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On the donor substrate dependence of group-transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase-catalyzed transglycosylation.

ABSTRACT: Chemical group-transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial-scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from sucrose by sucrose phosphorylase (SucP) to clarify a peculiar, yet generally important characteristic of this reaction: partitioning between glycosylation of glycerol and hydrolysis depends on the type and the concentration of the donor substrate used (here: sucrose, ?-d-glucose 1-phosphate (G1P)). We develop a kinetic framework to analyze the effect and provide evidence that, when G1P is used as donor substrate, hydrolysis occurs not only from the ?-glucosyl-enzyme intermediate (E-Glc), but additionally from a noncovalent complex of E-Glc and substrate which unlike E-Glc is unreactive to glycerol. Depending on the relative rates of hydrolysis of free and substrate-bound E-Glc, inhibition (Leuconostoc mesenteroides SucP) or apparent activation (Bifidobacterium adolescentis SucP) is observed at high donor substrate concentration. At a G1P concentration that excludes the substrate-bound E-Glc, the transfer/hydrolysis ratio changes to a value consistent with reaction exclusively through E-Glc, independent of the donor substrate used. Collectively, these results give explanation for a kinetic behavior of SucP not previously accounted for, provide essential basis for design and optimization of the synthetic reaction, and establish a theoretical framework for the analysis of kinetically analogous group-transfer reactions by hydrolytic enzymes.

SUBMITTER: Klimacek M

PROVIDER: S-EPMC7540478 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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On the donor substrate dependence of group-transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase-catalyzed transglycosylation.

Klimacek Mario M Sigg Alexander A Nidetzky Bernd B

Biotechnology and bioengineering 20200722 10

Chemical group-transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial-scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from sucrose by sucrose phosphorylase (SucP) to clarify a p ...[more]

PMID: 32573774

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Project description:The role of acid-base catalysis in the two-step enzymatic mechanism of alpha-retaining glucosyl transfer by Leuconostoc mesenteroides sucrose phosphorylase has been examined through site-directed replacement of the putative catalytic Glu237 and detailed comparison of purified wild-type and Glu237-->Gln mutant enzymes using steady-state kinetics. Reactions with substrates requiring Brønsted catalytic assistance for glucosylation or deglucosylation were selectively slowed at the respective step, about 10(5)-fold, in E237Q. Azide, acetate and formate but not halides restored catalytic activity up to 300-fold in E237Q under conditions in which the deglucosylation step was rate-determining, and promoted production of the corresponding alpha-glucosides. In situ proton NMR studies of the chemical rescue of E237Q by acetate and formate revealed that enzymatically formed alpha-glucose 1-esters decomposed spontaneously via acyl group migration and hydrolysis. Using pH profiles of kcat/K(m), the pH dependences of kinetically isolated glucosylation and deglucosylation steps were analysed for wild-type and E237Q. Glucosylation of the wild-type proceeded optimally above and below apparent pK(a) values of about 5.6 and 7.2 respectively whereas deglucosylation was dependent on the apparent single ionization of a group of pK(a) approximately 5.8 that must be deprotonated for reaction. Glucosylation of E237Q was slowed below apparent pK(a) approximately 6.0 but had lost the high pH dependence of the wild-type. Deglucosylation of E237Q was pH-independent. The results allow unequivocal assignment of Glu237 as the catalytic acid-base of sucrose phosphorylase. They support a mechanism in which the pK(a) of Glu237 cycles between approximately 7.2 in free enzyme and approximately 5.8 in glucosyl enzyme intermediate, ensuring optimal participation of the glutamate residue side chain at each step in catalysis. Enzyme deglucosylation to an anionic nucleophile took place with Glu237 protonated or unprotonated. The results delineate how conserved active-site groups of retaining glycoside hydrolases can accommodate enzymatic function of a phosphorylase.

Project description:Advanced biotransformation processes typically involve the upstream processing part performed continuously and interlinked tightly with the product isolation. Key in their development is a catalyst that is highly active, operationally robust, conveniently produced, and recyclable. A promising strategy to obtain such catalyst is to encapsulate enzymes as permeabilized whole cells in porous polymer materials. Here, we show immobilization of the sucrose phosphorylase from Bifidobacterium adolescentis (P134Q-variant) by encapsulating the corresponding E. coli cells into polyacrylamide. Applying the solid catalyst, we demonstrate continuous production of the commercial extremolyte 2-α-D-glucosyl-glycerol (2-GG) from sucrose and glycerol. The solid catalyst exhibited similar activity (≥70%) as the cell-free extract (~800 U g-1 cell wet weight) and showed excellent in-operando stability (40 °C) over 6 weeks in a packed-bed reactor. Systematic study of immobilization parameters related to catalyst activity led to the identification of cell loading and catalyst particle size as important factors of process optimization. Using glycerol in excess (1.8 M), we analyzed sucrose conversion dependent on space velocity (0.075-0.750 h-1) and revealed conditions for full conversion of up to 900 mM sucrose. The maximum 2-GG space-time yield reached was 45 g L-1 h-1 for a product concentration of 120 g L-1. Collectively, our study establishes a step-economic route towards a practical whole cell-derived solid catalyst of sucrose phosphorylase, enabling continuous production of glucosides from sucrose. This strengthens the current biomanufacturing of 2-GG, but also has significant replication potential for other sucrose-derived glucosides, promoting their industrial scale production using sucrose phosphorylase. KEY POINTS: • Cells of sucrose phosphorylase fixed in polyacrylamide were highly active and stable. • Solid catalyst was integrated with continuous flow to reach high process efficiency. • Generic process technology to efficiently produce glucosides from sucrose is shown.

Dataset Information

On the donor substrate dependence of group-transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase-catalyzed transglycosylation.

Publications

On the donor substrate dependence of group-transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase-catalyzed transglycosylation.

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