超声作用下脂肪酶催化豆油水解反应的研究
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摘要
当前应用最为广泛的高温高压油脂水解技术及传统的中压催化水解技术存在着副反应严重和污染环境等不可避免的缺陷,而超声波和酶催化技术都具有投资低、条件温和、耗能低、副反应少、环境友好等特点,本文结合两者的优势潜质,对超声波-脂肪酶协同催化油脂水解反应进行了研究。
选择具有安全无毒,成本低,可提高底物及产物体积浓度,产物分离纯化步骤少等优点的无溶剂体系,对常规恒温振荡水浴方式下的C. lipolytica脂肪酶催化豆油水解的反应进行了研究。得到C. lipolytica的反应特性为:最佳反应温度在45℃附近;该酶为碱性脂肪酶,从pH 6.1-8.3范围内酶活力呈波浪式变化;当C. lipolytica浓度超过1%(w/w)时,出现明显的酶浓度抑制现象;水油比超过0.675:1(w/w)后,反应速率的变化趋于平衡。在此基础上进行了响应面分析,建立了能够反映各条件因素对反应进程影响程度的数学模型,对实验数据进行的方差分析和典型分析结果显示:温度、加酶量对水解反应影响高度显著,而缓冲溶液pH、水油比在实验范围内对反应的影响不显著。C. lipolytica脂肪酶催化豆油水解反应的最优反应条件为:温度42.2℃、pH 7.51、加酶量0.85%(w/w)、水油比1.24(w/w)。
超声反应中,超声功率的大小是酶催化反应非常重要的影响因素,在本文所研究的功率范围内没有发现酶失活现象。实验结果显示随着超声强度的增加水解反应速率增大,当超声强度超过1.20W/cm2时,反应速率随功率的增加变化缓慢,但是通过精密度实验发现随着超声强度的增加实验结果的稳定性得到了提高,说明在较低超声强度下,输出功率的波动对反应速率的影响较大,综合考虑后选择了1.64W/cm2的超声波进行后续研究。
研究了超声波对C. lipolytica脂肪酶催化豆油水解反应的影响作用,通过与振荡水浴方式下实验结果的对比发现超声波使C. lipolytica的耐热性有了5℃左右的提高;可以避免或是减少酶在油水界面的聚集,没有出现酶浓度抑制反应现象;在超声反应中,水变得更加“有效”,即水解反应按照假单底物规律进行时水的浓度临界值较低;而pH的变化规律与振荡水浴中一致,表明超声作用没有改变该酶的离子化反应状态。对超声作用下的C. lipolytica脂肪酶催化豆油水解反应进行了响应面分析,建立了能够反映各条件因素对反应进程影响程度的数学模型,并得到超声作用下的最优反应条件为:温度47.0℃、pH 7.77、加酶量0.77%(w/w)、水油比0.71(w/w)。
复合酶反应是通过两种或多种酶的协同作用,使其催化能力得到明显提高的一种酶反应技术。本文对Sigma-3126,生工PPL,CLL,Novo435和Amano-G五种不同来源的脂肪酶进行了筛选和复配,得到了水解效果较好的CLL和Amano-G组合。对各种反应条件进行了研究,确定最优工艺条件为:CLL和Amano-G的加入量分别为1%(w/w)和0.1%(w/w);在CLL反应进行1h后加入Amano-G;反应温度45℃;去离子水直接加入反应体系作为水相,水油比1:1(w/w)。在此条件下,超声波对反应有一定的促进作用,在反应开始阶段其作用尤为明显,24h的豆油水解率可以由常规振荡水浴中的45.7%提高到94.2%。
酶催化反应动力学是了解酶作用机理的重要途径之一,动力学模型能够比较准确的反映某个酶反应系统中的各种相互作用对反应的影响程度。目前大多数脂肪酶油脂水解反应模型都是假设油水两相的界面面积为常数,这样的假设在无溶剂体系中是不恰当的。本文在建立了有效底物浓度模型的基础上,将有效底物浓度和油水界面面积的变化相结合,建立了一个新的动力学模型。
对所建立模型的适用范围应用亚甲基蓝染色-显微镜法进行了验证,证明该模型在油相体积分数Φ从0.1-0.9范围内适用;应用TLC扫描分析法证明有效底物浓度模型的假设条件在Φ=0.1-0.9范围内可靠。并将所得动力学模型应用于C. lipolytica脂肪酶催化的豆油水解反应中,在求得反应初速度和Sauter直径随Φ变化规律的基础上,应用非线性回归拟合技术分别求出了振荡水浴和超声作用下模型参数的具体数值。分别讨论了动力学参数Ke、k*cat和kd/kp对反应速率的影响作用,并结合具体反应对参数的实际物理意义进行了解释与分析。
应用所建立的有效底物浓度模型,对底物抑制现象给出了合理的解释,指出实验中出现的底物抑制现象只是“表观”底物浓度抑制,反应速率实际是由有效底物浓度所控制,超声波能够减小底物抑制,加快反应进行,其根本原因是由于超声波对有效底物浓度的影响而引起的。
热力学研究可以避免体系结构和过程机理所造成的局限,有利于对反应的宏观特征进行分析。本文对热力学公式进行了推导,测得了C. lipolytica脂肪酶催化豆油水解反应在200rpm振荡水浴和240V超声功率条件下的反应活化能Ea分别为10.33kJ/mol和10.24kJ/mol;指前因子A分别为2.85和3.35。并以45℃下的反应为例,计算并讨论了热力学常数Δ~≠H、Δ~≠G和Δ~≠S在振荡水浴和超声作用下的变化情况。
在综合前人的研究成果和本论文的实验基础上,对超声促进C. lipolytica脂肪酶催化豆油水解反应的作用机理进行分析,提出了超声波对脂肪酶催化水解反应有促进局部O/W乳化作用。
