Immobilized enzyme as well as preparation method and application thereof

Abstract

The invention relates to an immobilized enzyme and a preparation method and application thereof in the technical field of biology. The preparation method comprises the following steps: mixing an enzyme solution and Tween 80 to prepare a mixed emulsion; preparing the mixed emulsion and a carrier into an immobilized enzyme by adopting an adsorption method; under the detection wavelength of 600nm + / -3nm, the OD value of the mixed emulsion is equal to a preset value + / -6, the preset value is equal to (0.37 R <-1 >) * (C-0.4), R is equal to Mt / Mp, Mt is the mass value of the Tween 80, Mp is the mass value of the enzyme in the enzyme solution, the mass unit of the Tween 80 is the same as the mass unit of the enzyme in the enzyme solution, C is the mass concentration value of the enzyme in the enzyme solution, and M is the mass concentration value of the enzyme in the enzyme solution. The unit of the mass concentration of the enzyme in the enzyme liquid is expressed by a / b, and b = a * 10 < 3 >. According to the preparation method, the yield of the immobilized enzyme can be improved, and the application performance of the product is excellent.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and particularly to the application of an immobilized enzyme and its preparation method. Background Art

[0002] The enzyme immobilization technology can improve the structural stability and operational stability of biocatalysts, making it easy to separate from the reaction system and reuse, thereby reducing the use cost of bioenzymes. Therefore, it can effectively expand the application of bioenzymes in industries such as pharmaceuticals, food, and textiles. During the enzyme immobilization process, the properties of the carrier, the concentration and dosage of the enzyme, the type and addition method of the modifier, etc. will all affect the structure and distribution state of the bioenzyme on the carrier.

[0003] In order to improve the catalytic performance and shelf life of enzyme preparations, a common strategy is to add enzyme activators or protectants during the immobilization process or in liquid enzymes. The presence of activators or protectants can, on the one hand, adjust the configuration of bioenzymes to the optimal state, and on the other hand, form a protective layer for bioenzymes in the carrier pores, improving their tolerance to high temperature, acid-base, solvents, etc.

[0004] Traditional processes for preparing immobilized enzymes using enzyme activators or protectants, for example: the preparation of immobilized lipase using lipase, a carrier, and a co-immobilizer as described in CN200510112638.5, where the co-immobilizer is at least one of PEG6000, coconut oil, Tween 80, gelatin, lecithin, and magnesium sulfate. However, the performance of the immobilized enzymes prepared using traditional immobilized enzyme preparation processes needs to be improved. Summary of the Invention

[0005] Based on this, one or more embodiments of this application provide an application of an immobilized enzyme and its preparation method.

[0006] One or more embodiments of this application provide a method for preparing an immobilized enzyme, and the preparation method includes the following steps:

[0007] Mix an enzyme solution and Tween 80 to prepare a mixed emulsion; and,

[0008] Use the adsorption method to prepare an immobilized enzyme from the mixed emulsion and a carrier;

[0009] Wherein,

[0010] At a detection wavelength of 600 nm ± 3 nm, the OD value of the mixed emulsion = preset value ± 6, and the preset value = (0.37R - 1) × (C - 0.4),

[0011] R = Mt÷Mp, where Mt is the mass value of the Tween 80, and Mp is the mass value of the enzyme in the enzyme solution. The unit of the mass of the Tween 80 is the same as the unit of the mass of the enzyme in the enzyme solution.

[0012] C is the mass concentration value of the enzyme in the enzyme solution. The unit of the mass concentration of the enzyme in the enzyme solution is expressed as a / b, and b = a×10 3 。

[0013] In some embodiments of the present application, the enzyme solution satisfies one or more of the following conditions:

[0014] (1) The mass concentration of the enzyme in the enzyme solution does not exceed 25 mg / g; optionally, the mass concentration of the enzyme in the enzyme solution is 5 mg / g - 25 mg / g; and,

[0015] (2) The enzyme activity of the enzyme in the enzyme solution is 5500 U / mg - 11000 U / mg.

[0016] In some embodiments of the present application, R ≤ 20; optionally, R is 3 - 20; further optionally, R is 3 - 15.

[0017] In some embodiments of the present application, the dosage of the Tween 80 corresponding to every 100 g of the enzyme solution does not exceed 25 g; optionally, the dosage of the Tween 80 corresponding to every 100 g of the enzyme solution is 3.75 g - 22.5 g.

[0018] In some embodiments of the present application, the preparation method satisfies one or more of the following conditions:

[0019] 1) The enzyme includes lipase; optionally, the microbial source of the lipase includes Thermomyces lanuginosus, Rhizopus oryzae, Rhizomucor miehei, Aspergillus oryzae, Pichia pastoris or its genetically modified strains;

[0020] 2) The solvent of the enzyme solution includes a phosphate buffer solution with a concentration of 10 mM - 100 mM and a pH of 5.5 - 6.5; and,

[0021] 3) The method of mixing the enzyme solution and Tween 80 includes shaking or stirring;

[0022] Optionally, the conditions for shaking include: the temperature is 4°C - 30°C, the rotation speed is 120 rpm - 180 rpm, and the time is 10 min - 2 h;

[0023] Optionally, the conditions for stirring include: the frequency is 25 Hz - 35 Hz, and the time is 10 min - 2 h.

[0024] In some embodiments of the present application, the adsorption method includes physical adsorption; optionally, the carrier includes macroporous adsorption resin; further optionally, the macroporous adsorption resin includes macroporous methacrylic resin or / and macroporous polystyrene resin.

[0025] In some embodiments of the present application, the adsorption method includes ion adsorption; optionally, the carrier includes ion exchange resin; further optionally, the ion exchange resin includes methacrylic acid type anion exchange resin.

[0026] In some embodiments of the present application, the preparation method satisfies one or more of the following conditions:

[0027] (A) The conditions of the adsorption method include: performing under oscillation or stirring conditions until the change in the concentration of the enzyme in the mixture of the mixed emulsion and the carrier is < 0.1 mg / g within 1 h; optionally, the oscillation conditions include: a rotation speed of 120 rpm - 180 rpm and a time of 10 min - 2 h; optionally, the stirring conditions include: a frequency of 25 Hz - 35 Hz and a time of 10 min - 2 h;

[0028] (B) The conditions of the adsorption method include: an adsorption temperature of 4°C - 30°C;

[0029] (C) The ratio of the mass of the carrier to the mass of the enzyme in the enzyme solution is (25 - 50):1; and,

[0030] (D) The water content of the immobilized enzyme is 2 wt% - 20 wt%.

[0031] One or more embodiments of the present application also provide an immobilized enzyme prepared by the above preparation method.

