| Cyclopentadecanolide belongs to a kind of macrocycle musk, it is widely used in fields of perfume, fine chemicals, and food etc.. About 50% tetracosenoic acid was found in malana oleifera chum oil-a special plant in Guangxi provience, China. The acid is an ideal material to synthesize cyclopentadecanolide. Although a lot of chemical synthesis methods for cyclopentadecanolide have been reported successfully, such harsh conditions as high temperature and high pressure proved to have restricted its industrialization. As a biocatalyst, lipase was widely used in organic synthesis, fine chemical and food processing fields for their high efficiency, high selectivity and high specification in mild conditions. In recent decades years, more and more researches have turned to concentrate on the technology of enzyme catalysis in macrolide, while biological method for systhesis of cyclopentadecanolide has not received much attention.One kind of screened lipase was used to catalyze the synthesis of cyclopentadecanolide from methyl 15-hydroxy-pentadecanate,which was made from material of malana oleifera chum. The research mainly concluded the following five aspects:(1)Screening the strains and optimizing the fermentation conditions. (2) Seperating and purifying the lipase to a level of chromatography purity. (3) Caculating dynamics parameter and derivating dynamics equation. (4)Comparing the lipase-catalyzed reaction between organic system and biphasic system and optimizing reaction conditins. (5) Derivating reaction process during two systems.After uv-mutation,Candida sp.GXU08 out of ten lipase-produced strains was obtained to have high orienting lactonization abilities to synthesize cyclopentadecanolide. The fermentation conditions of Candida sp.GXU08 lipase were optimized through orthogonal experiments. It showed that:The compositions of optimal fermentation medium were sucrose 0.5%,starch0.5%, peptone 1.5%, K2HPO40.05%, MgSO40.15%, (NH4)2SO41%, camellia oil 1.5%, rapeseed oil 1.5%, pH=8.5,fermentation time 48h. And the fermentation time, peptone, camellia oil and pH had dramatical effect on lipase activity of fermentation. Under the optimal fermentation condition, the lipase activity would reach 27.53 U·mL-1, which was 3.74 times of the original culture medium.After (NH4)2SO4 fractionation precipitation, CM positive ion cellulose chromography, Sephacryl-S400 gel-chromography and Sephadex-G100 gel-chromography,the lipase was seperated and purified to a level of chromography purity with one protein peak and had high catalytic ability,which would help to the study of lipase character and dynamics parameter in the future. It was found that the specific activity of lipase was 14.50 U·mg-1 ,the purification fold was 1.27, and the recycle yield of liapse activity was 89.25% after (NH4)2SO4 fractionation precipitation. Three obvious protein peaks were found after CM ion-exchange cellulose chromography, and the specific activity of lipase was 49.50 U·mg-1,the purification fold was 4.33, the recycle yield of liapse activity was 70.49%. Two obvious protein peaks were found after Sephacryl-S400 gel-chromography, and the specific activity of lipase was improved to 154.50 U·mg-1,the purification fold was 13.48, and the recycle yield of liapse activity was 53.28%. Only one protein peak was found after Sephadex-G100 gel-chromography, and the specific activity of lipase achieved 344.97 U·mg-1,the purification fold was 30.10, and the recycle yield of liapse activity was 41.75%.The dynamics equation of catalytic reaction from GXU08 lipase was derivated as the following:v=(2.413x10-6[S]/1.86+[S] (the units of v and [S] were mol.L-1min-1 and mmol.L-1), It was caculated that the Michaelis-Menten constant Km was 1.86 mmol.L-1 and the maximal reaction velocity Vmaxwas 2.413×10-6 mol·-1.min-1. The result showed that real velocity met with the Michaelis-Menten model well. So the catalytic reaction accorded with the derivated dynamics equation.The catalytic reaction of GXU08 lipase in organic system was studied. The quality of cyclopentadecanolide would be the largest under the optimal reation conditions:Csubstrate= 8 mmol·L-1, T=40℃,ω=180 r·nin-1, pH=6.5, lipase activity=1600 U and t=72 h. It also showed that the larger the dielectric constant was, the smaller the quality of cyclopentadecanolide made by unit lipase was in organic solvent, so did the contrary. The lipase showed best catalytic capability in hydrophobic media of cyclohexane and had good speciality to methyl 15-hydroxy-pentadecanate.Ultrasonic technology was firstly used both in organic system and water/organic solvent biphasic system during the lipase catalytic reaction to systhesize cyclopentadecanolide and received good catalytic effect. The largest quality of cyclopentadecanolide would be 50.284×10-5 mg.U-1 under the optimal reation conditions:Csubstrate= 8 mmol.L-1, T=40℃,ω=180 r·min-1,pH=7.0,lipase concentration 2.0 g·mL-1,0.5 mL and t=72 h if the reaction was operated in cyclohexane. While the largest quality of cyclopentadecanolide in organic system was only 29.02×10-5 mg.U-1. The former was 1.733 times of that of the latter.It was found that the quality of cyclopentadecanolide would greatly increase if the lipase solution or reaction system was reused once. What's more,the quality of cyclopentadecanolide in biphasic system was always higher than that in organic system. So the lipase powder could be replaced by lipase solution to catalyze the synthesis of cyclopentadecanolide, which would save the process of freeze drying and improve the quality of cyclopentadecanolide.The lipase catalytic reaction in organic system was obtained through the experiment as following:It verified that methyl 15-hydroxy-pentadecanate would directly cyclize into cyclopentadecanolide in organic system.While the reaction in biphasic system was:It showed that methyl 15-hydroxy-pentadecanate would hydrolysis into hydroxypentadecanoic acid firstly, and then hydroxypentadecanoic acid directly cyclized into cyclopentadecanolide.
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