粗状假丝酵母脂肪酶的分离纯化水解乳脂的研究
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摘要
脂肪酶(triacylglycerol acylhydrolases, E.C. 3.1.1.3)是一类能催化长链脂肪酸甘油酯水解为甘油和长链脂肪酸(或者是酯合成反应)的生物催化剂,广泛应用于有机合成、脂肪和油脂的水解、油脂改性、精细化工和消旋体药物的手性拆分等诸多领域。目前,国内对脂肪酶制剂的需求不断增长,脂肪酶高产菌株的筛选,其发酵条件的优化以及适用于工业化大生产的分离纯化工艺成为研究的热点。
乳脂是一种用途极为广泛的食品配料,但其风味物质大多是一些易挥发的酸类、酮醛类和内酯类物质。利用脂肪酶对乳脂中甘油三酯的水解反应,增加乳脂中的中、短链脂肪酸,可达到增强风味的目的。
本课题对实验室保存的粗状假丝酵母菌种Candida valida1444诱变株进行筛选复壮,对其脂肪酶进行了分离纯化,并对该脂肪酶水解天然乳脂的特性进行了探讨。主要研究工作包括以下几个方面:
1.利用平板涂布法对粗状假丝酵母菌种Candida valida1444诱变株进行菌种复壮,筛选得到一株产脂肪酶活性相对较高且传代稳定的菌株Z8D3。
2.对粗状假丝酵母诱变株Z8D3产脂肪酶进行分离纯化,发酵液经超声波破碎得到粗酶液,然后经过双水相萃取、丙酮沉淀、DEAE-Sepharose FF阴离子交换层析、Phenyl Sepharose FF疏水层析等步骤的处理,比活力从8.19U/mg提高到170.43 U/mg,纯化倍数为20.81倍,活力回收率为19.8%。最后经过SDS-PAGE电泳,得到单一条带,分子量约为56.6kDa。
3.对假丝酵母脂肪酶水解天然乳脂的各影响因素进行条件优化,得到催化水解反应的最适条件为:摇床转速180rpm,质量分数1.0%的OP乳化剂,温度为45℃,缓冲体系pH值为8.0,加酶量为200U,反应21h,乳脂水解率达到最佳。并通过GC-MS分析鉴定了水解产物中主要的致香成分。
本论文对粗状假丝酵母诱变株产脂肪酶的分离纯化做了基础性研究,并对该脂肪酶水解天然乳脂的特性进行了探讨,为该酶的催化特性研究,高纯度酶制剂的制备工艺及在乳品工业中的应用提供了一定的理论指导。
Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is a kind of biocatalysts to hydrolysis long-chain triglycerides to glycerol and fang-chain fatty acids. It can hydrolysis Triglyceride to fatty acid and glycerol in the interface of oil-water. Lipases occur widely in nature, but only microbial lipases are commercially significant. Many applications of lipases include speciality organic syntheses, hydrolysis of fats and oils, modification of fats, flavor enhancement in food processing, chemical analyses and resolution of racemic mixtures.As the domestic demand for lipase was growing, screening of high-yield lipase strain, optimum conditions of fermentation and industrial large-scale purification of lipase become research focuses.
Natural milk fat is widely used in foods, but the volatile aroma of milk fat is mainly related with fatty acids, ketones, aldehydes, and lactones, which are apt to losses in the course of food processing. The flavor of milk fat could be improved by increasing the content of short- and medium-chain fatty acids by means of lipase catalyzed hydrolysis.
Lipase exhibits a different substrate specificity and selectivity relative to the structure or length of lipid chains depending on its source. In this research work, rejuvenation was used to improve the lipase production of Candida valida mutated with UV. The lipase from Candida valida was purified to homogeneity and the hydrolysis of natural milk fat by the lipase was also studied in the paper. The main results were shown below:
1. We used plate coating methods to rejuvenate Candida valida Z6 which was conserved by our laboratory. Finally, the high yield Candida valida Z8D3 strain was selected. Stability test demonstrated that the lipase activity remained the same level after five generations.
2. The lipase from Candida valida Z8D3 has been purified using ultrasonic fragmentation followed by aqueous two-phase extraction, acetone precipitation, DEAE-Sepharose FF anion-exchange chromatography and Phenyl Sepharose FF hydrophobic interaction chromatography. This protocol resulted in 20.81 fold purification with 19.8% final recovery, specific activity from 8.19U/mg to170.43 U/mg. The purified enzyme showed a prominent single band on SDS–PAGE. The molecular mass of the purified enzyme was determined to be approximately 56.6 kDa.
3. For the hydrolysis of natural milk fat by the lipase, the optimum hydrolysis conditions were: the speed of shaker 180 rpm, OP emulsifier content of 1.0% milk fat, temperature 45℃, pH value 8.0, quantity of lipase 200 U and hydrolysis time 21h. The main fragrant ingredients in the hydrolyzed natural milk fat were also determined by GC-MS.
