新疆天山冰川低温β-半乳糖苷酶的筛选及其在毕赤酵母细胞表面展示的酶学特性研究

At present, one of the effective ways to avoid lactose intolerance for lactose-intolerant people is by adding β-galactosidase (EC3.2.1.23), which is capable of hydrolyzing β-1, 4-D-galactoside linkages, to produce lactose reduced food. To satisfy the cold-chain requirement of milk industry, cold-adapted β-galactosidase with high activity at low temperature has special advantages and applications in food industry. However, lower expression level of cold-adapted β-galactosidase in the wild strains and lower thermal stability often lead to serious loss of enzyme activity during purification and production processes after recombinant expression. Based on previous results obtained by our research group during isolation and purification of cold-adapted β-galactosidase producing strains from No.1 Glacier in Xinjiang Tianshan Mountain, this project is going to clone the coding genes of cold-adapted β-galactosidase with high activity from No.1 Glacier in Xinjiang Tianshan Mountain by a combination of culture-based screening and metagenomics-based screening. Further, this project is going to construct recombinant Pichia pastoris strain displaying active cold-adapted β-galactosidase on its cell surface, and analyze the impact of yeast cell surface display on the catalytic activity and thermal stability of cold-adapted β-galactosidase, which will provide the basis for avoiding loss of enzyme activity during purification and production of recombinant cold-adapted β-galactosidase in order to achieve high efficient production and usage of cold-adapted β-galactosidase.

目前避免乳糖酶缺乏人群食用乳制品后产生乳糖不耐受症状主要通过外源添加能水解乳糖的β-半乳糖苷酶 (β-galactosidase, EC3.2.1.23)，俗称乳糖酶。为了满足现代乳品冷链加工和运输的需要，低温下高活性乳糖酶对低乳糖牛乳的高效制备具有重要意义，但低温乳糖酶在野生菌株中表达水平低，热稳定性差也导致其在重组表达后的纯化、制备过程中酶活损失严重。本项目在前期对天山1号冰川产低温β-半乳糖苷酶菌株的分离纯化和产酶特性的研究基础上，拟结合纯培养分离筛选和宏基因组文库筛选，从新疆天山1号冰川冻土微生物中筛选克隆高催化活性低温乳糖酶基因，结合对重组酶酶学特性的鉴定和分析，进一步构建毕赤酵母细胞表面展示低温乳糖酶的重组菌株，系统解析酵母细胞表面展示对低温乳糖酶催化活性和热稳定性等酶学性质的影响，为规避纯化、制备过程的酶活损失，实现其高效制备和利用提供依据。

为了更好地满足现代乳品冷链加工和运输的需要，筛选获得酶学特性适合低温下高效水解牛乳乳糖的乳糖酶对低乳糖牛乳的制备具有重要意义。本课题从新疆天山1 号冰川沉积层采样，分别通过纯培养筛选和宏基因组学的手段对冻土低温微生物中高活性低温β-半乳糖苷酶的编码基因进行了挖掘筛选。通过纯培养筛选获得低温β-半乳糖苷酶活性较高的菌株120株，克隆获得潜在低温β-半乳糖苷酶基因6个，完成了4个来源于Cryobacterium sp. LW097的低温β-半乳糖苷酶酶学特性的鉴定。对大肠杆菌表达的Cryobacterium sp. LW097来源的4个β-半乳糖苷酶的酶学特性分析表明，这4个β-半乳糖苷酶的最适反应温度在25~45 °C之间，4 °C时的相对酶活为最大酶活的40~60%，55 °C处理均易失活，均为典型的低温β-半乳糖苷酶筛。结合金属阳离子、水解产物葡萄糖及半乳糖对酶活性的影响、底物特异性分析、酶动力学参数的测定及牛乳乳糖水解活性的确认，最终确认Bgal435的酶学性质是较适合低温下水解牛乳乳糖的β-半乳糖苷酶，4 °C反应24 h牛乳乳糖的水解率约为67.9%，48 h可达约72.5%。本课题还针对新疆天山1号冰川沉积层冻土DNA的提取方法进行了比较和优化，为进一步构建功能宏基因组文库筛选高活性低温β-半乳糖苷酶奠定了基础。