Wan Xuerui, SunKang Yongjie, Chen Yijun, Zhang Zhao, Gou Huitian, Xue Yu, Wang Chuan, Wei Yaqin, Yang Yuze
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, China.
The Beijing Municipal Animal Husbandry Station, Beijing, China.
Front Microbiol. 2024 Sep 17;15:1442797. doi: 10.3389/fmicb.2024.1442797. eCollection 2024.
Endoglucanase (EG) and cellobiohydrolase (CBH) which produced by microorganisms, have been widely used in industrial applications.
In order to construct recombinant bacteria that produce high activity EG and CBH, in this study, (endoglucanase) and (cellobiohydrolase) were cloned from the rumen microbial genome of yak and subsequently expressed independently and co-expressed within NZ9000 ( NZ9000).
The recombinant strains NZ9000/pMG36e-usp45- (-), NZ9000/pMG36e-usp45- (-), and NZ9000/pMG36e-usp45--usp45- (--) were successfully constructed and demonstrated the ability to secrete EG, CBH, and EG-CBH. The sodium carboxymethyl cellulose activity of the recombinant enzyme EG was the highest, and the regenerated amorphous cellulose (RAC) was the specific substrate of the recombinant enzyme CBH, and EG-CBH. The optimum reaction temperature of the recombinant enzyme CBH was 60°C, while the recombinant enzymes EG and EG-CBH were tolerant to higher temperatures (80°C). The optimum reaction pH of EG, CBH, and EG-CBH was 6.0. Mn, Fe, Cu, and Co could promote the activity of CBH. Similarly, Fe, Ba, and higher concentrations of Ca, Cu, and Co could promote the activity of EG-CBH. The addition of engineered strains to whole-plant corn silage improved the nutritional quality of the feed, with the lowest pH, acid detergent fiber (ADF), and neutral detergent fiber (NDF) contents observed in silage from the group ( < 0.05), and the lowest ammonia nitrogen (NH-N), and highest lactic acid (LA) and crude protein (CP) contents in silage from the + group ( < 0.05), while the silage quality in the - group was not satisfactory.
Consequently, the recombinant strains , , and were successfully constructed, which could successfully expressed EG, CBH, and EG-CBH. promoted silage fermentation by degrading cellulose to produce sugar, enabling the secretory expression of EG, CBH, and EG-CBH for potential industrial applications in cellulose degradation.
微生物产生的内切葡聚糖酶（EG）和纤维二糖水解酶（CBH）已在工业应用中广泛使用。
为构建产生高活性EG和CBH的重组菌，本研究从牦牛瘤胃微生物基因组中克隆了（内切葡聚糖酶）和（纤维二糖水解酶），随后在NZ9000（NZ9000）中独立表达并共表达。
成功构建了重组菌株NZ9000/pMG36e-usp45-（-）、NZ9000/pMG36e-usp45-（-）和NZ9000/pMG36e-usp45--usp45-（--），并证明其具有分泌EG、CBH和EG-CBH的能力。重组酶EG的羧甲基纤维素钠活性最高，再生无定形纤维素（RAC）是重组酶CBH和EG-CBH的特异性底物。重组酶CBH的最适反应温度为60℃，而重组酶EG和EG-CBH对较高温度（80℃）具有耐受性。EG、CBH和EG-CBH的最适反应pH为6.0。锰、铁、铜和钴可促进CBH的活性。同样，铁、钡以及较高浓度的钙、铜和钴可促进EG-CBH的活性。将工程菌株添加到全株玉米青贮饲料中可提高饲料的营养品质，组青贮饲料的pH值、酸性洗涤纤维（ADF）和中性洗涤纤维（NDF）含量最低（<0.05），组青贮饲料的氨氮（NH-N）含量最低，乳酸（LA）和粗蛋白（CP）含量最高（<0.05），而组的青贮饲料质量不理想。
因此，成功构建了重组菌株、和，它们能够成功表达EG、CBH和EG-CBH。通过降解纤维素产生糖促进青贮发酵，使EG、CBH和EG-CBH能够分泌表达，在纤维素降解方面具有潜在的工业应用价值。
