Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Grønnegårdsvej 15, 1870 Frederiksberg C., Denmark.
Glycobiology. 2011 Apr;21(4):426-36. doi: 10.1093/glycob/cwq174. Epub 2010 Nov 9.
Salmonella contain genes annotated as chitinases; however, their chitinolytic activities have never been verified. We now demonstrate such an activity for a chitinase assigned to glycoside hydrolase family 18 encoded by the SL0018 (chiA) gene in Salmonella enterica Typhimurium SL1344. A C-terminal truncated form of chiA lacking a putative chitin-binding domain was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3) with an N-terminal (His)(6) tag. The purified enzyme hydrolyzes 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside, 4-nitrophenyl β-D-N,N',N″-triacetylchitotriose and carboxymethyl chitin Remazol Brilliant Violet but does not act on 4-nitrophenyl N-acetyl-β-D-glucosaminide, peptidoglycan or 4-nitrophenyl β-D-cellobioside. Enzyme activity was also characterized by directly monitoring product formation using (1)H-nuclear magnetic resonance which showed that chitin is a substrate with the release of N,N'-diacetylchitobiose. Hydrolysis occurs with the retention of configuration and the enzyme acts on only the β-anomers of chitooligosaccharide substrates. The enzyme also released N-acetyllactosamine disaccharide from Galβ1 → 4GlcNAcβ-O-(CH(2))(8)CONH(CH(2))(2)NHCO-tetramethylrhodamine, a model substrate for LacNAc terminating glycoproteins and glycolipids.
沙门氏菌含有被注释为几丁质酶的基因；然而，它们的几丁质酶活性从未得到验证。我们现在证明了这种活性存在于 SL1344 型鼠伤寒沙门氏菌的 SL0018（chiA）基因编码的糖苷水解酶家族 18 中的一种几丁质酶中。通过 PCR 扩增、克隆和表达，得到一个缺少假定几丁质结合结构域的 chiA 的 C 端截断形式，该蛋白在大肠杆菌 BL21（DE3）中带有 N 端（His）（6）标签。纯化后的酶水解 4-硝基苯基 N，N′-二乙酰基-β-D-壳二糖苷、4-硝基苯基 β-D-N，N′，N″-三乙酰壳三糖和羧甲基壳聚糖，但不作用于 4-硝基苯基 N-乙酰基-β-D-葡糖胺、肽聚糖或 4-硝基苯基 β-D-纤维二糖苷。酶活性也通过直接监测使用（1）H 核磁共振产生的产物来进行特征分析，表明壳聚糖是一种产物为 N，N′-二乙酰壳二糖的底物。水解发生时保留了构型，并且酶仅作用于壳寡糖底物的β-异构体。该酶还从 Galβ1 → 4GlcNAcβ-O-（CH（2））（8）CONH（CH（2））（2）NHCO-四甲基罗丹明释放 N-乙酰乳糖胺二糖，这是一种用于 LacNAc 末端糖蛋白和糖脂的模型底物。
