Sequence- and structure-guided improvement of the catalytic performance of a GH11 family xylanase from Bacillus subtilis

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 pH and (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, Structural of a glycoside hydrolase xylanase from Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). GH11 xylanases have been and characterized with a to various industrial most of GH11 xylanases are for industrial applications because of their and a mutant of Bacillus xylanase with catalytic activity by PubMed Scopus Google Scholar, C. Xu Y.Q. Li Q. Li X.T. Xiong K. Li Improving the and catalytic efficiency of GH11 xylanase by J. Biol. Macromol. 2019; PubMed Scopus Google Scholar). the engineering of GH11 xylanases by evolution and design is increasing design via site-directed mutagenesis been successfully used to the catalytic activity of GH11 a study mutations and molecular dynamics simulations of the GH11 xylanase from that the residues the a crucial in substrate and mutations in this site the increased the catalytic activity by Q. in catalytic activity of by site-directed mutagenesis of active site Microbiol. Biotechnol. 2018; 102: PubMed Scopus Google Scholar). study that the mutant of from had a increase in the catalytic activity with the WT enzyme Improving the catalytic performance of a GH11 xylanase by protein Microbiol. Biotechnol. PubMed Scopus Google Scholar). have been to a but library to develop better xylanases for XOS production by the diversity of xylanase sequences and XOS with a low of as xylobiose and xylotriose a higher while xylose is an that to (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). xylanases that are to produce high of XOS with a low of while the of have application Q. Sun B.G. J. Xiong K. Xu Y.Q. Li X.T. a xylanase from by to improve catalytic J. Biol. Macromol. 2017; PubMed Scopus Google Scholar). xylanases are from Bacillus the catalytic efficiency of a xylanase from Bacillus is of to the as this enzyme costs and of In a we and the GH11 xylanase XynLC9 from Bacillus The enzyme pH and hydrolysis characteristics, which and from corncob-extracted but negligible of Wu B. of xylanase from Bacillus subtilis in E. and application for xylooligosaccharides production from agro-industrial J. Biol. Macromol. 2017; 96: PubMed Scopus Google Scholar). Accordingly, XynLC9 is an attractive for commercial and thus as a for and In the we aimed to improve the catalytic performance of XynLC9. by a structural between XynLC9 and two GH11 xylanases, from Trichoderma reesei and Xyn11A from Thermobifida fusca, both of which had higher catalytic activity than XynLC9. 5-YWQN-8 in XynLC9 were as for protein engineering saturation and iterative mutagenesis. by simulations to the of the activity the mutants of XynLC9. The the of relationships in GH11 xylanases, which thus also for the design of GH11 an to structural and that an in the and of Q. in catalytic activity of by site-directed mutagenesis of active site Microbiol. Biotechnol. 2018; 102: PubMed Scopus Google Scholar). the of XynLC9 B. subtilis xylanase as the shown in XynLC9 displayed an β-jelly roll of GH11 xylanases, the of a and two and and a The of and B and B the and a of B with formed the The between the and the regarded as the active with two catalytic residues and on and between and the which for substrate to the active while between the and formed a that the with the of the The of in mutants of the GH11 xylanase from Bacillus J. Biol. Macromol. 2017; PubMed Scopus Google Scholar). The structural of XynLC9 used to residues and by with the of GH11 xylanases that had catalytic activity and products were are from T. reesei and Xyn11A from T. with activity of and M. S. of Trichoderma reesei xylanase by to surface J. Biol. Macromol. PubMed Scopus Google Scholar, Q. of high xylooligosaccharides from Thermobifida and in Biotechnol. PubMed Scopus Google respectively, which is and higher than that of XynLC9. Furthermore, the products formed by and Xyn11A were and M. S. of Trichoderma reesei xylanase by to surface J. Biol. Macromol. PubMed Scopus Google Scholar, J.A. A. E.N. Structural on the of for the production of xylooligosaccharides from 2018; Google Scholar). sequence that XynLC9 and sequence with and Structural revealed that XynLC9 to and Xyn11A with of and respectively, that were better conserved than were to the structural of the active site In contrast to xylanases and XynLC9 the N-terminal XynLC9 possessed that were as and the and and the that the of in the active site were more conserved because the of provided of and The active site between XynLC9 and the two xylanases on the of that residues 5-YWQN-8 in XynLC9 in and in we XynLC9 with 5-YWQN-8 residues as the In this a approach used to the of the active site of XynLC9. of and by iterative saturation mutagenesis to while the two residues and were subjected to saturation mutagenesis both and library for the of iterative saturation mutagenesis L.J. Wu L.R. the of for activity Google Scholar). were library library B and library the