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Tailoring pullulanase PulAR from Anoxybacillus sp. AR-29 for enhanced catalytic performance by a structure-guided consensus approach

Tailoring pullulanase PulAR from Anoxybacillus sp. AR-29 for enhanced catalytic performance by a... oligomers. In recent years, a few type I pullulanases from Introduction Fervidobacterium nodosum Rt17-B1(Yang et  al. 2020), Starch transformation can be accomplished by using an Bacillus methanolicus PB1 (Zhang et  al. 2020), Geoba- enzymatic process that involves two primary steps: liq- cillus thermocatenulatus DSMZ73010 (Li et  al. 2018), uefaction and saccharification (Hii et  al. 2012). Gener - Bacillus megaterium W1210 (Yang et  al. 2017), Anoxy- ally, the saccharification is conducted at 60 ℃ and pH bacillus sp. SK3-4 (Kahar et  al. 2016) and Paenibacillus 4.5–5.5 for 48–60 h, via pullulanase in combination with polymyxa Nws-pp2 (Wei et  al. 2015) have been cloned β-amylase or glucoamylase, producing maltose syrup and and characterized. However, most of the reported type glucose syrup. Pullulanase [EC 3.2.1.41] is a debranch- I pullulanases exhibit neutral or basic pH optimum, and ing enzyme that can specifically cleave α-1,6-glycosidic their stabilities under acidic or thermophilic conditions linkages in pullulan, starch, amylopectin, glycogen, and are usually poor. related oligosaccharides (Bertoldo and Antranikian Protein engineering is an efficient way to obtain the 2002). Addition of pullulanase would reduce the amount desirable enzymes (Böttcher and Bornscheuer 2010). As of glucoamylase or β-amylase used in the saccharification reported previously, many reports focused on enhanc- step and improve substrate concentration and conversion ing the thermostability or catalytic efficiency of the type (Duan et  al. 2013). High-purity maltose syrup is a low- I pullulanases. For example, Duan et  al. successfully calorie, low-sweetness sugar that is being widely used in improved the thermostability and catalytic efficiency of the food, medicine, and cosmetic industries (Bertoldo a Type I pullulanase from Bacillus deramificans by site- et al. 1999), in which the content of maltose is above 60%. directed mutagenesis (SDM) (Duan et  al. 2013). In a In recent years, enzymatic preparation of maltose syrups recent example, Bi and coworkers employed a computer- has drawn rising interest for its mild reaction condition, aided method to raise Tm of the thermophilic pullula- high selectivity, and high catalytic efficiency (Lin et  al. nase from Bacillus thermoleovorans by 3.8 ℃ (Bi et  al. 2013). In combination with β-amylase and/or maltase, 2020). Up to date, only a limited number of reports on pullulanase can raise starch hydrolysis efficiency to high- improving acidic adaptation of pullulanase are available purity maltose syrup and reduce production cost. (Wang et  al. 2017; Zeng et  al. 2019). Chen and cowork- Pullulanases are divided into type I pullulanase and ers improved the acidic adaptation of Bacillus acidopul- type II pullulanase based on substrate specificity and lulyticus pullulanase by altering hydrogen bonds network reaction products. Type II pullulanase hydrolyzes both near the catalytic residues, shifting its optimum pH from α-1,6-glycosidic linkages and α-1,4-glycosidic linkages 5.0 to 4.0 at the expense of activity reduction (Chen et al. (Kang et  al. 2011; Li et  al. 2012; Pang et  al. 2019). Com- 2019). Therefore, it is still needed to dig out the pullu - pared with type II pullulanase, type I pullulanase specifi - lanase with high catalytic efficiency and stability under cally hydrolyzes α-1,6-glycosidic linkages in pullulan and thermophilic and acidic conditions. other polysaccharides, forming maltotriose and linear Li  et al. Bioresources and Bioprocessing (2022) 9:25 Page 3 of 11 In this study, we identified a novel pullulanase from ampicillin and cultivated at 37  °C overnight. The over - Anoxybacillus sp. AR-29 (PulAR). Four residues A365, night cultures were then transferred into another 100 mL V401, H499, and T504 lining the catalytic pocket were of LB medium containing ampicillin (100  μg/mL), and identified as critical for the thermostability and acid cultivated for 3  h until the OD at 600  nm was between resistance by a structure-guided consensus approach. 0.6 and 0.8. The protein expression was then induced by The catalytic performance of PulAR under thermophilic adding IPTG at a final concentration of 0.5 mM for 16 h and acidic conditions was enhanced by SDM. In addition, at 16  °C. The cells were harvested by centrifugation at structural analysis and MD simulations were performed 8000g for 10  min at 4  °C and resuspended in 20  mL of to elucidate their roles. binding buffer (20 mM Tris–HCl, 250 mM NaCl, 20 mM imidazole). Cell lysates were prepared with a French Materials and methods press operating at 4 °C, and then centrifuged at 8000g for Bacterial strains, plasmids, and enzyme 30 min. The resultant soluble fraction was micro-filtrated, The Anoxybacillus sp. AR-29 strain was isolated and and loaded onto a Ni–NTA column which was pre-equil- stored in our laboratory.  The PulAR gene (GenBank ibrated with the binding buffer. The target protein was accession number KY273924.1) was cloned from Anoxy- eluted by a 20–250 mM imidazole gradient at a flow rate bacillus sp. AR-29. We have constructed the pET-32a of 1  mL/min. The protein was pooled and dialysed with ( +)-PulAR plasmid in our previous study. Escherichia Buffer C (20  mM Tris–HCl and 150  mM NaCl, pH 8.0). coli DH5α was used as the host for the cloning work, and The purified protein was estimated by SDS-PAGE, and E. coli BL21(DE3) was the host for the expression of the the concentration of the protein was determined by the enzymes. Phanta Super-Fidelity DNA Polymerase and the BCA protein assay kit. restriction enzyme Dpn I were purchased from Vazyme Biotech Co., Ltd (Nanjing, China). All other chemicals Characterization of WT‑PulAR and reagents were obtained from standard commercial Pullulanase activity was measured in 500  μL reaction sources. mixtures that contained 50 μL of pullulan (0.5%), 400 μL of sodium phosphate buffer (100  mM, pH 6.0), and the Genomic DNA extraction, amplification and bioinformatics appropriate amounts of the purified enzymes, and incu - analysis bated at 60 °C for 10 min. One unit of pullulanase activity The genomic DNA of Anoxybacillus sp. AR-29 was was defined as the amount of enzyme required to release extracted using TIANamp Bacteria DNA Kit (Tiangen, 1  μmol of reducing sugars per minute. Effects of pH on Beijing, China). And the genomic DNA of Anoxybacillus the purified PulAR were determined in 100  mM buffer sp. AR-29 was used as the template for the amplification over pH 3.6–9, including sodium acetate buffer (pH 3.6– of the pulAR-encoding gene, using the forward primer 5.8), sodium phosphate buffer (pH 5.8–7.5) and Tris–HCl 5ʹ-GCG ATA TCA TGT ATG AGG TCT TTT CC-3 ʹ and buffer (pH 7.5–9.0). The temperature optimum of PulAR reverse primer 5 ʹ -GCC TCG AGT TAT ATG TGA TTT was measured at temperatures between 45 and 95  °C in GCT TTTT-3 ʹ, respectively. PulAR gene was amplified 100 mM sodium acetate buffer (pH 6.0). by PCR according to the following protocol: denatura- The kinetic parameters of WT-PulAR were determined tion at 95 °C for 60 s, 20 cycles of (95 °C, 30 s; 55 °C, 30 s; according to the method as previously described (Li et al. 72 °C, 90 s), and a final extension at 72 °C for 10 min. The 2015), using pullulan at varying concentrations (1.0, 1.25, protein sequence and nucleotide sequence of PulAR were 1.33, 2.0, 2.5, 3.33, and 5.0 mg/mL) as substrate at 60 °C analyzed by using BLASTp and BLASTn (http:// www. for 10 min in 100 mM buffer (pH 6.0). Experiments were ncbi. nlm. nih. gov/), respectively. The MW and pI of this conducted in triplicates. The Michaelis–Menten equa - enzyme were predicted via the web server (http:// web. tion was fitted to the data points to determine K and expasy. org/ compu te_ pi/). v by nonlinear least-squares regression analysis using max Origin 8.5. Cloning, over‑expression and purification of PulAR The PCR products were double digested with the restric - Screening for hotspot residues by a structure‑guided tion enzymes EcoR V and Xho I and cloned into the pET- consensus approach 32a ( +), which was also digested by the same restriction The protein sequence of PulAR was aligned with the pul - enzymes, yielding the recombinant plasmid pET-32a lulanases from Anoxybacillus sp. 18–11 (pH 6.0) (PulA) opt ( +)-PulAR. For over-expression of PulAR in E. coli (Xu et  al. 2014), Bacillus acidopullulyticus (pH 5.0) opt BL21(DE3), the recombinant plasmid was transformed (Bapul) (Turkenburg et al. 2009), and Bacillus naganoen- into E. coli BL21(DE3). The transformant was picked into sis (pH 4.5) (Bnpul) (Nie et  al. 2013). The temperature opt the tube with 5  mL LB medium containing 100  μg/mL optimum range of these pullulanases was 55–65  °C. The Li et al. Bioresources and Bioprocessing (2022) 9:25 Page 4 of 11 putative structure of PulAR was obtained with the homol- of NaCl was set at 0.9%. After initial minimization ogy-modeling pipeline SWISS-MODEL server (http:// through the steepest descent and simulated annealing, swiss- model. expasy. org), using the structure of type I convergence was reached. The time step is 1 fs, and the pullulanase (PDB ID: 3WDH) from Anoxybacillus sp. track is saved every 100 ps. All independent MD simu- LM18-11 as the template. The structures were analyzed lations were repeated three times. and visualized by using PyMOL (http:// www. pymol. org/). The residues within 8  Å of the catalytic triad of PulAR were identified and the differences in these amino acid Results and discussion residues among the above pullulanases were explored. Sequence analysis of PulAR encoding gene Totally, five residues (A365, T399, V401, Y491, and T504) The pullulanase PulAR gene (2,259  bp long, GenBank different from those of the acidophilic pullulanases (Ba pul accession number KY273924.1) has a putative transla- and Bnpul) were selected for SDM. In addition, the muta- tional start site GTG with a G + C content of 78.4%, and tion Y477A could improve the thermostability of a Type encodes an enzyme with a predicted molecular mass of I pullulanase PulA in our previous report (Li et al. 2015). 85.0  kDa with a theoretical pI of 5.49. The structure of Therefore, the residue H499 of PulAR was also chosen for PulAR was constructed based on the crystal structure SDM, which was corresponding to Y477 of PulA. of the pullulanase PulA from Anoxybacillus sp. LM18- 11 (PDB ID: 3WDH), with which it shares 58.29% iden- tity (Fig. 1A). Analysis of the protein sequence of PulAR Construction of the mutants by NCBI BLASTp showed that it contains the YNW- The PulAR gene was cloned into the pET-32a( +) plas- GYDP motif and four conserved regions (I–IV) (Addi- mid, and the recombinant plasmid pET-32a( +)-PulAR tional file  1: Fig. S2), which are similar to those of type I was used as the template for site-directed mutagenesis. pullulanases and comprise a catalytic triad and several The PCR was conducted as follows: 95 °C for 5 min, then substrate binding sites. Therefore, the residues D435, 26 cycles (95  °C for 30  s, 50  °C for 30  s, and 72  °C for E464, and D554 of PulAR are inferred as the catalytic 8 min), and final extension at 72 °C for 10 min. The PCR residues. No signal peptide was found in the pullula- reaction system (25  μL) consisted of 12.5  μL 2 × Phanta −1 nase PulAR through analysis by Signal P (https:// servi buffer, 0.5  μL dNTP mixture (each at 10  mmol L ), −1 ces. healt htech. dtu. dk/ servi ce. php? Signa lP-4.1). Com- 1 μL forward primer (10 μmol L ), 1 μL reverse primer −1 parison with the pullulanase sequences in the GenBank (10 μmol L ), 1  μL plasmid template (50  ng), 8.5  μL −1 database, listed in Table  1, revealed that PulAR shares ultra-pure water, and 0.5 μL DNA polymerase (1 U μL ). 70.8%, 60.2%, 58.3%, 46.5%, 43.6%, 41.5%, 41.1% and The primers are listed in Additional file  1: Table  S1. The 38.1% identity with the thermostable pullulanases from PCR products were digested with Dpn I and then trans- Bacillus stearothermophilus (Kuriki et  al. 1990), Geo- formed into E. coli BL21(DE3). To verify that only the bacillus thermoleovorans (Zouari Ayadi et  al. 2008), designated mutations were inserted by the DNA poly- Anoxybacillus sp. LM18-11 (Xu et al. 2014), Bacillus sp. merase, the full plasmids containing the pullulanase gene CICIM 263 (Li et  al. 2012), Anaerobranca gottschalkii were sequenced. (Bertoldo et  al. 2004), Fervidobacterium pennivorans DSM 9078 (Bertoldo et  al. 1999), Thermotoga neapoli - Expression, purification and characterization of PulAR tana (Kang et  al. 2011), and Caldicellulosiruptor sac- mutants charolyticus (Albertson et al. 1997), respectively. Expression, purification, and characterization of the PulAR mutants were conducted according to the meth- ods described above as 2.3 and 2.4. The purified protein Screening for mutation hotspots was analyzed by SDS-PAGE (Additional file 1: Fig. S1). To identify the critical residues responsible for catalytic activity and stability of PulAR, we compared the pro- Structural and MD simulation analyses of PulAR tein sequences of neutrophilic type I pullulanases (PulA Homology modeling of the PulAR mutants was per- and PulAR) with the acidophilic pullulanases (BnPul formed with the same approach as used for WT. Dis- and BaPul) (Fig.  1a), which have temperature and pH covery Studio and DSSP web server (http:// www. cmbi. optima of 50–60  °C and pH 4.5–6.0. The differences in ru. nl/ xssp/) were adopted to analyze the structural the amino acid residues among the above pullulanases information. YASARA software was used for molecular within 8 Å of the catalytic triad were explored (Fig. 1b), dynamics simulation (MD simulation). MD simulations and six mutants A365V, T399S, V401T, V401C, Y491V, were performed at 60  °C and pH 5.0 for 20  ns. Dur- and T504V, were generated. Besides, the single mutant ing the dynamic simulation, the force field was Amber H499A was also constructed. 