CN113073090A

CN113073090A - Method for immobilizing lipase for enriching polyunsaturated fatty acids

Info

Publication number: CN113073090A
Application number: CN202110367775.2A
Authority: CN
Inventors: 王小三; 杨壮壮; 江聪; 程昕祎; 董喆; 黄健花; 常明
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-04-06
Filing date: 2021-04-06
Publication date: 2021-07-06

Abstract

The invention discloses a lipase immobilization method for enriching polyunsaturated fatty acids. The method comprises the steps of dissolving lipase in a buffer solution to obtain a lipase solution; and (3) putting the lipase liquid and resin into a reactor, separating a solid phase and a liquid phase after reaction, and drying the solid phase to obtain the immobilized lipase. According to the invention, the free lipase is immobilized by adopting the macroporous resin, the activity of the enzyme in the aspect of enriching n-3PUFA is improved after the lipase is immobilized, the hydrolysis or alcoholysis efficiency is enhanced, the n-3PUFA content in the product is increased, meanwhile, the product and the enzyme are easy to separate and can be recycled, and the cost is saved.

Description

Method for immobilizing lipase for enriching polyunsaturated fatty acids

Technical Field

The invention relates to the field of deep processing of grease, in particular to a lipase immobilization method for enriching polyunsaturated fatty acids.

Background

Lipases are commonly used biocatalysts in the oil industry, often for catalyzing the hydrolysis of oils and the synthesis of specific lipids. The source of the compound is wide, and the compound is commonly found in prokaryotes and eukaryotes. In recent years, most of lipases are mainly derived from bacteria and yeast, and compared with animal and plant lipases, microbial lipases have many advantages, such as easy availability, short enzyme production period, large-scale production, and wider pH adaptability and temperature tolerance.

In the oil industry, free enzyme is directly used, lipase is difficult to separate out for reuse after reaction, subsequent process operation is directly influenced, waste of lipase is caused, and higher cost is caused. And (3) enzyme immobilization, namely immobilizing free lipase on a carrier. On one hand, the separation of oil and enzyme after the enzymatic reaction is finished is easy, and the separated immobilized lipase can be reused. On the other hand, the immobilized enzyme has higher enzyme activity and stability than the free enzyme.

Polyunsaturated fatty acid (PUFA) has multiple physiological functions of resisting inflammation, treating coronary heart disease, improving intelligence, etc. The n-3 PUFAs products on the market are mainly in three forms, namely ethyl ester type, glyceride type and free fatty acid type. However, the ethyl ester type is difficult to digest and absorb in human bodies, the free fatty acid type has poor stability, the glyceride type is the best choice, the content of n-3 PUFAs in the glyceride type is relatively low, about 30%, the health care and medicinal requirements of people cannot be met, and the concentrated glyceride type n-3 PUFAs is very necessary.

Therefore, the method for enriching n-3 PUFAs by using the enzyme method which is green and efficient and can reduce the cost has important research significance and value.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a lipase immobilization method for enriching polyunsaturated fatty acids, and particularly relates to a method for immobilizing lipase by mixing and solidifying a lipase liquid and resin. The method can improve the repeated use times of the lipase to more than 5 times, and has higher catalytic efficiency compared with free enzyme. Provides important technical support for industrialization of enriching n-3 polyunsaturated fatty glyceride in grease by an enzyme method.

As one aspect of the invention, a method for immobilizing lipase rich in polyunsaturated fatty acids is provided, the method comprising dissolving lipase in a buffer to obtain a lipase solution; putting the lipase liquid and resin in a reactor, separating a solid phase and a liquid phase after reaction, and drying the solid phase to obtain immobilized lipase;

wherein the lipase comprises one or more of AY "Amano"400SD, AY "Amano"30SD derived from Candida cylindracea and Candida antarctica lipase A derived from Candida antarctica.

Further, the resin is styrene macroporous adsorption resin and comprises one or more of NKA-9, AB-8 and D101.

Further, the buffer solution comprises one or more of phosphate buffer solution, borate buffer solution and citrate buffer solution.

Further, the pH value of the lipase liquid is 5-7; the concentration of the lipase in the lipase liquid is 5-30 mg/mL.

Further, the mass ratio of the lipase liquid to the resin is 2: 1-150: 1.

Further, the reaction temperature is 20-50 ℃.

Further, the reaction time is 3-12 h.

Further, the reactor comprises a thermostatic stirrer or a thermostatic oscillator; wherein the stirring or shaking rate is 60-300 r/min.