of the mutant the mutant in library in the activity In library the N8Y mutant displayed the activity which 1.8-fold higher than that of WT XynLC9. The most mutant in library with increase in activity when with WT XynLC9 of the that the catalytic activity of XynLC9 to the mutations in the N-terminal the active mutagenesis to mutations have mutants and and mutant by the of enzyme of mutants more active than WT XynLC9, the mutant W6F/Q7H displayed the most of the activity the mutant N8Y and mutant W6F/Q7H were for The of mutants N8Y and W6F/Q7H were xylan as the shown in XynLC9 and N8Y and W6F/Q7H mutants xylanase activity pH the had pH more than of their when pH from to and W6F/Q7H had an of which to WT XynLC9. However, two mutants with the N8Y and W6F/Q7H mutants and of the activity for while XynLC9 activity as a of their increased activity and the two mutants of XynLC9 are for industrial applications. products of XOS and xylan by XynLC9 and two were the shown in of the were active and but a product range when and were used as as a XynLC9 and N8Y and displayed with as the major the hydrolysis of the products were and while smaller of and of were also in the The also xylan as a for of the and were the products formed while the of and in the were The of XynLC9 and mutants were by xylan as the substrate The for N8Y and W6F/Q7H were higher than that for XynLC9, the substrate S. of the catalytic performance of a GH10 xylanase from Talaromyces PubMed Scopus Google Scholar). However, the turnover rate of two mutants higher than that of XynLC9, that the mutants better than the WT to most of the substrate into product Improving the catalytic activity of xylanase from via mutagenesis of residues PubMed Scopus Google Scholar). to the in N8Y and W6F/Q7H displayed and higher catalytic efficiency respectively, than the which is with the enzyme activity of XynLC9 and mutants the from in a The the from Structural comparisons and simulations were for WT XynLC9 and the N8Y and W6F/Q7H mutants to the molecular that led to the catalytic efficiency of the demonstrated that the mutations in to had on the structural of XynLC9 simulations were a for WT XynLC9 and two The of the of the in the of the substrate and the of the 10 were for The were higher for two mutants than for WT XynLC9, that the structural by simulations more from the in the of mutants N8Y and W6F/Q7H than WT XynLC9 of a surface that active site in the GH11 xylanase from Bacillus 2012; PubMed Scopus Google Scholar). the of the The of to between XynLC9 and two mutants However, a increase in for the N8Y mutant 10 to that the residues were more with than the The of in mutants of the GH11 xylanase from Bacillus J. Biol. Macromol. 2017; PubMed Scopus Google Scholar). this for the mutant with smaller than that of XynLC9 in the of residues 10 to that the mutations of N8Y and W6F/Q7H the of the thus the between the enzyme and To the structural by mutations to 8, a of the of WT XynLC9 and the two mutants the from the 10 of the of the simulations as for the functional of the In XynLC9, residues to were on the active site and in of the N-terminal The to a to a which the of a bond between and In addition, a hydrophobic between this and the of WT XynLC9 with and of by with a smaller the active site the catalytic The major bond in of in the mutant W6F/Q7H and the in WT XynLC9 were two between the of and the of and between the of the and the of also the between residues to of XynLC9 mutants and the substrate the The revealed that and with because were to the while provided two with the The of an in the N8Y mutant to the of with the but this the of to the of of the two between this and the xylose of the substrate the W6F/Q7H provided bond to the of xylose in the that mutations ability to the when with WT XynLC9. the we the surface of the active site of WT XynLC9, and W6F/Q7H to and and The of the mutations led to both a surface area and with the mutant the most The volume and area of the active site to the substrate and product the catalytic activity of two and of the active site of XynLC9 and the from in a The the from To the potential of the xylanase for XOS the mutant W6F/Q7H with catalytic activity to corncob-extracted xylan. shown in 8, XOS production increased of xylan with WT XynLC9 mutant However, W6F/Q7H performance to that of WT XynLC9. of with the the of XOS which higher yield than that of WT XynLC9. The that the and with and the production of XOS from which with the catalytic catalytic activity is of the most to the xylanase engineering Q. in catalytic activity of by site-directed mutagenesis of active site Microbiol. Biotechnol. 2018; 102: PubMed Scopus Google Scholar, Improving the catalytic performance of a GH11 xylanase by protein Microbiol. Biotechnol. PubMed Scopus Google Scholar). for site-directed mutagenesis to improve the catalytic performance of xylanases are Q. in catalytic activity of by site-directed mutagenesis of active site Microbiol. Biotechnol. 