Current mainstream hydrolysis technology of oils and fats needs high temperature/high operation pressure and conventional hydrolysis technology needs catalyzer, which would produce serious by-reactions and pollute entironment. Ultrasonic technology, the same as enzymatic technology, is a technology with the characters of investment-saving, warmer condition, lower energy cost, less by-reactions, and safety to environment. This thesis combined the potential advantages of the two technologies to investigate oils and fats hydrolysis reaction catalyzed by ultrasound-enzymatic technology.
Solvent-free system is provide with the advantages of safety, low-cost, high volume concentration of substrates (or products) and easy to separate, which was selected to investigate hydrolysis of soybean oil catalyzed by C. lipolytica lipase in shaking bath. The reaction characters were obtained: optimum reaction temperature around 45℃, C. lipolytica is an alkali-lipase with wave-like changing of activity from pH 6.1-8.3, obvious enzyme concentration inhibition occurs when C. lipolytica concentration exceeds 1%(w/w), when water/oil ratio exceeds 0.675:1, raising of reaction rate tends to balance. Respond surface methodology (RSM) was used on the base of C. lipolytica reaction characters. A mathematics model was established to reflect influence of reaction conditions on the reaction course. Analysis of variance (ANOVA) and canonical analysis of experimental data showed the results that temperature and enzyme concentration are significant at 99% confidence level on the hydrolysis reaction; pH and water/oil ratio are non-significant on the reaction in the experimental range. The optimum reaction conditions are: temperature 42.2℃, pH 7.51, enzyme concentration 0.85(w/w) and water/oil ratio 1.24(w/w).
Power of ultrasound is an important influence on the reaction catalyzed by enzyme. Enzyme inactivation did not present in the selected ultrasonic power range. Experimental results showed that reaction rate increases with the increasing of ultrasonic power, when ultrasonic intensity exceeds 1.20W/cm2, increasing of reaction rate gradually slows. But stability of reaction is improved by increasing ultrasonic intensity, which indicates that the fluctuation of power output would induce a remarkable variation in reaction rate in lower ultrasonic intensity. Selected 1.64W/cm2 of ultrasonic intensity for the following experiments.