03, the TIP3P model was used, and the concentration Li  et al. Bioresources and Bioprocessing (2022) 9:25 Page 5 of 11 Fig. 1 a Sequence alignment of PulAR with Bacillus acidopullulyticus pullulanase (Bapul) and Bacillus naganoensis pullulanase (Bnpul). b Mutation hotspots A365, T399, V401, Y491, and T504 Li et al. Bioresources and Bioprocessing (2022) 9:25 Page 6 of 11 Table 1 Comparison of optimum temperatures between bacterial type I pullulanases Bacterial source Accession number Optimum Similarity Special activity References temperature with PulAR (℃) Bacillus stearothermophilus 1808262A 60 70.8% 0.214 U/mL Kuriki et al. (1990) Geobacillus thermoleovorans CAC85704.1 70 60.2% 36 U/mg Zouari Ayadi et al. (2008) Anoxybacillus sp. LM18‑11 AEW23439.1 60 58.3% v 750 U/mg Xu et al. (2014) max Bacillus sp. CICIM 263 AGA03915.1 70 46.5% 73 U/mg Li et al. (2012) Anaerobranca gottschalkii AAS47565.1 65–70 43.6% 56 U/mg Bertoldo et al. (2004) Fervidobacterium pennivorans DSM 9078 AAD30387.1 80 41.5% 78 U/mg Bertoldo et al. (1999) Thermotoga neapolitana ACN58254.1 80–85 41.1% 25.1 U/mg Kang et al. (2011) Caldicellulosiruptor saccharolyticus AAB06264.1 85 38.1% – Albertson et al. (1997) Anoxybacillus sp. AR‑2 KY273924 55 100.0% 24.4 U/mg This study KY273924 65 99.0% 87.8U/mg (PulAR‑A365V/ This study V401C/T504V/H499A) Generation of PulAR‑positive mutants and enzymatic PulAR-A365V, PulAR-V401C, PulAR-A365V-V401C, characterization PulAR-A365V-V401C-T504V, and PulAR-A365V- Firstly, the pullulanase activities of seven mutants were V401C-T504V-H499A were 4.4, 10.0, 14.4, 32.2, and assayed at pH 5.0 and pH 6.0, respectively, and then the 40.0  U/mg, respectively. Among them, the specific activity ratio of WT and its mutants at pH 5.0 to that at activity of the quadruple mutant PulAR-A365V-V401C- pH 6.0 (A /A ) were evaluated. As described in T504V-H499A was 8.1-fold higher than that of WT at pH5.0 pH6.0 Additional file  1: Table  S2, A /A of WT-PulAR 60 ℃, pH 5.0. pH5.0 pH6.0 and its mutants (A365V, T399S, V401T, V401C, Y491V, To evaluate the thermostabilities of PulAR and its T504V and H499A) were 0.20, 0.49, 0.12, 0, 0.75, mutants, the enzymes were incubated at 60 ℃ and pH 6.0, 0.19, 0.29 and 0.50, respectively. Therefore, we com - and then the residual activities were assayed after varying bined the positive mutations A365V, V401C, T504V, incubation times. As shown in Table  2, all the mutants and H499A, generating three triple mutants PulAR- PulAR-A365V, PulAR-V401C, PulAR-A365V-V401C, A365V-V401C, PulAR-A365V-V401C-T504V, and PulAR-A365V-V401C-T504V, and PulAR-A365V- PulAR-A365V-V401C-T504V-H499A. V401C-T504V-H499A displayed increased half-lives. At We characterized the three combined mutants 60 and 65  °C, the half-lives (t ) of PulAR were only 4.8 1/2 PulAR-A365V-V401C, PulAR-A365V-V401C-T504V, and 2.5  h, respectively, whereas those of the quadruple and PulAR-A365V-V401C-T504V-H499A, as well mutant were 17.5 and 10.3 h, which were 2.65 and 3.12- as two single mutants PulAR-A365V and PulAR- fold higher than those of PulAR, respectively. The stabili - V401C. As shown in Fig.  2, the optimum temperature ties of PulAR under the acidic conditions (pH 4.5 and 5.0) (T ) of PulAR was 55 ℃, and these of the mutants were also significantly enhanced. The half-lives of PulAR opt PulAR-A365V, PulAR-V401C, PulAR-A365V-V401C, were 5.4 and 6.1 h at pH 4.5 and 5.0, respectively, whereas PulAR-A365V-V401C-T504V, and PulAR-A365V- those of the quadruple mutant PulAR-A365V-V401C- V401C-T504V-H499A were 55, 60, 60, 60, and 65 ℃, T504V-H499A displayed longer half-lives of 13.9 and respectively. Compared with WT, the T of the 17.3 h, respectively (Table 3). The structures of PulAR and opt mutant PulAR-A365V-V401C-T504V-H499A was its mutants were modeled to investigate the mechanisms increased by 10 ℃. In addition, at 60 ℃ and pH 6.0, the of the enhanced thermostability and pH stability. Ala365 specific activities of WT and its mutants PulAR-A365V, is buried in the internal of the protein. As shown in Fig. 3, PulAR-V401C, PulAR-A365V-V401C, PulAR-A365V- the mutation A365V introduces two extra hydrophobic V401C-T504V, and PulAR-V401C-T504V-H499A interactions F432-V365 and F434-V365 while maintain- were 24.4, 37.8, 43.3, 48.9, 68.9, and 87.8  U/mg. The ing the two hydrogen bonds V365-R433 and D435-V365. optimum pH of PulAR-A365V, PulAR-V401C, PulAR- The residue V401 is located on the protein surface. The A365V-V401C, and PulAR-A365V-V401C-T504V was mutation V401C increased the hydrophilicity of the pro- 6.0, which was similar to that of the WT. At 60 ℃ and tein surface (Fig. 4), contributing to enhanced thermosta- pH 5.0, the specific activities of PulAR and its mutants bility (Yu et  al. 2012). Similar to the mutation Y477A in Li  et al. Bioresources and Bioprocessing (2022) 9:25 Page 7 of 11 Fig. 2 Temperature (a) and pH (b) optima of WT‑PulAR and its mutants our previous report, the solvent accessibility of the resi- 2015). Replacing T504 with Val possessing an extra CH due at the position 499 was reduced from 29.9 to 2.4 as group reinforces the hydrophobic interior of the struc- assayed by the DSSP web server, which might enable the ture, leading to enhancement of the thermostability and protein structure of PulAR more compact (Li et al. 2005, pH stability (Tai et al. 2011). Li et al. Bioresources and Bioprocessing (2022) 9:25 Page 8 of 11 Catalytic efficiency measurement and MD analysis Table 2 Half‑lives of WT ‑PulAR and its variants at 6065 °C and 65 °C WT-PulAR and its mutants were subjected to kinetic analysis at 60 ℃, pH 5.0 and 6.0, respectively. Compared Mutant 60 °C 65 °C with WT, at pH 6.0, 60 °C, the K values of PulAR-A365V, k (1/h) t (h) k (1/h) t (h) d 1/2 d 1/2 PulAR-V401C, PulAR-A365V-V401C, PulAR-A365V- V401C-T504V, and PulAR-A365V-V401C-T504V-H499A WT‑PulAR 0.14 4.8 ± 0.2 0.28 2.5 ± 0.1 decreased by 7.3%, 7.3%, 24.4%, 33.5%, and 53.0%, respec- PulAR‑A365V 0.12 5.9 ± 0.5 0.17 4.1 ± 0.2 tively, while the k values increased by 39.1%, 54.3%, PulAR‑ V401C 0.12 6.0 ± 0.1 0.17 4.1 ± 0.1 cat 168.9%, 193.7%, and 254.3%, respectively (Table  4). In PulAR‑A365V ‑ V401C 0.07 9.9 ± 0.2 0.10 6.7 ± 0.4 addition, at pH 5.0, 60 °C, the K values of PulAR-A365V, PulAR‑A365V ‑ V401C‑ T504V 0.05 13.2 ± 0.1 0.08 9.2 ± 0.2 PulAR-V401C, PulAR-A365V-V401C, PulAR-A365V- PulAR‑A365V ‑ V401C‑ T504V‑ 0.04 17.5 ± 0.1 0.07 10.3 ± 0.3 H499A V401C-T504V, and PulAR-A365V-V401C-T504V-H499A decreased by 15.2%, 19.3%, 31.7%, 43.7%, and 68.5%, respectively, while the k values increased by 37.7%, cat 45.7%, 154.8% 196.7%, and 230.7%, respectively (Table 5). Table 3 Half‑lives of WT ‑PulAR and its variants at pH 4.5 and 5.0 Resultantly, the catalytic efficiencies (k /K ) of the cat M Mutant pH 4.5 pH5.0 “best” quadruple mutant PulAR-A365V-V/V-H499A were 6.6- and 9.6-fold higher than those of PulAR, at k (1/h) t (h) k (1/h) t (h) d 1/2 d 1/2 pH 6.0 and 5.0, respectively. The catalytic efficiency of WT‑PulAR 0.13 5.4 ± 0.3 0.11 6.1 ± 0.5 PulAR was enhanced by mutations, which were identi- PulAR‑A365V 0.10 7.0 ± 0.4 0.08 8.5 ± 1.4 fied by sequence alignment of the acidophilic pullula - PulAR‑ V401C 0.10 7.1 ± 0.6 0.08 8.4 ± 0.3 nase and neutrophilic pullulanase. Further, the roles of PulAR‑A365V ‑ V401C 0.07 9.4 ± 0.3 0.06 10.8 ± 0.2 A365, V401, T504, and H499 in the structure–function PulAR‑A365V ‑ V401C‑ T504V 0.06 11.1 ± 0.4 0.05 13.6 ± 1.0 relationship were analyzed. A365, V401, T504 and H499 PulAR‑A365V ‑ V401C‑ T504V‑ 0.05 13.9 ± 1.2 0.04 17.3 ± 1.2 form the catalytic pocket, shown in Fig.  1b. They are H499A located within 8  Å of the catalytic residues D435, E464, Fig. 3 Structural analysis of PulAR before (a) and after (b) mutation A365V Li  et al. Bioresources and Bioprocessing (2022) 9:25 Page 9 of 11 Fig. 4 The hydrophobic residue V401 is shown in yellow (a) and the hydrophilic residue C401 is shown in blue (b) Table 4 Catalytic