CN110241109B

CN110241109B - Immobilized enzyme and preparation method thereof

Info

Publication number: CN110241109B
Application number: CN201810191556.1A
Authority: CN
Inventors: 毛相朝; 黄文灿; 薛长湖
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2018-03-08
Filing date: 2018-03-08
Publication date: 2023-05-16
Anticipated expiration: 2038-03-08
Also published as: CN110241109A

Abstract

The invention relates to an immobilized enzyme and a preparation method thereof, wherein chitin nanofiber is used as a carrier, and the enzyme is immobilized on the carrier through covalent connection; wherein the hydroxyl groups of the chitin nanofibers are oxidized to carboxylic acid groups for covalent attachment of enzymes to the chitin nanofibers. According to the invention, the hydroxyl groups of the chitin nanofibers are oxidized into carboxylic acid groups, so that enzymes can be covalently connected through the carboxylic acid groups, and are fixed on a carrier, the enzymes are further fixed by the aid of the cross-linking agent, and the two methods are matched, so that the fixed amount of the enzymes is further increased, the stability of the enzymes is improved, and the catalytic efficiency of the enzymes is also improved.

Description

Immobilized enzyme and preparation method thereof

Technical Field

The invention belongs to the field of nano materials, relates to an immobilized enzyme and a preparation method thereof, and in particular relates to an immobilized enzyme taking chitin as a carrier and a preparation method thereof.

Background

The enzyme is used as a biocatalyst, has wide research and application prospects in the fields of medicines, foods, chemical industry and the like, but the enzyme is unstable in the catalysis process, but the natural enzyme has poor stability, is easy to inactivate and can not be recovered, and the problems of mixing products after the reaction and the like limit the application of the enzyme. The immobilization of the enzyme is a technology of limiting enzyme molecules in a certain area by using a solid material, and the immobilized enzyme technology improves the stability of the enzyme, realizes the repeated use of the enzyme and reduces the cost.

Enzyme immobilization methods can be mainly classified into adsorption methods, covalent coupling methods, crosslinking methods, embedding methods, microcapsule methods, and the like, depending on the carrier, the method of operation, and the mechanism of immobilization. The adsorption method is a method for immobilizing the enzyme through the interaction of secondary bonds between the surface of the carrier and the surface of the enzyme, has simple operation and mild conditions, and can keep better enzyme activity, but the immobilized enzyme is easy to run off due to weaker physical adsorption acting force; the crosslinking method is a method of crosslinking a bridging immobilized enzyme between enzyme molecules by using a bifunctional or multifunctional reagent, and the immobilized enzyme is strong but the enzyme molecules are easily deactivated due to severe crosslinking conditions. More and more immobilization methods are not limited to single methods, but two or more immobilization methods are used in combination according to needs to achieve the purpose of preparing two immobilization materials.

Many different nanomaterials have been developed, including nanoparticles, nanotubes, nanofibers, and the like. The nanofiber has the advantages of high dispersibility in a reaction system, easiness in recovery, high surface area to volume ratio, excellent physical properties and the like, and therefore, the nanofiber is widely applied.

Chitin is a natural polymer compound which is present in the shells of aquatic crustaceans, such as shrimp and crab, and has very wide distribution. Chitin (chitin) is also called chitin, chitin and chitin, is a linear polysaccharide formed by connecting N-acetylglucosamine units through beta-1, 4-glycosidic bonds, is a white flaky solid, is nontoxic, tasteless, acid-base resistant, corrosion resistant, high-temperature resistant and sunlight resistant, and has very stable performance, thus far discovered as the only natural alkaline polysaccharide. It is present in large quantities in the crustaceans of lower animals, particularly arthropods, the second largest natural renewable resource in nature with reserves next to cellulose. Meanwhile, as glutaraldehyde has strong penetrability on substances, after the glutaraldehyde is used for treating the chitin, the surface of the chitin forms an organic porous structure, which is beneficial to the adsorption of enzymes. Glutaraldehyde is an active bifunctional reagent, which not only reacts with amino groups and carboxyl groups of enzyme proteins, but also activates amino groups of chitin. Furthermore, the chitin has the advantages of good biodegradability, reproducibility, sustainability and the like, so the chitin is a good enzyme immobilization carrier.

