Low-temperature Lipase Isolation, Purification And Enzymatic Properties

A pychrotrophic bacterial strain producing cold-adapted lipase was isolated from a refrigerator of a meat factory in Kunming. Based on morphological, physiobiochemical characteristics, the isolate strain was identified as one strain of Serratia species, and named as Serratia sp. KM1. The lipase produced by this strain was named as Lip-KM1.Lip-KM1 was an extracellular enzyme. We also find the optimal condition of the fermentation is: 13°C, pH 7.2, 52h.The lipase was purified to homogeneity by ultrafiltration, (NH₄)₂SO₄ precipition, Sephacry^?HRS-100 and Superdex G75 gel chromotograph. The specific activity of purified Lip-KM1 was increased from 42.06U/mg to 1093.4U/mg with yield of 10.3%. SDS-PAGE and Superdex G75 analysis indicated that the molecular mass of Lip-KMl is estimated to be 34.3kDa.The characterization of Lip-KMl was also completed. The enzyme was stable at temperature of 0°C～25°C and pH of 7.2-10. The optimal temperature and pH for this enzyme were 37°C and 9.0, respectively. Besides optimal substrate of pNPB, Lip-KM1 can hydrolyze olive oil, so Lip-KMl can be identified as a cold-adapted lipase. The enzyme also can hydrolyze some p-nitrophenyl esters, among used ester compounds, Lip-KM1showed high catalytic activity towards the esters with C₄～C₁₂. It indicated that the enzyme optimal ester substrates was the length of C₈.The activity of Lip-KM1 was activated by Na⁺, Mg²⁺, Ca²⁺, Mn²⁺, and Ca²⁺ was the best activator for Lip-KM1. Cu²⁺ Zn²⁺ inhibited the activity obviously. SDS, EDTA, PMSF partially inhibited the activity of Lip-KM1. Acetonitrile, methanol, ethanol and DMSO can slightly activate the enzyme at low concentration. The enzyme was stable in various organic solvents, it could keep nearly 50% activity even at 50% DMSO, methanol, ethanol.The enzyme has high affinity and catalytic activity towards pNPB at 37°C, The K_m,K_cat,, K_cat/K_m, E_a, were calculated as 16.58mmol/L, 1.03×10⁴S^-1, 6.21×10²mmol^-1·L·S^-1, 31.0 KJ/mol respectively.The enzyme showed an excellent tolerance to high concentration of organic solventsand metal ions, so the enzyme had valuable potential in the industrial applications.