efficiencies of WT ‑PulAR and its mutants at 60 °C and pH 6.0 −1 −1 −1 −1 Mutant v (μmol min  mg ) K (mg mL ) k (s ) k /K (mL max M cat cat M −1 −1 mg  s ) WT‑PulAR 31.6 ± 1.2 1.64 ± 0.20 52.7 32.1 PulAR‑A365V 44.0 ± 2.3 1.52 ± 0.12 73.3 48.2 PulAR‑ V401C 48.8 ± 1.6 1.52 ± 0.50 81.3 54.2 PulAR‑A365V ‑ V401C 85.0 ± 2.5 1.24 ± 0.32 141.7 114.3 PulAR‑A365V ‑ V401C‑ T504V 92.9 ± 2.0 1.09 ± 0.11 154.8 142.0 PulAR‑A365V ‑ V401C‑ T504V‑H499A 112.0 ± 3.1 0.77 ± 0.15 186.7 242.5 Table 5 Catalytic efficiencies of WT ‑PulAR and its mutants at 60 °C and pH 5.0 −1 −1 −1 −1 Mutant v (μmol min  mg ) K (mg mL ) k (s ) k /K (mL max M cat cat M −1 −1 mg  s ) WT‑PulAR 25.6 ± 1.1 4.67 ± 0.12 42.7 9.1 PulAR‑A365V 35.3 ± 0.3 3.96 ± 0.13 58.8 14.8 PulAR‑ V401C 37.3 ± 0.5 3.77 ± 0.20 62.2 16.8 PulAR‑A365V ‑ V401C 65.3 ± 1.2 3.19 ± 0.50 108.8 34.1 PulAR‑A365V ‑ V401C‑ T504V 76.0 ± 2.3 2.63 ± 0.28 126.7 48.2 PulAR‑A365V ‑ V401C‑ T504V‑H499A 84.7 ± 1.6 1.47 ± 0.05 141.2 96.1 and D554. And all the single mutation A365V, V401C, simulation analysis of PulAR and the quadruple mutant T504V, H499, and the superposition of mutations tends PulAR-A365V-V401C-T504V-H499A was conducted. to confer increased flexibility of the active sites, resulting As shown in Fig.  5, during the initial 6  ns for simula- in the increased catalytic efficiencies (Tables 4, 5). tion, both the structures of PulAR and quadruple mutant To further investigate the mechanism of the PulAR PulAR-A365V-V401C-T504V-H499A are unstable. The mutant against high temperature and acidic pH, MD entire protein conformation of the quadruple mutant Li et al. Bioresources and Bioprocessing (2022) 9:25 Page 10 of 11 Fig. 5 MD simulation analysis of WT‑PulAR and its quadruple mutant PulAR‑A365V ‑ V401C‑ T504V‑H499A became more stable than PulAR after 6  ns simulation Supplementary Information time, which was consistent with stability enhancement The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40643‑ 022‑ 00516‑4. under the thermophilic and acidic conditions. In this work, the catalytic performance of PulAR Additional file 1: Table S1. Primers design for site ‑ directed mutagenesis. under the thermophilic and acidic conditions was sig- Table S2. Activity ratios of PulAR and its mutants at pH 5.0 to at pH 6.0. nificantly enhanced by using a structure-guided con - Fig. S1. SDS‑PAGE analysis of WT ‑PulA and its mutants. M, Markers; Lane sensus approach. Four mutations A365V, V401C, 1, purified WT ‑PulA; Lane 2, purified PulA‑A365V; Lane 3, purified PulA‑ V401C; Lane 4, purified PulA‑A365V/V401C; Lane 5, purified PulA‑A365V/ T504V, and H499A were obtained by SDM. Finally, V401C/T504C; Lane 6, purified PulA‑A365V/V401C/T504C/H499A. Fig. S2. the “best” quadruple mutant PulAR-A365V/V401C/ Multiple sequence alignment of pullulanases from Anoxybacillus sp. AR‑29, T504V/H499A showed higher catalytic activity and Anoxybacillus sp. LM18‑11, Bacillus acidopullulyticus and Bacillus naganoen- sis. Conserved residues are indicated in frames. thermostabilities under the thermophilic and acidic conditions. Structural comparison indicated that the increased internal hydrophobic interactions, the Acknowledgements We are sincerely grateful for the help and pullulanase‑producing strain reduced solvent accessibility surface area and the provided by Professor Hui Song of Tianjin Institute of industrial biotechnology, increased hydrophilic of the protein surface are the Chinese Academy of Sciences. main reasons for the enhancement of thermostability Authors’ contributions and acid resistance. The “best” mutant PulAR-A365V/ YJW conceived of the study. SFL performed the experiment. SFL and SYX V401C/T504V/H499A exhibited great potential in the statistically analyzed the data, and collected the data. YJW and SYX drafted the production of high-purity maltose syrup and other manuscript. YGZ revised the manuscript. All authors read and approved the final manuscript. related starch processing industry. Li  et al. Bioresources and Bioprocessing (2022) 9:25 Page 11 of 11 Funding Kuriki T, Park JH, Imanaka T (1990) Characteristics of thermostable pullulanase This research was financially supported by National Natural Science Founda‑ from Bacillus stearothermophilus and the nucleotide sequence of the tion of China (31801466, 22178318). gene. J Ferment Bioeng 69(4):204–210 Li WF, Zhou XX, Lu P (2005) Structural features of thermozymes. Biotechnol Availability of data and materials Adv 23(4):271–281 The data and the materials are all available in this article and additional docu‑ Li Y, Zhang L, Niu D, Wang Z, Shi G (2012) Cloning, expression, characteriza‑ ment file. tion, and biocatalytic investigation of a novel bacilli thermostable type I pullulanase from Bacillus sp. CICIM 263. J Agric Food Chem 60(44):11164–11172 Declarations Li SF, Xu JY, Bao YJ, Zheng HC, Song H (2015) Structure and sequence analysis‑ based engineering of pullulanase from Anoxybacillus sp. LM18‑11 for Ethics approval and consent to participate improved thermostability. J Biotechnol 210:8–14 Not applicable. Li L, Dong F, Lin L, He D, Wei W, Wei D (2018) N‑terminal domain truncation and domain insertion‑based engineering of a novel thermostable type Consent for publication I pullulanase from Geobacillus thermocatenulatus. J Agric Food Chem All the authors have read and approved to submit it to Bioresources and 66(41):10788–10798 Bioprocessing. Lin Q, Xiao H, Liu GQ, Liu Z, Li L, Yu F (2013) Production of maltose syrup by enzymatic conversion of rice starch. Food Bioprocess Technol Competing interests 6(1):242–248 The authors declare no competing financial interests. Nie Y, Yan W, Xu Y, Chen WB, Mu XQ, Wang X, Xiao R (2013) High‑level expres‑ sion of Bacillus naganoensis pullulanase from recombinant Escherichia coli Author details with auto‑induction: effect of lac operator. PLoS ONE 8(10):e78416 Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College Pang B, Zhou L, Cui W, Liu Z, Zhou S, Xu J, Zhou Z (2019) A hyperthermostable of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 type II pullulanase from a deep‑sea microorganism Pyrococcus yayanosii Chaowang Road, Hangzhou 310014, People’s Republic of China. Engineering CH1. J Agric Food Chem 67(34):9611–9617 Research Center of Bioconversion and Biopurification of the Ministry of Educa‑ Tai H, Irie K, Mikami SI, Yamamoto Y (2011) Enhancement of the thermosta‑ tion, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, People’s bility of hydrogenobacter thermophilus Cytochrome c552 through Republic of China. The National and Local Joint Engineering Research Center introduction of an extra methylene group into its hydrophobic protein for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, interior. Biochemistry 50(15):3161–3169 Hangzhou 310014, People’s Republic of China. Turkenburg JP, Brzozowski AM, Svendsen A, Borchert TV, Davies GJ, Wilson KS (2009) Structure of a pullulanase from Bacillus acidopullulyticus. Proteins Received: 31 December 2021 Accepted: 7 March 2022 76(2):516–519 Wang QY, Xie NZ, Du QS, Qin Y, Li JX, Meng JZ, Huang RB (2017) Active hydro‑ gen bond network (AHBN) and applications for improvement of thermal stability and pH‑sensitivity of pullulanase from Bacillus naganoensis. PLoS ONE 12(1):e0169080 References Wei W, Ma J, Chen SQ, Cai XH, Wei DZ (2015) A novel cold‑adapted type I pul‑ Albertson GD, McHale RH, Gibbs