Earticle

초록

영어

Two intestinal bacterial enzymes, Escherichia coli α-xylosidase (YicI) and Lactobacillus johnsonii α-1,3-glucosidase (LJAG31), are classified to glycoside hydrolase family 31 (GH31) based on their amino acid sequence similarity. The molecular mechanism and applicability of the two enzymes were studied by analyzing reaction products and kinetics in combination with site-directed mutagenesis.The specificity of the aglycone-binding site of YicI was characterized by examining transxylosylation-catalyzing property of the enzyme. Acceptor specificity and regioselectivity were investigated. YicI preferred aldopyranosyl sugars with an equatorial 4-OH as the acceptor substrate. This observation suggests that 4-OH in the acceptor sugar ring is essential for the transxylosylation. The disaccharide transfer products formed by YicI inhibited intestinal α-glucosidases. Heterologous production of LJAG31 by E. coli was poor due to inclusion body formation. However, this problem was overcome by several strategies. LJAG31 displayed higher specificity toward nigerose, maltulose, and kojibiose than other natural substrates having anα-glucosidic linkage at the non-reducing end. To our knowledge, LJAG31 is the first bacterial α-1,3-glucosidase to be characterized with a high kcat/Km value for nigerose. By mutating Asp409, the chemical rescue and glycosynthase reactions were monitored. All D409 mutants lost their activity, but D409G among the mutants showed the restoration of activity by externally added sodium azide, resulting in the formation of β-glucosyl azide. This finding demonstrates that D409 is the catalytic nucleophile in LJAG31. In addition, glycosynthase activity of those Asp409 mutants was verified.

키워드

저자

• MINSUN KANG [ CJ Research Institute of Biotechnology. ]

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간행물

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• 간행물명

  한국생물공학회 학술대회

• 간기

  반년간

• 수록기간

  1985~2013

• 십진분류

  KDC 476 DDC 576