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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.
*Corresponding author. E-mail:
xueyemin@njnu.edu.cn.
Tel: +86-025-85891275.
Abbreviations:
AguA,
α-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 the aguA; aguA3, the
N-terminal plus interior mutated forms of the aguA;
aguA4, 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.
Accepted
9 June, 2008 |
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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. |