MD, Bergquist PL (1997) Cloning and lulanase of Paenibacillus polymyxa Nws‑pp2: in vivo functional expression sequence of a type I pullulanase from an extremely thermophilic anaero‑ and biochemical characterization of glucans hydrolyzates analysis. BMC bic bacterium, Caldicellulosiruptor saccharolyticus. Biochim Biophys Acta Biotechnol 15:96 Gene Struct Express 1354(1):35–39 Xu J, Ren F, Huang CH, Zheng Y, Zhen J, Sun H, Ko TP, He M, Chen CC, Chan HC, Bertoldo C, Antranikian G (2002) Starch‑hydrolyzing enzymes from thermo ‑ Guo RT, Song H, Ma Y (2014) Functional and structural studies of pullula‑ philic archaea and bacteria. Curr Opin Chem Biol 6(2):151–160 nase from Anoxybacillus sp. LM18–11. Proteins 82(9):1685–1693 Bertoldo C, Duffner F, Jorgensen PL, Antranikian G (1999) Pullulanase type Yang S, Yan Q, Bao Q, Liu J, Jiang Z (2017) Expression and biochemical I from Fervidobacterium pennavorans Ven5: cloning, sequencing, and characterization of a novel type I pullulanase from Bacillus megaterium. expression of the gene and biochemical characterization of the recombi‑ Biotechnol Lett 39(3):397–405 nant enzyme. 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Enzyme Microb Technol 48(3):260–266 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioresources and Bioprocessing Springer Journals

Tailoring pullulanase PulAR from Anoxybacillus sp. AR-29 for enhanced catalytic performance by a structure-guided consensus approach

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Abstract

oligomers. In recent years, a few type I pullulanases from Introduction Fervidobacterium nodosum Rt17-B1(Yang et  al. 2020), Starch transformation can be accomplished by using an Bacillus methanolicus PB1 (Zhang et  al. 2020), Geoba- enzymatic process that involves two primary steps: liq- cillus thermocatenulatus DSMZ73010 (Li et  al. 2018), uefaction and saccharification (Hii et  al. 2012). Gener - Bacillus megaterium W1210 (Yang et  al. 2017), Anoxy- ally, the saccharification is conducted at 60 ℃ and pH bacillus sp. SK3-4 (Kahar et  al. 2016) and Paenibacillus 4.5–5.5 for 48–60 h, via pullulanase in combination with polymyxa Nws-pp2 (Wei et  al. 2015) have been cloned β-amylase or glucoamylase, producing maltose syrup and and characterized. However, most of the reported type glucose syrup. Pullulanase [EC 3.2.1.41] is a debranch- I pullulanases exhibit neutral or basic pH optimum, and ing enzyme that can specifically cleave α-1,6-glycosidic their stabilities under acidic or thermophilic conditions linkages in pullulan, starch, amylopectin, glycogen, and are usually poor. related oligosaccharides (Bertoldo and Antranikian Protein engineering is an efficient way to obtain the 2002). Addition of pullulanase would reduce the amount desirable enzymes (Böttcher and Bornscheuer 2010). As of glucoamylase or β-amylase used in the saccharification reported previously, many reports focused on enhanc- step and improve substrate concentration and conversion ing the thermostability or catalytic efficiency of the type (Duan et  al. 2013). High-purity maltose syrup is a low- I pullulanases. For example, Duan et  al. successfully calorie, low-sweetness sugar that is being widely used in improved the thermostability and catalytic efficiency of the food, medicine, and cosmetic industries (Bertoldo a Type I pullulanase from Bacillus deramificans by site- et al. 1999), in which the content of maltose is above 60%. directed mutagenesis (SDM) (Duan et  al. 2013). In a In recent years, enzymatic preparation of maltose syrups recent example, Bi and coworkers employed a computer- has drawn rising interest for its mild reaction condition, aided method to raise Tm of the thermophilic pullula- high selectivity, and high catalytic efficiency (Lin et  al. nase from Bacillus thermoleovorans by 3.8 ℃ (Bi et  al. 2013). In combination with β-amylase and/or maltase, 2020). Up to date, only a limited number of reports on pullulanase can raise starch hydrolysis efficiency to high- improving acidic adaptation of pullulanase are available purity maltose syrup and reduce production cost. (Wang et  al. 2017; Zeng et  al. 2019). Chen and cowork- Pullulanases are divided into type I pullulanase and ers improved the acidic adaptation of Bacillus acidopul- type II pullulanase based on substrate specificity and lulyticus pullulanase by altering hydrogen bonds network reaction products. Type II pullulanase hydrolyzes both near the catalytic residues, shifting its optimum pH from α-1,6-glycosidic linkages and α-1,4-glycosidic linkages 5.0 to 4.0 at the expense of activity reduction (Chen et al. (Kang et  al. 2011; Li et  al. 2012; Pang et  al. 2019). Com- 2019). Therefore, it is still needed to dig out the pullu - pared with type II pullulanase, type I pullulanase specifi - lanase with high catalytic efficiency and stability under cally hydrolyzes α-1,6-glycosidic linkages in pullulan and thermophilic and acidic conditions. other polysaccharides, forming maltotriose and linear Li  et al. Bioresources and Bioprocessing (2022) 9:25 Page 3 of 11 In this study, we identified a novel pullulanase from ampicillin and cultivated at 37  °C overnight. The over - Anoxybacillus sp. AR-29 (PulAR). Four residues A365, night cultures were then transferred into another 100 mL V401, H499, and T504 lining the catalytic pocket were of LB medium containing ampicillin (100  μg/mL), and identified as critical for the thermostability and acid cultivated for 3  h until the OD at 600  nm was between resistance by a structure-guided consensus approach. 0.6 and 0.8. The protein expression was then induced by The catalytic performance of PulAR under thermophilic adding IPTG at a final concentration of 0.5 mM for 16 h and acidic conditions was enhanced by SDM. In addition, at 16  °C. The cells were harvested by centrifugation at structural analysis and MD simulations were performed 8000g for 10  min at 4  °C and resuspended in 20  mL of to elucidate their roles. binding buffer (20 mM Tris–HCl, 250 mM NaCl, 20 mM imidazole). Cell lysates were prepared with a French Materials and methods press operating at 4 °C, and then centrifuged at 8000g for Bacterial strains, plasmids, and enzyme 30 min. The resultant soluble fraction was micro-filtrated, The Anoxybacillus sp. AR-29 strain was isolated and and loaded onto a Ni–NTA column which was pre-equil- stored in our laboratory.  