Over-expression of xylanolytic ∝-glucuronidase from Thermotoga maritima in the pHsh system of Escherichia coli for the production of xylobiose from xylan

Abstract

The GH67∝-glucuronidase encoded by aguA of Thermotoga maritima is one of the most thermostable ∝- glucuronidases described to date and thus has considerable potential in industrial application. The
enzyme was higher expressed by using the novel pHsh expression vector than in pET vector in Echerichia coli. The site directed mutations changed codons for the first two arginines, a leucine and a
proline in the 5’ flanking region of aguA to CGU, CUG and CCG respectively, resulted in a maximum activity of 7.1 U mg-1 in pHsh system. The results of calculation using MFOLD showed the introduction of replacement of the nucleotides encoding the N-terminal region of the protein by optimizing rare codons based on reducing the mRNA secondary structures in TIR is a useful approach to increase the
expression level of heterologous proteins in E. coli cells. The ∝-glucuronidase of T. maritima was clearly able to remove 4-O-methylglucuronic acid groups from polymeric xylan and its fragment oligosaccharides. The enzyme acts synergistically with xylanase and beta-xylosidase in the hydrolysis of birchwood xylan and 4-O-methyl-D-glucuronoxylan. Enzymatic hydrolysis of corncob xylan examined by HPLC showed that more xylobiose was produced by xylanase hydrolysis in the presence of ∝- glucuronidase.