leu199 and Ile196 residues provides a hydro- other fungal lipases, as the rmsd values between the FAE- phobic environment. McAuley et al. (2004) have reported III and RmL (Rhizomucor miehei), FAE-III and TIL (Ther - that residues involved in binding to the ferulic acid were momyces lanuginosa) were 1.0  Å (over 217 Cα atoms) found to be similar in both native and complexed struc- and 1.3 Å (over 205 Cα atoms). Based on the conforma- tures of FAE-III. This study has also reported that feru - tion states, the lipases are classified as active and inac - lic acid binds to the proteins in an unproductive way as tive forms; the catalytic residues are exposed in active it contains third molecule of ferulic acid present in the form and in inactive forms, these residues are closed by FAE-III complex at approximately 20 Å distance from the a helical lid. FAE-III residues ranging from 71 to 77 are catalytic site. Ferulic acid interacts with AsnA223 of mol- involved in formation of the helical lid but it does not ecule A and AspB217 of molecule B of other asymmet- cover the active site and functions as an active form. ric unit through a water molecule (McAuley et  al. 2004) According to Aliwan et  al. (1999), the occurrence of the (Fig. 5). Aspergillus niger (PDBID: 1UWC) Feruloyl esterase Fig. 5 The protein secondary structure of Aspergillus niger feruloyl esterase (carbohydrate esterase class-I) (PDB ID:1UWC) (McAuley et al. 2004), where alpha-helices (red), beta strands (blue) and coils (gray). (Image was developed using Discovery Studio Visualizer ) Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 12 of 16 Myceliophthora thermophile (Glucuronoyl esterase) sequence (Ser–Asp–His). The Ser213 situated between Three-dimensional structure of glucuronoyl esterase the S6 strand and H6 helix (forming a characteristic isolated from Sporotrichum thermophile (StGE2) was nucleophilic elbow) performs nucleophilic attack on the explained by (Charavgi et  al. 2013). The StGE2 is a ser - substrate, His346 acts as acid/base suggesting the nature ine-type hydrolase family containing a three-layer αβα- of the substrate and Glu236 stabilizes the positive charge sandwich hydrolase fold with a Rossmann-fold topology of His346. The structural organization and conforma - (Charavgi et al. 2013). Though StGE2 structure belongs to tion of the catalytic triad are stabilized by the internal α/β hydrolase superfamily, it deviates from the standard hydrogen bonds formed between Ser213 hydroxyl group α/β hydrolase fold. The β-sheet of StGE2 is expanded due and NE2 of His346, OE1 of Glu236 and ND1 of His346 to the insertion of two antiparallel β-strands at the N-ter- (Charavgi et al. 2013). The catalytic triad is also stabilized minus of StGE2 forming a twisted β-sheet. This twisted by the direct and water-mediated polar linkages formed β-sheet is sandwiched between 18 helices arranged in by Arg214, Gly216, Lys217, Gln235, Phe304 and Asn306 two layers, eight α-helices and 3 helices are arranged on residues. Along with these interactions, the disulfide one side of the β-sheet and on the opposite side, another bridges formed by the Cys347 and Cys212 also enhance set of eight α-helices and 3 helices are equally divided. the rigidity of the catalytic site by bridging Ser213 and However, the standard α/β hydrolase fold exhibits eight His346 residues. Active site of StGE2 is located on the strands of β-sheets in the central core of the structure surface of the molecule and exposed to the solvent mol- sandwiched between two clusters of α-helices (Charavgi ecules and unaffected by other interactions. The site et  al. 2013; Ollis et  al. 1992) (Fig.  6). The StGE2 struc - directed mutagenesis experiments of Ser213 residue to ture is stabilized by three disulfide bonds (Cys31–Cys65, understand the structural and functional properties of Cys212–Cys347 and Cys244–Cys319). The catalytic triad StGE2 (Charavgi et al. 2013). However, the superposition of StGE2 is represented by Ser213, Glu236 and His346, of StGE2 with the S213A mutant structures on C atoms where it deviates from the standard catalytic triad with a rmsd value of 0.4  Å shows that both StGE2 and Sporotrichum thermophile (PDBID: 4G4G) glucuronoyl esterase Fig. 6 The protein secondary structure of Sporotrichum thermophile glucuronoyl esterase (carbohydrate esterase class-I) (PDB ID:4G4G) (Charavgi et al. 2013), where alpha-helices (red), beta strands (blue) and coils (gray). (Image was developed using Discovery Studio Visualizer ) Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 13 of 16 S213A mutants differ slightly. Similarly, the superposition dehydration, sterilization and pasteurization lead to of S213A mutant complexed with methyl 4-O-methyl- severe physical damage of plant cellular tissues including β-d-glucopyranuronate (MCU) with StGE2 and S213A primary cell wall and middle lamella. However, recently, mutant crystals showed that substrate analog of MCU these traditional methods were replaced with vacuum binds to the active site without disturbing the struc- infusion methods using pectin methyl esterases and cal- ture of the protein. The RMSD values obtained from the cium chloride mixture to overcome the negative effects superposition of S213A mutant bound to MCU with the of fruits- and vegetables-processing methods (Suutar- S213A mutant and StGE2 on C atoms was 0.40  Å and inen et  al. 