提高奶酪中白地霉Δ12脂肪酸脱饱和酶催化活性的分子机理研究

Soft cheese with white rind lacks essential fatty acids. Geotrichum candidum is the dominant strain in its fermentation, and there is structural gene of delta 12 fatty acid desaturase (GcFADS12) in its genome, which catalyzes oleic acid and linoleic acid to produce linoleic acid and alpha linolenic acid, respectively. The pretest results suggested that GcFADS12 had been translated by this gene, but its catalytic activity was low. To enhance the production of essential fatty acids of G. candidum, firstly, on the basis of preliminary work, the study will analyze the catalytic activity and substrate specificity of GcFADS12 by heterologous expression in Saccharomyces cerevisiae; moreover, the substrate binding domain or key sites of GcFADS12 will be located by enzyme molecular structure simulation and substrate docking technology, and the key sites will be replaced to screen the positive mutant enzyme; finally, the positive mutation vectors will be constructed by site-directed mutagenesis, and the effect of the corresponding amino acid sites in the positive mutant enzyme on the catalytic activity and substrate specificity for GcFADS12 will be determined and analyzed by gas chromatography, so it will clarify the molecular mechanism of enhancing catalytic activity of GcFADS12 for producing linoleic acid and alpha linolenic acid. This study will provide the theoretical basis and precondition for the development of dairy products rich in polyunsaturated fatty acids, especially essential fatty acids.

白霉奶酪脂质组成中缺乏必需脂肪酸。白地霉作为其发酵优势菌，其基因组中存在催化油酸和亚油酸分别生成亚油酸和α-亚麻酸的Δ12脂肪酸脱饱和酶（GcFADS12）的结构基因。预实验结果提示，该基因具备GcFADS12的功能，但催化活性较弱。为提高其生成必需脂肪酸的能力，本课题在前期工作基础上，首先通过酿酒酵母异源表达GcFADS12，分析其催化活性和底物选择性；进一步通过酶分子结构模拟与底物对接技术定位其与底物结合的结构域或关键位点，并对关键位点进行点饱和替换，从模拟结构中筛选GcFADS12正突变酶；在此基础上利用点突变技术构建GcFADS12正突变酶载体，采用气相色谱法测定并分析GcFADS12正突变酶对应氨基酸位点对其催化活性和底物选择性的影响，从而阐明提高GcFADS12合成亚油酸和α-亚麻酸的催化活性分子机理。本课题将为开发富含多不饱和脂肪酸尤其必需脂肪酸的乳品提供理论依据和前提条件。

奶酪作为一种传统的高脂肪的奶制品，其脂质组成中必需脂肪酸含量低的问题一直没有被解决。由于原料牛乳中固有的多不饱和脂肪酸含量比较少，其中必需脂肪酸仅占总脂肪酸质量的3.5%，且发酵奶酪的优势菌株大多不具有将饱和脂肪酸转化成大量多不饱和脂肪酸的能力或其转化率很低，因此奶酪中缺乏人体所需的必需脂肪酸。在奶酪发酵菌株的必需脂肪酸的合成过程中，Δ12脂肪酸脱饱和酶（FADS12）在调节必需脂肪酸含量起到关键作用。FADS12作为限速酶可以催化油酸和亚油酸合成必需脂肪酸，在不饱和脂肪酸代谢途径中意义重大，其活性高低将会直接影响生物体必需脂肪酸含量和分布。基于此，依托本项目支持，得到了以下成果：.1.鉴定了不同进口白霉奶酪样品中筛分出的生长优势霉菌及不同培养条件下生产亚油酸的情况；.2.解析了霉菌奶酪成熟过程中脂肪酸转化规律，并改进了霉菌奶酪中不饱和脂肪酸含量提高方法；.3.分析了白地霉FADS12（GcFADS12）催化活性和底物选择性，其对油酸和亚油酸的转化率分别达到20.40±0.66%和6.40 ± 0.57%；.4.通过分子改造手段提高了GcFADS12催化活性，解析了影响其催化特性的关键功能域和氨基酸位点，从而解析了其催化的分子机理；.5.阐明了FADS12/15s在EFA生物合成中的作用；.6.构建了G.candidum SW-FADS12工程菌，证明了在奶酪中添加外源豆渣，利用GcFADS12的过表达可提高亚油酸的产量；.7.揭示了FADS的结构域和关键位点与其活性的关系；.8.证明了工程菌G.candidum SW-FADS12的生物安全性。.该项目成果解释了白霉奶酪优势菌白地霉中FADS12活性低的原因，为脂肪酸脱饱和酶家族成员的催化机理研究提供参考，也为发酵食品工业的应用提供新思路，并第一次发现一种产品类似物可以抑制这种酶。为增强GcFADS12转化OA和LA生成LA和ALA的能力，提高白霉奶酪中必需脂肪酸的含量奠定了理论基础和前提条件。也为乳酸菌转化LA提供大量的底物，同时也为提高奶制品中共轭亚油酸含量提供一种新研究思路。