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Engineering of a Thermostable Biocatalyst for the Synthesis of 2-O-Glucosylglycerol.

ABSTRACT: 2-O-Glucosylglycerol is accumulated by various bacteria and plants in response to environmental stress. It is widely applied as a bioactive moisturising ingredient in skin care products, for which it is manufactured via enzymatic glucosylation of glycerol by the sucrose phosphorylase from Leuconostoc mesenteroides. This industrial process is operated at room temperature due to the mediocre stability of the biocatalyst, often leading to microbial contamination. The highly thermostable sucrose phosphorylase from Bifidobacterium adolescentis could be a better alternative in that regard, but this enzyme is not fit for production of 2-O-glucosylglycerol due to its low regioselectivity and poor affinity for glycerol. In this work, the thermostable phosphorylase was engineered to alleviate these problems. Several engineering approaches were explored, ranging from site-directed mutagenesis to conventional, binary, iterative or combinatorial randomisation of the active site, resulting in the screening of ∼3,900 variants. Variant P134Q displayed a 21-fold increase in catalytic efficiency for glycerol, as well as a threefold improvement in regioselectivity towards the 2-position of the substrate, while retaining its activity for several days at elevated temperatures.

SUBMITTER: Franceus J

PROVIDER: S-EPMC8518079 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Engineering of a Thermostable Biocatalyst for the Synthesis of 2-O-Glucosylglycerol.

Franceus Jorick J Ubiparip Zorica Z Beerens Koen K Desmet Tom T

Chembiochem : a European journal of chemical biology 20210602 18

2-O-Glucosylglycerol is accumulated by various bacteria and plants in response to environmental stress. It is widely applied as a bioactive moisturising ingredient in skin care products, for which it is manufactured via enzymatic glucosylation of glycerol by the sucrose phosphorylase from Leuconostoc mesenteroides. This industrial process is operated at room temperature due to the mediocre stability of the biocatalyst, often leading to microbial contamination. The highly thermostable sucrose pho ...[more]

PMID: 33991026

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Project description:BackgroundReplacing fossil fuels as energy carrier requires alternatives that combine sustainable production, high volumetric energy density, easy and fast refueling for mobile applications, and preferably low risk of hazard. Molecular hydrogen (H2) has been considered as promising alternative; however, practical application is struggling because of the low volumetric energy density and the explosion hazard when stored in large amounts. One way to overcome these limitations is the transient conversion of H2 into other chemicals with increased volumetric energy density and lower risk hazard, for example so-called liquid organic hydrogen carriers such as formic acid/formate that is obtained by hydrogenation of CO2. Many homogenous and heterogenous chemical catalysts have been described in the past years, however, often requiring high pressures and temperatures. Recently, the first biocatalyst for this reaction has been described opening the route to a biotechnological alternative for this conversion.ResultsThe hydrogen-dependent CO2 reductase (HDCR) is a highly active biocatalyst for storing H2 in the form of formic acid/formate by reversibly catalyzing the hydrogenation of CO2. We report the identification, isolation, and characterization of the first thermostable HDCR operating at temperatures up to 70 °C. The enzyme was isolated from the thermophilic acetogenic bacterium Thermoanaerobacter kivui and displays exceptionally high activities in both reaction directions, substantially exceeding known chemical catalysts. CO2 hydrogenation is catalyzed at mild conditions with a turnover frequency of 9,556,000 h-1 (specific activity of 900 µmol formate min-1 mg-1) and the reverse reaction, H2 + CO2 release from formate, is catalyzed with a turnover frequency of 9,892,000 h-1 (930 µmol H2 min-1 mg-1). The HDCR of T. kivui consists of a [FeFe] hydrogenase subunit putatively coupled to a tungsten-dependent CO2 reductase/formate dehydrogenase subunit by an array of iron-sulfur clusters.ConclusionsThe discovery of the first thermostable HDCR provides a promising biological alternative for a chemically challenging reaction and might serve as model for the better understanding of catalysts able to efficiently reduce CO2. The catalytic activity for reversible CO2 hydrogenation of this enzyme is the highest activity known for bio- and chemical catalysts and requiring only ambient temperatures and pressures. The thermostability provides more flexibility regarding the process parameters for a biotechnological application.

Dataset Information

Engineering of a Thermostable Biocatalyst for the Synthesis of 2-O-Glucosylglycerol.

Publications

Engineering of a Thermostable Biocatalyst for the Synthesis of 2-O-Glucosylglycerol.

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