[0032] One or more embodiments of the present application also provide a production method of an enzyme-catalyzed product, the production method including the following steps:

[0033] Preparing an immobilized enzyme by the above preparation method; and,

[0034] Using the immobilized enzyme to catalyze a substrate to prepare an enzyme-catalyzed product.

[0035] Compared with the traditional technology, the beneficial effects of the embodiments of the present application include:

[0036] The preparation method of the immobilized enzyme provided by the embodiments of the present application can ensure the sufficient interaction between Tween 80 and the enzyme protein, improve the effective doping of Tween 80 on the carrier, increase the finished product weight of the immobilized enzyme, and make the application performance of the product stable and excellent. Moreover, this method uses the OD value as the basis for adding the carrier, which is conducive to the efficient and accurate control of the process quality and provides a general basis for the quality control of the immobilized enzyme process. Description of the Drawings

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more comprehensively understand the present application and its beneficial effects, the following will briefly introduce the drawings required for use in the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0038] Figure 1 It is a derivation diagram of the linear relationship between OD600 of the present application and the mass of Tween 80, the mass of the enzyme, and the enzyme concentration. Detailed Embodiments

[0039] The following will further describe the present application in detail in conjunction with the drawings, embodiments, and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosed content of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more comprehensive understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs. The terms used in the description of the present application in this specification are only for the purpose of describing the embodiments and examples and are not intended to limit the present application.

[0041] Term

[0042] Unless otherwise stated or there are contradictions, the terms or phrases used herein have the following meanings:

[0043] As used herein, the terms "and / or", "or / and", and "and / or" include any one of two or more related listed items, and also include any and all combinations of the related listed items. The any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctive combinations selected from "and / or", "or / and", and "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example is the technical solution of "A, and / or, B, and / or, C, and / or, D", which includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the combination of the four items A, B, C, and D (that is, the technical solution connected by "logical AND").

[0044] In this application, terms such as "multiple", "multiple types", "multiple times", "multiple elements", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0045] As used herein, "their combinations", "any combination thereof", "any combination mode thereof", etc. include all suitable combination modes of any two or more than two items in the listed items.

[0046] In this article, the "suitable" in "suitable combination mode", "suitable mode", "any suitable mode", etc. is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0047] In this article, "preferred", "better", "more preferable", "it is advisable" are only used to describe embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of this application.

[0048] In this application, "further", "even further", "especially", etc. are used for descriptive purposes and indicate differences in content, but should not be construed as a limitation on the protection scope of this application.

[0049] In this application, "optionally", "optional", "optional" mean that it can be there or not, that is, it refers to any one of the two parallel solutions of "yes" or "no". If "optional" appears multiple times in a technical solution, unless otherwise specified and there are no contradictions or mutual restrictions, each "optional" is independent of each other.

[0050] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for descriptive purposes and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0051] In this application, among the technical features described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions containing the listed features.

[0052] In this application, regarding numerical intervals (i.e., numerical ranges), unless otherwise specified, the selectable numerical values are considered continuous within the above numerical intervals and include the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, when t is an integer selected from 1 to 10, it means that t is any integer selected from the integer group composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0053] The temperature parameter in this application, unless otherwise specified, allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuation within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C is allowed.

[0054] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0055] All documents mentioned in this application are incorporated herein by reference as if each document was individually incorporated by reference. Unless it conflicts with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited for all contents and all purposes. When referring to cited documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also incorporated by reference. When referring to cited documents in this application, examples and preferred methods of the relevant technical features cited can also be incorporated as references into this application, but only to the extent that this application can be implemented. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or be amended adaptively according to the description in this application.

[0056] For the preparation of immobilized enzymes by traditional adsorption methods, such as the content described in CN200510112638.5, there is a lack of effective means for quantitative comparison, which is not conducive to the control of production quality and cannot stably output high-quality immobilized enzymes. The main purposes of the embodiments of this application include establishing a method for preparing immobilized enzymes, selecting a suitable node for adding a carrier for adsorption and curing during the emulsification of Tween 80 and the enzyme solution, which is beneficial to the adsorption of enzymes and the doping of Tween 80, beneficial to the application effect and product yield of the immobilized enzyme product, and beneficial to the quality control of the immobilized enzyme product.

[0057] In this application, OD600 is the optical density value measured when the wavelength is set to 600 nm, which is a standard index for tracking the density of microorganisms in liquid cultures and is usually used to indicate the density of bacterial cells. It should be understood that the wavelength is allowed to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±3 nm, ±2 nm, ±1 nm are allowed.

[0058] The first aspect of the embodiments of the present application

[0059] The embodiments of this application provide a method for preparing immobilized enzymes, and the preparation method includes the following steps:

[0060] Mix the enzyme solution and Tween 80 to prepare a mixed emulsion; and,

[0061] Using the adsorption method, prepare the mixed emulsion and the carrier into immobilized enzymes;

[0062] Wherein,

[0063] At a detection wavelength of 600 nm ± 3 nm, the OD value of the mixed emulsion = preset value ± 6, and the preset value = (0.37R - 1) × (C - 0.4),

[0064] R = ml ÷ m2, m1 is the mass value of the Tween 80, m2 is the mass value of the enzyme in the enzyme solution, and the units of the mass of the Tween 80 and the mass of the enzyme in the enzyme solution are the same,

[0065] C is the mass concentration value of the enzyme in the enzyme solution, and the unit of the mass concentration of the enzyme in the enzyme solution is expressed as a / b, where b = a × 10 3 .

[0066] The above a and b can be any two units with a difference of 10 3 .

[0067] In some examples, a is g and b is kg, or a is mg and b is g.

[0068] In this application, the mass concentration refers to the mass of the substance contained in a unit volume or a unit mass. Generally, the mass of the substance contained in a unit volume and the mass of the substance contained in a unit mass can be converted. For example, when the enzyme solution concentration is 1 × 10 3 kg / m 3 , 1 mg / g can be converted to 1 mg / mL. The method for preparing the immobilized enzyme provided by the examples of this application can ensure the full interaction between Tween 80 and the enzyme protein, improve the effective doping of Tween 80 on the carrier, increase the finished product weight of the immobilized enzyme, and make the application performance of the product stable and excellent. Moreover, this method uses the OD value as the basis for adding the carrier, which is conducive to the efficient and accurate control of the process quality and provides a universal basis for the quality control of the immobilized enzyme process.

