Screening And Classification For High Cold-adapted Lipase-Producing Strains And Optimization Of Its Fermentation Conditions

Cold-adapted enzymes have recently received increasingly attention, due to theirrelevance for both basic and applied research. Most of them were produced bylow-temperature microorganism. It's very significative to exploiture valuableenzyme-yielding microbes of low-temperature environment for enriching the resource ofcold-adapted enzymes. In this thesis, 89 lipase-producing bacterial strains were isolatedfrom refrigeratory which stores meat by the combination of Tween-80 agar and oliveoil agar, in which, the lipases of 9 strains were proved to be cold'adapted lipase by therepeated screening of p-NPP. Finally, The high lipase-producing strain Lip35 wasisolated. Through comparation of the two screening methods, a simplier, faster and moreprecise screening method was established.The strain Lip35 was identified. Through optic microscope and scanning electronmicroscope, the Lip35's modality, size were observed. Modality, size, color of colonyboth in Tween-80 agar and agar were described.Through the traditional physiological andbiochemical test, together with the 16sRNA sequence and phylogenetic analysis, thestrain Lip35 was finally ascertained to bepseudomonas (pseudomonas Lip35).The fermentation conditions of pseudomonas Lip35, including carbon source, nitrogensource, inorganic salt, substrates, products and so on,. were investigated in shake flasklevel. It was found that the concentrations of yeast-extract, soybean powder, NaCl,Tween-80 had the more obvious influences. The orthogonal test of the four factorsshowed that the optimized culture medium was: yeast-extract 0.7ï¼…, soybean power 6.0ï¼…, NaCl 0.1ï¼…, Tween-80 0.5ï¼…. Through shake flask experiments, itwas acquired thatthe best culture conditions: pH 9.5, 20℃, 250 r/min, 40 mL/300 mL, and 5ï¼…(V/V) ofbacterium culture (OD₆₀₀=2.100-2.400), for 67 h. Under such conditions, the lipaseactivity reached 58.9 U/mL, which was 35～40 times of the initial strain.The lipase characters of the fermentation liquid was researched, obtained resultshowed that: the optimal reaction temperature of Lip35's cold-adapted lipase was 20℃and the enzyme was thermal lability, only 10ï¼…of its activity was remained after 5 minincubation at 60℃and higher temperature. The lipase showed high lipolytic activityfrom pH 7.5 to pH 8.0 and its optimal pH for activity was 8.0, belonged to the alkalinelipase. The test of lipase pH stability indicated that the lipase of Lip35 was more stable inpH 6.0-9.0. The lipase activity was inhibited by K⁺ (about 50ï¼…of the activity was lost),but was stimulated by Ca²⁺ (more than four times of the initial activity was achieved).The EDTA test testified that this lipase was not Ca²⁺ dependent enzyme.