110-4 |
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K.-W. OH and C.-S. Park. Department of Food Science and Technology, KyungHee University, 1 Secheon-ri, Kiheung, Yongin, 449-701, South Korea Maltogenic amylases (EC 3.2.1.133) are a group of enzyme belonging to a subgroup of glycoside hydrolase family 13 along with neopullulanase (EC 3.2.1.135) and cyclomltodextrinase (EC 3.2.1.54). The enzyme hydrolyzes b-cyclodextrin and starch mainly to maltose and pullulan to panose. The coupled transglycosylation and hydrolysis activities of maltogenic amylase were used for the production of branched oligosaccharides from liquefied starch, giving more efficient process than the traditional one. We have tried to find the maltogenic amylase from various lactic acid bacteria. Our objective was to clone and express the gene corresponding to the maltogenic amylase in lactic acid bacteria The presence of a maltogenic amylase in lactic acid bacteria was confirmed by PCR-based method. The cloned gene was analyzed by DNA sequencing and deduced amino acid sequence alignment. The expressed recombinant protein was efficiently purified by Ni-NTA affinity chromatography and analyzed by SDS-PAGE and western blot analysis. The gene encoding the maltogenic amylase from a gram-positive baterium, Lactobacillus delbrueckii subsp. lactis KCTC1058 (LDMA) was successfully cloned in E. coli DH5a. It was found the gene was composed of 1,746 nucleotides and encoded a polypeptide with 581 amino acids with a calculated molecular mass of 66,496 Da. The ldma gene was expressed using p6xHis119 and pTKNd6xHis119 expression vector in E. coli. The optimal reaction temperature and pH of purified LDMA were 45°ĘC and pH 5.5, respectively. Interestingly, the expression level of the recombinant protein was strongly correlated with the growth stage. The enzyme hydrolyzes a-1,4- and a-1,6-glucosidic linkages of various substrates such as b-CD, starch and pullulan.
Session 110, Biotechnology: General
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