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Novel Quantitative Method for the Degree of Branching in Dextran
김영민,Atsuo Kimura,김도만 한국식품과학회 2011 Food Science and Biotechnology Vol.20 No.2
A novel quantitative method for the determination of degree of branching in Leuconostoc mesenteroides B-512F dextran was developed by using the combination of 3 dextran-degrading enzymes. First, Paenibacillus sp. endo-dextranase was randomly degraded B-512F dextran into linear or branched isomalto-oligosaccharides with various degree of polymerization (2-8). Second, Streptococcus mutans dextran glucosidase hydrolyzed linear or branched isomalto-oligosaccharides into glucose and branched isomalto-penta-saccharides. Third, the branched isomaltopenta-saccharide was degraded into glucose by using Bacteroides thetaimicron α-glucosidase. The number of branching points in B-512F dextran (5.42%) was determined by the difference in the amount of glucose in the reaction digest between BTGase-PDex and DGase-PDex treatments.
김영민,김도만,Atsuo Kimura 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.5
Organic solvent-resistant Aspergillus niger α-glucosidase (ANGase) can synthesize α-2-deoxyglucosyl derivatives (2DDs) in water-organic solvent media by a trans-addition reaction from D-glucal to various acceptors. Herein, we studied the influence of four different solvents on ANGase stability and activity. ANGase exhibited 47 or 43% residual activity following incubation in 50% (v/v) or in 70% (v/v) acetone for 4 h, respectively. When various carbohydrates were used as acceptor molecules, ANGase catalyzed the addition reaction of four different sugar alcohols, glucose, sucrose, or trehalose to D-glucal. Among the acceptor molecules tested, xylitol was the best acceptor by producing the highest yield (87% addition). The concentra-tion of acetone/acceptor influenced the formation of 2DDs and the yields. We confirmed the molecular weight of five kinds of products by mass spectrometry and enzymatic hydrolysis. Current method is useful for the production of carbohydrates con-taining 2-deoxyglucose moiety.
Kang, Hee Kyoung,Kimura, Atsuo,Kim, Doman American Chemical Society 2011 Journal of agricultural and food chemistry Vol.59 No.8
<P>The variations in glucosidic linkage specificity observed in products of different glucansucrases appear to be based on relatively small differences in amino acid sequences in their sugar-binding acceptor subsites. Various amino acid mutations near active sites of DSRBCB4 dextransucrase from <named-content content-type='genus-species' xlink:type='simple'>Leuconostoc mesenteroides</named-content> B-1299CB4 were constructed. A triple amino acid mutation (S642N/E643N/V644S) immediately next to the catalytic D641 (putative transition state stabilizing residue) converted DSRBCB4 enzyme from the synthesis of mainly α-(1→6) dextran to the synthesis of α-(1→6) glucan containing branches of α-(1→3) and α-(1→4) glucosidic linkages. The subsequent introduction of mutation V532P/V535I, located next to the catalytic D530 (nucleophile), resulted in the synthesis of an α-glucan containing increased branched α-(1→4) glucosidic linkages (approximately 11%). The results indicate that mutagenesis can guide glucansucrase toward the synthesis of various oligosaccharides or novel polysaccharides with completely altered linkages without compromising high transglycosylation activity and efficiency.</P>
Maltopentaose 생산 균의 분리 및 생산 조건 연구
김영민,서은성,김도만,김도원,이진하,Atsuo Kimura 한국생물공학회 2001 KSBB Journal Vol.26 No.6
We isolated a bacterium that produces an extracellular maltopentaose(G5)-forming amylase from amylose and soluble starch. The bacterium was identified and assigned as a Bacillus sp. AIR-5. The amylase did not hydrolyze maltose, maltotriose, maltotetraose or maltopentaose. Optimum medium composition for maltopentaose production in flask culture was 2%(w/v) soluble starch, 0.4%(w/v) tryptone, 0.5%(w/v) NaCl, 0.5%(w/v) K$_2$HPO$_4$, and 3 mM CaCl$_2$at pH 8.0, 28$^{\circ}C$. The highest yield for maltopentaose production in this condition was 6.45 g/L and was 32.55% of theoretical yield.
