基于油脂脱胶的特异性耐热磷脂酶的发掘及分子改造

Phospholipases are hydrolytic enzymes that act on phospholipids and split them into different products. They not only have hydrolytic activity on phospholipids, but also show acyltransferase and transacylase activities. Phospholipases exhibit a wide range of application potentials in different fields, such as food, medical, feed and chemical industries. Oil degumming is a key process during oil refining in oil industry. Enzymatic oil degumming by phospholipaes have some advantages compared to traditional processes, such as high degumming yield, low energy consumption, low water consumption and low waste water discharge. However, due to the weakness of basic research on phospholipase filed, the excellent specific phospholipases for oil degumming are still lacking in domestic, which limite the development and technical progress in oil industry. Based on the demond in oil industry and the previous research work of phospholipases at our lab, the proposal will continue to isolate phospholipase producting microorganims, clone and high level express phospholipase genes, purify and characterize various phospholipases, resolve the crystal structures of novel phospholipases and elucidate their catalytic mechanisms, modify the enzyme moleculars to improve their properties for industrial uses, and finally apply the phospholipases in oil degumming process. Thus, the project will helpful for the understanding of the properties and catalysis mechanisms of novel phospholipases, as well as application potential in oil and other industries.

磷脂酶是能够特异水解甘油磷脂的一类酶，不仅具有水解磷脂的活性，而且还具有酰基转移酶和转磷脂酰活性。磷脂酶可广泛应用于食品、医药、饲料以及化工等不同领域。油脂工业中油脂脱胶工艺是油脂精炼的关键步骤，磷脂酶酶法脱胶具有精炼油脂得率高、脱胶完全、能耗低、用水量少和废水排放少等优点，是油脂脱胶领域的主要发展方向。目前，国内磷脂酶相关基础研究薄弱，仍然缺少特性优良的油脂脱胶用耐热磷脂酶，限制了我国油脂工业的发展和技术进步。基于我国油脂工业的实际应用需求和实验室前期磷脂酶相关研究基础，本项目拟继续从自然界筛选高产耐热磷脂酶的微生物，克隆和高效表达耐热磷脂酶基因，纯化并研究磷脂酶的酶学性质，解析新型磷脂酶的晶体结构并揭示其催化作用机理，分子改造磷脂酶的催化特性，最后研究耐热磷脂酶在油脂脱胶中的应用。项目的实施不仅有助于理解耐热磷脂酶的特性和催化机制，而且还将为磷脂酶在油脂等工业领域的应用提供理论依据。

磷脂酶是能够特异水解甘油磷脂的一类酶，不仅具有水解磷脂的活性，而且还具有酰基转移酶和转磷脂酰活性。磷脂酶可广泛应用于食品、医药、饲料以及化工等不同领域。油脂工业中油脂脱胶工艺是油脂精炼的关键步骤，磷脂酶酶法脱胶具有精炼油脂得率高、脱胶完全、能耗低、用水量少和废水排放少等优点，是油脂脱胶领域的主要发展方向。目前，国内磷脂酶相关基础研究薄弱，仍然缺少特性优良的油脂脱胶用耐热磷脂酶，限制了我国油脂工业的发展和技术进步。基于我国油脂工业的实际应用需求和实验室前期磷脂酶相关研究基础，本项目从自然界筛选高产耐热磷脂酶的微生物，克隆和高效表达耐热磷脂酶基因，纯化并研究磷脂酶的酶学性质，解析新型磷脂酶的晶体结构并揭示其催化作用机理，分子改造磷脂酶的催化特性，最后研究耐热磷脂酶在油脂脱胶中的应用。1）从土样和实验室现有菌种中筛选到4株产磷脂酶的真菌，经鉴定，为太瑞斯梭孢壳霉（Thielavia terrestris）CAU709、米曲霉（Aspergillus oryzae）、微孢根霉（Rhizopus microsporus）F518、毛壳霉（Chaetomium sp.）CQ31；2）成功从太瑞斯梭孢壳霉中克隆出2个磷脂酶基因，经过异源表达、分子伴侣共表达等获得了高产磷脂酶的基因工程菌株；3）成功完成了1种新型磷脂酶的分子改造，得到酶学性质提高的酯酶，获得了基因工程菌株；4）对新型磷脂酶进行了高效表达、纯化、酶学性质和应用研究，为其后续应用奠定了基础。项目成果不仅有助于理解磷脂酶的特性和催化机制，而且还将为磷脂酶在油脂等工业领域的应用提供理论依据。