基于脂肪酶催化的油脂高值化改造研究
发布时间：2018-08-31 20:59
【摘要】：油脂资源绿色可再生,天然产量丰富,是生物质资源的重要组成部分。生物催化在区域选择性、副反应控制等方面具有传统的油脂加工工艺不可比拟的优势,因而成为近年来的研究热点。然而,生物催化剂高昂的市场售价,限制了该类技术的应用与发展。本课题组开发的Candidasp.99-125脂肪酶系列产品具有成本低,比酶活高,稳定性良好等优点,性价比优于同类国外产品。目前,以Candidasp. 99-125脂肪酶为核心的绿色催化平台技术日渐成熟,并已成功应用于多类脂肪酸酯类化合物的合成与生产。但Candida sp. 99-125脂肪酶针对于结构型酯类化合物(糖醇酯,甘油结构酯等)的催化特性及催化区域选择性尚有深入挖掘潜力,相关工艺尚未系统性开发并进一步优化。另外,近年来脂肪酶催化的不饱和双键环氧化反应也逐渐成为油脂改造的研究热点之一,Candida sp. 99-125脂肪酶在该方面的应用尚需发展。为进一步完善以Candida sp. 99-125脂肪酶为基础的绿色平台催化技术,本论文针对结构型类化合物产品及环氧油脂类产品,分别选取具有产品代表性的木糖醇脂肪酸酯、人乳脂肪替代结构酯(Human Milk Fat Substitutes,HMFS)、环氧脂肪酸为目标产物,进行了合成过程分析及工艺开发的研究。论文通过探讨Candida sp. 99-125脂肪酶的催化区域选择性;优化反应水含量及底物摩尔比等参数,构建合理的反应微环境;结合过程反应机理及副反应进程,调控反应温度、过程传质等参数;最终设计开发了Candida sp. 99-125脂肪酶针对于相应产品的高效催化工艺。具体研究内容如下:1.Candida sp. 99-125脂肪酶催化木糖醇脂肪酸酯合成中,首先建立了叔丁醇溶剂催化反应体系。通过预加热处理制备木糖醇-叔丁醇过饱和溶液,有效地提高反应体系中木糖醇浓度,从而提高木糖醇脂肪酸酯的转化率。在该体系中反应6 h后,木糖醇转化率约为70%,产品中1(5)-O-木糖醇单脂肪酸酯含量约95%.在此基础上,本研究建立了更为绿色的木糖醇酯无溶剂反应体系。通过木糖醇等摩尔分批5次加入,解决反应底物溶解问题。反应120 h后,糖醇酯的转化率为70%。其中1(5)-O-木糖醇癸酸单酯相对含量为16%,1,5-0-木糖醇癸酸二酯相对含量为60%,木糖醇1,2,5-0-癸酸三酯与1,3,5-O-木糖醇癸酸三酯相对含量为24%.同时发现,体系最终产品构型同脂肪酶的催化选择性无关,反应能量壁垒和体系热力学特性决定了最终产品的构型和组成。2.母乳脂肪中甘油三酯具有特定结构,其从头合成对催化选择性及反应过程控制要求较高,极具挑战性。本研究结合酶催化过程中酰基转移副反应的机理实现了对反应过程进行调控,并有效控制了催化过程的位置选择性,得到了针对Candida sp. 99-125脂肪酶催化合成HMFS的最佳工艺条件。催化得到的产品经分离纯化后,产品的酸价为2 mgKOH/g,甘油三酯中sn-2位棕榈酸占总棕榈酸比例的含量为52%-55%,棕榈酸甘油三酯在总甘油三酯中的含量为8.4%-9.6%,目标产品型甘油三酯含量为55-59%,产品过氧化值1.41-1.89meq/g,产品的各项指标均可满足并超过国家标准要求。3.不饱和脂肪酸的环氧化过程中,由于游离羧基的作用,使得该反应过程相对于脂肪酸酯的环氧化更加复杂,副反应更难于控制。本研究首先针对化学催化脂肪酸环氧化过程中,环氧基团开环副反应严重的问题,通过过程分析与优化,建立了过程动力学模型,通过分析各反应的速率及能量问题,得到了化学催化过程各类开环反应难以避免的结论。在化学催化研究的基础上,设计并优化了Candida sp.99-125脂肪酶催化的不饱和脂肪酸环氧化反应体系。以油酸为底物,在30 ℃条件下,反应10 h后,产物环氧值可达4.12%,环氧转化率76.58%,环氧化选择性0.98。以无患子油混合脂肪酸为原料,验证了该催化工艺的稳定性和可靠性。最终,从反应条件、反应机理、反应动力学方面,对比了化学催化甲酸原位环氧化和Candida sp. 99-125脂肪酶催化环氧化过程的区别,讨论了两种方法的优缺点。综上所述,论文通过研究Candida sp. 99-125脂肪酶在结构型酯类化合物产品催化转化过程中,酰基转移副反应的机理、历程、相关调控措施、策略;以及不饱和脂肪酸环氧化过程中开环副反应的影响及控制技术,过氧化物对脂肪酶的毒性问题及其解决方案;进一步完善了以Candidasp.99-125脂肪酶为基础的绿色生物催化平台技术,有利于提高生物催化技术在工业生产应用中的竞争力。
[Abstract]:Biocatalysis is an important component of biomass resources because of its green and renewable resources and abundant natural production. Biocatalysis has incomparable advantages over traditional oil processing techniques in terms of regional selectivity and side reaction control, so it has become a research hotspot in recent years. However, the high market price of biocatalysts limits this kind of technology. The application and development of Candidasp.99-125 lipase series developed by our research group have the advantages of low cost, high specific enzyme activity, good stability and better cost performance than the same foreign products. At present, the green catalytic platform technology based on Candidasp.99-125 lipase is becoming more and more mature, and has been successfully applied to the esterification of fatty acids. However, the catalytic properties and regioselectivity of Candida sp.99-125 lipase for structural esters (glycol esters, glycerol esters, etc.) have not been systematically developed and optimized. In addition, the unsaturated double bond epoxidation catalyzed by lipase has also been studied in recent years. The application of Candida sp.99-125 lipase in this field needs further development. In order to further improve the green platform catalytic technology based on Candida sp.99-125 lipase, this paper selected the representative wood for structural compounds and epoxy oils respectively. The synthesis of glycol fatty acid esters, human Milk Fat Substitutes (HMFS) and epoxy fatty acids as target products was studied. The catalytic regioselectivity of Candida sp.99-125 lipase was investigated, and the water content