白地霉脂肪酶的制备、修饰改良及应用研究
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摘要
脂肪酶催化是绿色催化的重要组成部分。在非水相介质中,脂肪酶催化活力不高是制约脂肪酶非水相催化的主要因素。运用定向进化和理化技术对脂肪酶进行修饰改良,创制出高酶活力和高稳定性的酶制剂,是蛋白质工程和酶催化剂工程领域的重要研究方向。围绕脂肪酶修饰改良这一热点问题,本文立足于脂肪酶毕赤酵母重组工程菌的构建,以生产背景清晰的白地霉脂肪酶。分别以油酸甲酯合成和鱼油水解代表非水相催化和水相催化,运用基于双分子印迹的多重组合修饰、有机溶剂处理、交联聚集和酶蛋白包衣的新型固定化技术,对白地霉脂肪酶进行修饰改良,拟创制出适用于酯化合成和油脂水解的新型催化剂。本研究开发的双分子印迹技术及其多重修饰技术,丰富了物理化学修饰技术；并将两相固定化—鱼油处理、交联聚集和酶蛋白包衣修饰技术应用于水解鱼油富集PUFAs研究。创制出的修饰改良型白地霉脂肪酶水解鱼油富集PUFAs效果明显提高。这些非水相修饰和水相修饰方法还为其它酶的修饰改良提供了借鉴,可尝试性地将粗放型的酶液创制成高活力和高操作稳定性的精细型酶制剂。主要结果及创新点摘要如下：
1.构建了白地霉脂肪酶基因工程菌,生产背景清晰的脂肪酶,代替高成本且背景不清晰的商品酶,提供用于修饰改良的酶源。在硕士阶段构建重组工程菌的基础上,进一步比较了由四种载体pPIC9K、pPICZaA、pGAPZaA和GAP-pPIC9K构建的重组菌的产酶效果,以pPIC9K载体构建的重组菌酶活力最高。该重组脂肪酶对具有C9位顺式双键的长链不饱和脂肪酸甘油酯具有明显的底物特异性,且具有宽广的天然油脂底物谱；对正己烷等有机溶剂具耐受性；其最适温度范围和最适pH分别为40-50℃和8.0。独特的底物特异性和良好的有机溶剂、温度及pH耐受性表明该酶拥有良好工业应用潜力。
2.综合运用基于生物印迹的多重组合修饰技术,有效提高了脂肪酶的非水相催化活力和操作稳定性。以油酸甲酯合成为模式反应,以自制脂肪酶为研究材料,基于油酸和甲醇双分子生物印迹,联用pH记忆、添加剂激活、脂包衣及大孔树脂固定化等物理化学修饰技术进行酶的修饰改良。与未修饰酶相比,这些方法协同修饰使脂肪酶的酯化活力提高至18.4倍,重复使用10个批次仍能保留90%的相对转化率。
3.在常规大孔树脂固定化的基础上,引入了固定化前有机溶剂预处理和固定化后有机溶剂后处理；同时对脂肪酶进行了交联聚集和酶蛋白包衣固定化修饰,提高了脂肪酶的非水相催化活力和操作稳定性。先水相固定化,再用异丙醇处理,使脂肪酶的酯化活力提高至12.6倍；辛烷—水预处理,然后再固定化(即两相固定化)使酯化活力提高至14.8倍；交联聚集制备的修饰酶PEI-CLEAs使酯化活力提高至16.1倍；酶蛋白包衣修饰制备的微晶酶PCMCs使酯化活力提高至19.7倍。四种新型固定化酶重复使用5个批次仍能保留80%以上的相对转化率。
4.在非水相修饰的基础上,针对鱼油水解的水相催化,调整修饰策略,用鱼油处理代替油酸印迹,并偶联辛烷—水两相固定化修饰,应用于鱼油水解的水相催化,取得了良好效果。游离酶仅能达到12%的水解度,水相固定化修饰酶能将其提高至28%,两相固定化修饰酶可提高至31%,水相固定化与鱼油处理组合修饰酶可提高至36%,而两相固定化与鱼油处理组合修饰酶则可提高至40%。固定化—鱼油处理酶的酶促反应初速度和水解度高于固定化—油酸印迹酶,而鱼油处理和固定化分步与同步制备对酶促反应的初速度和水解度影响不大。强极性和非极性溶剂均弱化了固定化—鱼油处理酶的催化活力,弱极性溶剂则能维持较好的催化效果。重复使用5个批次后,固定化—鱼油处理酶仍能保留80%的水解度。辛烷—水两相固定化与鱼油处理组合修饰用于鱼油水解反应系本文首创。
5.将交联聚集修饰应用于鱼油水解的水相催化,PEI-CLEAs水解鱼油的初速度、水解度、操作稳定性、温度和有机溶剂耐受性均优于游离酶。PEI-CLEAs能获得42%的水解度,将EPA和DHA从原始鱼油的6.94%和0.97%分别富集到12.69%和3.88%,EPA和DHA (EPA+DHA)得率为77.51%。PEI-CLEAs在50-55℃仍能保留65%的相对水解度,经丙酮、叔丁醇和辛烷处理后仍能保留85%的相对水解度。重复使用5个批次,PEI-CLEAs相对水解度能保持72%。
6.将酶蛋白包衣修饰应用于鱼油水解的水相催化,PCMCs水解鱼油的初速度、水解度、操作稳定性、温度和有机溶剂耐受性也均优于游离酶。PCMCs能获得48%的水解度,将EPA和DHA分别富集到12.89%和3.98%,EPA和DHA得率为81.04%。PCMCs在45-50℃范围内能保持85%以上的相对水解度；在丙酮、丙醇、叔丁醇和辛烷中保留86%以上的相对水解度。重复使用5个批次,PCMCs仅能保持52%的相对水解度。
7.对于鱼油水解催化反应,固定化—鱼油处理酶、交联酶聚集体和酶蛋白包衣催化的初速度、水解度、获得最大水解度所需时间、温度和有机溶剂耐受性、操作稳定性存在较大差异。不同的修饰酶在不同方面表现出优势,应针对催化目的和条件,选择合适的修饰酶。
