An immobilized enzyme preparation and its preparation method and use method

Abstract

The present invention relates to the field of enzyme engineering technology, and in particular to an immobilized enzyme preparation and a preparation method and a use method thereof. The immobilized enzyme preparation comprises lipase, an auxiliary agent A and an auxiliary agent B, the mass ratio of the three being 5-8:20-50:60-100; the lipase is selected from a lipase derived from Aspergillus oryzae and a lipase derived from Rhizomucor miehei, the mass ratio of the two being 2-4:1; the auxiliary agent A is composed of rapeseed meal and diatomaceous earth; the auxiliary agent B comprises konjac glucomannan gel and an emulsifier. The present invention has the advantages of high reaction activity and can be recycled and reused multiple times.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to an immobilized enzyme preparation and a preparation method and a use method thereof. Background Art

[0002] Polyglycerol fatty acid esters, also known as polyglycerol esters, are a type of nonionic surfactants prepared by direct esterification of polyglycerol and fatty acids. There are many varieties of polyglycerol fatty acid esters, and their structures depend on the degree of polymerization of polyglycerol, the type of fatty acid, and the degree of esterification. These products have good emulsification, dispersibility, wetting and other properties, and are safe, green and environmentally friendly. Therefore, they are widely used in food additives, petrochemicals, medicine and health, cosmetics, plastics and other fields.

[0003] The use of lipase to participate in the synthesis of polyglycerol esters is a common synthesis method, but the reaction activity of lipase is low and it is not conducive to recycling. Immobilizing lipase can improve its reaction activity and recycle it for reuse.

[0004] Enzyme immobilization is a technology that improves the stability and reusability of enzymes during the catalytic process. After immobilization, lipase has the advantages of high stability, good continuity, convenient recycling, easy control, repeated use, and reduced cost, providing technical support for large-scale industrial applications.

[0005] Immobilization methods can be roughly divided into two categories, namely physical methods and chemical methods. Physical methods include adsorption and embedding; chemical methods include cross-linking and covalent bonding.

[0006] Adsorption is the process of adsorbing the enzyme to the surface of a carrier through tiny forces, such as van der Waals forces, hydrophobic forces, and dispersion forces. Adsorption has little effect on the catalytic activity of the enzyme, but the connection between the enzyme and the carrier is weak. When the catalytic conditions are harsh, the enzyme is easy to fall off the carrier, which will affect the catalytic effect, contaminate the catalytic reaction products, and is not conducive to the recovery and reuse of the enzyme.

[0007] Enzyme encapsulation is a method of capturing lipase into the internal voids of polymers or microcapsules. Carrageenan, sodium alginate gel, etc. are the main materials used in the encapsulation method. The encapsulation method has a relatively small effect on the catalytic activity of the enzyme, but due to the special structure of the encapsulation material, mass transfer is easily restricted. At the same time, the connection between the encapsulated and fixed enzyme and the carrier is also weak, and the enzyme is easy to fall off the carrier, affecting the enzyme activity.

[0008] Cross-linking refers to the formation of a three-dimensional network structure through cross-linking coupling between enzyme molecules or between enzyme molecules and carriers. The reaction conditions of the cross-linking method are usually more intense, making the enzyme activity relatively low.

[0009] Covalent bonding is a method of first introducing an active group onto the carrier, and then reacting this active group with a group on the enzyme molecule to form a covalent bond. The immobilized enzyme obtained by the covalent bonding method has good stability, but due to its harsh preparation conditions, the enzyme is easily inactivated during the fixation process, and some cross-linking agents are also toxic and are not suitable for use in food.

[0010] There is currently no research on the immobilization of lipases involved in the synthesis of polyglycerol esters.

[0011] Therefore, it is very necessary to develop an immobilized enzyme preparation and a preparation method and a use method thereof that can solve the above-mentioned technical problems. Summary of the invention

[0012] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an immobilized enzyme preparation with high reaction activity and capable of being recycled and reused multiple times, as well as a preparation method and a use method thereof.

