Xiaofeng Zhang,Xinqi Xu,Xuemei Yao,Rong Wang,Hengtao Tang,Xin Ju,Liangzhi Li
Ribose-5-phosphate isomerase B is of great importance for biocatalysis and biosynthesis, but the multifunctional residues in active sites hinder the research efforts. This study employed rational design strategies to locate the key residues of RpiB from Ochrobactrum sp. CSL1 (OsRpiB). A single-mutant S9T of a noncontact residue showed 80% activity improvement toward d-allose. A double-mutant S98H/S134H further increased the activity to 3.6-fold. The mutations were analyzed by kinetics and molecular dynamics analyses, indicating that S9T might enhance the substrate binding and catalysis by inducing a steric effect, and S98H/S134H could strengthen both ring opening and binding of d-allose. Though S98H/S134H showed low temperature stability, its potential was explored by isomerizing d-allose to d-psicose with higher conversion and in less reaction time. The findings of this study were beneficial for illustrating the complex functions of key residues in RpiBs and applying OsRpiB in preparing rare sugars.
Isomer conversion and separation are crucial in the chemical, pharmaceutical, and food industries. However, this process remains challenging due to the reversed conversion of isomers and their similar boiling points and kinetic diameters. Herein, an enzyme-catalyzed nanochannel membrane with a hierarchical structure is fabricated for selective and directional isomeric conversion and separation. The enzyme is embedded in a confined polymer network that shows high stability and retains 95% of its initial activity after 50 reaction cycles. Studying the transport mechanisms between isomers through the membrane shows that the hierarchical layers of the membrane impose unequal energy barriers to the forward and backward transport of products, promoting unidirectional diffusion of products and interrupting the equilibrium of the isomerization reaction to increase the conversion rate by 43%. Overall, this work opens a new avenue for the design of biocatalytic nanochannel membranes and the directional production and in situ separation of isomers.
Shuangshuang Duan,Yonghua Chen,Guodong Wang,Zebin Li,Shitong Dong,Yingshuai Wu,Yuanwei Wang,Chunling Ma,Ruiming Wang
D -Allose is a rare cis-caprose with a wide range of physiological functions, which has a wide range of applications in medicine, food, and other industries. L -Rhamnose isomerase ( L -Rhi) is the earliest enzyme found to catalyze the production of D -allose from D -psicose. This catalyst has a high conversion rate, but its specificity for substrates is limited; thus, it cannot fulfill the requirements of industrial production of D -allose. In this study, L -Rhi derived from Bacillus subtilis was employed as the research subject, and D -psicose as the conversion substrate. Two mutant libraries were constructed through alanine scanning, saturation mutation, and rational design based on the analysis of the secondary structure , tertiary structure , and interactions with ligands of the enzyme. The yield of D -allose produced by these mutants was assessed; it was found that the conversion rate of mutant D325M to D -allose was increased by 55.73 %, and the D325S improved by 15.34 %, while mutant W184H increased by 10.37 % at 55 °C, respectively. According to modeling analysis, manganese (Mn 2+ ) had no significant effect on the production of D -psicose from D -psicose by L -Rhi. The results of molecular dynamics simulation demonstrated that the mutants W184H, D325M, and D325S had more stable protein structures while binding with the substrate D -psicose, as evidenced by its root mean square deviation (RMSD), root mean square fluctuation (RMSF), and binding free energy values. It was more conducive to binding D -psicose and facilitating its conversion to D -allose, providing the basis for the production of D -allose.
危险品化学品经营许可证（带存储）