Enhancing catalytic activity of a hybrid xylanase through single substitution of Leu to Pro near the active site.
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.
College of Animal Science, Zhejiang University, Hangzhou 310058, China.
Qian
Q
Wang
Li-Li
LL
Zhao
Jian-Yi
JY
Sun
Jian-Xin
JX
Liu
Xiao-Yan
XY
Weng
D019943:Amino Acid Substitution; D020134:Catalytic Domain; D003470:Culture Media / Q000737:chemistry; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature; D014995:Xylosidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D003470:Culture Media; D011994:Recombinant Proteins; D014995:Xylosidases