Improvement of Alkaliphily of<i>Bacillus</i>Alkaline Xylanase by Introducing Amino Acid Substitutions Both on Catalytic Cleft and Protein Surface

Umemoto, Hirohito; Ihsanawati,; Inami, Mayuko; Yatsunami, Rie; Fukui, Toshiaki; Kumasaka, Takashi; Tanaka, Nobuo

doi:10.1271/bbb.80869

Cited by 38 publications

(25 citation statements)

References 10 publications

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“…5, wild type ∆XBD was most active at 55 C. ∆XBDK51R exhibited almost the same temperature profile and temperature optimum as wild type ∆XBD. On the other hand, the temperature optima of ∆XBDR5 and ∆XBDK51R R5 were both 60 C. 10) Although these mutants showed lower specific activity than wild type ∆XBD at 37 60 C, they showed apparently higher activity at 65 C. These results indicated that the introduction of excess Arg residues on the protein surface increased the thermostability of ∆XBD.…”

Section: Structural Comparison Of Catds From Gh Family 11 Xylanasesmentioning

confidence: 93%

“…3(B)). 10) These results suggested that the introduction of five Arg residues on the Ser Thr surface could also improve the alkaliphily of ∆XBD. It is noteworthy that the optimum pH of ∆XBD has been successfully shifted from 8.5 to 9.5 by combining the two methodologies presented in this study.…”

Section: Structural Comparison Of Catds From Gh Family 11 Xylanasesmentioning

confidence: 96%

“…3(B)). 10) On the other hand, ∆XBDK52R showed lower activity at alkaline pHs compared to wild type ∆XBD, and had a pH optimum of 8.0. These results indicated that reinforcement of the characteristic salt bridge formed by Asp14 and Lys51 caused an alkaliphilic shift in optimum pH.…”

Section: Structural Comparison Of Catds From Gh Family 11 Xylanasesmentioning

confidence: 98%

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Analysis of Functional Domains and Improvement of Alkaliphily of an Alkaline Xylanase on the Basis of Its Three-dimensional Structure

Umemoto¹,

Yazawa²,

Takakura³

et al. 2010

J. Appl. Glycosci.

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Xylanase J (XynJ) from alkaliphilic Bacillus sp. strain 41M-1 is a multi-domain enzyme consisting of a glycoside hydrolase (GH) family 11 catalytic domain and a carbohydrate-binding module family 36 xylan-binding domain (XBD). Structural comparison of the GH family 11 catalytic domains indicated that there were several specific salt bridges in the catalytic cleft of XynJ. Mutant enzymes were prepared by substituting several amino acid residues responsible for the characteristic salt bridges. Elimination of the salt bridges caused an acidophilic shift in optimum pH, suggesting that the characteristic salt bridges contributed to the alkaliphily of XynJ. On the other hand, reinforcing one of the characteristic salt bridges in the catalytic domain shifted the optimum pH of XynJ from 8.5 to 9.0. Furthermore, introducing excess Arg residues on the protein surface was also effective to improve the alkaliphily of XynJ. Xylan-binding properties of various XBD mutants were investigated as fusion proteins with glutathione-S-transferase. Furthermore, the same substitutions were introduced into the XBD region of XynJ and insoluble xylan-hydrolyzing activity was measured. The results indicated that some Asp, Trp and Tyr residues were important for xylan-binding activity of the XBD, and that xylan-hydrolyzing activity of XynJ was closely correlated to xylan-binding activity of the XBD region.

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Section: Structural Comparison Of Catds From Gh Family 11 Xylanasesmentioning

confidence: 93%

Section: Structural Comparison Of Catds From Gh Family 11 Xylanasesmentioning

confidence: 96%

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Analysis of Functional Domains and Improvement of Alkaliphily of an Alkaline Xylanase on the Basis of Its Three-dimensional Structure

Umemoto¹,

Yazawa²,

Takakura³

et al. 2010

J. Appl. Glycosci.

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“…Various microorganisms secrete xylanases into culture media and glycohydrolase family 10 or 11 xylanases are the most common [3]. Family 5,7,8,or 43 xylanases are also known but not much informations are available [1]. High crude oil prices drive development of alternative energy sources including bioenergy [4].…”

Section: Introductionmentioning

confidence: 99%

“…Isolation of microorganisms utilizing xylan efficiently is important since xylan occupies a significant portion of utilizable polymers in plants [4]. Isolation of novel xylanases [5] and improvement of their properties [6][7][8] are actively being studied. Here we report cloning of a family 11 xylanase gene from Bacillus amyloliquefaciens CH51.…”

Section: Introductionmentioning

confidence: 99%

Cloning of a Family 11 Xylanase Gene from Bacillus amyloliquefaciens CH51 Isolated from Cheonggukjang

et al. 2012

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Cloning, expression, and characterization of a novel alkali‐tolerant xylanase from alkaliphilic Bacillus sp. SN5

Bai

Xue

Zhou

et al. 2014

Biotech and App Biochem

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A xylanase gene (xyn11A) was cloned from the genomic library of alkalophilic Bacillus sp. SN5. It encoded a polypeptide of 366 amino acids, consisting of a family 11 glycoside hydrolase, a short linker region, and a family 36 carbohydrate-binding module (CBM). The intact xylanase Xyn11A and the CBM-linker-truncated Xyn11A-LC were expressed in Escherichia coli BL21 (DE3). Both purified recombinant proteins exhibited the highest activity at 55 °C. The optimal pH for Xyn11A activity was 7.5, whereas Xyn11A-LC showed a broad pH profile (>80% activity at pH 5.5-8.5) with optimal activity at pH 5.5 and 7.5-8.0. They had high alkali tolerance, retaining over 80% residual activity after preincubation at pH 8.5-11.0 at 37 °C for 1 H. Xyn11A-LC showed better thermal stability, lower affinity, and lower catalytic activity to insoluble xylan than Xyn11A, whereas its specific activity for soluble beechwood xylan (4,511.9 U/mg) was greater than that of Xyn11A (3,136.4 U/mg). These results implied that the CBM of Xyn11A could change the enzymatic properties and play a role in degrading insoluble xylan. Xyn11A-LC is a family 11 alkali-tolerant cellulase-free xylanase with high specific activity, which qualifies it as a potential candidate for industrial applications, especially in the paper industry.

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Improvement of Alkaliphily ofBacillusAlkaline Xylanase by Introducing Amino Acid Substitutions Both on Catalytic Cleft and Protein Surface

Cited by 38 publications

References 10 publications

Analysis of Functional Domains and Improvement of Alkaliphily of an Alkaline Xylanase on the Basis of Its Three-dimensional Structure

Analysis of Functional Domains and Improvement of Alkaliphily of an Alkaline Xylanase on the Basis of Its Three-dimensional Structure

Cloning of a Family 11 Xylanase Gene from Bacillus amyloliquefaciens CH51 Isolated from Cheonggukjang

Cloning, expression, and characterization of a novel alkali‐tolerant xylanase from alkaliphilic Bacillus sp. SN5

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