Immobilized enzyme, preparation method thereof and method for purifying oleic acid by using immobilized enzyme

Abstract

The invention provides an immobilized enzyme, a preparation method thereof and a method for purifying oleic acid by using the immobilized enzyme, and relates to the fields of preparation of oleic acid and bioengineering, in particular to a preparation method of the immobilized enzyme and composite catalysis application. The invention provides a method for purifying oleic acid by composite catalysis of immobilized enzymes, which comprises the following steps: respectively immobilizing lipoxygenase LOX and hydroperoxide lyase HPL, then filling the two immobilized enzymes into an enzyme reaction column in different stacking modes, and catalyzing a reaction when a substrate passes through the enzyme reaction column to obtain oleic acid. The polyunsaturated fatty acid is combined with O2 under the action of lipoxygenase to form fatty acid hydroperoxide; the latter is also used as a substrate of hydroperoxide lyase to be cracked, polyunsaturated fatty acid in the substrate reacts to form fatty acid hydroperoxide, and then the fatty acid hydroperoxide is cracked into chain scission aldehyde to respectively generate C6, C9 or C10 aldehyde; aldehydes are removed through molecular distillation purification, so that the purity of the oleic acid is improved, and the purity of the oleic acid with the purity of 80% can be improved to 85%.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of oleic acid preparation and bioengineering, in particular to a fixed enzyme preparation method and composite catalysis application, especially to a fixed enzyme, a preparation method thereof and a method for purifying oleic acid by using the fixed enzyme. BACKGROUND

[0002] Oleic acid is a colorless to light yellow oily liquid at room temperature, easy to oxidize, and relatively stable in chemical properties. High-purity oleic acid (≥85%) has high demand in medicine (such as drug carriers), high-end lubricants and fine chemicals, while industrial-grade oleic acid (70%-80%) is used in soap, plastic additives, etc.

[0003] Oleic acid often coexists with other fatty acids (such as linoleic acid, stearic acid, palmitic acid, etc.) in natural oils and fats, and needs to be separated by purification to obtain the target product. The main raw material is a mixture of fatty acids after hydrolysis of animal and vegetable oils and fats, or a crude fatty acid mixture obtained by soap foot acidification treatment.

[0004] The main purification methods of oleic acid include molecular distillation, crystallization, solvent extraction, enzyme separation and chromatography, etc. The composition of fatty acids in natural oils and fats is complex, and the boiling point and melting point of oleic acid and linoleic acid (double unsaturated fatty acid) are close, so the separation efficiency of traditional distillation or crystallization is low; solvent extraction method may produce organic waste, which does not meet the requirements of green chemistry; high-purity (≥85%) oleic acid needs to be combined with multiple processes, but the yield is low. Distillation method usually involves high temperature and high pressure parts, which has high energy consumption, and crystallization and solvent extraction may cause unnecessary side reactions, poor selectivity of target product, and may also pollute the environment.

[0005] Biological enzyme method is considered as a green and efficient alternative because of its mild reaction conditions, high catalytic efficiency and good specificity. However, the existing free enzyme catalysis technology has many bottlenecks in industrial application. First, free enzymes have poor stability in the reaction system and are easy to be inactivated, which cannot be reused, resulting in high production cost. Second, the reaction product is mixed with the enzyme, and the separation and purification steps are complicated, and the enzyme protein may pollute the product. Third, the existing biological enzymes are simply mixed in free state, and the catalytic efficiency and synergistic effect cannot be optimized, resulting in that the content of linoleic acid in the final product is still high, and the purity of oleic acid is limitedly improved.

[0006] Therefore, there is an urgent need in the art to develop a new enzyme catalysis technology which can realize efficient recovery and reuse of enzymes, accurately control the reaction process, effectively remove by-products, thereby significantly reduce the content of linoleic acid and improve the purity of oleic acid. SUMMARY

[0007] To improve the stability of enzymes in the reaction system and the quality of oleic acid in existing bioenzymatic methods, and to reduce enzyme protein contamination, this application provides immobilized enzymes, their preparation methods, and methods for purifying oleic acid using immobilized enzymes.

