蔗糖富集环境中蔗糖磷酸化酶的酶学性质研究与改造

Enzymatic properties and modification of sucrose phosphorylase from the metagenome of sucrose-enriched environment

摘要:

【背景】蔗糖富集土壤是蔗糖磷酸化酶的重要来源之一。此酶能以较廉价的蔗糖为底物进行转葡萄糖基反应，改善受体底物的理化性质，因而具有重要的应用价值。【目的】研究蔗糖富集土壤中蔗糖磷酸化酶的酶学性质和转糖苷活性，为其更优质的改造提供材料和理论基础。【方法】将宏基因组中获得的蔗糖磷酸化酶基因克隆到表达载体上构建重组大肠杆菌，诱导表达并进行镍亲和层析纯化蛋白。以蔗糖为底物测量重组酶的基本酶学性质，研究其对糖类底物的转糖苷活性。通过底物通道分析，利用反向PCR技术对其第155位点进行饱和突变，并测定突变体基本酶学性质和转糖苷活性。【结果】纯化的酶蛋白分子量大小约为56 kD，活性状态时以三聚体形式存在。在以蔗糖为底物时，最适温度和最适pH值分别为55 °C和6.5；Km和Vmax值分别为23.1±2.4 mmol/L和407.9±8.5 μmol/(mg·min)，对第155位点进行了定点饱和突变，获得了部分酶学性质或转糖苷活性改善的突变体。【结论】宏基因组中蔗糖磷酸化酶的研究丰富了酶学数据，并通过分子改造获得了部分性质更优的突变体，为转糖苷活性关键氨基酸的研究和该酶的工业应用奠定了基础。

Abstract:

[Background] The metagenome of sucrose-enriched soil is one of the important sources of sucrose phosphorylase. The enzyme has important applications in industries, as it can catalyze transglycosylation reaction to improve the properties of the receptors using cheap sucrose. [Objective] The properties and transglycosylation activity of sucrose phosphorylase isolated from the metagenome were studied, providing foundation and research materials for better modification. [Methods] A gene encoding sucrose phosphorylase from metagenomic library was cloned into the expression vector pSE380, and the recombinant strain was constructed. Then, the target protein was purified by nickel affinity chromatography. The properties were determined using sucrose as substrate, and transglycosylation activities of the carbohydrate receptors were also investigated. After protein modeling and substrate channel were analyzed, the site-saturation mutagenesis at the amino acid 155 was performed by reverse PCR. The enzymatic properties and transglycosylation activity of the mutants also were studied in detail. [Results] The molecular weight of purified protein is about 56 kD. And the recombinant protein exists in the form of a trimer in the active state. The optimum temperature and pH value were 55 °C and 6.5, respectively; Km and Vmax value were 23.1±2.4 mmol/L and 407.9±8.5 μmol/(mg·min) using sucrose as substrate. Among the site-directed saturation mutants of the amino acid 155, some mutants showed improved enzymatic properties or transglycosylation activities. [Conclusion] The study of sucrose phosphorylase from the metagenomics enriched the enzymatic data. The mutants with better properties were obtained, which provides theoretical foundation for researching on key amino acids related to transglycosylation and the applications of sucrose phosphorylase.