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A glycosynthase derived from an inverting chitinase with an extended binding cleft
Takayuki Ohnuma1, Satoshi Dozen1, Yuji Honda2
1Department of Advanced Bioscience, Kinki University, 3327-204, Nakamachi, Nara 631-8505, Japan;
Journal of Biochemistry
|February 25, 2016
Summary
Researchers engineered a novel glycosynthase enzyme from rye seed chitinase. This enzyme efficiently synthesizes longer sugar chains by adding three-sugar units without decomposition, advancing carbohydrate synthesis.
Area of Science:
- Biochemistry
- Enzymology
- Carbohydrate Chemistry
Background:
- Chitinases are enzymes that degrade chitin, a polysaccharide.
- Glycosynthases are engineered enzymes capable of synthesizing glycosidic bonds.
- The GH19 chitinase from rye seeds (RSC-c) possesses an extended binding cleft suitable for engineering.
Purpose of the Study:
- To engineer a glycosynthase from RSC-c for efficient synthesis of longer oligosaccharides.
- To investigate the role of catalytic residues Glu89 and Ser120 in enzyme activity.
- To characterize the hydrolytic and synthetic capabilities of RSC-c mutants.
Main Methods:
- Site-directed mutagenesis was used to create single (E89G, S120A) and double (E89G/S120A) mutants of RSC-c.
- Enzyme activity was assessed using α-(GlcNAc)3-F as a substrate in the presence of (GlcNAc)4.
- Product formation and decomposition were analyzed to determine glycosynthase efficiency.
Main Results:
- Wild-type RSC-c produced (GlcNAc)3 and fluoride from α-(GlcNAc)3-F.
- Mutant E89G showed limited synthesis, while S120A exhibited high fluoride release but product decomposition.
- The double mutant E89G/S120A demonstrated abolished hydrolytic activity and efficient synthesis of (GlcNAc)7 without decomposition.
Conclusions:
- The double mutant E89G/S120A functions as an efficient glycosynthase.
- This engineered enzyme enables the addition of a three-sugar unit to oligosaccharides.
- The study provides a valuable tool for carbohydrate synthesis and enzymatic engineering.
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