两种体系中的15-羟基十五烷酸甲酯酶催化合成环十五内酯及动力学研究
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摘要
环十五内酯属于大环内酯香料的一种,它在香水、精细化工品、食品等领域有广泛应用。中国广西特产植物蒜头果的油脂中含有50%的二十四碳-15-烯酸,它是合成麝香类香料—环十五内酯的理想原料。很多化学方法已经成功合成了环十五内酯,但能耗大,反应条件苛刻等缺点阻碍了其工业化生产。脂肪酶作为一种生物催化剂,在温和的条件下具有高效、高选择性及高特异性的催化作用,被广泛应用于有机合成、精细化工及食品加工等领域,有关脂肪酶催化合成大环内酯方面的研究已有几十年,但酶催化合成环十五内酯的研究一直未受到关注。
本文以自行筛选到的微生物产脂肪酶,催化蒜头果为原料制得15-羟基十五烷酸甲酯合成环十五内酯。主要开展了五个方面的研究：(1)脂肪酶菌株的筛选及正交法优化发酵条件；(2)将粗脂肪酶纯化为层析纯的酶；(3)酶催化动力学参数的求取及动力学方程的推导；(4)有机溶剂体系与水/有机溶剂两相体系中酶催化反应的对比及反应条件的优化；(5)两种体系中酶催化反应历程的推断。
实验筛选到10株高产脂肪酶菌株,其中经紫外诱变后的菌株Candida sp.GXU08(简称GXU08)对15—羟基十五烷酸甲酯转化为环十五内酯具有良好的催化性能。通过正交试验,得到菌株最佳培养基配方及发酵条件为：蔗糖0.5%,淀粉0.5%,蛋白胨1.5%,K2HPO40.05%,MgSO40.15%,(NH4)2SO41%,茶油1.5%,菜籽油1.5%,pH=8.5,发酵时间48 h,其中发酵时间、蛋白胨、茶油和pH值对发酵液酶活的影响较显著。该优化条件下所得发酵液的酶活为27.53 U·mL-1,是优化前的3.74倍。
实验分别采用硫酸铵分级沉淀、CM-阳离子纤维素柱层析、Sephacryl-S400凝胶层析及Sephadex-G100凝胶层析分离纯化GXUO8脂肪酶,纯化后只出现一个蛋白峰,得到了层析纯的酶,且分离出的脂肪酶具有较高的内酯化能力,可为今后研究酶学性质及动力学参数奠定基础。
发酵液经40%～70%的(NH4)2SO4分级沉淀粗分离后,酶比活力为14.50 U·mg-1,纯化倍数为1.27,酶活回收率为89.25%；经CM-离子交换层析分离后,出现了3个较明显的蛋白峰,酶比活力为49.50 U.mg-1,纯化倍数为4.33,酶活回收率为70.49%；经Sephacryl-S400凝胶层析分离后,出现了2个明显的蛋白峰,酶比活力提高到154.50 U·mg-1,纯化倍数为13.48,酶活回收率为53.28%；经Sephadex-G100凝胶层析纯化后只出现1个蛋白峰,酶比活力提高到344.97 U·mg-1,纯化倍数为30.10,酶活回收率为41.75%。
推导出GXU08脂肪酶催化反应的动力学方程为：ν=(?)(其中v的单位是mol·L-1min-1,[S]的单位是mmol·L-1),计算得出米氏常数Km=1.86 mmol·L-1,最大反应速度Vmax=2.413×10-6 mol.L-1·min-1验证结果表明：实际反应速度可以较好的与米氏模型相吻合,表明该酶催化反应符合推导出的动力学方程。
探讨了有机溶剂体系中的酶催化反应,得到最佳反应条件为：浓度为8 mmol。·L-1的底物,于40℃,180r·min-1, pH=6.5,酶活为1600 U酶粉的作用下,在环己烷中反应72 h,环十五内酯的生成量最大。实验证明：溶剂的介电常数越大,单位酶活生成环十五内酯的质量越小,反之亦然。实验结果表明：酶在疏水介质环己烷中表现出最佳催化能力,且GXU08脂肪酶对15—羟基十五烷酸甲酯具有很好的专一性。
首次将超声波振荡应用于有机溶剂体系和水/有机溶剂两相体系的酶催化合成环十五内酯的反应中,取得了较好的催化效果。两相体系的最适反应条件为：0.5 mL,2.0 g·mL-1的脂肪酶酶液,在超声波震荡下均匀分散于pH=7.0的8 mmol·L-1 -15羟基十五烷酸甲酯的环已烷溶液中,于40℃,180 r·min-1的摇床中反应72 h,其单位酶活所得环十五内酯的质量最大为50.284×10-5mg.U-1,而优化条件下的纯有机溶剂体系中仅为29.02×105mg.U-1,前者为后者的1.733倍。
研究还发现,两相体系中分别将脂肪酶和反应体系重复使用一次,均可较大地提高环十五内酯的产量。且在相同的反应条件下,酶液比酶粉具有更高的催化合成环十五内酯的能力。故可直接使用一定浓度的经硫酸铵沉淀后的酶液代替酶粉催化合成环十五内酯,既可省却冷冻干燥的过程,又可提高环十五内酯的产量。
此外,实验得出有机相中的酶催化反应为：
表明15-羟基十五烷酸甲酯在该体系中是直接关环生成环十五内酯。两相体系中的酶催化反应为：
表明15-羟基十五烷酸甲酯在该体系中是先水解为15-羟基十五烷酸,15-羟基十五烷酸再关环生成环十五内酯。
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.
