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。
[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°C and good stability within the ranges of pH 6.0–8.0 and temperatures below 45°C.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.
作者
张苍萍
李延啸
杨绍青
闫巧娟
江正强
ZHANG Cangping;LI Yanxiao;YANG Shaoqing;YAN Qiaojuan;JIANG Zhengqiang(College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China;College of Engineering,China Agricultural University,Beijing 100083,China;Food Laboratory of Zhongyuan,Luohe 462000,Henan,China;College of Food Science and Engineering,Nanjing University of Finance and Economics,Collaborative Innovation Center for Modern Grain Circulation and Safety,Nanjing 210023,Jiangsu,China)
出处
《微生物学通报》
CAS
CSCD
北大核心
2024年第9期3551-3561,共11页
Microbiology China
基金
国家重点研发计划(2022YFC2104900)。
