In-fusion Expression of Xylanase Genes XynA and XynB From Aspergillus sulphureus in E. coli and Characterization of The Recombinant Enzyme
- VernacularTitle:硫色曲霉木聚糖酶基因xynA与xynB在大肠杆菌中的融合表达及酶学性质分析
- Author:
Yihang LI
;
Jiayun QIAO
;
Yunhe CAO
- Publication Type:Journal Article
- Keywords:
Aspergillus sulphureus, xylanase, in-fusion expression
- From:
Progress in Biochemistry and Biophysics
2006;0(10):-
- CountryChina
- Language:Chinese
-
Abstract:
Two sets of primers were designed according to the sequences of xynA and xynB from Aspergillus sulphureus, and the DNA fragments composed of 574 bp and 594 bp were amplified by polymerase chain reaction (PCR), respectively. The two PCR products respectively digested with EcoRⅠ/BamHⅠ and BglⅡ/HindⅢ were ligated into multiple cloning sites of pET-28a(+). The resulting plasmid is pET-xynAB, in which xylanase A and B are ligated by a 7-amino acid peptide (GlyGlyGlySerGlyGlyGly). E. coli BL21 transformed with pET-xynAB was induced by IPTG, and a special protein band about 50 ku was detected by SDS-PAGE. The protein purified with Ni-NTA column was denatured by 8 mol/L urea and dialyzed for refolding. The recombinant xylanase showed optimal activity at 50℃ and pH 4.4. The enzyme retained above 75% of its activity at the range of pH 2.4～5.4. The xylanase displayed about 50% retained acitivity after incubating at 80℃ for 30 min. Various metal ions have different effects on activity of the recombinant xylanase.