2018; 102: PubMed Scopus Google Scholar, S. Q. Y. The of to catalytic in active site of a GH11 Microbiol. Biotechnol. PubMed Scopus Google Scholar). structural is in relationships and also into protein Previously, a structural with the xylanase from a mutant of the xylanase from B. subtilis which had both and increased catalytic activity of Bacillus subtilis GH11 xylanase by surface J. Biol. Macromol. PubMed Scopus Google Scholar). by the we the in the of the XynLC9 from B. subtilis with the xylanase from T. reesei and the xylanase Xyn11A from T. to the structural in the xylanase XynLC9 that to improve the catalytic activity of this enzyme. sequence that the residues the active site were conserved xylanases in with the functional of the active site However, the residues were to 5-YWQN-8 for XynLC9 with for and for we the of residues by GH11 The that the in of XynLC9 in GH11 xylanases, but the of a and in to and in XynLC9, respectively, is is that mutagenesis conserved in the active site to the residues in the active site are to the functional diversity of the Q. in catalytic activity of by site-directed mutagenesis of active site Microbiol. Biotechnol. 2018; 102: PubMed Scopus Google Scholar, S. S. specificity and of the enzyme families as revealed by PubMed Scopus Google Scholar). we on the of in library and xylanase activity for mutant which with the most in this However, we two N8Y and which 1.8-fold and higher catalytic activity than WT XynLC9, the in the catalytic activity of XynLC9 is than with for engineering of GH11 xylanases with site-directed mutagenesis functional the in xylanase led to increase in activity engineering of GH11 xylanase from for catalytic efficiency on 2019; PubMed Scopus Google Scholar). The of and with and the catalytic activity of A. xylanase by Q. in catalytic activity of by site-directed mutagenesis of active site Microbiol. Biotechnol. 2018; 102: PubMed Scopus Google Scholar). were also for the mutant of from which displayed increase in catalytic activity when with the WT Improving the catalytic performance of a GH11 xylanase by protein Microbiol. Biotechnol. PubMed Scopus Google Scholar). The catalytic activity of B. subtilis xylanase by two residues and a on the A. S. E. A. J.A. and the for substrate specificity of the of glycoside hydrolase PubMed Scopus Google Scholar). Li Q. Sun B.G. J. Xiong K. Xu Y.Q. Li X.T. a xylanase from by to improve catalytic J. Biol. Macromol. 2017; PubMed Scopus Google reported that the of N-terminal and site-directed mutagenesis in the of xylanase from led to increase in In addition, the residues in study were in Li Wu Q. Y. Wu Li Improving the and catalytic efficiency of a xylanase from by iterative mutagenesis on in 2017; Scopus Google also the catalytic efficiency of xylanase by 1.8-fold in with However, the the of GH11 xylanases and of the active site Q. in catalytic activity of by site-directed mutagenesis of active site Microbiol. Biotechnol. 2018; 102: PubMed Scopus Google the engineering GH11 xylanases in the to improve the catalytic activity are the N8Y and W6F/Q7H mutants of XynLC9 were for characterization because of their in both mutants also displayed is a between enzyme activity and the of mutations as enzyme is for while structural high catalytic mutants with increased catalytic activity have lower Wu J. B. The of two of the xylanase in an increase in but a in PubMed Scopus Google Scholar, Y. Li of a GH11 xylanase by and sequence 2019; PubMed Scopus Google Scholar). However, of increased catalytic activity and for the two XynLC9 mutants are for the mutant of B. subtilis xylanase also demonstrated a increase in activity and a in of Bacillus subtilis GH11 xylanase by surface J. Biol. Macromol. PubMed Scopus Google Scholar). The in with provided into the catalytic performance of the mutants in this In with WT XynLC9, both mutants had for In with molecular surface area that the for the of the mutants higher than that of XynLC9 and for N8Y and for XynLC9, However, both mutants had higher that mutations product release from the active the of increase for higher than that for in catalytic efficiency for two which in with the catalytic activity of two were to the of Li and 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, engineering of GH11 xylanase from for catalytic efficiency on 2019; PubMed Scopus Google Scholar). The simulations were with the to the of XynLC9 possessed from to the xylose had with XynLC9. In the and had with the while formed two with the xylose the In W6F/Q7H the the hydrophobic between and residues and in a of the active site the bond this between the enzyme and The of the surface area and volume of the active site also that the W6F/Q7H provided a space for substrate entry and product the substrate as shown by the higher The N8Y led to the of a bond between and as as a between and which the of the of the active thus increasing the volume of the active site feature by the surface area and volume of the active site of mutant In addition, for GH11 xylanases, the of the active site is between the conserved of the and the conserved of Q. in catalytic activity of by site-directed mutagenesis of active site Microbiol. Biotechnol. 