滇金丝猴粪便微生物来源的β-半乳糖苷酶基因的表达及酶学性质

Expression and characterization of β-galactosidase from fecal microbes of Rhinopithecus bieti

摘要:

[目的]对滇金丝猴粪便微生物来源的β-半乳糖苷酶进行异源表达和纯化，并研究其酶学性质。[方法]从滇金丝猴粪便微生物的宏基因组中克隆出一个β-半乳糖苷酶基因galRBM20_1，对该基因进行异源表达和酶学性质分析。构建含有T7强启动子的pEASY-E2-galRBM20_1质粒，转化至大肠杆菌BL21（DE3），经IPTG诱导表达后进行酶学性质研究。[结果]滇金丝猴粪便来源的β-半乳糖苷酶（galRBM20_1）最适pH为5.0，在pH 4-7之间能保留70%及其以上的活性。最适温度为45℃，在37℃和45℃下耐受1 h，酶活不变。特别的是，该酶具有良好的NaCl稳定性，经1-5 mol/L的NaCl作用1 h后，相对酶活均能超过初始酶活：当NaCl的作用浓度为4 mol/L时，β-半乳糖苷酶相对酶活最高（146%）；当NaCl的作用浓度为5 mol/L时，β-半乳糖苷酶的相对酶活仍达到135%。[结论]本研究从滇金丝猴粪便微生物的宏基因库中克隆得到β-半乳糖苷酶基因galRBM20_1，并成功在大肠杆菌BL21（DE3）表达，首次从动物胃肠道宏基因组中获得具有耐盐和转糖基产Galactooligosaccharides（GOS）性能的β-半乳糖苷酶。该酶具有良好的耐盐性，和较广的pH作用范围，使其在食品、生物技术领域和环保方面的发展具有良好的应用价值。

关键词:滇金丝猴;粪便微生物宏基因组;β-半乳糖苷酶;耐盐性;转糖基

Abstract:

[Objective] The study was to express and characterize β-galactosidase from the fecal microbes of Rhinopithecus bieti. [Methods] A β-galactosidase gene galRBM20_1 was cloned from the fecal microbial metagenome of Rhinopithecus bieti. The pEASY-E2-galRBM20_1 plasmid was constructed and transformed into Escherichia coli BL21 (DE3). The expression was induced by Isopropyl β-D-1-thiogalactopyranoside and the enzymatic properties were studied.[Results] The optimum pH of β-galactosidase galRBM20_1 was 5.0, and the enzyme retained more than 70% activity between pH 4 and 7. The optimum temperature is 45℃ and the enzyme was stable at 37℃ and 45℃, retaining nearly 100% activity after incubation for 1 h. In addition, the enzyme had strong NaCl stability. After 1 h of 1 to 5 mol/L NaCl treatment, the relative enzyme activity maintained 100%; when the concentration of NaCl was 4 mol/L, the activity of β-galactosidase galRBM20_1 was the highest (146%); it still had activity after treatment with 2.5 mol/L NaCl at 45℃ for 24 h. [Conclusion] β-galactosidase galRBM20_1 has good salt tolerance and wide pH range.

Key words:Rhinopithecus bieti;fecal microbial metagenome;β-galactosidase;salt-tolerant;transglycosylation