[0008] In one aspect, this application provides a method for preparing immobilized enzymes, wherein two enzyme solutions of lipoxygenase LOX and peroxidase HPL are respectively mixed with a carrier and immobilized to obtain immobilized enzyme LOX and immobilized enzyme HPL; then washed, filtered, and dried for later use.

[0009] The carrier includes any one or a combination of macroporous adsorption resin, amino resin, epoxy resin, diatomaceous earth, silica, sodium alginate, and chitosan.

[0010] The enzyme concentration range is 10-20 mg / mL, and the solvent is water.

[0011] Macroporous adsorption resins are a class of high molecular weight adsorption resins that do not contain exchange groups and have a macroporous structure. They have a good macroporous network structure and a large specific surface area, and mainly rely on adsorption to adsorb enzymes and proteins; while epoxy resins and amino resins mainly use chemical bonds to bind with enzymes and proteins.

[0012] Optionally, LOX can be extracted from soybean residue and soybean meal, purified, and then immobilized using a carrier to obtain immobilized LOX enzyme; HPL can be extracted from amaranth, purified, and then immobilized using a carrier to obtain immobilized HPL enzyme.

[0013] Furthermore, the volume-to-mass ratio of the two enzymes, LOX and HPL, to the carrier is 2-6:1.

[0014] Further optimization resulted in an enzyme-to-carrier ratio of 4:1.

[0015] Furthermore, the immobilization conditions are a temperature of 20-40 ℃, a rotation speed of 100-200 rpm, and an immobilization time of 4-16 h.

[0016] Further optimization was performed using the following conditions for immobilization: temperature 25 ℃, rotation speed 150 rpm, and immobilization time 10 h.

[0017] Secondly, this application provides an immobilized enzyme, including immobilized enzyme LOX and immobilized enzyme HPL, obtained by the immobilized enzyme preparation method described in this application.

[0018] Preferably, the immobilized enzyme LOX has an activity of 5-8 U / g, and the immobilized enzyme HPL has an activity of 2-5 U / g.

[0019] Method for determining the enzyme activity of immobilized LOX: Fatty acid hydroperoxides are prepared by LOX oxidation of fatty acids. A certain amount of 80% pure oleic acid is weighed and added to a borate buffer solution at pH 9.0. After thorough stirring at 4 °C, LOX is added and oxygen is introduced to initiate the reaction. The hydroperoxides generated in the reaction have conjugated double bonds and exhibit a characteristic absorption peak at 234 nm. Therefore, the absorbance of the reaction system at 234 nm is measured during the reaction. The reaction is considered complete when the absorbance no longer increases with increasing reaction time, and the mixture is then refrigerated at 4 °C for later use.

[0020] Method for determining the enzyme activity of immobilized HPL enzymes: Fatty acid hydroperoxides possess conjugated double bonds and exhibit a characteristic UV absorption peak at 234 nm, a characteristic not found in C6 aldehydes. As the substrate cleaves, its conjugated double bonds are disrupted, resulting in a decrease in UV absorbance at 234 nm. Enzyme activity is determined based on this change. 1 U of enzyme activity is defined as the amount of enzyme required to convert 1 μmol of substrate within 1 min at 25 °C and pH 6.0.

[0021] Thirdly, this application provides a method for purifying oleic acid using an immobilized enzyme, wherein the immobilized enzyme L described in this application is stacked with a molecular sieve in an enzyme catalytic column; oleic acid passes through the enzyme reaction column and is catalyzed by O2, and the unsaturated fatty acids in oleic acid are cleaved into aldehydes by the immobilized enzyme LOX and / or the immobilized enzyme HPL, and then the aldehydes are removed by distillation, thereby improving the purity of oleic acid.

[0022] Furthermore, in the enzyme catalytic column, in the direction of oleic acid passage, there are, in sequence, an immobilized enzyme LOX, a molecular sieve, a mixed enzyme consisting of immobilized enzyme LOX and immobilized enzyme HPL, a molecular sieve, an immobilized enzyme HPL, and a molecular sieve.

[0023] Preferably, the amount of immobilized enzyme LOX and immobilized enzyme HPL in the mixed enzyme is equal.

[0024] Furthermore, the molecular sieve type is any one of 3A molecular sieve, 4A molecular sieve, 5A molecular sieve, 13X molecular sieve, Y-type molecular sieve and oxygen-enriched molecular sieve.

