Optimized expression of an acid xylanase from <Emphasis Type="Italic">Aspergillus usamii</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its biochemical characterization |
| |
Authors: | Chenyan Zhou Dongfeng Li Minchen Wu Wu Wang |
| |
Institution: | (1) School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, P.R. China;(2) The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, P.R. China;(3) School of Medicine and Pharmaceutics, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, P.R. China |
| |
Abstract: | The xylanase gene xyn II from Aspergillus usamii E001 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K and integrated into the genome of a methylotrophic yeast, P. pastoris GS115, by electroporation. His+ transformants were screened for on the basis of their resistance to G418 and activity assay. A transformant, P. pastoris GSC12, which showed resistance to over 6 mg G418/ml and highest xylanase activity was selected. Recombinant xylanase was
secreted by P. pastoris GSC12 24 h after methanol induction of shake-flask cultures, and reached a final yield of 3139. About 68 U/mg 120 h after
the induction. The molecular mass of this xylanase was estimated to be 21 kDa by SDS-PAGE. The optimum pH and temperature
were 4.2 and 50 °C, respectively. Xylanase was stable below 50 °C and within pH 3.0–7.0. Its activity was increased by EDTA
and Co2+ ion and strongly inhibited by Mn2+, Li+ and Ag+ ions. The K
m and V
max values with birchwood xylan as the substrate were found to be 5.56 mg/ml and 216 μmol/mg/min, respectively. This is the first
report on expression and characterization of xylanase from A. usamii in P. pastoris. The hydrolysis products consisted of xylooligosaccharides together with a small amount of xylose. This property made the
enzyme attractive for industrial purposes, as relatively pure xylooligosaccharides could be obtained. |
| |
Keywords: | Aspergillus usamii Characterization Expression Pichia pastoris Xylanase Xylooligosaccharides |
本文献已被 SpringerLink 等数据库收录! |
|