Effects of ultrasound on soybean oil hydrolysis reaction catalyzed by C. lipolytica lipase were investigated. Compared with that in shaking bath, heat-durability is enhanced about 5℃in ultrasonic bath; enzyme concentration inhibition does not present, which probably could attribute to agglomeration of enzyme on the oil-water interface is avoided or reduced by ultrasound; water is more“efficient”in ultrasonic reaction, in another word, the critical point is lower at which the kinetics could be considered as a pseudo-mono-substrate reaction; ultrasound causes little change to the ionization state of C. lipolytica, as similar degree-pH pattern were presented in shaking and ultrasonic conditions. RSM was also utilized in ultrasound bath and a mathematics model was established to reflect influence of reaction conditions on the reaction course. The optimum reaction conditions in ultrasonic bath are: temperature 47.0℃, pH 7.77, enzyme concentration 0.77(w/w) and water/oil ratio 0.71(w/w).
Multiple-enzyme reaction is a technology of cooperating bi-enzyme or multi-enzyme to enhance the catalytic capability of enzymes. Selected five different source lipases of Sigma-3126, Bio-Eng PPL, CLL, Novo435 and Amano-G to investigate the complex effect. Combining CLL and Amano-G could give a better hydrolysis rate and yield. The optimum reaction condition was obtained: the concentration of CLL and Amano-G are 1%(w/w) and 0.1%(w/w), respectively; adding Amano-G after C. lipolytica acts 1h; temperature is 45℃; water-phase is de-ionized water, and water/oil ratio is 1:1(w/w). Under above condition, hydrolysis reaction could be accelerated by ultrasound, especially in the initial reaction stage. After 24h, reaction yield could be enhanced from 45.7% in conventional shaking bath to 94.2% catalyzed by ultrasonic-bi-enzyme.
Kinetics of enzymatic reaction is an important approach to mechanism. Kinetics model can present the infection of influence factors on the reaction accurately. At present, in most kinetics models, the interfacial area of water-oil interface is hypothesized to be a constant, which is not suitable for solvent-free system. In the thesis, we established a new kinetics model in solvent-free system based on a model of active-substrate. And the model combined interfacial area of water-oil interface and active substrate.
The applicability had been proved suitable in the range of volume fraction of oilΦ=0.1-0.9 by methylene blue dyeing-microscope technology, and the hypothesis had been also proved correct in the range ofΦ=0.1-0.9 by TLC. The kinetics model was used in hydrolysis reaction of soybean oil catalyzed by C. lipolytica lipase. Based on initial reaction rate and the principle of Sauter diameter changed with water/oil ratioΦ, parameters of the kinetics model were calculated using non-line regression fitting methodology in both shaking bath and ultrasonic bath. The influence of parameters Ke、k*cat and kd/kp on reaction rate were discussed, and explained the actual physical meaning of parameters.
Substrate inhibition phenomenon was explained using the active-substrate model. It indicated that substrate inhibition phenomenon occurred in hydrolysis reaction is an“apparent”substrate inhibition. Reaction rate is controlled by active-substrate concentration. Ultrasound could reduce substrate inhibition and accelerate reaction rate, which essential reason is the effect of ultrasound on the active-substrate.
Study on the thermodynamics can avoid the limitation of system structure and process mechanism, which is favored on the macroscopy characters of reaction. Based on the derivation of thermodynamics formula, activation energy Ea of soybean oil hydrolysis reaction catalyzed by C. lipolytica was determined, Ea in 200rpm shaking bath is 10.33 kJ/mol and in 240V ultrasonic bath is 10.24 kJ/mol, respectively. Additional, pre-exponential factor A was calculated, in 200rpm shaking bath is 2.85 and in 240V ultrasonic bath is 3.35, respectively. Parameters of thermodynamicsΔ~≠H,Δ~≠G, andΔ~≠S at 45℃, were also calculated and discussed in shaking bath and in ultrasonic bath, respectively.
