Increased yield of 2‐O‐α‐d‐glucopyranosyl‐l‐ascorbic acid synthesis by α‐glucosidase using rational design that regulating the ground state of enzyme and substrate complex

Abstract

Backgroundα‐Glucosidase (AG) is a bifunctional enzyme, it has a capacity to synthesize 2‐O‐α‐d‐glucopyranosyl‐l‐ascorbic acid (AA‐2G) from l‐ascorbic acid (L‐AA) and low‐cost maltose under mild conditions, but it can also hydrolyze AA‐2G, which leads to low synthesis efficiency of AA‐2G.Main Methods and Major ResultsThis study introduces a rational molecular design strategy to regulate enzymatic reactions based on inhibiting the formation of ground state of enzyme‐substrate complex. Y215 was analyzed as the key amino acid site affecting the affinity of AG to AA‐2G and L‐AA. For the purpose of reducing the hydrolysis efficiency of AA‐2G, the mutant Y215W was obtained by analyzing the molecular docking binding energy and hydrogen bond formation between AG and the substrates. Compared with the wild‐type, isothermal titration calorimetry (ITC) results showed that the equilibrium dissociation constant (KD) of the mutant for AA‐2G was doubled; the Michaelis constant (Km) for AA‐2G was reduced by 1.15 times; and the yield of synthetic AA‐2G was increased by 39%.Conclusions and ImplicationsOur work also provides a new reference strategy for the molecular modification of multifunctional enzymes and other enzymes in cascade reactions system.

Improvement of an enzymatic cascade synthesis of nicotinamide mononucleotide via protein engineering and reaction‐process reinforcement

“…The improvement in enzyme stability can be inferred from the changes in hydrogen bonds within the enzyme's simulated structure. [ 26,27 ] When the amino acid at position 15 is mutated from tyrosine to serine, hydrogen bonds were formed between the enzyme and the substrate PRPP (Figure S7C and S7D), which leaded to a better thermostability of the enzyme. The elevation in the T m value also suggested an enhanced stability of the Cp ‐NAMPT‐Y15S compared to the WT enzyme (Figure S3B).…”

“…Results showed that the t 1/2 of Cp-NAMPT-Y15S at 35, 37, and 40 within the enzyme's simulated structure. [26,27] When the amino acid at position 15 is mutated from tyrosine to serine, hydrogen bonds were formed between the enzyme and the substrate PRPP (Figure S7C and S7D), which leaded to a better thermostability of the enzyme.…”