College of Plant Sciences, Jilin University, Changchun, China.
Lett Appl Microbiol. 2013 Oct;57(4):336-43. doi: 10.1111/lam.12118. Epub 2013 Jul 8.
In this report, a glycoside hydrolase 13 family pullulanase gene (Tk0977) was cloned from a thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1 (Pul-Tk). Pul-Tk encodes a protein of 765 amino acids including a putative 22-residue signal peptide. The protein has four consensus motives and a catalytic triad of glycoside hydrolase 13 family in the deduced amino acid sequence. The recombinant enzyme was expressed in Escherichia coli and purified to homogeneity. Pul-Tk can hydrolyse both pullulan and soluble starch. The purified enzyme was optimal at pH 5·5-6·0 and 100°C and exhibited good stability over a broad pH range (4-8). The Vmax and Km values were 118·39 ± 1·76 μmol mg(-1 ) min(-1) and 0·37 ± 0·02 mg ml(-1) for pullulan and 53·19 ± 11·66 μmol mg(-1 ) min(-1) and 0·36 ± 0·05 mg ml(-1) for starch. All these favourable enzymatic properties make it valuable in various industries.
Pullulanases have a great potential in industrial applications including the starch industry, the production of maltose syrups and high-purity glucose and fructose. In this study, a pullulanase from hyperthermophilic archaeon Thermococcus kodakarensis KOD1 was successfully expressed in Escherichia coli and the recombinant enzyme can be purified and characterized. The high activity, broad pH range and stability implicate it as a potential enzyme for industrial applications.
在本报告中，从嗜热厌氧古菌 Thermococcus kodakarensis KOD1（Pul-Tk）中克隆了糖苷水解酶 13 家族普鲁兰酶基因（Tk0977）。Pul-Tk 编码一种 765 个氨基酸的蛋白质，包括一个假定的 22 个残基信号肽。该蛋白质在推导的氨基酸序列中具有四个共识基序和糖苷水解酶 13 家族的催化三联体。重组酶在大肠杆菌中表达并纯化至均一性。Pul-Tk 可以水解普鲁兰和可溶性淀粉。纯化酶在 pH 5.5-6.0 和 100°C 时最佳，在较宽的 pH 范围内（4-8）表现出良好的稳定性。Vmax 和 Km 值分别为 118.39±1.76μmol·mg(-1)·min(-1)和 0.37±0.02mg·ml(-1)用于普鲁兰和 53.19±11.66μmol·mg(-1)·min(-1)和 0.36±0.05mg·ml(-1)用于淀粉。所有这些有利的酶学性质使其在各个行业中具有很高的价值。
普鲁兰酶在工业应用中有很大的潜力，包括淀粉工业、麦芽糖糖浆的生产以及高纯度葡萄糖和果糖的生产。在本研究中，成功地在大肠杆菌中表达了来自嗜热古菌 Thermococcus kodakarensis KOD1 的普鲁兰酶，并且可以纯化和表征重组酶。高活性、宽 pH 范围和稳定性表明它是一种有潜力的工业应用酶。
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