Based on the conclusions of others and our experimental results, the enzymatic hydrolysis reaction mechanism of oils and fats irradiated by ultrasound was discussed. We proposed a“localized O/W emulsion”function for the enzymatic hydrolysis reaction of oils and fats accelerated by ultrasound.
选择具有安全无毒,成本低,可提高底物及产物体积浓度,产物分离纯化步骤少等优点的无溶剂体系,对常规恒温振荡水浴方式下的C. lipolytica脂肪酶催化豆油水解的反应进行了研究。得到C. lipolytica的反应特性为:最佳反应温度在45℃附近;该酶为碱性脂肪酶,从pH 6.1-8.3范围内酶活力呈波浪式变化;当C. lipolytica浓度超过1%(w/w)时,出现明显的酶浓度抑制现象;水油比超过0.675:1(w/w)后,反应速率的变化趋于平衡。在此基础上进行了响应面分析,建立了能够反映各条件因素对反应进程影响程度的数学模型,对实验数据进行的方差分析和典型分析结果显示:温度、加酶量对水解反应影响高度显著,而缓冲溶液pH、水油比在实验范围内对反应的影响不显著。C. lipolytica脂肪酶催化豆油水解反应的最优反应条件为:温度42.2℃、pH 7.51、加酶量0.85%(w/w)、水油比1.24(w/w)。
超声反应中,超声功率的大小是酶催化反应非常重要的影响因素,在本文所研究的功率范围内没有发现酶失活现象。实验结果显示随着超声强度的增加水解反应速率增大,当超声强度超过1.20W/cm2时,反应速率随功率的增加变化缓慢,但是通过精密度实验发现随着超声强度的增加实验结果的稳定性得到了提高,说明在较低超声强度下,输出功率的波动对反应速率的影响较大,综合考虑后选择了1.64W/cm2的超声波进行后续研究。
研究了超声波对C. lipolytica脂肪酶催化豆油水解反应的影响作用,通过与振荡水浴方式下实验结果的对比发现超声波使C. lipolytica的耐热性有了5℃左右的提高;可以避免或是减少酶在油水界面的聚集,没有出现酶浓度抑制反应现象;在超声反应中,水变得更加“有效”,即水解反应按照假单底物规律进行时水的浓度临界值较低;而pH的变化规律与振荡水浴中一致,表明超声作用没有改变该酶的离子化反应状态。对超声作用下的C. lipolytica脂肪酶催化豆油水解反应进行了响应面分析,建立了能够反映各条件因素对反应进程影响程度的数学模型,并得到超声作用下的最优反应条件为:温度47.0℃、pH 7.77、加酶量0.77%(w/w)、水油比0.71(w/w)。
复合酶反应是通过两种或多种酶的协同作用,使其催化能力得到明显提高的一种酶反应技术。本文对Sigma-3126,生工PPL,CLL,Novo435和Amano-G五种不同来源的脂肪酶进行了筛选和复配,得到了水解效果较好的CLL和Amano-G组合。对各种反应条件进行了研究,确定最优工艺条件为:CLL和Amano-G的加入量分别为1%(w/w)和0.1%(w/w);在CLL反应进行1h后加入Amano-G;反应温度45℃;去离子水直接加入反应体系作为水相,水油比1:1(w/w)。在此条件下,超声波对反应有一定的促进作用,在反应开始阶段其作用尤为明显,24h的豆油水解率可以由常规振荡水浴中的45.7%提高到94.2%。
酶催化反应动力学是了解酶作用机理的重要途径之一,动力学模型能够比较准确的反映某个酶反应系统中的各种相互作用对反应的影响程度。目前大多数脂肪酶油脂水解反应模型都是假设油水两相的界面面积为常数,这样的假设在无溶剂体系中是不恰当的。本文在建立了有效底物浓度模型的基础上,将有效底物浓度和油水界面面积的变化相结合,建立了一个新的动力学模型。
对所建立模型的适用范围应用亚甲基蓝染色-显微镜法进行了验证,证明该模型在油相体积分数Φ从0.1-0.9范围内适用;应用TLC扫描分析法证明有效底物浓度模型的假设条件在Φ=0.1-0.9范围内可靠。并将所得动力学模型应用于C. lipolytica脂肪酶催化的豆油水解反应中,在求得反应初速度和Sauter直径随Φ变化规律的基础上,应用非线性回归拟合技术分别求出了振荡水浴和超声作用下模型参数的具体数值。分别讨论了动力学参数Ke、k*cat和kd/kp对反应速率的影响作用,并结合具体反应对参数的实际物理意义进行了解释与分析。
应用所建立的有效底物浓度模型,对底物抑制现象给出了合理的解释,指出实验中出现的底物抑制现象只是“表观”底物浓度抑制,反应速率实际是由有效底物浓度所控制,超声波能够减小底物抑制,加快反应进行,其根本原因是由于超声波对有效底物浓度的影响而引起的。
热力学研究可以避免体系结构和过程机理所造成的局限,有利于对反应的宏观特征进行分析。本文对热力学公式进行了推导,测得了C. lipolytica脂肪酶催化豆油水解反应在200rpm振荡水浴和240V超声功率条件下的反应活化能Ea分别为10.33kJ/mol和10.24kJ/mol;指前因子A分别为2.85和3.35。并以45℃下的反应为例,计算并讨论了热力学常数Δ~≠H、Δ~≠G和Δ~≠S在振荡水浴和超声作用下的变化情况。
在综合前人的研究成果和本论文的实验基础上,对超声促进C. lipolytica脂肪酶催化豆油水解反应的作用机理进行分析,提出了超声波对脂肪酶催化水解反应有促进局部O/W乳化作用。
