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KMID : 1094720140190050900
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Biotechnology and Bioprocess Engineering
2014 Volume.19 No. 5 p.900 ~ p.907
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An alkaline pH control strategy for methionine adenosyltransferase production in Pichia pastoris fermentation
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Hu Xiaoqing
Chu Ju
Zhang Siliang
Zhuang Yingping
Wu Xin
Chen Huaxin
Lv Zhongyuan
Yuan Zhongyi
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Abstract
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Pichia pastoris is a successful system for expressing heterologous proteins and its fermentation pH is always maintained below 7.0. However, particular proteins are unstable under acidic conditions, such as methionine adenosyltransferase (MAT), and thus fermentation under acidic pH conditions is unsuitable because protein activity is lost owing to denaturation. Here, a strategy employing alkaline pH in the late fermentation period was developed to improve MAT production. Initially, P. pastoris KM71 was transformed with the mat gene to overexpress MAT. After 72 h of in vitro incubation at different pH values, the expressed MAT displayed highest stability at pH 8.0; however, pH 8.0 inhibited cell growth and induced cell rupture, thus affecting protein production. To balance MAT stability and Pichia cell viability, different pH control strategies were compared. In strategy A (reference), the induction pH was maintained at 6.0, whereas in strategy B, it was gradually elevated to 8.0 through a 25 h transition period (80 ∼ 105 h). MAT activity was 0.86 U/mg (twofold higher than the control). However, MAT content was reduced by 50% when compared with strategy A, because of proteases released upon cell lysis. To improve cell viability under alkaline conditions, glycerol was added in addition to methanol (strategy C). When compared with strategy B, the MAT-specific activity remained nearly constant, whereas the expression level increased to 1.27 g/L. The alkaline pH control strategy presented herein for MAT production represents an excellent alternative for expressing proteins that are stable only under alkaline conditions.
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KEYWORD
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methionine adenosyltransferase, alkaline pH, residual activity, cell lysis, Pichia pastoris
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