[0013] The present invention is achieved through the following technical solutions:

[0014] An immobilized enzyme preparation comprises lipase, an auxiliary agent A and an auxiliary agent B, wherein the mass ratio of the three is 5-8:20-50:60-100; the lipase is selected from lipase derived from Aspergillus oryzae and lipase derived from Rhizomucor miehei, wherein the mass ratio of the two is 2-4:1; the auxiliary agent A comprises rapeseed meal and diatomaceous earth; and the auxiliary agent B comprises konjac glucomannan gel and an emulsifier.

[0015] In some embodiments of the present invention, the mass ratio of rapeseed meal to diatomaceous earth is 1-3:1.

[0016] In some embodiments of the present invention, the mass ratio of the konjac glucomannan gel to the emulsifier is 100:0.5-1.

[0017] In some embodiments of the present invention, the emulsifier is selected from at least one of glyceryl monostearate and sucrose fatty acid ester.

[0018] The present invention also relates to a method for preparing the above-mentioned immobilized enzyme preparation, comprising the following steps:

[0019] (1) mixing an auxiliary agent A with a phosphate buffer solution containing lipase to obtain a mixture 1;

[0020] (2) Add additive B to water, adjust the pH to alkaline, heat and stir, add mixture 1, cast the obtained mixed solution into a film, and obtain the film after drying.

[0021] In some embodiments of the present invention, the concentration of lipase in the phosphate buffer solution containing lipase in step (1) is 8-12 wt %.

[0022] In some embodiments of the present invention, the pH of the phosphate buffer solution containing lipase in step (1) is 7.5.

[0023] In some embodiments of the present invention, the mass ratio of auxiliary agent B to water in step (2) is 2-6:100.

[0024] In some embodiments of the present invention, the pH in step (2) is 8-12.

[0025] In some embodiments of the present invention, the heating temperature in step (2) is 75-85°C.

[0026] In some embodiments of the present invention, the thickness of the film in step (2) is 4-6 mm.

[0027] The present invention also relates to a method for using the above-mentioned immobilized enzyme preparation, comprising the following steps:

[0028] After the fatty acid and oligoglycerol are mixed, an immobilized enzyme preparation is added to react, centrifuged, and the upper layer material is molecularly distilled twice to obtain polyglycerol esters.

[0029] The upper layer obtained after centrifugation is the oil phase, i.e., a mixture of polyglycerol esters and fatty acids, and the lower layer is the oligoglycerol that has not participated in the reaction.

[0030] In some embodiments of the invention, the fatty acid comprises oleic acid.

[0031] In some embodiments of the present invention, the mass ratio of the fatty acid to the oligoglycerol is 1:1-3.

[0032] In some embodiments of the present invention, the mass ratio of the immobilized enzyme preparation to the oligoglycerol is 2-4:100.

[0033] In some embodiments of the present invention, the reaction temperature is 35-50° C. and the reaction time is 4-6 h.

[0034] In some embodiments of the present invention, the parameters of the first molecular distillation are: pressure 5-10Pa, temperature 75-85°C. The heavy phase product obtained by the first molecular distillation is subjected to a second molecular distillation, and the parameters are: pressure 2-5Pa, temperature 90-95°C, and the light phase product obtained by distillation is polyglycerol ester.

[0035] The beneficial effects of the present invention are:

[0036] The invention adopts rapeseed meal and diatomaceous earth as auxiliary agent A, adopts konjac glucomannan gel and emulsifier as auxiliary agent B, and the two auxiliary agents are combined to fix lipase, so that the synergistic effect is remarkable, and the reaction activity of lipase is significantly improved. The esterification rate of the prepared polyglycerol ester can reach more than 98%, and the polyglycerol ester still has high reaction activity after being recycled and reused 30 times, and the esterification rate of the polyglycerol ester is maintained at more than 96%. At the same time, the prepared polyglycerol ester maintains a high yield and purity.

