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• W3204643380 abstract "Xylanases produce xylooligosaccharides from xylan and have thus attracted increasing attention for their usefulness in industrial applications. Previously, we demonstrated that the GH11 xylanase XynLC9 from Bacillus subtilis formed xylobiose and xylotriose as the major products with negligible production of xylose when digesting corncob-extracted xylan. Here, we aimed to improve the catalytic performance of XynLC9 via protein engineering. Based on the sequence and structural comparisons of XynLC9 with the xylanases Xyn2 from Trichoderma reesei and Xyn11A from Thermobifida fusca, we identified the N-terminal residues 5-YWQN-8 in XynLC9 as engineering hotspots and subjected this sequence to site saturation and iterative mutagenesis. The mutants W6F/Q7H and N8Y possessed a 2.6- and 1.8-fold higher catalytic activity than XynLC9, respectively, and both mutants were also more thermostable. Kinetic measurements suggested that W6F/Q7H and N8Y had lower substrate affinity, but a higher turnover rate (kcat), which resulted in increased catalytic efficiency than WT XynLC9. Furthermore, the W6F/Q7H mutant displayed a 160% increase in the yield of xylooligosaccharides from corncob-extracted xylan. Molecular dynamics simulations revealed that the W6F/Q7H and N8Y mutations led to an enlarged volume and surface area of the active site cleft, which provided more space for substrate entry and product release and thus accelerated the catalytic activity of the enzyme. The molecular evolution approach adopted in this study provides the design of a library of sequences that captures functional diversity in a limited number of protein variants. Xylanases produce xylooligosaccharides from xylan and have thus attracted increasing attention for their usefulness in industrial applications. Previously, we demonstrated that the GH11 xylanase XynLC9 from Bacillus subtilis formed xylobiose and xylotriose as the major products with negligible production of xylose when digesting corncob-extracted xylan. Here, we aimed to improve the catalytic performance of XynLC9 via protein engineering. Based on the sequence and structural comparisons of XynLC9 with the xylanases Xyn2 from Trichoderma reesei and Xyn11A from Thermobifida fusca, we identified the N-terminal residues 5-YWQN-8 in XynLC9 as engineering hotspots and subjected this sequence to site saturation and iterative mutagenesis. The mutants W6F/Q7H and N8Y possessed a 2.6- and 1.8-fold higher catalytic activity than XynLC9, respectively, and both mutants were also more thermostable. Kinetic measurements suggested that W6F/Q7H and N8Y had lower substrate affinity, but a higher turnover rate (kcat), which resulted in increased catalytic efficiency than WT XynLC9. Furthermore, the W6F/Q7H mutant displayed a 160% increase in the yield of xylooligosaccharides from corncob-extracted xylan. Molecular dynamics simulations revealed that the W6F/Q7H and N8Y mutations led to an enlarged volume and surface area of the active site cleft, which provided more space for substrate entry and product release and thus accelerated the catalytic activity of the enzyme. The molecular evolution approach adopted in this study provides the design of a library of sequences that captures functional diversity in a limited number of protein variants. Endo-β-1,4-xylanase (EC. 3.2.1.8) randomly cleaves the β-D-xylopyranose bond between two D-xylopyranosyl residues linked by β-(1,4) bond and is a crucial enzyme in xylan degradation (1Nakamichi Y. Fouquet T. Ito S. Watanabe M. Matsushika A. Inoue H. Structural and functional characterization of a bifunctional GH30-7 xylanase B from the filamentous fungus Talaromyces cellulolyticus.J. Biol. Chem. 2019; 294: 4065-4078Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). To date, xylanases have been successfully used in a wide range of industrial applications, including pulp bleaching, animal feed manufacture, food preparation, and biofuel production (2Juturu V. Wu J.C. Microbial xylanases: Engineering, production and industrial applications.Biotechnol. Adv. 2012; 30: 1219-1227Crossref PubMed Scopus (290) Google Scholar). Accordingly, a crucial application of xylanases, supported by their high specificity and limited impact on the environment, is in the production of emerging prebiotics xylooligosaccharides (XOS) from various agro-industrial wastes (3Karlsson E.N. Schmitz E. Linares-Pasten J.A. Adlercreutz P. Endo-xylanases as tools for production of substituted xylooligosaccharides with probiotic properties.Appl. Microbiol. Biotechnol. 