Identification And Culture Conditions Of A Lipase-Producing Halophilic Strain And Its Halophilic Mechanism

Halophiles are important communities of Extremophiles group. As a new kind of microbiology resources with application foreground, halophiles possess peculiar physiological structure and metabolism mechanism, and they also can produce many componds with bioactivities. Lipase, the important industrial enzyme, is obtained from extreme environment that has become a method to meet the industrial different needs.In this thesis, by means of Rhodamine B plate coloring method and Tween 80 plate depositing method, a kind of producing lipase halophile JNPH-3 was screened from the samples isolated from Hypersaline lakes of Inner Mongolia. Its cells are round and Gram-negative. Colonies on agar plates containing 20% (w/v) total salts were red, smooth-faced, elevated and round (dia 0.2~0.8 mm). This microbe could grow in medium containing 9~30% NaCl, optimal salinity at 12~27%, optimal temperature at 45℃, pH at 6.0~10.0 while optimally at 7~9.5, and it also can bear high concentration of Mg2+. After its morphology, physiology, biochemical features and 16S rDNA sequences were characterized, this organism was identified as Halorubrum aidingense JNPH-3, which belonged to the group of extreme halophiles.The optimum temperature of the lipase produced by JNPH-3 was 37℃, and the optimum pH was 7.5~9.5, while the presence of high salinity was necessary to show its activities. This lipase was a kind of constitutive enzyme, namely when there weren't any oleaginous revulsants in the surroundings, this enzyme also could be produced. However, the presence of most revulsants restrained the growth of JNPH-3 which led to the lipase production declined. The yield of this enzyme was positive correlation with the growth of its generator, and it could come to maximum when the cultivation time was 60 h and then decline. In all the carbon and nitrogen resources which had been investigated, the organic carbon and nitrogen resources could better advance JNPH-3's growth and lipase produce, while single inorganic carbon and nitrogen could slower this microbe's growth and the lipase produce.The growth of JNPH-3 specially depended on high concentration of Na+ which meant that other positive ions couldn't sustain its growth, but when other negative ions instead of Cl-, it could faintly grow. The variety of K+ concentration in culture medium had no obvious effect on its growth, but in the cytoplast, the concentration of K+was much higher than outside and it also rose following the increase of salinity in culture medium. Dissociative acidic amino acids (Glu, Asp) were high proportion in cytoplast, and Glu could be concerned with the regulation of osmotic pressure. Through analysis of HPLC, JNPH-3 could produce (or accumulate) ectoine in the complex medium and the production of ectoine could regulate the osmotic pressure in the low salinity.