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

Yanhong Peng,Xianqing Liao,Ronghua Zhou,Lixin Ma,Yong Min,Ben Rao,Ying Wang,Xiaoyan Liu,Yaping Wang

doi:10.3390/molecules27030800

Yanhong Peng, Xianqing Liao + Show 7 more

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https://doi.org/10.3390/molecules27030800

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Abstract

Chitosanase hydrolyzes β-(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 °C and 5.0, respectively. CDA was stable at 10 °C and 20 °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)-AGα1. Further studies showed that the expression of CDA(1-3)-AGα1 in multi-copy strains was increased, and that strain PCDA3-AGα1 was chosen for high-density fermentation and enzyme activity studies. By using a multi-copy expression and high-density fermentation approach, we observed CDA-AGα1 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.

Highlights

Chitin is a linear homopolymer of acetylated β-(1,4)-linked glucosamine residues, is one of the most abundant polysaccharides in the world [1], and is found primarily in insects, crustaceans, nematodes, and fungi
Chitosanases effectively convert chitosan to chiton oligosaccharides
We identified a Bacillus glycinifermentans BT2019 strain that exhibited the highest enzyme activity at 20 ◦ C

Summary

Introduction

Chitin is a linear homopolymer of acetylated β-(1,4)-linked glucosamine residues, is one of the most abundant polysaccharides in the world [1], and is found primarily in insects, crustaceans, nematodes, and fungi. Derived chitosan oligosaccharides are water soluble and exhibit multiple biological functions, including anti-tumor, anti-bacterial, and anti-fungal activities. These features make chitosan and associated oligosaccharides useful in the agriculture, food, and pharmaceutical industries. Chemical treatment and enzymatic hydrolysis are two primary methods used to derive oligosaccharides from chitosan. Families 46, 75, and 80 include most of the known chitosanase enzymes which have been isolated and heterologously expressed, based on their ability to efficiently process chitosan into oligosaccharide mixtures for industrial applications [7,8,9,10,11]. To generate chitooligosaccharide mixtures with specific polymerization levels, enzymatic hydrolysis processes have to be stringently regulated. Cold-active enzymes have been used under mild reaction conditions to generate different mixtures, suggesting this approach could be valid in producing specific chitooligosaccharide mixtures

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