Screening Of A Low-temperature Lipase-producing Pseudomonas Sp. Strain And Culture Condition Optimization For Producing The Low-temperature Lipase

Since 80% of the chemosphere was lower than 5 ℃, the low temperature environment has a more extensively influence than the high temperature. Low temperature microorganisms, including psychrophiles and psychrotrophs, were widely distributed in natural environment, such as, rivers, seas, iceberg, mountain, arctic and Antarctic, deep holes and refrigeratory, cold storage and freezing food. These low temperature microorganisms were diverse in both amount and species. Most of the low temeratature microorganisms could produce low temperature enzymes. Low temperature enzymes performed better than any other normal temperature enzymes under low temperature. This leads to their widely potential application in bioengineering. It was very important for the research on the low temperature microorganisms both in theory and application.The 25 low-temperature strains used in this research were colleted from 4 soil samples of Huazhong Agricultural University, 6 soil samples of Yangtze River Wuhan region, 30 water samples of Yangtze River Wuhan region, 45 water samples of Taohua River, Guilin, Guangxi Province.Low-temperature lipase producing strains distributed widely in the natural environment. Lipid hydrolysis test proved that the 12 of 25 screened low-temperature strains could hydrolyse lipid under 10 ℃ and couldn't do that under 37 ℃. A low-temperature lipase producing strain, YT34-8, was got by the primary screening and the re-screening and was used in the following researches.The colony of the strain YT34-8 on LB culture medium after cultured under 15 ℃ for 72 h, was white, round, uplifted, opaque, smooth, moist and has a wave like edge with transparent halo around the colony (the dimension was about 5 mm). The strain was gram negative, rod shape and locomotion, facultative anaerobic. Biochemistry and physiology test showed that the best temperature for its growth was 28 ℃. It could also grow well at 4 ℃, and it could not grow above 37 ℃. It could grow with out NaCl. It was oxidase positive, haptoase positive, gelatin liquefaction positive, starch hydrolysis negative, denitrification positive and lipase positive. It could utilize glucose and fructose. The 16S rRNA has a 98% similarity with Pseudomonas. The strain was proved belonged to Pseudomonas genus by the further biochemistry and physiology tests.The culture condition of the Pseudomonas YT34-8 for the cold-adapated lipase producing was optimized. Firstly, 11 factors for enzyme producing were screened by single-factor experiment and the former research. Secondly, 3 most important factors (olive oil concentration, volume coefficient, flask rotate rate) for enzyme producing were screened by Plackett-Burman method. Finally, the 3 facotrs was furtherly optimized by response surface analysis method. The optimum composition of the culture medium was: 2% corn steep liquor, 2.5% soybean meal, 1% sugar, 0.57% olive oil, 0.05%...