L-天冬酰胺酶能催化L-天冬酰胺水解生成L-天冬氨酸,可有效抑制食品高温加工中丙烯酰胺(2A类致癌物)的形成。尽管I型L-天冬酰胺酶热稳定性较好,但其催化活性仍有待提高。该研究对吉利热球菌(Thermococcus zilligii)I型L-天冬酰胺酶 (TzI) 进行分子改造,以期提高其催化活性。首先,利用分子对接和虚拟饱和突变筛选了10个活性可能提高的TzI突变体,并对上述突变体进行表达、纯化及酶学性质分析。结果显示,突变酶L265W的比酶活力达到2 751.13 U/mg,较原始酶TzI提升29%。尽管最适反应温度仍为90 ℃,但突变酶L265W在80、85及90 ℃的半衰期较TzI分别提升1.5、1.56和1.43倍。其次,在80 ℃热烫条件下用等量的酶处理薯条,并测定其油炸后的丙烯酰胺含量。结果表明,L265W可使油炸薯条中丙烯酰胺下降65%,优于商品酶和野生型TzI的处理效果。因此,该研究构建的突变酶L265W具有良好的应用潜力,有助于推动L-天冬酰胺酶的工业化应用。
L-asparaginase catalyze the hydrolysis of L-asparagine to produce L-aspartic acid, which can effectively inhibit the formation of acrylamide (class 2A carcinogen) in high-temperature food processing.Although type I L-asparaginase has good thermal stability, its catalytic activity still needs to be improved.This study aimed to improve the catalytic activity of Thermococcus zilligii type I L-asparaginase (TzI) through molecular modification.First, 10 TzI mutants with potential increased activity were screened using molecular docking and virtual saturation mutation, and their expression, purification, and enzymatic properties were analyzed.The results showed that the specific enzyme activity of mutant enzyme L265W reached 2 751.13 U/mg, 29% higher than the original enzyme TzI.Although the optimal reaction temperature was still 90 ℃, the half-lives of mutant enzyme L265W at 80, 85, and 90 ℃ were increased by 1.5, 1.56, and 1.43 times compared to TzI, respectively.Second, the potato chips were treated with an equal amount of enzyme under 80 ℃ blanching conditions, and the acrylamide content after frying was measured.The results showed that L265W can reduce acrylamide in fried French fries by 65%, which is better than the treatment with commercial enzymes and original enzyme TzI.Therefore, the mutated enzyme L265W constructed in this study has good potential for application, which will help promote the industrial application of L-asparaginase.
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