Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Improvement in the catalytic performance of a phenylpyruvate reductase from Lactobacillus plantarum by site-directed and saturation mutagenesis based on the computer-aided design.

ABSTRACT: To enhance the specific activity and catalytic efficiency (k _cat/K _m) of an NADH-dependent LpPPR, its directed modification was performed based on the computer-aided design using molecular docking simulation and multiple sequence alignment. Firstly, five single-site variants of an LpPPR-encoding gene (lpppr) were amplified and expressed in E. coli BL21 (DE3). The asymmetric reduction of 20 mM phenylpyruvic acid (PPA) was carried out using 50 mg/mL E. coli/lpppr ^R53Q or /lpppr ^A79V whole wet cells at 37 °C for 20 min, giving d-phenyllactic acid (PLA) with 41.1 or 44.3% yield, being 1.17- or 1.26-fold that by E. coli/lpppr. Secondly, double-site variants were obtained by saturation mutagenesis of Ala79 in LpPPR^R53Q. Among all tested E. coli transformants, E. coli/lpppr ^R53Q/A79V exhibited the highest d-PLA yield of 85.3%. The specific activity and k _cat/K _m of the purified LpPPR^R53Q/A79V increased to 67.5 U/mg and 169.8 mM^-1 s^-1, which were 3.0- and 13.2-fold those of LpPPR, respectively. Finally, the catalytic mechanism analysis of LpPPR^R53Q/A79V by molecular docking simulation indicated that the replacement of Arg53 in LpPPR with Gln expanded its substrate-binding pocket, while that Ala79 with Val formed an additional π-sigma interaction with phenyl group of PPA.

Supplementary material

The online version of this article (10.1007/s13205-020-02633-3) contains supplementary material, which is available to authorized users.

SUBMITTER: Zhang D

PROVIDER: S-EPMC7806690 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Computer-Aided Saturation Mutagenesis of Arabidopsis thaliana Ent-Copalyl Diphosphate Synthase.

Project description:Ent-copalyl diphosphate synthase controls the biosynthesis of gibberellin plant hormones, which in turn coordinate the expression of numerous enzymes. Some gibberellin-dependent genes encode enzymes coordinating the biosynthesis of tanshinones: diterpene derivatives with broad medical applications. New biotechnological approaches, such as metabolic engineering using naturally occurring or mutated enzymes, have been proposed to meet the growing demand for tanshinones which is currently met by the Chinese medicinal plant Salvia miltiorrhiza Bunge. These mutants may be prepared by directed evolution, saturation mutagenesis or rational enzyme design. In the presented paper, 15,257 non-synonymous variants of Arabidopsis thaliana ent-copalyl diphosphate synthase were obtained using the SNAP2 tool. The obtained forms were screened to isolate variants with potentially improved biological functions. A group of 455 mutants with potentially improved stability was isolated and subjected to further screening on the basis of ligand-substrate affinity, and both secondary structure and active site structure stability. Finally, a group of six single mutants was obtained, which were used to construct double mutants with potentially improved stability and ligand affinity. The potential influence of single mutations on protein stability and ligand affinity was evaluated by double mutant cycle analysis. Finally, the procedure was validated by in silico assessment of the experimentally verified enzyme mutants with reduced enzymatic activity.

| S-EPMC7007437 | biostudies-literature

Improvement of Lactobacillus plantarum aerobic growth as directed by comprehensive transcriptome analysis.

Project description:An aerobic Lactobacillus plantarum culture displayed growth stagnation during early growth. Transcriptome analysis revealed that resumption of growth after stagnation correlated with activation of CO(2)-producing pathways, suggesting that a limiting CO(2) concentration induced the stagnation. Analogously, increasing the CO(2) gas partial pressure during aerobic fermentation prevented the temporal growth stagnation.

| S-EPMC2519335 | biostudies-literature

Improvement of Lactobacillus plantarum Aerobic Growth by Comprehensive Transcriptome Analysis

Project description:An aerobic Lactobacillus plantarum culture displayed growth stagnation during early growth. Transcriptome analysis revealed that regain of growth after stagnation correlated with activation of CO2-producing pathways suggesting that limiting CO2-concentration induced stagnation. Analogously providing increased CO2 gas partial pressure during aerobic fermentation prevented the temporal growth stagnation. Keywords: cell type comparison

2008-01-18 | GSE10194 | GEO

Improvement of Lactobacillus plantarum Aerobic Growth by Comprehensive Transcriptome Analysis

2010-05-26 | E-GEOD-10194 | biostudies-arrayexpress

<i>CADEE</i>: Computer-Aided Directed Evolution of Enzymes.

Project description:The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.

| S-EPMC5331465 | biostudies-literature

Patch cloning method for multiple site-directed and saturation mutagenesis.