[0025] Furthermore, in the enzyme catalytic column, the mass ratio of immobilized enzyme LOX, molecular sieve, mixed enzyme composed of immobilized enzyme LOX and immobilized enzyme HPL, and immobilized enzyme HPL is 1:1-4:1.

[0026] Furthermore, the immobilized enzyme LOX in the enzyme catalytic column accounts for 50 wt% of the total immobilized enzyme mass in the enzyme catalytic column.

[0027] Furthermore, the enzyme-catalyzed reaction temperature is 20-40 °C, and the flow rate of oleic acid through the enzyme reaction column is 0.5-2.5 kg / kg enzyme / h.

[0028] Further optimization resulted in an enzyme-catalyzed reaction temperature of 30 °C.

[0029] Further optimization yielded an enzyme-catalyzed reaction flow rate of 1.5 kg / kg enzyme / h.

[0030] Beneficial Effects: 1. This invention provides a method for purifying oleic acid using immobilized enzyme composite catalysis. LOX and HPL are immobilized separately, and then the two immobilized enzymes are packed into an enzyme reaction column using different stacking methods. The substrate undergoes a catalytic reaction as it passes through the enzyme reaction column. Specifically, using lipoxygenase (LOX) and hydroperoxide lyase (HPL) as enzyme catalysts, and 80% pure oleic acid (5% C16:0, 80% C18:1, and 12% C18:2) as the substrate, O2 is introduced to catalyze the reaction. Polyunsaturated fatty acids combine with O2 under the action of lipoxygenase (LOX) to form fatty acid hydroperoxides. The enzymatic hydrolysis product of lipoxygenase (LOX) is then cleaved as a substrate by hydroperoxide lyase (HPL). As LOX and HPL... The different enzyme specificities react the polyunsaturated fatty acids in the substrate into fatty acid hydroperoxides, which are then cleaved into easily volatile short-chain aldehydes, producing C6, C9, or C10 aldehydes respectively. The aldehydes are then removed by molecular distillation, which improves the purity of oleic acid, increasing the purity from 80% to 85%. The enzyme reaction column is compatible with the complex enzyme, which can intercept the immobilized enzyme, and the reaction solution contains almost no enzyme.

[0031] 2. This invention uses macroporous adsorption resin as a carrier, and immobilized composite enzymes with lipoxygenase (LOX) and hydroperoxide lyase (HPL) as enzyme catalysts are packed in an enzyme reaction column in different stacking ways. The substrate can catalyze the reduction of polyunsaturated fatty acids in oleic acid by passing through the enzyme reaction column, thereby improving the purity of oleic acid. At the same time, oxygen-enriched molecular sieves are laid at the bottom of the immobilized enzyme to increase the oxygen content in the reaction system and further promote the reaction.

[0032] 3. The enzyme source of the present invention is safe and easy to obtain. The preparation conditions of the immobilized composite enzyme are simple, the reaction conditions are mild, the production stability is good, it is green and environmentally friendly, and no toxic or harmful substances are added during the reaction process, making it suitable for industrial production. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the stacking and packing method of immobilized enzyme LOX and immobilized enzyme HPL in the enzyme reaction column in Embodiment 2-1 of the present invention. Detailed Implementation

[0034] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] Example 1-1, a method for preparing an immobilized enzyme, comprising the following preparation process:

[0036] LOX and HPL were immobilized using macroporous adsorption resin (LXTE-1000, Xi'an Lanxiao Technology New Materials Co., Ltd.). An enzyme solution (concentration 15 mg / mL) at a volume-to-mass ratio of 4:1 (mL / g) was mixed with the macroporous adsorption resin, and immobilization was carried out at 25 ℃ and 150 rpm for 10 h. After immobilization, the immobilized enzymes were washed and dried. The enzyme activities of immobilized LOX and HPL were measured to be 6.4 U / g and 3.8 U / g, respectively.

[0037] Examples 1-2 illustrate a method for preparing an immobilized enzyme, comprising the following preparation steps:

[0038] LOX and HPL were immobilized using epoxy resin. An enzyme solution (15 mg / mL) was mixed with epoxy resin at a volume-to-mass ratio of 2:1, and immobilized for 4 h at 20 ℃ and 100 rpm. After immobilization, the immobilized enzymes were washed and dried.

