Lin C S, Chen H C, Lin F P
Institute of Marine Food Science, National Taiwan Ocean University, Keelung, Taiwan, Republic of China.
Enzyme Microb Technol. 1997 Nov 15;21(7):472-8. doi: 10.1016/s0141-0229(96)00249-9.
The gene encoding a chitinase from Aeromonas caviae was cloned by PCR techniques. Its recombinant gene expression was performed using pET20b(+) in Escherichia coli BL21 (DE3). The recombinant chitinase with the extra 33 and 13 amino acids in its N- and C-termini, respectively, was purified to near homogeneity using His-Tag affinity chromatography. The recombinant chitinase was found to be present in both the culture medium and the cytoplasm. A single protein band on the native polyacrylamide gel was confirmed by both the activity staining and protein staining. The optimum pH and temperature of the recombinant chitinase were determined to be 6.25-6.5 and 42.5 degrees C, respectively. It was stable within the pH range of 5-7. Significant activity stimulation by Cu2+ and inhibition by Fe3+ and Hg2+ were observed. Detergents such as SDS and Triton X-100 strongly inhibited the enzyme activity. Substrates such as 4-methylumbelliferyl-N,N'-diacetylchitobioside and 4-methylumbelliferyl-N,N',N"-triacetylchitotriose were hydrolyzed by the recombinant chitinase; however, 4-methylumbelliferyl-N-acetylglucosaminide was not cleaved during the activity assay periods. When chitin power was suspended in buffer with the chitinase (pH 6.5 and 42.5 degrees C), N-acetylchitooligosaccharides [(GlcNAc)n, n = 1-4] were detected at 24 h.
通过PCR技术克隆了豚鼠气单胞菌编码几丁质酶的基因。使用pET20b(+)在大肠杆菌BL21(DE3)中进行其重组基因表达。分别在其N端和C端带有额外33个和13个氨基酸的重组几丁质酶，通过His-Tag亲和层析纯化至接近均一。发现重组几丁质酶存在于培养基和细胞质中。通过活性染色和蛋白质染色均证实了天然聚丙烯酰胺凝胶上的单一蛋白条带。重组几丁质酶的最适pH和温度分别确定为6.25 - 6.5和42.5℃。它在pH 5 - 7范围内稳定。观察到Cu2+对其有显著的活性刺激作用，而Fe3+和Hg2+对其有抑制作用。SDS和Triton X-100等去污剂强烈抑制该酶的活性。重组几丁质酶可水解4-甲基伞形酮基-N,N'-二乙酰壳二糖和4-甲基伞形酮基-N,N',N"-三乙酰壳三糖等底物；然而，在活性测定期间4-甲基伞形酮基-N-乙酰葡糖胺未被裂解。当几丁质粉与几丁质酶一起悬浮于缓冲液中（pH 6.5和42.5℃）时，在24小时检测到N-乙酰壳寡糖[(GlcNAc)n, n = 1 - 4]。
