Constructions Of Lipase Producing Strains Library And Screening Of PLD Producing Strains

As one of the major enzyme in industry, lipase can catalyze the hydrolysis, synthesis, transesterification, alcoholysis, acidolysis, ammonolysis reaction, is widely used in the fields of food, medicine, textile, feed, washing, papermaking, biodiesel and environmental protection. Compared with animal and plant sources with lipase, microbial lipases have advantages for extensive sources, easy preparation, strong substrate specificity, high catalytic efficiency, mild reaction conditions, less by-products. Along with the expansion of the scope of application of lipase, the specific requirements also gradually improve; screening and obtaining specific lipase strain from nature is a simple and feasible method.Screened the 538 lipase stains from the 19 copies of inland soil samples and 22 sediment samples collected from the South China Sea using the of Victoria blue and saponification calcium circle. Rescreening depended on the specific enzyme activity assay, the hydrolytic activity of lipase by p-NP method, phospholipase D enzymes activities use the enzyme linked colorimetric method, esterification by palmitic acid ethyl ester method, the transesterification activity of the DHA glyceride method. The results of rescreening show that 295 strains have the hydrolysis activity of lipase,293 strains have the PLD hydrolysis activity; 138 strains with the ester synthesis activity; 23 strains with the transesterification activity; 57 strains with high hydrolytic activity; 55 strains have high PLD hydrolysis activity; 3 strains with the higher ester synthesis activity. The degree of correlation hydrolase and PLD enzyme was 45.08%, hydrolase and ester synthase related degree is 27.5%, ester synthase and transesterification correlation degree is 82.1%.We use 16S rDNA method to identify the strains screening from the samples of South China Sea. The statistical results of systematic distribution showed Gamma proteobacteria was the absolutely dominant micro flora of lipase producing strain in South China Sea. Psychrohacter sp.. Bacillus sp., Pseudoalteromonas sp. was the dominant genera for lipase producing.No.538 strain in the lipases library was identified as Stenotrophomonas maltophilia by 16S rDNA. By means of ammonium sulfate precipitation. DEAE-sepharose fast flow and gel filtration chromatography.we gain a single pure protein band. With method of protein sequencing, the single protein was identified as I lipase with GxSxG conserved sequence, whose relative molecular mass is 41.8 KD and isoelectric point is 8.86. Study for the enzymatic properties indicates that the optimum temperature for the lipase catalyzing is 40? and the optimum pH ranges from 7 to 8. The enzyme activity maintains stability under 40?, and the lipase maintains high enzyme activity in the range of pH 5-12. But Fe3+ and SDS can remarkably restrain the activity of the lipase.Phospholipase D (EC 3.1.4.4, PLD). can catalyze the hydrolysis of the fourth ester bond which was constructed by phosphate and organic base (such as choline, ethanolamine etc.) in phospholipid molecules. At the same time, it can also be use in phospholipid modified. Through catalyze the basic group exchange between various hydroxyl compounds and phospholipid form new phospholipid. But at present most of the report PLDs have high hydrolysis activity and low acyl transfer activity. This situation limits PLDs use in the preparation of single phospholipid and rare phospholipid and other phospholipids modified process. This study screens the stain producing PLD with acryl transfer activity from lipases stain library and natural environment. Screening from lipases library is divided into two steps. The first step is the determination of the hydrolytic activity; the second step is PS synthesis detection. Screening from soil samples consists of three steps. The first step plates detection; the second step is determination of fermentation liquor hydrolysis activity; the third step is synthesis of PS detection. Through a large number of screenings, we obtained a total of 6 strains with ability of synthesizing PS activity. Of which a2 has the application prospect. The 16S rDNA identification result shows it is Acinetobacter radioresistens.Using H103 ions exchange chromatography partial purification of a2 PLD.the success of the relative molecular mass of about 40KD protein by one-step purification.