一株产耐热耐碱木聚糖酶菌株的筛选及酶学性质研究

摘要

目的 对分离得到的一株产耐热耐碱木聚糖酶细菌ZH-07进行鉴定,并对其产木聚糖酶进行酶学性质分析.方法 根据培养基上透明圈大小筛选产酶量高的菌株,通过菌落形态、生理生化特征及16S rDNA序列同源性比对等分析鉴定菌株种属.并通过一系列单因素试验、聚丙烯酰胺凝胶电泳(SDS-PAGE)和薄层色谱(TLC)进行酶学性质研究.结果 ZH-07菌株鉴定为枯草芽孢杆菌(Bacillus subtilis).该菌能以农业废弃物玉米芯为碳源,液体发酵产耐热耐碱木聚糖酶,50℃培养36 h,木聚糖酶的最高酶活性达1664.9 U/mL;木聚糖酶的相对分子质量(Mr)约24×103,无纤维素酶活性;酶促反应最适温度为70℃,最适pH 10,70℃、pH 10处理3 h仍保持64.7%的活性.结论 Bacillus subtilis ZH-07产木聚糖酶有较好的热稳定性和碱稳定性,在低聚木糖工业生产中有巨大应用潜力.%Objective To screen a strain with high production of thermoalkaliphilic xylanase and analyze the enzymatic properties of the secreted xylanase. Methods The xylanase high-producing strain was selected based on the size of the hydrolytic ring on the selective medium. The strain was identified on the basis of the morphological features, physiological and biochemical characteristics and homologous sequence analysis of 16S rDNA. The enzymatic characterization was made through single-factor experiments, SDS-PAGE and TLC analysis. Results The strain ZH-07 was identified as Bacillus subtilis, producing a thermoalkaliphilic xylanase with the maximum activity of 1664.9 U/mL after 36 hours of cultivation in the medium containing corncob as the carbon source at 50 ℃. SDS-PAGE and TLC analysis revealed that the relative molecular weight (Mr) of the xylanase was about 24×103, which showed no cellulase activity. The optimal temperature and pH of the xylanase were 70 ℃ and pH 10, respectively. The remained activity was 64.7 % of the initial activity after incubation at 70 ℃ and pH 10 for 3 h. Conclusion Xylanase produced by Bacillus subtilis ZH-07 is thermo-stable and alkali-stable. The favorable properties of xylanase suggest that it has great application potential in xylo-oligosaccharides industries.