新型金黄色葡萄球菌 D-阿洛酮糖 3-差向异构酶的设计改造及其用于 D-阿洛酮糖高效生物催化生产。
Redesign of a novel D-allulose 3-epimerase from Staphylococcus aureus for thermostability and efficient biocatalytic production of D-allulose.
机构信息
Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, National Engineering Laboratory for Industrial Enzymes, Tianjin University of Science and Technology, Tianjin, 300457, People's Republic of China.
Laboratory of Basic Science on Healthy Longevity, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 113-8657, Japan.
出版信息
Microb Cell Fact. 2019 Mar 25;18(1):59. doi: 10.1186/s12934-019-1107-z.
BACKGROUND
A novel D-allulose 3-epimerase from Staphylococcus aureus (SaDAE) has been screened as a D-allulose 3-epimerase family enzyme based on its high specificity for D-allulose. It usually converts both D-fructose and D-tagatose to respectively D-allulose and D-sorbose. We targeted potential biocatalysts for the large-scale industrial production of rare sugars.
RESULTS
SaDAE showed a high activity on D-allulose with an affinity of 41.5 mM and catalytic efficiency of 1.1 s mM. Four residues, Glu146, Asp179, Gln205, and Glu240, constitute the catalytic tetrad of SaDAE. Glu146 and Glu240 formed unique interactions with substrates based on the structural model analysis. The redesigned SaDAE_V105A showed an improvement of relative activity toward D-fructose of 68%. The conversion rate of SaDAE_V105A reached 38.9% after 6 h. The triple mutant S191D/M193E/S213C showed higher thermostability than the wild-type enzyme, exhibiting a 50% loss of activity after incubation for 60 min at 74.2 °C compared with 67 °C for the wild type.
CONCLUSIONS
We redesigned SaDAE for thermostability and biocatalytic production of D-allulose. The research will aid the development of industrial biocatalysts for D-allulose.
背景
基于对 D-阿洛酮糖的高特异性，从金黄色葡萄球菌中筛选出一种新型 D-阿洛酮糖 3-差向异构酶（SaDAE），将其归类为 D-阿洛酮糖 3-差向异构酶家族酶。它通常将 D-果糖和 D-塔格糖分别转化为 D-阿洛酮糖和 D-山梨糖。我们以潜在的生物催化剂为目标，用于大规模工业生产稀有糖。
结果
SaDAE 对 D-阿洛酮糖表现出高活性，亲和力为 41.5mM，催化效率为 1.1s mM。四个残基Glu146、Asp179、Gln205 和 Glu240 构成了 SaDAE 的催化四联体。根据结构模型分析，Glu146 和 Glu240 与底物形成了独特的相互作用。重新设计的 SaDAE_V105A 对 D-果糖的相对活性提高了 68%。SaDAE_V105A 的转化率在 6 小时后达到 38.9%。三重突变体 S191D/M193E/S213C 比野生型酶具有更高的热稳定性，在 74.2°C 孵育 60 分钟后，其活性丧失 50%，而野生型酶的活性丧失 67°C。
结论
我们重新设计了 SaDAE 以提高耐热性和生物催化生产 D-阿洛酮糖的能力。该研究将有助于开发用于 D-阿洛酮糖的工业生物催化剂。
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