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Biochemical properties of a new thermo- and solvent-stable xylanase recovered using three phase partitioning from the extract of Bacillus oceanisediminis strain SJ3

Biochemical properties of a new thermo- and solvent-stable xylanase recovered using three phase... The present study investigates the production and partial biochemical characterization of an extracellular thermo‑ stable xylanase from the Bacillus oceanisediminis strain SJ3 newly recovered from Algerian soil using three phase partitioning ( TPP). The maximum xylanase activity recorded after 2 days of incubation at 37 °C was 20.24 U/ml in the presence of oat spelt xylan. The results indicated that the enzyme recovered in the middle phase of TPP system using the optimum parameters were determined as 50% ammonium sulfate saturation with 1.0:1.5 ratio of crude extract: t‑ butanol at pH and temperature of 8.0 and 10 °C, respectively. The xylanase was recovered with 3.48 purification fold and 107% activity recovery. The enzyme was optimally active at pH 7.0 and was stable over a broad pH range of 5.0–10. The optimum temperature for xylanase activity was 55 °C and the half‑ life time at this temperature was of 6 h. At this time point the enzyme retained 50% of its activity after incubation for 2 h at 95 °C. The crude enzyme resist to sodium dodecyl sulfate and β‑ mercaptoethanol, while all the tested ions do not affect the activity of the enzyme. The recovered enzyme is, at least, stable in tested organic solvents except in propanol where a reduction of 46.5% was observed. Further, the stability of the xylanase was higher in hydrophobic solvents where a maximum stability was observed with cyclohexane. These properties make this enzyme to be highly thermostable and may be suggested as a potential candidate for application in some industrial processes. To the best of our knowledge, this is the first report of xylanase activity and recoverey using three phase partitioning from B. oceanisediminis. Keywords: Bacillus oceanisediminis, Xylanase, Thermostability, Hydrophobic solvents, Industrial processes, Three phase partitioning requires the concerted action of xylanolytic enzymes Background (Trajano et al. 2014; Zhang and Viikari 2014). Xylans are Hemicellulose is the second most abundant renewable heterogeneous polysaccharides with a backbone consist- biomass after cellulose in nature (Collins et  al. 2005). ing of β-1,4 linked d-xylosyl residues. Xylan is the major component of hemicelluloses in wood Endo-β-1,4 xylanases (EC 3.2.1.8) are the main from angiosperms, where it accounts for 15–30% of the enzymes responsible for cleavage of the linkages within total dry weight. In gymnosperms, however, xylans con- the xylan backbone (Collins et  al. 2005), to which short tribute only 7–12% of the total dry weight. The structure side chains of O-acetyl, α-l-arabinofuranosyl, d-α glu- of xylan is complex, and its complete biodegradation curonic, and phenolic acid residues are attached (Col- lins et  al. 2005; Terrasan et  al. 2010; Xie et  al. 2015). *Correspondence: mohammed.gagaoua@inra.fr; gmber2001@yahoo.fr Xylanases have been used in a wide range of industrial UMR1213 Herbivores, INRA, VetAgro Sup, Clermont Université, Université applications and processes. They have been applied in de Lyon, 63122 Saint‑Genès‑Champanelle, France Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/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. Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 2 of 12 the bioconversion of lignocellulosic material and agro- organic phase that usually contains t-butanol (Gagaoua wastes to fermentative products, clarification of juices, and Hafid 2016). improvement in consistency of beer, and the digest- ibility of animal feed stock (Badhan et  al. 2007; Elgharbi Methods et  al. 2015a, b; Shameer 2016; Jain and Krishnan 2017). Substrates, reagents, and chemicals Due to their important activity at alkaline pH (8.0–11) Birchwood xylan, oat spelt xylan, starch, carboxymethyl and high temperature (50–90  °C), thermostable alkaline cellulose (CMC, low viscosity), tert-butanol, ammonium xylanases have attracted special attention in the pulp bio- sulfate, and 3,5-dinitrosalicylic acid (DNS) were pur- bleaching industry (Techapun et al. 2003; Bouacem et al. chased from Sigma Chemical Company (St. Louis, MO, 2014; Boucherba et  al. 2014; Bouanane-Darenfed et  al. USA). Unless otherwise specified, all other reagents and 2016). Xylanase, together with other hydrolytic enzymes, chemicals were of the analytical grade or highest level of have also proved useful for the generation of bio-fuels, purity available. including ethanol, from lignocellulosic biomass. Xyla- nases are used in pulp pre-bleaching process to remove Collection of samples and culture conditions the hemicelluloses, which bind to the pulp. The hydroly - of microorganisms sis of pulp bound hemicelluloses releases the lignin in The garden soil samples were collected from Bejaia north the pulp, reducing the amount of chlorine required for east of Algeria (Kabylia region) in March 2015. The soil conventional chemical bleaching and minimizing the was collected from five places and samples were pooled. toxic, chloroorganic waste. Therefore, xylanases from Sub-samples of approximately 1  g were suspended in alkalophilic bacteria and actinomycetes and fungi have 100 ml sterile distilled water. Mixtures were allowed to set- been studied widely (Perez-Rodriguez et  al. 2014; Wang tle and serial dilutions were prepared. From each dilution, et al. 2014). However, large scale cultivation of fungi and 0.1  ml was taken and spread on agar plates of medium actinomycetes is often difficult because of their slow gen - containing in g/l oat spelt xylan 10, yeast extract 2, NaCl eration time, coproduction of highly viscous polymers, 2.5, NH Cl 5, K H PO 15, Na HPO 30, MgSO ·7H O 4 2 4 2 4 4 2 and poor oxygen transfer (Wong et  al. 1997; Garg et  al. 0.25, and bacteriological agar 15. In this medium, there 2011). Bacillus genus is used more extensively than other is a little modification of the main carbon source, the oat bacteria in industrial fermentations, since they produce spelt xylan was used instead the birchwood xylan (Viet most of their enzymes. Some Bacillus strains have been et  al. 1991). The plates were incubated at pH 7 and 37 °C reported as xylanolytic enzymes producers (Lindner et al. for 2 days at 250 rpm. Those colonies that grew well under 1994; Seo et  al. 2013; Tarayre et  al. 2013; Elgharbi et  al. such conditions and showed an orange zone around the 2015a; Zouari et al. 2015). colonies after red Congo were retained for second screen- Bacillus oceanisediminis sp. nov. was first isolated ing. Colonies with a clear zone formation following the from a marine sediment collected in the South Sea of hydrolysis of xylan were evaluated as xylanase producers. China (Zhang et  al. 2010). Considering the above, the Several xylanlolytic strains were isolated and SJ3, which present study was undertaken to described, for the first exhibited a large clear zone of hydrolysis, was selected and time, the production of a thermostable xylanase from B. retained for further experimental study. oceanisediminis strain SJ3 recently isolated by our labo- ratory from Algerian soil, an attempt was made to bio- Bacterial identification of the isolate SJ3 chemically characterize the xylanase activity secreted by Analytical profile index (API) strip tests and 16S rRNA this strain. Also, preliminary investigation using three gene sequencing were carried out for the identification of phase partitioning (TPP) system (Gagaoua et  al. 2014; the genus to which the strain belong. Gagaoua and Hafid 2016) for xylanase purification was API 50 CHB/E and the API 20E strips (bioMérieux, SA, performed. In TPP process, firstly an inorganic salt (gen - Marcy-l’Etoile, France) were used to investigate the phys- erally ammonium sulfate) is added to the crude extract iological and biochemical characteristics of strain SJ3, containing proteins then mixted with tert-butanol in an as recommended elsewhere (Logan and Berkeley 1984). appropriate amount (Gagaoua et  al. 2015, 2016, 2017). The growth temperature (4, 10, 15, 20, 25, 30, 35, 40, and When t-butanol is added in the presence of ammo- 45 °C), pH level values (4, 5, 6, 7, 8, 9, 10, 11, and 12) and nium sulfate, it pushes the protein out of the solution. sodium chloride regimes were determined. In this process t-butanol binds to hydrophobic part of The 16S rRNA gene was amplified by PCR using for - the proteins to reduce the density of the proteins, lead- ward primer F-d1 5′-AGAGTTTGATCCTGGCTCA ing to float above the denser aqueous salt phase. Within G-3′, and reverse primer R-d1 5′-AAGGAGGTGATCCAA approximately an hour, it forms an interfacial (mid- GCC-3′, designed from base positions 8–27 and 1541– dle) precipitate between the lower aqueous and upper 1525, respectively, which were the conserved zones Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 3 of 12 within the rRNA operon of Escherichia coli (Gurtler and then boiled for 5 min and cooled. Absorption was meas- Stanisich 1996). The genomic DNA of strain SJ3 was ured at 540 nm. purified using the Wizard Genomic DNA Purification One unit of xylanase activity was defined as the amount Kit (Promega, Madison, WI, USA) and then used as a of enzyme that released 1 µmol of reducing sugar equiva- template for PCR amplification (30 cycles, 94 °C for 45 s lent to xylose per min under the assay conditions. denaturation, 60 °C for 45 s primer annealing, and 72 °C for 60  s extension). The amplified  ~1.5  kb PCR prod - Xylanase production uct was cloned in the pGEM-T Easy vector (Promega, Gowth condition of the xylanase activity Madison, WI, USA), leading to pSJ3-16S plasmid (this To study the properties of the xylanase activity produc- study). The E. coli DH5α (F supE44 Φ80 δlacZ ΔM15 tion, the isolates having high xylanase activities were − + Δ(lacZYA-argF) U169 endA1 recA1 hsdR17 (r , m ) cultivated in 250  ml shake-flasks containing 50  ml basic k k deoR thi-1 λ gyrA96 relA1) (Invitrogen, Carlsbad, CA, xylanase production medium at 37 °C. The basic xylanase USA) was used as a host strain. All recombinant clones production medium was prepared at pH 7.0 containing of E. coli were grown in Luria–Bertani (LB) broth media oat spelt xylan. The culture was harvested after 48 h, and with the addition of ampicillin, isopropyl-thio-β-d- centrifuged (10,000 rpm for 10 min). Growth was meas- galactopyranoside (IPTG), and X-gal for screening. DNA ured by determining absorbance at 600  nm. The sample electrophoresis, DNA purification, restriction, ligation, was then kept at 4 °C in the refrigerator. and transformation were all performed according to the method previously described elsewhere (Sambrook et al. Effect of incubation time on xylanase production 1989). Pre-culture (2%) was used to inoculate 250  ml xylan defined medium at 37  °C for 72  h. culture samples were DNA sequencing and molecular phylogenetic analysis collected each 4  h during the cultivation period. Imme- The nucleotide sequences of the cloned 16S rRNA gene diately after collection, the samples were centrifuged at were determined on both strands using BigDye Termina- 4 °C and 10,000g for 20 min. Supernatants were analyzed tor Cycle Sequencing Ready Reaction kits and the auto- for xylanase activity as described above. mated DNA sequencer ABI PRISM 3100-Avant Genetic Analyser (Applied Biosystems, Foster City, CA, USA. The Partial biochemical characterization of the recovered RapidSeq36_POP6 run module was used, and the sam- enzyme by TPP ples were analyzed using the ABI sequencing analysis Extraction and partial purification of xylanase by TPP software v. 3.7 NT. Aqueous systems such as three phase partitioning (TPP), The sequences obtained were compared to those pre - known as simple, economical and quick methods, were sent in the public sequence databases and with the described for the fast recovery of enzymes (Gagaoua and EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/), Hafid 2016). This elegant non-chromatographic tool may a web-based tool for the identification of prokaryotes be performed in a purification process to be used suc - based on 16S rRNA gene sequences from type strains cessfully in food or other industries. For its application in (Kim et al. 2012). this study, the crude extract was first collected after 48 h Phylogenetic and molecular evolutionary genetic anal- of batch incubation (Boucherba et  al. 2014). The culture yses were conducted via the the molecular evolutionary supernatant containing secreted xylanases was concen- genetics analysis (MEGA) software version 5 (http:// trated using Sartorius membranes (with 10-kDa cutoff www.megasoftware.net). Distances and clustering were membrane; Millipore) after a centrifugation at 10,000 rpm calculated using the neighbor-joining method. The tree for 10  min. Then, TPP experiments were carried out fol - topology of the neighbor-joining data was evaluated by lowing the recommendations of Gagaoua et  al. (2015). Bootstrap analysis with 100 re-samplings. The enzyme exclusively recovered in the interfacial phase was gently separated from the other phases and dissolved Xylanase assay in 50  mM Tris–HCl buffer (pH 8.5) and dialyzed over - Xylanase activity was determined by measuring the night at 4–5 °C and used for enzyme characterization. release of reducing sugar from soluble xylan using the DNS method (Miller 1959). In brief, 0.9  ml buffer A Effect of temperature and pH on xylanase activity (10  mg/ml oat spelt xylan in 50  mM sodium-phosphate Optimal temperature was determined by assaying the buffer at pH 7) were mixed with 0.1 ml of the recovered enzyme activity between 20 and 100 °C, by incubating the enzyme solution (1 mg/ml). After incubation at 55 °C for enzyme along with the substrate for 10 min at the respec- 10 min, the reaction was terminated by adding 1.5 ml of tive temperature. Relative xylanase activity was deter- the DNS reagent (Maalej et  al. 2009). The mixture was mined using 10 mg/ml oat spelt xylan at various pHs. The Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 4 of 12 pH range used varied from 4 to 10. Three different buff - experimental results were expressed as the mean of the rep- ers (50  mM) were used. Sodium acetate buffer was used licate determinations and standard deviation (mean ± SD). for pH 4–6; Sodium-phosphate buffer was used for pH The statistical significance was evaluated using t tests for from 6 to 7 and Tris–HCl buffer for pH 7–10. two-sample comparison and one-way analysis of variance (ANOVA) followed by Duncan test. The results were con - Effect of temperature on xylanase stability sidered statistically significant for P values of less than 0.05. The thermostability was determined at temperatures of The statistical analysis was performed using the R package 50, 55, 60, and 95 °C, after incubation with the substrate Version 3.1.1 (Vanderbilt University, USA). for different times (from 0.5 to 7  h); remaining xylanase activity was measured under standard assay conditions. Nucleotide sequence accession number The non-heated enzyme, which was left at room temper - The data reported in this work for the nucleotide ature, was considered as control (100%). sequence of the 16S rRNA (1089  bp) gene of the isolate SJ3 have been deposited in the DDBJ/EMBL/GenBank Effect of pH on xylanase stability databases under Accession Number KT222887. For pH stability, the enzyme was incubated with differ - ent buffers viz. 50  mM acetate buffer for pH range 4–6, Results and discussion 50  mM phosphate buffer for pH range 6–7, and 50  mM Screening of xylanase‑producing bacteria from Algerain Tris–HCl buffer for pH range 7–10 at 55  °C for 1  h. soil and molecular characterization of the target Thereafter, enzyme activity was determined using the microorganism enzyme assay as described above. In the current study, ten candidates were obtained from the first screening as xylanase producers. Among them, Effect of metal ions and reagents on activity a bacterium called SJ3, displayed the highest extracellu- The effect of metallic ions at concentration of 5  mM, lar xylanase activity after 2  days incubation in an initial chelating agents, surfactants, and inhibitors on the activ- medium (data not shown) and was, therefore, retained ity of crude xylanase were determined by preincubating for all subsequent studies. + 2+ 2+ 2+ the enzyme in the presence of Na, Mg, Ca, Mn , The physiological and biochemical characteristics of 2+ 2+ 2+ + 2+ 2+ Fe, Zn, Cu , K , Hg , and Cd , EDTA (5  Mm), the SJ3 isolate presented in this study were investigated SDS (1%), β-mercaptoethanol (20 mM), and Triton X-100 according to well-established protocols and criteria (1%) for 30  min at 55  °C before adding the substrate described in the Bergey’s Manual of Systematic Bacteri- (Ozcan et al. 2011). Subsequently, relative xylanase activi- ology as well as the API 50 CHB/E and the API 20E gal- ties were measured at standard enzyme assay conditions. leries for representative strains. The findings indicated Relative activity was expressed as the percentage of the that the SJ3 isolate was Gram-stain-positive, motile, activity observed in the absence of any compound. rod-shaped, catalase-positive, aerobic, and endospore forming microorganism. Optimal growth temperature Activity of crude enzyme on various carbohydrate substrate was 37  °C; optimal pH was 7.0. According to the results The presence of other carbohydrase was analyzed using obtained using the API 50 CHB/E medium and the API oat spelt xylan, birchwood xylan, starch, and CMC 20E strips, the characteristics strongly confirmed that the (10 mg/ml). The reducing sugar released during the assay isolate belongs to Bacillaceae order and Bacillus genus. was quantified by spectroscopy at λ . The physiological and some biochemical properties of the isolate SJ3 are given in Table 1. Effect of organic solvents on xylanase activity The 16S rRNA gene sequence (KT222887) obtained Cell free supernatant having maximum xylanase activ- was submitted to GenBank BLAST search analyses, ity was incubated with 30% (v/v) of different organic which yielded a strong homology of up to 99% with those solvents, namely, acetone, propanol, ethanol, metha- of several cultivated strains of Bacillus. From the analy- nol, chloroform, heptane, cyclohexane, and toluene sis of the almost-complete 16S rRNA gene sequence, for 30  min at 55  °C. The residual xylanase activity was this strain was found to be similar to B. oceanisediminis estimated against the control, in which solvent was not strain H (99.16% sequence identity). Through the align - present. ment of homologous nucleotide sequence of known bac- teria, phylogenetic relationships could be inferred, and Statistical analysis the phylogenetic position of the strain and related strains All determinations were performed at least in three inde- based on the 16S rDNA sequence is shown in Fig.  1. pendent replicates, and the control experiment without Taken together, the results suggest that this isolate may xylanase was carried out under the same conditions. The be assigned as B. oceanisediminis strain SJ3. Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 5 of 12 Table 1 Morphological, physiological, and some biochem- Time course of xylanase production showed maximum ical properties of the isolate Bacillus oceanisediminis strain enzyme activity at 48  h of incubation and thereafter, it SJ3 remained less constant till 72 h (Fig. 2). It is the same case with Bacillus subtilis strain ASH Characteristics Bacillus oceanisediminis strain SJ3 (Sanghi et al. 2009). The optimum time resulting in maxi - Isolation source Soil mum enzyme titre is likely to depend on several factors Motility + including the microbial strain. A survey of the literature Morphology Spore forming rods revealed the highest enzyme production from Bacillus Gram‑stain + pumilus strain SV-85S after 36 h (Nagar et al. 2010) and Temperature for growth 37 Bacillus sp. strain SSP-34 after 96  h (Subramaniyan and Temperature optimum range 25–45 Prema 2000) and B. pumilus strain VLK-1 after 56  h of pH for growth 7 incubation (Kumar et al. 2014). In the above reports, the pH optimum range 6–9 activity of xylanase exhibited a decline after reaching a NaCl for growth (%) 0–12 maximum value, which might be due to proteolysis of the Indole − enzyme. However, in the present study, though the incu- Methyl red + bation period for xylanase production from B. oceanised- Voges‑proskauer − iminis strain SJ3 was shorter than some other Bacillus sp. catalase + yet it did not decline after attaining the highest level. Glycerol + Erythritol − Some biochemical properties of the crude enzyme d ‑Arabinose − Xylanase activity from B. oceanisediminis strain SJ3 was l ‑Arabinose − efficiently recovered using the TPP technique. A puri - Ribose + fication