[0069] In some examples, the mass concentration of the enzyme in the enzyme solution does not exceed 25 mg / g (for example, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25 mg / g); optionally, the mass concentration of the enzyme in the enzyme solution is 5 mg / g - 25 mg / g. Further, the mass concentration of the enzyme in the enzyme solution is 5 mg / g - 15 mg / g. The inventors of this application conducted the following basic research (Mt: the mass of Tween; Mp is the mass of lipase, R = Mt / Mp, C is the concentration of lipase):

[0070] 1. The relationship between OD600, oscillation time and rate:

[0071] Tw = 6 g, 40 g of RML solution, C = 10 mg / g, Mt / Mp = 15, adjusting the oscillation rates of 100 rpm and 150 rpm, and finding that the relationship between the OD600 of the system and time is as Figure 1As shown. Conclusion: (1) OD600 first increases and then decreases with the prolongation of the oscillation time, reaching the maximum between 40 min and 200 min. (2) At 150 rpm, both the growth rate and the decline rate of OD600 are higher than those at 100 rpm.

[0072] 2. Relationship between OD600 and the parameters of the enzyme solution and Tween emulsion system:

[0073] (1) Keeping R(Mt / Mp) = 3.75 and R(Mt / Mp) = 15 unchanged, with the volume of the lipase solution remaining the same, adjust the concentration of protein (C) and the mass of Tween (Mt), and mix and oscillate the Tween and the enzyme solution for 1 h.

[0074] Under both conditions, OD600 shows a linear relationship with the protein concentration C:

[0075] When R(Mt / Mp) = 3.75, OD600 = 4.6014C - 1.8626 = 4.6014(C - 0.4);

[0076] When R(Mt / Mp) = 15, OD600 = 0.5475C - 0.2146 = 0.5475(C - 0.4);

[0077] Based on this, it is inferred that there is the following relationship between OD600 and the protein concentration C: OD600 = K(C - 0.4); where when R(Mt / Mp) = 3.75, K = 0.5475. When R(Mt / Mp) = 15, K = 4.6014. See Figure 1 Figure B in

[0078] (2) Controlling C between 0 - 10 mg / mL, by the same method as above, it is measured that when R(Mt / Mp) = 6, K = 1.032, and when R(Mt / Mp) = 10, K = 2.837. According to the above data, it is found that there is a linear relationship between the slope K and R(Mt / Mp): K = 0.373R - 0.986. See Figure 1 Figure C in

[0079] (3) When C is between 6 - 15 mg / g, by adjusting the value of R(Mt / Mp), it is found that there is also a linear relationship between the slope K (OD600 / C - 0.4) and R(Mt / Mp): K = 0.3726R - 1.0099. See Figure 1 Figure D in

[0080] Based on (2) and (3), it is concluded that within the range of C = 0 - 15, there is a linear relationship between the slope K and R(Mt / Mp) as follows: K = 0.37R - 1. And there is a relationship between OD600 and the enzyme solution concentration as OD600 = K(C - 0.4). Therefore, the relationship between OD600, Mt / Mp, and the enzyme concentration C is OD600 = (0.37R - 1)(C - 0.4).

[0081] In some of these examples, the enzyme activity of the enzyme in the enzyme solution is 5500 U / mg - 11000 U / mg (such as 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000 U / mg).

[0082] In some of these examples, R ≤ 20 (such as 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20); optionally, R is between 3 - 20; further optionally, R is between 3 - 15.

[0083] In some of these examples, the dosage of Tween 80 corresponding to every 100 g of the enzyme solution does not exceed 25 g (such as 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25 g). Optionally, the dosage of Tween 80 corresponding to every 100 g of the enzyme solution is 3.75 g - 22.5 g.

[0084] This application does not make specific limitations on the type of enzyme, including but not limited to lipase, such as lipase produced by Thermomyces lanuginosus, Rhizopus oryzae, Rhizomucor miehei, Aspergillus oryzae, Pichia pastoris, or their genetically modified strains.

[0085] The present application does not particularly limit the solvent of the enzyme solution. For example, a phosphate buffer solution with a concentration of 10 mM - 100 mM (such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mM) and a pH of 5.5 - 6.5 (such as 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5) is used as the solvent.

[0086] The present application does not particularly limit the method of mixing the enzyme solution and Tween 80, including but not limited to shaking or stirring. Shaking can be carried out under the following conditions: the temperature is 4°C - 30°C (such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30°C), the rotation speed is 120 rpm - 180 rpm (such as 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180 rpm), and the time is 10 min - 2 h (such as 10 min, 0.5 h, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 h). Stirring can also be carried out under the following conditions: the frequency is 25 Hz - 35 Hz (such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 Hz), and the time is 10 min - 2 h (such as 10 min, 0.5 h, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 h).

[0087] In some of these examples, the adsorption method includes physical adsorption; optionally, the carrier includes macroporous adsorption resin; further optionally, the macroporous adsorption resin includes macroporous methacrylic resin or / and macroporous polystyrene resin. The resin surface can include alkyl chains of different lengths, can also include alkyl chains with epoxy groups at the ends, or can be free of functional groups. For example, ECR1030, 8804, 8806, 1090, 8285, 5587 of Purolite Company and LX201A of Xi'an BlueSail Company, etc.

[0088] In some of these examples, the adsorption method includes an ion adsorption method; optionally, the carrier includes an ion exchange resin; further optionally, the ion exchange resin refers to a methacrylic acid type anion exchange resin, and the resin surface may include an alkyl chain with functional groups such as primary amine, secondary amine, or tertiary amine at the end. For example, ECR8404, 8409, 8415 of Purolite Company, LX1000HAA of Xi'an Lanchao Company, etc.

[0089] In some of these examples, the conditions of the adsorption method include: performing under oscillation or stirring conditions until the change in the concentration of the enzyme in the mixture of the mixed emulsion and the carrier is <0.1 mg / g within 1 h. Optionally, the oscillation conditions include: the rotation speed is 120 rpm - 180 rpm (for example, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180 rpm), and the time is 10 min - 2 h (for example, 10 min, 0.5 h, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 h). Optionally, the stirring conditions include: the frequency is 25 Hz - 35 Hz (for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 Hz), and the time is 10 min - 2 h (for example, 10 min, 0.5 h, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 h).

[0090] In some of these examples, the conditions of the adsorption method include: the adsorption temperature is 4°C - 30°C (for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30°C).

[0091] In the embodiments of the present application, in the step of preparing the immobilized enzyme from the mixed emulsion and the carrier by using the adsorption method, after sufficient adsorption, a suitable method can be selected to perform solid-liquid separation on the adsorption system, and the separated solid can also be further dried. The present application does not particularly limit the method of solid-liquid separation. For example, it can be vacuum filtration or cloth bag centrifugal filtration. The present application also does not particularly limit the drying method. It can use room temperature drying, oven drying, or fluidized bed drying, etc. Correspondingly, the present application does not particularly limit the drying time either. It can be adjusted according to the required water content of the product and the selected drying method. For example, if it is necessary to prepare an immobilized enzyme with a water content of 2 wt% - 20 wt%, it can be left at room temperature for, for example, 10 min - 72 h, that is, air-dried.