As a second aspect of the present invention, there is provided an immobilized lipase obtained by any one of the above-mentioned methods for immobilizing a polyunsaturated fatty acid-enriched lipase.

As a third aspect of the invention, the invention provides an application of immobilized lipase, which is applied to the enrichment of polyunsaturated fatty acid, wherein in the reaction of the enrichment of the polyunsaturated fatty acid, the addition amount of the immobilized lipase is 0.1-10% of the mass of a reaction substrate.

The invention has the following beneficial effects:

(1) compared with the traditional phenomenon that the immobilized lipase (comprising powdered enzyme and liquid enzyme) is not easy to separate after the catalytic reaction of free lipase is finished, and the phenomenon of troubles on the purification of target products at the later stage is caused, the immobilized lipase prepared by the method is easy to separate after the reaction, can be recycled, and saves the cost.

(2) The immobilized lipase provided by the invention can form stable acting forces such as hydrogen bonds between the cylindrical Candida albicans AY 'Amano' 400SD, AY 'Amano' 30SD and Candida antarctica lipase A selected from and styrene macroporous adsorption resin (including NKA-9, AB-8 or D101), so that the active center of the lipase is adsorbed and fixed on the surface of the resin, the activity of the lipase in enriching n-3PUFA is improved after immobilization, the hydrolysis or alcoholysis efficiency is enhanced, the fatty acid specificity of the lipase is improved, the loss of a target product is reduced, the n-3PUFA content in the product is further improved, and the yield of the product is improved.

Drawings

Fig. 1 is gas chromatogram of glyceride fatty acid components before and after the fish oil reaction in example 14 and comparative example 4 of the present invention, wherein (a) is before the fish oil reaction, (b) is after the fish oil and immobilized enzyme reaction, and (c) is after the fish oil and free enzyme reaction.

FIG. 2 is a liquid chromatography (differential detector) chart for analyzing the composition of an oil phase product obtained after hydrolysis of the fish oil of the present invention, wherein (a) is a spectrum for analyzing the components of the hydrolysate of example 14, and (b) is a spectrum for analyzing the components of the hydrolysate of comparative example 4.

Detailed Description

1. Method for measuring immobilized enzyme protein immobilization amount

(1) Preparation of protein standards

a. 1.2ml of the protein standard preparation solution was added to a tube of protein standard (30mg BSA), and after sufficient dissolution, 25mg/ml of the protein standard solution was prepared. Can be used immediately after preparation, or stored at-20 deg.C for a long time.

b. Taking a proper amount of 25mg/ml protein standard, and diluting to a final concentration of 0.5 mg/ml. For example, 20. mu.l of 25mg/ml protein standard is added with 980. mu.l of diluent to prepare 0.5mg/ml protein standard. The protein sample and the standard should be diluted with the same solution. For simplicity, standards may also be diluted with 0.9% NaCl or PBS. The diluted 0.5mg/ml protein standard can be stored for a long time at the temperature of 20 ℃ below zero.

(2) Preparation of BCA working solution

According to the number of samples, a proper amount of BCA working solution is prepared by adding 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), and the mixture is fully mixed. For example, 5ml of BCA reagent A and 100. mu.l of BCA reagent B are mixed and mixed to prepare 5.1ml of BCA working solution. The BCA working solution is stable within 24 hours at room temperature.

(3) Protein concentration determination

a. Adding standard substance into standard substance well of 96-well plate in an amount of 0, 1, 2, 4, 8, 12, 16, 20 μ l, and adding standard substance diluent to make up to 20 μ l, wherein the concentrations of the standard substance are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mg/ml respectively.

b. Add the appropriate volume of sample to the sample well of a 96-well plate. If the sample is less than 20. mu.l, the sample volume is recorded by adding standard diluent to make up to 20. mu.l.

c. Add 200. mu.l BCA working solution to each well and leave at 37 ℃ for 20-30 minutes.

Note that the mixture may be left at room temperature for 2 hours or at 60 ℃ for 30 minutes. When the BCA method is used for measuring the protein concentration, the color is continuously deepened along with the time. And the color reaction is accelerated by the increase of temperature. If the concentration is low, incubation at a higher temperature is appropriate, or the incubation time is suitably extended.

d. The absorbance at other wavelengths between A562, or 540-595nm was measured with a microplate reader.

e. The protein concentration of the sample was calculated from the standard curve and the sample volume used.

