Screening Of A Thermostable Lipase Producing Bacterium And Its Biological Characterization

Lipase is a special ester hydrolase, which can catalyze hydrolysis of triglyceride ester to generate diglyceride, monoglyceride, glycerol and fatty acids. Lipase has been widely used in oil processing, food industry, Pharmaceuticals and fine chemical synthesis due to its function of esterification, interesterification, and transesterification reaction. In the1990s, scientists began to study biological preparation of biodiesel, and developed a variety of lipase mediated conversion process instead of chemical catalyst. Lipase has becoming one of the most important groups of the industrial enzymes.A total of eighty-seven lipase producing bacteria strains were isolated from the different soil samples by the Tween-80nutrient agar plate method, nineteen of which was proved to produce lipase more effectively. Strain Mhy-1was selected and characterized further. On the screening medium plate, the strain formed circular, smooth, transparent, yellow colonies after one day incubation at30℃. It was Gram-negative and no spore was observed. The strain Mhy-1was preliminarily identified as Aeromonas salmonicida by morphological characteristics, physiological and biochemical, and16S rDNA sequence analysis.The cultural condition and fermentation technology of adapted lipase produced by stain Mhy-1was studied, The growth curve of the bacterium was obtained in LB liquid medium with0to10h of logarithmic phase, with the best culture time for the lipase production at36h, The optimum temperature and pH for cell growth were35℃and pH6.0-9.0. The optimum temperature and pH for the lipase production were30℃and pH9.0. The enzyme was preliminarily identified as an alkaline lipase.The lipase was purified from the culture supernatant of the strain Mhy-1by means of ammonium sulfate precipitation, column chromatography on DEAE-Sepharose fast flow. Studying characterization of the lipase showed that the enzyme appeared to be a single peptide of34kDa on SDS-PAGE. The optimal temperature and pH for the enzyme reactivity were45℃and8.0respectively. The enzyme was stable at temperatures of 30℃-70℃and pH7.0-9.0. The enzyme showed excellent thermostability when it was remained at70℃for half an hour. Addition of O.lmM of Ca2+increased the activity to115.25%, but addition of1mM of Zn2+、Co2+and Mg2+decreased the activity to88.81%、97.45%and94.91%respectively. Addition of1mM of SDS, EGTA and EDTA also inhibited the enzyme activity, but PMSF showed no significant effect on the activity of the enzyme.The immobilization conditions for purified enzyme by silica alginate were optimized. The studies have shown that the purified enzyme from stain Mhy-1could be used to catalyze the production of biodiesel with a transesterification rate of21.48%.