本文以自行筛选到的微生物产脂肪酶,催化蒜头果为原料制得15-羟基十五烷酸甲酯合成环十五内酯。主要开展了五个方面的研究：(1)脂肪酶菌株的筛选及正交法优化发酵条件；(2)将粗脂肪酶纯化为层析纯的酶；(3)酶催化动力学参数的求取及动力学方程的推导；(4)有机溶剂体系与水/有机溶剂两相体系中酶催化反应的对比及反应条件的优化；(5)两种体系中酶催化反应历程的推断。
实验筛选到10株高产脂肪酶菌株,其中经紫外诱变后的菌株Candida sp.GXU08(简称GXU08)对15—羟基十五烷酸甲酯转化为环十五内酯具有良好的催化性能。通过正交试验,得到菌株最佳培养基配方及发酵条件为：蔗糖0.5%,淀粉0.5%,蛋白胨1.5%,K2HPO40.05%,MgSO40.15%,(NH4)2SO41%,茶油1.5%,菜籽油1.5%,pH=8.5,发酵时间48 h,其中发酵时间、蛋白胨、茶油和pH值对发酵液酶活的影响较显著。该优化条件下所得发酵液的酶活为27.53 U·mL-1,是优化前的3.74倍。
实验分别采用硫酸铵分级沉淀、CM-阳离子纤维素柱层析、Sephacryl-S400凝胶层析及Sephadex-G100凝胶层析分离纯化GXUO8脂肪酶,纯化后只出现一个蛋白峰,得到了层析纯的酶,且分离出的脂肪酶具有较高的内酯化能力,可为今后研究酶学性质及动力学参数奠定基础。
发酵液经40%～70%的(NH4)2SO4分级沉淀粗分离后,酶比活力为14.50 U·mg-1,纯化倍数为1.27,酶活回收率为89.25%；经CM-离子交换层析分离后,出现了3个较明显的蛋白峰,酶比活力为49.50 U.mg-1,纯化倍数为4.33,酶活回收率为70.49%；经Sephacryl-S400凝胶层析分离后,出现了2个明显的蛋白峰,酶比活力提高到154.50 U·mg-1,纯化倍数为13.48,酶活回收率为53.28%；经Sephadex-G100凝胶层析纯化后只出现1个蛋白峰,酶比活力提高到344.97 U·mg-1,纯化倍数为30.10,酶活回收率为41.75%。
推导出GXU08脂肪酶催化反应的动力学方程为：ν=(?)(其中v的单位是mol·L-1min-1,[S]的单位是mmol·L-1),计算得出米氏常数Km=1.86 mmol·L-1,最大反应速度Vmax=2.413×10-6 mol.L-1·min-1验证结果表明：实际反应速度可以较好的与米氏模型相吻合,表明该酶催化反应符合推导出的动力学方程。
探讨了有机溶剂体系中的酶催化反应,得到最佳反应条件为：浓度为8 mmol。·L-1的底物,于40℃,180r·min-1, pH=6.5,酶活为1600 U酶粉的作用下,在环己烷中反应72 h,环十五内酯的生成量最大。实验证明：溶剂的介电常数越大,单位酶活生成环十五内酯的质量越小,反之亦然。实验结果表明：酶在疏水介质环己烷中表现出最佳催化能力,且GXU08脂肪酶对15—羟基十五烷酸甲酯具有很好的专一性。
首次将超声波振荡应用于有机溶剂体系和水/有机溶剂两相体系的酶催化合成环十五内酯的反应中,取得了较好的催化效果。两相体系的最适反应条件为：0.5 mL,2.0 g·mL-1的脂肪酶酶液,在超声波震荡下均匀分散于pH=7.0的8 mmol·L-1 -15羟基十五烷酸甲酯的环已烷溶液中,于40℃,180 r·min-1的摇床中反应72 h,其单位酶活所得环十五内酯的质量最大为50.284×10-5mg.U-1,而优化条件下的纯有机溶剂体系中仅为29.02×105mg.U-1,前者为后者的1.733倍。
研究还发现,两相体系中分别将脂肪酶和反应体系重复使用一次,均可较大地提高环十五内酯的产量。且在相同的反应条件下,酶液比酶粉具有更高的催化合成环十五内酯的能力。故可直接使用一定浓度的经硫酸铵沉淀后的酶液代替酶粉催化合成环十五内酯,既可省却冷冻干燥的过程,又可提高环十五内酯的产量。
此外,实验得出有机相中的酶催化反应为：
表明15-羟基十五烷酸甲酯在该体系中是直接关环生成环十五内酯。两相体系中的酶催化反应为：
表明15-羟基十五烷酸甲酯在该体系中是先水解为15-羟基十五烷酸,15-羟基十五烷酸再关环生成环十五内酯。
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.
引文
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