[0092] In the embodiments of the present application, the step of preparing the immobilized enzyme by using the adsorption method with the mixed emulsion and the carrier can be carried out by relying on a water bath shaker, an air bath shaker, or mechanical stirring, etc. The present application does not particularly limit the adsorption time (for example, 1 h - 24 h), and it is only necessary that the protein concentration change per hour in the obtained adsorption system is less than 0.1 mg / mL when the mixed emulsion and the carrier are in full contact.

[0093] In some examples, the ratio of the mass of the carrier to the mass of the enzyme in the enzyme solution is (25 - 50):1, such as 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1.

[0094] In some examples, the water content of the immobilized enzyme is 2 wt% - 20 wt% (for example, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%).

[0095] The present application does not particularly limit the detection method of the water content of the immobilized enzyme. For example, it can be monitored by the drying and weighing method or a moisture meter.

[0096] The second aspect of the embodiments of the present application

[0097] The embodiments of the present application provide an immobilized enzyme prepared by the preparation method described in the first aspect.

[0098] The third aspect of the embodiments of the present application

[0099] The embodiments of the present application provide a production method of an enzyme-catalyzed product. The production method includes the following steps:

[0100] Preparing an immobilized enzyme by using the preparation method described in the first aspect; and,

[0101] Using the immobilized enzyme to catalyze a substrate to prepare an enzyme-catalyzed product.

[0102] The implementation scheme of the present application will be described in detail below in conjunction with embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions in the following embodiments, the guidance given in the present application is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or referring to the experimental methods known in the art.

[0103] In the following specific embodiments, regarding the measurement parameters of raw material components, if there is no special instruction, there may be slight deviations within the weighing accuracy range. Regarding the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0104] In Examples 1 to 15 and Comparative Examples 1 to 8, the Rhizomucor miehei lipase (RML) solution is prepared by Aspergillus oryzae fermentation of the production strain BC4 (deposit number: CGMCC No. 18825). The fermentation method refers to Patent CN201410822387.9 (invention name: A method for fermenting lipase). The obtained fermentation broth is pretreated by adding 2 times of pure water and 5% diatomaceous earth RS300, and then prepared into liquid enzymes with different enzyme contents (25mg / g, 20mg / g, 15mg / g, 10mg / g, 5mg / g) after plate and frame filtration, vertical plate and frame secondary filtration, ultrafiltration concentration, and sterilizing filtration for subsequent enzyme immobilization. The solvent of the lipase solution is phosphate buffer with a concentration of 10mM - 100mM and a pH value of 5.5 - 6.5, without preservatives and protectants.

[0105] In Example 16, the Thermomyces lanuginosus lipase (TLL) solution from Thermomyces lanuginosus is prepared by Aspergillus oryzae fermentation of the production strain BC4 (deposit number CGMCC No. 40406). The fermentation method and the post-extraction method are the same as those of RML, and different enzyme contents of TLL liquid enzymes (enzyme activity is 10000U / mg) can be prepared for subsequent enzyme immobilization. The solvent of the lipase solution is phosphate buffer with a concentration of 10mM - 100mM and a pH value of 5.5 - 6.5, without preservatives and protectants.

[0106] In Example 17, the Rhizopus oryzae lipase (ROL) solution is prepared by Pichia pastoris fermentation of the m316H strain (deposit number CGMCC No. 19221). The fermentation method refers to Patent CN201410822387.9 (invention name: A method for fermenting lipase). The obtained fermentation broth is post-extracted by the same method as RML to prepare liquid enzymes with different enzyme contents (enzyme activity is 11000U / mg) for subsequent enzyme immobilization. The solvent of the lipase solution is phosphate buffer with a concentration of 10mM - 100mM and a pH value of 5.5 - 6.5, without preservatives and protectants.

[0107] Example 1. Preparation of Immobilized Enzyme A

[0108] (1) Weigh 400 g of RML lipase (Rhizomucor miehei lipase) solution (15 mg / g, 90000 U / g) and 90 g of Tween 80 into a 2-L Erlenmeyer flask. Place the Erlenmeyer flask in a constant-temperature shaker at 18 °C and 150 rpm and shake for 1 h. Measure the OD600 of the mixed emulsion every 10 min. Among them, the solvent of the lipase solution is 20 mM phosphate buffer at pH 6.0.

[0109] (2) Under the condition that the OD600 reaches 67.2, add 300 g of ion exchange resin LX1000HAA to the mixed emulsion and continue to shake (the temperature and rotation speed of shaking are the same as in the previous step) until the change in the enzyme concentration in the resulting mixture is <0.1 mg / g within 1 h (it takes about 24 h). Then, use a Buchner funnel to separate the solid-liquid mixture. Wash the upper solid with 100 mL of phosphate buffer (20 mM, pH 6.0) three times to wash the enzyme, and then air-dry at room temperature.

[0110] (3) Use a Sartorius MA150 moisture analyzer to measure the moisture content of the immobilized enzyme. Under the condition that it drops to about 12%, pack and store it, and weigh it. The finished product weight of immobilized enzyme A is about 200 g, and the transesterification activity is 448 IUN / g.

[0111] The transesterification activity of the immobilized enzyme is measured according to the following method (unless otherwise specified, the transesterification activities in the examples and comparative examples of this application are all measured by this method):

[0112] Use a mixture of refined soybean oil (Wilmar (Shanghai) Food Industry Co., Ltd.) and fully hydrogenated soybean oil (Wilmar (Shanghai) Specialty Oils Co., Ltd.) as the substrate (w / w = 73:27). With the mass ratio of the immobilized enzyme to the substrate being 1:20, react under the conditions of shaking at 70 °C and 150 rpm for 30 min. After the reaction is completed, pour out the upper layer of oil. For the lower-layer immobilized enzyme, add the same mass of fresh substrate oil, and continue to react under the same conditions for 30 min. Measure the solid fat content at 40 °C (i.e., the SFC content) of the second reaction product.

[0113] The method for measuring the SFC content at 40 °C is as follows: Place the solid fat tubes filled with samples in an oven at 100 °C for 15 min, in a water bath at 60 °C for 5 min, in a water bath at 0 °C for 60 min, and in a water bath at 40 °C for 30 min, and then measure the solid fat content with a nuclear magnetic resonance instrument.

[0114] The calculation formula for transesterification activity: Transesterification activity / IUN = (SFCBlank - SFCsample) / 30 * 1260; where,

[0115] SFCBlank refers to the SFC value of the substrate without transesterification reaction at 40 °C;

[0116] The SFC sample refers to the SFC value of the product sample after the enzymatic transesterification reaction.

