Method for immobilizing lipase by using carboxyl resin and immobilized lipase prepared by method
阅读说明：本技术 一种使用羧基树脂固定化脂肪酶的方法及由该方法制得的固定化脂肪酶 (Method for immobilizing lipase by using carboxyl resin and immobilized lipase prepared by method ) 是由 胡云峰 朱衡 张云 于 2019-10-11 设计创作，主要内容包括：本发明公开了一种使用羧基树脂固定化脂肪酶的方法及由该方法制得的固定化脂肪酶。该方法使用EDC·HCl作为交联剂,将羧基载体和游离酶通过酰胺键连接,达到固定化的目的。经酶学性质测定发现,与游离脂肪酶比较,本发明制得的固定化脂肪酶IDA-LIPASE在热耐受性、可重复利用性、保存稳定性方面具有极佳的优势,此外,在该方法下,进行模拟10倍扩大化,当载体量扩大为实验条件的10倍时,固定化酶酶活获得提高,因此具备实际扩大化生产的潜力,利用本发明制备的固定化酶可以在实际应用中发挥较好的催化能力,并且可以为生产降低成本,具有方便、实用、经济的特点,具有重要的研究价值和实用价值。(The invention discloses a method for immobilizing lipase by using carboxyl resin and immobilized lipase prepared by the method. The method uses EDC & HCl as a cross-linking agent, and connects a carboxyl carrier and a free enzyme through an amide bond to achieve the purpose of immobilization. The enzyme property determination shows that compared with free LIPASE, the immobilized LIPASE IDA-LIPASE prepared by the invention has excellent advantages in the aspects of heat tolerance, reusability and storage stability, in addition, under the method, 10 times of amplification is simulated, and when the carrier quantity is amplified to 10 times of experimental conditions, the enzyme activity of the immobilized enzyme is improved, so that the immobilized enzyme has the potential of actual amplification production.)
1. A method for immobilizing lipase by using carboxyl resin, which is characterized by comprising the following steps: and mixing the carboxyl carrier, the cross-linking agent and the lipase solution, carrying out immobilization reaction, and after the reaction is finished, carrying out suction filtration to remove residual enzyme solution and the cross-linking agent, thus obtaining the immobilized enzyme.
2. The method of claim 1, wherein the carboxyl carrier is LX-1000 IDA.
3. The method of claim 1, wherein the cross-linking agent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.
4. The method for immobilizing lipase using carboxyl resin according to any one of claims 1 to 3, comprising the steps of: mixing a dried LX-1000IDA carboxyl resin carrier with a 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride solution, adding a lipase solution, uniformly mixing, oscillating a table concentrator at 25 ℃ to perform immobilization reaction for 6 hours, performing suction filtration, and removing residual cross-linking agent and enzyme solution to obtain the immobilized lipase, wherein the mixing ratio of the carboxyl resin carrier to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride solution to the lipase solution is 2g:100mL:100 mL.
5. The method of claim 4, wherein the ratio of the amount of the carboxyl resin carrier to the lipase is 0.2g: 800U.
6. The method of claim 4, wherein the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride solution is 1.2% by mass, and the solvent is phosphate buffer solution having pH 4.5 and 0.1% by mass.
7. The method of claim 4, wherein the concentration of the lipase solution is 2mg/mL, the solvent is phosphate buffer solution with pH 4.5 and mass fraction of 0.1%.
8. The immobilized lipase obtained by the method for immobilizing a lipase on a carboxyl resin according to any one of claims 1 to 7.
Technical Field
The invention belongs to the technical field of biochemical engineering and biocatalysis, and particularly relates to a method for immobilizing lipase by using carboxyl resin and immobilized lipase prepared by the method.
