High-level expression of an alpha-L-arabinofuranosidase from Thermotoga maritima in Escherichia coli for the production of xylobiose from xylan.
To efficiently produce xylobiose from xylan, high-level expression of an alpha-L-arabinofuranosidase gene from Thermotoga maritima was carried out in Escherichia coli. A 1.5-kb DNA fragment, coding for an alpha-L-arabinofuranosidase of T. maritima, was inserted into plasmid pET-20b without the pelB signal sequence leader, and produced pET-20b-araA1 with 8 nt spacing between ATG and Shine-Dalgarno sequence. A maximum activity of 12 U mg(-1) was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-20b-araA1. The over-expressed alpha-L-arabinofuranosidase was purified 13-fold with a 94% yield from the cellular extract of E. coli by a simple heat treatment. Production of xylooligosaccharides from corncob xylan by endoxylanase and alpha-L-arabinofuranosidase was examined by TLC and HPLC: xylobiose was the major product from xylan at 90 degrees C and its proportion in the xylan hydrolyzates increased with the reaction time. Hydrolysis with in the xylanase absence of alpha-L-arabinofuranosidase gave only half this yield.
Xue
Yemin
Y
Jiangsu Key Laboratory for Biodiversity and Bio-resources, The Key Laboratory of Microbial Engineering, Nanjing Normal University, Nanjing, P.R. China.
Wu
Ailian
A
Zeng
Hongyan
H
Shao
Weilan
W
D004187:Disaccharides / Q000138:chemical synthesis*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D014990:Xylans / Q000737:chemistry*
D004187:Disaccharides; D011994:Recombinant Proteins; D014990:Xylans; C004173:xylobiose