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Park, Yeo-Ul,Jung, Jong-Hyun,Seo, Dong-Ho,Jung, Dong-Hyun,Kim, Jae-Han,Seo, Ean-Jeong,Baek, Nam-In,Park, Cheon-Seok Elsevier 2018 Enzyme and microbial technology Vol.114 No.-
<P><B>Abstract</B></P> <P> <I>Desulfurococcus amylolyticus</I> is an anaerobic and hyperthermophilic crenarchaeon that can use various carbohydrates as energy sources. We found a gene encoding a glycoside hydrolase family 57 amylolytic enzymes (DApu) in a putative carbohydrate utilization gene cluster in the genome of <I>D. amylolyticus</I>. This gene has an open reading frame of 1,878 bp and consists of 626 amino acids with a molecular mass of 71 kDa. Recombinant DApu (rDApu) completely hydrolyzed pullulan to maltotriose by attacking α-1,6-glycosidic linkages, and was able to produce glucose and maltose from soluble starch and amylopectin. Although rDApu showed no activity toward α-cyclodextrin (CD) and β-CD, maltooctaose (G8) was detected from reaction with γ-CD. The highest activity of rDApu was measured at pH 5.0 and 95 °C. The half-life of rDApu was 12.7 h at 95 °C and 27 min at 98 °C. Interestingly, rDApu was able to transfer a maltose unit to 6-<I>O</I>-α-maltosyl-β-CD via transglycosylation. Structure analysis using MALDI-TOF/TOF MS and nuclear magnetic resonance revealed that the new transglycosylated products were 6<SUP>1</SUP>, 6<SUP>4</SUP>-di-<I>O</I>-maltosyl-β-CD and 6<SUP>1</SUP>, 6<SUP>3</SUP>, 6<SUP>5</SUP>-tri-<I>O</I>-maltosyl-β-CD.</P> <P><B>Highlights</B></P> <P> <UL> <LI> GH57 amylopullulanase from <I>Desulfurococcus amylolyticus</I> (DApu) was characterized. </LI> <LI> DApu exhibits not only hydrolysis activity but also transglycosylation activity. </LI> <LI> Branched cyclodextrins were formed by transfer of a maltose unit by DApu. </LI> </UL> </P>
( Myo-deok Kim ),( Dong-hyun Jung ),( Dong-ho Seo ),( Jong-hyun Jung ),( Ean-jeong Seo ),( Nam-in Baek ),( Sang-ho Yoo ),( Cheon-seok Park ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.11
The transglycosylation activity of amylosucrase (ASase) has received significant attention owing to its use of an inexpensive donor, sucrose, and broad acceptor specificity, including glycone and aglycone compounds. The transglycosylation reaction of recombinant ASase from Deinococcus radiopugnans (DRpAS) was investigated using various phenolic compounds, and quercetin-3-O-rutinoside (rutin) was found to be the most suitable acceptor molecule used by DRpAS. Two amino acid residues in DRpAS variants (DRpAS Q299K and DRpAS Q299R), assumed to be involved in acceptor binding, were constructed by site-directed mutagenesis. Intriguingly, DRpAS Q299K and DRpAS Q299R produced 10-fold and 4-fold higher levels of rutin transglycosylation product than did the wild-type (WT) DRpAS, respectively. According to in silico molecular docking analysis, the lysine residue at position 299 in the mutants enables rutin to more easily position inside the active pocket of the mutant enzyme than in that of the WT, due to conformational changes in loop 4.