(4) Protein immobilization amount measurement

2. Method for measuring n-3PUFA content

Putting 50mg of sample into a 10mL graduated tube, adding 2mL of 0.5mol/L potassium hydroxide-methanol solution, saponifying at 65 ℃ for 30min, cooling, adding 2mL of 25% volume fraction boron trifluoride-methanol solution, and carrying out water bath at 70 ℃ for 5 min; adding 2mL of n-hexane, oscillating for 3-4min to extract fatty acid methyl ester, adding 4mL of saturated NaCl solution, taking the upper layer solution, adding anhydrous sodium sulfate, oscillating (centrifuging at 10000rpm for 5min), sucking by a syringe, passing through a membrane, and detecting by using a gas chromatography, wherein the gas chromatography has the following operation parameters: selecting a 7890 gas chromatograph and a Flame Ionization Detector (FID); the gas chromatographic column is 60m × 0.32mm × 2.5 μm; the nitrogen flow rate was set to 1.0mL/min and the temperature of the injector and detector were set to 250 ℃. The initial column temperature was maintained at 80 ℃ for 0.5min and then increased from 80 ℃ to 165 ℃ at a rate of 40 ℃/min. The column temperature was raised to 230 ℃ at a rate of 4 ℃/min and held at 230 ℃ for 4 min. And calculating the content of n-3 PUFAs by using a peak area normalization method.

3. Method for analyzing hydrolysate

Taking 20mg of the hydrolyzed mixed product, adding 1mL of mobile phase (normal hexane: isopropanol: formic acid: 15:1:0.003) for dissolving, passing through a membrane, and detecting by liquid chromatography, wherein the operating parameters of the liquid chromatography are as follows: HPLC, Sepax HP silica gel column (aperture 5m, 4.6mm x 250mm) differential detector; elution was performed with hexane, isopropanol and formic acid (15:1:0.003, v/v/v) at a rate of 1.0 mL/min. And calculating the content of free fatty acid under hydrolysis by using a peak area normalization method.

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof will be described in detail with reference to the following examples.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

The lipases AY "Amano"400SD (400000U/g) and AY "Amano"30SD (30000U/g) used in the present invention were purchased from Japan Korea biological enzyme preparations, Inc.; lipase Candida antarctica lipase A (6000U/g) was purchased from Novoxin Biotechnology Ltd.

The resins used in the invention comprise styrene series macroporous adsorption resins NKA-9, AB-8 and D101, acrylic weak-acid cation exchange resin D113 and styrene series weak-base anion exchange resins D301 and D392, which are all purchased from Tianjin City photo-recovery fine chemical research institute.

The oil and fat used in the invention are all sold in the market, wherein the n-3 PUFAs of the fish oil (tuna oil) is 34.3 percent. Other reagents are not specifically indicated and are all commercially available.

The hydrolysis rate calculation formula is as follows:

wherein the glyceride specific gravity is obtained by the following method or a calculation formula:

mass% of glyceride ═ mass% of triglyceride + mass% of diglyceride + mass% of monoglyceride obtained by liquid phase detection.

Example 1

Taking lipase AY 'Amano' 400SD and phosphate buffer solution in a 50mL conical flask to obtain 10g of lipase solution (wherein the concentration of the lipase is 15mg/mL, the pH value is 6), adding 0.5g of NKA-9 resin, and sealing. Then placing the mixture into a constant temperature oscillator, rotating at the speed of 300r/min, keeping the water temperature at the constant temperature of 37 ℃ and keeping the temperature for 7 hours. And after the reaction is finished, carrying out suction filtration to separate a solid phase and a liquid phase, and drying the obtained solid phase in a vacuum drier for 24 hours to obtain the AY 'Amano' 400SD-NKA-9 immobilized enzyme. Taking out and storing at 4 ℃. The protein immobilization amount of the immobilized enzyme is calculated to be 154.1 mg/g.

The experimental conditions and results for examples 2-13 are shown in Table 1 below, with the operating parameters being the same as in example 1 except for the conditions noted.

Comparative example 1

Taking lipase AY "Amano"400SD and phosphate buffer solution in a 50mL conical flask to obtain 10g of lipase solution (wherein the lipase concentration is 15mg/mL, the pH is 6), adding 0.5g of D301 resin, and sealing. Then placing the mixture into a constant temperature oscillator at the rotation speed of 300r/min, keeping the water temperature at the constant temperature of 37 ℃ for 7 h. And after the reaction is finished, carrying out suction filtration to separate a solid phase and a liquid phase, and drying the obtained solid phase in a vacuum drier for 24 hours to obtain the AY 'Amano' 400SD-301D immobilized enzyme. Taking out and storing at 4 ℃. The protein immobilization amount of the immobilized enzyme is calculated to be 25.3 mg/g.

