Department of Microbiology, Pusan National University, Busan 609-735, Republic of Korea.
J Biosci Bioeng. 2009 Dec;108(6):455-9. doi: 10.1016/j.jbiosc.2009.06.003.
The genes encoding beta-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 beta-N-acetylglucosaminidases were found to be 65-75 degrees 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-beta-D-glucose (pNPGlc), p-nitrophenyl N-acetyl beta-D-glucosamine (pNPGlcNAc), p-nitrophenyl di-N-acetyl beta-D-chitobiose (pNPGlcNAc(2)) and p-nitrophenyl tri-N-acetyl beta-D-chitotriose (pNPGlcNAc(3)). However, the enzymes had no activity against p-nitrophenyl-beta-D-galactose (pNPGal) and p-nitrophenyl N-acetyl beta-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 beta-N-acetylglucosaminidases. However, NagA and CbsA can be distinguished from other GH3-type beta-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 beta-N-acetylglucosamine to be further metabolized in Thermotoga sp.
从栖热菌属（Thermotoga maritima）和海栖热袍菌属（Thermotoga neapolitana）中克隆并在大肠杆菌中表达了编码β-N-乙酰氨基葡萄糖苷酶（nagA 和 cbsA）的基因，以研究栖热菌属是否能够将壳聚糖作为碳源利用。通过 HiTrap Q HP 和 Sephacryl S-200 HR 柱层析将 NagA 和 CbsA 纯化至均一性。两种酶均为同源二聚体，含有家族 3 糖苷水解酶（GH3）催化结构域，单体分子量为 54 kDa。β-N-乙酰氨基葡萄糖苷酶的最适温度和 pH 分别为 65-75°C 和 7.0-8.0。两种酶均水解壳寡糖，如二乙酰壳二糖和三乙酰壳三糖，以及合成底物，如对硝基苯基-β-D-葡萄糖（pNPGlc）、对硝基苯基 N-乙酰-β-D-葡萄糖胺（pNPGlcNAc）、对硝基苯基二乙酰基-β-D-壳二糖（pNPGlcNAc（2））和对硝基苯基三乙酰基-β-D-壳三糖（pNPGlcNAc（3））。然而，这些酶对 p-硝基苯基-β-D-半乳糖（pNPGal）和 p-硝基苯基 N-乙酰-β-D-半乳糖胺（pNPGalNAc）或高度聚合的壳聚糖没有活性。测定了 pNPGlcNAc、pNPGlcNAc（2）和 pNPGlcNAc（3）的 k(cat)和 K(m) 值。pNPGlcNAc 的 k(cat)/K(m) 值在三种合成底物中最高。NagA 和 CbsA 最初将 p-硝基苯基底物水解为 GlcNAc，表明这些酶具有从非还原端开始对壳寡糖的外切活性，与其他β-N-乙酰氨基葡萄糖苷酶类似。然而，NagA 和 CbsA 可以与其他 GH3 型β-N-乙酰氨基葡萄糖苷酶区分开来，因为它们对二乙酰基壳二糖具有高活性。因此，本研究结果表明，两种酶的生理作用是在栖热菌属中，将壳聚糖水解为β-N-乙酰氨基葡萄糖后，将通过膜运输的壳寡糖降解，以进一步代谢。