[0039] Examples 1-3 illustrate a method for preparing immobilized enzymes, comprising the following preparation steps:

[0040] LOX and HPL were immobilized using amino resins. The enzyme solution (15 mg / mL) was mixed with amino resin at a volume-to-mass ratio of 6:1, and immobilization was performed for 16 h at 30 ℃ and 200 rpm. After immobilization, the immobilized enzymes were washed and dried.

[0041] Examples 1-4 describe a method for preparing immobilized enzymes, which differs from Examples 1-1 in that an equal amount of epoxy resin with the same average pore size and specific surface area is used to replace the LXTE-1000 macroporous adsorption resin.

[0042] Examples 1-5 describe a method for preparing immobilized enzymes, which differs from Examples 1-1 in that an equal amount of amino resin with the same average pore size and specific surface area is used to replace the LXTE-1000 macroporous adsorption resin.

[0043] Example 2-1, a method for purifying oleic acid using immobilized enzyme composite catalysis, comprising the following preparation process:

[0044] This embodiment uses the immobilized enzyme-packed enzyme reaction column prepared in Example 1-1 of the present invention (the stacking method is shown in the attached figure). Figure 1 The enzyme consists of an immobilized enzyme LOX and a mixed enzyme of immobilized enzyme LOX and HPL (with a mass ratio of 1:1:1:1, and a mass ratio of 1:2:1 for immobilized enzyme HPL). The lower layer is immobilized enzyme HPL, the middle layer is the mixed enzyme of immobilized enzyme LOX and HPL, and the upper layer is immobilized enzyme LOX. Each immobilized enzyme layer is lined with an oxygen-enriched molecular sieve (5A modified molecular sieve). Oleic acid with a purity of 80% (5% C16:0, 80% C18:1, and 12% C18:2) is used as the substrate. Oxygen is introduced to maintain the system pressure at 0.2±0.1MPa, and the catalytic reaction is carried out at a flow rate of 1.5 kg / kg enzyme / h and a reaction temperature of 30℃. Through this process, the polyunsaturated fatty acids in the substrate are reacted into fatty acid hydroperoxides, which are then cleaved into chain-broken aldehydes. The aldehydes are then purified and removed by molecular distillation, thereby improving the purity of oleic acid.

[0045] Example 2-2, a method for purifying oleic acid using immobilized enzyme composite catalysis, differs from Example 2-1 in that it uses the immobilized enzymes LOX and HPL obtained in Example 1-2.

[0046] Examples 2-3 describe a method for purifying oleic acid using immobilized enzyme composite catalysis, which differs from Example 2-1 in that it uses the immobilized enzymes LOX and HPL obtained in Examples 1-3.

[0047] Examples 2-4 describe a method for purifying oleic acid using immobilized enzyme composite catalysis, which differs from Example 2-1 in that it uses the immobilized enzymes LOX and HPL from Examples 1-4.

[0048] Examples 2-5 describe a method for purifying oleic acid using immobilized enzyme composite catalysis, which differs from Example 2-1 in that it uses the immobilized enzymes LOX and HPL from Examples 1-5.

[0049] Examples 2-6 illustrate a method for purifying oleic acid using immobilized enzyme composite catalysis, without employing a mixed enzyme of immobilized enzymes LOX and HPL. The preparation process is as follows:

[0050] This comparative example uses an enzyme reaction column filled with immobilized enzyme LOX and HPL prepared in Examples 1-1 of this invention. The lower layer is filled with immobilized enzyme HPL and the upper layer is filled with immobilized enzyme LOX (each layer accounts for 50%, and the total mass of the immobilized enzyme is consistent with that in Examples 2-1). Oleic acid with a purity of 80% is used as the substrate, and the catalytic reaction is carried out at a flow rate of 1.5 kg / kg enzyme / h and a reaction temperature of 30°C.

[0051] Examples 2-7 describe a method for purifying oleic acid using immobilized enzyme composite catalysis, which differs from Example 2-1 in that an equal amount of immobilized enzyme LOX and immobilized enzyme HPL are used in a 1:1 mass ratio mixture.

