African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12193

Full Length Research Paper

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

Yemin Xue*, Jinjin Yu, Xiangfei Song, Jingjing Peng, Weilan Shao
Jiangsu Key Laboratory for Biodiversity and Bio-resources, the Key Laboratory of Microbial Engineering, Nanjing Normal University, 1 Wenyuan Rd, Nanjing 210046, P.R. China.
Email: [email protected]

  •  Accepted: 09 June 2008
  •  Published: 18 July 2008

Abstract

 

The GH67α-glucuronidase encoded by aguA of Thermotoga maritima is one of the most thermostable α-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 α-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 α-glucuronidase.

 

Key words: α-Glucuronidase, free energy, overexpression, pHsh, rare codon, secondary structure xylobiose.

Abbreviation

AguAα-glucuronidase; Ara, α-L-arabinofuranosidase; Xyl, β-xylosidase; XynB, endoxylanase B; aguA, the α-glucuronidase gene; aguA1, the N-terminal mutated forms of the aguA; aguA2, the interior mutated forms of theaguA; aguA3, the N-terminal plus interior mutated forms of the aguAaguA4, the deleted forms of four ammonia acids at N-terminals of AguA; GluA, glucuronic acid; TIR, translational initiation region; RBS, ribosome binding site; XOSs,xylooligosaccharides; PPB, potassium phosphate buffer.