2000, 2002). Based on its degree of esterifica - 0.12  Å. The residues present around and in the catalytic tion, pectin is divided into low-methoxylated (LM) and 2 2 triad with surface areas 27.1 Å (Ser213), 2.7 Å (Glu236) high-methoxylated (HM) (Kohli et  al. 2015). The HM and 75.7 Å (His346) are exposed to the solvent. The site- pectin is generally used for the production of high-sugar directed mutagenesis experiment on Ser213 with Ala jams and jellies. As the HM pectin follows a hydropho- residue has confirmed that lack of the hydroxyl group did bic interaction and dehydration at low pH for its gelling not affect the catalytic site conformation. and to achieve proper gelling of the HM pectin, higher The structure analysis of S213A mutant bound with sugar concentrations are essential (Kohli et  al. 2015). MCU has revealed the catalytic function of the StGE2 Contrastingly, LM pectin follows different gelling mecha - protein. The substrate MCU is bound to the catalytic nisms through ionic interactions as it contains calcium site residues through seven direct, four indirect and 59 (divalent cations) which interacts with the free carboxylic van der Waals interactions (Charavgi et  al. 2013). The acid groups resulting in a successful cross linkage; there- MCU analog binds to the cavity through its O atom of fore, LM pectin does not require higher concentrations the hydroxyl group present on the glucopyranose ring; of sugar for gelling. Thus, industrially LM pectin is used the side chains of Gln259 and Glu267 residues of S213A for the production of low-sugar jellies and jams suit- mutant interact with O1, O2 and O3 of sugar by hydro- able for the consumption of diabetic patients (Kohli et al. gen bonding. When compared to the structure of StGE2, 2015). The apple- and orange-peel wastes are rich in pec - the Glu267 side chain residue conformation in S213A tin; thus, it is industrially used as gelling and thickening mutant differs by dihedral angle of ~ 74° supporting the agents. However, the degree of esterification significantly strong binding of the substrate analog, whereas StGE2 affects the thickening and gelling property of the pec - Glu267 is arranged at dihedral angle of ~ 127°. The oxy - tin. Pectin methyl esterases are used for retrieving pec- gen groups present on the substrate interact with resi- tin with lower degree of esterification (Kohli et  al. 2015; dues Trp310 NE1 (O2) and Lys217 NZ (O3 and O4) of Morris et al. 2000). the protein (Charavgi et  al. 2013). Similarly, O6a which Industrially, feruloyl esterases are applied in various relates to the ester bond interacts with NE2 atom present biotechnological processes such as production of plat- on the imidazole ring of His 346. The interactions among form chemicals, fuel, animal feed, textile, paper and pulp, the N atom of Arg214 and its side chains with the meth- food processing, agriculture and pharmaceutical indus- oxy group (O4 atom) and ester group (O6 atom) which tries (Fazary and Ju 2013). In the last few decades, several further forms hydrogen bonds with Ser213 OG suggests studies were being conducted to understand and increase that Arg214 is involved in the oxyanion hole formation the activity of enzymes used in paper and pulp indus- (Charavgi et al. 2013) (Fig. 6). tries. Enzyme mixtures containing feruloyl esterases and acetyl xylan esterases enhance the enzymatic activity of Commercial significance of pectin and lignin de‑esterases hemicellulases and cellulases, by breaking down the sub- Commercially, pectin methyl esterases are majorly stitutions and linkages between lignin and carbohydrate applied in association with pectinases in food and bev- complexes and solubilize the complex (Fazary and Ju erage processing industries. The presence of pectin leads 2013; Mathew and Abraham 2004; Topakas et  al. 2004). to cloudiness of the fruit juices; pectinase preparations The lignocellulolytic enzyme mixtures including feruloyl are applied industrially for clarifying and reducing the esterases, secreted by fungi, were being applied for the viscosity of fruit juices and eases the process of concen- pretreatment of animal feedstock. Pretreatment of ani- tration and filtration (Demir et  al. 2001). Pectinases are mal feedstock with the lignocellulolytic enzyme mixtures applied in combinations with cellulases; hemicellulases were significantly found to increase the activity of rumen were applied for the disruption of plant cell walls and microbiota by 80%, thus increasing the rate of digestion commercially used to produce juices from tropical fruits (Mathew and Abraham 2004; Tarbouriech et  al. 2005; (Alkorta et  al. 1998; Massiot et  al. 1997; Wicker et  al. Topakas et al. 2004). Major applications of feruloyl ester- 2002). Industrial processes used for preservation of fruits ases were reported in food and pharmaceutical indus- and vegetables such as blanching, washing, freezing, tries by converting cinnamic acid/ferulic acid and other Sista Kameshwar and Qin Bioresour. Bioprocess. (2018) 5:43 Page 14 of 16 phenolic compounds into commercially valuable prod- these de-esterifying enzymes are highly significant as ucts like vanillin (Fazary and Ju 2013). Feruloyl esterases they are involved in the production of commercially are also applied for improving the quality of bread, prepa- valuable products ranging from food, beverage, tex- ration and