High Throughput Screening Platform for a FAD-Dependent L-Sorbose Dehydrogenase

Shan, Xiaoyu; Liu, Li; Zeng, Weizhu; Chen, Jian; Zhou, Jingwen

doi:10.3389/fbioe.2020.00194

Cited by 11 publications

(9 citation statements)

References 36 publications

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“…The genome of G. oxidans WSH-004 was extracted using the FastPure Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit (Vazyme Biotech Co., Ltd.), and sndhsdh from G. oxidans WSH-004 was cloned into the plasmid pBBR1MCS5 15 to verify the performance of SNDH and its mutants in G. oxidans WSH-004. The SNDH gene was optimized by GenScript Biotech Corporation and expressed in E. coli BL21(DE3) expressing L-sorbose dehydrogenase 18 using the pET28a-(+) plasmid to verify the activity of SNDH and its mutants. Plasmid P3−2-Sm was used for overexpression of peroxidase HPI from E. coli BL21 in glucose-oxidizing bacteria.…”

Section: Strains and Plasmidsmentioning

confidence: 99%

Computer-Aided Semi-Rational Design to Enhance the Activity of l-Sorbosone Dehydrogenase from Gluconobacter oxidans WSH-004

Li,

Wang,

Huo

et al. 2024

J. Agric. Food Chem.

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The titer of the microbial fermentation products can be increased by enzyme engineering. L-Sorbosone dehydrogenase (SNDH) is a key enzyme in the production of 2-keto-L-gulonic acid (2-KLG), which is the precursor of vitamin C. Enhancing the activity of SNDH may have a positive impact on 2-KLG production. In this study, a computer-aided semirational design of SNDH was conducted. Based on the analysis of SNDH's substrate pocket and multiple sequence alignment, three modification strategies were established: (1) expanding the entrance of SNDH's substrate pocket, (2) engineering the residues within the substrate pocket, and (3) enhancing the electron transfer of SNDH. Finally, mutants S453A, L460V, and E471D were obtained, whose specific activity was increased by 20, 100, and 10%, respectively. In addition, the ability of Gluconobacter oxidans WSH-004 to synthesize 2-KLG was improved by eliminating H 2 O 2 . This study provides mutant enzymes and metabolic engineering strategies for the microbial-fermentation-based production of 2-KLG.

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Section: Strains and Plasmidsmentioning

confidence: 99%

Computer-Aided Semi-Rational Design to Enhance the Activity of l-Sorbosone Dehydrogenase from Gluconobacter oxidans WSH-004

Li,

Wang,

Huo

et al. 2024

J. Agric. Food Chem.

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“…However, the possibility of bearing in mind these changes along the reaction course is usually not considered in the selection of a biocatalyst for a specific process. In high-throughput screening, for example, which is normally used in directed evolution [110][111][112], analyzing the whole reaction course will add some difficulties in a screening that by definition must be very rapid [113][114][115][116]. That way, the selection of an optimal enzyme as catalyst for a specific process may not be as simple as it looks, and in some cases, it may be that there is no real "optimal" enzyme.…”

Section: Figurementioning

confidence: 99%

One Pot Use of Combilipases for Full Modification of Oils and Fats: Multifunctional and Heterogeneous Substrates

et al. 2020

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Lipases are among the most utilized enzymes in biocatalysis. In many instances, the main reason for their use is their high specificity or selectivity. However, when full modification of a multifunctional and heterogeneous substrate is pursued, enzyme selectivity and specificity become a problem. This is the case of hydrolysis of oils and fats to produce free fatty acids or their alcoholysis to produce biodiesel, which can be considered cascade reactions. In these cases, to the original heterogeneity of the substrate, the presence of intermediate products, such as diglycerides or monoglycerides, can be an additional drawback. Using these heterogeneous substrates, enzyme specificity can promote that some substrates (initial substrates or intermediate products) may not be recognized as such (in the worst case scenario they may be acting as inhibitors) by the enzyme, causing yields and reaction rates to drop. To solve this situation, a mixture of lipases with different specificity, selectivity and differently affected by the reaction conditions can offer much better results than the use of a single lipase exhibiting a very high initial activity or even the best global reaction course. This mixture of lipases from different sources has been called “combilipases” and is becoming increasingly popular. They include the use of liquid lipase formulations or immobilized lipases. In some instances, the lipases have been coimmobilized. Some discussion is offered regarding the problems that this coimmobilization may give rise to, and some strategies to solve some of these problems are proposed. The use of combilipases in the future may be extended to other processes and enzymes.

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“…[2,14] A previous study has shown that heterologous overexpression of FAD dependent SDH can produce a small amount of 2-KLG, while co-expression with NAD(P) + -dependent SNDH can greatly increase the output of 2-KLG. [2] Therefore, elucidating the structure and the catalytic mechanism is essential for improving SNDH performance and the efficiency of 2-KLG production. The metabolic pathway of one step route of G. oxydans WSH-004 to synthesize 2-KLG.…”

Section: Introductionmentioning

confidence: 99%

Structural Insight into the Catalytic Mechanisms of an L‐Sorbosone Dehydrogenase

Li,

Deng,

Hou

et al. 2023

Advanced Science

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L‐Sorbosone dehydrogenase (SNDH) is a key enzyme involved in the biosynthesis of 2‐keto‐L‐gulonic acid , which is a direct precursor for the industrial scale production of vitamin C. Elucidating the structure and the catalytic mechanism is essential for improving SNDH performance. By solving the crystal structures of SNDH from Gluconobacter oxydans WSH‐004, a reversible disulfide bond between Cys295 and the catalytic Cys296 residues is discovered. It allowed SNDH to switch between oxidation and reduction states, resulting in opening or closing the substrate pocket. Moreover, the Cys296 is found to affect the NADP+ binding pose with SNDH. Combining the in vitro biochemical and site‐directed mutagenesis studies, the redox‐based dynamic regulation and the catalytic mechanisms of SNDH are proposed. Moreover, the mutants with enhanced activity are obtained by extending substrate channels. This study not only elucidates the physiological control mechanism of the dehydrogenase, but also provides a theoretical basis for engineering similar enzymes.

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High Throughput Screening Platform for a FAD-Dependent L-Sorbose Dehydrogenase

Cited by 11 publications

References 36 publications

Computer-Aided Semi-Rational Design to Enhance the Activity of l-Sorbosone Dehydrogenase from Gluconobacter oxidans WSH-004

Computer-Aided Semi-Rational Design to Enhance the Activity of l-Sorbosone Dehydrogenase from Gluconobacter oxidans WSH-004

One Pot Use of Combilipases for Full Modification of Oils and Fats: Multifunctional and Heterogeneous Substrates

Structural Insight into the Catalytic Mechanisms of an L‐Sorbosone Dehydrogenase

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