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- 저자 : Zhongyuan Lv (검색결과 1 건)
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Xiaoqing Hu,Ju Chu,Si-Liang Zhang,Ying-ping Zhuang,Xin Wu,Huaxin Chen,Zhongyuan Lv,Zhongyi Yuan 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.5
Pichia pastoris is a successful system forexpressing heterologous proteins and its fermentation pH isalways maintained below 7.0. However, particular proteinsare unstable under acidic conditions, such as methionineadenosyltransferase (MAT), and thus fermentation underacidic pH conditions is unsuitable because protein activityis lost owing to denaturation. Here, a strategy employingalkaline pH in the late fermentation period was developedto improve MAT production. Initially, P. pastoris KM71was transformed with the mat gene to overexpress MAT. After 72 h of in vitro incubation at different pH values, theexpressed MAT displayed highest stability at pH 8.0;however, pH 8.0 inhibited cell growth and induced cellrupture, thus affecting protein production. To balance MATstability and Pichia cell viability, different pH controlstrategies were compared. In strategy A (reference), theinduction pH was maintained at 6.0, whereas in strategy B,it was gradually elevated to 8.0 through a 25 h transitionperiod (80 ~ 105 h). MAT activity was 0.86 U/mg (twofoldhigher than the control). However, MAT content wasreduced by 50% when compared with strategy A, becauseof proteases released upon cell lysis. To improve cellviability under alkaline conditions, glycerol was added inaddition to methanol (strategy C). When compared withstrategy B, the MAT-specific activity remained nearlyconstant, whereas the expression level increased to 1.27 g/L. The alkaline pH control strategy presented herein for MATproduction represents an excellent alternative for expressingproteins that are stable only under alkaline conditions.
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