Trimbur D E, Gutshall K R, Prema P, Brenchley J E
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park.
Appl Environ Microbiol. 1994 Dec;60(12):4544-52. doi: 10.1128/aem.60.12.4544-4552.1994.
Enzymes with high specific activities at low temperatures have potential uses for chemical conversions when low temperatures are required, as in the food industry. Psychrotrophic microorganisms which grow at low temperatures may be a valuable source of cold-active enzymes that have higher activities at low temperatures than enzymes found for mesophilic microorganisms. To find cold-active beta-galactosidases, we isolated and characterized several psychrotrophic microorganisms. One isolate, B7, is an Arthrobacter strain which produces beta-galactosidase when grown in lactose minimal media. Extracts have a specific activity at 30 degrees C of 2 U/mg with o-nitrophenyl-beta-D-galactopyranoside as a substrate. Two isozymes were detected when extracts were subjected to electrophoresis in a nondenaturing polyacrylamide gel and stained for activity with 5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (X-Gal). When chromosomal DNA was prepared and transformed into Escherichia coli, three different genes encoding beta-galactosidase activity were obtained. We have subcloned and sequenced one of these beta-galactosidase genes from the Arthrobacter isolate B7. On the basis of amino acid sequence alignment, the gene was found to have probable catalytic sites homologous to those from the E. coli lacZ gene. The gene encoded a protein of 1,016 amino acids with a predicted molecular mass of 111 kDa. The enzyme was purified and characterized. The beta-galactosidase from isolate B7 has kinetic properties similar to those of the E. coli lacZ beta-galactosidase but has a temperature optimum 20 degrees C lower than that of the E. coli enzyme.
在低温下具有高比活性的酶在需要低温的化学转化中具有潜在用途，如在食品工业中。能在低温下生长的嗜冷微生物可能是冷活性酶的宝贵来源，这些酶在低温下的活性高于嗜温微生物的酶。为了寻找冷活性β-半乳糖苷酶，我们分离并鉴定了几种嗜冷微生物。其中一个分离株B7是一株节杆菌，当在乳糖基本培养基中生长时会产生β-半乳糖苷酶。以邻硝基苯基-β-D-吡喃半乳糖苷为底物时，提取物在30℃下的比活性为2 U/mg。当提取物在非变性聚丙烯酰胺凝胶中进行电泳并用5-溴-4-氯-吲哚基-β-D-吡喃半乳糖苷（X-Gal）进行活性染色时，检测到两种同工酶。当制备染色体DNA并转化到大肠杆菌中时，获得了三个编码β-半乳糖苷酶活性的不同基因。我们已经从节杆菌分离株B7中对其中一个β-半乳糖苷酶基因进行了亚克隆和测序。基于氨基酸序列比对，发现该基因具有与大肠杆菌lacZ基因可能的催化位点同源的位点。该基因编码一种1016个氨基酸的蛋白质，预测分子量为111 kDa。对该酶进行了纯化和表征。分离株B7的β-半乳糖苷酶的动力学性质与大肠杆菌lacZβ-半乳糖苷酶相似，但最适温度比大肠杆菌酶低20℃。
