普鲁兰酶是淀粉糖工业中的重要酶制剂,现工业上使用的普鲁兰酶PulA与葡糖淀粉酶在酸性pH工业应用属性上存在差异,影响淀粉高葡糖浆的酶法制备质量和效率。为此,对普鲁兰酶PulA实施定向进化,获得了普鲁兰酶突变体N9。该突变体的最适作用条件为60 ℃和pH 4.5,在65 ℃下保留95%以上酶活力,在pH 4.3或pH 4.0下分别保留96%和85%酶活力。与现工业葡糖淀粉酶进行复配及应用于高葡糖浆的制备,在30%(质量分数)底物(淀粉液化液)溶液中加入25 U/g普鲁兰酶N9和125~150 U/g葡糖淀粉酶,于61~65 ℃和pH 4.0~4.3糖化24 h,糖液中葡萄糖当量值最高达97.51%,较对照提高了1.73%。该研究获得的普鲁兰酶新分子的应用酶学性能得到有益提升,在淀粉糖工业中具有潜在应用价值。
Pullulanase is a crucial enzyme in the starch sugar industry, significantly influencing the enzymatic production of high-glucose syrups.The varying industrial application properties of pullulanase PulA and glucoamylase at acidic pH levels impact the quality and efficiency of this process.In this study, directed evolution was utilized to derive the pullulanase PulA mutant, N9.Optimal conditions for mutant N9 were determined to be at 60 ℃ and pH 4.5, with the enzyme retaining over 95% activity at 65 ℃, and 96% and 85% activity at pH 4.3 and 4.0, respectively.When combined with currently-used industrial glucoamylase preparation for high-glucose syrup production, a formulation of 25 U/g N9 and 125-150 U/g glucoamylase in a 30% liquefied starch, subjected to saccharification at 61-65 ℃ and pH 4.0-4.3 for 24 h, yielded a maximum glucose equivalent value of 97.51%, an increase of 1.73% compared to the control.Consequently, the enhanced enzymatic properties of mutant N9 demonstrate substantial potential for improved efficiency and productivity in the starch sugar industry.
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