[0037] The present invention optimizes the composition of lipase, adopts lipase from Aspergillus oryzae and lipase from Rhizomucor miehei for catalysis, and further improves the esterification rate of polyglycerol ester. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0039] The oligoglycerol used in each embodiment of the present invention is triglycerol; the lipase derived from Aspergillus oryzae was purchased from Sigma-Aldrich, item number SAE0065; the lipase derived from Rhizomucor miehei was purchased from Novozymes, model number Palatase 20000L.

[0040] Example 1

[0041] An immobilized enzyme preparation is composed of lipase, auxiliary agent A and auxiliary agent B, and the mass ratio of the three is 7:35:80; the lipase is selected from lipase derived from Aspergillus oryzae and lipase derived from Rhizomucor miehei, and the mass ratio of the two is 3:1; the auxiliary agent A is composed of rapeseed meal and diatomaceous earth, and the mass ratio is 2:1; the auxiliary agent B includes konjac glucomannan gel and glyceryl monostearate, and the mass ratio is 100:0.75.

[0042] The method for preparing the immobilized enzyme preparation comprises:

[0043] (1) mixing the auxiliary agent A with a phosphate buffer solution containing lipase (lipase concentration in the solution is 10 wt %, pH 7.5) to obtain a mixture 1;

[0044] (2) Add additive B to water at a mass ratio of 4:100, adjust the pH to 10, heat and stir in a water bath at 80°C, add mixture 1, cast the resulting mixed solution into a 5 mm thick layer, and dry to obtain the product.

[0045] Example 2

[0046] An immobilized enzyme preparation consists of lipase, auxiliary agent A and auxiliary agent B, the mass ratio of the three being 5:20:100; the lipase is selected from lipase derived from Aspergillus oryzae and lipase derived from Rhizomucor miehei, the mass ratio of the two being 2:1; the auxiliary agent A consists of rapeseed meal and diatomaceous earth, the mass ratio being 1:1; the auxiliary agent B comprises konjac glucomannan gel and glyceryl monostearate, the mass ratio being 100:0.5.

[0047] The preparation method of the immobilized enzyme preparation comprises:

[0048] (1) mixing the auxiliary agent A with a phosphate buffer solution containing lipase (lipase concentration in the solution is 8 wt %, pH 7.5) to obtain a mixture 1;

[0049] (2) Add additive B to water at a mass ratio of 2:100, adjust the pH to 8, heat and stir in a 75°C water bath, add mixture 1, cast the resulting mixed solution into a 4 mm thick layer, and dry it to obtain the product.

[0050] Example 3

[0051] An immobilized enzyme preparation is composed of lipase, auxiliary agent A and auxiliary agent B, and the mass ratio of the three is 8:50:60; the lipase is selected from lipase derived from Aspergillus oryzae and lipase derived from Rhizomucor miehei, and the mass ratio of the two is 4:1; the auxiliary agent A is composed of rapeseed meal and diatomaceous earth, and the mass ratio is 3:1; the auxiliary agent B includes konjac glucomannan gel and sucrose fatty acid ester, and the mass ratio is 100:1.

[0052] The preparation method of the immobilized enzyme preparation comprises:

[0053] (1) mixing the auxiliary agent A with a phosphate buffer solution containing lipase (lipase concentration in the solution is 12 wt %, pH 7.5) to obtain a mixture 1;

[0054] (2) Add additive B to water at a mass ratio of 6:100, adjust the pH to 12, heat and stir in a water bath at 85°C, add mixture 1, cast the resulting mixed solution into a 6 mm thick layer, and dry to obtain the product.

