Calcium-sensitive extracellular poly(alpha-L-guluronate)lyase from a marine bacterium Pseudomonas sp strain F6: Purification and some properties

Poly(alpha -L-guluronate)lyase, as one of alginate lyases, was purified fro m the culture medium of a marine bacterium, Pseudomonas sp. strain F6, to a n electrophoretically homogeneous state. The enzyme was shown to have a mol ecular mass of 36 kDa by sodium dodecylsulfate-polyacrylamide gel electroph oresis (SDS-PAGE), and was most active at around pH 7.5 and was stable betw een pH 6.5 and pH 8.5. In the thermal stability experiments, the enzyme's a ctivity diminished through an intermediate state with increasing incubation temperatures and was finally lost when heated at 100 degreesC for 15 min. The addition of hen egg-white lysozyme to the enzyme decreased thermal stab ility dramatically. The apparent retention of enzyme activity (approximatel y 50%) was observed after the addition of 6 M guanidine hydrochloride and 8 M urea. Enzyme activity was lost completely with 10 mMSDS, while the order ed structure, which is considered likely to be beta -structure, was markedl y created. The similar conformational feature has also been created in mari ne bacterial and mollusc enzymes and the beta -structure is commonly observ ed in polyuronate lyases. The divalent cation (Ca2+) promoted the activity of the calcium chelator-treated enzyme significantly, suggesting that Ca2is involved in the formation of the active intermediate between the acidic uronate(s) and amino acid side-chain(s) of the enzyme.