[0117] Example 2: Preparation of Immobilized Enzyme B

[0118] (1) Weigh 400 g of RML lipase solution (10 mg / g, 60000 U / g) and 60 g of Tween 80 into a 2 L Erlenmeyer flask. Place the Erlenmeyer flask in a constant temperature shaker at 18 °C and 150 rpm and shake for 1 h. Measure the OD600 of the mixed emulsion every 10 min. Among them, the solvent of the lipase solution is 20 mM phosphate buffer at pH 6.0.

[0119] (2) Under the condition that OD600 reaches 44.4, add 200 g of ion exchange resin LX1000HAA to the mixed emulsion and continue to shake (the temperature and rotation speed of shaking are the same as the previous step) until the change in the enzyme concentration in the resulting mixture is <0.1 mg / g within 1 h (about 24 h). Then, use a Buchner funnel to separate the solid-liquid mixture. Wash the upper solid with 100 mL of phosphate buffer (20 mM, pH 6.0) three times, and then air-dry at room temperature.

[0120] (3) Use a Sartorius MA150 moisture analyzer to measure the moisture content of the immobilized enzyme. Under the condition of reducing to about 12%, pack and store, and weigh. The finished product weight of immobilized enzyme B is about 126 g, and the transesterification activity is 448 IUN / g.

[0121] Example 3: Preparation of Immobilized Enzyme C

[0122] (1) Weigh 400 g of RML lipase solution (5 mg / g, 30000 U / g) and 30 g of Tween 80 into a 2 L Erlenmeyer flask. Place the Erlenmeyer flask in a constant temperature shaker at 18 °C and 150 rpm and shake for 1 h. Measure the OD600 of the mixed emulsion every 10 min. Among them, the solvent of the lipase solution is 20 mM phosphate buffer at pH 6.0.

[0123] (2) Under the condition that OD600 reaches 21.0, add 100 g of ion exchange resin LX1000HAA to the mixed emulsion and continue to shake (the temperature and rotation speed of shaking are the same as the previous step) until the change in the enzyme concentration in the resulting mixture is <0.1 mg / g within 1 h (about 24 h). Then, use a Buchner funnel to separate the solid-liquid mixture. Wash the upper solid with 100 mL of phosphate buffer (20 mM, pH 6.0) three times, and then air-dry at room temperature.

[0124] (3) The moisture content of the immobilized enzyme was determined using a Sartorius MA150 moisture analyzer. After the moisture content dropped to about 12%, it was bagged and stored, and weighed. The finished product weight of immobilized enzyme C was about 62.8 g, and the transesterification activity was 450 IUN / g.

[0125] Example 4: Preparation of immobilized enzyme D

[0126] (1) Weigh 400 g of RML lipase solution (10 mg / g, 60000 U / g) and 15 g of Tween 80 into a 2 L Erlenmeyer flask. Place the Erlenmeyer flask in a constant temperature shaker at 18 °C and 150 rpm and shake for 1 h. Measure the OD600 of the mixed emulsion every 10 min. Among them, the solvent of the lipase solution is 20 mM phosphate buffer at pH 6.0.

[0127] (2) When OD600 reaches 5.3, add 200 g of ion exchange resin LX1000HAA to the mixed emulsion and continue shaking (the temperature and rotation speed are the same as the previous step) until the enzyme concentration change in the resulting mixture is <0.1 mg / g within 1 h (about 24 h). Then, use a Buchner funnel to separate the solid-liquid mixture. Wash the upper solid with 100 mL of phosphate buffer (20 mM, pH 6.0) three times, and then air-dry at room temperature.

[0128] (3) The moisture content of the immobilized enzyme was determined using a Sartorius MA150 moisture analyzer. After the moisture content dropped to about 12%, it was bagged and stored, and weighed. The finished product weight of immobilized enzyme D was about 110 g, and the transesterification activity was 450 IUN / g.

[0129] Example 5: Preparation of immobilized enzyme E

[0130] (1) Weigh 400 g of RML lipase solution (10 mg / g, 60000 U / g) and 40 g of Tween 80 into a 2 L Erlenmeyer flask. Place the Erlenmeyer flask in a constant temperature shaker at 18 °C and 150 rpm and shake for 1 h. Measure the OD600 of the mixed emulsion every 10 min. Among them, the solvent of the lipase solution is 20 mM phosphate buffer at pH 6.0.

[0131] (2) When OD600 reaches 31.7, add 200 g of ion exchange resin LX1000HAA to the mixed emulsion and continue shaking (the temperature and rotation speed are the same as the previous step) until the enzyme concentration change in the resulting mixture is <0.1 mg / g within 1 h (about 24 h). Then, use a Buchner funnel to separate the solid-liquid mixture. Wash the upper solid with 100 mL of phosphate buffer, and wash the enzyme three times, and then air-dry at room temperature.

[0132] (3) Use the Sartorius MA150 moisture analyzer to measure the moisture content of the immobilized enzyme. Under the condition of reducing to about 12%, bag and store it, and weigh it. The finished product weight of the immobilized enzyme E is about 120 g, and the transesterification activity is 450 IUN / g.

[0133] Example 6. Preparation of immobilized enzyme F

[0134] (1) Weigh 400 g of RML lipase solution (20 mg / g, 120000 U / g) and 30 g of Tween 80 into a 2 L Erlenmeyer flask. Place the Erlenmeyer flask in a constant temperature shaker at 18 °C and 150 rpm and shake for 1 h. Measure the OD600 of the mixed emulsion every 10 min. Among them, the solvent of the lipase solution is 20 mM phosphate buffer at pH 6.0.

[0135] (2) Under the condition that OD600 reaches 35, add 200 g of ion exchange resin LX1000HAA to the mixed emulsion, and continue to shake (the temperature and rotation speed of shaking are the same as the previous step) until the change in the enzyme concentration in the resulting mixture is <0.1 mg / g within 1 h (24 h). Then, use a Buchner funnel to separate the solid-liquid mixture. Wash the upper solid with 100 mL of phosphate buffer three times and dry it at room temperature.

[0136] (3) Use the Sartorius MA150 moisture analyzer to measure the moisture content of the immobilized enzyme. Under the condition of reducing to about 12%, bag and store it, and weigh it. The finished product weight of the immobilized enzyme F is about 125 g, and the transesterification activity is 450 IUN / g.

[0137] Example 7. Preparation of immobilized enzyme G

[0138] (1) Set the cooling water temperature <15 °C and pass cooling water into the jacket of an 800 L adsorption tank. Add 400 kg of RML lipase solution (10 mg / g, 100000 U / g) to the adsorption tank, and then add 60 kg of Tween 80. Stir for 1 h at a stirring motor frequency of 30 Hz through a ribbon agitator, and measure the OD of the mixture to be about 43.9. Among them, the solvent of the lipase solution is 20 mM phosphate buffer at pH 6.0.

