Strain Breeding For High Lipase Productivity And Kinetic Research On Enzymatic Biodiesel Synthesis

Microorganism screening for high lipase productivity, lipase purification, lipaseimmobilization, lipase application in biodiesel synthesis were discussed.The main points were as follows,1. Lipase high-yield microorganism(Candida, Bacillus subtilis, Aspergillus oryzae,Pseudomonas sp.) were screened, which were selected from oil-polluted soil. Micro-organisms were cultured for 2 days after the elution of 100, 000-fold, of which the bestshowed high ability of hydrolysis of fatty acid glycerides. Lipase productivity was im-proved 10-fold by mutation methods of UV/NTG treatment. Several strains were mutage-nized by the treatment of ultraviolet radiation and the most suitable time for the treatmentwas 30 s. In some cases, NTG was used to strengthen the effect of mutageni-zaton. Fourstrains with high lipase productivity were selected and assigned as Candida 05, Bacillussubtilis 04, Aspergillus oryzae 09 and Pseudomonas sp. 07, respectively.2. Microorganisms were cultured at the best conditions, which resulted in high lipaseproduction(10.8 IU/mL in 13 samples of 20, 11.97 IU/mL in 5 samples of 20) after oneday fermentation in 50 mL container at 35℃if incubated with 0.1 million strains.3. Strains with high lipase productivity were analyzed at nucleic acid level withspecial primers.4. Extracellular lipases were purified with sodium sulfate and columnchromatography.A column chromatography method was designed to get rid of the impure proteinsmixed with lipase from fermentation. The results showed that the primary treatment ofthe crude samples with ammonia sulfite precipitation was effective, and about 20%impure proteins were removed in the precipitation step. Further, continuous elutioncolumn chromatography was also applied as a preparative technique for attaining thehighly pure lipase. During the continuous elution column chromatography, the enzymeswas eluted and 5, 7-fold purification was achieved in a yield of about 55%. The two stepsmay finally yielded an electrophoretically homogeneous enzyme.5. Carriers were selected for lipase immobilization, of which diatomite, ion-exchange resin were better than DEAE-cellulose, Al₂O₃, nano-carbon tube.Mathematical models were used in explaining the mechanism of adsorption competition.6. The optimum conditions for immobilization were similar: dosage of lipase 500u/g carrier, temperature 30℃, pH 8.0, 120 min.7. Synthesis of biodiesel catalyzed by lipase from Candida, Bacillus subtilis,Aspergillus oryzae, Pseudomonas sp. were investigated, especially on effects of factorssuch as species of lipases, reaction temperature, initial ratio of reactants, dosage of lipase,pH value, composition of oils.The optimum reaction conditions for transesterification catalyzed by lipases fromCandida, Bacillus subtilis, Aspergillus oryzae, and Pseudomonas sp. were similar: dosageof lipase 500 u/g oil, molar ratio of rapeseed oil to methanol 1/3, reaction temperature 40～50℃. Di-glyceride was favored to mono-glyceride in initial product.Reaction course, theory of lipase adsorption, lipase specificity on substrate, kinetics,and the application of enzymatic synthesis in biodiesel were investigated. The tests oflipase productivity, lipase purification, lipase immobilization, and lipase application inbiodiesel were carried out, which indicated that lipases from the above microorganismswere able to catalyze the transesterification reaction of rapeseed oil with moderate oleicacid content.Lipase from Candida exhibited certain 1, 3-specificity on the substrate of glyceridein this order, C_12:0,C_14:0＞C_16:0,C_18:0＞C_18:1＞C_18:2,C_18:3; The reaction rate constantscorrelates well with the temperature. Lipase specificity to oleic group was observed in thereaction catalyzed by lipases from Bacillus subtilis, Aspergillus oryzae, Pseudomonas sp.Achievements of this study:1. Four strains with high lipase productivity were selected, whose lipase productivitywere improved with mutagenization method; The optimum culture conditions wereachieved;2. Four strains with high lipase productivity were analyzed on nucleic acidsequences with special primers;3. Lipases from Candida, Bacillus subtilis, Aspergillus oryzae, and Pseudomonas sp. were effective in biodiesel preparation from rapeseed oils with moderate oleic acidcontent; 80% activity was retained after 10 days in operation at the preferential conditions(low than 45℃, low stirring speed, etc.).4. Mathematical models were used in explaining the mechanisms of competitiveadsorption and enzymatic reactions, which illustrated the kinetic equations.