Expression of β-xylosidase An-xyl from Aspergillus niger and characterization of its xylose tolerance / 生物工程学报

The hydrolysis of xylo-oligosaccharides catalyzed by β-xylosidase plays an important role in the degradation of lignocellulose. However, the enzyme is easily inhibited by its catalytic product xylose, which severely limits its application. Based on molecular docking, this paper studied the xylose affinity of Aspergillus niger β-xylosidase An-xyl, which was significantly differentially expressed in the fermentation medium of tea stalks, through cloning, expression and characterization. The synergistic degradation effect of this enzyme and cellulase on lignocellulose in tea stems was investigated. Molecular docking showed that the affinity of An-xyl to xylose was lower than that of Aspergillus oryzae β-xylosidase with poor xylose tolerance. The Ki value of xylose inhibition constant of recombinant-expressed An-xyl was 433.2 mmol/L, higher than that of most β-xylosidases of the GH3 family. The Km and Vmax towards pNPX were 3.6 mmol/L and 10 000 μmol/(min·mL), respectively. The optimum temperature of An-xyl was 65 ℃, the optimum pH was 4.0, 61% of the An-xyl activity could be retained upon treatment at 65 ℃ for 300 min, and 80% of the An-xyl activity could be retained upon treatment at pH 2.0-8.0 for 24 h. The hydrolysis of tea stem by An-xyl and cellulase produced 19.3% and 38.6% higher reducing sugar content at 2 h and 4 h, respectively, than that of using cellulase alone. This study showed that the An-xyl mined from differential expression exhibited high xylose tolerance and higher catalytic activity and stability, and could hydrolyze tea stem lignocellulose synergistically, which enriched the resource of β-xylosidase with high xylose tolerance, thus may facilitate the advanced experimental research and its application.

High-level expression and characterization of Selenomonas ruminantium β-xylosidase in Pichia pastoris / 生物工程学报

β-xylosidase (EC 3.2.1.37) is an important part of the xylanolytic enzymes system. In the present research, β-xylosidase gene Sxa derived from Selenomonas ruminantium was expressed in Pichia pastoris GS115. According to the codon bias and rare codons of P. pastoris, mRNA secondary structure and GC content, Sxa gene was optimized. The optimized full-length gene mSxa was obtained by gene synthesis technique and the recombinant yeast expression vector pPIC9K-mSxa was constructed. After being digested by restriction enzyme BglⅡ, the mSxa gene was transformed into P. pastoris GS115. Then, phenotype and geneticin G418 resistance screening, and PCR were adopted to identify the positive transformants. Finally, the recombinant P. pastoris GS115-pPIC9K-mSxa was obtained. Based on enzymatic activity assay, a high-level expression clone was picked up and then the enzymatic characteristics of the recombinant β-xylosidase were studied. The results showed that the molecular weight of the mSxa expressed in P. pastoris G115 was about 66 kDa. The maximum activity was achieved 287.61 IU/mL at fermenter level. Enzymatic characterization showed the β-xylosidase was stable between 40 ℃ and 60 ℃, and pH between 5.0 and 7.0. The optimal reaction temperature and pH were 55 ℃ and 6.0, and preferentially degrading the β-xylose glycosidic bond. The enzymatic activity was activated by Mn²⁺ and Ca²⁺, and inhibited by Fe³⁺, Cu²⁺, Co²⁺, Mg²⁺, EDTA and SDS. The study indicates that the modified β-xylosidase gene mSxa from Selenomonas ruminantium can express successfully with high activity in P. pastoris. The study lays a foundation for further industrial application of the β-xylosidase.

Isolation and Identification of Xylanolytic Enzyme from an Effective Strain Bacillus Licheniformis Isolated from the Decaying Wood.

A new species of Bacillus licheniformis produced extracellular xylanase under submerged fermentation when wheat bran is used as carbon source. The xylan is the most common hemicellulosic polysaccharide in food industry and agricultural wastes, comprising a backbone of xylose residues linked by β-1,4 glycosidic bonds. Bacillus licheniformis has been shown to be a promising organism for enhanced production of xylanases & β-xylosidase under submerged fermentation (SmF). The optimization of cultural conditions and carbon, nitrogen sources for enzymes production. The bacterial strain Bacillus licheniformis was cultivated using as substrate xylan, wheat bran, corn straw, corncob, and sugarcane bagasse. Wheat bran has been a good xylanase (16.8U/ml) & β xylosidase (5.6U/ml) activity after 48h of fermentation. Maximum enzyme activity was observed in xylan as carbon source and peptone as nitrogen source. Both crude enzymes were characterized and a bacterial xylanase shows optimum pH for xylanase activity at 6.5 & β xylosidase were found to be 6.0. The optimum temperatures were 450C for both and they were thermally stable up to 500C. The parameters of Vmax and Km obtained using Line weaver-Burk plot method were 277.7μmol / min/mg and 5.26 mg /L correspondingly.