阴沟肠杆菌GX-3的β-木糖苷酶的酶学性质及木糖耐受性分子改造

Characterization and molecular engineering of a β-xylosidase from Enterobacter cloacae GX-3

摘要:

目的 克隆表达来自阴沟肠杆菌GX-3中注释为糖苷水解酶家族3的β-木糖苷酶，并深入探究其酶学特性。针对该酶木糖耐受性较低的问题，对相关氨基酸残基进行分子改造以提高其木糖耐受性。方法 根据阴沟肠杆菌GX-3基因组中注释为糖苷水解酶家族3的β-木糖苷酶序列设计引物，通过PCR扩增目的基因，以pQE30为载体构建重组质粒，将其转化至大肠杆菌M15中进行诱导表达，采用镍亲和层析法纯化重组酶，研究重组酶的酶学性质，并对木糖耐受性相关的氨基酸残基进行定点突变。结果 从阴沟肠杆菌GX-3中成功克隆出属于糖苷水解酶家族3的β-木糖苷酶基因，并实现了在大肠杆菌中的异源表达。底物特异性分析表明，重组酶EXYL是一种多功能酶，具有β-木糖苷酶、β-葡萄糖苷酶和α-L-阿拉伯呋喃糖苷酶3种酶活性。重组酶的最适底物为对硝基苯基-β-D-吡喃木糖苷(p-nitrophenyl-β-D-xylopyranoside, pNPX)，其最适pH和温度分别为5.5和45 ℃，K_m和V_max值分别为(0.73±0.06) mmol/L和(130.00±6.85) μmol/(mg·min)，木糖的抑制常数K_i值为(51.95±2.36) mmol/L。当重组酶EXYL作用于木寡糖(X3-X5)时，主要产物为木糖和木二糖，产量各约占50%。对EXYL木糖耐受性相关的氨基酸位点进行定点突变，获得正向突变体W138C和W138A，突变酶W138C和W138A的木糖耐受性分别提高了2.38倍和1.83倍。结论 本研究为探究β-木糖苷酶的多功能酶活性提供了参考，也为增强GH3家族β-木糖苷酶的木糖耐受性提供了新的思路。

Abstract:

Objective To clone and express a gene encoding the β-xylosidase from Enterobacter cloacae GX-3, a putative member of the glycoside hydrolase family 3, systematically characterize the recombinant enzyme, and improve the xylose tolerance by molecular engineering of key amino acid residues involved in xylose binding.Methods On the basis of the whole-genome sequencing data of E. cloacae GX-3, primers were designed to amplify the β-xylosidase gene annotated as GH3. The target gene was amplified by PCR and cloned into the pQE30 expression vector, and the resulting recombinant plasmid was transformed into Escherichia coli M15 for induced expression. The recombinant enzyme was purified by nickel-affinity chromatography, and its enzymatic properties were studied. Site-directed mutagenesis was conducted on amino acid residues associated with xylose tolerance.Results The β-xylosidase gene belonging to the GH3 family was successfully cloned from E. cloacae GX-3 and heterologously expressed in E. coli M15. Substrate specificity analysis revealed that the recombinant enzyme EXYL was a multifunctional enzyme exhibiting β-xylosidase, β-glucosidase, and α-L-arabinofuranosidase activities. EXYL showed the optimal performance with the substrate of pNPX and at pH 5.5 and 45 ℃. The K_m and V_max values of this enzyme were (0.73±0.06) mmol/L and (130.00±6.85) μmol/(mg·min), respectively. The inhibition constant (K_i) for xylose was (51.95±2.36) mmol/L. When EXYL acted on xylooligosaccharides (X3-X5), the main products were xylose and xylobiose, each accounting for approximately 50% of the yield. Site-directed mutagenesis of xylose tolerance-related residues yielded positive mutants W138C and W138A, which showed 2.38-fold and 1.83-fold improvements in xylose tolerance, respectively.Conclusion This study provides insights into the multifunctional activities of β-xylosidases and offers new strategies for enhancing the xylose tolerance of β-xylosidases in the GH3 family.