The experimental conditions and results of comparative examples 2-3 are shown in Table 1 below, with the operating parameters being the same as in example 1 except for the noted conditions.

TABLE 1 conditions and results of lipase immobilization

The data of examples 1-5 in Table 1 show that the protein immobilization capacity of lipase AY 'Amano' 400SD, AY 'Amano' 30SD and Candida antarctica lipase A on styrene macroporous resin carriers NKA-9, AB-8 and D101 is above 90 mg/g; examples 6-13 also show that the protein immobilization amount of 3 enzymes on the carrier in 3 under the condition of changing partial immobilization is above 60mg/g, wherein the protein immobilization amount of the AY 'Amano' 400SD-NKA-9 immobilized enzyme reaches 154.1mg/g, which is far higher than the common requirement level (50mg/g) of the protein immobilization amount of the immobilized enzyme adsorbed by the resin in industry; the ion exchange resins D113, D392 and D301 of comparative examples 1 to 3 have poor adsorption effect on lipase AY "Amano"400 SD.

Since different lipase proteins generally consist of different amino acids and different lipases have different amino acid sequences, the active center of most lipases is a triplet consisting of three amino acids Ser-Asp-His, and the active center of a few lipases, such as Candida cylindracea lipase, consists of Ger-Glu-His. The spatial structure of lipases is often a hydrophobic beta-sheet, surrounded by an amphipathic alpha-helix. The resin absorbs the active center of the lipase on the surface of the resin by forming stable acting force such as hydrogen bond and the like with amino acid peptide bond, and the capacity of the resin for absorbing different amino acids is strong or weak, and the difference is mainly reflected by the difference of the dissociation constant of the amino acid and the difference of specific affinity to the resin. Therefore, the lipase immobilization effect has a large correlation with the properties of the alpha-helix around the active center, i.e., has a close relationship with the properties of the amino acids constituting the alpha-helix. The styrene macroporous adsorption resins NKA-9, AB-8 and D101 are hydrogen bond type adsorption resins, have adsorption forces such as van der Waals attraction and hydrogen bond effect, and have specific affinity (especially alpha-spiral Ger amino acid) with Ger-Glu-His amino acid of lipase such as AY 'Amano' 400SD and AY 'Amano' 30SD, so that the adsorption of the resin and the lipase is enhanced, and the solidification rate of the lipase is further improved.

Example 14

Accurately weighing 3.0g of fish oil (the fatty acid composition of a fish oil sample is shown in figure 1(a) of the specification, wherein n-3 PUFAs is 34.3%), 3g of phosphate buffer solution (pH 6) and AY 'Amano' 400SD-NKA-9 immobilized lipase obtained in example 1, wherein the addition amount of the immobilized lipase is 0.5% of the total mass of a reaction substrate, adding the immobilized lipase into a reaction kettle, placing a magnetic rotor and sealing the reaction kettle. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 10 hours. The hydrolysis rate was 55.2% (as shown in FIG. 2(a) of the specification). After the reaction is finished, removing the hydrolyzed free fatty acid by using KOH-ethanol aqueous solution, then washing by water for 3 times, taking the upper clear oil phase, and evaporating the solvent to obtain the fish oil glyceride rich in n-3 PUFAs. The fatty acid composition of the glyceride product has n-3 PUFAs increased from 34.3% of the crude oil to 62.3% of the crude oil after hydrolysis (as shown in the figure 1(b) in the specification).

Example 15

The same procedure as in example 14 was repeated except that the immobilized enzyme in the reaction system was changed to AY "Amano"400SD-AB-8 immobilized lipase obtained in example 2, and the hydrolysis ratio after the reaction and the content of n-3 PUFAs in the obtained product were as shown in Table 2.

Example 16

The same procedure as in example 14 was repeated except that the immobilized enzyme in the reaction system was changed to AY "Amano"400SD-D101 immobilized lipase obtained in example 3, and the hydrolysis ratio after the reaction and the content of n-3 PUFAs in the obtained product were as shown in Table 2.