2018; 102: PubMed Scopus Google Scholar). To the of the active site in XynLC9 and two the between and on The that the active site in two mutants more than in the WT enzyme 10 for the The of the active site in two mutants also to to their higher catalytic the mutations of W6F/Q7H and N8Y provided a space and for the active as as the between the substrate and which the higher rate and catalytic activity of two also reported for the engineering of a GH11 xylanase from the of residues by increased the of the active site to substrate to the catalytic with a of the catalytic efficiency Li Improving the of a GH11 xylanase via site-directed mutagenesis by sequence and structural 2017; PubMed Scopus Google Scholar). by the structural and of two GH11 xylanases, J. G. C. T. study to the structural and dynamics the activity and of GH11 J. PubMed Scopus Google also reported that a and more catalytic a major in the activity of GH11 xylanases by substrate and product In addition, substrate the of a xylanase for a which an in biotechnological as S. A. J.A. of the site of xylanases the impact of substrate on Chem. PubMed Scopus Google Scholar). To the potential applications of N8Y and the of mutations on substrate in In enzyme to a but library a In this on sequence and N-terminal residues 5-YWQN-8 in the GH11 xylanase XynLC9 from B. subtilis were identified as the hotspots for activity engineering of this enzyme. W6F/Q7H and with catalytic activity and better were by mutagenesis and iterative as for of and production of functional Structural and simulations provided an into the that to the of the catalytic performance of two that the of the diversity of protein sequences and is an approach to design The sequences of and Xyn11A were from the protein The of and Xyn11A were from the A. J. Structural of two major from Trichoderma PubMed Scopus Google Scholar, A. S. of a GH11 xylanase from Thermobifida Biol. 2012; PubMed Scopus Google Scholar). sequence were The of XynLC9 with the the of the xylanase from B. subtilis as the of a xylanase from Bacillus subtilis PubMed Scopus Google Scholar). The of and The of XynLC9 to the of and Xyn11A and the of were the the by from into the active site of the xylanase Q. Q. S. K. M. P. A. of xylanase and product for the catalytic Biol. PubMed Scopus Google Scholar). residues in the active site of xylanases were identified mutagenesis were and B by extension the as the are in saturation mutagenesis library site with the and The sequences of are also in XynLC9 mutant to a the to via The mutations were by and the into for the were in the pH and by The for enzyme with higher enzyme activity than WT XynLC9 were mutants and XynLC9 were in a a to the The molecular and of the were on a The of the the and for the of of protein the of PubMed Scopus Google and the activity of the The activity of the mutants and with that of WT XynLC9. activity of the XynLC9 and mutants the as Wu B. of xylanase from Bacillus subtilis in E. and application for xylooligosaccharides production from agro-industrial J. Biol. Macromol. 2017; 96: PubMed Scopus Google Scholar). The of of xylan and of a enzyme pH and for 10 of xylanase activity as the of enzyme of from xylose The pH for WT XynLC9 and mutants with a pH range from to 10 and The of on xylanase activity in from to To the pH of the WT XynLC9 and mutants were in the various pH for The of XynLC9 mutants by of the enzyme pH for The that turnover rate (kcat), and catalytic efficiency for WT XynLC9 and were and pH to of xylan as the The were by the to the To the hydrolysis of XynLC9 and XOS and xylan were used as the the hydrolysis of of the substrate were with of the enzyme for the used for the activity the hydrolysis of the of substrate and enzyme 10 and 10 the products were by with a of as the The on the were with by for 10 The of WT XynLC9 and mutants in 10 pH were on a with the of to a and a of The and were and The structural of the protein were the WT as the the of the mutant and by simulations were with and the E. P. the of protein the of a GH11 Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. S. J.C. B. E. performance molecular simulations from to Full Text Full Text PDF Scopus Google Scholar). The in a of and the of the 10 and were for of The used to the The of in mutants of the GH11 xylanase from Bacillus J. Biol. Macromol. 2017; PubMed Scopus Google Scholar). The were with the with and the of the with and simulations were in The to with a of with a of the for analyses, a of and to increase the of the The and were from the were and by The of the of XynLC9 mutants and the by The surface area and volume of the active site in WT XynLC9 and mutants were from with for to the B. Q. G. of from Bacillus subtilis and production of Chem. 2019; PubMed Scopus Google Scholar). production of XOS by of XynLC9 mutant in in a volume of xylan. The and for were and used to by the are the The of in mutants of the GH11 xylanase from Bacillus J. Biol. Macromol. 2017; PubMed Scopus Google Scholar). 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