CN 101050457A discloses a method for preparing chitin-glutaraldehyde crosslinking adsorption immobilized chitosanase, which comprises the steps of refining chitin carrier, preparing crosslinking agent, crosslinking chitin-glutaraldehyde, extracting chitosanase, immobilizing, etc. CN 103966277A discloses a method for preparing phosphatidylserine by catalyzing immobilized phospholipase D, which uses chitin as a carrier, glutaraldehyde as a crosslinking reagent, and immobilizes free phospholipase D to obtain immobilized enzyme of phospholipase D. The method adopts glutaraldehyde crosslinking to fix the corresponding enzyme, has small fixed quantity and is unstable, and recycling can not be realized.

Therefore, it is important to provide a novel environment-friendly functionalized magnetic chitin nanofiber for immobilizing enzymes thereon.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention aims to provide an immobilized enzyme and a preparation method thereof, wherein the immobilized enzyme adopts chitin nanofiber as a carrier and is fixedly connected through covalent bonds, so that the enzyme is more stable to fix, and the fixing amount is increased.

To achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an immobilized enzyme, which uses chitin nanofibers as a carrier, and the enzyme is immobilized on the carrier through covalent linkage;

wherein the hydroxyl groups of the chitin nanofibers are oxidized to carboxylic acid groups for covalent attachment of enzymes to the chitin nanofibers.

In the invention, the hydroxyl of the chitin nanofiber is oxidized into the carboxylic acid group, so that the enzyme can be covalently connected through the carboxylic acid group, and thus, the enzyme is fixed on the carrier, the enzyme fixing amount is improved, the enzyme stability is improved, and the enzyme catalytic reaction efficiency is improved.

According to the present invention, the present inventors have found that oxidation of the primary hydroxyl group on the C6 of the chitin nanofiber to a carboxylic acid group is most easily achieved, and the best effect of connection with the enzyme is achieved, and the enzyme can be immobilized on the carrier to the maximum extent.

Preferably, the chitin nanofibres have a length of 30-300nm, which is determined by the pH of the reaction and the concentration of sodium hypochlorite, and may be, for example, 30nm, 32nm, 35nm, 38nm, 40nm, 42nm, 45nm, 48nm, 50nm, 51nm, 52nm, 53nm, 55nm, 56nm, 58nm, 60nm, 62nm, 65nm, 68nm, 70nm, 72nm, 75nm, 78nm, 80nm, 85nm, 90nm, 95nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 220nm, 250nm, 280nm or 300nm, preferably 50-200nm.

In the invention, the inventor finds that when the chitin nanofiber is too long, the dispersibility of the chitin nanofiber in an aqueous solution is poor, the subsequent catalytic reaction of enzyme is affected, and when the chitin nanofiber is too short, the enzyme amount on the chitin nanofiber is small, and the catalytic effect is poor.

Preferably, the enzyme is any one or a combination of at least two of chymotrypsin, alpha agarase, beta agarase, phospholipase D, lipase or astaxanthin esterase, and the combination may be, for example, a combination of chymotrypsin and alpha agarase, a combination of alpha agarase and beta agarase, a combination of phospholipase D and lipase, a combination of lipase and astaxanthin esterase, a combination of chymotrypsin, alpha agarase and beta agarase, a combination of chymotrypsin, alpha agarase, beta agarase and phospholipase D, a combination of alpha agarase, beta agarase, phospholipase D and lipase, a combination of chymotrypsin, alpha agarase, beta agarase, phospholipase D, lipase and astaxanthin esterase.

According to the invention, the carrier is further modified with an aminated magnetic nanomaterial.

Preferably, the magnetic nanomaterial is any one or a combination of at least two of ferroferric oxide, silicon oxide or aluminum oxide, preferably ferroferric oxide.

According to the invention, the immobilized enzyme can be reused by modifying the aminated magnetic nano material on the carrier, so that the enzyme can repeatedly react for a plurality of times, can be effectively separated from the product, and avoids the pollution of the enzyme to the product.

Preferably, the enzyme is further linked to the chitin nanofibril carrier by cross-linking, the cross-linking being achieved by a cross-linking agent, the cross-linking agent being glutaraldehyde.

In the invention, the enzyme is further immobilized through the cross-linking agent, and the two methods are matched, so that the immobilized amount of the enzyme is further increased, and the catalytic efficiency of the enzyme is improved.

In a second aspect, the present invention provides a method for preparing an immobilized enzyme as described in the first aspect, comprising the steps of:

(1) Preparing chitin nano-fiber, oxidizing hydroxyl of the chitin nano-fiber, and activating the chitin nano-fiber;

(2) And (3) adding enzyme to react with the chitin nanofiber after the activation in the step (1) to fix, thereby obtaining the immobilized enzyme.