Nonaqueous biocatalysis with lipase has advantages over aqueous biocatalysis owing to its multiple reaction types. For a given lipase-catalyzed transformation process, it is very important to obtain suitable and effective enzyme preparations, since the catalytic activity and enzyme stability are key factors affecting biocatalysis efficiency. The major obstacles restricting lipase application in non-aqueous biocatalysis are low reaction rates and poor stability. Modifying or improving enzyme preparations with desirable catalytic properties through directed evolution or physical/chemical methodology has been a focus of recent research. In this study, constructions of recombinant strains for producing clear background Geotrichum candidum lipase were performed. Based on these, double imprint molecule bioimprinting coupled to other methods, as well as novel immobilization techniques including organic treatment, cross-linked enzyme aggregates (CLEAs), protein-coated micro-crystals (PCMCs) were employed to modify free lipase. These modified lipase preparations were successfully applied in esterification of oleic acid and methanol and hydrolysis of fish oil for enrichment of PUFAs. These approaches provided reference for the preparation of other enzyme biocatalysts by upgrading crude enzymes to refined biocatalysts with high activity and stability.
1. Based on my previous work for master degree, which mainly focused on cloning of Geotrichum candidum lipase gene, constructions of recombinant strains of Pichia pastoris with pPIC9K, pPICZaA, pGAPZaA and GAP-pPIC9K were further performed here. The results showed that pPIC9K vector was favourable for expression of Geotrichum candidum lipase. Recombinant lipase was produced, purified and characterized. Characterization of the properties showed that the lipase exhibited maximum activity at 40-50℃and pH 8.0, and was fairly stable between pH 6.0-10.0 and below the temperature 60℃. The lipase was compatible with the presence of organic solvents such as n-heptane, hexane, cyclohexane, benzene and diethyl ether. The lipase showed a notable hydrolysis preference for vegetable oils and triacylglycerol substrates containing cis-9 unsaturated fatty acid. The lipase also exhibited a good hrdrolysis activity towards a wide range of natural oils. The above properties indicate that the lipase is a promising candidate for applications in biocatalysis.