fold of 3.48 and a recovery yield of 107% were d ‑ Xylose + obtained. Using macroaffinity ligand-facilitated TPP, Galactose + Sharma and Gupta (2002) purified a xylanase from Glucose + Aspergillus niger with a recovery yield of 60% and a Fructose − 95-fold purification. The authors reported other recovery d ‑Mannose − parameters using the denatured xylanase and the optimal Mannitol − parameters were 93% and a purification factor of 21 (Roy Sorbitol − et  al. 2004). TPP has been reported to recover different Cellobiose − enzyme activities (e.g., xylanase, cellulase, cellobiase, Maltose − β-glucosidase, and α-chymotrypsin) from their inacti- Lactose − vated/denatured forms (Roy et  al. 2004, 2005; Sardar Saccharose − et  al. 2007). These findings suggest that TPP may be a Inulin − valuable technique for the simultaneous renaturation/ Strach − purification of the multiple enzymes present in a protein Gelatin + mixture. Concerning the high yield recovery obtained in this preliminary study several studies reported high recovery yields (>100%) for the purification of enzymes using the TPP system (Gagaoua and Hafid 2016; Gagaoua Optimization of xylanase production by strain SJ3 et al. 2017). In the current study, the bacterial strains were newly iso- lated from Algerain soil samples (Bejaia north east, Alge- Effect of temperature on xylanase activity ria), were screened for their xylanase activities. Using the The effect of temperature on the xylanase activity from B. ratio of the clear zone diameter (onto xylan agar plates) oceanisediminis strain SJ3 is shown in Fig. 3a, for 10 min and that of the colony, five isolates exhibiting the high - reaction the optimum temperature was 55 °C (assayed in est ratio were tested for xylanase production in liquid the range 20–100 °C), the xylanase produced by Bacillus culture. Among those strains, a bacterium called strain brevis is also optimally active at the same temperature SJ3, displayed the highest extracellular xylanase activ- (Goswami et  al. 2013). The optimum temperature of the ity (20.24 U/ml) after 48  h incubation in an optimized enzyme is near to that of the xylanases from B. subtilis medium (Fig.  2) and was, therefore, retained for all sub- strain CXJZ isolated from the degumming line (60  °C) sequent studies. (Guo et  al. 2012) and Bacillus sp. strain 41M-1 which Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 6 of 12 Bacillus oceanisediminis strain SJ3 (KT222887) 84 T Bacillus firmus strain NCIMB 9366 (NR_118955) Bacillus oceanisediminis strain H2 (CG292772) Bacillus infantis strain SMC 4352-1 (AY904032) Bacillus nealsonii strain DSM 15077 (NR_044546) 59 T Bacillus beringensis strain BR035 (NR_116849) Bacillus lentus strain IAM 12466 (D16268) Bacillus acidicola strain 105-2 (NR_041942) Bacillus gottheilii strain WCC 4585 (NR_108491) Bacillus bataviensis strain LMG 21833 (AJ542508) 100 T Bacillus niacini strain IFO 15566 (NR 024695) Bacillus muralis strain LMG 20238 (NR_104284) Bacillus salsus strain A24 (HQ433466) Bacillus selenatarsenatis strain SF-1 (NR 041465) Bacillus thermophilus strain SgZ-10 (NR_109677) Escherichia coli strain ATCC 11775 (X80725) 0.02 Fig. 1 Phylogenetic tree based on 16S rRNA gene sequences showing the position of strain SJ3 within the radiation of the genus Bacillus. The sequence of E. coli strain ATCC 11775 (Accession No. X80725) was chosen arbitrarily as an outgroup. Bar 0.02 nt substitutions per base. Numbers at nodes (>50%) indicate support for the internal branches within the tree obtained by bootstrap analysis (percentages of 100 bootstraps). NCBI accession numbers are presented in parentheses Effect of pH on xylanase activity 12 25 Absorbance at 600 nm The optimum pH of B. oceanisediminis strain SJ3 xyla- Xylanase activity (U/mL) nase activity (assayed in the range 4–10) is 7 (Fig.  3b). Other xylanases from Bacillus strains so far character- ized generally show wide differences in their optimal pH, going from acidic values, such as 4 for the glycosyl hydrolase family 11 xylanase from B. amyloliquefaciens strain CH51 (Baek et al. 2012), 5 for the xylanase activity produced by B. subtilis strain GN156 (Pratumteep et  al. 2010), 5.8 for the xylanase from B. subtilis strain CXJZ (Gang et al. 2012), up to 9 in the case of the endoxylanase 0 0 activity from B. halodurans strain TSEV (Kumar and 04 812162024283236404448525660646872768082869094 Satyanarayana 2013, 2014). Incubation time (h) Fig. 2 Time course of Bacillus oceanisediminis strain SJ3 cell growth Thermostability profile of the xylanase activity (open diamond) monitored by measuring the OD at 600 nm and xylanase production (closed diamond). Vertical bars indicate standard Thermal stability was carried out by preincubating xyla - error of the mean (n = 3) nase up to 7  h at 50, 55, 60, and 95  °C (Fig.  4), at 50  °C there was no significant decrease in xylanase activity dur - ing 4  h. The enzyme was stable at 50  °C, with a half-life showed maximum activity at 50  °C (Nakamura et  al. time of 9  h, a half-life time of 6 and 4.72  h was respec- 1995) and Bacillus sp. strain BP-23 (50 °C) (Blanco et al. tively observed at 55 and 60 °C. B. brevis xylanase is less 1995) but distant from that of the xylanases produced by thermostable, it showed a half-life time of 3  h at 55  °C Bacillus halodurans strain TSEV (80  °C) (Kumar and 1 (Goswami et al. 2013). Satyanarayana 2014), Caldicoprobacter algeriensis strain At 95  °C the profile obtained for thermostability TH7C1 (Bouacem et  al. 2014), B. subtilis strain GN156 showed that 50% of the original activity was retained after (40 °C) (Pratumteep et al. 2010), and Bacillus amylolique- 2 h exposure, the results clearly indicated that the suitable faciens strain CH51 (25 °C) (Baek et al. 2012). temperature range for industrial application for xylanase Cell growth (OD 600 nm) Xylanase activity (U/mL) Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 7 of 12 50°C 55°C 60°C 95°C 25 0,51 234567 Time (h) Fig. 4 Thermostability profile of Bacillus oceanisediminis strain SJ3 xylanase at pH 7 at different temperatures. (closed diamond): 50 °C, 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 (closed square): 55 °C, (closed triangle): 60 °C, (closed circle): 95 °C. Sam‑ Temperature (°C) ples were taken at 1 h interval and relative activity was determined. The activity of the non‑heated enzyme was taken to be 100%. Each point represents the mean of three independent experiments. Verti- cal bars indicate standard error of the mean (n = 3) The xylanase from Pseudomonas macquariensis had half-life time of 2 h at 50 °C whereas it had a half-life time of 1 h at 60 °C. At high temperatures, enzyme gets partly unfolded (Sharma et al. 2008). The xylanase of B. oceani - sediminis strain SJ3 is highly thermostable, such enzymes with high thermostability and an ability to function at wide pH range are desirable for many industrial pro- 44,5 55,5 66,5 77,5 88,5 99,5 10 cesses which take place at very high or low pH and high pH temperature. With this respect, the strain could be a good Fig. 3 Eec ff ts of temperature (a) and pH (b) on xylanase activity pro ‑ source for industrial and biotechnological applications. duced by Bacillus oceanisediminis strain SJ3 and recovered by three phase partitioning. a The enzyme activity was determined by incu‑ bating the enzyme with 10 mg/ml oat spelt xylan dissolved in 50 mM pH stability profile of the xylanase activity phosphate buffer at pH 7. The activity of the enzyme at 55 °C was It is observed that the highest xylanase activity was estab- taken as 100%. b The enzyme was incubated at 55 °C with 10 mg/ lished at pH 7.0; on the other hand, it was found to be ml oat spelt xylan dissolved in different buffer. Buffer solutions used most stable at pH 7.0–8.0 but it was also stable in a range for pH activity are presented in “Results and discussion”. The activity of the enzyme at pH 7.0 was taken as 100%. Each point represents of pH 5–10 and at pH 10 approximately 80% of its activ- the mean of three independent experiments. Vertical bars indicate ity was retained (Fig.  5). The enzyme stable in alkaline standard error of the mean (n = 3) conditions were characterized by a decreased number of acidic residues and an increased number of arginines (Hakulinen et al. 2003). The similar pattern of pH stabil - from B. oceanisediminis strain SJ3 was 50–95  °C. This ity was also found in Bacillus vallismortis strain RSPP-15 xylanase is more thermostable than B. amyloliquefaciens (Gaur et al. 2015). strain XR44A xylanase activity which showed a half-life time of 5 min at 70 °C, 15 min at both 50 and 60 °C, and Effect of metallic ions, reagents, and inhibitors on xylanase 2  h at 40  °C. Interestingly, it retained 90% of activity for activity at least 2 days at 30 °C, with a half-life time of 7 days. The We investigated the effects of metallic ions and other enzyme immediately loses activity at temperatures higher reagents on the activities of the crude xylanase (Table 2). than 70 °C (Amore et al. 2015), the xylanase produced by Most of the metallic ions (at concentration of 5  mM) Bacillus aerophilus strain KGJ2, retained more than 90% tested had little influence on the activity, the same results activity after incubation at 80–90  °C for 60  min (Gowd- were obtained with the xylanases produced by Bacillus haman et al. 2014). The enzyme produced by Bacillus sp. sp. strain SPS-0 (Bataillon et  al. 2000); in this experi- strain DM-15 was stable for 15 min at 60 °C while 95% of ment, maximum xylanase production was reported in 2+ the original activity was lost at 90 °C (Ozcan et al. 2011). the presence of Ca (138%); some other researchers Relative xylanase activity (%) Relative xylanase activity (%) Relative xylanase activity (%) Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 8 of 12 (Hmida-Sayari et  al. 2012; Chang et  al. 2017). Xylanase 2+ was strongly inhibited in the presence of Hg . Similar results were observed in case of B. subtilis (Sanghi et  al. 2010) and Bacillus halodurans strain PPKS-2 (Prakash et al. 2012), it has been reported that the xylanase activity was inhibited by mercury ion, which might be due to its interaction with sulfhydryl groups of cysteine residue in or close to the active site of the enzyme (Bastawde 1992). 