2018; 102: 9081-9088Crossref PubMed Scopus (58) Google Scholar). Among the nondigestible oligosaccharides, XOS exhibit higher resistance to acidic pH and heat and a better ability to stimulate the growth of Bifidobacterium. In addition, XOS have also been shown to improve calcium absorption, bowel function, and lipid metabolism and were also reported to offer protection against cardiovascular disease and reduce the risk to develop colon cancer (4Amorim C. Silverio S.C. Prather K.L.J. Rodrigues L.R. From lignocellulosic residues to market: Production and commercial potential of xylooligosaccharides.Biotechnol. Adv. 2019; 37: 107397Crossref PubMed Scopus (81) Google Scholar). However, the major current limitations for the wide applications of XOS are high production costs and low yields (4Amorim C. Silverio S.C. Prather K.L.J. Rodrigues L.R. From lignocellulosic residues to market: Production and commercial potential of xylooligosaccharides.Biotechnol. Adv. 2019; 37: 107397Crossref PubMed Scopus (81) Google Scholar, 5Li Q. Sun B.G. Xiong K. Teng C. Xu Y.Q. Li L.J. Li X.T. Improving special hydrolysis characterization into Talaromyces thermophilus F1208 xylanase by engineering of N-terminal extension and site-directed mutagenesis in C-terminal.Int. J. Biol. Macromol. 2017; 96: 451-458Crossref PubMed Scopus (14) Google Scholar). Numerous xylanases from bacteria, fungi, and yeasts have been isolated, purified, and characterized to date (6Alokika Singh B. Production, characteristics, and biotechnological applications of microbial xylanases.Appl. Microbiol. Biotechnol. 2019; 103: 8763-8784Crossref PubMed Scopus (39) Google Scholar). Based on the sequence homologies and hydrophobic cluster analyses, most of the xylanases belong to glycoside hydrolase (GH) families 10 and 11, while a smaller number belongs to families 5, 8, and 30. GH10 xylanases typically have a high molecular mass and feature a (β/α)8-barrel fold, while GH11 xylanases display a conserved β-jelly roll fold (7Paes G. Berrin J.G. Beaugrand J. GH11 xylanases: Structure/function/properties relationships and applications.Biotechnol. Adv. 2012; 30: 564-592Crossref PubMed Scopus (260) Google Scholar). In contrast to their counterparts from the GH10 family, GH11 xylanases are regarded as “true xylanases” and are attractive because of their small size, strict substrate specificity, and a range of optimal pH values and temperatures (7Paes G. Berrin J.G. Beaugrand J. GH11 xylanases: Structure/function/properties relationships and applications.Biotechnol. Adv. 2012; 30: 564-592Crossref PubMed Scopus (260) Google Scholar, 8Cheng Y.S. Chen C.C. Huang C.H. Ko T.P. Luo W.H. Huang J.W. Liu J.R. Guo R.T. Structural analysis of a glycoside hydrolase family 11 xylanase from Neocallimastix partriciarum.J. Biol. Chem. 2014; 289: 11020-11028Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Although numerous GH11 xylanases have been mined and characterized with a view to meet various industrial demands, most of these naturally occurring GH11 xylanases are unsuitable for industrial applications because of their poor specific activities and biochemical properties (9Xu X. Liu M.Q. Huo W.K. Dai X.J. Obtaining a mutant of Bacillus amyloliquefaciens xylanase A with improved catalytic activity by directed evolution.Enzyme Microb. Technol. 2016; 86: 59-66Crossref PubMed Scopus (30) Google Scholar, 10Teng C. Jiang Y.F. Xu Y.Q. Li Q. Li X.T. Fan G.S. Xiong K. Yang R. Zhang C.N. Ma R. Zhu Y.P. Li J.L. Wang C.T. Improving the thermostability and catalytic efficiency of GH11 xylanase PjxA by adding disulfide bridges.Int. J. Biol. Macromol. 