嗜冷节杆菌F2菌株冷活性β-半乳糖苷酶的过表达及功能分析
Overexpression and functional analysis of cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2.
作者信息
Nakagawa Tomoyuki, Ikehata Ryoko, Myoda Takao, Miyaji Tatsuro, Tomizuka Noboru
机构信息
Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, Hokkaido 099-2493, Japan.
出版信息
Protein Expr Purif. 2007 Aug;54(2):295-9. doi: 10.1016/j.pep.2007.03.010. Epub 2007 Mar 21.
Cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2 was overexpressed in Escherichia coli using the Cold expression system and the recombinant enzyme, rBglAp, was characterized. The purified rBglAp exhibited similar enzymatic properties to the native enzyme, e.g., (i) it had high activity at 0 degrees C, (ii) its optimum temperature and pH were 10 degrees C and 8.0, respectively, and (iii) it was possible to rapidly inactivate the rBglAp at 50 degrees C in 5 min. Moreover, rBglAp was able to hydrolyze both ONPG and lactose with K(m) values of 2.7 and 42.1mM, respectively, at 10 degrees C. One U of rBglAp could hydrolyze about 70% of the lactose in 1 ml of milk in 24h, and the enzyme produced trisaccharide from lactose. We conclude that rBglAp is a cold-active enzyme that is extremely heat labile and has significant potential application to the food industry.
使用冷表达系统在大肠杆菌中对嗜冷乳糖节杆菌F2菌株的冷活性β-半乳糖苷酶进行了过量表达，并对重组酶rBglAp进行了表征。纯化后的rBglAp表现出与天然酶相似的酶学性质，例如：（i）在0℃时具有高活性；（ii）其最适温度和pH分别为10℃和8.0；（iii）在50℃下5分钟内可使rBglAp迅速失活。此外，rBglAp能够水解ONPG和乳糖，在10℃时其米氏常数（K(m)）分别为2.7和42.1mM。1单位的rBglAp在24小时内可水解1毫升牛奶中约70%的乳糖，并且该酶可由乳糖产生三糖。我们得出结论，rBglAp是一种冷活性酶，对热极其不稳定，在食品工业中具有显著的潜在应用价值。
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