Title |
Glucoamylase of Caulobacter crescentus CB15: cloning and expression in Escherichia coli and functional identification
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Published in |
AMB Express, January 2014
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DOI | 10.1186/2191-0855-4-5 |
Pubmed ID | |
Authors |
Masayoshi Sakaguchi, Yudai Matsushima, Toshiyuki Nankumo, Junichi Seino, Satoshi Miyakawa, Shotaro Honda, Yasusato Sugahara, Fumitaka Oyama, Masao Kawakita |
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. |
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