Homologous Expression and Characterization of <i>Pichia pastoris</i> Kex2

doi:10.13523/j.cb.20190105

China Biotechnology

2019, Vol. 39

Issue (1): 38-45 DOI: 10.13523/j.cb.20190105

Homologous Expression and Characterization of Pichia pastoris Kex2

Tong WANG,Yan XU,Xiao-wei YU(

)

1 Key Laboratory of Industrial Biotechnology of Jiangnan University, Wuxi　214122, China
2 Suqian Industrial Technology Research Institute of Jiangnan University,Suqian　223814, China

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Abstract

Kex2 protease is a precursor-processing protease from yeast. Kex2 protease from Pichia pastoris (PPKex2) was homologous expressed in Pichia pastoris. Then the expression and enzymatic characteristics of PPKex2 were studied and further compared with the Kex2 protease from Saccharomyces cerevisiae (SCKex2) expressed in Pichia pastoris. Firstly, these Kex2 genes were cloned from the genomes of Pichia pastoris and Saccharomyces cerevisiae, inserted into pPIC9K expression vector, and then transformed into Pichia pastoris GS115. The expression of PPKex2 and SCKex2 were induced by methanol and purified with anion exchange chromatography (Q-FF). Finally, the enzymatic characteristics of these Kex2 protease were characterized. The specific enzyme activity of PPKex2 in the supernatant was seven times higher than that of SCKex2. Enzymatic characterization showed that PPKex2 was similar to SCKex2 and had the optimal activity at pH 8.0-9.0 and 37℃. In terms of stability, PPKex2 was most stable at pH 7.0. PPKex2 was more stable than SCKex2 in alkaline pH and less stable than SCKex2 in acidic pH. Meanwhile, the thermostability of PPKex2 is lower compared with that of SCKex2. The kinetic analysis showed that the k_cat and k_cat/K_m of PPKex2 was 4.8- and 3.3-fold higher than that of SCKex2, respectively. The first time report the expression and enzymatic characteristics of PPKex2 homologous expressed in Pichia pastoris, which shows potential application in the future.

Key words： Kex2 Pichia pastoris Homologous expression Enzymatic characteristics

Received: 17 August 2018 Published: 28 February 2019

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Corresponding Authors: Xiao-wei YU E-mail: bioyuxw@aliyun.com

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Tong WANG,Yan XU,Xiao-wei YU. Homologous Expression and Characterization of Pichia pastoris Kex2. China Biotechnology, 2019, 39(1): 38-45.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190105 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I1/38

Fig.1 Alignment of amino acid sequences for the PPKex2 and SCKex2 Identical amino acid residues were highlighted in dark blue,the predicted secondary structure of PPKex2 above the sequence

Fig.2 Plasmid map of pPIC9K-PPKex2 and pPIC9K-SCKex2

Fig.3 SDS-PAGE of the supernatant of PPKex2 and SCKex2 at different induction time

Fig.4 SDS-PAGE analysis of the purified PPKex2 and SCKex2

Fig.5 The optimal pH and temperature of PPKex2 and SCKex2 (a)The optimal pH of PPKex2 and SCKex2 (b) The initial reaction rate of PPKex2 and SCKex2 at different temperature with method 1 (c) The optimal temperature of PPKex2 and SCKex2 with method 2

Fig.6 The pH- stability and thermostability of PPKex2 and SCKex2 (a)The pH-stability of PPKex2 and SCKex2 (b) The thermostability of PPKex2 (c) The thermostability of SCKex2

Table 1 The kinetic constant of PPKex2 and SCKex2


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