[0139] (2) After adding 200 kg of ion exchange resin LX1000HAA, continue to stir (the stirring conditions are the same as the previous step) until the change in the enzyme concentration in the resulting mixture is <0.1 mg / g within 1 h (about 22 h). Use a bag centrifuge to separate the solid-liquid mixture, and add the solid material in batches to a 420 L fluidized bed dryer for drying.

[0140] (3) Use the Sartorius MA150 moisture analyzer to measure the moisture content of the immobilized enzyme irregularly. When it drops to about 12%, bag it for storage and weigh it. The finished product weight of immobilized enzyme G is about 125 kg, and the transesterification activity is 458 IUN / g.

[0141] Example 8. Preparation of immobilized enzyme B1

[0142] This example is a variant of Example 2. The main differences from Example 2 are mainly in steps (1) and (2). Specifically: in step (1), the dosage of Tween 80 is 80 g; correspondingly, the OD600 in step (2) reaches 61.5.

[0143] The finished product weight of immobilized enzyme B1 is about 131 g, and the transesterification activity is 444 IUN / g.

[0144] Example 9. Preparation of immobilized enzyme B2

[0145] This example is a variant of Example 2. The main differences from Example 2 are mainly in steps (1) and (2). Specifically: in step (1), the dosage of Tween 80 is 12 g; correspondingly, the OD600 in step (2) reaches 3.5.

[0146] The finished product weight of immobilized enzyme B2 is about 100 g, and the transesterification activity is 435 IUN / g.

[0147] Example 10. Preparation of immobilized enzyme B3

[0148] This example is a variant of Example 2. The main differences from Example 2 are mainly in steps (1) and (2). Specifically: in step (1), the lipase solution is 400 g of RML lipase solution (25 mg / g, 150000 U / g), and correspondingly, when the OD600 in step (2) reaches 31, 500 g of ion exchange resin LX1000HAA is added to the mixed emulsion.

[0149] The finished product weight of immobilized enzyme B3 is about 310 g, and the transesterification activity is 465 IUN / g.

[0150] Example 11. Preparation of immobilized enzyme B4

[0151] This example is a variant of Example 2. The main differences from Example 2 are mainly in steps (2) and (3). Specifically, the oscillation conditions in steps (1) and (2) include: the temperature is 25 °C and the rotation speed is 180 rpm; the oscillation time in step (1) is 10 min. For comparison with Example 2, when the OD600 is detected to be close to 44.4, the detection frequency is increased. When the OD600 reaches 44.4, the next experimental operation is carried out.

[0152] The finished product weight of the immobilized enzyme B4 is about 125 g, and the transesterification activity is 440 IUN / g.

[0153] Example 12. Preparation of immobilized enzyme G1

[0154] This example is a variant of Example 7. The main differences from Example 7 are mainly in steps (2) and (3). Specifically, the stirring conditions in steps (1) and (2) include: a frequency of 25 Hz and a time of 2 h. For comparison with Example 7, when the OD600 is detected to be close to 43.9, the detection frequency is increased, and when the OD600 reaches 43.9, the next experimental operation is carried out.

[0155] The finished product weight of the immobilized enzyme G1 is about 125×10 3 g, and the transesterification activity is 465 IUN / g.

[0156] Example 13. Preparation of immobilized enzyme B5

[0157] This example is a variant of Example 2. The main difference from Example 2 is mainly in step (2). Specifically, in step (2), an ion exchange resin is used, and it is YKT120 of Tianjin Yunkai Company. For comparison with Example 2, when the OD600 is detected to be close to 44.4, the detection frequency is increased, and when the OD600 reaches 44.4, the next experimental operation is carried out.

[0158] The finished product weight of the immobilized enzyme B5 is about 125 g, and the transesterification activity is 470 IUN / g.

[0159] Example 14. Preparation of immobilized enzyme B6

[0160] This example is a variant of Example 2. The main difference from Example 2 is mainly in step (2). Specifically, in step (2), a macroporous adsorption resin is used, and it is ECR8806M of Purolite Company. For comparison with Example 2, when the OD600 is detected to be close to 44.4, the detection frequency is increased, and when the OD600 reaches 44.4, the next experimental operation is carried out.

[0161] The finished product weight of the immobilized enzyme B6 is about 105 g, and the transesterification activity is 472 IUN / g.

[0162] Example 15. Preparation of immobilized enzyme B7

[0163] This example is a variant of Example 2, and the main difference from Example 2 lies in step (2). Specifically, in step (2), macroporous adsorption resin, namely LX201A from Xi'an Lanhxiao Company, is used. To compare with Example 2, when the OD600 is detected to be close to 44.4, the detection frequency is increased, and when OD600 reaches 44.4, the next experimental operation is carried out.

[0164] The finished product weight of immobilized enzyme B7 is about 105 g, and the transesterification activity is 466 IUN / g.

[0165] Example 16. Preparation of immobilized enzyme B8

[0166] This example is a variant of Example 2, and the main difference from Example 2 lies in step (1). Specifically, in step (1), the source of lipase is Thermomyces lanuginosus, the enzyme activity is 10000 U / mg, and the enzyme solution solvent is phosphate buffer with 60 mM and pH 5.5. To compare with Example 2, when the OD600 is detected to be close to 44.4, the detection frequency is increased, and when OD600 reaches 44.4, the next experimental operation is carried out.

[0167] The finished product weight of immobilized enzyme B8 is about 127 g, and the transesterification activity is 580 IUN / g.

[0168] Example 17. Preparation of immobilized enzyme B9

[0169] This example is a variant of Example 2, and the main difference from Example 2 lies in step (1). Specifically, in step (1), the source of lipase is Rhizopus oryzae, the enzyme activity is 11000 U / mg, and the enzyme solution solvent is phosphate buffer with 90 mM and pH 6.5. To compare with Example 2, when the OD600 is detected to be close to 44.4, the detection frequency is increased, and when OD600 reaches 44.4, the next experimental operation is carried out.

[0170] The finished product weight of immobilized enzyme B9 is about 105 g, and the transesterification activity is 590 IUN / g.

[0171] Example 18. Preparation of immobilized enzyme B10

[0172] This example is a variant of Example 2, and the main difference from Example 2 lies in step (2). Specifically, in step (2), ion exchange resin LX1000HAA is added under the condition that OD600 reaches 37.68. To compare with Example 2, when the OD600 is detected to be close to 44.4, the detection frequency is increased, and when OD600 reaches 44.4, the next experimental operation is carried out.

[0173] The finished product weight of immobilized enzyme B10 is about 120 g, and the transesterification activity is 450 IUN / g.

[0174] Comparative Example 1: Preparation of immobilized enzyme H

[0175] (1) Weigh 400 g of RML lipase solution (15 mg / g, 90000 U / g) and 90 g of Tween 80 into a 2 L Erlenmeyer flask. Place the Erlenmeyer flask in a constant temperature shaker at 18 °C and 150 rpm and shake for 30 min. Measure the OD600 of the mixed emulsion every 10 min.

