Purification and characterization of a new neutral metalloprotease from marine Exiguobacterium sp. SWJS2.

Among the protease-producing bacterial strains isolated from deep-sea sediments, SWJS2 was finally selected and identified as genus Exiguobacterium. Plackett-Burman and orthogonal array designs were applied to optimize the fermentation conditions, and the results are as follows: Glucose 5g, yeast extract 15g, glycerin 2g and CaCl(2)2H(2)O 0.5g dissolved in 1 L artificial seawater; temperature 25 degrees C, original pH 7, inoculum rate 2%, seed age 12 H, loading volume 25mL (250-mL Erlenmeyer flask), shaking speed 150 rpm, and fermentation time 44 H. The protease activity production was improved from about 80 to 660 U/mL under the optimized parameters. The protease was purified fourfold with specificity activity of 30,654.1 U/mg protein and a total yield of 16.2%. The protease exhibited the maximum activity at 40-45 degrees C and pH 7. Moreover, the enzyme activity was found to be inhibited by Cu2+, Ba2+, Cd2+, Hg2+, and Al3+ at 5 mM, whereas it can be increased by Mg2+, Mn2+, and Ca2+ at 0.5-5 mM. The enzyme was totally inactivated by 1 or 5 mM ethylenediaminetetraacetic acid but not by phenylmethanesulfonyl fluoride, tyrpsin inhibitor from Glycine max (STI), benzamidine, 5,5-dithio-bis-(2-nitro benzoic acid), or pepstatin A, suggesting that it belonged to metalloprotease.