The PulAR gene (GenBank ibrated with the binding buffer. The target protein was accession number KY273924.1) was cloned from Anoxy- eluted by a 20–250 mM imidazole gradient at a flow rate bacillus sp. AR-29. We have constructed the pET-32a of 1  mL/min. The protein was pooled and dialysed with ( +)-PulAR plasmid in our previous study. Escherichia Buffer C (20  mM Tris–HCl and 150  mM NaCl, pH 8.0). coli DH5α was used as the host for the cloning work, and The purified protein was estimated by SDS-PAGE, and E. coli BL21(DE3) was the host for the expression of the the concentration of the protein was determined by the enzymes. Phanta Super-Fidelity DNA Polymerase and the BCA protein assay kit. restriction enzyme Dpn I were purchased from Vazyme Biotech Co., Ltd (Nanjing, China). All other chemicals Characterization of WT‑PulAR and reagents were obtained from standard commercial Pullulanase activity was measured in 500  μL reaction sources. mixtures that contained 50 μL of pullulan (0.5%), 400 μL of sodium phosphate buffer (100  mM, pH 6.0), and the Genomic DNA extraction, amplification and bioinformatics appropriate amounts of the purified enzymes, and incu - analysis bated at 60 °C for 10 min. One unit of pullulanase activity The genomic DNA of Anoxybacillus sp. AR-29 was was defined as the amount of enzyme required to release extracted using TIANamp Bacteria DNA Kit (Tiangen, 1  μmol of reducing sugars per minute. Effects of pH on Beijing, China). And the genomic DNA of Anoxybacillus the purified PulAR were determined in 100  mM buffer sp. AR-29 was used as the template for the amplification over pH 3.6–9, including sodium acetate buffer (pH 3.6– of the pulAR-encoding gene, using the forward primer 5.8), sodium phosphate buffer (pH 5.8–7.5) and Tris–HCl 5ʹ-GCG ATA TCA TGT ATG AGG TCT TTT CC-3 ʹ and buffer (pH 7.5–9.0). The temperature optimum of PulAR reverse primer 5 ʹ -GCC TCG AGT TAT ATG TGA TTT was measured at temperatures between 45 and 95  °C in GCT TTTT-3 ʹ, respectively. PulAR gene was amplified 100 mM sodium acetate buffer (pH 6.0). by PCR according to the following protocol: denatura- The kinetic parameters of WT-PulAR were determined tion at 95 °C for 60 s, 20 cycles of (95 °C, 30 s; 55 °C, 30 s; according to the method as previously described (Li et al. 72 °C, 90 s), and a final extension at 72 °C for 10 min. The 2015), using pullulan at varying concentrations (1.0, 1.25, protein sequence and nucleotide sequence of PulAR were 1.33, 2.0, 2.5, 3.33, and 5.0 mg/mL) as substrate at 60 °C analyzed by using BLASTp and BLASTn (http:// www. for 10 min in 100 mM buffer (pH 6.0). Experiments were ncbi. nlm. nih. gov/), respectively. The MW and pI of this conducted in triplicates. The Michaelis–Menten equa - enzyme were predicted via the web server (http:// web. tion was fitted to the data points to determine K and expasy. org/ compu te_ pi/). v by nonlinear least-squares regression analysis using max Origin 8.5. Cloning, over‑expression and purification of PulAR The PCR products were double digested with the restric - Screening for hotspot residues by a structure‑guided tion enzymes EcoR V and Xho I and cloned into the pET- consensus approach 32a ( +), which was also digested by the same restriction The protein sequence of PulAR was aligned with the pul - enzymes, yielding the recombinant plasmid pET-32a lulanases from Anoxybacillus sp. 18–11 (pH 6.0) (PulA) opt ( +)-PulAR. For over-expression of PulAR in E. coli (Xu et  al. 2014), Bacillus acidopullulyticus (pH 5.0) opt BL21(DE3), the recombinant plasmid was transformed (Bapul) (Turkenburg et al. 2009), and Bacillus naganoen- into E. coli BL21(DE3). The transformant was picked into sis (pH 4.5) (Bnpul) (Nie et  al. 2013). The temperature opt the tube with 5  mL LB medium containing 100  μg/mL optimum range of these pullulanases was 55–65  °C. The Li et al. Bioresources and Bioprocessing (2022) 9:25 Page 4 of 11 putative structure of PulAR was obtained with the homol- of NaCl was set at 0.9%. After initial minimization ogy-modeling pipeline SWISS-MODEL server (http:// through the steepest descent and simulated annealing, swiss- model. expasy. org), using the structure of type I convergence was reached. The time step is 1 fs, and the pullulanase (PDB ID: 3WDH) from Anoxybacillus sp. track is saved every 100 ps. All independent MD simu- LM18-11 as the template. The structures were analyzed lations were repeated three times. and visualized by using PyMOL (http:// www. pymol. org/). The residues within 8  Å of the catalytic triad of PulAR were identified and the differences in these amino acid Results and discussion residues among the above pullulanases were explored. Sequence analysis of PulAR encoding gene Totally, five residues (A365, T399, V401, Y491, and T504) The pullulanase PulAR gene (2,259  bp long, GenBank different from those of the acidophilic pullulanases (Ba pul accession number KY273924.1) has a putative transla- and Bnpul) were selected for SDM. In addition, the muta- tional start site GTG with a G + C content of 78.4%, and tion Y477A could improve the thermostability of a Type encodes an enzyme with a predicted molecular mass of I pullulanase PulA in our previous report (Li et al. 2015). 85.0  kDa with a theoretical pI of 5.49. The structure of Therefore, the residue H499 of PulAR was also chosen for PulAR was constructed based on the crystal structure SDM, which was corresponding to Y477 of PulA. of the pullulanase PulA from Anoxybacillus sp. LM18- 11 (PDB ID: 3WDH), with which it shares 58.29% iden- tity (Fig. 1A). Analysis of the protein sequence of PulAR Construction of the mutants by NCBI BLASTp showed that it contains the YNW- The PulAR gene was cloned into the pET-32a( +) plas- GYDP motif and four conserved regions (I–IV) (Addi- mid, and the recombinant plasmid pET-32a( +)-PulAR tional file  1: Fig. S2), which are similar to those of type I was used as the template for site-directed mutagenesis. pullulanases and comprise a catalytic triad and several The PCR was conducted as follows: 95 °C for 5 min, then substrate binding sites. Therefore, the residues D435, 26 cycles (95  °C for 30  s, 50  °C for 30  s, and 72  °C for E464, and D554 of PulAR are inferred as the catalytic 8 min), and final extension at 72 °C for 10 min. The PCR residues. No signal peptide was found in the pullula- reaction system (25  μL) consisted of 12.5  μL 2 × Phanta −1 nase PulAR through analysis by Signal P (https:// servi buffer, 0.5  μL dNTP mixture (each at 10  mmol L ), −1 ces. healt htech. dtu. dk/ servi ce. php? Signa lP-4.1). Com- 1 μL forward primer (10 μmol L ), 1 μL reverse primer −1 parison with the pullulanase sequences in the GenBank (10 μmol L ), 1  μL plasmid template (50  ng), 8.5  μL −1 database, listed in Table  1, revealed that PulAR shares ultra-pure water, and 0.5 μL DNA polymerase (1 U μL ). 70.8%, 60.2%, 58.3%, 46.5%, 43.6%, 41.5%, 41.1% and The primers are listed in Additional file  1: Table  S1. The 38.1% identity with the thermostable pullulanases from PCR products were digested with Dpn I and then trans- Bacillus stearothermophilus (Kuriki et  al. 1990), Geo- formed into E. coli BL21(DE3). To verify that only the bacillus thermoleovorans (Zouari Ayadi et  al. 2008), designated mutations were inserted by the DNA poly- Anoxybacillus sp. LM18-11 (Xu et al. 2014), Bacillus sp. merase, the full plasmids containing the pullulanase gene CICIM 263 (Li et  al. 2012), Anaerobranca gottschalkii were sequenced. (Bertoldo et  al. 2004), Fervidobacterium pennivorans DSM 9078 (Bertoldo et  al. 1999), Thermotoga neapoli - Expression, purification and characterization of PulAR tana (Kang et  al. 2011), and Caldicellulosiruptor sac- mutants charolyticus (Albertson et al. 1997), respectively. Expression, purification, and characterization of the PulAR mutants were conducted according to the meth- ods described above as 2.3 and 2.4. The purified protein Screening for mutation hotspots was analyzed by SDS-PAGE (Additional file 1: Fig. S1). To identify the critical residues responsible for catalytic activity and stability of PulAR, we compared the pro- Structural and MD simulation analyses of PulAR tein sequences of neutrophilic type I pullulanases (PulA Homology modeling of the PulAR mutants was per- and PulAR) with the acidophilic pullulanases (BnPul formed with the same approach as used for WT. Dis- and BaPul) (Fig.  1a), which have temperature and pH covery Studio and DSSP web server (http:// www. cmbi. optima of 50–60  °C and pH 4.5–6.0. The differences in ru. nl/ xssp/) were adopted to analyze the structural the amino acid residues among the above pullulanases information. YASARA software was used for molecular within 8 Å of the catalytic triad were explored (Fig. 1b), dynamics simulation (MD simulation). MD simulations and six mutants A365V, T399S, V401T, V401C, Y491V, were performed at 60  °C and pH 5.0 for 20  ns. Dur- and T504V, were generated. Besides, the single mutant ing the dynamic simulation, the force field was Amber H499A was also constructed. 