25 The chelating agent EDTA enveloping metal ions extensively did not change the xylanase activity (Table 2) that means the enzyme did not require metal ions for its 44,5 55,5 66,5 77,5 88,5 99,5 10 catalysis. pH Triton X-100 and β-mercaptoethanol had little effect Fig. 5 pH stability of the xylanase activity produced by Bacillus on the xylanase activity (Table  2) whereas the Bacillus oceanisediminis strain SJ3 and recovered by three phase partitioning. DM-15 xylanase is sensitive (Ozcan et al. 2011). The crude enzyme was incubated with 50 mM buffers at 55 °C for 1 h and relative activity was measured under the standard assay condi‑ Total inactivation due to SDS has already been reported tions. The activity of the enzyme at optimum pH was taken as 100%. for xylanases of different origins (Fujimoto et al. 1995), in Buffer solutions used for pH stability are presented in “Results and contrast to the resistance to SDS was found in this study, discussion”. Each point represents the mean of three independent with 87% relative activity after 10 min at 55 °C (Table 2). experiments. Vertical bars indicate standard error of the mean (n = 3) Activity of the crude xylanase on various carbohydrate substrates Table 2 Eec ff t of  different metallic ions, surfactants, Activity of the crude enzyme on some carbohydrate was chelating agents, and inhibitors on xylanase activity showed at Fig.  6, the crude enzyme mainly contained xylanase as indicated by the highest activity on birch- Chemical additives Concentration Relative enzyme activity (%) wood xylan (25 U/ml) and oat spelt xylan (20 U/ml). The Control – 100 ± 2.5 crude enzyme did not contain amylase but hardly cel- 2+ Mg (MgCl ) 5 mM 106 ± 2.6 lulase (1.99 U/ml). Crude enzymes produced by Bacil- 2+ Ca (CaCl ) 5 mM 138 ± 4.1 lus sp. strain AQ1 not only showed xylanolytic activity 2+ Fe (FeSO ) 5 mM 88 ± 2.2 but also amylolytic and cellulolytic activity (Wahyuntari K (KCl) 5 mM 98 ± 2.4 et al. 2009). Comparisons to the large literature studies as 2+ Cu (CuCl ) 5 mM 92 ± 2.3 summarized in Table 3. Na (NaCl) 5 mM 94 ± 2.3 Based on the available data from this experiment, the 2+ Mn (MnCl ) 5 mM 99 ± 2.5 difference in crude enzyme on the different xylan sub - 2+ Cd (CdCl ) 5 mM 83 ± 2.0 strate could not be explained yet. It is still needed more 2+ Zn (ZnCl ) 5 mM 95 ± 2.3 2+ Hg (HgCl ) 5 mM 20 ± 0.6 Triton X‑100 1% 93 ± 2.3 SDS 1% 87 ± 2.2 EDTA 5 mM 88 ± 2.2 100 β‑Mercaptoethanol 20 mM 86 ± 2.2 Xylanase activity measured in the absence of any chemical additives was taken as control (100%). The non-treated and dialyzed enzyme was considered as 100% for metallic ion assay. Residual activity was measured at pH 7.0 and 55 °C Values represent means of three independent replicates, and ±standard errors are reported 20 Birchwood xylanOat spelt xylanCMC Starch 2+ Carbohydrate substrate (10 mg/mL) also reported that C a ion strongly stimulated xylanase Fig. 6 The effect of the carbohydrate substrate source on the activity. Slightly stimulation was also observed by addi- 2+ xylanase activity produced by Bacillus oceanisediminis strain SJ3 and tion of Mg (106%) (Mamo et  al. 2006; Lv et  al. 2008; recovered by three phase partitioning. The enzyme was incubated Ozcan et al. 2011). with 10 mg/ml of substrate at 55 °C and pH 7.0. Each point represents On the other hand, the inhibition of xylanases by cal- the mean of three independent experiments. Vertical bars indicate cuim and magnesium ions have also been reported standard error of the mean (n = 3) Redisual xylanase activity (%) Relative xylanase activity (%) Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 9 of 12 Table 3 Production of xylanases from various bacteria, namely Bacillus genus and comparisons to our findings Organism Substrate Cultivation conditions Xylanase activity (U/ml or References (temperature and pH) U/mg) Bacillus oceanisediminis SJ3 Oat spelt xylan 55 °C; pH 7.0 20.24 U/ml Present study Jonesia denitrificans BN‑13 Birchwood xylan 50 °C; pH 7.0 77 U/mg Boucherba et al. (2014) Bacillus pumilus MTCC 8964 Oat spelt xylan 60 °C; pH 6 241 U/ml Kumar et al. (2010) Bacillus brevis Birchwood xylan 55 °C; pH 7.0 1.52 U/ml Goswami et al. (2013) Bacillus brevis wheat straw 55 °C; pH 7.0 4380 U/mg Goswami et al. (2014) Bacillus sp. strain BP‑23 Birchwood xylan 50 °C; pH 5.5 40.2 U/mg Blanco et al. (1995) Bacillus halodurans TSEV Cane molasses 80 °C; pH 9.0 15 U/ml Kumar and Satyanarayana (2014) Bacillus amyloliquefaciens Birchwood xylan 25 °C; pH 4.0 701.1 U/mg Baek et al. (2012) CH51 Bacillus subtilis CXJZ birchwood and oat spelt 60 °C; pH 5.8 36,633 U/mg Gang et al. (2012) xylan Bacillus pumilus SSP‑34 Oat spelts xylan 50 °C; pH 6.0 1723 U/mg Subramaniyan (2012) Bacillus pumilus SV‑205 Wheat bran 60 °C; pH 10.0 7382.7 U/ml Nagar et al. (2012) Bacillus subtilus BS05 Sugarcane bagasse 50 °C; pH 5.0 17.58 U/ml Irfan et al. (2012) Gracilibacillus sp. TSCPVG Birchwood xylan pH 7.5 1667 U/mg Poosarla and Chandra (2014) Paenibacillus sp. NF1 Oat spelt xylan 60 °C; pH 6.0 3081.05 U/mg Zheng et al. (2014) Paenibacillus macerans Beechwood xylan 60 °C; pH 4.5 4170 U/mg Dheeran et al. (2012) IIPSP3 Anoxybacillus flavithermus Oat spelt xylan 65 °C; pH 6.0 and pH 8.0 117.64 U/mg Ellis and Magnuson (2012) TWXYL3 complete studies to elaborate the type of xylanolytic (v/v) and less than 60% of its initial activity at 30% (Yang activities present in the crude enzyme of B. oceani- et al. 2010). sediminis strain SJ3. From preliminary study, it can be In some cases, the presence of solvents enhanced the observed that the strain SJ3 was able to grow and pro- xylanase activity, for example the xylanase of B. val- duce xylanases using commercial xylan. The pH and lismortis is extraordinarily stable in the presence of all temperature optima of the preparation were 7 and 55 °C, organic solvents under study. After incubation with respectively, and the enzyme was stable in a range of pH n-dodecane, isooctane, n-decane, xylene, toluene, n-hex- 5–10 retained 50% of its activity during 6 h at 55 °C. The ane, n-butanol, and cyclohexane, the xylanase activity enzyme is also resistant to hydrophobic solvents, these increased to 230.8, 137.7, 219.8, 107, 190.5, 194.7, 179.3, properties place this enzyme as promising for industrial and 111.6%, respectively (Gaur and Tiwari 2015). and biotechnological applications especially lignocellu- lose bioconversion and bioethanol production. Conclusion In conclusion, a new extracellular thermostable xyla- Eec ff t of organic solvents of the xylanase activity nase from B. oceanisediminis strain SJ3 was produced The xylanase from B. oceanisediminis strain SJ3 is resist- and characterized in this study. The preliminary results ant to hydrophobic solvents: heptan, chloroform, toluene, of the use of Three phase partitioning for the recovery and cyclohexane (the relative activity is 99.2%) but a loss of the xylanase were presented. The time course for xyla - of the enzyme activity was observed by addition of 30% nase accumulation by strain SJ3 in xylan-based medium (v/v) of methanol, ethanol, propanol, and acetone (Fig. 7). showed that the highest xylanase activity reached 20.24 These alcohols completely inhibited the enzyme from U/ml in an optimized medium with oats spelt xylan used Termitomyces sp. and Macrotermes subhyalinus at 30% as a substrate after 48  h of cultivation. The crude xyla - (v/v) and 60% (v/v), respectively. Primary alcohols includ- nase from strain SJ3 was biochemically characterized. ing methanol, ethanol, and isopropanol as well as poly- The results revealed that the enzyme was highly sta - hydric alcohol containing glycol and glycerol, all showed ble and active at high temperature (55  °C) and alkaline inhibitory effects on A. niger strain C3486 xylanase activ - pH 7.0. Properties of this enzyme such as high specific ity which retained around 90% at the concentration of 2% activity, wide range of pH optimum and stability, and Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 10 of 12 ControlHepaneCyclohexane Toluene Chloroform Propnol AcetoneEthanol Methanol Organic solvents 30% (v/v) Fig. 7 Eec ff t of organic solvents on xylanase activity produced by Bacillus oceanisediminis strain SJ3 and recovered by three phase partitioning. Relative xylanase activity was expressed as a percentage of the control reaction without solvent. Each point represents the mean of three independ‑ ent experiments. Vertical bars indicate standard error of the mean (n = 3) thermostability at elevated temperature as well as organic Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ solvents tolerance, are appropriate for industrial and lished maps and institutional affiliations. biotechnological applications. Interestingly, this enzyme presented high xylanolytic activity with oats spelt xylan, Received: 8 May 2017 Accepted: 1 July 2017 and was very effective in the pulp bleaching industry, thus offering a potential promising candidate for applica - tion in biotechnological bioprocesses. Accordingly, fur- ther studies, some of which are currently underway, are References needed to investigate the purification to homogeneity Amore A, Parameswaran B, Kumar R, Birolo L, Vinciguerra R, Marcolongo L, Ionata E, La Cara F, Pandey A, Faraco V (2015) Application of a new and encoding gene, perform site-directed mutagenesis, xylanase activity from Bacillus amyloliquefaciens XR44A in brewer’s spent and determine its structure–function relationships. grain saccharification. J Chem Technol Biotechnol 90:573–581 Badhan AK, Chadha BS, Kaur J, Saini HS, Bhat MK (2007) Production of multiple Authors’ contributions xylanolytic and cellulolytic enzymes by thermophilic fungus Mycelioph- NB, MG, and SB designed this research plan and discussed with ABD. NB, MG, thora sp. IMI 387099. Bioresour Technol 98:504–510 CB, KB, MYC performed all the research experiments and NB and MG wrote the Baek CU, Lee SG, Chung YR, Cho I, Kim JH (2012) Cloning of a family 11 xyla‑ draft paper. 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Int J Syst Evol Microbiol Biotechnol 166(7):1831–1842 60:2924–2929 Subramaniyan S, Prema P (2000) Cellulase‑free xylanases from Bacillus and Zheng HC, Sun MZ, Meng LC, Pei HS, Zhang XQ, Yan Z, Sun JS (2014) Purifica‑ other microorganisms. FEMS Microbiol Lett 183:1–7 tion and characterization of a thermostable xylanase from Paenibacillus Tarayre C, Brognaux A, Brasseur C, Bauwens J, Millet C, Matteotti C, Destain J, sp. NF1 and its application in xylooligosaccharides production. J Micro‑ Vandenbol M, Portetelle D, De Pauw E, Haubruge E, Francis F, Thonart P biol Biotechnol 24:489–496 (2013) Isolation and cultivation of a xylanolytic Bacillus subtilis extracted Zouari Ayadi D, Hmida Sayari A, Ben Hlima H, Ben Mabrouk S, Mezghani M, from the gut of the termite Reticulitermes santonensis. Appl Biochem Bejar S (2015) Improvement of Trichoderma reesei xylanase II thermal Biotechnol 171:225–245 stability by serine to threonine surface mutations. 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Biochemical properties of a new thermo- and solvent-stable xylanase recovered using three phase partitioning from the extract of Bacillus oceanisediminis strain SJ3