[0055] Example 4

[0056] A method for preparing polyglycerol ester comprises the following steps:

[0057] After oleic acid and oligoglycerol were mixed in a mass ratio of 1:2, the immobilized enzyme preparation of Example 1 was added for reaction, the mass ratio of the immobilized enzyme preparation to the oligoglycerol was 3:100, the reaction temperature was 40°C, the time was 5h, centrifuged, and the upper layer was molecularly distilled twice, the parameters of the first molecular distillation were: pressure 8Pa, temperature 80°C. The heavy phase product obtained by the first molecular distillation was molecularly distilled for the second time, the parameters were: pressure 3Pa, temperature 92°C, and the light phase product obtained by distillation was polyglycerol ester.

[0058] Among them, the esterification rate (%) of the upper material tested after centrifugation = (AV0-AV1) / AV0*100%, where AV0 is the oleic acid value, AV1 is the acid value of the upper material, and the acid value is determined in accordance with GB / T5530-2005.

[0059] The esterification rate of the upper layer after centrifugation was measured to be 99.5%. The immobilized enzyme preparation was recovered and reused, and was reused 30 times according to the above preparation method, and the esterification rate was maintained at 96.8%.

[0060] The final yield of polyglycerol ester was 87.4% and the purity was 99.8%.

[0061] Example 5

[0062] A method for preparing polyglycerol ester comprises the following steps:

[0063] After oleic acid and oligoglycerol were mixed in a mass ratio of 1:1, the immobilized enzyme preparation of Example 2 was added for reaction, the mass ratio of the immobilized enzyme preparation to the oligoglycerol was 2:100, the reaction temperature was 35°C, the time was 4h, centrifuged, and the upper layer was molecularly distilled twice, the parameters of the first molecular distillation were: pressure 5Pa, temperature 75°C. The heavy phase product obtained by the first molecular distillation was molecularly distilled for the second time, the parameters were: pressure 2Pa, temperature 90°C, and the light phase product obtained by distillation was polyglycerol ester.

[0064] After centrifugation, the esterification rate of the upper layer was 99.3%. The immobilized enzyme preparation was recovered and reused. It was reused 30 times according to the above preparation method, and the esterification rate was maintained at 96.1%.

[0065] The final yield of polyglycerol ester was 86.5% and the purity was 99.6%.

[0066] Example 6

[0067] A method for preparing polyglycerol ester comprises the following steps:

[0068] After oleic acid and oligoglycerol were mixed at a mass ratio of 1:3, the immobilized enzyme preparation of Example 3 was added for reaction, the mass ratio of the immobilized enzyme preparation to the oligoglycerol was 4:100, the reaction temperature was 50°C, the time was 6h, centrifuged, and the upper layer was molecularly distilled twice, the parameters of the first molecular distillation were: pressure 10Pa, temperature 85°C. The heavy phase product obtained by the first molecular distillation was molecularly distilled for the second time, the parameters were: pressure 5Pa, temperature 95°C, and the light phase product obtained by distillation was polyglycerol ester.

[0069] After centrifugation, the esterification rate of the upper layer was 99.6%. The immobilized enzyme preparation was recovered and reused, and was reused 30 times according to the above preparation method, and the esterification rate was maintained at 97.0%.

[0070] The final yield of polyglycerol ester was 87.1% and the purity was 99.5%.

[0071] Comparative Example 1

[0072] The only difference from Example 4 is that the immobilized enzyme preparation used is different, specifically, the auxiliary agent A used in preparing the immobilized enzyme preparation is different, which is only rapeseed meal, the total amount of auxiliary agent A remains unchanged, and the other conditions are the same.

[0073] The esterification rate of the upper layer after centrifugation was measured to be 95.7%. The immobilized enzyme preparation was recovered and reused, and was reused 30 times according to the above preparation method, and the esterification rate was maintained at 89.2%.

[0074] The final yield of polyglycerol ester was 80.5% and the purity was 97.1%.

[0075] Comparative Example 2

[0076] The only difference from Example 4 is that the immobilized enzyme preparation used is different, specifically, the auxiliary agent A used in preparing the immobilized enzyme preparation is different, which is only diatomaceous earth, and the total amount of auxiliary agent A remains unchanged, and the other conditions are the same.