2019; 128: 354-362Crossref PubMed Scopus (15) Google Scholar). Therefore, the engineering of GH11 xylanases by directed evolution and rational design is gaining increasing attention. Rational design via site-directed mutagenesis or peptide segment substitution has already been successfully used to enhance the catalytic activity of several GH11 xylanases. For instance, a study using virtual mutations and molecular dynamics (MD) simulations of the GH11 xylanase AnXynB from Aspergillus niger indicated that the amino acid residues at the −3 subsite played a crucial role in substrate binding, and specific mutations in this site (in particular, the S41N/T43E double mutant) increased the catalytic activity by 1.7-fold (11Wu X.Y. Tian Z.N. Jiang X.K. Zhang Q. Wang L.S. Enhancement in catalytic activity of Aspergillus niger XynB by selective site-directed mutagenesis of active site amino acids.Appl. Microbiol. Biotechnol. 2018; 102: 249-260Crossref PubMed Scopus (27) Google Scholar). A similar study showed that the double mutant W125F/F163W of XynCDBFV from Neocallimastix patriciarum had a 20% increase in the catalytic activity compared with the WT enzyme (12Cheng Y.S. Chen C.C. Huang J.W. Ko T.P. Huang Z.Y. Guo R.T. Improving the catalytic performance of a GH11 xylanase by rational protein engineering.Appl. Microbiol. Biotechnol. 2015; 99: 9503-9510Crossref PubMed Scopus (34) Google Scholar). Although some impressive results have been achieved, learning how to construct a “small but smart” library to develop better xylanases for XOS production by utilizing the natural diversity of xylanase sequences and structures has remained challenging. XOS with a low degree of polymerization, such as xylobiose (X2) and xylotriose (X3), present a higher prebiotic activity, while xylose (X1) itself is an undesirable component that needs to be removed (4Amorim C. Silverio S.C. Prather K.L.J. Rodrigues L.R. From lignocellulosic residues to market: Production and commercial potential of xylooligosaccharides.Biotechnol. Adv. 2019; 37: 107397Crossref PubMed Scopus (81) Google Scholar). Therefore, xylanases that are able to produce high levels of XOS with a low degree of polymerization, while minimizing the occurrence of xylose, have great application value (13Li Q. Sun B.G. Jia H.Y. Hou J. Yang R. Xiong K. Xu Y.Q. Li X.T. Engineering a xylanase from Streptomyce rochi L10904 by mutation to improve its catalytic characteristics.Int. J. Biol. Macromol. 2017; 101: 366-372Crossref PubMed Scopus (21) Google Scholar). Currently, commercially useful xylanases are mainly obtained from Bacillus species. Thus, enhancing the catalytic efficiency of a xylanase from Bacillus species is undoubtedly of great significance to the biotechnology sector as this will decrease enzyme costs and broaden its scope of application. In a previous study, we cloned and expressed the GH11 xylanase XynLC9 from Bacillus subtilis. The recombinant enzyme exhibited effective alkaline pH tolerance and specific hydrolysis characteristics, which mainly liberated X2 and X3 from corncob-extracted xylan, but only negligible amounts of X1 (14Chang S.Y. Guo Y.L. Wu B. He B.F. Extracellular expression of alkali tolerant xylanase from Bacillus subtilis Lucky9 in E. coli and application for xylooligosaccharides production from agro-industrial waste.Int. J. Biol. Macromol. 2017; 96: 249-256Crossref PubMed Scopus (46) Google Scholar). Accordingly, XynLC9 is an attractive candidate for commercial utilization and thus appropriate as a starting point for further exploration and development. In the present study, we aimed to improve the catalytic performance of XynLC9. Our strategy was guided by a detailed structural comparison between XynLC9 and two well-known GH11 xylanases, xyn2 from Trichoderma reesei and Xyn11A from Thermobifida fusca, both of which had higher catalytic activity than XynLC9. Residues 5-YWQN-8 in XynLC9 were then selected as focal points for protein engineering using saturation and iterative mutagenesis. Experimental work was accompanied by MD simulations to rationalize the observed improvements of the activity among the engineered mutants of XynLC9. The present findings significantly advance the understanding of structure–function relationships in GH11 xylanases, which may thus also be applicable for the rational design of other GH11 xylanases. Protein structure alignments provide an excellent tool to identify amino acid side chains and/or structural regions and patches that play an instrumental role in the function and mechanism of enzymes (11Wu X.Y. Tian Z.N. Jiang X.K. Zhang Q. Wang L.S. Enhancement in catalytic activity of Aspergillus niger XynB by selective site-directed mutagenesis of active site amino acids.Appl. Microbiol. Biotechnol. 