2. Geotrichum candidum lipase with enhanced activity and operational stability was prepared for use in esterification of oleic acid and methanol for the first time. A combined strategy comprising bioimprinting with dual imprint molecules and a co-solvent coupled to pH tuning, KCl salt activation, lecithin coating and immobilization on macroporous resin effectively enhanced the activity and operational stability of Geotrichum candidum lipase. The modified lipase enhanced 18.4-fold esterification activity towards methyl oleate synthesis, and retained 90% relative conversion by repeated usage of 10 times.
3. Based on conventional immobilization, organic solvent pretreatment before immobilization and organic solvent treatment after immobilization, as well as cross-linking of enzyme aggregates and protein-coating were introduced. For reaction of esterification of oleic acid and methanol, compared to immobilized lipase in buffer, polar isopropanol and propanol treatment after immobilization gave higher esterification activity. Isopropanol-buffer pretreatment and octane-buffer pretreatment before immobilization gave higher esterification activity than immobilized lipase in buffer. Especially, the effect of octane-buffer pretreatment was more obvious. PEI-CLEAs and PCMCs also showed better biocatalysis efficiency (esterification activity, activity recovery and operational stability) than free lipase in esterification reaction of oleic acid and methanol.
4. Based on non-aqueous modifications, adjustment of modification methods was performed. Namely, fish oil treatment substituted for oleic acid bioimprinting, and coupled to immobilization were carried out, and then applied these modified lipases into hydrolysis of fish oil successfully. For free lipase, being liable to agglomeration in reaction medium was overcome by immobilization. Interfacial activation was introduced by immobilization in octane-buffer mixture. Substrate-binding pockets were activated by treatment of fish oil. These modification procedures resulted in different enhancement in initial reaction rate and hydrolysis degree. For hydrolysis of fish oil, free lipase without any modification only gave 12% of hydrolysis degree. Lipase immobilized in buffer and immobilized in octane-buffer mixture gave 28% and 31% of hydrolysis degree, respectively. Lipase immobilized in buffer coupled to treatment by fish oil gave 36% of hydrolysis degree, while lipase immobilized in octane-buffer mixture coupled to treatment by fish oil gave 40% of hydrolysis degree. Initial reaction rate and hydrolysis degree by immobilized lipase coupled to treatment by fish oil were both higher than those of immobilized lipase coupled to bioimprinting using oleic acid as imprint molecule. However, modified by immobilization and treatment of fish oil stepwisely or simultaneously had no significant effect on initial reaction rate and hydrolysis degree. Strong polar and hydrophobic solvents had negative impact on immobilization-fish oil treatment lipase, low polar solvents were helpful to maintain the modification effect of immobilization-fish oil treatment lipases. After 5 batch of usage, the immobilization-fish oil treatment lipases still maintained more than 80% of relative hydrolysis degree.
5. Cross-linking of enzyme aggregates was applied into hydrolysis of fish oil successfully. Based on acetone precipitation, PEI and glutaraldehyde and enzyme interaction, stable cross-linked enzyme aggregates PEI-CLEAs was prepared. PEI-CLEAs had more excellent temperature and organic solvent tolerance than free lipase and CLEAs, which could maintain more than 65% of hydrolysis degree in the temperature range of 50-55℃, and maintain more than 85% of hydrolysis degree after being treated by acetone, tertiary butanol and octane. PEI-CLEAs increased hydrolysis degree to 42%. After 5 batch reactions, PEI-CLEAs still maintained more than 72% of relative hydrolysis degree. PEI-CLEAs had advantages over CLEAs and free lipase in initial reaction rate, hydrolysis degree and operational stability.
