Directed Transformation Of Xylosidase In The Thermophilic Anaerobic Bacteria

The gene xylC encoding aβ-xylosidase from Thermoanaerobacterium sp. strain JW/SL YS485 was subcloned into pHsh, resulting in pHsh-xy1C and was overexpressed in E. coli JM109.The secondary structure of the translation initiation region (TIR) is a crucial factor for translation initiation rate. Meanwhile, codon preference plays an important role. According to the two principles, the TIR of the gene xylC was optimized. Five mutants pHsh-xyl I , pHsh-xyl II, pHsh-xyl III, pHsh-xylIV, pHsh-xylV were obtained by optimizing the secondary structure of mRNA's TIR of the gene xylC to release ATG from pairing bases, and furthermore to release RBS from pairing bases, by changing GAA to AAA next to ATG, by using more preferred codons downstream of the TIR, and by adding a DB sequence at the 15th base downstream of the ATG. All the plasmids were transformed into E. coli JM109. The recombinant strains harboring these plasmids were obtained, and induced to expressβ-xylosidase. Theβ-xylosidase activities of E. coli JM109 harbouring pHsh-xylC,pHsh-xyl I , pHsh-xylII,pHsh-xylIII,pHsh-xylIV, pHsh-xylV were 1.35 U/ml,4.5U/ml,3.012 U/ml,5.5 U/ml,5.49 U/ml and 5.018 U/ml, respectively. E. coli JM109 harbouring pHsh-xylIII produced the highestβ-xylosidase activity at 5.5 U/ml. This was 4.07 fold higher thanβ-xylosidase produced by E. coli JM109 harbouring pHsh-xylC (1.35 U/ml) and about 55 fold higher thanβ-xylosidase produced by E. coli JM109 harboring pxylo-2.2 (0.1 U/ml). In the resulting strain harboring pHsh-xylIII which exhibited the greatestβ-xylosidase activity, the recombinant enzyme made up about 34% of the total proteins present in the intracellular soluble fraction on the basis of densitometer scanning of the SDS-PAGE gel.The recombinant enzyme was purified by four major purification steps including heat treatment, DEAE-sepharose, Gel filtration and Hydrophobic interaction. The purified enzyme showed a single protein band on SDS-PAGE. The molecular mass calculated from the deduced amino acid sequence was in reasonable agreement with 72-73 kDa estimated by SDS-PAGE (FIG.2). After the last chromatographic step the specific activity of the purified enzyme was 62.9 U per mg protein. The enzyme was purified 3.6 fold with recovery of 21.6% of the initial activity from crude extract. The result showed that lots of other proteins can be eliminated by heat treatment and the recovery was up to 95%, indicating that we can obtain the enzyme cheaply and simply by heat treatment in industry.Characterization of the purified recombinantβ-xylosidase showed a similarity to the natural enzyme. The purified recombinant enzyme had a high K_m value of 28.4 mM, which was much higher than anyβ-xylosidase reported and a V_max value of 275.8 U/mg. The enzyme was not sensitive to xylose which inhibits mostβ-xylosidase at a concentration up to 67 mM. And even 70% of activity was retained when xylose was up to 200 mM. The purified recombinant enzyme showed optimal activity at pH 6.0 at 65℃and was stable at 65℃for 2 h at pH 6.0 with a residual activity of 87%. Ca²⁺, Na⁺, Mg²⁺, K⁺ or DTT did not have any effect on its activity, but the presence of Hg²⁺ and Ag⁺ could completely reduce enzymatic activity. 50mM NaCl or KCl slightly stimulated the enzyme activity. 100 mM NaCl or KCl had no effect on the activity. However, high concentrations of NaCl or KCl in the reaction mixture caused inhibition of its activity. Its high substrate and xylose tolerance and thermal stability qualify this enzyme for industrial application.