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

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 (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.