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首页> 外文期刊>BMC Biotechnology >Cloning and characterization of a novel cold-active glycoside hydrolase family 1 enzyme with β-glucosidase, β-fucosidase and β-galactosidase activities
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Cloning and characterization of a novel cold-active glycoside hydrolase family 1 enzyme with β-glucosidase, β-fucosidase and β-galactosidase activities

机译：具有β-葡萄糖苷酶，β-岩藻糖苷酶和β-半乳糖苷酶活性的新型冷活性糖苷水解酶家族1酶的克隆和鉴定

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Background Cold-active enzymes, sourced from cold-adapted organisms, are characterized by high catalytic efficiencies at low temperatures compared with their mesophilic counterparts, which have poor activity. This property makes them advantageous for biotechnology applications as it: (i) saves energy costs, (ii) shortens the times for processes operated at low temperatures, (iii) protects thermosensitive substrates or products of the enzymatic reaction, (iv) prevents undesired chemical transformations, and (v) prevents the loss of volatile compounds. Results A bglMKg gene that encodes a monomeric cold-active glycoside hydrolase family 1 enzyme with an apparent molecular mass of 50 kDa was isolated by the functional screening of a marine metagenomic library. The BglMKg enzyme was expressed in E. coli, purified by FPLC and characterized. The recombinant BglMKg could effectively hydrolyze various chromogenic substrates and β-linked oligosaccharides, and had remarkably high β-galactosidase, β-glucosidase and β-fucosidase activities. Because of the lack of information about the usefulness of β-fucosidases in industry, further characterization of the enzymatic properties of BglMKg was only carried out with substrates specific for β-glucosidase or β-galactosidase. The BglMKg had maximal β-galactosidase and β-glucosidase activities at approximately 40°C and 45°C, respectively. The optimum pH for β-galactosidase activity was 6.5, whereas the optimum pH for β-glucosidase activity was 7.5. In general, the enzyme was stable below 30°C and from pHs 6.0 to 8.0. The results of the kinetic studies revealed that BglMKg more efficiently hydrolyzed β-glucosidase substrates than β-galactosidase ones. Conclusions BglMKg is a small, monomeric, cold-active β-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications.
机译：背景技术与低温适应性生物相比，冷适应性生物来源的冷活性酶与低温亲热性酶相比，在低温下具有较高的催化效率。该特性使它们在生物技术应用中具有优势，因为：（i）节省了能源成本;（ii）缩短了低温操作过程的时间;（iii）保护了热敏性底物或酶促反应产物;（iv）防止了不希望的化学物质（v）防止挥发性化合物的损失。结果通过海洋宏基因组文库的功能筛选分离到一个编码bglMKg基因，该基因编码表观分子量为50 kDa的单体冷活性糖苷水解酶家族1酶。 Bg1MKg酶在大肠杆菌中表达，通过FPLC纯化并表征。重组BglMKg可以有效地水解各种发色底物和β-连接的寡糖，并具有很高的β-半乳糖苷酶，β-葡萄糖苷酶和β-岩藻糖苷酶活性。由于缺乏有关β-岩藻糖苷酶在工业上有用性的信息，仅使用对β-葡萄糖苷酶或β-半乳糖苷酶具有特异性的底物对BglMKg的酶学性质进行进一步表征。 BglMKg分别在大约40°C和45°C时具有最大的β-半乳糖苷酶和β-葡萄糖苷酶活性。 β-半乳糖苷酶活性的最佳pH为6.5，而β-葡萄糖苷酶活性的最佳pH为7.5。通常，该酶在30°C以下和pH 6.0至8.0下稳定。动力学研究的结果表明，与β-半乳糖苷酶相比，BglMKg更有效地水解了β-葡萄糖苷酶底物。结论BglMKg是一种小的单体冷活性β-葡萄糖苷酶，具有附加的酶促活性。它在大肠杆菌中有效表达，表明BglMKg可能是工业应用的候选者。

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