Costantino H R, Brown S H, Kelly R M
Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland 21218.
J Bacteriol. 1990 Jul;172(7):3654-60. doi: 10.1128/jb.172.7.3654-3660.1990.
Pyrococcus furiosus is a strictly anaerobic hyperthermophilic archaebacterium with an optimal growth temperature of about 100 degrees C. When this organism was grown in the presence of certain complex carbohydrates, the production of several amylolytic enzymes was noted. These enzymes included an alpha-glucosidase that was located in the cell cytoplasm. This alpha-glucosidase has been purified 310-fold and corresponded to a protein band of 125 kilodaltons as resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited optimum activity at pH 5.0 to 6.0 and over a temperature range of 105 to 115 degrees C. Kinetic analysis conducted at 108 degrees C revealed hydrolysis of the substrates p-nitrophenyl-alpha-D-glucopyranoside (PNPG), methyl-alpha-D-glucopyranoside, maltose, and isomaltose. Trace activity was detected towards p-nitrophenyl-beta-D-glucopyranoside, and no activity could be detected towards starch or sucrose. Inhibition studies conducted at 108 degrees C with PNPG as the substrate and maltose as the inhibitor yielded a Ki for maltose of 14.3 mM. Preincubation for 30 min at 98 degrees C in 100 mM dithiothreitol and 1.0 M urea had little effect on enzyme activity, whereas preincubation in 1.0% sodium dodecyl sulfate and 1.0 M guanidine hydrochloride resulted in significant loss of enzyme activity. Purified alpha-glucosidase from P. furiosus exhibited remarkable thermostability; incubation of the enzyme at 98 degrees C resulted in a half life of nearly 48 h.
嗜热栖热菌是一种严格厌氧的超嗜热古细菌，最佳生长温度约为100摄氏度。当这种微生物在某些复合碳水化合物存在的情况下生长时，会产生几种淀粉分解酶。这些酶包括一种位于细胞质中的α-葡萄糖苷酶。这种α-葡萄糖苷酶已被纯化310倍，在10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中对应于一条125千道尔顿的蛋白带。该酶在pH 5.0至6.0以及105至115摄氏度的温度范围内表现出最佳活性。在108摄氏度进行的动力学分析表明，底物对硝基苯基-α-D-吡喃葡萄糖苷（PNPG）、甲基-α-D-吡喃葡萄糖苷、麦芽糖和异麦芽糖会发生水解。对硝基苯基-β-D-吡喃葡萄糖苷检测到微量活性，而对淀粉或蔗糖未检测到活性。以PNPG为底物、麦芽糖为抑制剂在108摄氏度进行的抑制研究得出麦芽糖的Ki为14.3 mM。在100 mM二硫苏糖醇和1.0 M尿素中于98摄氏度预孵育30分钟对酶活性影响不大，而在1.0%十二烷基硫酸钠和1.0 M盐酸胍中预孵育会导致酶活性显著丧失。从嗜热栖热菌中纯化的α-葡萄糖苷酶表现出显著的热稳定性；将该酶在98摄氏度孵育导致半衰期接近48小时。