Example 17

The same procedure as in example 14 was repeated except that the immobilized enzyme in the reaction system was changed to AY "Amano"30SD-NKA-9 immobilized lipase obtained in example 4, and the hydrolysis ratio after the reaction and the content of n-3 PUFAs in the obtained product were as shown in Table 2.

Comparative example 4

Accurately weighing 3.0g of fish oil, 3g of phosphoric acid buffer solution (pH 6) and 960U of AY 'Amano' 400SD free lipase, adding into a reaction kettle, placing into a magnetic rotor, and sealing. The reaction kettle is placed on a magnetic stirrer, circulating water is introduced into the reaction kettle, the temperature of the water is constant at 37 ℃, and the reaction kettle is kept for 10 hours. The hydrolysis rate was 39.1% (as shown in FIG. 2(b) of the specification). After the reaction is finished, removing the hydrolyzed free fatty acid by using KOH-ethanol aqueous solution, then washing by water for 3 times, taking the upper clear oil phase, and evaporating the solvent to obtain the fish oil glyceride rich in n-3 PUFAs. The fatty acid composition of the glyceride product has n-3 PUFAs increased from 34.3% in crude oil to 58.4% in hydrolyzed form (as shown in FIG. 1 c).

Comparative example 5

The same as in comparative example 4, except that the enzyme in the reaction system was changed to AY "Amano"30SD lipase, the operation was the same, and the hydrolysis ratio after the reaction and the content of n-3 PUFAs in the obtained product were as shown in Table 2.

TABLE 2 conditions and results of reactions for the lipase hydrolysis of fish oils

The results of examples 14-16 and comparative example 4 expressing the effect of lipase AY "Amano"400SD on the aspect of enriching n-3PUFA in fish oil hydrolysis before and after immobilization show that the hydrolysis effect of AY "Amano"400SD after immobilization by using resins NKA-9, AB-8 and D101 is improved compared with that of free enzyme, wherein the hydrolysis effect of NKA-9 immobilized AY "Amano"400SD lipase is obviously improved; the lipase AY 'Amano' 30SD has obviously improved enrichment effect of n-3 PUFAs compared with the free enzyme after being immobilized by NKA-9 resin. The method is characterized in that the activity of the lipase in the aspect of enriching n-3PUFA is improved after the lipase is immobilized, the hydrolysis or alcoholysis efficiency is enhanced, the fatty acid specificity of the lipase is improved, the loss of a target product is reduced, and the content of the n-3PUFA in the product is further improved; and the immobilized lipase is easy to separate after hydrolysis reaction, and can be recycled and reused for at least 5 times, so that the cost is saved.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims

1. A method for immobilizing lipase for enriching polyunsaturated fatty acids, which is characterized by comprising the following steps: dissolving lipase in a buffer solution to obtain a lipase solution; putting the lipase liquid and resin in a reactor, separating a solid phase and a liquid phase after reaction, and drying the solid phase to obtain immobilized lipase;

2. The method for immobilizing lipase rich in polyunsaturated fatty acids according to claim 1, wherein the resin is a styrene-based macroporous adsorption resin comprising one or more of NKA-9, AB-8 and D101.

3. The method of claim 1, wherein the buffer comprises one or more of phosphate buffer, borate buffer, and citrate buffer.

4. The method for immobilizing lipase to enrich for polyunsaturated fatty acids according to claim 1, wherein the pH of the lipase solution is 5 to 7; the concentration of the lipase in the lipase liquid is 5-30 mg/mL.

5. The method for immobilizing lipase in an enriched form of polyunsaturated fatty acids according to claim 1, wherein the mass ratio of the lipase liquid to the resin is 2:1 to 150: 1.

6. The method for immobilizing lipase rich in polyunsaturated fatty acids according to claim 1, wherein the reaction temperature is 20 to 50 ℃.

7. The method for immobilizing lipase to enrich for polyunsaturated fatty acids according to claim 1, wherein the reaction time is 3 to 12 hours.

8. The method of claim 1, wherein the reactor comprises a constant temperature stirrer or a constant temperature shaker; wherein the stirring or shaking rate is 60-300 r/min.

9. An immobilized lipase prepared by the method for immobilizing a polyunsaturated fatty acid-enriched lipase according to any one of claims 1 to 8.

10. The use of the immobilized lipase in the polyunsaturated fatty acid enrichment according to claim 9, wherein the addition amount of the immobilized lipase in the polyunsaturated fatty acid enrichment is 0.1-10% of the mass of the reaction substrate.