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.