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Springer Journals
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2017 The Author(s)
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2197-4365
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10.1186/s40643-017-0161-9
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Abstract

The present study investigates the production and partial biochemical characterization of an extracellular thermo‑ stable xylanase from the Bacillus oceanisediminis strain SJ3 newly recovered from Algerian soil using three phase partitioning ( TPP). The maximum xylanase activity recorded after 2 days of incubation at 37 °C was 20.24 U/ml in the presence of oat spelt xylan. The results indicated that the enzyme recovered in the middle phase of TPP system using the optimum parameters were determined as 50% ammonium sulfate saturation with 1.0:1.5 ratio of crude extract: t‑ butanol at pH and temperature of 8.0 and 10 °C, respectively. The xylanase was recovered with 3.48 purification fold and 107% activity recovery. The enzyme was optimally active at pH 7.0 and was stable over a broad pH range of 5.0–10. The optimum temperature for xylanase activity was 55 °C and the half‑ life time at this temperature was of 6 h. At this time point the enzyme retained 50% of its activity after incubation for 2 h at 95 °C. The crude enzyme resist to sodium dodecyl sulfate and β‑ mercaptoethanol, while all the tested ions do not affect the activity of the enzyme. The recovered enzyme is, at least, stable in tested organic solvents except in propanol where a reduction of 46.5% was observed. Further, the stability of the xylanase was higher in hydrophobic solvents where a maximum stability was observed with cyclohexane. These properties make this enzyme to be highly thermostable and may be suggested as a potential candidate for application in some industrial processes. To the best of our knowledge, this is the first report of xylanase activity and recoverey using three phase partitioning from B. oceanisediminis. Keywords: Bacillus oceanisediminis, Xylanase, Thermostability, Hydrophobic solvents, Industrial processes, Three phase partitioning requires the concerted action of xylanolytic enzymes Background (Trajano et al. 2014; Zhang and Viikari 2014). Xylans are Hemicellulose is the second most abundant renewable heterogeneous polysaccharides with a backbone consist- biomass after cellulose in nature (Collins et  al. 2005). ing of β-1,4 linked d-xylosyl residues. Xylan is the major component of hemicelluloses in wood Endo-β-1,4 xylanases (EC 3.2.1.8) are the main from angiosperms, where it accounts for 15–30% of the enzymes responsible for cleavage of the linkages within total dry weight. In gymnosperms, however, xylans con- the xylan backbone (Collins et  al. 2005), to which short tribute only 7–12% of the total dry weight. The structure side chains of O-acetyl, α-l-arabinofuranosyl, d-α glu- of xylan is complex, and its complete biodegradation curonic, and phenolic acid residues are attached (Col- lins et  al. 2005; Terrasan et  al. 2010; Xie et  al. 2015). *Correspondence: mohammed.gagaoua@inra.fr; gmber2001@yahoo.fr Xylanases have been used in a wide range of industrial UMR1213 Herbivores, INRA, VetAgro Sup, Clermont Université, Université applications and processes. They have been applied in de Lyon, 63122 Saint‑Genès‑Champanelle, France Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/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. Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 2 of 12 the bioconversion of lignocellulosic material and agro- organic phase that usually contains t-butanol (Gagaoua wastes to fermentative products, clarification of juices, and Hafid 2016). improvement in consistency of beer, and the digest- ibility of animal feed stock (Badhan et  al. 2007; Elgharbi Methods et  al. 2015a, b; Shameer 2016; Jain and Krishnan 2017). Substrates, reagents, and chemicals Due to their important activity at alkaline pH (8.0–11) Birchwood xylan, oat spelt xylan, starch, carboxymethyl and high temperature (50–90  °C), thermostable alkaline cellulose (CMC, low viscosity), tert-butanol, ammonium xylanases have attracted special attention in the pulp bio- sulfate, and 3,5-dinitrosalicylic acid (DNS) were pur- bleaching industry (Techapun et al. 2003; Bouacem et al. chased from Sigma Chemical Company (St. Louis, MO, 2014; Boucherba et  al. 2014; Bouanane-Darenfed et  al. USA). Unless otherwise specified, all other reagents and 2016). Xylanase, together with other hydrolytic enzymes, chemicals were of the analytical grade or highest level of have also proved useful for the generation of bio-fuels, purity available. including ethanol, from lignocellulosic biomass. Xyla- nases are used in pulp pre-bleaching process to remove Collection of samples and culture conditions the hemicelluloses, which bind to the pulp. The hydroly - of microorganisms sis of pulp bound hemicelluloses releases the lignin in The garden soil samples were collected from Bejaia north the pulp, reducing the amount of chlorine required for east of Algeria (Kabylia region) in March 2015. The soil conventional chemical bleaching and minimizing the was collected from five places and samples were pooled. toxic, chloroorganic waste. Therefore, xylanases from Sub-samples of approximately 1  g were suspended in alkalophilic bacteria and actinomycetes and fungi have 100 ml sterile distilled water. Mixtures were allowed to set- been studied widely (Perez-Rodriguez et  al. 2014; Wang tle and serial dilutions were prepared. From each dilution, et al. 2014). However, large scale cultivation of fungi and 0.1  ml was taken and spread on agar plates of medium actinomycetes is often difficult because of their slow gen - containing in g/l oat spelt xylan 10, yeast extract 2, NaCl eration time, coproduction of highly viscous polymers, 2.5, NH Cl 5, K H PO 15, Na HPO 30, MgSO ·7H O 4 2 4 2 4 4 2 and poor oxygen transfer (Wong et  al. 1997; Garg et  al. 0.25, and bacteriological agar 15. In this medium, there 2011). Bacillus genus is used more extensively than other is a little modification of the main carbon source, the oat bacteria in industrial fermentations, since they produce spelt xylan was used instead the birchwood xylan (Viet most of their enzymes. Some Bacillus strains have been et  al. 1991). The plates were incubated at pH 7 and 37 °C reported as xylanolytic enzymes producers (Lindner et al. for 2 days at 250 rpm. Those colonies that grew well under 1994; Seo et  al. 2013; Tarayre et  al. 2013; Elgharbi et  al. such conditions and showed an orange zone around the 2015a; Zouari et al. 2015). colonies after red Congo were retained for second screen- Bacillus oceanisediminis sp. nov. was first isolated ing. Colonies with a clear zone formation following the from a marine sediment collected in the South Sea of hydrolysis of xylan were evaluated as xylanase producers. China (Zhang et  al. 2010). Considering the above, the Several xylanlolytic strains were isolated and SJ3, which present study was undertaken to described, for the first exhibited a large clear zone of hydrolysis, was selected and time, the production of a thermostable xylanase from B. retained for further experimental study. oceanisediminis strain SJ3 recently isolated by our labo- ratory from Algerian soil, an attempt was made to bio- Bacterial identification of the isolate SJ3 chemically characterize the xylanase activity secreted by Analytical profile index (API) strip tests and 16S rRNA this strain. Also, preliminary investigation using three gene sequencing were carried out for the identification of phase partitioning (TPP) system (Gagaoua et  al. 2014; the genus to which the strain belong. Gagaoua and Hafid 2016) for xylanase purification was API 50 CHB/E and the API 20E strips (bioMérieux, SA, performed. In TPP process, firstly an inorganic salt (gen - Marcy-l’Etoile, France) were used to investigate the phys- erally ammonium sulfate) is added to the crude extract iological and biochemical characteristics of strain SJ3, containing proteins then mixted with tert-butanol in an as recommended elsewhere (Logan and Berkeley 1984). appropriate amount (Gagaoua et  al. 2015, 2016, 2017). The growth temperature (4, 10, 15, 20, 25, 30, 35, 40, and When t-butanol is added in the presence of ammo- 45 °C), pH level values (4, 5, 6, 7, 8, 9, 10, 11, and 12) and nium sulfate, it pushes the protein out of the solution. sodium chloride regimes were determined. In this process t-butanol binds to hydrophobic part of The 16S rRNA gene was amplified by PCR using for - the proteins to reduce the density of the proteins, lead- ward primer F-d1 5′-AGAGTTTGATCCTGGCTCA ing to float above the denser aqueous salt phase. Within G-3′, and reverse primer R-d1 5′-AAGGAGGTGATCCAA approximately an hour, it forms an interfacial (mid- GCC-3′, designed from base positions 8–27 and 1541– dle) precipitate between the lower aqueous and upper 1525, respectively, which were the conserved zones Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 3 of 12 within the rRNA operon of Escherichia coli (Gurtler and then boiled for 5 min and cooled. Absorption was meas- Stanisich 1996). The genomic DNA of strain SJ3 was ured at 540 nm. purified using the Wizard Genomic DNA Purification One unit of xylanase activity was defined as the amount Kit (Promega, Madison, WI, USA) and then used as a of enzyme that released 1 µmol of reducing sugar equiva- template for PCR amplification (30 cycles, 94 °C for 45 s lent to xylose per min under the assay conditions. denaturation, 60 °C for 45 s primer annealing, and 72 °C for 60  s extension). The amplified  ~1.5  kb PCR prod - Xylanase production uct was cloned in the pGEM-T Easy vector (Promega, Gowth condition of the xylanase activity Madison, WI, USA), leading to pSJ3-16S plasmid (this To study the properties of the xylanase activity produc- study). The E. coli DH5α (F supE44 Φ80 δlacZ ΔM15 tion, the isolates having high xylanase activities