6. Protein-coating was applied into hydrolysis of fish oil successfully. In the temperature range of 45-50℃, PCMCs showed better thermostability than free lipase, and retained at least 86% of relative hydrolysis degree after treatment by acetone, octane, tertiary butanol and propanol. PCMCs increased hydrolysis degree to 48%. After continuous usage of 5 batches, PCMCs still maintained more than 52% of relative hydrolysis degree.
7. For hydrolysis of fish oil, lipase preparations modified by immobilization-fish oil treatment, aggregation and cross-linking, and enzyme coating exhibited different modification effect. Different modification methods gave different degree of initial reaction rate, hydrolysis degree, required time of achieving the highest hydrolysis degree, thermostability, organic solvent tolerance and operational stability. Different lipase preparations showed advantage in different aspects, thus, when being applied, special lipase preparation matches specific target and condition.
1.构建了白地霉脂肪酶基因工程菌,生产背景清晰的脂肪酶,代替高成本且背景不清晰的商品酶,提供用于修饰改良的酶源。在硕士阶段构建重组工程菌的基础上,进一步比较了由四种载体pPIC9K、pPICZaA、pGAPZaA和GAP-pPIC9K构建的重组菌的产酶效果,以pPIC9K载体构建的重组菌酶活力最高。该重组脂肪酶对具有C9位顺式双键的长链不饱和脂肪酸甘油酯具有明显的底物特异性,且具有宽广的天然油脂底物谱；对正己烷等有机溶剂具耐受性；其最适温度范围和最适pH分别为40-50℃和8.0。独特的底物特异性和良好的有机溶剂、温度及pH耐受性表明该酶拥有良好工业应用潜力。
2.综合运用基于生物印迹的多重组合修饰技术,有效提高了脂肪酶的非水相催化活力和操作稳定性。以油酸甲酯合成为模式反应,以自制脂肪酶为研究材料,基于油酸和甲醇双分子生物印迹,联用pH记忆、添加剂激活、脂包衣及大孔树脂固定化等物理化学修饰技术进行酶的修饰改良。与未修饰酶相比,这些方法协同修饰使脂肪酶的酯化活力提高至18.4倍,重复使用10个批次仍能保留90%的相对转化率。
3.在常规大孔树脂固定化的基础上,引入了固定化前有机溶剂预处理和固定化后有机溶剂后处理；同时对脂肪酶进行了交联聚集和酶蛋白包衣固定化修饰,提高了脂肪酶的非水相催化活力和操作稳定性。先水相固定化,再用异丙醇处理,使脂肪酶的酯化活力提高至12.6倍；辛烷—水预处理,然后再固定化(即两相固定化)使酯化活力提高至14.8倍；交联聚集制备的修饰酶PEI-CLEAs使酯化活力提高至16.1倍；酶蛋白包衣修饰制备的微晶酶PCMCs使酯化活力提高至19.7倍。四种新型固定化酶重复使用5个批次仍能保留80%以上的相对转化率。
4.在非水相修饰的基础上,针对鱼油水解的水相催化,调整修饰策略,用鱼油处理代替油酸印迹,并偶联辛烷—水两相固定化修饰,应用于鱼油水解的水相催化,取得了良好效果。游离酶仅能达到12%的水解度,水相固定化修饰酶能将其提高至28%,两相固定化修饰酶可提高至31%,水相固定化与鱼油处理组合修饰酶可提高至36%,而两相固定化与鱼油处理组合修饰酶则可提高至40%。固定化—鱼油处理酶的酶促反应初速度和水解度高于固定化—油酸印迹酶,而鱼油处理和固定化分步与同步制备对酶促反应的初速度和水解度影响不大。强极性和非极性溶剂均弱化了固定化—鱼油处理酶的催化活力,弱极性溶剂则能维持较好的催化效果。重复使用5个批次后,固定化—鱼油处理酶仍能保留80%的水解度。辛烷—水两相固定化与鱼油处理组合修饰用于鱼油水解反应系本文首创。
5.将交联聚集修饰应用于鱼油水解的水相催化,PEI-CLEAs水解鱼油的初速度、水解度、操作稳定性、温度和有机溶剂耐受性均优于游离酶。PEI-CLEAs能获得42%的水解度,将EPA和DHA从原始鱼油的6.94%和0.97%分别富集到12.69%和3.88%,EPA和DHA (EPA+DHA)得率为77.51%。PEI-CLEAs在50-55℃仍能保留65%的相对水解度,经丙酮、叔丁醇和辛烷处理后仍能保留85%的相对水解度。重复使用5个批次,PEI-CLEAs相对水解度能保持72%。
6.将酶蛋白包衣修饰应用于鱼油水解的水相催化,PCMCs水解鱼油的初速度、水解度、操作稳定性、温度和有机溶剂耐受性也均优于游离酶。PCMCs能获得48%的水解度,将EPA和DHA分别富集到12.89%和3.98%,EPA和DHA得率为81.04%。PCMCs在45-50℃范围内能保持85%以上的相对水解度；在丙酮、丙醇、叔丁醇和辛烷中保留86%以上的相对水解度。重复使用5个批次,PCMCs仅能保持52%的相对水解度。
7.对于鱼油水解催化反应,固定化—鱼油处理酶、交联酶聚集体和酶蛋白包衣催化的初速度、水解度、获得最大水解度所需时间、温度和有机溶剂耐受性、操作稳定性存在较大差异。不同的修饰酶在不同方面表现出优势,应针对催化目的和条件,选择合适的修饰酶。
