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http://dx.doi.org/10.4014/jmb.0908.08028

High-Level Expression of an Aspergillus niger Endo-$\beta$-1,4-Glucanase in Pichia pastoris Through Gene Codon Optimization and Synthesis  

Zhao, Shumiao (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University)
Huang, Jun (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University)
Zhang, Changyi (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University)
Deng, Ling (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University)
Hu, Nan (College of Life Science and Pharmacy, Nanjing University of Technology)
Liang, Yunxiang (State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University)
Publication Information
Journal of Microbiology and Biotechnology / v.20, no.3, 2010 , pp. 467-473 More about this Journal
Abstract
To improve the expression efficiency of recombinant endo-$\beta$-1,4-glucanase in P. pastoris, the endo-$\beta$-1,4-glucanase (egI) gene from Aspergillus niger was synthesized using optimized codons. Fourteen pairs of oligonucleotides with 15 bp overlap were designed and the full-length syn-egI gene was generated by two-step PCR-based DNA synthesis. In the synthesized endo-$\beta$-1,4-glucanase gene syn-egI, 193 nucleotides were changed, and the G+C content was decreased from 54% to 44.2%. The syn-egI gene was inserted into pPIC9K and transformed into P. pastoris GS115 by electroporation. The enzyme activity of recombinant P. pastoris stain 2-7# reached 20.3 U/ml with 1% barley $\beta$-glucan and 3.3 U/ml with 1% carboxymethylcellulose (CMC) as substrates in shake flasks versus 1,270.3 U/ml and 220.7 U/ml for the same substrates in 50-1 fermentors. The molecular mass of the recombinant protein was approximately 40 kDa as determined by SDS-PAGE analysis, the optimal temperature for recombinant enzyme activity was $70^{\circ}C$, and the optimal pH was 5.0 when CMC was used as the substrate.
Keywords
Aspergillus niger; codon optimization; endo-$\beta$-1,4-glucanase; expression; Pichia pastoris;
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