2018; 102: 249-260Crossref PubMed Scopus (27) Google Scholar). We first established the model structure of XynLC9 using B. subtilis 168 xylanase BsXynA (1XXN) as the template. As shown in Fig. S1, XynLC9 displayed an overall β-jelly roll structure typical of GH11 xylanases, resembling the shape of a partially closed right hand and comprised two twisted β-sheets (named A and B) and a single α-helix. The β-sheet A was composed of five antiparallel β-strands (A2–A6), and β-sheet B contained eight β-strands (B2-B9). Both β-strands A and B constituted the “fingers”, and a twisted pair of β-strands B together with α-helix formed the “palm.” The large cleft between the “finger” and the “palm” domains was regarded as the active site, with two catalytic glutamate residues (Glu78 and Glu172) residing on B6 and B4. A loop between β-strands B7 and B8 created the “thumb” domain, which was critical for controlling substrate access to the active site, while another loop between the B6 and B9 strands formed a “cord” that connected the “fingers” with the base of the “thumb” (15Silva S.B. Pinheiro M.P. Fuzo C.A. Silva S.R. Ferreira T.L. Lourenzoni M.R. Nonato M.C. Vieira D.S. Ward R.J. The role of local residue environmental changes in thermostable mutants of the GH11 xylanase from Bacillus subtilis.Int. J. Biol. Macromol. 2017; 97: 574-584Crossref PubMed Scopus (9) Google Scholar). The structural model of XynLC9 was used to identify functionally relevant residues and regions by comparing it with the structures of related GH11 xylanases that had superior catalytic activity and whose primary reaction products were X2 and/or X3. Suitable candidates are xyn2 from T. reesei and Xyn11A from T. fusca with specific activity of 1080 U/mg and 960 U/mg (16Ayadi D.Z. Sayari A.H. Hlima H.B. Mabrouk S.B. Mezghani M. Bejar S. Improvement of Trichoderma reesei xylanase Ⅱ thermal stability by serine to threonine surface mutations.Int. J. Biol. Macromol. 2015; 72: 163-170Crossref PubMed Scopus (29) Google Scholar, 17Wang Q. Du W. Weng X.Y. Liu M.Q. Wang J.K. Liu J.X. Recombination of thermo-alkalistable, high xylooligosaccharides producing endo-xylanase from Thermobifida fusca and expression in Pichia pastoris.Appl. Biochem. Biotechnol. 2015; 175: 1318-1329Crossref PubMed Scopus (12) Google Scholar), respectively, which is approximately 1.8- and 1.6-fold higher than that of XynLC9. Furthermore, the main products formed by xyn2 and Xyn11A were X3 and X2, respectively (16Ayadi D.Z. Sayari A.H. Hlima H.B. Mabrouk S.B. Mezghani M. Bejar S. Improvement of Trichoderma reesei xylanase Ⅱ thermal stability by serine to threonine surface mutations.Int. J. Biol. Macromol. 2015; 72: 163-170Crossref PubMed Scopus (29) Google Scholar, 18Linares-Pasten J.A. Aronsson A. Karlsson E.N. Structural considerations on the use of endo-xylanases for the production of prebiotic xylooligosaccharides from biomass.Curr. Protein Pept. Sci. 2018; 19: 48-67PubMed Google Scholar). A multiple sequence alignment illustrated that XynLC9 shared 47% and 62% sequence identity with xyn2 and Xyn11A, respectively (Fig. 1A). Structural superimposition revealed that XynLC9 shared similar structure to xyn2 and Xyn11A with RMSD values of 1.86 Å and 2.09 Å, respectively, demonstrating that structures were better conserved than sequences. Moreover, three xylanase–xylohexaose complexes were aligned to assess the structural conservation of the active site regions. In contrast to xylanases xyn2 and Xyn11A, XynLC9 lacked the N-terminal β-strand B1 region; thus, XynLC9 only possessed five subsites that were categorized as +1, +2, and +3, toward the reducing (aglycone) end, and −1 and −2, toward the nonreducing (glycone) end, where subsite −3 was missing. Moreover, our results indicated that the structures of three enzymes in the active site were more conserved because the superimposition of three specific regions provided RMSD values of 1.34 Å and 0.43 Å, respectively. The significant active site variation between XynLC9 and the other two xylanases was mainly focused on the edge of glycone subsites (Fig. 1B) that harbored four consecutive amino acid residues 5-YWQN-8 in XynLC9 (FYSY in xyn2 and FYSF in Xyn11A). Consequently, we engineered XynLC9 with 5-YWQN-8 residues as the