and molar ratio of substrate were optimized to construct a reasonable lipase. Combining the reaction mechanism and side reaction process, adjusting the reaction temperature and mass transfer parameters, a high efficient catalytic process of Candida sp.99-125 lipase for the corresponding products was designed and developed. The xylitol-tert-butanol supersaturated solution was prepared by pre-heating treatment, which effectively increased the xylitol concentration in the reaction system and thus increased the conversion of xylitol fatty acid esters. After 6 h reaction, the conversion of xylitol was about 70%, and the content of 1(5)-O-xylitol monofatty acid esters in the product was about 95%. On this basis, a greener solvent-free reaction system of xylitol esters was established. The substrate dissolution was solved by adding xylitol in equal mole five times. After 120 hours, the conversion of glycol esters was 70%. The relative content of 1(5)-O-xylitol decanoic acid monoester was 16%, and the relative content of 1,5-0-xylitol decanoic acid diester was 60%. The relative content of xylitol 1,2,5-0-decanoate and 1,3,5-O-xylitol tridecanoate was 24%. It was also found that the final product configuration was independent of the catalytic selectivity of lipase. The reaction energy barrier and thermodynamic properties of the system determined the configuration and composition of the final product. 2. Triglyceride in breast milk fat had a specific structure and was synthesized ab initio. This study combined the mechanism of acyl transfer side reaction in enzyme catalytic process to control the reaction process, and effectively controlled the position selectivity of the catalytic process. The optimum conditions for the synthesis of HMFS catalyzed by Candida sp.99-125 lipase were obtained. After separation and purification, the acid value of the product was 2 mg KOH/g, the content of sn-2 palmitic acid in triglyceride was 52% - 55%, the content of triglyceride in triglyceride was 8.4% - 9.6%, the content of target product was 55-59%, and the peroxide value of the product was 1.41-1.89meq/g. The epoxidation process of unsaturated fatty acids is more complicated than that of fatty acid esters because of the action of free carboxyl groups, and the side reactions are more difficult to control. Through process analysis and optimization, a process kinetic model was established. By analyzing the rate and energy of each reaction, it was concluded that all kinds of ring opening reactions in the chemical catalytic process were unavoidable. On the basis of the study of chemical catalysis, the epoxidation of unsaturated fatty acids catalyzed by Candida sp.99-125 lipase was designed and optimized. With oleic acid as substrate, the epoxy value of the product reached 4.12%, the conversion of epoxy was 76.58%, and the selectivity of epoxidation was 0.98. The stability and reliability of the catalytic process were verified by using mixed fatty acids of Non-saponin oil as raw material. Finally, the reaction conditions, reaction mechanism and reaction kinetics were compared. The difference between in situ epoxidation of formic acid and epoxidation catalyzed by Candida sp.99-125 lipase was discussed, and the advantages and disadvantages of the two methods were discussed. And the influence and control technology of ring-opening reaction in epoxidation of unsaturated fatty acids, the toxicity of peroxides to lipase and its solution, and the green biocatalytic platform technology based on Candidasp.99-125 lipase were further improved to improve the competitiveness of biocatalytic technology in industrial production.