were − + Δ(lacZYA-argF) U169 endA1 recA1 hsdR17 (r , m ) cultivated in 250  ml shake-flasks containing 50  ml basic k k deoR thi-1 λ gyrA96 relA1) (Invitrogen, Carlsbad, CA, xylanase production medium at 37 °C. The basic xylanase USA) was used as a host strain. All recombinant clones production medium was prepared at pH 7.0 containing of E. coli were grown in Luria–Bertani (LB) broth media oat spelt xylan. The culture was harvested after 48 h, and with the addition of ampicillin, isopropyl-thio-β-d- centrifuged (10,000 rpm for 10 min). Growth was meas- galactopyranoside (IPTG), and X-gal for screening. DNA ured by determining absorbance at 600  nm. The sample electrophoresis, DNA purification, restriction, ligation, was then kept at 4 °C in the refrigerator. and transformation were all performed according to the method previously described elsewhere (Sambrook et al. Effect of incubation time on xylanase production 1989). Pre-culture (2%) was used to inoculate 250  ml xylan defined medium at 37  °C for 72  h. culture samples were DNA sequencing and molecular phylogenetic analysis collected each 4  h during the cultivation period. Imme- The nucleotide sequences of the cloned 16S rRNA gene diately after collection, the samples were centrifuged at were determined on both strands using BigDye Termina- 4 °C and 10,000g for 20 min. Supernatants were analyzed tor Cycle Sequencing Ready Reaction kits and the auto- for xylanase activity as described above. mated DNA sequencer ABI PRISM 3100-Avant Genetic Analyser (Applied Biosystems, Foster City, CA, USA. The Partial biochemical characterization of the recovered RapidSeq36_POP6 run module was used, and the sam- enzyme by TPP ples were analyzed using the ABI sequencing analysis Extraction and partial purification of xylanase by TPP software v. 3.7 NT. Aqueous systems such as three phase partitioning (TPP), The sequences obtained were compared to those pre - known as simple, economical and quick methods, were sent in the public sequence databases and with the described for the fast recovery of enzymes (Gagaoua and EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/), Hafid 2016). This elegant non-chromatographic tool may a web-based tool for the identification of prokaryotes be performed in a purification process to be used suc - based on 16S rRNA gene sequences from type strains cessfully in food or other industries. For its application in (Kim et al. 2012). this study, the crude extract was first collected after 48 h Phylogenetic and molecular evolutionary genetic anal- of batch incubation (Boucherba et  al. 2014). The culture yses were conducted via the the molecular evolutionary supernatant containing secreted xylanases was concen- genetics analysis (MEGA) software version 5 (http:// trated using Sartorius membranes (with 10-kDa cutoff www.megasoftware.net). Distances and clustering were membrane; Millipore) after a centrifugation at 10,000 rpm calculated using the neighbor-joining method. The tree for 10  min. Then, TPP experiments were carried out fol - topology of the neighbor-joining data was evaluated by lowing the recommendations of Gagaoua et  al. (2015). Bootstrap analysis with 100 re-samplings. The enzyme exclusively recovered in the interfacial phase was gently separated from the other phases and dissolved Xylanase assay in 50  mM Tris–HCl buffer (pH 8.5) and dialyzed over - Xylanase activity was determined by measuring the night at 4–5 °C and used for enzyme characterization. release of reducing sugar from soluble xylan using the DNS method (Miller 1959). In brief, 0.9  ml buffer A Effect of temperature and pH on xylanase activity (10  mg/ml oat spelt xylan in 50  mM sodium-phosphate Optimal temperature was determined by assaying the buffer at pH 7) were mixed with 0.1 ml of the recovered enzyme activity between 20 and 100 °C, by incubating the enzyme solution (1 mg/ml). After incubation at 55 °C for enzyme along with the substrate for 10 min at the respec- 10 min, the reaction was terminated by adding 1.5 ml of tive temperature. Relative xylanase activity was deter- the DNS reagent (Maalej et  al. 2009). The mixture was mined using 10 mg/ml oat spelt xylan at various pHs. The Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 4 of 12 pH range used varied from 4 to 10. Three different buff - experimental results were expressed as the mean of the rep- ers (50  mM) were used. Sodium acetate buffer was used licate determinations and standard deviation (mean ± SD). for pH 4–6; Sodium-phosphate buffer was used for pH The statistical significance was evaluated using t tests for from 6 to 7 and Tris–HCl buffer for pH 7–10. two-sample comparison and one-way analysis of variance (ANOVA) followed by Duncan test. The results were con - Effect of temperature on xylanase stability sidered statistically significant for P values of less than 0.05. The thermostability was determined at temperatures of The statistical analysis was performed using the R package 50, 55, 60, and 95 °C, after incubation with the substrate Version 3.1.1 (Vanderbilt University, USA). for different times (from 0.5 to 7  h); remaining xylanase activity was measured under standard assay conditions. Nucleotide sequence accession number The non-heated enzyme, which was left at room temper - The data reported in this work for the nucleotide ature, was considered as control (100%). sequence of the 16S rRNA (1089  bp) gene of the isolate SJ3 have been deposited in the DDBJ/EMBL/GenBank Effect of pH on xylanase stability databases under Accession Number KT222887. For pH stability, the enzyme was incubated with differ - ent buffers viz. 50  mM acetate buffer for pH range 4–6, Results and discussion 50  mM phosphate buffer for pH range 6–7, and 50  mM Screening of xylanase‑producing bacteria from Algerain Tris–HCl buffer for pH range 7–10 at 55  °C for 1  h. soil and molecular characterization of the target Thereafter, enzyme activity was determined using the microorganism enzyme assay as described above. In the current study, ten candidates were obtained from the first screening as xylanase producers. Among them, Effect of metal ions and reagents on activity a bacterium called SJ3, displayed the highest extracellu- The effect of metallic ions at concentration of 5  mM, lar xylanase activity after 2  days incubation in an initial chelating agents, surfactants, and inhibitors on the activ- medium (data not shown) and was, therefore, retained ity of crude xylanase were determined by preincubating for all subsequent studies. + 2+ 2+ 2+ the enzyme in the presence of Na, Mg, Ca, Mn , The physiological and biochemical characteristics of 2+ 2+ 2+ + 2+ 2+ Fe, Zn, Cu , K , Hg , and Cd , EDTA (5  Mm), the SJ3 isolate presented in this study were investigated SDS (1%), β-mercaptoethanol (20 mM), and Triton X-100 according to well-established protocols and criteria (1%) for 30  min at 55  °C before adding the substrate described in the Bergey’s Manual of Systematic Bacteri- (Ozcan et al. 2011). Subsequently, relative xylanase activi- ology as well as the API 50 CHB/E and the API 20E gal- ties were measured at standard enzyme assay conditions. leries for representative strains. The findings indicated Relative activity was expressed as the percentage of the that the SJ3 isolate was Gram-stain-positive, motile, activity observed in the absence of any compound. rod-shaped, catalase-positive, aerobic, and endospore forming microorganism. Optimal growth temperature Activity of crude enzyme on various carbohydrate substrate was 37  °C; optimal pH was 7.0. According to the results The presence of other carbohydrase was analyzed using obtained using the API 50 CHB/E medium and the API oat spelt xylan, birchwood xylan, starch, and CMC 20E strips, the characteristics strongly confirmed that the (10 mg/ml). The reducing sugar released during the assay isolate belongs to Bacillaceae order and Bacillus genus. was quantified by spectroscopy at λ . The physiological and some biochemical properties of the isolate SJ3 are given in Table 1. Effect of organic solvents on xylanase activity The 16S rRNA gene sequence (KT222887) obtained Cell free supernatant having maximum xylanase activ- was submitted to GenBank BLAST search analyses, ity was incubated with 30% (v/v) of different organic which yielded a strong homology of up to 99% with those solvents, namely, acetone, propanol, ethanol, metha- of several cultivated strains of Bacillus. From the analy- nol, chloroform, heptane, cyclohexane, and toluene sis of the almost-complete 16S rRNA gene sequence, for 30  min at 55  °C. The residual xylanase activity was this strain was found to be similar to B. oceanisediminis estimated against the control, in which solvent was not strain H (99.16% sequence identity). Through the align - present. ment of homologous nucleotide sequence of known bac- teria, phylogenetic relationships could be inferred, and Statistical analysis the phylogenetic position of the strain and related strains All determinations were performed at least in three inde- based on the 16S rDNA sequence is shown in Fig.  1. pendent replicates, and the control experiment without Taken together, the results suggest that this isolate may xylanase was carried out under the same conditions. The be assigned as B. oceanisediminis strain SJ3. Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 5 of 12 Table 1 Morphological, physiological, and some biochem- Time course of xylanase production showed maximum ical properties of the isolate Bacillus oceanisediminis strain enzyme activity at 48  h of incubation and thereafter, it SJ3 remained less constant till 72 h (Fig. 2). It is the same case with Bacillus subtilis strain ASH Characteristics Bacillus oceanisediminis strain SJ3 (Sanghi et al. 2009). The optimum time resulting in maxi - Isolation source Soil mum enzyme titre is likely to depend on several factors Motility + including the microbial strain. A survey of the literature Morphology Spore forming rods revealed the highest enzyme production from Bacillus Gram‑stain + pumilus strain SV-85S after 36 h (Nagar et al. 2010) and Temperature for growth 37 Bacillus sp. strain SSP-34 after 96  h (Subramaniyan and Temperature optimum range 25–45 Prema 2000) and B. pumilus strain VLK-1 after 56  h of pH for growth 7 incubation (Kumar et al. 2014). In the above reports, the pH optimum range 6–9 activity of xylanase exhibited a decline after reaching a NaCl for growth (%) 0–12 maximum value, which might be due to proteolysis of the Indole − enzyme. However, in the present study, though the incu- Methyl red + bation period for xylanase production from B. oceanised- Voges‑proskauer − iminis strain SJ3 was shorter than some other Bacillus sp. catalase + yet it did not decline after attaining the highest level. Glycerol + Erythritol − Some biochemical properties of the crude enzyme d ‑Arabinose − Xylanase activity from B. oceanisediminis strain SJ3 was l ‑Arabinose − efficiently recovered using the TPP technique. A puri - Ribose + fication fold of 3.48 and a recovery yield of 107% were d ‑ Xylose + obtained. Using macroaffinity ligand-facilitated TPP, Galactose + Sharma and Gupta (2002) purified a xylanase from Glucose + Aspergillus niger with a recovery yield of 60% and a Fructose − 95-fold purification. The authors reported other recovery d ‑Mannose − parameters using the denatured xylanase and the optimal Mannitol − parameters were 93% and a purification factor of 21 (Roy Sorbitol − et  al. 2004). TPP has been reported to recover different Cellobiose − enzyme activities (e.g., xylanase, cellulase, cellobiase, Maltose − β-glucosidase, and α-chymotrypsin) from their inacti- Lactose − vated/denatured forms (Roy et  al. 2004, 2005; Sardar Saccharose − et  al. 2007). These findings suggest that TPP may be a Inulin − valuable technique for the simultaneous renaturation/ Strach − purification of the multiple enzymes present in a protein Gelatin + mixture. Concerning the high yield recovery obtained in this preliminary study several studies reported high recovery yields (>100%) for the purification of enzymes using the TPP system (Gagaoua and Hafid 2016; Gagaoua Optimization of xylanase production by strain SJ3 et al. 2017). In the current study, the bacterial strains were newly iso- lated from Algerain soil samples (Bejaia north east, Alge- Effect of temperature on xylanase activity ria), were screened for their xylanase activities. Using the The effect of temperature on the xylanase activity from B. ratio of the clear zone diameter (onto xylan agar plates) oceanisediminis strain SJ3 is shown in Fig. 3a, for 10 min and that of the colony, five isolates exhibiting the high - reaction the optimum temperature was 55 °C (assayed in est ratio were tested for xylanase production in liquid the range 20–100 °C), the xylanase produced by Bacillus culture. Among those strains, a bacterium called strain brevis is also optimally active at the same temperature SJ3, displayed the highest extracellular xylanase activ- (Goswami et  al. 2013). The optimum temperature of the ity (20.24 U/ml) after 48  h incubation in an optimized enzyme is near to that of the xylanases from B. subtilis medium (Fig.  2) and was, therefore, retained for all sub- strain CXJZ isolated from the degumming line (60  °C) sequent studies. (Guo et  al. 2012) and Bacillus sp. strain 41M-1 which Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 6 of 12 Bacillus oceanisediminis strain SJ3 (KT222887) 84 T Bacillus firmus strain NCIMB 9366 (NR_118955) Bacillus oceanisediminis strain H2 (CG292772) Bacillus infantis strain SMC 4352-1 (AY904032) Bacillus nealsonii strain DSM 15077 (NR_044546) 59 T Bacillus beringensis strain BR035 (NR_116849) Bacillus lentus strain IAM 12466 (D16268) Bacillus acidicola strain 105-2 (NR_041942) Bacillus gottheilii strain WCC 4585 (NR_108491) Bacillus bataviensis strain LMG 21833 (AJ542508) 100 T Bacillus niacini strain IFO 15566 (NR 024695) Bacillus muralis strain LMG 20238 (NR_104284) Bacillus salsus strain A24 (HQ433466) Bacillus selenatarsenatis strain SF-1 (NR 041465) Bacillus thermophilus strain SgZ-10 (NR_109677) Escherichia coli strain ATCC 11775 (X80725) 0.02 Fig. 1 Phylogenetic tree based on 16S rRNA gene sequences showing the position of strain SJ3 within the radiation of the genus Bacillus. The sequence of E. coli strain ATCC 11775 (Accession No. X80725) was chosen arbitrarily as an outgroup. Bar 0.02 nt substitutions per base. Numbers at nodes (>50%) indicate support for the internal branches within the tree obtained by bootstrap analysis (percentages of 100 bootstraps). NCBI accession numbers are presented in parentheses Effect of pH on xylanase activity 12 25 Absorbance at 600 nm The optimum pH of B. oceanisediminis strain SJ3 xyla- Xylanase activity (U/mL) nase activity (assayed in the range 4–10) is 7 (Fig.  3b). Other xylanases from Bacillus strains so far character- ized generally show wide differences in their optimal pH, going from acidic values, such as 4 for the glycosyl hydrolase family 11 xylanase from B. amyloliquefaciens strain CH51 (Baek et al. 2012), 5 for the xylanase activity produced by B. subtilis strain GN156 (Pratumteep et  al. 2010), 5.8 for the xylanase from B. subtilis strain CXJZ (Gang et al. 2012), up to 9 in the case of the endoxylanase 0 0 activity from B. halodurans strain TSEV (Kumar and 04 812162024283236404448525660646872768082869094 Satyanarayana 2013, 2014). Incubation time (h) Fig. 2 Time course of Bacillus oceanisediminis strain SJ3 cell growth Thermostability profile of the xylanase activity (open diamond) monitored by measuring the OD at 600 nm and xylanase production (closed diamond). Vertical bars indicate standard Thermal stability was carried out by preincubating xyla - error of the mean (n = 3) nase up to 7  h at 50, 55, 60, and 95  °C (Fig.  4), at 50  °C there was no significant decrease in xylanase activity dur - ing 4  h. The enzyme was stable at 50  °C, with a half-life showed maximum activity at 50  °C (Nakamura et  al. time of 9  h, a half-life time of 6 and 4.72  h was respec- 1995) and Bacillus sp. strain BP-23 (50 °C) (Blanco et al. tively observed at 55 and 60 °C. B. brevis xylanase is less 1995) but distant from that of the xylanases produced by thermostable, it showed a half-life time of 3  h at 55  °C Bacillus halodurans strain TSEV (80  °C) (Kumar and 1 (Goswami et al. 2013). Satyanarayana 2014), Caldicoprobacter algeriensis strain At 95  °C the profile obtained for thermostability TH7C1 (Bouacem et  al. 2014), B. subtilis strain GN156 showed that 50% of the original activity was retained after (40 °C) (Pratumteep et al. 2010), and Bacillus amylolique- 2 h exposure, the results clearly indicated that the suitable faciens strain CH51 (25 °C) (Baek et al. 2012). temperature range for industrial application for xylanase Cell growth (OD 600 nm) Xylanase activity (U/mL) Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 7 of 12 50°C 55°C 60°C 95°C 25 0,51 234567 Time (h) Fig. 4 Thermostability profile of Bacillus oceanisediminis strain SJ3 xylanase at pH 7 at different temperatures. (closed diamond): 50 °C, 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 (closed square): 55 °C, (closed triangle): 60 °C, (closed circle): 95 °C. Sam‑ Temperature (°C) ples were taken at 1 h interval and relative activity was determined. The activity of the non‑heated enzyme was taken to be 100%. Each point represents the mean of three independent experiments. Verti- cal bars indicate standard error of the mean (n = 3) The xylanase from Pseudomonas macquariensis had half-life time of 2 h at 50 °C whereas it had a half-life time of 1 h at 60 °C. At high temperatures, enzyme gets partly unfolded (Sharma et al. 2008). The xylanase of B. oceani - sediminis strain SJ3 is highly thermostable, such enzymes with high thermostability and an ability to function at wide pH range are desirable for many industrial pro- 44,5 55,5 66,5 77,5 88,5 99,5 10 cesses which take place at very high or low pH and high pH temperature. With this respect, the strain could be a good Fig. 3 Eec ff ts of temperature (a) and pH (b) on xylanase activity pro ‑ source for industrial and biotechnological applications. duced by Bacillus oceanisediminis strain SJ3 and recovered by three phase partitioning. a The enzyme activity was determined by incu‑ bating the enzyme with 10 mg/ml oat spelt xylan dissolved in 50 mM pH stability profile of the xylanase activity phosphate buffer at pH 7. The activity of the enzyme at 55 °C was It is observed that the highest xylanase activity was estab- taken as 100%. b The enzyme was incubated at 55 °C with 10 mg/ lished at pH 7.0; on the other hand, it was found to be ml oat spelt xylan dissolved in different buffer. Buffer solutions used most stable at pH 7.0–8.0 but it was also stable in a range for pH activity are presented in “Results and discussion”. The activity of the enzyme at pH 7.0 was taken as 100%. Each point represents of pH 5–10 and at pH 10 approximately 80% of its activ- the mean of three independent experiments. Vertical bars indicate ity was retained (Fig.  5). The enzyme stable in alkaline standard error of the mean (n = 3) conditions were characterized by a decreased number of acidic residues and an increased number of arginines (Hakulinen et al. 2003). The similar pattern of pH stabil - from B. oceanisediminis strain SJ3 was 50–95  °C. This ity was also found in Bacillus vallismortis strain RSPP-15 xylanase is more thermostable than B. amyloliquefaciens (Gaur et al. 2015). strain XR44A xylanase activity which showed a half-life time of 5 min at 70 °C, 15 min at both 50 and 60 °C, and Effect of metallic ions, reagents, and inhibitors on xylanase 2  h at 40  °C. Interestingly, it retained 90% of activity for activity at least 2 days at 30 °C, with a half-life time of 7 days. The We investigated the effects of metallic ions and other enzyme immediately loses activity at temperatures higher reagents on the activities of the crude xylanase (Table 2). than 70 °C (Amore et al. 2015), the xylanase produced by Most of the metallic ions (at concentration of 5  mM) Bacillus aerophilus strain KGJ2, retained more than 90% tested had little influence on the activity, the same results activity after incubation at 80–90  °C for 60  min (Gowd- were obtained with the xylanases produced by Bacillus haman et al. 2014). The enzyme produced by Bacillus sp. sp. strain SPS-0 (Bataillon et  al. 2000); in this experi- strain DM-15 was stable for 15 min at 60 °C while 95% of ment, maximum xylanase production was reported in 2+ the original activity was lost at 90 °C (Ozcan et al. 2011). the presence of Ca (138%); some other researchers Relative xylanase activity (%) Relative xylanase activity (%) Relative xylanase activity (%) Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 8 of 12 (Hmida-Sayari et  al. 2012; Chang et  al. 2017). Xylanase 2+ was strongly inhibited in the presence of Hg . Similar results were observed in case of B. subtilis (Sanghi et  al. 2010) and Bacillus halodurans strain PPKS-2 (Prakash et al. 2012), it has been reported that the xylanase activity was inhibited by mercury ion, which might be due to its interaction with sulfhydryl groups of cysteine residue in or close to the active site of the enzyme (Bastawde 1992). 