[0052] Its preparation process is as follows:

[0053] This comparative example uses an enzyme reaction column filled with a mixture of immobilized enzyme LOX and HPL prepared in Example 1-1 of this invention (the mass ratio of immobilized enzyme HPL to LOX is 1:1, and the total mass of the immobilized enzyme is consistent with that in Example 2-1). Oleic acid with a purity of 80% is used as the substrate, and the catalytic reaction is carried out at a flow rate of 1.5 kg / kg enzyme / h and a reaction temperature of 30 °C.

[0054] Examples 2-8 illustrate a method for purifying oleic acid using immobilized enzyme composite catalysis, comprising the following preparation process:

[0055] This comparative example uses an enzyme reaction column filled with immobilized enzyme LOX and HPL prepared in Example 1-1 of this invention. The lower layer is filled with immobilized enzyme HPL, and the upper layer is filled with a mixture of immobilized enzyme LOX and HPL (each layer accounts for 50%, the mass ratio of immobilized enzyme LOX to HPL in the upper layer is 1:1, and the total mass of immobilized enzyme is consistent with that in Example 2-1). Oleic acid with a purity of 80% is used as the substrate, and the catalytic reaction is carried out at a flow rate of 1.5 kg / kg enzyme / h and a reaction temperature of 30 °C.

[0056] Examples 2-9 illustrate a method for purifying oleic acid using immobilized enzyme composite catalysis, comprising the following preparation process:

[0057] This comparative example uses an enzyme reaction column filled with immobilized enzyme LOX and HPL prepared in Example 1-1 of this invention. The lower layer is filled with a mixture of immobilized enzyme LOX and HPL, and the upper layer is filled with immobilized enzyme LOX (the upper and lower layers each account for 50%, the mass ratio of immobilized enzyme LOX to HPL in the lower layer is 1:1, and the total mass of immobilized enzyme is consistent with that in Example 2-1). Oleic acid with a purity of 80% is used as the substrate, and the catalytic reaction is carried out at a flow rate of 1.5 kg / kg enzyme / h and a reaction temperature of 30 °C.

[0058] Examples 2-10 illustrate a method for purifying oleic acid using immobilized enzyme composite catalysis. The difference between this method and Example 2-1 is that the immobilized enzyme in Example 2-10 is not placed under an oxygen-enriched molecular sieve.

[0059] The preparation process is as follows:

[0060] This comparative example uses an enzyme reaction column filled with immobilized enzyme LOX and HPL prepared in Example 1-1 of this invention. The lower layer is filled with mixed immobilized enzyme HPL, the middle layer is filled with mixed immobilized enzyme LOX and HPL, and the upper layer is filled with immobilized enzyme LOX (the upper and lower layers each account for 25%, the middle layer accounts for 50%, the mass ratio of immobilized enzyme LOX to HPL in the middle layer is 1:1, and the total mass of immobilized enzyme is consistent with that in Example 2-1). Oleic acid with a purity of 80% is used as the substrate, and the catalytic reaction is carried out at a flow rate of 1.5 kg / kg enzyme / h and a reaction temperature of 30 °C.

[0061] Comparative Example 1, a method for purifying oleic acid using immobilized enzyme composite catalysis, differs from Example 2-1 in that: immobilized enzyme LOX is used to replace the middle layer of immobilized enzyme LOX and HPL mixed enzyme in equal amounts, and immobilized enzyme LOX is used to replace the lower layer of immobilized enzyme HPL in equal amounts.

[0062] Its preparation process is as follows:

[0063] This comparative example uses the immobilized enzyme LOX packed enzyme reaction column prepared in Example 1-1 of this invention (the mass of the immobilized enzyme packed is consistent with that in Example 2-1), with oleic acid of 80% purity as substrate, and the catalytic reaction is carried out at a flow rate of 1.5 kg / kg enzyme / h and a reaction temperature of 30 °C.

[0064] Comparative Example 2, a method for purifying oleic acid using immobilized enzyme composite catalysis, differs from Example 2-1 in that: immobilized enzyme HPL is used to replace the upper layer immobilized enzyme LOX in equal amounts, and immobilized enzyme HPL is used to replace the middle layer immobilized enzyme LOX and HPL mixed enzyme in equal amounts.

