Isolation, Identification And Optimization Of Fermentation Conditions For The Lipase Production Of Serratia Marcescens Lipa1318 And Expression Of Its Lipase Gene

Since the extensive application of lipase in food, medicine, detergent, cosmetics,biodiesel synthesis and other fields, it has attracted much attention. Discovery of new microbial resources with high potential yield of lipase and lipase gene, exploration the utilization of lipase will be of great importance for research on lipase and its application.This paper mainly research on the following aspects:(1) Screening and identification of lipase producing strain Serratia marcescens Lipa1318: Using the assay methods on tween plate and olive oil plate, 57 lipase producing strains were isolated from 122 soil samples collected from Hebei province. A lipase producing strain with the activity of 2.56 U/m L, named Lipa1318, was obtained by rescreening with p-NPP assay method. It was identified preliminarily as S. marcescens according to the results of morphological features, the physiological and biochemical characteristics. A 1535 bp long 16 S r DNA was amplified by PCR and sequenced. The 16 S r DNA sequence was compared with that of others published on website of NCBI resulting in the generation of a phylogenetic tree through the software MEGA 4.1, which clearly confirmed the former conclusion that this strain was S. marcescens. Therefore, the strain was designated as S. marcescens Lipa1318. The optimum temperature and p H value,thermostability and p H stability of crude enzyme of the strain Lipa1318 were tested. The results demonstrated that the optimal temperature and p H were 60 °C and p H8,respectively. The highest activity of lipase reached 3.56 U/m L under this condition. The lipase would retain about 90% activity after incubation at 50 °C for 1 h and 70% activity after incubated at 30 °C for 2.5 h under p H value of 5-11. All the evidence demonstrated that this lipase was a thermostable alkaline lipase with good thermal stability and p H stability.(2) Optimization of the fermentation conditions for lipase production of S. marcescens Lipa1318: The fermentation conditions for lipase production were optimized using single factor test and response surface design software(Design-Expert V8.0.6.1). The results showed that the optimal fermentation medium for the strain Lipa1318 to produce lipase contained 1% beef extract, 0.31% glucose, 0.15% yeast powder, 0.54% Triton X-100, 0.50mmol/L Ca2+. The highest lipase activity was up to 13.95+0.07 U/m L when it was cultured at 30 °C for 96 h with an inoculation amount of 4% in a 300 m L Erlenmeyer flask with 60 m L fermentation medium. When the stain was cultured in a 10 L fermenter under the optimal conditions described above, the lipase activity increased to 25.32 U/m L which was nearly 2 times of that obtained in the flask culture.(3) Cloning and expression of S. marcescens Lipa1318 lipase gene: According to lipase gene sequence of S. marcescens which have been release in Gen Bank, the primers for amplification of lipase gene of S. marcescens Lipa1318 were designed. Then the gene was cloned and named lip A with a Gen Bank accession number KJ669102. Lip A was 1845 bp long and the sequence alignment results indicated that it shared as high as 94-99%homology with other lipase gene of S. marcescens strains. The lip A gene encoded a protein consisting of 614 amino acids and the molecular weight was 65 k Da. Lip A protein had at least 2 amino acids that were different from that of other S. marcescens strains. The secondary structure and tertiary structure of lip A were predicted using molecular biology software. The results showed that the residues 484-539 located in the posterior segment of the lip A were conserved. The lip A gene was inserted into expression vector p ET-21 b and p ET-22 b and transformed into E. coli BL21(DE3). The results showed that lip A in vector p ET-21 b were likely expressed as inactive inclusion bodies, whereas it in vector p ET-22 b was expressed as the soluble protein with obviously activity. The expression of positive transformants containing the recombinant plasmid p ET-21b-lip A and p ET-22b-lip A could obtained under the optimal conditions that it was induced with IPTG at 25°C for 3 h and30°C for 5 h, respectively.After the p ET-22b-lip A induce total activity reached 6.6 U / m L,compared with the original strain increased by 2.5 times.