米黑根毛霉β-半乳糖苷酶基因的克隆、分泌表达及重组酶的性质

β-Galactosidase (EC 3.2.1.23, lactase) is a kind of glycosyl hydrolytic enzymes capable of catalyzing the hydrolysis of β-galactosidic bond in β-galactosides, it can also synthesis galacto-oligosaccharides by transgalactosylation reaction. β-Galactosidases display the potential applications in various fields such as dairy, functional food additive and feed industries. Recently thermostable β-galactosidases have been the research focus, while the research on β-galactosidases from thermophilic fungi in China are still few. The aim of the present proposal is to clone and express extracellularly at least 2 β-galactosidase genes from thermophilic fungi Rhizomucor meihei, which was isolated in our lab, and investigate the characterization and transgalactosylation mechanism of the recombinant enzyme. The implementation of the proposal will not only supply basic theoretical knowledge and evidence for the study, production and application of β-galactosidases, but will also promote the development of enzyme and functional oligosaccharide production industries.

β-半乳糖苷酶（EC 3.2.1.23，乳糖酶）能够催化水解β-半乳糖苷类化合物中的β-半乳糖苷键，也会发生转糖苷反应生成低聚半乳糖。因此，该酶在乳品工业、功能性食品添加剂以及饲料等领域有广泛的应用价值。近年来，耐热β-半乳糖苷酶的研究在国际上成为热点。国内关于嗜热真菌β-半乳糖苷酶的研究相对较少。本项目拟从自行筛选的嗜热真菌米黑根毛霉中克隆2种以上不同糖苷键水解酶家族β-乳糖苷酶基因，在毕赤酵母等真核系统中高效分泌表达，研究所得重组酶的酶学性质及其转糖苷反应的作用机制。本课题的实施将为β-半乳糖苷酶的研究、生产和应用提供依据，推动我国酶制剂及功能性低聚糖行业的发展。

摘要：半乳糖苷酶能够催化水解半乳糖苷类化合物中的半乳糖苷键，也会发生转糖苷反应生成低聚半乳糖。因此，该酶在乳品工业、功能性食品添加剂以及饲料等领域有广泛的应用价值。近年来，耐热半乳糖苷酶的研究在国际上成为热点。国内关于耐热半乳糖苷酶的研究相对较少。本项目从自行筛选的嗜热真菌米黑根毛霉CAU432中克隆和高效表达2种糖苷键水解酶35家族的β-乳糖苷酶基因（RmBgalA和RmBgalB）。氨基酸序列分析表明与其它已知β-乳糖苷酶相比，两个酶的最高序列同源性均低于60%。高效表达米黑根毛霉β-乳糖苷酶基因B（RmBgalB），完成了重组β-乳糖苷酶B的纯化和性质。重组β-乳糖苷酶B的最适pH和温度分别为7.0和45 ºC，具有优良的水解和转糖苷能力。发表SCI论文1篇，申报发明专利2项（授权1项）。研究完成了预期目标，成果为半乳糖苷酶的研究、生产和应用提供了科学依据。