25 The chelating agent EDTA enveloping metal ions extensively did not change the xylanase activity (Table 2) that means the enzyme did not require metal ions for its 44,5 55,5 66,5 77,5 88,5 99,5 10 catalysis. pH Triton X-100 and β-mercaptoethanol had little effect Fig. 5 pH stability of the xylanase activity produced by Bacillus on the xylanase activity (Table  2) whereas the Bacillus oceanisediminis strain SJ3 and recovered by three phase partitioning. DM-15 xylanase is sensitive (Ozcan et al. 2011). The crude enzyme was incubated with 50 mM buffers at 55 °C for 1 h and relative activity was measured under the standard assay condi‑ Total inactivation due to SDS has already been reported tions. The activity of the enzyme at optimum pH was taken as 100%. for xylanases of different origins (Fujimoto et al. 1995), in Buffer solutions used for pH stability are presented in “Results and contrast to the resistance to SDS was found in this study, discussion”. Each point represents the mean of three independent with 87% relative activity after 10 min at 55 °C (Table 2). experiments. Vertical bars indicate standard error of the mean (n = 3) Activity of the crude xylanase on various carbohydrate substrates Table 2 Eec ff t of  different metallic ions, surfactants, Activity of the crude enzyme on some carbohydrate was chelating agents, and inhibitors on xylanase activity showed at Fig.  6, the crude enzyme mainly contained xylanase as indicated by the highest activity on birch- Chemical additives Concentration Relative enzyme activity (%) wood xylan (25 U/ml) and oat spelt xylan (20 U/ml). The Control – 100 ± 2.5 crude enzyme did not contain amylase but hardly cel- 2+ Mg (MgCl ) 5 mM 106 ± 2.6 lulase (1.99 U/ml). Crude enzymes produced by Bacil- 2+ Ca (CaCl ) 5 mM 138 ± 4.1 lus sp. strain AQ1 not only showed xylanolytic activity 2+ Fe (FeSO ) 5 mM 88 ± 2.2 but also amylolytic and cellulolytic activity (Wahyuntari K (KCl) 5 mM 98 ± 2.4 et al. 2009). Comparisons to the large literature studies as 2+ Cu (CuCl ) 5 mM 92 ± 2.3 summarized in Table 3. Na (NaCl) 5 mM 94 ± 2.3 Based on the available data from this experiment, the 2+ Mn (MnCl ) 5 mM 99 ± 2.5 difference in crude enzyme on the different xylan sub - 2+ Cd (CdCl ) 5 mM 83 ± 2.0 strate could not be explained yet. It is still needed more 2+ Zn (ZnCl ) 5 mM 95 ± 2.3 2+ Hg (HgCl ) 5 mM 20 ± 0.6 Triton X‑100 1% 93 ± 2.3 SDS 1% 87 ± 2.2 EDTA 5 mM 88 ± 2.2 100 β‑Mercaptoethanol 20 mM 86 ± 2.2 Xylanase activity measured in the absence of any chemical additives was taken as control (100%). The non-treated and dialyzed enzyme was considered as 100% for metallic ion assay. Residual activity was measured at pH 7.0 and 55 °C Values represent means of three independent replicates, and ±standard errors are reported 20 Birchwood xylanOat spelt xylanCMC Starch 2+ Carbohydrate substrate (10 mg/mL) also reported that C a ion strongly stimulated xylanase Fig. 6 The effect of the carbohydrate substrate source on the activity. Slightly stimulation was also observed by addi- 2+ xylanase activity produced by Bacillus oceanisediminis strain SJ3 and tion of Mg (106%) (Mamo et  al. 2006; Lv et  al. 2008; recovered by three phase partitioning. The enzyme was incubated Ozcan et al. 2011). with 10 mg/ml of substrate at 55 °C and pH 7.0. Each point represents On the other hand, the inhibition of xylanases by cal- the mean of three independent experiments. Vertical bars indicate cuim and magnesium ions have also been reported standard error of the mean (n = 3) Redisual xylanase activity (%) Relative xylanase activity (%) Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 9 of 12 Table 3 Production of xylanases from various bacteria, namely Bacillus genus and comparisons to our findings Organism Substrate Cultivation conditions Xylanase activity (U/ml or References (temperature and pH) U/mg) Bacillus oceanisediminis SJ3 Oat spelt xylan 55 °C; pH 7.0 20.24 U/ml Present study Jonesia denitrificans BN‑13 Birchwood xylan 50 °C; pH 7.0 77 U/mg Boucherba et al. (2014) Bacillus pumilus MTCC 8964 Oat spelt xylan 60 °C; pH 6 241 U/ml Kumar et al. (2010) Bacillus brevis Birchwood xylan 55 °C; pH 7.0 1.52 U/ml Goswami et al. (2013) Bacillus brevis wheat straw 55 °C; pH 7.0 4380 U/mg Goswami et al. (2014) Bacillus sp. strain BP‑23 Birchwood xylan 50 °C; pH 5.5 40.2 U/mg Blanco et al. (1995) Bacillus halodurans TSEV Cane molasses 80 °C; pH 9.0 15 U/ml Kumar and Satyanarayana (2014) Bacillus amyloliquefaciens Birchwood xylan 25 °C; pH 4.0 701.1 U/mg Baek et al. (2012) CH51 Bacillus subtilis CXJZ birchwood and oat spelt 60 °C; pH 5.8 36,633 U/mg Gang et al. (2012) xylan Bacillus pumilus SSP‑34 Oat spelts xylan 50 °C; pH 6.0 1723 U/mg Subramaniyan (2012) Bacillus pumilus SV‑205 Wheat bran 60 °C; pH 10.0 7382.7 U/ml Nagar et al. (2012) Bacillus subtilus BS05 Sugarcane bagasse 50 °C; pH 5.0 17.58 U/ml Irfan et al. (2012) Gracilibacillus sp. TSCPVG Birchwood xylan pH 7.5 1667 U/mg Poosarla and Chandra (2014) Paenibacillus sp. NF1 Oat spelt xylan 60 °C; pH 6.0 3081.05 U/mg Zheng et al. (2014) Paenibacillus macerans Beechwood xylan 60 °C; pH 4.5 4170 U/mg Dheeran et al. (2012) IIPSP3 Anoxybacillus flavithermus Oat spelt xylan 65 °C; pH 6.0 and pH 8.0 117.64 U/mg Ellis and Magnuson (2012) TWXYL3 complete studies to elaborate the type of xylanolytic (v/v) and less than 60% of its initial activity at 30% (Yang activities present in the crude enzyme of B. oceani- et al. 2010). sediminis strain SJ3. From preliminary study, it can be In some cases, the presence of solvents enhanced the observed that the strain SJ3 was able to grow and pro- xylanase activity, for example the xylanase of B. val- duce xylanases using commercial xylan. The pH and lismortis is extraordinarily stable in the presence of all temperature optima of the preparation were 7 and 55 °C, organic solvents under study. After incubation with respectively, and the enzyme was stable in a range of pH n-dodecane, isooctane, n-decane, xylene, toluene, n-hex- 5–10 retained 50% of its activity during 6 h at 55 °C. The ane, n-butanol, and cyclohexane, the xylanase activity enzyme is also resistant to hydrophobic solvents, these increased to 230.8, 137.7, 219.8, 107, 190.5, 194.7, 179.3, properties place this enzyme as promising for industrial and 111.6%, respectively (Gaur and Tiwari 2015). and biotechnological applications especially lignocellu- lose bioconversion and bioethanol production. Conclusion In conclusion, a new extracellular thermostable xyla- Eec ff t of organic solvents of the xylanase activity nase from B. oceanisediminis strain SJ3 was produced The xylanase from B. oceanisediminis strain SJ3 is resist- and characterized in this study. The preliminary results ant to hydrophobic solvents: heptan, chloroform, toluene, of the use of Three phase partitioning for the recovery and cyclohexane (the relative activity is 99.2%) but a loss of the xylanase were presented. The time course for xyla - of the enzyme activity was observed by addition of 30% nase accumulation by strain SJ3 in xylan-based medium (v/v) of methanol, ethanol, propanol, and acetone (Fig. 7). showed that the highest xylanase activity reached 20.24 These alcohols completely inhibited the enzyme from U/ml in an optimized medium with oats spelt xylan used Termitomyces sp. and Macrotermes subhyalinus at 30% as a substrate after 48  h of cultivation. The crude xyla - (v/v) and 60% (v/v), respectively. Primary alcohols includ- nase from strain SJ3 was biochemically characterized. ing methanol, ethanol, and isopropanol as well as poly- The results revealed that the enzyme was highly sta - hydric alcohol containing glycol and glycerol, all showed ble and active at high temperature (55  °C) and alkaline inhibitory effects on A. niger strain C3486 xylanase activ - pH 7.0. Properties of this enzyme such as high specific ity which retained around 90% at the concentration of 2% activity, wide range of pH optimum and stability, and Boucherba et al. Bioresour. Bioprocess. (2017) 4:29 Page 10 of 12 ControlHepaneCyclohexane Toluene Chloroform Propnol AcetoneEthanol Methanol Organic solvents 30% (v/v) Fig. 7 Eec ff t of organic solvents on xylanase activity produced by Bacillus oceanisediminis strain SJ3 and recovered by three phase partitioning. Relative xylanase activity was expressed as a percentage of the control reaction without solvent. Each point represents the mean of three independ‑ ent experiments. Vertical bars indicate standard error of the mean (n = 3) thermostability at elevated temperature as well as organic Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ solvents tolerance, are appropriate for industrial and lished maps and institutional affiliations. biotechnological applications. Interestingly, this enzyme presented high xylanolytic activity with oats spelt xylan, Received: 8 May 2017 Accepted: 1 July 2017 and was very effective in the pulp bleaching industry, thus offering a potential promising candidate for applica - tion in biotechnological bioprocesses. Accordingly, fur- ther studies, some of which are currently underway, are References needed to investigate the purification to homogeneity Amore A, Parameswaran B, Kumar R, Birolo L, Vinciguerra R, Marcolongo L, Ionata E, La Cara F, Pandey A, Faraco V (2015) Application of a new and encoding gene, perform site-directed mutagenesis, xylanase activity from Bacillus amyloliquefaciens XR44A in brewer’s spent and determine its structure–function relationships. grain saccharification. J Chem Technol Biotechnol 90:573–581 Badhan AK, Chadha BS, Kaur J, Saini HS, Bhat MK (2007) Production of multiple Authors’ contributions xylanolytic and cellulolytic enzymes by thermophilic fungus Mycelioph- NB, MG, and SB designed this research plan and discussed with ABD. NB, MG, thora sp. IMI 387099. Bioresour Technol 98:504–510 CB, KB, MYC performed all the research experiments and NB and MG wrote the Baek CU, Lee SG, Chung YR, Cho I, Kim JH (2012) Cloning of a family 11 xyla‑ draft paper. 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Journal

"Bioresources and Bioprocessing" – Springer Journals

Published: Dec 1, 2017

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

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