Isolation And Identification Of Burkholderia Sp. SYBC LIP-Y And Its Cold-active Lipase Production By Fermentation

Lipase, which catalyses the hydrolysis of triacylglycerides played an important role in industrial catalyzation. To screen and investigate cold adapted lipase producing microorganisms has certain theoretical significance and potential applicable value.In this study, a new cold-adapted lipase producing strain was isolated and identified. Lipase produced by Burkholderia sp. SYBC LIP-Y were purified and characterized. The Effect and mechanism of alkanes on activity of lipase of SYBC LIP-Y were studied. Tthe lipase gene clone were reported in this study. The results were as follows:1) A producing strain, named SYBC LIP-Y, was isolated from the decayed seeds of Ginkgo biloba L. by screening with plates containing Victoria Blue B and with the flask-shaking fermentation. It was identified as Burkholderia species. The GenBank accession number of the 16S rRNA gene sequence was FJ392830.2) Response Surface Methodology (RSM) was used to optimize immersion fermentation conditions for cold-adapted lipase production by Burkholderia sp. SYBC LIP-Y. Through Plackett-Burman design some process factors of lipase production were evaluated and three factors had prominent effect on lipase production were picked out, which were beef extract, olive, and Triton X-100 respectively. The path of steepest ascent was used to approach the optimal region of lipase production subsequently. Then, the optimal combined concentration for maximum enzyme activity were further optimized by response surface methodology and determined as follows:beef extract 31.8 g/L, olive 21 mL/L, and Triton X-100 36.55 mL/L. The lipase activity reached 61.52 U/mL, which was 2.62 folds of that before optimization. The production of extracellular lipase from Burkholderia sp. SYBC LIP-Y was studied in 5 L fermentor. The optimizations of fermentation conditions were determined as follows:the seeded volume 10%, Triton X-100 5 mL/L, olive 10 mL/L, beef extract 40 g/L, fermentor agitation rate 300 r/min, ventilation amount 3.5 L/min. The maximum lipase activity was achieved in 5 L fermentor with fermentation time 36 h which was equivalent to that obtained in the flask fermentation. The fermentation time shortened 6 hours.3) The lipase properties were determined after purification by aqueous two-phase system, DEAE -52, and Sephadex G-75 filtration. The optimum pH and temperature of the purified enzyme were 10.0 and 30℃respectively. About 80% of the original activity is maintained by heating at 40℃for 60 minutes. The lipase belonged to the cold-active lipase. It was proved to have good temperature stability and was resistant to alcohol; the molecular mass of lipase was 36 kDa; lipase was strongly inhibited by Cu2+ and Zn2+, but slightly promoted by Na+ and Mg2+; nonionic surfactants Tween-40 and Tween-60; the best substrates of the lipase was p-NPB; the Km value, Vmax, and Kcat was 0.120 mmol/L, 400 U/mg, and 240 S-1, respectively.4) The impact of the short-chain alkanes and long chain alkanes on the lipase production of the strain Burkholderia sp. SYBC LIP-Y by liquid state fermentation was investigated. The results showed that lipase production was inhibited obviously by short-chain alkanes heptane, octane and decane but significantly promoted by long-chain alkanes dodecane, tetradecane, hexadecane. Lipase production of the strain Burkholderia sp. SYBC LIP-Y by liquid state fermentation when 4% (v/v) dodecane was added was about 1 fold of that of the control. May be due to the role of long-chain alkanes in the cell membrane, thus starting the antioxidant mechanism to facilitate the signal transmission, the mechanism of lipase high production of Burkholderia sp. SYBC LIP-Y maybe related to antioxidant mechanisms.5) The lipase gene was obtained by PCR. Its nucleotide sequence showed that the lipase was encoded by a 1,095 bp major open reading frame, and the deduced protein contained 364 amino acid residues. The deduced amino acid sequence contained a lipase consensus sequence GXSXG. The lipase gene had high homology with those of strains in genus Burkholderia. The lipase had a signal peptide. The tertiary structures were got by SWISS-MODEL. The gene encoding lipase of Burkholderia sp. SYBC LIP-Y was amplified by PCR and cloned into vector pET-22b. The Recombinant plasmid pET22b-lipase was transferred into E. coli BL21 and expressed successfully, although some of the expressed protein was desipotted in inclusion bodies.