嗜热新芽孢杆菌来源D-阿洛酮糖3-差向异构酶的高效表达和酶学性质

Efficient expression and characterization of D-allulose 3-epimerase from Novibacillus thermophilus

摘要:

【背景】 d-阿洛酮糖具有高甜度、低热量的特性，是一种优良的代糖产品。d-阿洛酮糖3-差向异构酶(d-allulose 3-epimerase, DPEase)可以催化d-果糖异构化生成d-阿洛酮糖，是酶法制备d-阿洛酮糖中的关键酶。【目的】 为提高DPEase的工业应用潜力，对其进行异源表达，并研究其酶学性质。【方法】 利用甘油醛三磷酸脱氢酶启动子将嗜热新芽孢杆菌(Novibacillus thermophilus)来源的DPEase (NtDPEase)在毕赤酵母(Komagataella phaffii)中组成型表达，并系统研究该酶的酶学性质。【结果】 重组菌在5 L发酵罐中经108 h高密度发酵，酶活最高为201.3 U/mL。经过纯化得到电泳纯DPEase，分子量为35 kDa。该酶的最适pH值为7.0，最适温度为60 ℃，在pH 6.0–8.0和45 ℃以下具有良好的稳定性。该酶用于转化不同浓度d-果糖(100–500 g/L)制备d-阿洛酮糖，最高转化率达29.0%。【结论】 本研究实现了DPEase在毕赤酵母中的高效表达，为d-阿洛酮糖的酶法合成提供了理论和实践依据。

Abstract:

[Background] d-allulose is an excellent sugar substitute with high sweetness and low calories. d-allulose 3-epimerase (DPEase) catalyzes the epimerization of d-fructose to produce d-allulose, being an essential enzyme in the enzymatic production of d-allulose. [Objective] To improve the potential of DPEase for industrial application, we realized heterologous expression of this enzyme and characterized the enzymatic properties. [Methods] A DPEase (NtDPEase) from Novibacillus thermophilus was expressed in Komagataella phaffii under the regulation of the glyceraldehyde-3-phosphatedehydrogenase (GAP) constitutive promoter. The enzymatic properties of the recombinant protein were then characterized. [Results] The transformant was incubated in a 5 L fermenter for high cell density fermentation (108 h), with the highest enzyme activity of 201.3 U/mL. The recombinant enzyme was purified to reach the electrophoretic purity, with a molecular weight of 35 kDa. This enzyme showed the best performance at pH 7.0 and 60 ℃ and good stability within the ranges of pH 6.0-8.0 and temperatures below 45 ℃. Furthermore, the enzyme was used to convert d-fructose with different concentrations (100-500 g/L) to produce d-allulose, which scored the highest conversion rate of 29.0%. [Conclusion] This study achieves the efficient expression of NtDPEase in K. phaffii for the first time, providing a theoretical and practical basis for the enzymatic production of d-allulose.