Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin, 300457, PR China.
Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, National Engineering Laboratory for Industrial Enzymes, Tianjin, 300457, PR China.
Enzyme Microb Technol. 2020 Jan;132:109441. doi: 10.1016/j.enzmictec.2019.109441. Epub 2019 Oct 9.
D-allulose has received increasing attention due to its excellent physiological properties and commercial potential. The D-allulose 3-epimerase from Rhodopirellula baltica (RbDAEase) catalyzes the conversion of D-fructose to D-allulose. However, its poor thermostability has hampered its industrial application. Site-directed mutagenesis based on homologous structures in which the residuals on high flexible regions were substituted according to B-factors analysis, is an effective way to improve the thermostability and robustness of an enzyme. RbDAEase showed substrate specificity toward D-allulose with a K of 58.57 mM and k of 1849.43 min. It showed a melting temperature (T) of 45.7 °C and half-life (t) of 52.3 min at pH 8.0, 60 °C with 1 mM Mn. The Site-directed mutation L144 F strengthened the thermostability to a Δt of 50.4 min, ΔTm of 12.6 °C, and ΔT of 22 °C. It also improved the conversion rate to 28.6%. Structural analysis reveals that a new hydrophobic interaction was formed by the mutation. Thus, site-directed mutagenesis based on B-factors analysis would be an efficient strategy to enhance the thermostability of designed ketose 3-epimerases.
D-allo 果糖因其优异的生理特性和商业潜力而受到越来越多的关注。来自波罗的海红冬孢酵母（Rhodopirellula baltica）的 D-allo 果糖 3-差向异构酶（RbDAEase）能够催化 D-果糖向 D-allo 果糖的转化。然而，其较差的热稳定性限制了其工业应用。基于同源结构的定点突变，根据 B 因子分析替换高柔性区域的残基，是提高酶热稳定性和鲁棒性的有效方法。RbDAEase 对 D-allo 果糖具有底物特异性，K 值为 58.57 mM，k 值为 1849.43 min-1。它在 pH 值为 8.0、60°C、1 mM Mn 下的熔点（T）为 45.7°C，半衰期（t）为 52.3 min。定点突变 L144 F 增强了热稳定性，t 值提高了 50.4 min，ΔTm 提高了 12.6°C，ΔT 提高了 22°C。转化率也提高到了 28.6%。结构分析表明，突变形成了新的疏水相互作用。因此，基于 B 因子分析的定点突变将是增强设计的酮糖 3-差向异构酶热稳定性的有效策略。
