| Bacillus sp. BI-19 strain was isolated from hot spring water of Indonesia saved in the laboratory. The thermophilic microorganisms produce thermophilicenzyme in the hot spring water, and the thermophilic enzyme has excellent thermostability. In addition, it can avoid microbial contamination efficient, reduce reaction solution viscosity, and improve the solubility of the substrate and reaction rate. As a biological catalyst, thermophilic enzyme has unique characteristics in industrial production. The thermophilic microorganisms have particular genetic types, physiological mechanism and metabolites. Therefore, there are lots of theoretical and practical meanings through studying the microorganism of hot springs and its thermophilic enzyme.A kind of strain, named BI-19 with high ability of lipase-producing, was screened by a series of processes including diluted water samples, the streaked plate, rhodamineB plate test in Indonesia hot spring water saved in the laboratory. Depending on the identification of the gene and phylogenetic analysis of the phylogenetic tree, BI-19 was identified as Bacillus. sp. In order to improve proportion of Bacillus sp.BI-19 in the fermentation conditions, a range of individual factors (temperature, pH, carbon sources, nitrogen sources, metal ions, bacterial load, and tributyrase) have been selected one for each time testing first. Furthermore, three most influential factors for lipase activity, ammonium sulfate, sucrose and tributyrase, were identified through Plackett-Burman experiments designed by Minitab software. The path of steepest ascent was undertaken to approach the optimal region of the three significant factors. Based on designing and responding surface analysis, Box-Behnken would be adopted to further investigate the mutual interaction between the variables and obtain optimal values that bring maximum lipase activity. The optimal conditions for the maximal activity discriminated from the regression equation were: ammonium acid mass fraction of 0.65%, tributyrase mass fraction of 0.03%, and the concentration of Kcl tendency for 5.57/L.In order to improve the lipase activity and the proportion, two lipase genes were chose through using the method of constructing genomic DNA plasmid library, respectively named lip-0256 and lip-1954. In terms of lip-0256 and lip-1954, they were cloned and expressed by genetic engineering method first; then, the recombinant enzyme enzymatic was analyzed. We found the lip-1954 recombinant enzyme activity is higher, then run the SDS-PAGE electrophoresis, found 50% of the recombinant protein was soluble of recombinant expression vectors(pET-30(a+)+lip-1954), so the recombinant bacteria pET-30(a+)+lip-1954 as the strain to research enzymology properties. Because expression vector pET-30 (a+) with His label on, so purified by Ni-NTA chromatographythe, purified protein revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 30 kDa.The optimum temperature for the lip-1954 was 70 ℃, and enzyme activity remains at about 50% at 60-75℃. The lip-1954 prefers to active in a bit of acid environment. The optimum pH was 9.0 which illustrated that the enzyme belongs to alkaline lipase, and it was used more among washing liquid. The activity of lip-1954 was promoted by the addition of Na+、Fe3+、Cd2+、K+、Fe2+, and Fe3+ strongly promoted at 200% lipase activities. The activity of lip-1954 was inhibited by the addition of Cu2+,Sr2+,Mg2+, Ca2+,Pb2+espectively. Researching the organic solvent tolerance of lip-1954, the activity of lip-1954 was promoted by glycerol and n-butyl alcohol, and lipase activities were increased by about 2-fold by n-butyl. The activity of lip-1954 remains at about 60% when addition other organic solvents. |
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