[0065] Its preparation process is as follows:

[0066] This comparative example uses the immobilized enzyme HPL packed enzyme reaction column prepared in Example 1-1 of this invention (the mass of the immobilized enzyme packed is consistent with that in Example 2-1), with oleic acid of 80% purity as substrate, and the catalytic reaction is carried out at a flow rate of 1.5 kg / kg enzyme / h and a reaction temperature of 30 °C.

[0067] The results of Examples 2-1 to 2-10 and Comparative Example 1-2 are shown in the table below:

[0068] Table 1. Effect of Immobilized Enzyme Packing Method on Oleic Acid Purity

[0069] Filling method C18:1 content % Example 2-1 85.4 Example 2-2 81.5 Example 2-3 81.3 Example 2-4 83.2 Example 2-5 80.5 Example 2-6 82.3 Example 2-7 83.1 Example 2-8 82.6 Example 2-9 83.5 Example 2-10 84.3 Comparative Example 1 80.7 Comparative Example 2 80.1

[0070] The results in the table above show that the immobilized enzyme packing and stacking method in Example 2-1 has the best effect, and the oleic acid purity can reach more than 85%.

[0071] Example 3, a method for purifying oleic acid using immobilized enzyme composite catalysis, comprising the following preparation process:

[0072] This embodiment uses the immobilized enzyme-packed enzyme reaction column prepared in Example 1-1 of the present invention, and uses the stacking method of Example 2-1 for packing. Different types of molecular sieves are used as a base under each layer of immobilized enzyme. Oleic acid with a purity of 80% is used as the substrate, and the catalytic reaction is carried out at a flow rate of 1.5 kg / kg enzyme / h and a reaction temperature of 30 °C.

[0073] Table 2. Effect of different types of molecular sieves on oleic acid purity

[0074] Packing type of molecular sieve C18:1 content % 3A molecular sieve 84.1 4A molecular sieve 84.3 5A molecular sieve 84.7 13X molecular sieve 84.8 Y-type molecular sieve 84.4 Oxygen-rich molecular sieve 85.4

[0075] The results in the table above show that the use of oxygen-enriched molecular sieves as a lining has the best effect, with oleic acid purity reaching 85.4%. 5A and 13X molecular sieves have a slight improvement effect, while 3A, 4A, and Y-type molecular sieves have no significant improvement effect.

[0076] Due to their pore size, oxygen-enriched molecular sieves possess the ability to enrich oxygen, which is beneficial for the stability of oxygen in enzyme reaction columns and enhances the reaction between enzymes and unsaturated fatty acids in oleic acid. Furthermore, molecular sieves exhibit adsorption properties, adsorbing certain impurities in the system and improving substrate dispersion, allowing for more thorough contact between the immobilized enzyme and the substrate, thereby promoting the reaction.

[0077] As the substrate passes through the enzyme reaction column, the polyunsaturated fatty acids first combine with O2 under the action of LOX lipoxygenase to form fatty acid hydroperoxides. The latter then serves as the substrate for HPL hydroperoxide lyase, which cleaves the polyunsaturated fatty acids in the substrate into fatty acid hydroperoxides, which are then cleaved into chain-broken aldehydes, producing C6, C9, or C10 aldehydes, respectively. In the packing method of Example 2-1, when the oleic acid substrate passes through the upper layer, the reaction stops after a certain extent due to product accumulation and reaction inhibition. After the oleic acid substrate reaches the middle layer, the fatty acid hydroperoxides react with HPL and are consumed, the product inhibition is relieved, and easily volatile aldehydes are generated. The LOX in the middle layer can further react with the unsaturated fatty acids in the substrate and, synergistically with the HPL in the lower layer, further reduce the content of polyunsaturated fatty acids in the system, thereby increasing the oleic acid content.

[0078] Example 4, a method for purifying oleic acid using immobilized enzyme composite catalysis, comprising the following preparation process:

[0079] In this embodiment, the immobilized enzyme was packed using the stacked packing method of Example 2-1 of the present invention, with oleic acid of 80% purity as the substrate, and the catalytic reaction was carried out at a flow rate of 1.5 kg / kg enzyme / h, with variations in the reaction temperature. The results are shown in the table below:

[0080] Table 3 Effect of different reaction temperatures on oleic acid purity

[0081] Reaction temperature °C C18:1 content % 20 82.9 25 84.1 30 85.3 35 84.7 40 83.6

[0082] The results in the table above show that as the reaction temperature increases, the purity of the oleic acid product gradually increases and then decreases. The oleic acid purity reaches its optimal value of 85.3% at 30 °C.

