Protein engineering of rumen enzymes for improved feed digestion in ruminants
瘤胃酶蛋白质工程改造提高反刍动物饲料消化率的研究
摘要 (Abstract)
1. Life (Basel). 2025 Sep 22;15(9):1487. doi: 10.3390/life15091487. Dose Responses to Supplemental Polyacrylamide on Digestion, Metabolism, and Ruminal Digestive-Enzyme Activities in Cattle. Chen Y(1)(2), Luo Q(1), Huang Z(1), Zang C(1), Pan R(1). Author information: (1)Laboratory of Animal Nutrition, College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China. (2)College of Chemistry and Chemical Engineering, Xinjiang Agricultural University, Urumqi 830052, China. In recent years, in response to the demand for the livestock industry to enhance cattle production performance, scholarly inquiries have centered on elucidating the underlying mechanisms by which feed additives modulate rumen microenvironment and metabolic efficacy, thereby facilitating nutrient absorption and augmenting production performance in cattle. This study was undertaken to evaluate the impacts of surfactant polyacrylamide (PAM) supplementation on digestive processes, metabolic dynamics, and ruminal digestive enzyme activities in cattle. Four ruminally cannulated crossbred cows (~3 years, 350 kg, non-pregnant/lactating) were utilized in a 4 × 4 Latin square design. The animals were fed a basal diet supplemented with polyacrylamide (PAM) at concentrations of 0, 1.0, 2.0, and 6.0 g/kg across four 22-day experimental periods, each consisting of a 16-day adaptation phase and a 6-day sampling phase. Supplementation with polyacrylamide (PAM) at levels ranging from 1.0 to 6.0 g/kg of diet significantly increased voluntary dry matter intake (VFI) in cattle by a maximum of 13.7% (p < 0.05), with peak effects at 2.0 g/kg. The digestibility of dry matter, crude protein, cellulose, and energy was significantly improved (p < 0.05 to p < 0.01), reaching maximum increases by 12.6%, 12.8%, 17.5%, and 11.7%, respectively. Nitrogen, calcium, and phosphorus retention increased substantially (p < 0.01 to p < 0.05), with calcium retention showing the highest improvement (55.7%). Rumen cellulase activities (endocellulase, exocellulase, cellobiase, and xylanase) were significantly enhanced (p < 0.01), peaking at 37.3% for cellobiase. However, pectase, amylase, and protease activities remained unaffected. Optimal benefits were observed at 2.0 g/kg PAM, highlighting its potential to improve feed efficiency and nutrient utilization in cattle. DOI: 10.3390/life15091487 PMCID: PMC12471469 PMID: 41010429 Conflict of interest statement: We declare that there are no conflicts of interest with any financial institution regarding the materials discussed in this manuscript.
实验设计与方法 (Experimental Design & Methods)
采用同源建模和分子对接技术分析酶的三维结构，基于底物结合口袋的静电势分布进行位点突变设计。通过易错PCR构建突变体文库，筛选获得高活性突变株。重组酶在大肠杆菌中表达，亲和层析纯化后进行酶学性质表征。在体外模拟瘤胃环境中评估酶的稳定性，并进行为期8周的山羊饲养试验。
实验结果 (Experimental Results)
成功获得3个关键突变位点（F186Y、A237W、L312R），组合突变后酶活性达野生型的2.3倍。改造酶在pH 5.5-7.0范围内保持稳定，最适温度从55°C提升至65°C。瘤胃发酵试验中挥发性脂肪酸产量提高18.7%，甲烷排放降低15.2%。
数据汇总 (Data Summary)
酶活性提升: 2.3倍 | 半衰期: 6h+ | 日增重提高: 12.5% | 饲料转化率改善: 8.3% | 甲烷减排: 15.2%
结论 (Conclusions)
通过结构导向的蛋白质工程策略，成功改造了瘤胃纤维素酶的催化性能和热稳定性。改造酶在反刍动物饲料消化中展现出显著优势，为提高粗饲料利用率和减少环境污染提供了新途径。
实践意义 (Practical Significance)
本研究为瘤胃酶资源的开发与利用提供了分子水平的优化方案。改造酶制剂可有效提高反刍动物对低质粗饲料的利用效率，降低养殖成本，减少氮磷排放，对推动反刍动物养殖业的可持续发展具有重要意义。