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Semi-rational design and molecular dynamics simulations study of the thermostability enhancement of cellobiose 2-epimerases.

ABSTRACT: Directed evolution using random mutation in vast sequence space leads to the low probability of obtaining target proteins. Emerging engineering strategies with computational tools are developed for more trustable outcomes. We used some semi-rational design methods to modify an industrial enzyme, namely cellobiose 2-epimerase (CE). A mutant was selected for its better thermostability and isomerization activity. The tradeoffs between thermostability, epimerization activity and isomerization activity of the CE mutants were different. To investigate the computational prediction performance of protein stability upon point mutations, molecular dynamics (MD) simulation analyses were conducted. The root mean square deviation (RMSD) and hydrogen bond analyses reproduced the correct trends in stability changes of the wild-type and mutated CEs with relatively high accuracy (correlation coefficients r ~ 0.5-0.8). The simulation temperature and time are important factors that influence the prediction performance. Our result shows that thermostability predictors calculated from MD simulation do better in predicting the thermostability changes of the mutated enzymes than the predictors using static-state information of the enzymes.

SUBMITTER: Chen Q

PROVIDER: S-EPMC8985420 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Semi-rational design and molecular dynamics simulations study of the thermostability enhancement of cellobiose 2-epimerases.

Chen Qiuming Q Xiao Yaqin Y Shakhnovich Eugene I EI Zhang Wenli W Mu Wanmeng W

International journal of biological macromolecules 20191113

Directed evolution using random mutation in vast sequence space leads to the low probability of obtaining target proteins. Emerging engineering strategies with computational tools are developed for more trustable outcomes. We used some semi-rational design methods to modify an industrial enzyme, namely cellobiose 2-epimerase (CE). A mutant was selected for its better thermostability and isomerization activity. The tradeoffs between thermostability, epimerization activity and isomerization activi ...[more]

PMID: 31733243

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Project description:BackgroundThe ability of fungal cellobiose dehydrogenase (CDH) to generate H2O2 in-situ is highly interesting for biotechnological applications like cotton bleaching, laundry detergents or antimicrobial functionalization of medical devices. CDH's ability to directly use polysaccharide derived mono- and oligosaccharides as substrates is a considerable advantage compared to other oxidases such as glucose oxidase which are limited to monosaccharides. However CDH's low activity with oxygen as electron acceptor hampers its industrial use for H2O2 production. A CDH variant with increased oxygen reactivity is therefore of high importance for biotechnological application. Uniform expression levels and an easy to use screening assay is a necessity to facilitate screening for CDH variants with increased oxygen turnover.ResultsA uniform production and secretion of active Myriococcum thermophilum CDH was obtained by using Saccharomyces cerevisiae as expression host. It was found that the native secretory leader sequence of the cdh gene gives a 3 times higher expression than the prepro leader of the yeast α-mating factor. The homogeneity of the expression in 96-well deep-well plates was good (variation coefficient <15%). A high-throughput screening assay was developed to explore saturation mutagenesis libraries of cdh for improved H2O2 production. A 4.5-fold increase for variant N700S over the parent enzyme was found. For production, N700S was expressed in P. pastoris and purified to homogeneity. Characterization revealed that not only the kcat for oxygen turnover was increased in N700S (4.5-fold), but also substrate turnover. A 3-fold increase of the kcat for cellobiose with alternative electron acceptors indicates that mutation N700S influences the oxidative- and reductive FAD half-reaction.ConclusionsSite-directed mutagenesis and directed evolution of CDH is simplified by the use of S. cerevisiae instead of the high-yield-host P. pastoris due to easier handling and higher transformation efficiencies with autonomous plasmids. Twelve clones which exhibited an increased H2O2 production in the subsequent screening were all found to carry the same amino acid exchange in the cdh gene (N700S). The sensitive location of the five targeted amino acid positions in the active site of CDH explains the high rate of variants with decreased or entirely abolished activity. The discovery of only one beneficial exchange indicates that a dehydrogenase's oxygen turnover is a complex phenomenon and the increase therefore not an easy target for protein engineering.

Dataset Information

Semi-rational design and molecular dynamics simulations study of the thermostability enhancement of cellobiose 2-epimerases.

Publications

Semi-rational design and molecular dynamics simulations study of the thermostability enhancement of cellobiose 2-epimerases.

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