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Characterization of glycosyl hydrolase family 3 β-N-acetylglucosaminidases from Thermotoga maritima and Thermotoga neapolitana

Paper ID Volume ID Publish Year Pages File Format Full-Text
21587 43230 2009 5 PDF Available
Title
Characterization of glycosyl hydrolase family 3 β-N-acetylglucosaminidases from Thermotoga maritima and Thermotoga neapolitana
Abstract

The genes encoding β-N-acetylglucosaminidase (nagA and cbsA) from Thermotoga maritima and Thermotoga neapolitana were cloned and expressed in Escherichia coli in order to investigate whether Thermotoga sp. is capable of utilizing chitin as a carbon source. NagA and CbsA were purified to homogeneity by HiTrap Q HP and Sephacryl S-200 HR column chromatography. Both enzymes were homodimers containing a family 3 glycoside hydrolase (GH3) catalytic domain, with a monomer molecular mass of 54 kDa. The optimal temperatures and pHs for the activities of the β-N-acetylglucosaminidases were found to be 65−75 °C and 7.0−8.0, respectively. Both enzymes hydrolyzed chitooligomers such as di-N-acetylchitobiose and tri-N-acetylchitotriose, and synthetic substrates such as p-nitrophenyl-β-d-glucose (pNPGlc), p-nitrophenyl N-acetyl β-d-glucosamine (pNPGlcNAc), p-nitrophenyl di-N-acetyl β-d-chitobiose (pNPGlcNAc2) and p-nitrophenyl tri-N-acetyl β-d-chitotriose (pNPGlcNAc3). However, the enzymes had no activity against p-nitrophenyl-β-d-galactose (pNPGal) and p-nitrophenyl N-acetyl β-d-galactosamine (pNPGalNAc) or highly polymerized chitin. The kcat and Km values were determined for pNPGlcNAc, pNPGlcNAc2 and pNPGlcNAc3. The kcat/Km value for pNPGlcNAc was the highest among three synthetic substrates. NagA and CbsA initially hydrolyzed p-nitrophenyl substrates to give GlcNAc, suggesting that the enzymes have exo-activity with chitin oligosaccharides from the non-reducing ends, like other β-N-acetylglucosaminidases. However, NagA and CbsA can be distinguished from other GH3-type β-N-acetylglucosaminidases in that they are highly active against di-N-acetylchitobiose. Thus, the present results suggest that the physiological role of both enzymes is to degrade the chitooligosaccharides transported through membrane following hydrolysis of chitin into β-N-acetylglucosamine to be further metabolized in Thermotoga sp.

Keywords
β-N-acetylglucosaminidase; Glycosyl hydrolase family 3; Kinetics; Thermostability; Thermotoga species
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Characterization of glycosyl hydrolase family 3 β-N-acetylglucosaminidases from Thermotoga maritima and Thermotoga neapolitana
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 108, Issue 6, December 2009, Pages 455–459
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us