High-level Expression of an α-l-arabinofuranosidase from Thermotoga maritima in Escherichia coli for the Production of Xylobiose from Xylan

Abstract

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.

The genusThermotoga: recent developments

“…Xylooligosaccharides resulting from xylan degradation have been shown to increase the numbers of beneficial gut bacteria, such as Bifidobacterium species [71]. Production of xylooligosaccharides, especially xylobiose, has been demonstrated using immobilized xylanase B (XynB) from T. maritima (TM0070), as well as a combination of arabinofuranosidase (TM0281) and XynB [72, 73]. The combination of both enzymes resulted in twice as much xylan degradation as XynB alone.…”

Overexpression of β-glucosidase from Thermotoga maritima for the production of highly purified aglycone isoflavones from soy flour

“…Thermotoga maritima (ATCC43589) was grown anaerobically at 80°C in modified Luria-Bertani (LB) medium (pH 7.0), which contained (per litre): 10 g Tryptone, 5 g Yeast extract, 27 g NaCl, 1 mg resazurin, 15 ml trace minerals (as in Difco Maritima Broth medium), 0.5 g Na 2 S. The anaerobic condition was generated by degassing, boiling and adding Na 2 S (Xue et al 2006). Escherichia coli JM109 (DE3) (Promega,Wisconsin,USA) and BL21-CodonPlus(DE3)-RIL (Novagen, USA) were used as host for the expression of b-glucosidase gene from T. maritima, via the T 7 RNA polymerase expression system with pET-20b plasmids (Novagen).…”

Cloning, expression and characterization of glycoside hydrolase family 11 endoxylanase from Bacillus pumilus ARA

“…Complete digestion of grass xylan needs involvements of xylanase, arabinosidase, a-glucuronidase and xylosidase activities. Usually the glycosidases are intracellular enzymes with high molecular masses and prefer oligosaccharides to those substrates of high degree of polymerization although synergistic reactions have been observed by adding an arabinosidase to a xylanase in vitro (Xue et al 2006). While we were searching for an extracellular arabinosidase having significant ability to release arabinose residues from oat-spelt xylan, B. pumilus ARA was found from a culture of T. lanuginosus as a contaminant releasing arabinosidase into the culture filtrate .…”

“…As a typical GH11 xylanase, XynA from T. lanuginosus has been found cellulosefree while greater catalytic versatility is found in GH10 xylanases (Singh et al 2003). However, the main products are xylose and xylobiose after oat-spelt xylan is extensively digested by either GH10 xylanse from Thermotoga maritima (Xue et al 2006) or GH11 xylanase from T. lanuginosus (Singh et al 2003). Nevertheless, the main products obtained from Bpu XynA digestion were various oligosaccharides with two or more residues while those from Tla XynA digestion were large quantities of xylose and xylobiose; the Bpu XynA digestion could produce large quantities of xylose and xylobiose only in the presence of arabinodase (Fig.…”