1. Complete secretion of recombinant Bacillus subtilis levansucrase in Pichia pastoris for production of high molecular weight levan
- Author
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Shuochang, Chen, Qiuping, Tong, Xiaolei, Guo, Hao, Cong, Zi, Zhao, Wenfeng, Liang, Jiemin, Li, Ping, Zhu, and Hui, Yang
- Subjects
Molecular Weight ,Hexosyltransferases ,Tandem Mass Spectrometry ,Structural Biology ,Saccharomycetales ,General Medicine ,Molecular Biology ,Biochemistry ,Pichia ,Bacillus subtilis ,Fructans - Abstract
Three signal peptides from α-mating factor (α-MF), inulinase (INU) and native levansucrase (LS) were compared for secretion efficiency of Bacillus subtilis levansucrase SacB-T305A in Pichia pastoris GS115. The first complete secretion of bacterial levansucrase in yeasts under methanol induction was achieved while using α-MF signal. The secreted recombinant Lev(α-MF) proved to be glycosylated by combination of NanoLC-MS/MS and Endo H digestion. Interestingly, glycosylation not only improved significantly the polymerase thermostability, but also reversed the products profiles to favor synthesis of high molecular weight (HMW) levan which accounted for approximately 73 % to total levan-type polysaccharides. It indicated for the first time that the glycosylation of recombinant B. subtilis levansucrase affected significantly the products molecular weight distribution. It also provided a promising enzymatic way to effectively product HMW levan from sucrose resources.
- Published
- 2022