棉铃虫棉酚降解酶的高活性筛选及其酶学特性与降解机理研究

In Xinjiang, a large amount of cottonseed meal was produced in the oil industry and used as animal's protein feed source, but the presence of toxic free gossypol greatly limited its high-efficient utilization in animal feed. Our previous studies have proved that microbial detoxification of cottonseed meal can effectively eliminate its toxic effect, but the production efficiency is low for a long time of fermentation, and the gossypol degrading enzyme plays key role during detoxification. The most important characteristic of enzymatic reaction is fast speed and high efficiency. Therefor, if gossypol degrading enzyme could be highly expressed by heterologous translational system, made into enzyme production and added to cottonseed meal substrate directly, an enzyme-catalyzed detoxification process would occur. This method would greatly increase the rate of gossypol degrading and feed biological safety. However, the research literatures about gossypol degrading enzyme and its function gene were scarce, so the target enzyme protein couldn't be obtained from heterologous translational system according to the function gene of that microbial enzyme. But some indications were given to us from the research literatures on resistance of Helicoverpa Armigera to free gossypol, we found that gossypol degrading correlated closely with cytochrome P450 enzyme system (CYP450s). Therefore, the gene integration technology would be applied to this project. The co-expressing vector of H. armigera CYP (cytochrome P450) and its CPR (Cytochrome P450 Reductase) would be constructed, and transferred into the cell of Pichia Pastoris through electroporation method. Then the gene-engineering bacteria, which could highly degradate gossypol, would be screened and obtained, and the target enzyme protein, i.e., CYP450s, could be acquired from this heterologous translational system. Through assaying the activity of recombinant CYP450s, investigating the molecular structure of gossypol degrading products and their metabolic pathways, the effect of recombinant CYP450s on gossypol degrading metabolism would be revealed. At last, the detoxification effect of recombinant CYP450s on cottonseed meal and its biological safety test and the effect of growth performance on the yellow-feathered broilers would be conducted, and which would provide the theoretical basis for that detoxified cottonseed meal to be safely and highly utilized.

新疆棉粕资源丰富，因其含有毒物游离棉酚，极为限制了其在饲料中的高效利用。前期研究已证明棉粕微生物发酵脱毒可有效消除其毒性，但发酵时间长效率低，其发挥关键作用的是棉酚降解酶。酶促反应的最大特点是速度快效率高，若能将该酶异源高效表达并提取纯化，直接对棉粕进行酶解脱毒，则可快速高效地提高脱毒效果。但因微生物源棉酚降解酶及其基因研究尚无突破性进展，故无法从该酶基因出发，异源表达获得目的酶。本项目从有关棉铃虫对棉酚的抗性研究中得到启示，发现棉酚降解与细胞色素P450酶系有密切联系。因此，本项目拟采用基因整合技术，构建棉铃虫P450还原酶基因CPR和棉酚诱导活性基因CYP共表达载体，电转毕赤酵母后筛选高效降解棉酚工程菌，异源表达P450酶系蛋白；系统研究该重组酶活性、酶学特性及对棉酚降解途径，揭示其对棉酚的降解机理；精确评估重组酶对棉粕酶解脱毒效果、安全性以及对黄羽肉鸡生产性能的影响，为棉粕生物酶解脱毒提供理论依据。

新疆棉粕资源丰富，因其含有毒物游离棉酚，极为限制了其在饲料中的高效利用。前期研究已证明棉粕微生物发酵脱毒可有效消除其毒性，但发酵时间长效率低，其发挥关键作用的是棉酚降解酶。酶促反应的最大特点是速度快效率高，若能将该酶异源高效表达并提取纯化，直接对棉粕进行酶解脱毒，则可快速高效地提高脱毒效果。本项目针对棉籽粕中的有毒物质游离棉酚，团队从有关棉铃虫对棉酚的抗性研究中得到启示，发现棉酚降解与细胞色素P450酶系有密切联系。项目组先后构建棉铃虫CPR-CYP9A12和酯酶表达质粒，通过毕赤酵母成功获得具有功能活性的蛋白酶，初步阐明其酶学性质。建立一种高效液相色谱测定酶液和菌液中游离棉酚含量，通过液相和液相质谱对棉酚的降解及质谱行为分析发现，添加棉酚降解酶CYP9A12作用于棉酚时，棉酚含量会显著性下降，同时棉酚中间代谢物会加速降解。在外源酶作用下，棉酚能够经脱羟和脱羧形成化合物G3，质荷比为m/z 437；添加棉酚降解酶CYP9A12后，棉酚代谢产物G2，m/z 293能够完全脱羧进一步氧化形成化合物G0 m/z，209和G0’ m/z，249，从而加速棉酚的体外降解。研究成果发表在《Pesticide Biochemistry and Physiology》2019年第155卷。在酯酶作用下棉酚代谢产物为M1和M2，化学式为C31H36O5，C34H36N2O8，质荷比m/z 为489.26和601.25，研究成果发表在《Toxins》2022年第14卷。通过单因素与Box-Behnken试验设计优化棉酚降解酶酶解脱毒条件，最终优化结果的棉酚含量达到40.91 mg/kg，较优化之前的棉酚含量352.94 mg/kg的水平降低了88.4 %，优化效果显著。研究成果发表在《AMB express》2019年第9卷。本项目从分子生物学角度研究了棉酚脱毒的代谢机制及棉籽粕在动物中的应用前景，对深入开发饲料蛋白质资源具有重要科学价值和指导意义。