硫色曲霉葡萄糖氧化酶催化机理与应用研究

Glucose oxidase (GOD) is a new kind of emerging enzyme additivity for feed industry. GOD promotes animal intestinal health, enhances probiotics in the intestine, inhibits harmful bacteria, and mitigates the liver injury toward toxins. Two major limitations for GOD wide applying in feed industry are: 1. fermentation of Aspergillus sp., the dominate GOD production process, is deficient in productivity; 2. the mechanism of GOD as feed additive is not clear. Thus, based on two GOD gene sequences, named god1 and god2, that predicted from a high GOD producible Aspergillus sulphureus, our lab designs this proposal to study the enzymatic mechanism of GOD, its productivity enhancement, and its effects on intestinal digesta in vitro and in vivo. Experiments of enzymatic mechanism of god1 and god2 are designed to figure out the key amino acid sites of these two GOD enzymes through site-directed-mutagenesis technique and enzymatic characterization study; productivity enhancement and secreted expression in Pichia pastoris should be achieved by codon optimization, gene multiple copies construction, high efficient signal peptide selection, as well as fermentation technology research of engineering strains, etc.; to predict the best dosages of the god1 and god2 in vivo, their effects on piglets digesta should be investigated by analysis of microbial community composition and short-chain fatty acids composition after the anaerobic cultivation with god1 and god2 in vitro; animal trial would further justify the benefits of god1 and god2 in vivo, such as ensuring gut health by balancing microbial community composition and short-chain fatty acids composition, as well as the liver anti-oxidation effect, based on the previous prediction. Therefore, this study shall be able to answer the enzymatic mechanism of GOD, to increase the GOD productivity, to illuminate the effects of the GOD toward intestinal microbe and short-chain fatty acids, and ultimately construct a solid foundation for its practical application in feed industry.

葡萄糖氧化酶（GOD）是一种新型饲用酶制剂，在促进动物肠道健康、肠道有益菌增殖、抑制有害菌，以及缓解毒素肝脏损伤等方面都有重要作用。GOD主要由曲霉等发酵生产，发酵活力低、作用机制不明确等因素限制了其在饲料中的广泛使用。本课题组完成了一株高产GOD的硫色曲霉全基因组测序，发现2条新型GOD编码基因god1和god2。拟在此基础上，通过定点突变，明确关键氨基酸位点，阐明催化机理；通过密码子优化、多拷贝串联等技术组合应用，实现god1和god2在毕赤酵母中的高效分泌表达；体外厌氧培养健康仔猪肠道食糜，测定这两种重组酶对于肠道微生物种类和数量的影响，以及短链脂肪酸含量的差异，预估酶的作用效果和最佳添加剂量；在体外试验的基础上开展动物饲养试验，进一步评价重组酶对于动物肠道健康、肠道微生物种类和数量、短链脂肪酸含量，以及肝脏抗氧化能力的影响，从而确定酶的作用效果，为其实际应用奠定基础。

葡萄糖氧化酶（glucose oxidase, GOD）是一种主要存在于真菌与昆虫内的氧化还原酶，它能特异性地催化β-D-葡萄糖，产生过氧化氢与葡萄糖酸，因此在食品饮料、医药以及饲料等行业有着广泛的应用。在工业上，GOD主要由黑曲霉和青霉属真菌产生。毕赤酵母是一种能够高效表达异源蛋白的真核表达系统，有着可高密度发酵、杂蛋白少等优势，在生产GOD方面有较大的应用前景。本研究通过共表达FAD合成酶构建了高效表达GOD的毕赤酵母工程菌株KGGF，使GOD酶活和比活力分别提高到原菌株的5.52倍和3.29倍。此结果验证了FAD在GOD催化反应机理中起到的重要作用。对菌株进行了高密度发酵，甲醇诱导132 h时，发酵上清中酶活达到最高值，为2183.42 U/mL。重组GOD具有良好的热稳定性，85℃下处理5 min仍具有大于70%的活性。进一步地探究了12种毕赤酵母内源基因的共表达对该重组GOD表达的影响，结果显示：共表达KAR2对GOD表达水平提高效果最好，使得酶活和比活分别提高到KGGF中的2.80倍和1.92倍。体外模拟结果显示：重组GOD与葡萄糖的共同添加对仔猪结、盲肠菌群组成有明显的影响，可以提高菌群多样性，减少有害菌丰度。GOD在仔猪日粮中的应用可改善断奶仔猪生产性能与抗氧化指标，并有助于优化食糜中短链脂肪酸的产生。GOD在肉鸡日粮中的应用可提高免疫水平与抗氧化指标，这可能是肠道微生物群落改变的结果。项目的研究成果不仅为提高毕赤酵母异源表达的GOD表达水平提供了新方法，更可以为其实际应用奠定基础。