差异柠檬酸杆菌GXW-1β-葡萄糖苷酶的酶学性质及分子改造

Characterization and molecular modification of β-glucosidase from Citrobacter koser GXW-1

摘要:

[目的]筛选鉴定1株产β-葡萄糖苷酶的菌株，克隆、表达该菌株中的β-葡萄糖苷酶基因，研究重组酶的酶学性质并进行分子改造。[方法]在自然界中采集土样，筛选到1株具有β-葡萄糖苷酶活性的菌株，对野生菌进行16S rDNA鉴定，比对分析GenBank数据库中与野生菌同属的β-葡萄糖苷酶基因序列，设计简并引物PCR扩增基因保守区；设计引物扩增目的基因，以pQE30为表达载体构建重组质粒，转化至大肠杆菌中进行诱导表达；采用镍亲和层析对重组酶进行纯化，研究其酶学性质；采用易错PCR和定点随机突变相结合的方法对野生型β-葡萄糖苷酶进行分子改造。[结果]一个来自于差异柠檬酸杆菌GXW-1的β-葡萄糖苷酶基因被克隆并在大肠杆菌中表达。酶学性质研究结果表明该β-葡萄糖苷酶CBGL的最适温度为45℃，最适pH为6.0，V_max值是（0.1704±0.0073）μmol/（mg·min），K_cat值为（0.2380±0.0102）/s。CBGL能水解α-pNPG、甜菊苷、黄豆苷和染料木苷。对野生酶进行分子改造，获得V_max是野生酶2.54倍的突变体W147F。[结论]CBGL不仅具有β-1，4-糖苷键水解能力，还可能具有一定的α-糖苷键水解酶活性。此外，CBGL还能够水解天然底物甜菊苷、黄豆苷和染料木苷。这些特性表明该β-葡萄糖苷酶在理论研究及在工业中有一定的应用价值。

Abstract:

[Objective] The aim of this study was to characterize β-glucosidase from Citrobacter koser GXW-1 isolated from soil and to improve the enzyme by molecular modification. [Methods] A bacterial strain with β-glucosidase activity was screened from the soil around Wuming sugar mill in Guangxi by esculin-ferric ammonium citrate selecting plate. The 16S rDNA of the strain was obtained and analyzed. By searching GenBank database, the genes encoding β-glucosidase from the same genus Citrobacter were found. These sequences were aligned. Then, a gene encoding β-glucosidase was amplified by PCR. The recombinant plasmid pQE-cbgl was constructed. The recombinant protein was purified with Ni-NTA. The enzyme properties of the recombinant protein CBGL were studied in detail. At last, the wild enzyme CBGL was reformed by error-prone PCR and site-directed random mutagenesis. [Results] C. koser GXW-1 with β-glucosidase activity was isolated from the soil. A gene encoding β-glucosidase was cloned from the wild strain GXW-1. The properties of CBGL were identified. Its optimal pH and temperature were 6.0 and 45℃. Its K_m and V_max value were (11.280±1.073) mmol/L and (0.1704±0.0073) μmol/(mg·min), respectively. Its K_i values was (66.84±3.40) mmol/L. CBGL can hydrolyze α-pNPG, stevioside, daidzin and genistin. CBGL was modified by error-prone PCR and site directed random mutagenesis. A positive mutant W147F was obtained successfully. Its V_max was 2.54 times that of the wild enzyme CBGL. [Conclusion] CBGL not only can hydrolyze β-glycosidic bond, but also can hydrolyze the α-glycosidic bond in α-pNPG. Furthermore, CBGL can hydrolyze stevioside, daidzin and genistin. These characteristics indicate that the β-glucosidase CBGL has important applications in theoretical research and in industry.