A novel thermoacidophilic pullulan-hydrolyzing enzyme (PUL) from hyperthermophilic archaeon Thermococcus kodakarensis (TK-PUL) that efficiently hydrolyzes starch under industrial conditions in the absence of any additional metal ions was cloned and characterized. TK-PUL possessed both pullulanase and α-amylase activities. The highest activities were observed at 95 to 100°C. Although the enzyme was active over a broad pH range (3.0 to 8.5), the pH optima for both activities were 3.5 in acetate buffer and 4.2 in citrate buffer. TK-PUL was stable for several hours at 90°C. Its half-life at 100°C was 45 min when incubated either at pH 6.5 or 8.5. The Km value toward pullulan was 2 mg ml−1, with a Vmax of 109 U mg−1. Metal ions were not required for the activity and stability of recombinant TK-PUL. The enzyme was able to hydrolyze both α-1,6 and α-1,4 glycosidic linkages in pullulan. The most preferred substrate, after pullulan, was γ-cyclodextrin, which is a novel feature for this type of enzyme. Additionally, the enzyme hydrolyzed a variety of polysaccharides, including starch, glycogen, dextrin, amylose, amylopectin, and cyclodextrins (α, β, and γ), mainly into maltose. A unique feature of TK-PUL was the ability to hydrolyze maltotriose into maltose and glucose.
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机译：克隆并鉴定了一种新型的嗜热支链淀粉水解酶（PUL），该酶来自超嗜热古细菌Thermococcus kodakarensis（TK-PUL），可在工业条件下在不存在任何其他金属离子的情况下有效地水解淀粉。 TK-PUL同时具有支链淀粉酶和α-淀粉酶活性。在95至100°C时观察到最高的活性。尽管该酶在很宽的pH范围（3.0至8.5）内均具有活性，但两种活性的最适pH值在乙酸盐缓冲液中均为3.5，在柠檬酸盐缓冲液中为4.2。 TK-PUL在90°C下可稳定几个小时。在pH 6.5或8.5下孵育时，其在100°C的半衰期为45分钟。支链淀粉的Km值为2 mg ml -1 ，Vmax为109 U mg -1 。重组TK-PUL的活性和稳定性不需要金属离子。该酶能够水解支链淀粉中的α-1,6和α-1,4糖苷键。在支链淀粉之后，最优选的底物是γ-环糊精，这是这类酶的新特征。另外，该酶主要将多种多糖水解成麦芽糖，包括淀粉，糖原，糊精，直链淀粉，支链淀粉和环糊精（α，β和γ）。 TK-PUL的独特功能是将麦芽三糖水解成麦芽糖和葡萄糖的能力。
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