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Expression and Surface Display of an Acidic Cold-Active Chitosanase in Pichia pastoris Using Multi-Copy Expression and High-Density Cultivation

文献类型： 外文期刊

作者： Peng, Yanhong 1 ; Wang, Yaping 1 ; Liu, Xiaoyan 1 ; Zhou, Ronghua 1 ; Liao, Xianqing 1 ; Min, Yong 1 ; Ma, Lixin 2 ; Wang, Ying 1 ; Rao, Ben 1 ;

作者机构： 1.Hubei Acad Agr Sci, Natl Biopesticide Engn Technol Res Ctr, Hubei Biopesticide Engn Res Ctr, Biopesticide Branch Hubei Innovat Ctr Agr Sci & T, Wuhan 430068, Peoples R China

2.Hubei Univ, Biol Fac Hubei Univ, Engn Hubei Collaborat Innovat Ctr Green Transform, State Key Lab Biocatalysis & Enzyme,Hubei Key Lab, Wuhan 430062, Peoples R China

关键词： chitosanase; chiton oligosaccharides; high-density fermentation; multi-copy strains; Pichia pastoris; displayed enzyme

期刊名称：MOLECULES （ 影响因子：4.927； 五年影响因子：5.11 ）

ISSN：

年卷期： 2022 年 27 卷 3 期

页码：

收录情况： SCI

摘要： Chitosanase hydrolyzes beta-(1,4)-linked glycosidic bonds are used in chitosan chains to release oligosaccharide mixtures. Here, we cloned and expressed a cold-adapted chitosanase (CDA, Genbank: MW094131) using multi-copy expression plasmids (CDA1/2/3/4) in Pichia pastoris. We identified elevated CDA expression levels in multi-copy strains, with strain PCDA4 selected for high-density fermentation and enzyme-activity studies. The high-density fermentation approach generated a CDA yield of 20014.8 U/mL, with temperature and pH optimization experiments revealing the highest CDA activity at 20 degrees C and 5.0, respectively. CDA was stable at 10 degrees C and 20 degrees C. Thus, CDA could be used at low temperatures. CDA was then displayed on P. pastoris using multi-copy expression plasmids. Then, multi-copy strains were constructed and labelled as PCDA(1-3)-AG alpha 1. Further studies showed that the expression of CDA(1-3)-AG alpha 1 in multi-copy strains was increased, and that strain PCDA3-AG alpha 1 was chosen for high-density fermentation and enzyme activity studies. By using a multi-copy expression and high-density fermentation approach, we observed CDA-AG alpha 1 expression yields of 102415 U/g dry cell weight. These data showed that the displayed CDA exhibited improved thermostability and was more stable over wider temperature and pH ranges than free CDA. In addition, displayed CDA could be reused. Thus, the data showed that displaying enzymes on P. pastoris may have applications in industrial settings.

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