99A-24 |
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J. -H. LEE and T. Kwon. Department of Food Science and Biotechnology, Kyonggi University, Yeongtong-Gu, Suwon, 442-760, South Korea Cloning of a gene encoding a-glycoside hydrolyzing enzyme (glycoside hydrolase) from the probiotic microorganism Bifidobacterium longum was conducted for future use in food industry. A 8.6-kb DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG, and 5-bromo-4-chloro-3-indoxyl-a-D-galactoside, was isolated from the genomic library of B. longum digested with EcoRI. In the cloned DNA fragment, 5 open reading frames were identified by sequence analysis. Three of them are the gene cluster of sucrose utilization encoding a sucrose phosphorylase (ScrP), a sucrose transporter (ScrT), and a GalR-LacI-type transcriptional regulator (ScrR). The scrP gene was expressed in E. coli, and its product was partially purified by DEAE-ion exchange chromatography. Synthesis of a 56-kDa protein corresponding to the size estimated from DNA sequence (56,411 Da), was identified using SDS-PAGE. Partially purified recombinant sucrose phosphorylase did not react with melibiose, melezitose, and raffinose, while it reacted with sucrose. Glucose, fructose and 3 by-products were detected as reaction products by TLC and HPLC when sucrose was used as a substrate. The transfer of glucose to 1,2-dihydroxybenzene, 1,4-dihydroxybenzene, 1,2,3-trihydroxybenzene, and 2-hydroxybenzyl alcohol by the recombinant enzyme was also identified. The enzymatic transglucosylation to synthesize a chemically stable form of L-ascorbic acid was further investigated with the recombinant sucrose phosphorylase. The reaction product supposed as 2-O-a-D-glucopyranosyl-L-ascorbic acid (AA-2G) was detected by HPLC.
Session 99A, Biotechnology: General
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