Breeding Of Strains With High Activity Of Organic Solvent Tolerant Lipase Production And Its Characteristics

In 1984, a report about catalytic reaction and thermal stability of lipase on organic phase by ZaksA and KlibanovA.M was attracted considerable public concern.The use of enzymes in organic media with low water content has been one of the most exiciting facets of enzymology in recent times. and then, Domestic and foreign scholars Studyed on the isolation and screening of strains which can produce Lipase, Lipase Preparation, Cloning of lipase gene. The biodiesel is a kind of liquid fuel which produced by transformation Using the zoology and botany fat as material,it is enviromental protection renewable energy. Biodiese Employed by enzymatic synthesis Has the advantage of mild reaction conditions, no pollutant discharge, etc., developments of biodiesel have great significant to promote the energy substitute, control air pollution,protect the ecological environment.But in process of enzyme preparation for biodiesel reaction,enzyme is very easy to deactivation,half-life is short,thus caused the production costs too much, This paper On the basis of isolating of high activity organic solvent tolerant lipase,studied the ferment conditions,Enzymatic properties and biodiesel production process,and then carried out the following aspects:(1) Isolation and screening of organic solvent tolerant lipase strains:we collected 30 soil samples from Daqing Oilfield and Engineering and Technology college Repair shop of Jilin agriculture university, through a series of steps inculding plate isolation using olive oil as substrate bromocresol purple and rhodamine B as indicator,20% toluene as the pressure of selection,flask fermnetation and detection the lipase activity by pNPP method, six lipase producing strains with higher lipase activity was screenen,named sw1, sw2, sw3, sw4, sw5 and sw6 respectively.(2) mutation breeding:Selection of organic solvent tolerant lipase producing strain with UV+DES. screened a mutant strain UD-8 of high lipase activity by UV of 354nm,vertical distance 20cm,treatment time 90s.and then treated with DES 0.5% of 40min on sw6.the UD-8 liase activity was 5.89 times than sw6.the strain of UD-8 was confirmed as Bacillus cereus according to 16SrDNA, morphological characteristics, physiological and biochemical characteristics,etc.(3) To improve Bacillus cereus UD-8 lipase production for later study,flask fermentation conditions of Bacillus cereus UD-8 lipase was studied by Response Surface Method and Orthogonal design,the Optimum medium was as follows:1% starch; yeast extract 0.64%; NH4NO3 0.384%; MgSO4·7H2O 0 .1%; NaCl 0.25%.the Optimum culture conditions was as follows:pH 6.5, temperature is 35℃,55ml medium in 250ml flask,4%inoculation, Bacillus cereus UD-8 lipase was 5.26 times then That before optimization(4) Bacillus cereus UD-8 liPase was Purified to homogeneiy detected by SDS-APGE.The proceed including amminum sulphate fraction precipitation,ultra filtration, DEAE-sephrose FF,molecular weight o UD-8 liPase was 34kd. The optimum temperature is 40℃, the optimum pH was 7.0, it was stable in pH 6.0-10.0. Various compounds influenced the lipase activity. k+, Na+, Mg2+, Ba2+were activator, Al3+, Cu2+ Zn2+, Ca2+, SDS and EDTA were inhibitor.Fe2+had no effect on UD-8 lipase.the lipase had good tolerance o toluene, cyclohexane, ethanol.UD-8 lipase was not a metal dependant enzyme. disulfide bonds act a littl action onmaintaining the structure of UD-8 lipase, it may be a serine proteases.(5) Studied the factors of Making Biodiesel by lipase,the results showed that:tertbutyl alcohol was th best reaction medium, ratio of methanol and oil was 3:1, the reaction temperature was 35℃,10% addition o lipase, reaction time 6h.