Low-temperature Protease Separation, Purification And Enzymatic Properties

A strain producing cold-adapted protease was isolated from a refrigerator of a meat factory. It was identified as a strain of Serratia based on morphological, physiological characteristics and phylogenetic analysis of 16S rDNA. The new isolated strain was named as Serratia sp. WJ 39.Pro-WJ39 was an extracellular enzyme. The protease was purified to homogeneity from fermentation liquor by ultrafiltration, (NH₄)₂SO₄ precipitation, DEAE Sepharose^? FF and Superdex 75 gel filtration. Pro-WJ39 was 46-fold purified with yield of 6.5%.Pro-WJ39 was stable at temperature of 0°C to 25°C, the optimal reaction temperature of pro-WJ39 was determined at 37°C. The activity of pro-WJ39 was stable at pH10. The enzyme had wide temperature adaptability; it remained 35% residual activity even at 0°C.The molecular mass of pro-WJ39 was determined by SDS-PAGE to be 47.6kD, which is same as Superdex 75 done, so it should be a monomeric protein.Characterization of the pro-WJ39 showed that it required certain actions such as K⁺, Na⁺, Ca²⁺ for activating enzyme and was partially inhibited by Cd²⁺, Cu²⁺, Ni²⁺, Mn²⁺, but there were no ions could obviously activate or inhibit its activity. SDS, acetonitrile and acetone also could partially inhibit the enzyme activity. Pro-WJ39 was strongly inhibited by lmmol/L PMSF, so it should be a serine protease.At 37°C, the Km of pro-WJ39 was 0.875mol·L^-1 and Kcat/Km was 2.313×10⁵ mol^-1·L·S^-1. Compared to the reported cold-adapted protease, pro-WJ39 had higher activity to combine and catalyze casein. The Ea of pro-WJ39 was calculated to be 23.0kJ/mol, which is not only obviously lower than the mesothermal protease, but also lower than the reported cold-adapted proteases of 36.9-38.0kJ/mol. All data showed that pro-WJ39 had a very high catalytic efficiency at low temperature.In this study we also found the enzyme had high catalytic activity towards the peptide which has a methyl group on its first amino acid, and the enzyme had low catalytic activity towards the peptide which has a phenyl group on its first amino acid.Pro-WJ39 not only had wide temperature adaptability, but also had a high catalyticactivity with low Ea even at low temperature. In additional, it had an excellent tolerance toward many heavy metal ions. Based on such characteristics, the enzyme should have a potential value in the industrial applications.