03, the TIP3P model was used, and the concentration Li  et al. Bioresources and Bioprocessing (2022) 9:25 Page 5 of 11 Fig. 1 a Sequence alignment of PulAR with Bacillus acidopullulyticus pullulanase (Bapul) and Bacillus naganoensis pullulanase (Bnpul). b Mutation hotspots A365, T399, V401, Y491, and T504 Li et al. Bioresources and Bioprocessing (2022) 9:25 Page 6 of 11 Table 1 Comparison of optimum temperatures between bacterial type I pullulanases Bacterial source Accession number Optimum Similarity Special activity References temperature with PulAR (℃) Bacillus stearothermophilus 1808262A 60 70.8% 0.214 U/mL Kuriki et al. (1990) Geobacillus thermoleovorans CAC85704.1 70 60.2% 36 U/mg Zouari Ayadi et al. (2008) Anoxybacillus sp. LM18‑11 AEW23439.1 60 58.3% v 750 U/mg Xu et al. (2014) max Bacillus sp. CICIM 263 AGA03915.1 70 46.5% 73 U/mg Li et al. (2012) Anaerobranca gottschalkii AAS47565.1 65–70 43.6% 56 U/mg Bertoldo et al. (2004) Fervidobacterium pennivorans DSM 9078 AAD30387.1 80 41.5% 78 U/mg Bertoldo et al. (1999) Thermotoga neapolitana ACN58254.1 80–85 41.1% 25.1 U/mg Kang et al. (2011) Caldicellulosiruptor saccharolyticus AAB06264.1 85 38.1% – Albertson et al. (1997) Anoxybacillus sp. AR‑2 KY273924 55 100.0% 24.4 U/mg This study KY273924 65 99.0% 87.8U/mg (PulAR‑A365V/ This study V401C/T504V/H499A) Generation of PulAR‑positive mutants and enzymatic PulAR-A365V, PulAR-V401C, PulAR-A365V-V401C, characterization PulAR-A365V-V401C-T504V, and PulAR-A365V- Firstly, the pullulanase activities of seven mutants were V401C-T504V-H499A were 4.4, 10.0, 14.4, 32.2, and assayed at pH 5.0 and pH 6.0, respectively, and then the 40.0  U/mg, respectively. Among them, the specific activity ratio of WT and its mutants at pH 5.0 to that at activity of the quadruple mutant PulAR-A365V-V401C- pH 6.0 (A /A ) were evaluated. As described in T504V-H499A was 8.1-fold higher than that of WT at pH5.0 pH6.0 Additional file  1: Table  S2, A /A of WT-PulAR 60 ℃, pH 5.0. pH5.0 pH6.0 and its mutants (A365V, T399S, V401T, V401C, Y491V, To evaluate the thermostabilities of PulAR and its T504V and H499A) were 0.20, 0.49, 0.12, 0, 0.75, mutants, the enzymes were incubated at 60 ℃ and pH 6.0, 0.19, 0.29 and 0.50, respectively. Therefore, we com - and then the residual activities were assayed after varying bined the positive mutations A365V, V401C, T504V, incubation times. As shown in Table  2, all the mutants and H499A, generating three triple mutants PulAR- PulAR-A365V, PulAR-V401C, PulAR-A365V-V401C, A365V-V401C, PulAR-A365V-V401C-T504V, and PulAR-A365V-V401C-T504V, and PulAR-A365V- PulAR-A365V-V401C-T504V-H499A. V401C-T504V-H499A displayed increased half-lives. At We characterized the three combined mutants 60 and 65  °C, the half-lives (t ) of PulAR were only 4.8 1/2 PulAR-A365V-V401C, PulAR-A365V-V401C-T504V, and 2.5  h, respectively, whereas those of the quadruple and PulAR-A365V-V401C-T504V-H499A, as well mutant were 17.5 and 10.3 h, which were 2.65 and 3.12- as two single mutants PulAR-A365V and PulAR- fold higher than those of PulAR, respectively. The stabili - V401C. As shown in Fig.  2, the optimum temperature ties of PulAR under the acidic conditions (pH 4.5 and 5.0) (T ) of PulAR was 55 ℃, and these of the mutants were also significantly enhanced. The half-lives of PulAR opt PulAR-A365V, PulAR-V401C, PulAR-A365V-V401C, were 5.4 and 6.1 h at pH 4.5 and 5.0, respectively, whereas PulAR-A365V-V401C-T504V, and PulAR-A365V- those of the quadruple mutant PulAR-A365V-V401C- V401C-T504V-H499A were 55, 60, 60, 60, and 65 ℃, T504V-H499A displayed longer half-lives of 13.9 and respectively. Compared with WT, the T of the 17.3 h, respectively (Table 3). The structures of PulAR and opt mutant PulAR-A365V-V401C-T504V-H499A was its mutants were modeled to investigate the mechanisms increased by 10 ℃. In addition, at 60 ℃ and pH 6.0, the of the enhanced thermostability and pH stability. Ala365 specific activities of WT and its mutants PulAR-A365V, is buried in the internal of the protein. As shown in Fig. 3, PulAR-V401C, PulAR-A365V-V401C, PulAR-A365V- the mutation A365V introduces two extra hydrophobic V401C-T504V, and PulAR-V401C-T504V-H499A interactions F432-V365 and F434-V365 while maintain- were 24.4, 37.8, 43.3, 48.9, 68.9, and 87.8  U/mg. The ing the two hydrogen bonds V365-R433 and D435-V365. optimum pH of PulAR-A365V, PulAR-V401C, PulAR- The residue V401 is located on the protein surface. The A365V-V401C, and PulAR-A365V-V401C-T504V was mutation V401C increased the hydrophilicity of the pro- 6.0, which was similar to that of the WT. At 60 ℃ and tein surface (Fig. 4), contributing to enhanced thermosta- pH 5.0, the specific activities of PulAR and its mutants bility (Yu et  al. 2012). Similar to the mutation Y477A in Li  et al. Bioresources and Bioprocessing (2022) 9:25 Page 7 of 11 Fig. 2 Temperature (a) and pH (b) optima of WT‑PulAR and its mutants our previous report, the solvent accessibility of the resi- 2015). Replacing T504 with Val possessing an extra CH due at the position 499 was reduced from 29.9 to 2.4 as group reinforces the hydrophobic interior of the struc- assayed by the DSSP web server, which might enable the ture, leading to enhancement of the thermostability and protein structure of PulAR more compact (Li et al. 2005, pH stability (Tai et al. 2011). Li et al. Bioresources and Bioprocessing (2022) 9:25 Page 8 of 11 Catalytic efficiency measurement and MD analysis Table 2 Half‑lives of WT ‑PulAR and its variants at 6065 °C and 65 °C WT-PulAR and its mutants were subjected to kinetic analysis at 60 ℃, pH 5.0 and 6.0, respectively. Compared Mutant 60 °C 65 °C with WT, at pH 6.0, 60 °C, the K values of PulAR-A365V, k (1/h) t (h) k (1/h) t (h) d 1/2 d 1/2 PulAR-V401C, PulAR-A365V-V401C, PulAR-A365V- V401C-T504V, and PulAR-A365V-V401C-T504V-H499A WT‑PulAR 0.14 4.8 ± 0.2 0.28 2.5 ± 0.1 decreased by 7.3%, 7.3%, 24.4%, 33.5%, and 53.0%, respec- PulAR‑A365V 0.12 5.9 ± 0.5 0.17 4.1 ± 0.2 tively, while the k values increased by 39.1%, 54.3%, PulAR‑ V401C 0.12 6.0 ± 0.1 0.17 4.1 ± 0.1 cat 168.9%, 193.7%, and 254.3%, respectively (Table  4). In PulAR‑A365V ‑ V401C 0.07 9.9 ± 0.2 0.10 6.7 ± 0.4 addition, at pH 5.0, 60 °C, the K values of PulAR-A365V, PulAR‑A365V ‑ V401C‑ T504V 0.05 13.2 ± 0.1 0.08 9.2 ± 0.2 PulAR-V401C, PulAR-A365V-V401C, PulAR-A365V- PulAR‑A365V ‑ V401C‑ T504V‑ 0.04 17.5 ± 0.1 0.07 10.3 ± 0.3 H499A V401C-T504V, and PulAR-A365V-V401C-T504V-H499A decreased by 15.2%, 19.3%, 31.7%, 43.7%, and 68.5%, respectively, while the k values increased by 37.7%, cat 45.7%, 154.8% 196.7%, and 230.7%, respectively (Table 5). Table 3 Half‑lives of WT ‑PulAR and its variants at pH 4.5 and 5.0 Resultantly, the