mutation targets. In this study, a semirational approach was used to engineer the entrance region of the active site of XynLC9. One pair of spatially adjacent residues, Tyr5 and Gln7, was mutated by iterative saturation mutagenesis to capture possible synergistic conformational effects, while the other two residues (Trp6 and Asn8) were subjected to single-site saturation mutagenesis (Fig. 2A). Considering both screening effort and library coverage, NDT codon degeneracy was applied for the construction of Tyr5-Gln7 iterative saturation mutagenesis (19Yin X.J. Liu Y.Y. Meng L.J. Zhou H.S. Wu J.P. Yang L.R. Semi-rational hinge engineering: Modulating the conformational transformation of glutamate dehydrogenase for enhanced reductive amination activity towards non-natural substrates.Catal. Sci. Technol. 2020; 10: 3376-3386Crossref Google Scholar). Thus, three focused libraries were constructed: library A (W6X), library B (N8X), and library C (Y5X/Q7X). After the initial screening of the mutant libraries, only the mutant W6F in library exhibited marginal improvement in the specific activity (nearly 10% increase). In library B, the N8Y mutant displayed the highest specific activity (1087 U/mg), which was about 1.8-fold higher than that of WT XynLC9. The most efficient mutant in library C was Y5/Q7H, with about 20% increase in activity when compared with WT XynLC9 (Fig. 2B). Moreover, each of the three libraries contained one positive mutant, indicating that the catalytic activity of XynLC9 was vulnerable to the mutations in the N-terminal region near the active site. Site-directed mutagenesis was performed to probe if these mutations might have synergetic effects. We generated three double mutants (W6F/Q7H, W6F/N8Y, and Q7H/N8Y) and one triple mutant (W6F/Q7H/N8Y) (Fig. S2), followed by the assay of enzyme activity. While each of these mutants was more active than WT XynLC9, the double mutant W6F/Q7H displayed the most significant enhancement of the specific activity (260%, Fig. 2B). Thus, the single mutant N8Y and double mutant W6F/Q7H were selected for subsequent analyses. The enzymatic properties of mutants N8Y and W6F/Q7H were investigated using beechwood xylan as the substrate. As shown in Figure 3A, XynLC9 and its N8Y and W6F/Q7H mutants exhibited maximal xylanase activity at pH 7.0. Also, the three enzymes had similar pH stability, retaining more than 60% of their initial activities when incubated over 12 h at pH values ranging from 6 to 9 (Fig. 3B). Mutants N/8Y and W6F/Q7H had an optimum temperature of 60 °C (Fig. 3C), which was identical to WT XynLC9. However, two mutants showed improved thermostability, with the N8Y and W6F/Q7H mutants retaining 51.3% and 62.2% of the original activity after incubation at 50 °C for 4 h, while XynLC9 only maintained about 35.5% activity (Fig. 3D). Hence, as a result of their increased specific activity and thermostability, the two mutants of XynLC9 are promising candidates for industrial applications. Hydrolysis products of XOS and beechwood xylan by XynLC9 and two variants were analyzed using the TLC method. As shown in Figure 4, none of the enzymes were active toward X2 and X3 but showed a similar product range when X4 and X5 were used as substrates. When using X4 as a substrate, XynLC9 and its mutants, N8Y and W6F/Q7H, displayed similar distribution with X3 as the major products. During the hydrolysis of X5, the primary products were X2 and X3, while smaller quantities of X4 and trace amounts of undigested X5 were also present in the reaction mixture. The same analysis was also performed using beechwood xylan as a substrate. Again, for each of the three enzymes, X2 and X3 were the primary products formed (Fig. 4), while the amounts of X4 and X5 in the mixture were minimal. The apparent kinetic parameters of XynLC9 and its mutants were determined by using beechwood xylan as the substrate (Table 1). The apparent Km values for N8Y and W6F/Q7H were higher than that for XynLC9, indicating the reduced substrate affinity (20Wang X.Y. Huang H.Q. Xie X.M. Ma R. Bai Y.G. Zheng F. You S. Zhang B.Y. Xie H.F. Luo H.Y. Improvement of the catalytic performance of a hyperthermostable GH10 xylanase from Talaromyces leycettanus JCM12802.Bioresour. Technol. 