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KMID : 0545120160260050837
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Journal of Microbiology and Biotechnology
2016 Volume.26 No. 5 p.837 ~ p.845
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Identification of Catalytic Amino Acid Residues by Chemical Modification in Dextranase
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Ko Jin-A
Nam Seung-Hee
Kim Do-Man
Lee Jun-Ho
Kim Young-Min
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Abstract
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A novel endodextranase isolated from Paenibacillus sp. was found to produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides with a degree of polymerization of 7?14 from dextran. To determine the active site, the enzyme was modified with 1-ethyl-3-[3- (dimethylamino)-propyl]-carbodiimide (EDC) and α-epoxyalkyl α-glucosides (EAGs), an affinity labeling reagent. The inactivation followed pseudo first-order kinetics. Kinetic analysis and chemical modification using EDC and EAGs indicated that carboxyl groups are essential for the enzymatic activity. Three Asp and one Glu residues were identified as candidate catalytic amino acids, since these residues are completely conserved across the GH family of 66 enzymes. Replacement of Asp189, Asp340, or Glu412 completely abolished the enzyme activity, indicating that these residues are essential for catalytic activity.
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KEYWORD
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dextranase, catalytic amino acids, chemical modification, carboxyl group
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