catalytic efficiencies (k /K ) of the cat M Mutant pH 4.5 pH5.0 “best” quadruple mutant PulAR-A365V-V/V-H499A were 6.6- and 9.6-fold higher than those of PulAR, at k (1/h) t (h) k (1/h) t (h) d 1/2 d 1/2 pH 6.0 and 5.0, respectively. The catalytic efficiency of WT‑PulAR 0.13 5.4 ± 0.3 0.11 6.1 ± 0.5 PulAR was enhanced by mutations, which were identi- PulAR‑A365V 0.10 7.0 ± 0.4 0.08 8.5 ± 1.4 fied by sequence alignment of the acidophilic pullula - PulAR‑ V401C 0.10 7.1 ± 0.6 0.08 8.4 ± 0.3 nase and neutrophilic pullulanase. Further, the roles of PulAR‑A365V ‑ V401C 0.07 9.4 ± 0.3 0.06 10.8 ± 0.2 A365, V401, T504, and H499 in the structure–function PulAR‑A365V ‑ V401C‑ T504V 0.06 11.1 ± 0.4 0.05 13.6 ± 1.0 relationship were analyzed. A365, V401, T504 and H499 PulAR‑A365V ‑ V401C‑ T504V‑ 0.05 13.9 ± 1.2 0.04 17.3 ± 1.2 form the catalytic pocket, shown in Fig.  1b. They are H499A located within 8  Å of the catalytic residues D435, E464, Fig. 3 Structural analysis of PulAR before (a) and after (b) mutation A365V Li  et al. Bioresources and Bioprocessing (2022) 9:25 Page 9 of 11 Fig. 4 The hydrophobic residue V401 is shown in yellow (a) and the hydrophilic residue C401 is shown in blue (b) Table 4 Catalytic efficiencies of WT ‑PulAR and its mutants at 60 °C and pH 6.0 −1 −1 −1 −1 Mutant v (μmol min  mg ) K (mg mL ) k (s ) k /K (mL max M cat cat M −1 −1 mg  s ) WT‑PulAR 31.6 ± 1.2 1.64 ± 0.20 52.7 32.1 PulAR‑A365V 44.0 ± 2.3 1.52 ± 0.12 73.3 48.2 PulAR‑ V401C 48.8 ± 1.6 1.52 ± 0.50 81.3 54.2 PulAR‑A365V ‑ V401C 85.0 ± 2.5 1.24 ± 0.32 141.7 114.3 PulAR‑A365V ‑ V401C‑ T504V 92.9 ± 2.0 1.09 ± 0.11 154.8 142.0 PulAR‑A365V ‑ V401C‑ T504V‑H499A 112.0 ± 3.1 0.77 ± 0.15 186.7 242.5 Table 5 Catalytic efficiencies of WT ‑PulAR and its mutants at 60 °C and pH 5.0 −1 −1 −1 −1 Mutant v (μmol min  mg ) K (mg mL ) k (s ) k /K (mL max M cat cat M −1 −1 mg  s ) WT‑PulAR 25.6 ± 1.1 4.67 ± 0.12 42.7 9.1 PulAR‑A365V 35.3 ± 0.3 3.96 ± 0.13 58.8 14.8 PulAR‑ V401C 37.3 ± 0.5 3.77 ± 0.20 62.2 16.8 PulAR‑A365V ‑ V401C 65.3 ± 1.2 3.19 ± 0.50 108.8 34.1 PulAR‑A365V ‑ V401C‑ T504V 76.0 ± 2.3 2.63 ± 0.28 126.7 48.2 PulAR‑A365V ‑ V401C‑ T504V‑H499A 84.7 ± 1.6 1.47 ± 0.05 141.2 96.1 and D554. And all the single mutation A365V, V401C, simulation analysis of PulAR and the quadruple mutant T504V, H499, and the superposition of mutations tends PulAR-A365V-V401C-T504V-H499A was conducted. to confer increased flexibility of the active sites, resulting As shown in Fig.  5, during the initial 6  ns for simula- in the increased catalytic efficiencies (Tables 4, 5). tion, both the structures of PulAR and quadruple mutant To further investigate the mechanism of the PulAR PulAR-A365V-V401C-T504V-H499A are unstable. The mutant against high temperature and acidic pH, MD entire protein conformation of the quadruple mutant Li et al. Bioresources and Bioprocessing (2022) 9:25 Page 10 of 11 Fig. 5 MD simulation analysis of WT‑PulAR and its quadruple mutant PulAR‑A365V ‑ V401C‑ T504V‑H499A became more stable than PulAR after 6  ns simulation Supplementary Information time, which was consistent with stability enhancement The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40643‑ 022‑ 00516‑4. under the thermophilic and acidic conditions. In this work, the catalytic performance of PulAR Additional file 1: Table S1. Primers design for site ‑ directed mutagenesis. under the thermophilic and acidic conditions was sig- Table S2. Activity ratios of PulAR and its mutants at pH 5.0 to at pH 6.0. nificantly enhanced by using a structure-guided con - Fig. S1. SDS‑PAGE analysis of WT ‑PulA and its mutants. M, Markers; Lane sensus approach. Four mutations A365V, V401C, 1, purified WT ‑PulA; Lane 2, purified PulA‑A365V; Lane 3, purified PulA‑ V401C; Lane 4, purified PulA‑A365V/V401C; Lane 5, purified PulA‑A365V/ T504V, and H499A were obtained by SDM. Finally, V401C/T504C; Lane 6, purified PulA‑A365V/V401C/T504C/H499A. Fig. S2. the “best” quadruple mutant PulAR-A365V/V401C/ Multiple sequence alignment of pullulanases from Anoxybacillus sp. AR‑29, T504V/H499A showed higher catalytic activity and Anoxybacillus sp. LM18‑11, Bacillus acidopullulyticus and Bacillus naganoen- sis. Conserved residues are indicated in frames. thermostabilities under the thermophilic and acidic conditions. Structural comparison indicated that the increased internal hydrophobic interactions, the Acknowledgements We are sincerely grateful for the help and pullulanase‑producing strain reduced solvent accessibility surface area and the provided by Professor Hui Song of Tianjin Institute of industrial biotechnology, increased hydrophilic of the protein surface are the Chinese Academy of Sciences. main reasons for the enhancement of thermostability Authors’ contributions and acid resistance. The “best” mutant PulAR-A365V/ YJW conceived of the study. SFL performed the experiment. SFL and SYX V401C/T504V/H499A exhibited great potential in the statistically analyzed the data, and collected the data. YJW and SYX drafted the production of high-purity maltose syrup and other manuscript. YGZ revised the manuscript. All authors read and approved the final manuscript. related starch processing industry. Li  et al. Bioresources and Bioprocessing (2022) 9:25 Page 11 of 11 Funding Kuriki T, Park JH, Imanaka T (1990) Characteristics of thermostable pullulanase This research was financially supported by National Natural Science Founda‑ from Bacillus stearothermophilus and the nucleotide sequence of the tion of China (31801466, 22178318). gene. J Ferment Bioeng 69(4):204–210 Li WF, Zhou XX, Lu P (2005) Structural features of thermozymes. Biotechnol Availability of data and materials Adv 23(4):271–281 The data and the materials are all available in this article and additional docu‑ Li Y, Zhang L, Niu D, Wang Z, Shi G (2012) Cloning, expression, characteriza‑ ment file. tion, and biocatalytic investigation of a novel bacilli thermostable type I pullulanase from Bacillus sp. CICIM 263. J Agric Food Chem 60(44):11164–11172 Declarations Li SF, Xu JY, Bao YJ, Zheng HC, Song H (2015) Structure and sequence analysis‑ based engineering of pullulanase from Anoxybacillus sp. LM18‑11 for Ethics approval and consent to participate improved thermostability. J Biotechnol 210:8–14 Not applicable. Li L, Dong F, Lin L, He D, Wei W, Wei D (2018) N‑terminal domain truncation and domain insertion‑based engineering of a novel thermostable type Consent for publication I pullulanase from Geobacillus thermocatenulatus. J Agric Food Chem All the authors have read and approved to submit it to Bioresources and 66(41):10788–10798 Bioprocessing. Lin Q, Xiao H, Liu GQ, Liu Z, Li L, Yu F (2013) Production of maltose syrup by enzymatic conversion of rice starch. Food Bioprocess Technol Competing interests 6(1):242–248 The authors declare no competing financial interests. Nie Y, Yan W, Xu Y, Chen WB, Mu XQ, Wang X, Xiao R (2013) High‑level expres‑ sion of Bacillus naganoensis pullulanase from recombinant Escherichia coli Author details with auto‑induction: effect of lac operator. 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Journal

Bioresources and Bioprocessing – Springer Journals

Published: Mar 21, 2022

Keywords: Pullulanase; Structure-guided consensus approach; Site-directed mutagenesis; Catalytic pocket; Stability; Catalytic efficiency

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