[0083] Example 5, a method for purifying oleic acid using immobilized enzyme composite catalysis, comprising the following preparation process:

[0084] In this embodiment, the immobilized enzyme was packed using the stacked packing method of Example 2-1 of the present invention, with oleic acid of 80% purity as the substrate. The reaction temperature was 30 °C, and the reaction flow rate was varied. The results are shown in the table below:

[0085] Table 4 Effect of reaction flow rate on oleic acid purity

[0086] Reaction flow rate kg / kg of enzyme / h C18:1 content % 0.5 85.2 1.0 85.3 1.5 85.1 2.0 83.8 2.5 82.4

[0087] The results in the table above show that as the reaction flow rate increases, the purity of oleic acid gradually decreases from a stable level. When the reaction flow rate exceeds 1.5 kg / kg enzyme / h, the purity of oleic acid decreases. At a flow rate of 1.5 kg / kg enzyme / h, the purity of the product oleic acid is 85.1%.

[0088] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing an immobilized enzyme, characterized in that, The two enzyme solutions of lipoxygenase LOX and peroxidase HPL were mixed with the carrier and immobilized to obtain immobilized enzyme LOX and immobilized enzyme HPL, respectively; then washed, filtered and dried for later use. The carrier includes any one or a combination of macroporous adsorption resin, amino resin, epoxy resin, diatomaceous earth, silica, sodium alginate, and chitosan.

2. The method for preparing an immobilized enzyme according to claim 1, characterized in that, The volume-to-mass ratio of the two enzymes, LOX and HPL, to the carrier is 2-6:

1.

3. A method for preparing an immobilized enzyme according to claim 1 or 2, characterized in that, The immobilization conditions are: temperature 20-40 ℃, rotation speed 100-200 rpm, and immobilization time 4-16 h.

4. An immobilized enzyme, characterized in that, The immobilized enzymes include LOX and HPL, which are obtained using the immobilized enzyme preparation methods described in any one of 1-3.

5. A method for purifying oleic acid using immobilized enzymes, characterized in that, The immobilized enzyme described in claim 4 is stacked with molecular sieve in an enzyme catalytic column; oleic acid passes through the enzyme reaction column and is catalyzed by O2. The unsaturated fatty acids in oleic acid are cleaved into aldehydes by the immobilized enzyme LOX and / or the immobilized enzyme HPL, and then the aldehydes are removed by distillation, thereby improving the purity of oleic acid.

6. The method for purifying oleic acid using an immobilized enzyme according to claim 5, characterized in that, In the enzyme catalytic column, in the direction of oleic acid passage, there are, in sequence, immobilized enzyme LOX, molecular sieve, a mixed enzyme consisting of immobilized enzyme LOX and immobilized enzyme HPL, molecular sieve, immobilized enzyme HPL and molecular sieve.

7. A method for purifying oleic acid using an immobilized enzyme according to claim 5 or 6, characterized in that, The molecular sieve type is any one of 3A molecular sieve, 4A molecular sieve, 5A molecular sieve, 13X molecular sieve, Y-type molecular sieve and oxygen-enriched molecular sieve.

8. The method for purifying oleic acid using an immobilized enzyme according to claim 7, characterized in that, In the enzyme catalytic column, the mass ratio of immobilized enzyme LOX, molecular sieve, mixed enzyme consisting of immobilized enzyme LOX and immobilized enzyme HPL, and immobilized enzyme HPL is 1:1-4:

1.

9. The method for purifying oleic acid using an immobilized enzyme according to claim 8, characterized in that, The immobilized enzyme LOX in the enzyme catalytic column accounts for 50 wt% of the total immobilized enzyme mass in the enzyme catalytic column.

10. A method for purifying oleic acid using an immobilized enzyme according to any one of claims 5-6, 8-9, characterized in that, The enzyme-catalyzed reaction temperature is 20-40 ℃, and the flow rate of oleic acid through the enzyme reaction column is 0.5-2.5 kg / kg enzyme / h.

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