[0176] (2) Under the condition that the OD600 reaches 30.0, add 300 g of ion exchange resin LX1000HAA to the mixed emulsion, continue to shake for 24 h, then use a Buchner funnel to separate the solid-liquid mixture. Wash the upper solid with 100 mL of phosphate buffer solution three times, and then air-dry at room temperature.

[0177] (3) Use a Sartorius MA150 moisture analyzer to measure the moisture content of the immobilized enzyme. Under the condition that it drops to about 12%, pack and store, and weigh. The finished product weight of immobilized enzyme H is about 155 g, and the transesterification activity is 428 IUN / g.

[0178] Comparative Example 2: Preparation of immobilized enzyme I

[0179] (1) Weigh 400 g of RML lipase solution (10 mg / g, 60000 U / g) and 60 g of Tween 80 into a 2 L Erlenmeyer flask. Place the Erlenmeyer flask in a constant temperature shaker at 4 °C and 100 rpm and shake for 20 min. Measure the OD600 of the mixed emulsion every 10 min.

[0180] (2) Under the condition that the OD600 reaches 20.2, add 200 g of ion exchange resin LX1000HAA to the mixed emulsion, continue to shake for 24 h, then use a Buchner funnel to separate the solid-liquid mixture. Wash the upper solid with 100 mL of phosphate buffer solution three times, and then air-dry at room temperature.

[0181] (3) Use a Sartorius MA150 moisture analyzer to measure the moisture content of the immobilized enzyme. Under the condition that it drops to about 12%, pack and store, and weigh. The finished product weight of immobilized enzyme I is about 100 g, and the transesterification activity is 400 IUN / g.

[0182] Comparative Example 3: Preparation of immobilized enzyme J

[0183] (1) Weigh 400 g of RML lipase solution (10 mg / g, 60000 U / g) and 40 g of Tween 80 into a 2-L Erlenmeyer flask. Place the flask in a constant-temperature shaker at 10°C and 100 rpm and shake for 10 min. Measure the OD600 of the mixed emulsion every 10 min.

[0184] (2) Under the condition that the OD600 reaches 15.7, add 200 g of ion exchange resin LX1000HAA to the mixed emulsion. Continue shaking for 24 h, then use a Buchner funnel to separate the solid-liquid mixture. Wash the upper solid with 100 mL of phosphate buffer three times, and then air-dry at room temperature.

[0185] (3) Use a Sartorius MA150 moisture analyzer to measure the moisture content of the immobilized enzyme. Under the condition that it drops to about 12%, pack and store it, and weigh it. The finished product weight of immobilized enzyme J is about 80 g, and the transesterification activity is 415 IUN / g.

[0186] Comparative Example 4: Preparation of Immobilized Enzyme K

[0187] (1) Weigh 400 g of RML lipase solution (10 mg / g, 60000 U / g) and 60 g of Tween 80 into a 2-L Erlenmeyer flask. Place the flask in a constant-temperature shaker at 10°C and 200 rpm and shake for 6 h, then measure the OD600 of the mixed emulsion.

[0188] (2) Under the condition that the OD600 reaches 30.4, add 200 g of ion exchange resin LX1000HAA to the mixed emulsion. Continue shaking for 24 h, then use a Buchner funnel to separate the solid-liquid mixture. Wash the upper solid with 100 mL of phosphate buffer three times, and then air-dry at room temperature.

[0189] (3) Use a Sartorius MA150 moisture analyzer to measure the moisture content of the immobilized enzyme. Under the condition that it drops to about 12%, pack and store it, and weigh it. The finished product weight of immobilized enzyme K is about 115 g, and the transesterification activity is 250 IUN / g.

[0190] Comparative Example 5: Preparation of Immobilized Enzyme L

[0191] (1) Set the cooling water temperature <15°C and pass cooling water through the jacket of an 800-L adsorption tank. Add 400 kg of RML lipase solution (10 mg / g, 60000 U / g) to the adsorption tank, then add 60 kg of Tween 80, and stir for 30 min at a stirring motor frequency of 20 Hz through a ribbon agitator. Measure the OD of the mixture to be approximately 25.1.

[0192] (2) After adding 200 kg of ion exchange resin LX1000HAA, continue stirring for 22 h, and use a bag - type centrifuge to separate the solid - liquid mixture. The solid material is added to a 420 L fluidized - bed dryer in batches for drying.

[0193] (3) Use a Sartorius MA150 moisture analyzer to measure the moisture content of the immobilized enzyme irregularly. Under the condition that the moisture content drops to about 12%, bag it for storage and weigh it. The finished product weight of immobilized enzyme L is about 85 kg, and the transesterification activity is 423 IUN / g.

[0194] Comparative Example 6: Preparation of immobilized enzyme B11

[0195] This example is a comparative example of Example 2. The main difference from Example 2 lies in step (1). Specifically, in step (1), gelatin is used instead of Tween 80. To compare with Example 2, when the OD600 is detected to be close to 44.4, increase the detection frequency. When OD600 reaches 44.4, carry out the next experimental operation.

[0196] The finished product weight of immobilized enzyme B11 is about 110 g, and the transesterification activity is 250 IUN / g.

[0197] Comparative Example 7: Preparation of immobilized enzyme B12

[0198] This example is a comparative example of Example 2. The main difference from Example 2 is that the immobilized enzyme is prepared by the traditional adsorption method, including the following steps:

[0199] (1) Weigh 200 g of ion exchange resin LX1000HAA and 60 g of Tween 80 into a container, and oscillate in a constant - temperature shaker at 18 °C and 150 rpm for 1 h, then air - dry at room temperature to obtain an activated carrier.

[0200] (2) Mix 400 g of RML lipase solution (10 mg / g, 60000 U / g) with the activated carrier, and continue oscillating until the change in the enzyme concentration in the resulting mixture is < 0.1 mg / g within 1 h. Then use a Buchner funnel to separate the solid - liquid mixture. Wash the upper - layer solid with 100 mL of phosphate buffer solution 3 times, and then air - dry at room temperature.

[0201] (3) Use a Sartorius MA150 moisture analyzer to measure the moisture content of the immobilized enzyme. Under the condition that the moisture content drops to about 12%, bag it for storage and weigh it. The finished product weight of immobilized enzyme B12 is about 110 g, and the transesterification activity is 210 IUN / g.

[0202] Comparative Example 8: Preparation of immobilized enzyme B13

[0203] This comparative example is the comparative example of Example 2, and the main differences from Example 2 are mainly in steps (1) and (2). Specifically: in step (1), the dosage of Tween 80 is 100 g; correspondingly, OD600 in step (2) reaches 65.

