基于白酒发酵过程多组学数据挖掘大曲淀粉糖化酶及其协同应用效果解析

Mining of starch-saccharifying enzymes in Daqu from multi-omics data of the Baijiu fermentation process and analysis of their synergistic effects in application

摘要:

大曲是中国传统白酒生产中的关键糖化发酵剂，研究大曲糖化酶及其协同应用对酿酒工业的现代化升级具有重要意义。然而，单纯针对大曲体系开展酶资源挖掘，面临着目标范围广泛、工作量大、适用性不足等挑战。基于此，本研究通过对白酒发酵过程的宏蛋白质组及宏基因组数据进行解析，筛选出17种优势淀粉糖化酶蛋白并依据其基因序列设计引物，成功从大曲中克隆出细菌、真菌和昆虫来源的8种淀粉糖化酶基因。本研究成功表达了象牙色克罗彭施泰特氏菌(Kroppenstedtia eburnea)来源的α-1,4-葡萄糖苷酶KeGA5及微小根毛霉(Rhizomucor pusillus)来源的α-淀粉酶RpAM11，其中KeGA5为K. eburnea功能酶异源表达的首次报道。KeGA5和RpAM11的最适反应温度分别为55 ℃和75 ℃，最适pH分别为5.0和7.0–8.0，且在50 ℃以下和pH 5.0–8.0范围内表现出较好的稳定性。白酒发酵过程中的温度和乙醇浓度对酶活性影响显著，但对稳定性影响较小；发酵产生的酸性环境对酶活性和稳定性均有较大影响。此外，KeGA5和RpAM11在水解多种含有α-1,4-葡萄糖苷键的底物时表现出协同作用，尤其对可溶性淀粉和糊精显示出最高水解活性。高粱底物孵育实验表明，重组酶组合在释放麦芽糖、葡萄糖和葡萄糖醛酸方面的效果与大曲粗酶体系相似。本研究提出了大曲糖化酶理性挖掘及功能特性研究的新策略，为未来白酒工业中功能酶制剂和菌酶混合制剂的开发提供了科学参考。

Abstract:

Daqu plays a role as an agent of saccharification and fermentation in the traditional production of Baijiu. Investigating the saccharifying enzymes in Daqu and their synergistic applications is vital for the modernization of the brewing industry. However, the enzyme resource mining focusing solely on Daqu faces challenges such as a broad target range, heavy workload, and limited applicability. To address these issues, we mined 17 dominant starch-saccharifying enzymes from the metaproteomic and metagenomic data of the Baijiu fermentation process and designed primers based on their gene sequences. A total of eight starch-saccharifying enzyme genes, originating from bacteria, fungi, and insects, were successfully cloned from Daqu. Additionally, an α-1,4-glucosidase KeGA5 from Kroppenstedtia eburnea and an α-amylase RpAM11 from Rhizomucor pusillus were successfully expressed, with KeGA5 representing the first reported functional enzyme from K.eburnea. KeGA5 and RpAM11 demonstrated the highest activities at 55 ℃ and 75 ℃ and pH 5.0 and 7.0–8.0, respectively. Both enzymes exhibited good stability below 50 ℃ and within the range of pH 5.0–8.0. The temperature and ethanol concentration in the fermentation process of Baijiu significantly affected enzyme activities but had little impact on enzyme stability, while the acidic environment had considerable impacts on the activities and stability of both enzymes. Furthermore, KeGA5 and RpAM11 exhibited synergistic effects in hydrolyzing the substrates containing α-1,4-glucosidic bonds, with the highest hydrolytic activity observed on soluble starch and dextrin. The incubation experiment with sorghum as the substrate demonstrated that the pure enzyme combination had similar effects to the Daqu crude enzyme system in releasing maltose, glucose, and glucuronic acid. This study presents a novel strategy for the rational exploration and functional characterization of saccharifying enzymes in Daqu, providing scientific insights for developing enzyme preparations and microorganism-enzyme mixtures in the future Baijiu industry.