Dataset Information

Expression and characterization of thermostable glycogen branching enzyme from Geobacillus mahadia Geo-05.

ABSTRACT: The glycogen branching enzyme (EC 2.4.1.18), which catalyses the formation of ?-1,6-glycosidic branch points in glycogen structure, is often used to enhance the nutritional value and quality of food and beverages. In order to be applicable in industries, enzymes that are stable and active at high temperature are much desired. Using genome mining, the nucleotide sequence of the branching enzyme gene (glgB) was extracted from the Geobacillus mahadia Geo-05 genome sequence provided by the Malaysia Genome Institute. The size of the gene is 2013 bp, and the theoretical molecular weight of the protein is 78.43 kDa. The gene sequence was then used to predict the thermostability, function and the three dimensional structure of the enzyme. The gene was cloned and overexpressed in E. coli to verify the predicted result experimentally. The purified enzyme was used to study the effect of temperature and pH on enzyme activity and stability, and the inhibitory effect by metal ion on enzyme activity. This thermostable glycogen branching enzyme was found to be most active at 55 °C, and the half-life at 60 °C and 70 °C was 24 h and 5 h, respectively. From this research, a thermostable glycogen branching enzyme was successfully isolated from Geobacillus mahadia Geo-05 by genome mining together with molecular biology technique.

SUBMITTER: Mohtar NS

PROVIDER: S-EPMC5144683 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Expression and characterization of thermostable glycogen branching enzyme from Geobacillus mahadia Geo-05.

Mohtar Nur Syazwani NS Abdul Rahman Mohd Basyaruddin MB Raja Abd Rahman Raja Noor Zaliha RN Leow Thean Chor TC Salleh Abu Bakar AB Mat Isa Mohd Noor MN

PeerJ 20161206

The glycogen branching enzyme (EC 2.4.1.18), which catalyses the formation of α-1,6-glycosidic branch points in glycogen structure, is often used to enhance the nutritional value and quality of food and beverages. In order to be applicable in industries, enzymes that are stable and active at high temperature are much desired. Using genome mining, the nucleotide sequence of the branching enzyme gene (glgB) was extracted from the Geobacillus mahadia Geo-05 genome sequence prov ...[more]

PMID: 27957389

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Project description:In bacteria, glycogen plays important roles in carbon and energy storage. Its structure has recently been linked with bacterial environmental durability. Among the essential genes for bacterial glycogen metabolism, the glgB-encoded branching enzyme GBE plays an essential role in forming α-1,6-glycosidic branching points, and determines the unique branching patterns in glycogen. Previously, evolutionary analysis of a small sets of GBEs based on their N-terminal domain organization revealed that two types of GBEs might exist: (1) Type 1 GBE with both N1 and N2 (also known as CBM48) domains and (2) Type 2 GBE with only the N2 domain. In this study, we initially analyzed N-terminal domains of 169 manually reviewed bacterial GBEs based on hidden Markov models. A previously unreported group of GBEs (Type 3) with around 100 amino acids ahead of the N1 domains was identified. Phylogenetic analysis found clustered patterns of GBE types in certain bacterial phyla, with the shorter, Type 2 GBEs predominantly found in Gram-positive species, while the longer Type 1 GBEs are found in Gram-negative species. Several in vitro studies have linked N1 domain with transfer of short oligosaccharide chains during glycogen formation, which could lead to small and compact glycogen structures. Compact glycogen degrades more slowly and, as a result, may serve as a durable energy reserve, contributing to the enhanced environmental persistence for bacteria. We were therefore interested in classifying GBEs based on their N-terminal domain via large-scale sequence analysis. In addition, we set to understand the evolutionary patterns of different GBEs through phylogenetic analysis at species and sequence levels. Three-dimensional modeling of GBE N-termini was also performed for structural comparisons. A further study of 9,387 GBE sequences identified 147 GBEs that might belong to a possibly novel group of Type 3 GBE, most of which fall into the phylum of Actinobacteria. We also attempted to correlate glycogen average chain length (ACL) with GBE types. However, no significant conclusions were drawn due to limited data availability. In sum, our study systematically investigated bacterial GBEs in terms of domain organizations from evolutionary point of view, which provides guidance for further experimental study of GBE N-terminal functions in glycogen structure and bacterial physiology.

Dataset Information

Expression and characterization of thermostable glycogen branching enzyme from Geobacillus mahadia Geo-05.

Publications

Expression and characterization of thermostable glycogen branching enzyme from <i>Geobacillus mahadia</i> Geo-05.

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