CHARACTERIZATION OF A PSYCHROTROPHIC ARTHROBACTER GENE AND ITS COLD-ACTIVE BETA-GALACTOSIDASE

Enzymes with high specific activities at low temperatures have potenti al uses for chemical conversions when low temperatures are required, a s in the food industry. Psychrotrophic microorganisms which grow at lo w temperatures may be a valuable source of cold-active enzymes that ha ve higher activities at low temperatures than enzymes found for mesoph ilic microorganisms. To find cold-active beta-galactosidases, we isola ted and characterized several psychrotrophic microorganisms. One isola te, B7, is an Arthrobacter strain which produces beta-galactosidase wh en grown in lactose minimal media. Extracts have a specific activity a t 30 degrees C of 2 U/mg with o-nitrophenyl-beta-D-galactopyranoside a s 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 Escheric hia coil, three different genes encoding beta-galactosidase activity w ere obtained. We have subcloned and sequenced one of these beta-galact osidase 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 encode d a protein of 1,016 amino acids with a predicted molecular mass of 11 1 kDa. The enzyme was purified and characterized. The beta-galactosida se from isolate B7 has kinetic properties similar to those of the E. c oil lacZ beta-galactosidase but has a temperature optimum 20 degrees C lower than that of the E. coli enzyme.