Current mainstream hydrolysis technology of oils and fats needs high temperature/high operation pressure and conventional hydrolysis technology needs catalyzer, which would produce serious by-reactions and pollute entironment. Ultrasonic technology, the same as enzymatic technology, is a technology with the characters of investment-saving, warmer condition, lower energy cost, less by-reactions, and safety to environment. This thesis combined the potential advantages of the two technologies to investigate oils and fats hydrolysis reaction catalyzed by ultrasound-enzymatic technology.
Solvent-free system is provide with the advantages of safety, low-cost, high volume concentration of substrates (or products) and easy to separate, which was selected to investigate hydrolysis of soybean oil catalyzed by C. lipolytica lipase in shaking bath. The reaction characters were obtained: optimum reaction temperature around 45℃, C. lipolytica is an alkali-lipase with wave-like changing of activity from pH 6.1-8.3, obvious enzyme concentration inhibition occurs when C. lipolytica concentration exceeds 1%(w/w), when water/oil ratio exceeds 0.675:1, raising of reaction rate tends to balance. Respond surface methodology (RSM) was used on the base of C. lipolytica reaction characters. A mathematics model was established to reflect influence of reaction conditions on the reaction course. Analysis of variance (ANOVA) and canonical analysis of experimental data showed the results that temperature and enzyme concentration are significant at 99% confidence level on the hydrolysis reaction; pH and water/oil ratio are non-significant on the reaction in the experimental range. The optimum reaction conditions are: temperature 42.2℃, pH 7.51, enzyme concentration 0.85(w/w) and water/oil ratio 1.24(w/w).
Power of ultrasound is an important influence on the reaction catalyzed by enzyme. Enzyme inactivation did not present in the selected ultrasonic power range. Experimental results showed that reaction rate increases with the increasing of ultrasonic power, when ultrasonic intensity exceeds 1.20W/cm2, increasing of reaction rate gradually slows. But stability of reaction is improved by increasing ultrasonic intensity, which indicates that the fluctuation of power output would induce a remarkable variation in reaction rate in lower ultrasonic intensity. Selected 1.64W/cm2 of ultrasonic intensity for the following experiments.