Nonaqueous biocatalysis with lipase has advantages over aqueous biocatalysis owing to its multiple reaction types. For a given lipase-catalyzed transformation process, it is very important to obtain suitable and effective enzyme preparations, since the catalytic activity and enzyme stability are key factors affecting biocatalysis efficiency. The major obstacles restricting lipase application in non-aqueous biocatalysis are low reaction rates and poor stability. Modifying or improving enzyme preparations with desirable catalytic properties through directed evolution or physical/chemical methodology has been a focus of recent research. In this study, constructions of recombinant strains for producing clear background Geotrichum candidum lipase were performed. Based on these, double imprint molecule bioimprinting coupled to other methods, as well as novel immobilization techniques including organic treatment, cross-linked enzyme aggregates (CLEAs), protein-coated micro-crystals (PCMCs) were employed to modify free lipase. These modified lipase preparations were successfully applied in esterification of oleic acid and methanol and hydrolysis of fish oil for enrichment of PUFAs. These approaches provided reference for the preparation of other enzyme biocatalysts by upgrading crude enzymes to refined biocatalysts with high activity and stability.
1. Based on my previous work for master degree, which mainly focused on cloning of Geotrichum candidum lipase gene, constructions of recombinant strains of Pichia pastoris with pPIC9K, pPICZaA, pGAPZaA and GAP-pPIC9K were further performed here. The results showed that pPIC9K vector was favourable for expression of Geotrichum candidum lipase. Recombinant lipase was produced, purified and characterized. Characterization of the properties showed that the lipase exhibited maximum activity at 40-50℃and pH 8.0, and was fairly stable between pH 6.0-10.0 and below the temperature 60℃. The lipase was compatible with the presence of organic solvents such as n-heptane, hexane, cyclohexane, benzene and diethyl ether. The lipase showed a notable hydrolysis preference for vegetable oils and triacylglycerol substrates containing cis-9 unsaturated fatty acid. The lipase also exhibited a good hrdrolysis activity towards a wide range of natural oils. The above properties indicate that the lipase is a promising candidate for applications in biocatalysis.
2. Geotrichum candidum lipase with enhanced activity and operational stability was prepared for use in esterification of oleic acid and methanol for the first time. A combined strategy comprising bioimprinting with dual imprint molecules and a co-solvent coupled to pH tuning, KCl salt activation, lecithin coating and immobilization on macroporous resin effectively enhanced the activity and operational stability of Geotrichum candidum lipase. The modified lipase enhanced 18.4-fold esterification activity towards methyl oleate synthesis, and retained 90% relative conversion by repeated usage of 10 times.