Background
Lipases (EC 3.1.1.3) are important industrial enzymes capable of catalyzing reactions such as ester hydrolysis, ester exchange and ester synthesis, and in recent years, researchers find that lipases have important application potential in the aspect of producing biodiesel, and immobilized enzymes enable free lipases to resist high-strength actual industrial conditions such as high temperature and stirring in order to promote the application of lipases in industries such as chemical industry, environmental protection and medicine. The method has the advantages of operability, easy separation, economy, convenience, reutilization, stability, high mechanical strength and the like, and common immobilization methods comprise an embedding method, an adsorption method, a crosslinking method and a covalent bonding method. The related carrier types include natural carriers (chitosan, chitin, diatomite and the like), inorganic carriers (glass, metal, alumina, bentonite, silicon dioxide) and novel carriers (graphene and magnetic particles). At present, no relevant reports of using carboxyl resin to immobilize lipase exist.
Disclosure of Invention
The invention aims to provide a method for immobilizing lipase by using a carboxyl resin carrier and immobilized lipase prepared by the method, wherein the immobilized lipase has good heat resistance, operation stability and storage stability and has the potential of practical industrial production.
The technical scheme adopted by the invention is as follows:
the first object of the present invention is to provide a method for immobilizing lipase using carboxyl resin, comprising the steps of:
and mixing the carboxyl carrier, the cross-linking agent and the lipase solution, carrying out immobilization reaction, and after the reaction is finished, carrying out suction filtration to remove residual enzyme solution and the cross-linking agent, thus obtaining the immobilized enzyme.
The carboxyl carrier is LX-1000IDA, and the tail part of the carboxyl carrier is provided with two carboxyl active groups.
The cross-linking agent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC & HCl). The cross-linking agent has two C ═ N double bond structures and can react with carboxyl.
Specifically, the method for immobilizing the lipase by using the carboxyl resin comprises the following steps: mixing a dried LX-1000IDA carboxyl resin carrier with a 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride solution, adding a lipase solution, uniformly mixing, oscillating a table concentrator at 25 ℃ to perform immobilization reaction for 6 hours, performing suction filtration, and removing residual cross-linking agent and enzyme solution to obtain the immobilized lipase, wherein the mixing ratio of the carboxyl resin carrier to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride solution to the lipase solution is 2g:100mL:100 mL.
Preferably, the amount of the carboxyl resin carrier/lipase ratio is 0.2g: 800U.
The mass fraction of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride solution is 1.2%, and phosphate (Na) with pH of 4.5 and mass fraction of 0.1% is utilized2HPO4-NaH2PO4) And (4) dissolving the buffer solution.
The free lipase is Candida marinus lipase (CAS: 9001-62-1; Assay is more than or equal to 50000U/g, and molecular weight is 67000 Da). A lipase solution with a concentration of 2mg/mL was prepared using a phosphate buffer solution with a pH of 4.5 and a mass fraction of 0.1%.
The second object of the present invention is to provide an immobilized lipase obtained by the above method for immobilizing a lipase on a carboxyl resin.
The immobilized lipase (Candida marinus lipase) is prepared by covalent bonding of a carboxyl carrier (LX-1000IDA), and a crosslinking agent EDC & HCl with two C ═ N double bonds is used. The carboxyl at the tail of the carboxyl carrier reacts with C ═ N of EDC & HCl to form a compound, then the compound reacts with the amino of LIPASE, the LIPASE is fixed on the carboxyl carrier, the immobilized LIPASE IDA-LIPASE is prepared, and meanwhile, EDC & HCl structure is dropped, so that the structural complexity is reduced.
Further, in order to determine the enzymatic properties of free lipase, the reusability and various stability were improved. And (3) performing characterization of enzymology properties on the obtained immobilized LIPASE IDA-LIPASE, and determining the optimal reaction temperature, the optimal reaction pH, the thermal stability, the acid-base stability, the operation stability and the storage stability of the immobilized LIPASE IDA-LIPASE. Compared with free LIPASE, the enzymatic properties of the immobilized enzyme are improved, the immobilized enzyme IDA-LIPASE prepared under the optimal conditions is improved from 114U/g to 210U/g after the actual amplification and the ten-fold amplification of the carrier amount are simulated, and the enzyme has better actual industrial preparation and application potentials.
