Xing Tong
State Key Laboratory of Food Science and Technology of China, Key Laboratory of Industrial Biotechnology of Education Ministry, School of Environment And Civil Engineering, Jiangnan University, Wuxi 214122, ChinaQiusong Tang
State Key Laboratory of Food Science and Technology of China, Key Laboratory of Industrial Biotechnology of Education Ministry, School of Environment And Civil Engineering, Jiangnan University, Wuxi 214122, ChinaYufei Wu
State Key Laboratory of Food Science and Technology of China, Key Laboratory of Industrial Biotechnology of Education Ministry, School of Environment And Civil Engineering, Jiangnan University, Wuxi 214122, ChinaJing Wu
State Key Laboratory of Food Science and Technology of China, Key Laboratory of Industrial Biotechnology of Education Ministry, School of Environment And Civil Engineering, Jiangnan University, Wuxi 214122, ChinaJian Chen
State Key Laboratory of Food Science and Technology of China, Key Laboratory of Industrial Biotechnology of Education Ministry, School of Environment And Civil Engineering, Jiangnan University, Wuxi 214122, ChinaSupported by the National Science Foundation for Distinguished Young Scholars (20625619), the Research Program of State Key Laboratory of Food Science and Technology (SKLF-MB-200802), and the Major Project of China National Programs for Fundamental Resear
[Objective] α-glucosidase from Aspergillus niger SG136 was expressed in Pichia pastoris. [Methods] cDNA of mature α-glucosidase(aglu) was amplified from the total DNA of A. niger SG136 by PCR and overlap-PCR with primers designed based on the sequence of A. niger CBS 513.88 in NCBI database. The gene was cloned into pMD18-T simple vector. The sequencing result showed that the gene encoded for a protein of 960 amino acids residues, which was 1 amino acid different from that of A. niger CBS 513.88. The expression vector pPIC9K-aglu was constructed by subcloning the gene into plasmid pPIC9K, and then transformed into P. pastoris through electroporation after linearized by BglⅡ digestion. The recombinant P. pastoris KM71/pPIC9K-aglu were screened in MD and YPD/G418 plates, and identified by PCR. In shaking culture condition, methanol was added to a final concentration of 1 % to induce the secretion of α-glucosidase. [Results] Electrophoresis analysis of KM71/pPIC9K-aglu culture supernatant showed that there were two major protein bands corresponding to 98 kDa and 33 kDa respectively in SDS-PAGE, and there was only one band in Native PAGE; while in the control experiment of KM71/pPIC9K, there were no visible bands. Transglucosidation reaction from crude enzyme revealed that contents of isomaltooligosaccharides were up to 26.0 % under the optimal conditions of pH 5 and 60 ℃ at 24 h. [Conclusion] A. niger α-glucosidase was expressed in P. pastoris with transglucosidation activity.
Xing Tong, Qiusong Tang, Yufei Wu, Jing Wu, Jian Chen. Cloning of the gene encoding α-glucosidase from Aspergillus niger and its expression in Pichia pastoris. [J]. Acta Microbiologica Sinica, 2009, 49(2): 262-268
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