Yanman Zhu1,2, Yongmei He2,3,Bo Wang 2 , Rihe Peng2*, Quanhong Yao2*

 

1.Biochemistry and Molecular Biology, Shanghai Normal University ,Shanghai, China

2.Agro-Biotechnology Research Center, Shanghai Academy of Agricultural Sciences, Shanghai, China

3.College of Food Science, Shanghai Ocean University ,Shanghai, China

 

Abstract:

Pectobacterium wasabiae is a dreadful causal agent of potato soft rot. It relied mainly on the production a wide range of plant cell wall-degrading enzymes to cause disease. Of the different enzymes, the endo-β-1,4-glucanase may be important in pathogenesis, being responsible for hydrolyzing the cellulose. In the process of pathogen infection, cellulase secreted by the pathogen is major determinant of pathogenicity, which played an important role in softening and decomposition of cell wall. But so far, the related research very rarely. However, most characteristic of the cellulase in the soft rot bacteria was not clear so far. Here, a putative endo-β-1,4-glucanase gene from P. wasabiae SCC3193 was previously synthesized according the bias codon of yeast, heteroexpressed in Pichia pastoris, and further investigated for its characteristic. Pectobacterium wasabiae SCC3193 is a genetically well characterized model in soft rot research. SDS-PAGE analysis showed that the recombinant endo-β-1,4-glucanase (PcegAI) produced in P. pastoris has a molecular mass of approximately 30kDa. When CMC was used as substrate, it exhibited maximal activity at pH 4.0 with an optimum temperature of 40°C, and stable at pH 5-8. The Km and specific activity values for CMC are 64.5 mM and 7 ×103 U mg-1 respectively. Most metallic ions had no influence on the activity of endo-β-1,4-glucanase at a concentration of 1 mM except for Cu2+ and Mn2+. These characteristics suggested that PcegAI may play a major role in degrading of cellulose polymers. The high activity of this enzyme also has potential in biomass opening, such as the bioconversion of lignocellulosic materials and bioethanol production.

Keywords: endo-β-1,4-glucanase; Pectobacterium wasabiae; heterologous expression; Pichia pastorias; enzymatic characteristics