[0077] The esterification rate of the upper layer after centrifugation was measured to be 96.6%. The immobilized enzyme preparation was recovered and reused, and was reused 30 times according to the above preparation method, and the esterification rate was maintained at 89.4%.

[0078] The final yield of polyglycerol ester was 81.3% and the purity was 97.8%.

[0079] Comparative Example 3

[0080] The only difference from Example 4 is that the immobilized enzyme preparation used is different, specifically, the auxiliary agent A used to prepare the immobilized enzyme preparation is different, the rapeseed meal is replaced by soybean meal of equal mass, and the other conditions are the same.

[0081] The esterification rate of the upper layer after centrifugation was measured to be 97.0%. The immobilized enzyme preparation was recovered and reused, and was reused 30 times according to the above preparation method, and the esterification rate was maintained at 90.1%.

[0082] The final yield of polyglycerol ester was 83.8% and the purity was 98.4%.

[0083] Comparing Comparative Examples 1 to 3 with Example 4, it can be seen that in Comparative Examples 1 and 2, the auxiliary agent A uses a single component, in Comparative Example 3, soybean meal and diatomaceous earth are used in combination, and in Example 4, rapeseed meal and diatomaceous earth are used in combination. As a result, the esterification rate, polyglycerol ester yield and purity of Example 4 are significantly higher than those of Comparative Examples 1 to 3, indicating that the rapeseed meal and diatomaceous earth in the auxiliary agent A have a significant synergistic effect, which significantly improves the reaction activity of lipase, and still has a high reaction activity after being recycled and reused 30 times, and the prepared polyglycerol ester maintains a high yield and purity.

[0084] Comparative Example 4

[0085] The only difference from Example 4 is that different immobilized enzyme preparations are used. Specifically, only lipase and auxiliary agent A are used to prepare the immobilized enzyme preparation, and the mass ratio of lipase: auxiliary agent A is 7:115. The other conditions are the same.

[0086] The esterification rate of the upper layer after centrifugation was measured to be 92.7%. The immobilized enzyme preparation was recovered and reused, and was reused 30 times according to the above preparation method, and the esterification rate was maintained at 85.4%.

[0087] The final yield of polyglycerol ester was 78.5% and the purity was 96.3%.

[0088] Comparative Example 5

[0089] The only difference from Example 4 is that different immobilized enzyme preparations are used. Specifically, only lipase and auxiliary agent B are used to prepare the immobilized enzyme preparation, and the mass ratio of lipase to auxiliary agent B is 7:115. The other conditions are the same.

[0090] The esterification rate of the upper layer after centrifugation was measured to be 93.4%. The immobilized enzyme preparation was recovered and reused, and was reused 30 times according to the above preparation method, and the esterification rate was maintained at 87.9%.

[0091] The final yield of polyglycerol ester was 79.8% and the purity was 95.6%.

[0092] By comparing Comparative Examples 4-5 with Example 4, it can be seen that a single auxiliary agent A or auxiliary agent B is used in Comparative Examples 4 and 5, but the total amount of the auxiliary agent remains unchanged. In Example 4, auxiliary agent A and auxiliary agent B are used in combination. As a result, the esterification rate, polyglycerol ester yield and purity of Example 4 are significantly higher than those of Comparative Examples 4-5, indicating that auxiliary agent A and auxiliary agent B have a significant synergistic effect, which significantly improves the reaction activity of lipase, and still has a high reaction activity after being recycled and reused 30 times. At the same time, the prepared polyglycerol ester maintains a high yield and purity.

[0093] Comparative Example 6

[0094] The only difference from Example 4 is that the immobilized enzyme preparation used is different, specifically, the lipase used to prepare the immobilized enzyme preparation is different, only the lipase from Aspergillus oryzae is used, the total amount of lipase remains unchanged, and the other conditions are the same.

