Characterization of glycosyl hydrolase family 3 β-N-acetylglucosaminidases from Thermotoga maritima and Thermotoga neapolitana(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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概要

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• 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 54kDa. The optimal temperatures and pHs for the activities of the β-N-acetylglucosaminidases were found to be 65-75℃ 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 (pNPGlcNAc_2) and p-nitrophenyl tri-N-acetyl β-D-chitotriose (pNPGlcNAc_3). 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 k_<cat> and K_m values were determined for pNPGlcNAc, pNPGlcNAc_2 and pNPGlcNAc_3. The k_<cat>/K_m 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.

著者

• Choi Kyoung-hwa

  Department Of Microbiology Pusan National University

• CHA Jaeho

  Department of Microbiology, Pusan National University

• Cha Jaeho

  Department Of Microbiology Pusan National University

• PARK Cheon-Seok

  Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University

• Seo Ja

  Department of Microbiology, Pusan National University

• Park Kyung-Min

  Department of Microbiology, Pusan National University

• Park Cheon-seok

  Graduate School Of Biotechnology And Institute Of Life Science And Resources Kyung Hee University

• Seo Ja

  Department Of Microbiology Pusan National University

• Park Kyung-min

  Department Of Microbiology Pusan National University

• Park Cheon-seok

  Graduate School Of Biotechnology And Inst. Of Life Sci. And Resources Kyung Hee Univ.

関連論文

• Isolation of an Exopolysaccharide-producing Bacterium, Sphingomonas sp. CS101, Which Forms an Unusual Type of Sphingan
• Characterization of glycosyl hydrolase family 3 β-N-acetylglucosaminidases from Thermotoga maritima and Thermotoga neapolitana(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
• Molecular Cloning and Functional Expression of a New Amylosucrase from Alteromonas macleodii
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