【摘要】：油脂資源綠色可再生,天然產(chǎn)量豐富,是生物質(zhì)資源的重要組成部分。生物催化在區(qū)域選擇性、副反應控制等方面具有傳統(tǒng)的油脂加工工藝不可比擬的優(yōu)勢,因而成為近年來的研究熱點。然而,生物催化劑高昂的市場售價,限制了該類技術的應用與發(fā)展。本課題組開發(fā)的Candidasp.99-125脂肪酶系列產(chǎn)品具有成本低,比酶活高,穩(wěn)定性良好等優(yōu)點,性價比優(yōu)于同類國外產(chǎn)品。目前,以Candidasp. 99-125脂肪酶為核心的綠色催化平臺技術日漸成熟,并已成功應用于多類脂肪酸酯類化合物的合成與生產(chǎn)。但Candida sp. 99-125脂肪酶針對于結構型酯類化合物(糖醇酯,甘油結構酯等)的催化特性及催化區(qū)域選擇性尚有深入挖掘潛力,相關工藝尚未系統(tǒng)性開發(fā)并進一步優(yōu)化。另外,近年來脂肪酶催化的不飽和雙鍵環(huán)氧化反應也逐漸成為油脂改造的研究熱點之一,Candida sp. 99-125脂肪酶在該方面的應用尚需發(fā)展。為進一步完善以Candida sp. 99-125脂肪酶為基礎的綠色平臺催化技術,本論文針對結構型類化合物產(chǎn)品及環(huán)氧油脂類產(chǎn)品,分別選取具有產(chǎn)品代表性的木糖醇脂肪酸酯、人乳脂肪替代結構酯(Human Milk Fat Substitutes,HMFS)、環(huán)氧脂肪酸為目標產(chǎn)物,進行了合成過程分析及工藝開發(fā)的研究。論文通過探討Candida sp. 99-125脂肪酶的催化區(qū)域選擇性;優(yōu)化反應水含量及底物摩爾比等參數(shù),構建合理的反應微環(huán)境;結合過程反應機理及副反應進程,調(diào)控反應溫度、過程傳質(zhì)等參數(shù);最終設計開發(fā)了Candida sp. 99-125脂肪酶針對于相應產(chǎn)品的高效催化工藝。具體研究內(nèi)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劑催化反應體系。通過預加熱處理制備木糖醇-叔丁醇過飽和溶液,有效地提高反應體系中木糖醇濃度,從而提高木糖醇脂肪酸酯的轉化率。在該體系中反應6 h后,木糖醇轉化率約為70%,產(chǎn)品中1(5)-O-木糖醇單脂肪酸酯含量約95%.在此基礎上,本研究建立了更為綠色的木糖醇酯無溶劑反應體系。通過木糖醇等摩爾分批5次加入,解決反應底物溶解問題。反應120 h后,糖醇酯的轉化率為70%。其中1(5)-O-木糖醇癸酸單酯相對含量為16%,1,5-0-木糖醇癸酸二酯相對含量為60%,木糖醇1,2,5-0-癸酸三酯與1,3,5-O-木糖醇癸酸三酯相對含量為24%.同時發(fā)現(xiàn),體系最終產(chǎn)品構型同脂肪酶的催化選擇性無關,反應能量壁壘和體系熱力學特性決定了最終產(chǎn)品的構型和組成。2.母乳脂肪中甘油三酯具有特定結構,其從頭合成對催化選擇性及反應過程控制要求較高,極具挑戰(zhàn)性。本研究結合酶催化過程中�；D移副反應的機理實現(xiàn)了對反應過程進行調(diào)控,并有效控制了催化過程的位置選擇性,得到了針對Candida sp. 99-125脂肪酶催化合成HMFS的最佳工藝條件。催化得到的產(chǎn)品經(jīng)分離純化后,產(chǎn)品的酸價為2 mgKOH/g,甘油三酯中sn-2位棕櫚酸占總棕櫚酸比例的含量為52%-55%,棕櫚酸甘油三酯在總甘油三酯中的含量為8.4%-9.6%,目標產(chǎn)品型甘油三酯含量為55-59%,產(chǎn)品過氧化值1.41-1.89meq/g,產(chǎn)品的各項指標均可滿足并超過國家標準要求。3.不飽和脂肪酸的環(huán)氧化過程中,由于游離羧基的作用,使得該反應過程相對于脂肪酸酯的環(huán)氧化更加復雜,副反應更難于控制。本研究首先針對化學催化脂肪酸環(huán)氧化過程中,環(huán)氧基團開環(huán)副反應嚴重的問題,通過過程分析與優(yōu)化,建立了過程動力學模型,通過分析各反應的速率及能量問題,得到了化學催化過程各類開環(huán)反應難以避免的結論。在化學催化研究的基礎上,設計并優(yōu)化了Candida sp.99-125脂肪酶催化的不飽和脂肪酸環(huán)氧化反應體系。以油酸為底物,在30 ℃條件下,反應10 h后,產(chǎn)物環(huán)氧值可達4.12%,環(huán)氧轉化率76.58%,環(huán)氧化選擇性0.98。以無患子油混合脂肪酸為原料,驗證了該催化工藝的穩(wěn)定性和可靠性。最終,從反應條件、反應機理、反應動力學方面,對比了化學催化甲酸原位環(huán)氧化和Candida sp. 99-125脂肪酶催化環(huán)氧化過程的區(qū)別,討論了兩種方法的優(yōu)缺點。綜上所述,論文通過研究Candida sp. 99-125脂肪酶在結構型酯類化合物產(chǎn)品催化轉化過程中,�；D移副反應的機理、歷程、相關調(diào)控措施、策略;以及不飽和脂肪酸環(huán)氧化過程中開環(huán)副反應的影響及控制技術,過氧化物對脂肪酶的毒性問題及其解決方案;進一步完善了以Candidasp.99-125脂肪酶為基礎的綠色生物催化平臺技術,有利于提高生物催化技術在工業(yè)生產(chǎn)應用中的競爭力。
[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.
【學位授予單位】：北京化工大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：TQ641;O643.36

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本文編號：2216145