【学位授予单位】：北京化工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ641;O643.36
本文编号：2216145
[Abstract]:Biocatalysis is an important component of biomass resources because of its green and renewable resources and abundant natural production. Biocatalysis has incomparable advantages over traditional oil processing techniques in terms of regional selectivity and side reaction control, so it has become a research hotspot in recent years. However, the high market price of biocatalysts limits this kind of technology. The application and development of Candidasp.99-125 lipase series developed by our research group have the advantages of low cost, high specific enzyme activity, good stability and better cost performance than the same foreign products. At present, the green catalytic platform technology based on Candidasp.99-125 lipase is becoming more and more mature, and has been successfully applied to the esterification of fatty acids. However, the catalytic properties and regioselectivity of Candida sp.99-125 lipase for structural esters (glycol esters, glycerol esters, etc.) have not been systematically developed and optimized. In addition, the unsaturated double bond epoxidation catalyzed by lipase has also been studied in recent years. The application of Candida sp.99-125 lipase in this field needs further development. In order to further improve the green platform catalytic technology based on Candida sp.99-125 lipase, this paper selected the representative wood for structural compounds and epoxy oils respectively. The synthesis of glycol fatty acid esters, human Milk Fat Substitutes (HMFS) and epoxy fatty acids as target products was studied. The catalytic regioselectivity of Candida sp.99-125 lipase was investigated, and the water content and molar ratio of substrate were optimized to construct a reasonable lipase. Combining the reaction mechanism and side reaction process, adjusting the reaction temperature and mass transfer parameters, a high efficient catalytic process of Candida sp.99-125 lipase for the corresponding products was designed and developed. The xylitol-tert-butanol supersaturated solution was prepared by pre-heating treatment, which effectively increased the xylitol concentration in the reaction system and thus increased the conversion of xylitol fatty acid esters. After 6 h reaction, the conversion of xylitol was about 70%, and the content of 1(5)-O-xylitol monofatty acid esters in the product was about 95%. On this basis, a greener solvent-free reaction system of xylitol esters was established. The substrate dissolution was solved by adding xylitol in equal mole five times. After 120 hours, the conversion of glycol esters was 70%. The relative content of 1(5)-O-xylitol decanoic acid monoester was 16%, and the relative content of 1,5-0-xylitol decanoic acid diester was 60%. The relative content of xylitol 1,2,5-0-decanoate and 1,3,5-O-xylitol tridecanoate was 24%. It was also found that the final product configuration was independent of the catalytic selectivity of lipase. The reaction energy barrier and thermodynamic properties of the system determined the configuration and composition of the final product. 2. Triglyceride in breast milk fat had a specific structure and was synthesized ab initio. This study combined the mechanism of acyl transfer side reaction in enzyme catalytic process to control the reaction process, and effectively controlled the position selectivity of the catalytic process. The optimum conditions for the synthesis of HMFS catalyzed by Candida sp.99-125 lipase were obtained. After separation and purification, the acid value of the product was 2 mg KOH/g, the content of sn-2 palmitic acid in triglyceride was 52% - 55%, the content of triglyceride in triglyceride was 8.4% - 9.6%, the content of target product was 55-59%, and the peroxide value of the product was 1.41-1.89meq/g. The epoxidation process of unsaturated fatty acids is more complicated than that of fatty acid esters because of the action of free carboxyl groups, and the side reactions are more difficult to control. Through process analysis and optimization, a process kinetic model was established. By analyzing the rate and energy of each reaction, it was concluded that all kinds of ring opening reactions in the chemical catalytic process were unavoidable. On the basis of the study of chemical catalysis, the epoxidation of unsaturated fatty acids catalyzed by Candida sp.99-125 lipase was designed and optimized. With oleic acid as substrate, the epoxy value of the product reached 4.12%, the conversion of epoxy was 76.58%, and the selectivity of epoxidation was 0.98. The stability and reliability of the catalytic process were verified by using mixed fatty acids of Non-saponin oil as raw material. Finally, the reaction conditions, reaction mechanism and reaction kinetics were compared. The difference between in situ epoxidation of formic acid and epoxidation catalyzed by Candida sp.99-125 lipase was discussed, and the advantages and disadvantages of the two methods were discussed. And the influence and control technology of ring-opening reaction in epoxidation of unsaturated fatty acids, the toxicity of peroxides to lipase and its solution, and the green biocatalytic platform technology based on Candidasp.99-125 lipase were further improved to improve the competitiveness of biocatalytic technology in industrial production.
【学位授予单位】：北京化工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ641;O643.36
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1 寇秀芬,徐家立;酶法合成糖及糖醇酯[J];微生物学报;2000年02期
相关博士学位论文 前1条
1 陈必强;脂肪酶固定化及其在化学品合成中的应用[D];北京化工大学;2008年
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