Abstract：
The current study aimed to improve the acid resistance and thermostability of Bacillus velezensis α-amylase through site-directed mutagenesis, with a specific focus on its applicability to the feed industry. Four mutation sites, P546E, H572D, A614E, and K622E, were designed in the C domain of α-amylase, and three mutants, Mut1 (E), Mut2 (ED), and Mut3 (EDEE), were produced. The results showed that the specific activity of Mut3 was 50 U/mg higher than the original α-amylase (Ori) after incubation at 40 °C for 4 h. Compared to Ori, the acid resistance of Mut3 showed a twofold increase in specific activity at pH 2.0. Moreover, the results of preliminary feed hydrolysis were compared between Ori and Mut3 by designing three factors, three levels of orthogonal experiment for enzymatic hydrolysis time, feed quantity, and amount of amylase. It was observed that the enzymatic hydrolysis time and feed quantity showed an extremely significant difference (p < 0.01) in Mut3 compared to Ori. However, the amount of enzyme showed significant (p < 0.05) improvement in the enzymatic hydrolysis in Mut3 as compared to Ori. The study identified Mut3 as a promising candidate for the application of α-amylase in the feed industry.
摘要：
本研究旨在通过定点诱变提高巴西芽孢杆菌α-淀粉酶的耐酸性和热稳定性,特别关注其对饲料工业的适用性。四个突变位点,P546E,H572D,A614E,和K622E,被设计在α-淀粉酶的C域，还有三个变种人,Mut1(E),Mut2(ED),和Mut3（EDEE），是生产出来的。结果表明，在40°C孵育4h后，Mut3的比活性比原始α-淀粉酶（Ori）高50U/mg。与Ori相比，在pH2.0时，Mut3的耐酸性显示比活性增加了两倍。此外，通过设计三个因素，比较了Ori和Mut3的初步饲料水解结果，酶解时间的三个水平正交实验,饲料数量,和淀粉酶的量。观察到,与Ori相比,Mut3中的酶促水解时间和进料量显示出极其显著的差异(p<0.01)。然而,与Ori相比,酶的量在Mut3的酶水解中显示出显著(p<0.05)的改善。该研究确定Mut3是α-淀粉酶在饲料工业中应用的有希望的候选物。