The optimum reaction temperature of IDA-LIPASE is increased by 10 ℃, the thermal stability is excellent, and the immobilized enzyme is improved by about 40% at most compared with the initial enzyme activity after incubation for 3 hours at the temperature of 40, 50, 60 and 70 ℃. The hydrolysis reaction can be repeated for 7 times, wherein 50% of enzyme activity is remained when the hydrolysis reaction is repeated for 4 times, and about 40% of enzyme activity is remained when the hydrolysis reaction is repeated for 7 times; incubating in a water bath at 70 deg.C for 3 hr, taking out part of the solution every 0.5 hr to measure residual enzyme activity, and finding that 60% of the immobilized enzyme still remains after 3 hr. And the wide temperature adaptation range is found; the storage stability is excellent, and 84.6 percent of enzyme activity is still kept after the enzyme is stored for 30 days at 4 ℃.
The invention has the beneficial effects that: the enzyme property test shows that compared with free LIPASE (CRL), the immobilized LIPASE IDA-LIPASE prepared by the method of using carboxyl carrier resin to immobilize LIPASE improves the optimum reaction temperature, the optimum reaction pH, the thermal stability, the operation repeatability and the storage stability. IDA-LIPASE is incubated for 3 hours in a high-temperature environment (40, 50, 60 and 70 ℃), and then the immobilized enzyme is found to be improved to different degrees compared with the initial enzyme activity, and the highest enzyme activity is improved by about 40 percent. The hydrolysis reaction can be repeated for 7 times, wherein 50% of enzyme activity is remained when the hydrolysis reaction is repeated for 4 times, and about 40% of enzyme activity is remained when the hydrolysis reaction is repeated for 7 times; incubating in a water bath at 70 deg.C for 3 hr, taking out part of the solution every 0.5 hr to measure residual enzyme activity, and detecting that 60% of the immobilized enzyme still remains after 3 hr. After 30 days of storage, 84.6% of enzyme activity remained. The immobilized enzyme IDA-LIPASE prepared by the method has the advantages of catalytic capability expression and preservation under the condition of high-temperature stirring, so that the immobilized enzyme has wide application potential, can be used for actual expanded production, and has important research and practical values.
Drawings
FIG. 1 is a schematic diagram of the preparation process of immobilized enzyme IDA-LIPASE.
FIG. 2 is a pH characterization of the optimum reaction for immobilized enzyme IDA-LIPASE and free enzyme CRL.
FIG. 3 is a graph showing the optimum reaction temperatures for immobilized enzyme IDA-LIPASE and free enzyme CRL.
FIG. 4 is a thermal stability characterization of immobilized enzymes IDA-LIPASE and free enzyme CRL, wherein (a) is the enzyme activity results of incubating the immobilized enzymes IDA-LIPASE and free enzyme CRL in an incubation environment at 70 ℃ for 3h and measuring every 30 min; (b) the enzyme activity result is measured after the immobilized enzyme IDA-LIPASE and the free enzyme CRL are incubated for 3 hours in water bath environments with different temperatures (40 ℃, 50 ℃, 60 ℃ and 70 ℃).
FIG. 5 shows the acid-base stability characterization of immobilized enzyme IDA-LIPASE and free enzyme CRL.
FIG. 6 is a graph showing the operational stability of immobilized enzyme IDA-LIPASE and free enzyme CRL.
FIG. 7 shows the storage stability of immobilized enzyme IDA-LIPASE and free enzyme CRL.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
In the following examples, lipase immobilized on a carboxyl resin carrier was used, and the free lipase was Candida marinus lipase (CAS: 9001-62-1; Assay. gtoreq.50000U/g; molecular weight: 67000 Da). 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC & HCl) is taken as a coupling agent, and LX-1000IDA is taken as a carboxyl resin carrier.