蛹虫草半乳糖苷酶基因的克隆表达和重组酶的性质研究

Food intolerance, a major health concern is affecting a large human population world-wide. Anti-nutritional factors present in food pose major health concern for which enzymes are the solutions as remedial measures. Both α-D-galactosidase and β-D-galactosidase catalyze a wide range of substrates to release α or β-linked D-galactose respectively. α-D-galactosidase is used in the hydrolysis of raffinose and stachyose present in soy beans and other leguminous food and feed that cause intestinal discomfort. Treatment of milk and milk products with the enzyme β-galactosidase to remove lactose is an efficient method to solve the problem of lactose intolerance. .Cordyceps militaris is a popular Chinese medicinal mushroom. Cordyceps militaris possesses various properties such as antifungal, antibacterial, anticancer, anti-inflammatory and antioxidant activities. There are so far no reports on isolation of α-galactosidase and β-galactosidase from C. militaris. The genome sequence of C. militaris has been completed and analyzed by comparative genomics. The genes for α-galactosidase and β-galactosidase have been identified in the genome of C. militaris. Hence, the project aims at production of α-galactosidase and β-galactosidase by cloning the respective genes from C. militaris based on its genome sequence and expression of the recombinant enzymes in heterologous host such as Escherichia coli in order to achieve high levels of the enzymes. Cloning and efficient expression of the recombinant galactosidases will enable and greatly simplify the downstream purification processes resulting in greater yield of the enzymes. Therefore, simple and fast chromatographic purification methods will be designed resulting in a cost-effective process. Finally, the biochemical characterization of the enzymes will be performed to study their properties which will make them useful in the food and other industries. Furthermore, the study of their applications in the food industry will be undertaken.

食物不耐受问题会对大量人口的健康产生影响，这主要由食物中的抗营养因子如棉子糖、乳糖等引起。在食品加工中进行恰当的酶解处理是解决该问题的有效途径。α-半乳糖苷酶和β-半乳糖苷酶分别催化多种底物释放α-或 β-连接的D-半乳糖，这两种酶分别是解决大豆制品和乳制品食物不耐受的关键酶。真菌蛹虫草具有抑菌、抗衰老、抑制肿瘤、增强免疫力等生物功能。NCBI数据表明，蛹虫草基因组序列中含有编码α-半乳糖苷酶和β-半乳糖苷酶的基因。但迄今为止，未见有关于蛹虫草α-半乳糖苷酶和β-半乳糖苷酶研究的相关报道。本项目基于蛹虫草基因组序列，采用分子生物学方法分别克隆其α-半乳糖苷酶和β-半乳糖苷酶的基因，在异源宿主如大肠杆菌中进行重组酶的高效表达，建立高收率、高效率的重组酶分离纯化方法，开展这两种酶的性质及其在食品工业的应用研究，为解决食物不耐受问题做贡献。

β-半乳糖苷酶和α-半乳糖苷酶是解决乳制品和大豆制品食物不耐受的关键酶。为了低成本生产酶，有必要克隆酶基因，在异源宿主超表达，研究酶的性质，探索酶高效利用方法。.以蛹虫草(Cordyceps militaris)基因组DNA为模板，用PCR方法扩增了β-半乳糖苷酶（No.XM_006668035）基因。以pET28a 为载体，转化E.coli BL21，用异丙基-β-d-硫代半乳糖苷（IPTG）或乳糖诱导表达。优化了E.coli表达酶的条件，成功扩增了3kb的酶基因，并在E. coli中克隆，酶以包涵体形式被表达。.以C.militaris 基因组DNA为模板，用PCR法进行了两个α-半乳糖苷酶基因CMAG1（No. XM_006674208）和CMAG2（No. EGX94484.1）的扩增，但基因均未能被扩增。采用基因合成方法将基因CMAG2合成，成功转化E.coli BL21，用IPTG或乳糖诱导表达。但合成的基因未能在E. coli中表达。.用海藻酸钠和壳聚糖为载体，成功地将源自Aspergillus oryzae、 Kluyveromyces lactis (Maxilact LG5000, DSM) 和Kluyveromyces fragilis (Lactozym, Novozymes)的三个β-半乳糖苷酶及一个源自A. oryzae 的α-半乳糖苷酶固定化。 .研究了游离的和固定化的β-半乳糖苷酶和α-半乳糖苷酶的生化性质。发现酶的固定化轻微改变了A. oryzae 的β-半乳糖苷酶的最适反应pH值，而Maxilact和Lactozym酶的最适pH未改变。游离的和固定化的α-半乳糖苷酶的最适反应pH值相同。与游离酶相比，壳聚糖固定化β-半乳糖苷酶和α-半乳糖苷酶均显示了较高的最适反应温度。.研究了游离和固定化β-半乳糖苷酶水解牛乳中乳糖的性能。在40°C用酶水解牛乳中的乳糖，用HPLC的SugarD柱分析乳糖含量，发现所有固定化酶均可水解牛乳中大于90%的乳糖。研究了游离的和固定化的A. oryzae的α-半乳糖苷酶水解豆乳中水苏糖及棉子糖的性能。发现固定化酶在8 h内能完全水解豆乳中棉子糖系列寡糖（RFOs），而游离酶在相同时间内能水解79%的RFOs。固定化酶重复利用5次后其活力没有显著下降。 .本项目成果为半乳糖苷酶高效利用奠定了基础。.