[0204] The finished product weight of the immobilized enzyme B13 is about 135 g, and the transesterification activity is 426 IUN / g.

[0205] Table 1. Comparison of immobilized enzyme preparation parameters, yields and activities

[0206]

[0207]

[0208] For Example 1 and Comparative Example 1, using the same preparation raw materials and controlling the OD600 of the mixture to be about 67.2, close to the preset value (0.37R - 1)(C - 0.4) (about 66.43), the finished product weight of the immobilized enzyme can be increased by 29%, and the enzyme activity of the immobilized enzyme is excellent.

[0209] For Example 2 and Comparative Example 2, using the same preparation raw materials, when the OD600 of the mixture deviates significantly from the preset value (0.37R - 1)(C - 0.4) (about 43.7), the finished product weight of the immobilized enzyme decreases by 26%, and the enzyme activity of the immobilized enzyme also decreases by 12%.

[0210] For Example 5 and Comparative Example 3, using the same preparation raw materials, when the OD600 of the mixture does not reach the preset value (0.37R - 1)(C - 0.4) and the deviation is about 10, the finished product weight of the immobilized enzyme decreases by 33%, and the enzyme activity of the immobilized enzyme also decreases by 8%.

[0211] For Example 2 and Comparative Example 4, using the same preparation raw materials, when the reaction time of the Tween 80 and enzyme solution mixture is too long, the OD600 of the mixture will also deviate from the preset value (0.37R - 1)(C - 0.4) (about 43.68) and drop to 30.4. Although the finished product weight of the immobilized enzyme only decreases by 10%, the enzyme activity of the immobilized enzyme drops by more than 40%.

[0212] For Example 7 and Comparative Example 5, using the same equipment and the same preparation raw materials, when the reaction of the Tween 80 and enzyme solution mixture is insufficient and the OD600 is only 25.1, significantly deviating from the preset value (0.37R - 1)(C - 0.4) (about 43.68), the finished product weight of the immobilized enzyme drops by 32%, and the enzyme activity of the immobilized enzyme also drops by 10%.

[0213] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity of description, not all possible combinations of the various technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0214] The above-described embodiments merely represent several implementation manners of the present application, which are convenient for understanding the technical solutions of the present application specifically and in detail. However, it should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning, or limited experiments based on the technical solutions provided in the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the content of the appended claims, and the specification and the drawings can be used to explain the content of the claims.

Claims

1. A method for preparing an immobilized enzyme, characterized in that, The preparation method includes the following steps: Mix the enzyme solution and Tween 80 to prepare a mixed emulsion; and, Using the adsorption method, prepare the mixed emulsion and the carrier into an immobilized enzyme; Wherein, At a detection wavelength of 600nm ± 3nm, the OD value of the mixed emulsion = preset value ± 6, and the preset value = (0.37R - 1) × (C - 0.4), R = Mt÷Mp, Mt is the mass value of the Tween 80, Mp is the mass value of the enzyme in the enzyme solution, and the unit of the mass of the Tween 80 is the same as the unit of the mass of the enzyme in the enzyme solution, C is the mass concentration value of the enzyme in the enzyme solution, and the unit of the mass concentration of the enzyme in the enzyme solution is expressed as a / b, where b = a×10 3 .

2. The preparation method of the immobilized enzyme according to claim 1, wherein The enzyme solution satisfies one or more of the following conditions: (1) The mass concentration of the enzyme in the enzyme solution does not exceed 25mg / g; optionally, the mass concentration of the enzyme in the enzyme solution is 5mg / g - 25mg / g; and, (2) The enzyme activity of the enzyme in the enzyme solution is 5500U / mg - 11000U / mg.

3. The preparation method of the immobilized enzyme according to claim 1, wherein The R ≤ 20; Optionally, the R is 3 - 20; Further optionally, the R is 3 - 15.

4. The preparation method of the immobilized enzyme according to claim 1, wherein, The dosage of the Tween 80 corresponding to every 100g of the enzyme solution does not exceed 25g; Optionally, the dosage of the Tween 80 corresponding to every 100g of the enzyme solution is 3.75g - 22.5g.

5. The preparation method of the immobilized enzyme according to any one of claims 1 to 4, characterized in that, The preparation method satisfies one or more of the following conditions: 1) The enzyme includes lipase; optionally, the microbial source of the lipase includes Thermomyces lanuginosus, Rhizopus oryzae, Rhizomucor miehei, Aspergillus oryzae, Pichia pastoris or their genetically modified strains; 2) The solvent of the enzyme solution includes a phosphate buffer solution with a concentration of 10mM - 100mM and a pH of 5.5 - 6.5; And, 3) The method of mixing the enzyme solution and Tween 80 includes shaking or stirring; Optionally, the conditions for shaking include: the temperature is 4℃ - 30℃, the rotation speed is 120rpm - 180rpm, and the time is 10min - 2h; Optionally, the conditions for stirring include: the frequency is 25Hz - 35Hz, and the time is 10min - 2h.

6. The method for preparing the immobilized enzyme according to any one of claims 1 to 4, characterized in that, The adsorption method includes physical adsorption method; Optionally, the carrier includes macroporous adsorption resin; Further optionally, the macroporous adsorption resin includes macroporous methacrylic resin or / and macroporous polystyrene resin.

7. The preparation method of the immobilized enzyme according to any one of claims 1 to 4, characterized in that, The adsorption method includes ion adsorption method; Optionally, the carrier includes ion exchange resin; Further optionally, the ion exchange resin includes methacrylic acid type anion exchange resin.

8. The preparation method of the immobilized enzyme according to any one of claims 1 to 4, characterized in that, The preparation method satisfies one or more of the following conditions: (A) The conditions of the adsorption method include: under the conditions of shaking or stirring until the change in the concentration of the enzyme in the mixture of the mixed emulsion and the carrier within 1h < 0.1mg / g; optionally, the conditions for shaking include: the rotation speed is 120rpm - 180rpm, and the time is 10min - 2h; optionally, the conditions for stirring include: the frequency is 25Hz - 35Hz, and the time is 10min - 2h; (B) The conditions of the adsorption method include: the adsorption temperature is 4℃ - 30℃; (C) The ratio of the mass of the carrier to the mass of the enzyme in the enzyme solution is (25 - 50):1; and, (D) The water content of the immobilized enzyme is 2 wt% - 20 wt%.

9. An immobilized enzyme prepared by the preparation method according to any one of claims 1 to 8.

10. A method for producing an enzyme-catalyzed product, characterized in that, The production method comprises the following steps: Preparing an immobilized enzyme by using the preparation method according to any one of claims 1 to 8; and Using the immobilized enzyme to catalyze a substrate to prepare an enzyme-catalyzed product.

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