粪便微生物宏基因组来源GH1 β-葡萄糖苷酶的重组表达及酶学性质

Purification and characterization of GH1 β-glucosidase from fecal microbes metagenome

摘要:

[背景] β-葡萄糖苷酶是一类重要的纤维素分解酶类。目前较多可培养微生物来源的β-葡萄糖苷酶（Bgl）存在热稳定性和酸碱稳定性差及作用范围窄的问题。[目的] 从滇金丝猴粪便微生物宏基因组中挖掘新型β-葡萄糖苷基因，异源表达并研究其酶学性质，为食品等领域提供新型酶资源。[方法] 从粪便微生物宏基因组出发，扩增和异源表达β-葡萄糖苷酶基因BglRBS_26和BglRBS_9，并进行酶学性质研究。[结果] 获得GH1家族重组β-葡萄糖苷酶BglRBS_26和BglRBS_9，分子量分别为60 kD和50 kD。BglRBS_26的最适作用条件为pH 6.0、45 ℃；BglRBS_9的最适作用条件为pH 5.0、40 ℃。BglRBS_26和BglRBS_9的K_m分别为(0.681 6±0.164 2) μmol/L和(3.317 0±0.871 4) μmol/L。BglRBS_26具有较好的酸碱耐受性，在pH 5.0–6.0下处理1 h，剩余酶活大于110%；pH 7.0-8.0范围内，剩余酶活仍然保持在100%以上。此外，蔗糖对BglRBS_26和BglRBS_9有不同程度的激活，当反应体系中加入20%（质量体积分数）蔗糖，BglRBS_26酶活力可提高至140%；10%（质量体积分数）蔗糖可将BglRBS_9酶活力提高至180%。此外，BglRBS_26具有较好的NaCl耐受性和稳定性，其在37 ℃、2.5 mol/L的NaCl下处理1 h后保留80%活性。[结论] 从滇金丝猴粪便微生物宏基因组中获得2个新型β-葡萄糖苷酶基因BglRBS_26和BglRBS_9，并成功在大肠杆菌BL21（DE3）中表达。BglRBS_26和BglRBS_9具有较好的pH稳定性和蔗糖耐受性，在食品、发酵等行业具有潜在应用价值。

Abstract:

[Background] β-glucosidase is an important class of cellulolytic enzymes. At present, β-glucosidase (Bgl) derived from cultivable microorganisms has the problems of poor thermal stability and acid-base stability and a narrow range of action. [Objective] Discover new β-glucoside genes from the fecal microbial metagenomics of Rhinopithecus bieti, express heterologously and study its enzymatic properties, and provide new enzyme resources for food and other fields. [Methods] Starting from the fecal microbial metagenome, the β-glucosidase genes BglRBS_26 and BglRBS_9 were amplified and heterologously expressed, and the enzymatic properties were studied. [Results] The GH1 family recombinant β-glucosidase BglRBS_26 and BglRBS_9 were obtained, with molecular weights of 60 kD and 50 kD, respectively. The optimal conditions for BglRBS_26 are pH 6.0 and 45 ℃; the optimal conditions for BglRBS_9 are pH 5.0 and 40 ℃. The K_m of BglRBS_26 and BglRBS_9 are (0.681 6±0.164 2) μmol/L and (3.317 0±0.871 4) μmol/L, respectively. BglRBS_26 has good acid-base tolerance. After treatment at pH 5.0–6.0 for 1 h, the remaining enzyme activity is greater than 110%; in the range of pH 7.0–8.0, the remaining enzyme activity remains above 100%. In addition, sucrose can activate BglRBS_26 and BglRBS_9 to varying degrees. When 20% (M/V) sucrose is added to the reaction system, the enzyme activity of BglRBS_26 can be increased to 140%; 10% (M/V) sucrose can increase the enzyme activity of BglRBS_9 to 180%. In addition, BglRBS_26 has better NaCl tolerance and stability. It retains 80% enzyme activity after being treated at 37 ℃ and 2.5 mol/L NaCl for 1 h. [Conclusion] In this study, two novel β-glucosidase genes BglRBS_26 and BglRBS_9 were obtained from the R. bieti fecal microbial metagenome, and they were successfully expressed in E. coli BL21(DE3). BglRBS_26 and BglRBS_9 have good pH stability and sucrose tolerance, making them have potential applications in food, fermentation and other industries.