Dataset Information

Glucoamylase of Caulobacter crescentus CB15: cloning and expression in Escherichia coli and functional identification.

ABSTRACT: The biochemical properties of the maltodextrin-hydrolyzing enzymes of cold-tolerant proteobacterium Caulobacter crescentus CB15 remain to be elucidated, although whose maltodextrin transport systems were well investigated. We cloned the putative glucoamylase of C. crescentus CB15 (CauloGA) gene. The CauloGA gene product that was expressed in E. coli was prone to forming inclusion bodies; however, most of the gene product was expressed in a soluble and active form when it was expressed as a fusion protein with Staphylococcus Protein A. The fusion protein was purified using an IgG Sepharose column and was identified as the active GA. The optimum temperature and pH for the activity of this GA toward maltotriose as a substrate were approximately 40°C and 5.0, respectively, and a differential scanning fluorimetry (DSF) analysis revealed that the melting temperature (Tm) of CauloGA was 42.9°C. The kinetic analyses with maltotriose and other maltodextrins as the substrates indicated that CauloGA has higher kcat and smaller Km values at 30°C with both substrates compared with other GAs at lower substrate concentration. However, the enzyme activities toward the substrates decreased as the substrate concentrations increased at concentrations higher than approximately 10-fold the Km. The function-based identification of thermolabile Caulobacter GA contributes to the understanding of the maltodextrin-degradation system of C. crescentus as well as the bacterial GA's function-structure relationship.

SUBMITTER: Sakaguchi M

PROVIDER: S-EPMC3917699 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Glucoamylase of Caulobacter crescentus CB15: cloning and expression in Escherichia coli and functional identification.

Sakaguchi Masayoshi M Matsushima Yudai Y Nankumo Toshiyuki T Seino Junichi J Miyakawa Satoshi S Honda Shotaro S Sugahara Yasusato Y Oyama Fumitaka F Kawakita Masao M

AMB Express 20140127 1

The biochemical properties of the maltodextrin-hydrolyzing enzymes of cold-tolerant proteobacterium Caulobacter crescentus CB15 remain to be elucidated, although whose maltodextrin transport systems were well investigated. We cloned the putative glucoamylase of C. crescentus CB15 (CauloGA) gene. The CauloGA gene product that was expressed in E. coli was prone to forming inclusion bodies; however, most of the gene product was expressed in a soluble and active form when it was expressed as a fusio ...[more]

PMID: 24468405

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Project description:Expression of the catalase-peroxidase of Caulobacter crescentus, a gram-negative member of the alpha subdivision of the Proteobacteria, is 50-fold higher in stationary-phase cultures than in exponential cultures. To identify regulators of the starvation response, Tn5 insertion mutants were isolated with reduced expression of a katG::lacZ fusion on glucose starvation. One insertion interrupted an open reading frame encoding a protein with significant amino acid sequence identity to TipA, a helix-turn-helix transcriptional activator in the response of Streptomyces lividans to the peptide antibiotic thiostrepton, and lesser sequence similarity to other helix-turn-helix regulators in the MerR family. The C. crescentus orthologue of tipA was named skgA (stationary-phase regulation of katG). Stationary-phase expression of katG was reduced by 70% in the skgA::Tn5 mutant, and stationary-phase resistance to hydrogen peroxide decreased by a factor of 10. Like the wild type, the skgA mutant exhibited starvation-induced cross-resistance to heat and acid shock, entered into the helical morphology that occurs after 9 to 12 days in stationary phase, and during exponential growth induced katG in response to hydrogen peroxide challenge. Expression of skgA increased 5- to 10-fold in late exponential phase. skgA is the first regulator of a starvation-induced stress response identified in C. crescentus. SkgA is not a global regulator of the stationary-phase stress response; its action encompasses the oxidative stress-hydrogen peroxide response but not acid or heat responses. Moreover, SkgA is not an alternative sigma factor, like RpoS, which controls multiple aspects of starvation-induced cross-resistance to stress in enteric bacteria. These observations raise the possibility that regulation of stationary-phase gene expression in this member of the alpha subdivision of the Proteobacteria is different from that in Escherichia coli and other members of the gamma subdivision.

Dataset Information

Glucoamylase of Caulobacter crescentus CB15: cloning and expression in Escherichia coli and functional identification.

Publications

Glucoamylase of Caulobacter crescentus CB15: cloning and expression in Escherichia coli and functional identification.

Similar Datasets

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