99A-31 |
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D.-H. SHIN and C.-S. Park. Department of Food Science and Technology, KyungHee University, 1 Secheon-ri, Kiheung, Yongin, 449-701, South Korea Many types of amylases with unique properties have been isolated and characterized for various applications in the starch industry. These proteins share many structural and mechanistic characteristics. However, amylases can be divided into several groups according to substrate specificities, patterns of starch cleavage, transglycosylation or cyclization activities, and structural features. Maltogenic amylases (MAases EC 3.2.1.133) exhibit unique characteristics that are different from other a-amylases. MAases are located in intracellular and they has a dual activity of a-D-(1,4)- or a-D-(1,6)- glycosidic bond cleavages and an activity of transglycosylation via formation of a-D-(1,3)-, a-D-(1,4)-, a-D-(1,6)-glycisidic bond. The enzyme prefer cyclodextrins to starch or pullulan as substrate, which hydrolyze a-1,4-glucosidic bonds of substrates to produce glucose, maltose and panose. The goal of our study was to clone and express the maltogenic amylase from Lactococcus lactis subsp. lactis IL1403 (LCMA). Also, enzymatic properties and the specific activity toward various substrates were investigated. The gene corresponding to a maltogenic amylase in Lactococcus lactis subsp. lactis IL1403 was cloned and overexpressed in Escherichia coli using PCR-based method. The enzyme was overexpressed with p6xHis119, an expression vector having Bacillus licheniformis maltogenic amylase promoter, and efficiently purified by Ni-NTA affinity column. The open reading frame of LCMA consisted of 1752 nucleotides and encoded a polypeptide with 584 amino acids and a calculated molecular mass of 68,034 Da. The purified enzyme exhibited its optimum temperature and pH at 35°ĘC and pH 6, respectively. The relative hydrolytic activity are cyclodextrin (CD) > pullulan > starch (240:42:1). The gel permeation chromatography analysis revealed that the LCMA existed in dimeric form. This result showed that the MAase of Lactococcus lactis subsp. lactis is a typical MAase by showing its substrate specificity toward CD. However, the optimum temperature was quite low compared to other MAases found in various Bacillus strains.
Session 99A, Biotechnology: General
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