重组根瘤农杆菌葡萄糖耐受型β-葡萄糖苷酶性质

Enzymatic Characterization of a Novel Glucose-tolerant β-glucosidase from Agrobacterium tumefaciens

摘要:

目前已发现的葡萄糖耐受型β-葡萄糖苷酶均来源于真菌, 尚无原核细胞来源的相关报道。从根瘤农杆菌LBA4404中克隆β-葡萄糖苷酶基因bg1, 将其构建在表达载体pET-28b上, 转化Escherichia coli RP (DE3), IPTG诱导表达。重组β-葡萄糖苷酶的比活高达36.7 μmol/(min·mg)。对经过Ni柱纯化的重组酶进行酶学分析发现: 该酶是糖基水解酶家族1的成员, 底物亲和力高, 专一性低, 在温度为40°C和pH在5-8之间时具有较高的酶活, 在低于40°C和pH 5-10之间时可稳定保存。以pNP-β-Glc为底物, 该酶的最适pH为6.4, 最适温度为60°C, 在37°C和pH 6.4的反应体系中, 该酶的Km为0.09 mmol/L, 竞争性抑制剂葡萄糖酸-δ-内酯和葡萄糖的Ki分别为0.03 mmol/L和75 mmol/L, 具有很高的葡萄糖耐受性, 当金属离子Ag+和Zn2+存在时, 酶活被明显抑制。该酶对pNP-β-Gal和pNP-α-Glc的Km分别为3.61 mmol/L和14.31 mmol/L。

Abstract:

To date, the glucose-tolerant β-glucosidase has not been found in prokaryocyte. In the present study, the β-glucosidase gene bg1 from the Agrobacterium tumefaciens str. LBA4404 was cloned into the expression vector pET-28b and transformed into Escherichia coli RP (DE3). Bacteria containing positive clone were routinely grown and IPTG was added to induce the expression of recombinant protein. The β-glucosidase activity of crude extracts was found up to 36.7 μmol/(min·mg). Enzymatic study was performed on the recombinant β-glucosidase purified with Ni column and found that this Bg1 bore high substrate affinity and low substrate specificity, which belonged to the carbohydrate hydrolase superfamily 1. This β-glucosidase displayed quite high activity at pH 5-8 and 40°C, and was able to be stored quite stablely at pH 5-10 and under 40°C. Using pNP-β-Glc as the substrate, the optimum pH and temperature of the hydrolysis reaction were revealed to be 6.4 and 60°C, and the Km of Bg1 was 0.09 mmol/L at pH 6.4 and 37°C. It was inhibited by the competitive inhibitor glucono-δ-lactone (Ki 0.03 mmol/L) and resistant to the inhibition of glucose (Ki 75 mmol/L). We also found that Ag+ and Zn2+ strongly inhibited the activity of Bg1. Its Km for pNP-β-Gal and pNP-α-Glc were respectively 3.61 mmol/L and 14.31 mmol/L.