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Statistical optimization of culture conditions of mesophillic gamma-glutamyl transpeptidase from Bacillus altitudinis IHB B1644.

ABSTRACT: Microbial gamma-glutamyl transpeptidase (GGT) is a key enzyme in production of several γ-glutamyl compounds with food and pharmaceutical applications. Bacterial GGTs are not commercially available in the market owing to their low production from various sources. Thus, the study was focused on achieving the higher GGT production from B. altitudinis IHB B1644 by optimizing the culture conditions using one-variable-at-a-time (OVAT) strategy. A mesophillic temperature of 28 °C, agitation 200 rpm and neutral pH 7 were found to be optimal for higher GGT titre. Among the medium components, the monosaccharide glucose served as the best carbon source over disaccharides, and yeast extract was the preferred organic nitrogen source over inorganic nitrogen sources. The statistical approaches (Plakett-Burman and response surface methodology) were further employed for the optimization of medium components. Medium composition: 0.1% w/v glucose, 0.3% w/v yeast extract, 0.03% w/v magnesium sulphate, 0.20% w/v potassium dihydrogen phosphate and 2.5% w/v sodium chloride with inoculum size (1% v/v) was suitable for higher GGT titres (449 U ml^-1). Time kinetics showed the stability of enzyme up to 96 h of incubation suggesting its application in the industrial use. The proposed strategy resulted in 2.6-fold increase in the GGT production compared to that obtained in the unoptimized medium. The results demonstrated that RSM was fitting to identify the optimum production conditions and this finding should be of great importance for commercial GGT production.

SUBMITTER: Sharma E

PROVIDER: S-EPMC7239960 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Backgroundγ-glutamyltranspeptidase (GGT) is a bi-substrate enzyme conserved in all three domains of life. It catalyzes the cleavage and transfer of γ-glutamyl moiety of glutathione to either water (hydrolysis) or substrates like peptides (transpeptidation). GGTs exhibit great variability in their enzyme kinetics although the mechanism of catalysis is conserved. Recently, GGT has been shown to be a virulence factor in microbes like Helicobacter pylori and Bacillus anthracis. In mammalian cells also, GGT inhibition prior to chemotherapy has been shown to sensitize tumors to the therapy. Therefore, lately both bacterial and eukaryotic GGTs have emerged as potential drug targets, but the efforts directed towards finding suitable inhibitors have not yielded any significant results yet. We propose that delineating the residues responsible for the functional diversity associated with these proteins could help in design of species/clade specific inhibitors.ResultsIn the present study, we have carried out phylogenetic analysis on a set of 47 GGT-like proteins to address the functional diversity. These proteins segregate into various subfamilies, forming separate clades on the tree. Sequence conservation and motif prediction studies show that even though most of the highly conserved residues have been characterized biochemically in previous studies, a significant number of novel putative sites and motifs are discovered that vary in a clade specific manner. Many of the putative sites predicted during the functional divergence type I and type II analysis, lie close to the known catalytic residues and line the walls of the substrate binding cavity, reinforcing their role in modulating the substrate specificity, catalytic rates and stability of this protein.ConclusionThe study offers interesting insights into the evolution of GGT-like proteins in pathogenic vs. non-pathogenic bacteria, archaea and eukaryotes. Our analysis delineates residues that are highly specific to each GGT subfamily. We propose that these sites not only explain the differences in stability and catalytic variability of various GGTs but can also aid in design of specific inhibitors against particular GGTs. Thus, apart from the commonly used in-silico inhibitor screening approaches, evolutionary analysis identifying the functional divergence hotspots in GGT proteins could augment the structure based drug design approaches.

Project description:Background and aimsPredictive, noninvasive tools are needed to monitor key features of nonalcoholic fatty liver disease (NAFLD) in children that relate to improvement in liver histology. The purpose of this study was to evaluate the relationship between liver chemistries and liver histology using data from the CyNCh (Cysteamine Bitartrate Delayed-Release for the Treatment of NAFLD in Children) clinical trial.Approach and resultsThis study included 146 children. Improvement in liver histology, defined as decrease in nonalcoholic fatty liver disease (NAFLD) Activity Score ≥2 points without worsening of fibrosis, occurred in 43 participants (30%). There were 46 participants with borderline zone 1 nonalcoholic steatohepatitis (NASH) at baseline, with resolution in 28% (12 of 46). Multivariate models were constructed using baseline and change in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT) at 52 weeks, for improvement in (1) liver histology primary outcome, (2) borderline zone 1 NASH, and (3) fibrosis. For improvement in histology, the model (P < 0.0001) retained baseline and change in GGT (area under the receiver operating characteristic [AUROC], 0.79; 95% confidence interval [CI], 0.71-0.87). For borderline zone 1 NASH, the model (P = 0.0004) retained baseline and change in ALT (AUROC, 0.80; 95% CI, 0.67-0.93). For fibrosis, the model (P < 0.001) retained baseline and change in ALT (AUROC, 0.80; 95% CI, 0.67-0.93). Additional clinical parameters were added to the models using Akaike's information criterion selection, and significantly boosted performance: improvement in histology with AUROC of 0.89 (95% CI, 0.82-0.95), borderline zone 1 NASH with AUROC of 0.91 (95% CI, 0.83-0.99), and fibrosis with AUROC of 0.89 (95% CI, 0.82-0.94). Models were validated using data from the TONIC (Treatment of Nonalcoholic Fatty Liver Disease in Children) trial.ConclusionsIn children with NAFLD, dynamic changes in serum ALT and GGT are associated with change in liver histology and appear to be powerful indicators of histological response.

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Statistical optimization of culture conditions of mesophillic gamma-glutamyl transpeptidase from Bacillus altitudinis IHB B1644.

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