Sparse Cotton-like Trichosporon Of Thermophilic Bacteria Xylanase A Is Highly Expressed In Escherichia Coli

Xylanase is the most important enzyme in the degradation of xylan. It hydrolyzes xylan into xylooligosaccharide and xylose. Thermomyces lanuginosus DSM 5826 produces a high level of cellulase-free, thermostable xylanase, which is catalytically active over a broad pH range. In addition, its hydrolysis products consist of xylooligosaccharide and without any xylose.The gene xynA was analyzed according to the codon usage table of E. coli. A new gene xynA1 was synthesized by primer-splicing with preferred codons. The gene xynA1 was inserted into vectors pET-20b and pHsh to construct pET-20b-xynA and pHsh-xynA1. The mRNA secondary structure of recombinant vector pHsh-xynA1 was optimized by online-analysis, and resulted into the vector pHsh-xynA2. The three vectors were transformed into E. coli strain, respectively. Recombinant strains containing pHsh-xynA2 produced a highest xylanase activity of 47.1 U/mL after 6h, and the expression level was 10 times higher than the strains containing pHsh-xynA1. Recombinant strains containing pET-20b-xynA got a highest xylanase activity of 4.1 U/mL at 37℃after 3 h, and 50.0 U/mL at 16℃after 12 h.The recombinant enzyme (pHsh-xynA2) was purified 21.5-fold by heat treatment, DEAE-Sepharose FF column and t-Butyl-HIC column, and the properties of purified recombinant xylanse were determined. The optimal conditions for the xylanase reaction were pH6.0 at a temperature of 65℃. The purified enzyme was stable for 30 min over the pH range of 5.0-8.0, and had nearly 50% activities lost after 3 h at 65℃. The major xylooligomers found after hydrolysis of xylan by the xylanase (pHsh-xynA2) were xylotriose and xylibiose, and none xylose was found.