Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase

Kim, Seok Hwan; Pokhrel, Subarna; Yoo, Young Je

doi:10.1016/j.molcatb.2008.02.006

Cited by 25 publications

(13 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It was reported that the residues in the eight-residue loop were important in determining the pH optima of family 11 xylanase [7, 27]. The residue Glu 135 was located at the edge of the eight-residue loop on the protein surface (Fig.…”

Section: Discussionmentioning

confidence: 99%

Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis

et al. 2016

View full text Add to dashboard Cite

BackgroundFamily 11 alkaline xylanases have great potential economic applications in the pulp and paper industry. In this study, we would improve the alkalophilicity of family 11 alkaline xylanase Xyn11A-LC from Bacillus sp. SN5, for the better application in this field.ResultsA random mutation library of Xyn11A-LC with about 10,000 clones was constructed by error-prone PCR. One mutant, M52-C10 (V116A and E135V), with improved alkalophilicity was obtained from the library. Site-directed mutation showed that the mutation E135V was responsible for the alkalophilicity of the mutant. The variant E135V shifted the optimum pH of the wild-type enzyme from 7.5 to 8.0. Compared to the relative activities of the wild type enzyme, those of the mutant E135V increased by 17.5, 18.9, 14.3 and 9.5 % at pH 8.5, 9.0, 9.5 and 10.0, respectively. Furthermore, Glu135 saturation mutagenesis showed that the only mutant to have better alkalophilicity than E135V was E135R. The optimal pH of the mutant E135R was 8.5, 1.0 pH units higher than that of the wild-type. In addition, compared to the wild-type enzyme, the mutations E135V and E135R increased the catalytic efficiency (k cat/K m) by 57 and 37 %, respectively. Structural analysis showed that the residue at position 135, located in the eight-residue loop on the protein surface, might improve the alkalophilicity and catalytic activity by the elimination of the negative charge and the formation of salt-bridge.ConclusionsMutants E135V and E135R with improved alkalophilicity were obtained by directed evolution and site saturation mutagenesis. The residue at position 135 in the eight-residue loop on the protein surface was found to play an important role in the pH activity profile of family 11 xylanases. This study provided alkalophilic mutants for application in bleaching process, and it was also helpful to understand the alkaline adaptation mechanism of family 11 xylanases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0310-9) contains supplementary material, which is available to authorized users.

show abstract

Section: Discussionmentioning

confidence: 99%

Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis

et al. 2016

View full text Add to dashboard Cite

show abstract

“…The plasmids were chemically transformed into competent E. coli BL21 (DE3), and the cells were induced by adding 1 mM IPTG (isopropyl ␤-d-1-thiogalactopyranoside) and were cultured at 20 • C for 24 h to express AKR7A5. The wild-type and mutant enzymes were purified as previously described (Kim et al, 2008). The enzyme purity was estimated using 12 % SDS-PAGE, and the enzyme concentration was determined using the Bradford assay (Bradford, 1976).…”

Section: Enzyme Mutation Expression and Purificationmentioning

confidence: 99%

Engineering substrate specificity of succinic semialdehyde reductase (AKR7A5) for efficient conversion of levulinic acid to 4-hydroxyvaleric acid

Yeon

Park

Yoo

2015

Journal of Biotechnology

Self Cite

View full text Add to dashboard Cite

“…Taken together, the substitution L49P could possibly generate weaker hydrogen bond, hydrophobic interactions and van der Waals force with residues nearby (especially for V174), contributing to structure flexibility of the acid catalyst (E177). Consequently, the catalytic residues involved in substrate binding could easily approach to the substrate, which was beneficial for the acid catalyst to protonate the substrate (Kim et al 2008). The k cat /K m of FSI-A124 was 51.7% higher than that of its parent, suggesting the catalytic efficiency or the affinity to xylan substrate was dramatically improved ( Table 1).…”

Section: Sequence Analysismentioning

confidence: 99%

Enhancing catalytic activity of a hybrid xylanase through single substitution of Leu to Pro near the active site

Qian

Zhao

Sun

et al. 2011

World J Microbiol Biotechnol

View full text Add to dashboard Cite

A modified error-prone PCR and high-throughout screening system based on 96-well plate were employed to improve catalytic activity of a hybrid xylanase (ATx). The mutant (FSI-A124) with enhanced activity was further heterologously expressed in Pichia pastoris under the control of GAP promoter. The recombinant xylanase driven by the Saccharomyces cerevisiae α-mating factor was secreted into culture medium. After growth in YPD medium for 96 h, xylanase activity in the culture supernatant reached 66.1 U ml(-1), which was 2.92 times as that of its parent. 6 × His-tagged purification increased the specific activity to 1557.61 U mg(-1). The optimum temperature and pH of recombinant xylanase were 55°C and 6.0, respectively. A single amino acid substitution (L49P) was observed within sequence of the mutant. Insight of the three dimensional structure revealed that proline possibly produced weaker hydrogen bond, van der Waals force and hydrophobic interaction with other residues nearby than leucine, especially for V174, contributing to the flexibility of catalytic residue E177. In this study, FSI-A124 exhibited higher xylanase activity but poorer thermostability than its parent, indicating that activity and stability might be negatively correlated.

show abstract

Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase

Cited by 25 publications

References 36 publications

Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis

Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis

Engineering substrate specificity of succinic semialdehyde reductase (AKR7A5) for efficient conversion of levulinic acid to 4-hydroxyvaleric acid

Enhancing catalytic activity of a hybrid xylanase through single substitution of Leu to Pro near the active site

Contact Info

Product

Resources

About