Department of Food Science, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa 921-8836, Japan.
Glycobiology. 2011 Apr;21(4):503-11. doi: 10.1093/glycob/cwq191. Epub 2010 Nov 22.
A glycoside hydrolase (GH) gene from Photobacterium profundum SS9 (PBPRA0520) belonging to GH family 9 was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it is an intracellular enzyme. The recombinant protein showed hydrolytic activity toward chitobiose [(GlcN)(2)] and cellobiose (CG(2)) in various disaccharides. This protein also released 4-nitrophenol (PNP) from both 4-nitrophenyl-β-D-glucosaminide (GlcN-PNP) and 4-nitrophenyl-β-D-glucoside (Glc-PNP). The hydrolytic pattern observed in chitooligosaccharides and cellooligosaccharides suggested that the reaction proceeded from the nonreducing end in an exo-type manner. Time-dependent (1)H-nuclear magnetic resonance (NMR) analysis of the anomeric form of the enzymatic reaction products indicated that the protein is an inverting enzyme. k(cat)/K(m) of (GlcN)(2) hydrolysis was 14 times greater than that of CG(2) hydrolysis. These results suggested that the protein is an exo-β-D-glucosaminidase (EC 3.2.1.165) rather than a glucan 1,4-β-D-glucosidase (EC 3.2.1.74). Based on the results, we suggest that the function of conserved GH9 proteins in the chitin catabolic operon is to cleave a (GlcN)(2)-phosphate derivative by hydrolysis during intracellular chitooligosaccharide catabolism in Vibrionaceae.
来自深海发光杆菌 SS9（PBPRA0520）的糖苷水解酶（GH）基因属于 GH 家族 9，在大肠杆菌中表达。该蛋白表达时有完整的 N 端序列，表明它是一种细胞内酶。重组蛋白对各种二糖中的壳二糖[(GlcN)（2）]和纤维二糖（CG（2））具有水解活性。该蛋白还从 4-硝基苯-β-D-葡糖胺（GlcN-PNP）和 4-硝基苯-β-D-葡萄糖苷（Glc-PNP）释放 4-硝基苯酚（PNP）。在壳寡糖和纤维寡糖中观察到的水解模式表明反应以外向型方式从非还原端进行。对酶反应产物的端基形式的时间依赖性（1）H-核磁共振（NMR）分析表明，该蛋白是一种反转酶。（GlcN）（2）水解的 k(cat)/K(m)比 CG（2）水解的大 14 倍。这些结果表明，该蛋白是一种外切-β-D-葡糖胺酶（EC 3.2.1.165），而不是葡聚糖 1,4-β-D-葡萄糖苷酶（EC 3.2.1.74）。基于这些结果，我们认为保守的 GH9 蛋白在几丁质分解代谢操纵子中的功能是在弧菌属的细胞内壳寡糖分解代谢过程中通过水解切割（GlcN）（2）-磷酸衍生物。
