Department of Animal Biosciences, University of Guelph, Guelph, ON, N1G 2W1, Canada.
Canadian Food Inspection Agency (CFIA) - Ontario Operation, Guelph, ON, N1G 2W1, Canada.
Sci Rep. 2022 Jun 1;12(1):9135. doi: 10.1038/s41598-022-13124-1.
Development of highly efficacious exogenous fibre degradation enzymes can enhance efficiency of dietary fibre utilization and sustainability of global pork production. The objectives of this study were to investigate in vitro stability for two processive endoglucanases, referred to as GH5-tCel5A1 and GH5-p4818Cel5_2A that were overexpressed in CLEARCOLIBL21(DE3). Three-dimensional models predicted presence of Cys residues on the catalytic site surfaces of GH5-tCel5A1 and GH5-p4818Cel5_2A; and time course experimental results shown that both cellulases were susceptible to auto-oxidation by airborne O and were unstable. Furthermore, we examined these endoglucanases' stability under the mimicked in vitro porcine gastric and the small intestinal pH and proteases' conditions. Eadie-Hofstee inhibition kinetic analyses showed that GH5-tCel5A1 and GH5-p4818Cel5_2A respectively lost 18 and 68% of their initial activities after 2-h incubations under the gastric conditions and then lost more than 90% of their initial activities after 2-3 h of incubations under the small intestinal conditions. Therefore, further enzyme protein engineering to improve resistance and alternatively post-fermentation enzyme processing such as coating to bypass the gastric-small intestinal environment will be required to enable these two processive endoglucanases as efficacious exogenous fibre enzymes in pig nutrition application.
高效外源纤维降解酶的开发可以提高膳食纤维的利用效率和全球猪肉生产的可持续性。本研究的目的是研究两种可进行性内切葡聚糖酶 GH5-tCel5A1 和 GH5-p4818Cel5_2A 的体外稳定性，这两种酶在 CLEARCOLIBL21(DE3)中过表达。三维模型预测 GH5-tCel5A1 和 GH5-p4818Cel5_2A 的催化部位表面存在 Cys 残基；并且时程实验结果表明这两种纤维素酶都容易被空气中的 O 自动氧化，并且不稳定。此外，我们还研究了这些内切葡聚糖酶在模拟的体外猪胃和小肠 pH 值和蛋白酶条件下的稳定性。Eadie-Hofstee 抑制动力学分析表明，GH5-tCel5A1 和 GH5-p4818Cel5_2A 分别在胃条件下孵育 2 小时后初始活性损失 18%和 68%，然后在小肠条件下孵育 2-3 小时后初始活性损失超过 90%。因此，需要进一步进行酶蛋白工程以提高其抗性，或者在发酵后进行酶处理（如包被）以绕过胃-小肠环境，从而使这两种可进行性内切葡聚糖酶能够成为猪营养应用中的有效外源纤维酶。