Aglycone specificity of <i>Escherichia coli</i>α‐xylosidase investigated by transxylosylation
Kang, Min‐,Sun,Okuyama, Masayuki,Yaoi, Katsuro,Mitsuishi, Yasushi,Kim, Young‐,Min,Mori, Haruhide,Kim, Doman,Kimura, Atsuo BLACKWELL 2007 FEBS JOURNAL Vol.274 No.23
<P>The specificity of the aglycone‐binding site of <I>Escherichia coli</I>α‐xylosidase (YicI), which belongs to glycoside hydrolase family 31, was characterized by examining the enzyme's transxylosylation‐catalyzing property. Acceptor specificity and regioselectivity were investigated using various sugars as acceptor substrates and α‐xylosyl fluoride as the donor substrate. Comparison of the rate of formation of the glycosyl–enzyme intermediate and the transfer product yield using various acceptor substrates showed that glucose is the best complementary acceptor at the aglycone‐binding site. YicI preferred aldopyranosyl sugars with an equatorial 4‐OH as the acceptor substrate, such as glucose, mannose, and allose, resulting in transfer products. This observation suggests that 4‐OH in the acceptor sugar ring made an essential contribution to transxylosylation catalysis. Fructose was also acceptable in the aglycone‐binding site, producing two regioisomer transfer products. The percentage yields of transxylosylation products from glucose, mannose, fructose, and allose were 57, 44, 27, and 21%, respectively. The disaccharide transfer products formed by YicI, α‐<SMALL>d</SMALL>‐Xyl<I>p</I>‐(1→6)‐<SMALL>d</SMALL>‐Man<I>p</I>, α‐<SMALL>d</SMALL>‐Xyl<I>p</I>‐(1→6)‐<SMALL>d</SMALL>‐Fru<I>f</I>, and α‐<SMALL>d</SMALL>‐Xyl<I>p</I>‐(1→3)‐<SMALL>d</SMALL>‐Fru<I>p</I>, are novel oligosaccharides that have not been reported previously. In the transxylosylation to cello‐oligosaccharides, YicI transferred a xylosyl moiety exclusively to a nonreducing terminal glucose residue by α‐1,6‐xylosidic linkages. Of the transxylosylation products, α‐<SMALL>d</SMALL>‐Xyl<I>p</I>‐(1→6)‐<SMALL>d</SMALL>‐Man<I>p</I> and α‐<SMALL>d</SMALL>‐Xyl<I>p</I>‐(1→6)‐<SMALL>d</SMALL>‐Fru<I>f</I> inhibited intestinal α‐glucosidases.</P>
LEE, Jin-Ha,SAITO, Saori,MORI, Haruhide,NISHIMOTO, Mamoru,OKUYAMA, Masayuki,KIM, Doman,WONGCHAWALIT, Jintanart,KIMURA, Atsuo,CHIBA, Seiya Japan Society for Bioscience, Biotechnology, and A 2007 Bioscience, Biotechnology, and Biochemistry Vol.71 No.9
<P>cDNA encoding the bound type trehalase of the European honeybee was cloned. The cDNA (3,001 bp) contained the long 5′ untranslated region (UTR) of 869 bp, and the 3′ UTR of 251 bp including a poly(A) tail, and the open reading frame of 1,881 bp consisting of 626 amino acid residues. The <I>M</I><SUB>r</SUB> of the mature enzyme comprised of 591 amino acids, excluded a signal sequence of 35 amino acid residues, was 69,177. Six peptide sequences analyzed were all found in the deduced amino acid sequence. The amino acid sequence exhibited high identity with trehalases belonging to glycoside hydrolase family 37. A putative transmembrane region similar to trehalase-2 of the silkworm was found in the C-terminal amino acid sequence. Recombinant enzyme of the trehalase was expressed in the methylotrophic yeast <I>Pichia pastoris</I> as host, and displayed properties identical to those of the native enzyme except for higher sugar chain contents. This is the first report of heterologous expression of insect trehalase.</P>
Thi Thanh Hanh Nguyen,Sun-Hwa Jung,Sun Lee,류화자,강희경,Young-Hwan Moon,김영민,Atsuo Kimura,김도만 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.5
Human intestinal maltase (HMA) is an α-glucosidase responsible for the hydrolysis of α-1,4-linkages from the non-reducing end of malto-oligosaccharides. HMA has become an important target in the treatment of type-2 diabetes. In this study, epigallocatechin gallate (EGCG) and EGCG glucoside (EGCG-G1) were identified as inhibitors of HMA by an in vitro assay with IC50 of 20± 1.0 and 31.5 ± 1.0 μM, respectively. A Lineweaver-Burk plot confirmed that EGCG and EGCG-G1 were competitive inhibitors of maltose substrate against HMA and inhibition kinetic constants (Ki) calculated from a Dixon plot were 5.93 ± 0.26 and 7.88 ± 0.57 μM, respectively. Both EGCG and EGCG-G1 bound to the active site of HMA with numerous hydrophobic and hydrogen bond interactions.