This thesis has done some research on Candida valida lipase, including purification and the hydrolysis of natural milk fat by the lipase. This will be a foundation for the study of its catalysed mechanism, large-scale purification and application in dairy industry.
乳脂是一种用途极为广泛的食品配料,但其风味物质大多是一些易挥发的酸类、酮醛类和内酯类物质。利用脂肪酶对乳脂中甘油三酯的水解反应,增加乳脂中的中、短链脂肪酸,可达到增强风味的目的。
本课题对实验室保存的粗状假丝酵母菌种Candida valida1444诱变株进行筛选复壮,对其脂肪酶进行了分离纯化,并对该脂肪酶水解天然乳脂的特性进行了探讨。主要研究工作包括以下几个方面:
1.利用平板涂布法对粗状假丝酵母菌种Candida valida1444诱变株进行菌种复壮,筛选得到一株产脂肪酶活性相对较高且传代稳定的菌株Z8D3。
2.对粗状假丝酵母诱变株Z8D3产脂肪酶进行分离纯化,发酵液经超声波破碎得到粗酶液,然后经过双水相萃取、丙酮沉淀、DEAE-Sepharose FF阴离子交换层析、Phenyl Sepharose FF疏水层析等步骤的处理,比活力从8.19U/mg提高到170.43 U/mg,纯化倍数为20.81倍,活力回收率为19.8%。最后经过SDS-PAGE电泳,得到单一条带,分子量约为56.6kDa。
3.对假丝酵母脂肪酶水解天然乳脂的各影响因素进行条件优化,得到催化水解反应的最适条件为:摇床转速180rpm,质量分数1.0%的OP乳化剂,温度为45℃,缓冲体系pH值为8.0,加酶量为200U,反应21h,乳脂水解率达到最佳。并通过GC-MS分析鉴定了水解产物中主要的致香成分。
本论文对粗状假丝酵母诱变株产脂肪酶的分离纯化做了基础性研究,并对该脂肪酶水解天然乳脂的特性进行了探讨,为该酶的催化特性研究,高纯度酶制剂的制备工艺及在乳品工业中的应用提供了一定的理论指导。
Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is a kind of biocatalysts to hydrolysis long-chain triglycerides to glycerol and fang-chain fatty acids. It can hydrolysis Triglyceride to fatty acid and glycerol in the interface of oil-water. Lipases occur widely in nature, but only microbial lipases are commercially significant. Many applications of lipases include speciality organic syntheses, hydrolysis of fats and oils, modification of fats, flavor enhancement in food processing, chemical analyses and resolution of racemic mixtures.As the domestic demand for lipase was growing, screening of high-yield lipase strain, optimum conditions of fermentation and industrial large-scale purification of lipase become research focuses.
Natural milk fat is widely used in foods, but the volatile aroma of milk fat is mainly related with fatty acids, ketones, aldehydes, and lactones, which are apt to losses in the course of food processing. The flavor of milk fat could be improved by increasing the content of short- and medium-chain fatty acids by means of lipase catalyzed hydrolysis.
Lipase exhibits a different substrate specificity and selectivity relative to the structure or length of lipid chains depending on its source. In this research work, rejuvenation was used to improve the lipase production of Candida valida mutated with UV. The lipase from Candida valida was purified to homogeneity and the hydrolysis of natural milk fat by the lipase was also studied in the paper. The main results were shown below:
1. We used plate coating methods to rejuvenate Candida valida Z6 which was conserved by our laboratory. Finally, the high yield Candida valida Z8D3 strain was selected. Stability test demonstrated that the lipase activity remained the same level after five generations.
2. The lipase from Candida valida Z8D3 has been purified using ultrasonic fragmentation followed by aqueous two-phase extraction, acetone precipitation, DEAE-Sepharose FF anion-exchange chromatography and Phenyl Sepharose FF hydrophobic interaction chromatography. This protocol resulted in 20.81 fold purification with 19.8% final recovery, specific activity from 8.19U/mg to170.43 U/mg. The purified enzyme showed a prominent single band on SDS–PAGE. The molecular mass of the purified enzyme was determined to be approximately 56.6 kDa.
3. For the hydrolysis of natural milk fat by the lipase, the optimum hydrolysis conditions were: the speed of shaker 180 rpm, OP emulsifier content of 1.0% milk fat, temperature 45℃, pH value 8.0, quantity of lipase 200 U and hydrolysis time 21h. The main fragrant ingredients in the hydrolyzed natural milk fat were also determined by GC-MS.
This thesis has done some research on Candida valida lipase, including purification and the hydrolysis of natural milk fat by the lipase. This will be a foundation for the study of its catalysed mechanism, large-scale purification and application in dairy industry.
引文
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