2016; 222: 277-284Crossref PubMed Scopus (27) Google Scholar). However, the turnover rate (kcat) of two mutants was significantly higher than that of XynLC9, indicating that the mutants performed better than the WT to convert most of the bound substrate into product (21Yang J.K. Ma T.F. Shang-guan F. Han Z.G. Improving the catalytic activity of thermostable xylanase from Thermotoga maritima via mutagenesis of non-catalytic residues at glycone subsites.Enzyme Microb. Technol. 2020; 139: 109579Crossref PubMed Scopus (10) Google Scholar). Owing to the considerable improvement in kcat values, N8Y and W6F/Q7H displayed about 22% and 47% higher catalytic efficiency (kcat/Km), respectively, than the WT, which is consistent with the enzyme activity results described above.Table 1Kinetic values of XynLC9 and its mutants toward beechwood xylanEnzymeKm (mg/ml)kcat (/s)kcat/Km (ml/mg/s)XynLC97.2 ± 0.655.9 ± 4.87.8 ± 0.7N8Y11.2 ± 0.9105.7 ± 11.29.5 ± 0.8W6F/Q7H11.6 ± 0.9133.1 ± 11.711.4 ± 0.9The error bar represents the SDs from three replicates. Open table in a new tab The error bar represents the SDs from three replicates. Structural comparisons and MD simulations were performed for WT XynLC9 and the N8Y and W6F/Q7H mutants to explore the possible molecular basis that has led to the improved catalytic efficiency of the mutants. CD spectroscopy demonstrated that the mutations in positions 6 to 8 had no measurable effect on the secondary structural elements of XynLC9 (Fig. S3). Next, MD simulations were performed over a 50-nm time scale for WT XynLC9 and two mutants. The RMSD of the α-carbon atoms of the three enzymes in the presence of the substrate reached equilibrium after 40 ns, and thus, only the MD trajectories of the last 10 ns were selected for further analysis (Fig. 5A). The RMSD values were slightly higher for two mutants than for WT XynLC9, suggesting that the structural ensemble generated by MD simulations differed more from the initial structure in the case of mutants N8Y and W6F/Q7H than WT XynLC9 (22Vieira D.S. Ward R.J. Conformation analysis of a surface loop that controls active site access in the GH11 xylanase A from Bacillus subtilis.J. Mol. Model. 2012; 18: 1473-1479Crossref PubMed Scopus (13) Google Scholar). Root mean square fluctuation (RMSF) reflects the flexibility of each residue during the simulations. The analysis of RMSF values per residue did not show any significant changes at positions 5 to 8 between XynLC9 and two mutants (Fig. 5B). However, a slight increase in RMSF values was observed for the N8Y mutant over positions 10 to 12, indicating that the neighboring residues were more flexible with Tyr at position 8 than Asn at the same position (15Silva S.B. Pinheiro M.P. Fuzo C.A. Silva S.R. Ferreira T.L. Lourenzoni M.R. Nonato M.C. Vieira D.S. Ward R.J. The role of local residue environmental changes in thermostable mutants of the GH11 xylanase from Bacillus subtilis.Int. J. Biol. Macromol. 2017; 97: 574-584Crossref PubMed Scopus (9) Google Scholar). Nevertheless, this situation was reversed for the double mutant W6F/Q7H, with RMSF values smaller than that of XynLC9 in the region of residues 10 to 15. These results indicated that the mutations of N8Y and W6F/Q7H altered the local backbone structure flexibility of the enzyme, thus affecting the interactions between the enzyme and substrate. To further probe the structural changes caused by mutations at positions 6 to 8, a superimposition of the structures of WT XynLC9 and the two mutants was performed using the average structures extracted from the final 10 ns of the equilibrated state of the MD simulations as references for the final functional forms of the proteins. In native XynLC9, residues 6 to 8 were located on the active site cleft and in β-strand B2 of the N-terminal region. The Asn to Tyr substitution at residue 8 involved a neutral charge amino acid exchange to a bulky aromatic amino acid, which facilitated the formation of a new hydrogen bond between Tyr8 and Val16. In addition, there was a hydrophobic stacking interaction between this tyrosine and Trp6 (Fig. 6). On the other hand, Trp6 of WT XynLC9 interacted with Asn8, Ala18, and Asn20 (Fig. 6). Replacement of Trp6 by phenylalanine with a relatively smaller side group eliminated these interactions (Fig. 6). Gln7 was oriented toward the active site cleft near the catalytic site. The major