Enzymatic preparation of rare sugars as an alternative to traditional sweeteners is an effective strategy to achieve a low-calorie healthy diet. Ribose-5-phosphate isomerase B (RpiB) is a key enzyme in the non-oxidative branch of the catalytic pentose phosphate pathway. Here, we investigated the potential of Curtobacterium flaccumfaciens ZXL1 ( C. flaccumfaciens ZXL1) derived RpiB (CfRpiB) in D-allose preparation. The optimal reaction conditions for recombinant CfRpiB were found experimentally to be pH 7.0, 55 °C, and no metal ions. The kinetic parameters K m , k cat , and catalytic efficiency k ca t / K m were 320 mM, 4769 s −1 , and 14.9 mM −1 s −1 respectively. The conversion of D-allulose by purified enzyme (1 g L −1 ) to D-allose was 13% within 1 h. In addition, homology modeling and molecular docking were used to predict the active site residues: Asp13, Asp14, Cys72, Gly73, Thr74, Gly77, Asn106, and Lys144.
Fanjin Meng,Qian Zheng,Yuqiu Wu,Lishi Yan,Zhi Chen,Xin Ju,Liangzhi Li
The enzymes used for D-allose production generally failed to meet the requirements of industrial production of D-allose in terms of enzyme activity and catalytic efficiency. In this study, 13 single mutants of CfRpiB (Ribose-5-phosphate isomerase from Curtobacterium flaccumfaciens ) were constructed by combining site-specific mutations and ring-open residue mutations with rational design. It was found that the enzyme activities of all 13 single mutants were positively changed relative to the wild type. Among them, the single mutant with the highest increase in enzyme activity was C72M, which increased its enzyme activity by 30 %. On this basis, three iterative mutant strains were constructed, among which C72M/S105H increased the yield of D-allose by 2.62 times. Especially, enzymatic property measurements showed that its enzyme activity increased by 50 %. Molecular docking of the C72M/S105H mutant with D-allulose revealed that the increased enzymatic activity of the mutant C72M/S105H could be attributed to the smaller substrate binding site and tighter binding to the substrate. This study further reveals the relationship between mutation sites and enzymatic properties, demonstrating the feasibility of improving the efficiency of preparing rare sugars through enzyme molecular modification.
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