Project description:BackgroundVarious DNA manipulation methods have been developed to prepare mutant genes for protein engineering. However, development of more efficient and convenient method is still demanded. Homologous DNA assembly methods, which do not depend on restriction enzymes, have been used as convenient tools for cloning and have been applied to site-directed mutagenesis recently. This study describes an optimized homologous DNA assembly method, termed as multiple patch cloning (MUPAC), for multiple site-directed and saturation mutagenesis.ResultsTo demonstrate MUPAC, we introduced five back mutations to a mutant green fluorescent protein (GFPuv) with five deleterious mutations at specific sites and transformed Escherichia coli (E. coli) with the plasmids obtained. We observed that the over 90% of resulting colonies possessed the plasmids containing the reverted GFPuv gene and exhibited fluorescence. We extended the test to introduce up to nine mutations in Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RT) by assembling 11 DNA fragments using MUPAC. Analysis of the cloned plasmid by electrophoresis and DNA sequencing revealed that approximately 30% of colonies had the objective mutant M-MLV RT gene. Furthermore, we also utilized this method to prepare a library of mutant GFPuv genes containing saturation mutations at five specific sites, and we found that MUPAC successfully introduced NNK codons at all five sites, whereas other site remained intact.ConclusionsMUPAC could efficiently introduce various mutations at multiple specific sites within a gene. Furthermore, it could facilitate the preparation of experimental gene materials important to molecular and synthetic biology research.

| S-EPMC3829206 | biostudies-literature

Application of Site-Identification by Ligand Competitive Saturation in Computer-Aided Drug Design.

Project description:Site Identification by Ligand Competitive Saturation (SILCS) is a molecular simulation approach that uses diverse small solutes in aqueous solution to obtain functional group affinity patterns of a protein or other macromolecule. This involves employing a combined Grand Canonical Monte Carlo (GCMC)-molecular dynamics (MD) method to sample the full 3D space of the protein, including deep binding pockets and interior cavities from which functional group free energy maps (FragMaps) are obtained. The information content in the maps, which include contributions from protein flexibilty and both protein and functional group desolvation contributions, can be used in many aspects of the drug discovery process. These include identification of novel ligand binding pockets, including allosteric sites, pharmacophore modeling, prediction of relative protein-ligand binding affinities for database screening and lead optimization efforts, evaluation of protein-protein interactions as well as in the formulation of biologics-based drugs including monoclonal antibodies. The present article summarizes the various tools developed in the context of the SILCS methodology and their utility in computer-aided drug design (CADD) applications, showing how the SILCS toolset can improve the drug-development process on a number of fronts with respect to both accuracy and throughput representing a new avenue of CADD applications.

| S-EPMC8863107 | biostudies-literature

Improvement of biocatalysts for industrial and environmental purposes by saturation mutagenesis.

Project description:Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of costs, impacts, drawbacks and relevance to biotechnology. A technique that convincingly compromises the extremes of fully randomized vs. rational mutagenesis, with a high benefit/cost ratio, is saturation mutagenesis. Here we will present and discuss this approach in its many facets, also tackling the issue of randomization, statistical evaluation of library completeness and throughput efficiency of screening methods. Successful recent applications covering different classes of enzymes will be presented referring to the literature and to research lines pursued in our group. The focus is put on saturation mutagenesis as a tool for designing novel biocatalysts specifically relevant to production of fine chemicals for improving bulk enzymes for industry and engineering technical enzymes involved in treatment of waste, detoxification and production of clean energy from renewable sources.

| S-EPMC4030971 | biostudies-literature

Bioprinting of Multimaterials with Computer-aided Design/Computer-aided Manufacturing.

Project description:Multimaterials deposition, a distinct advantage in bioprinting, overcomes material's limitation in hydrogel-based bioprinting. Multimaterials are deposited in a build/support configuration to improve the structural integrity of three-dimensional bioprinted construct. A combination of rapid cross-linking hydrogel has been chosen for the build/support setup. The bioprinted construct was further chemically cross-linked to ensure a stable construct after print. This paper also proposes a file segmentation and preparation technique to be used in bioprinting for printing freeform structures.

| S-EPMC7294690 | biostudies-literature

Computer aided screening of inhibitors to 5-α reductase type 2 for prostate cancer.

Project description:Traditionally, drugs are discovered by testing compounds synthesized in time consuming multi-step processes against a battery of invivo biological screens. Promising compounds are then further studied in development, where their pharmacokinetic properties, metabolism and potential toxicity were investigated. Here, we present a study on herbal lead compounds and their potential binding affinity to the effectors molecules of major disease like Prostate Cancer. Clinical studies demonstrate a positive correlation between the extent of 5-α reductase type 2 (isoform 2) and malignant progression of precancerous lesions in prostate. Therefore, identification of effective, well-tolerated 5-α reductase inhibitors represents a rational chemo preventive strategy. This study has investigated the effects of naturally occurring nonprotein compounds berberine and monocaffeyltartaric acid that inhibits 5-α reductase type 2. Our results reveal that these compounds use less energy to bind to 5-α reductase and inhibit its activity. Their high ligand binding affinity to 5-α reductase introduces the prospect for their use in chemopreventive applications. In addition, they are freely available natural compounds that can be safely used to prevent prostate cancer.

| S-EPMC3124690 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data