hydrogen bond interactions in β-strand B2 of His7 in the double mutant W6F/Q7H and the equivalent residue (Gln7) in WT XynLC9 were conserved. These included two intramolecular hydrogen bonds between the main-chain carbonyl oxygen of position 7 and the main-chain amide nitrogen of Gly39 and between the side chain of the residue (His or Gln) and the side chain –OH group of Tyr5 (Fig. 6). We also analyzed the interactions between residues 6 to 8 of XynLC9 or its mutants and the substrate xylohexaose using the DS 3.5 software. The XynLC9–xylohexaose complex structure revealed that Trp6 and Asn8 might not interact directly with xylohexaose because they were distal to the substrate, while Gln7 provided two hydrogen bonds with O3 at the −2 subsite (Fig. 7). The introduction of an aromatic side chain in the N8Y mutant did not lead to the formation of CH-π stacking interactions with the substrate, but this mutation altered the conformation of Gln7 significantly, leading to the removal of one of the two hydrogen bonds between this glutamine and the −2 xylose moiety of the substrate (Fig. 7). For the W6F/Q7H double mutant, His7 only provided one hydrogen bond to the O2 of xylose residue in the −2 subsite (Fig. 7). These results indicated that mutations caused impaired binding ability to the ligand when compared with WT XynLC9. Using the entire simulation trajectories, we calculated the surface area/volume of the active site cavity of WT XynLC9, N8Y, and W6F/Q7H to be 607 Å2/879 Å3, 705 Å2/1004 Å3, and 730 Å2/1032 Å3, respectively (Fig. S4 and Table 2). The introduction of the mutations led to both a greater surface area and volume, with the double mutant having the most significant effect. The larger volume and area of the active site might be conducive to the substrate entrance and product dissociation, thereby improving the catalytic activity of two mutants.Table 2Geometrical and topological properties of the active site of XynLC9 and its mutantsEnzymeVolume (Å3)Area (Å2)Minimal distance (Trp9–Pro116) (Å)XynLC9607 ± 15879 ± 4310.3 ± 0.5N8Y705 ± 181004 ± 4111.9 ± 0.8W6F/Q7H730 ± 121032 ± 3712.1 ± 0.5The error bar represents the SDs from three replicates. Open table in a new tab The error bar represents the SDs from three replicates. To assess the potential of the engineered xylanase for XOS production, the double mutant W6F/Q7H with highest catalytic activity was selected to hydrolyze corncob-extracted xylan. As shown in Figure 8, XOS production increased over time irrespective of whether corncob xylan was incubated with WT XynLC9 or mutant W6F/Q7H. However, at every time point, W6F/Q7H exhibited performance superior to that of WT XynLC9. After 14 h of incubation with the double mutant, the concentration of XOS reached 10.6 mg/ml, which was about 1.6-fold higher yield than that of WT XynLC9. The results above indicated that the substitutions at positions 6 and 7 with Phe and His promoted the production capacity of XOS from corncob, which was consistent with the previously determined catalytic efficiency. Enhanced catalytic activity is one of the most desirable properties related to the xylanase engineering perspective (11Wu X.Y. Tian Z.N. Jiang X.K. Zhang Q. Wang L.S. Enhancement in catalytic activity of Aspergillus niger XynB by selective site-directed mutagenesis of active site amino acids.Appl. Microbiol. Biotechnol. 2018; 102: 249-260Crossref PubMed Scopus (27) Google Scholar, 12Cheng Y.S. Chen C.C. Huang J.W. Ko T.P. Huang Z.Y. Guo R.T. Improving the catalytic performance of a GH11 xylanase by rational protein engineering.Appl. Microbiol. Biotechnol. 2015; 99: 9503-9510Crossref PubMed Scopus (34) Google Scholar). Identifying suitable sites for site-directed or site-saturation mutagenesis to improve the catalytic performance of xylanases are challenging (11Wu X.Y. Tian Z.N. Jiang X.K. Zhang Q. Wang L.S. Enhancement in catalytic activity of Aspergillus niger XynB by selective site-directed mutagenesis of activ" @default.
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• W3204643380 title "Sequence- and structure-guided improvement of the catalytic performance of a GH11 family xylanase from Bacillus subtilis" @default.
• W3204643380 cites W1031578623 @default.
• W3204643380 cites W1993625852 @default.
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