Effects of ultrasound on soybean oil hydrolysis reaction catalyzed by C. lipolytica lipase were investigated. Compared with that in shaking bath, heat-durability is enhanced about 5℃in ultrasonic bath; enzyme concentration inhibition does not present, which probably could attribute to agglomeration of enzyme on the oil-water interface is avoided or reduced by ultrasound; water is more“efficient”in ultrasonic reaction, in another word, the critical point is lower at which the kinetics could be considered as a pseudo-mono-substrate reaction; ultrasound causes little change to the ionization state of C. lipolytica, as similar degree-pH pattern were presented in shaking and ultrasonic conditions. RSM was also utilized in ultrasound bath and a mathematics model was established to reflect influence of reaction conditions on the reaction course. The optimum reaction conditions in ultrasonic bath are: temperature 47.0℃, pH 7.77, enzyme concentration 0.77(w/w) and water/oil ratio 0.71(w/w).
Multiple-enzyme reaction is a technology of cooperating bi-enzyme or multi-enzyme to enhance the catalytic capability of enzymes. Selected five different source lipases of Sigma-3126, Bio-Eng PPL, CLL, Novo435 and Amano-G to investigate the complex effect. Combining CLL and Amano-G could give a better hydrolysis rate and yield. The optimum reaction condition was obtained: the concentration of CLL and Amano-G are 1%(w/w) and 0.1%(w/w), respectively; adding Amano-G after C. lipolytica acts 1h; temperature is 45℃; water-phase is de-ionized water, and water/oil ratio is 1:1(w/w). Under above condition, hydrolysis reaction could be accelerated by ultrasound, especially in the initial reaction stage. After 24h, reaction yield could be enhanced from 45.7% in conventional shaking bath to 94.2% catalyzed by ultrasonic-bi-enzyme.
Kinetics of enzymatic reaction is an important approach to mechanism. Kinetics model can present the infection of influence factors on the reaction accurately. At present, in most kinetics models, the interfacial area of water-oil interface is hypothesized to be a constant, which is not suitable for solvent-free system. In the thesis, we established a new kinetics model in solvent-free system based on a model of active-substrate. And the model combined interfacial area of water-oil interface and active substrate.
The applicability had been proved suitable in the range of volume fraction of oilΦ=0.1-0.9 by methylene blue dyeing-microscope technology, and the hypothesis had been also proved correct in the range ofΦ=0.1-0.9 by TLC. The kinetics model was used in hydrolysis reaction of soybean oil catalyzed by C. lipolytica lipase. Based on initial reaction rate and the principle of Sauter diameter changed with water/oil ratioΦ, parameters of the kinetics model were calculated using non-line regression fitting methodology in both shaking bath and ultrasonic bath. The influence of parameters Ke、k*cat and kd/kp on reaction rate were discussed, and explained the actual physical meaning of parameters.
Substrate inhibition phenomenon was explained using the active-substrate model. It indicated that substrate inhibition phenomenon occurred in hydrolysis reaction is an“apparent”substrate inhibition. Reaction rate is controlled by active-substrate concentration. Ultrasound could reduce substrate inhibition and accelerate reaction rate, which essential reason is the effect of ultrasound on the active-substrate.
Study on the thermodynamics can avoid the limitation of system structure and process mechanism, which is favored on the macroscopy characters of reaction. Based on the derivation of thermodynamics formula, activation energy Ea of soybean oil hydrolysis reaction catalyzed by C. lipolytica was determined, Ea in 200rpm shaking bath is 10.33 kJ/mol and in 240V ultrasonic bath is 10.24 kJ/mol, respectively. Additional, pre-exponential factor A was calculated, in 200rpm shaking bath is 2.85 and in 240V ultrasonic bath is 3.35, respectively. Parameters of thermodynamicsΔ~≠H,Δ~≠G, andΔ~≠S at 45℃, were also calculated and discussed in shaking bath and in ultrasonic bath, respectively.
Based on the conclusions of others and our experimental results, the enzymatic hydrolysis reaction mechanism of oils and fats irradiated by ultrasound was discussed. We proposed a“localized O/W emulsion”function for the enzymatic hydrolysis reaction of oils and fats accelerated by ultrasound.
引文
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