[0095] The esterification rate of the upper layer after centrifugation was measured to be 95.5%. The immobilized enzyme preparation was recovered and reused, and was reused 30 times according to the above preparation method, and the esterification rate was maintained at 93.4%.

[0096] The final yield of polyglycerol ester was 84.2% and the purity was 98.1%.

[0097] Comparative Example 7

[0098] The only difference from Example 4 is that the immobilized enzyme preparation used is different, specifically, the lipase used to prepare the immobilized enzyme preparation is different, and only the lipase from Rhizomucor miehei is used, the total amount of lipase remains unchanged, and the other conditions are the same.

[0099] The esterification rate of the upper layer after centrifugation was measured to be 96.8%. The immobilized enzyme preparation was recovered and reused, and was reused 30 times according to the above preparation method, and the esterification rate was maintained at 92.7%.

[0100] The final yield of polyglycerol ester was 85.9% and the purity was 98.4%.

[0101] Comparing Comparative Examples 6-7 with Example 4, it can be seen that in Comparative Examples 6 and 7, a single lipase derived from Aspergillus oryzae or a lipase derived from Rhizomucor miehei is used, but the total amount of the lipase remains unchanged. In Example 4, two lipases are used in combination. As a result, the esterification rate, polyglycerol ester yield and purity of Example 4 are significantly higher than those of Comparative Examples 6-7, indicating that the lipase derived from Aspergillus oryzae and the lipase derived from Rhizomucor miehei have significant synergistic effects, which significantly improve the reaction activity of the lipase, and after being recycled and reused 30 times, it still has a high reaction activity, and the prepared polyglycerol ester maintains a high yield and purity.

[0102] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. An immobilized enzyme preparation, characterized in that It comprises lipase, auxiliary agent A and auxiliary agent B, and the mass ratio of the three is 5-8:20-50:60-100; The lipase is selected from lipase derived from Aspergillus oryzae and lipase derived from Rhizomucor miehei, and the mass ratio of the two is 2-4:1; The auxiliary agent A is composed of rapeseed meal and diatomaceous earth; the auxiliary agent B includes konjac glucomannan gel and an emulsifier; The mass ratio of the rapeseed meal to diatomaceous earth is 1-3:1; the mass ratio of the konjac glucomannan gel to the emulsifier is 100:0.5-1; and the emulsifier is selected from at least one of monostearate glyceryl and sucrose fatty acid ester.

2. The method for preparing the immobilized enzyme preparation according to claim 1, characterized in that: The steps include: (1) mixing an auxiliary agent A with a phosphate buffer solution containing lipase to obtain a mixture 1; (2) Add additive B to water, adjust the pH to alkaline, heat and stir, add mixture 1, cast the obtained mixed solution into a film, and obtain the film after drying.

3. The preparation method according to claim 2, characterized in that: The concentration of lipase in the phosphate buffer solution containing lipase in step (1) is 8-12 wt % and the pH is 7.

5.

4. The preparation method according to claim 2, characterized in that: In step (2), the mass ratio of auxiliary agent B to water is 2-6:100, and the pH is 8-12.

5. The preparation method according to claim 2, characterized in that: The heating temperature in step (2) is 75-85° C., and the thickness of the film is 4-6 mm.

6. The method for using the immobilized enzyme preparation according to claim 1, characterized in that: The steps include: After the fatty acid and oligoglycerol are mixed, an immobilized enzyme preparation is added to react, centrifuged, and the upper layer material is molecularly distilled twice to obtain polyglycerol esters.

7. The method of use according to claim 6, characterized in that: The fatty acid includes oleic acid; the mass ratio of the fatty acid to oligoglycerol is 1:1-3; the mass ratio of the immobilized enzyme preparation to the oligoglycerol is 2-4:100; the reaction temperature is 35-50°C and the reaction time is 4-6h.