3. Based on conventional immobilization, organic solvent pretreatment before immobilization and organic solvent treatment after immobilization, as well as cross-linking of enzyme aggregates and protein-coating were introduced. For reaction of esterification of oleic acid and methanol, compared to immobilized lipase in buffer, polar isopropanol and propanol treatment after immobilization gave higher esterification activity. Isopropanol-buffer pretreatment and octane-buffer pretreatment before immobilization gave higher esterification activity than immobilized lipase in buffer. Especially, the effect of octane-buffer pretreatment was more obvious. PEI-CLEAs and PCMCs also showed better biocatalysis efficiency (esterification activity, activity recovery and operational stability) than free lipase in esterification reaction of oleic acid and methanol.
4. Based on non-aqueous modifications, adjustment of modification methods was performed. Namely, fish oil treatment substituted for oleic acid bioimprinting, and coupled to immobilization were carried out, and then applied these modified lipases into hydrolysis of fish oil successfully. For free lipase, being liable to agglomeration in reaction medium was overcome by immobilization. Interfacial activation was introduced by immobilization in octane-buffer mixture. Substrate-binding pockets were activated by treatment of fish oil. These modification procedures resulted in different enhancement in initial reaction rate and hydrolysis degree. For hydrolysis of fish oil, free lipase without any modification only gave 12% of hydrolysis degree. Lipase immobilized in buffer and immobilized in octane-buffer mixture gave 28% and 31% of hydrolysis degree, respectively. Lipase immobilized in buffer coupled to treatment by fish oil gave 36% of hydrolysis degree, while lipase immobilized in octane-buffer mixture coupled to treatment by fish oil gave 40% of hydrolysis degree. Initial reaction rate and hydrolysis degree by immobilized lipase coupled to treatment by fish oil were both higher than those of immobilized lipase coupled to bioimprinting using oleic acid as imprint molecule. However, modified by immobilization and treatment of fish oil stepwisely or simultaneously had no significant effect on initial reaction rate and hydrolysis degree. Strong polar and hydrophobic solvents had negative impact on immobilization-fish oil treatment lipase, low polar solvents were helpful to maintain the modification effect of immobilization-fish oil treatment lipases. After 5 batch of usage, the immobilization-fish oil treatment lipases still maintained more than 80% of relative hydrolysis degree.
5. Cross-linking of enzyme aggregates was applied into hydrolysis of fish oil successfully. Based on acetone precipitation, PEI and glutaraldehyde and enzyme interaction, stable cross-linked enzyme aggregates PEI-CLEAs was prepared. PEI-CLEAs had more excellent temperature and organic solvent tolerance than free lipase and CLEAs, which could maintain more than 65% of hydrolysis degree in the temperature range of 50-55℃, and maintain more than 85% of hydrolysis degree after being treated by acetone, tertiary butanol and octane. PEI-CLEAs increased hydrolysis degree to 42%. After 5 batch reactions, PEI-CLEAs still maintained more than 72% of relative hydrolysis degree. PEI-CLEAs had advantages over CLEAs and free lipase in initial reaction rate, hydrolysis degree and operational stability.
6. Protein-coating was applied into hydrolysis of fish oil successfully. In the temperature range of 45-50℃, PCMCs showed better thermostability than free lipase, and retained at least 86% of relative hydrolysis degree after treatment by acetone, octane, tertiary butanol and propanol. PCMCs increased hydrolysis degree to 48%. After continuous usage of 5 batches, PCMCs still maintained more than 52% of relative hydrolysis degree.
7. For hydrolysis of fish oil, lipase preparations modified by immobilization-fish oil treatment, aggregation and cross-linking, and enzyme coating exhibited different modification effect. Different modification methods gave different degree of initial reaction rate, hydrolysis degree, required time of achieving the highest hydrolysis degree, thermostability, organic solvent tolerance and operational stability. Different lipase preparations showed advantage in different aspects, thus, when being applied, special lipase preparation matches specific target and condition.
引文
[1]Hasan F, Shah AA, Hameed A. Industrial applications of microbial lipases[J]. Enzymey and Microbial Technology,2006,39:235-251
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