clarification of juices, synthesis of oligosaccha - tile, pulp and paper, biofuel and pharmaceutical indus- rides and production of medicinal compounds (Fazary tries. Continuous studies were being conducted to and Ju 2013). Glucuronoyl esterases are active against a understand and reveal the catalytic mechanisms and to wide range of natural and synthetic lignin–carbohydrate develop an efficient set of de-esterifying enzymes with complexes (Hüttner et al. 2017). Specifically, glucuronoyl high rate of substrate hydrolysis. In this article, we have esterases cleave ester linkages between the lignin and discussed specifically about the structural and func - glucuronoxylan complexes (Arnling Bååth et  al. 2016). tional aspects of pectin (pectin methyl esterase) and uTh s, glucuronoyl esterases share various industrial lignin–carbohydrate complex (feruloyl/cinnamoyl and applications in common with feruloyl esterases. Glucu- glucuronoyl esterases) de-esterifying enzymes. We have ronoyl esterases are primarily used in biofuel industries extensively conversed by comparing the structures of E. in combinations with other lignocellulolytic enzymes chrysanthemi and A. aculeatus for pectin esterases, L. for conversion of plant biomass into fuel. Studies have johnsonii, A. niger for feruloyl/cinnamoyl esterases and reported that the presence of glucuronoyl esterases in S. thermophile for glucuronoyl esterase. Understand- the enzyme mixture has significantly improved the rate ing the active catalytic residues of these de-esterifying of substrate hydrolysis (d’Errico et al. 2016). Glucuronoyl enzymes will significantly help in developing recom - esterases are also applied in food and beverage industries binantly active enzymes with higher substrate hydrol- for the clarification and concentration of fruit juices and ysis rates. It is highly necessary to understand the wine. Glucuronoyl esterases are also applied for syntheti- structural and functional properties of plant biomass cally altering glucuronic acid derivatives for developing de-esterifying enzymes, as they stand on first place in nonionic surfactants and other bioactive substances with bioconversion of plant biomass into commercially valu- great applications in the pharmaceutical (anti tumorous able products. prodrugs) and cosmetic preparations (De Graaf et  al. 2004). Studies were continuously being conducted for Abbreviations developing recombinant plants-vulnerable plant cell-wall CE: carbohydrate esterases; CAZy: carbohydrate active enzymes; PDB: protein components (Lionetti et  al. 2010; Sticklen 2006); paral- data bank; PME: pectin methyl esterase; FAE: ferulic acid esterases (or) feruloyl esterases; CAE: cinnamic acid esterase (or) cinnamoyl esterase; StGE2: Sporotri- lelly, studies were also reported to develop the recombi- chum thermophile glucuronoyl esterase 2; HM: highly methoxylated; LM: low nant enzymes with higher rate of hydrolysis and greater methoxylated. substrate-binding abilities (Himmel et  al. 2007; Mar- Authors’ contributions tinez et  al. 2009). In this article, we have listed some of AKS is a Ph.D. research fellow wrote the manuscript. WQ was the principal the state-of-the-art articles based on enzyme engineering supervisor who supervised in writing the manuscript and provided comments and developing efficient recombinant enzymes (Bloom and revisions to the manuscript. Both authors read and approved the final manuscript. et  al. 2005; Chen et  al. 2018; Dalby 2007; Damborsky and Brezovsky 2014; Gaj et  al. 2013; Jørgensen et  al. 2007; Martinez et al. 2009; Taylor et al. 2001; Tischer and Acknowledgements Not applicable. Wedekind 1999). Competing interests The authors declare that they have no competing interests. Conclusions In nature, plant cell-wall components occur in esterified Availability of data and materials The data presented and supporting the conclusion of our manuscript were form a type defensive mechanism adapted for inhibit- mostly presented in the form of text; structural images and functional proper- ing the activity of enzymes secreted by invading micro- ties were previously reported and published. The PDB IDs and structural and organisms. However, these invading microorganisms functional properties reported in our manuscript were cited in the manuscript. have evolved over the course of time and developed an Consent for publication efficient enzyme system for the de-esterification of the Not applicable. plant cell-wall components. Pectin methyl esterases, Ethics approval and consent to participate feruloyl/cinnamoyl esterases and glucuronoyl ester- Not applicable. ases play a significant role in plant biomass conversion by de-esterifying pectin and lignin–carbohydrate com- Funding This work was supported by Natural Sciences and Engineering Research plexes and provide an accessible substrate for the act- Council of Canada Funding (RGPIN-2017-05366) to Wensheng Qin and Ontario ing enzymes secreted by microorganisms. Industrially, Trillium Scholarship (OTS) to Ayyappa Kumar Sista Kameshwar. Sista Kameshwar and Qin Bioresour. Bioprocess. 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"Bioresources and Bioprocessing"Springer Journals

Published: Dec 1, 2018

Keywords: Biochemical Engineering; Environmental Engineering/Biotechnology; Industrial and Production Engineering

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