According to the invention, step (1) comprises in particular the following steps:

(1') mixing chitin, TEMPO (tetramethyl piperidine oxynitride), sodium bromide and sodium hypochlorite, and stirring for reaction;

(2') centrifuging the reacted mixture, collecting precipitate, purifying and crushing;

(3') the crushed chitin nanofibers were activated by EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride)/NHS (N-hydroxysuccinimide) reaction.

According to the invention, the mass concentration of the chitin is 8-15g/L, for example, 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L or 15g/L, preferably 9-12g/L.

Preferably, the molar concentration of TEMPO is 0.5-3mM, e.g., 0.5mM, 0.6mM, 0.8mM, 1mM, 1.2mM, 1.3mM, 1.5mM, 1.8mM, 2mM, 2.1mM, 2.3mM, 2.5mM, 2.6mM, 2.8mM or 3mM, preferably 0.8-2mM.

Preferably, the molar concentration of sodium bromide is 5-15mM, for example, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM or 15mM, preferably 8-12mM.

Preferably, the molar concentration of sodium hypochlorite is 5-15mM, for example, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM or 15mM, preferably 8-12mM.

Preferably, the pH of the reaction is from 9 to 11, for example, it may be from 9, 9.2, 9.5, 9.8, 10, 10.1, 10.2, 10.5, 10.8 or 11, preferably from 9.5 to 10.5.

Preferably, the rotational speed of centrifugation in step (2') is 6000 to 15000rpm, which may be, for example, 6000rpm, 6200rpm, 6500rpm, 6800rpm, 7000rpm, 7200rpm, 7500rpm, 7800rpm, 8000rpm, 8200rpm, 8500rpm, 8800rpm, 9000rpm, 9200rpm, 9500rpm, 10000rpm, 11000rpm, 12000rpm, 13000rpm, 14000rpm or 15000rpm, preferably 8000 to 12000rpm.

Preferably, the centrifugation in step (2') is carried out for a period of time of 5 to 15min, for example, 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min or 15min, preferably 8 to 12min.

Preferably, the crushing in step (2 ') adopts ultrasonic crushing, and the power of the ultrasonic crushing in step (2') is 200-500W, for example, 200W, 210W, 220W, 230W, 240W, 250W, 260W, 270W, 280W, 300W, 320W, 350W, 360W, 380W, 400W, 420W, 450W, 460W, 480W or 500W, preferably 300-400W.

Preferably, the time of the ultrasonic disruption in the step (2') is 20-40min, for example, 20min, 21min, 23min, 25min, 26min, 28min, 30min, 32min, 33min, 34min, 35min, 36min, 38min or 40min, preferably 25-35min.

Preferably, the mass concentration of EDC is 5-15mg/mL, for example, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, 11mg/mL, 12mg/mL, 13mg/mL, 14mg/mL or 15mg/mL, and preferably 8-12mg/mL.

Preferably, the mass concentration of NHS is 40-60mg/mL, for example, 40mg/mL, 42mg/mL, 45mg/mL, 48mg/mL, 50mg/mL, 52mg/mL, 55mg/mL, 56mg/mL, 58mg/mL or 60mg/mL, preferably 45-55mg/mL.

Preferably, the temperature of the activation is 15-25deg.C, such as 15deg.C, 16deg.C, 17deg.C, 18deg.C, 19deg.C, 20deg.C, 21deg.C, 22deg.C, 23deg.C, 24deg.C or 25deg.C, preferably 18-23deg.C.

Preferably, the time of activation is 0.5 to 5 hours, for example, 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours, preferably 1 to 3 hours.

According to the invention, the step (2) is preceded by a step of magnetic modification, wherein the magnetic modification is carried out by adopting an aminated magnetic nanomaterial.

Preferably, the preparation method of the aminated magnetic nanomaterial comprises the following steps: preparing a magnetic nano material, and amination the magnetic nano material.

Preferably, the amination of the magnetic nanomaterial specifically comprises the following steps: adding an ethanol solution into the magnetic nano material reaction system, adjusting the pH, placing the mixture on a magnetic stirrer for stirring under the protection of protective gas, adding 3% volume of APTES (3-aminopropyl triethoxysilane), continuously stirring, performing magnetic separation, and cleaning to obtain the aminated magnetic nano material.

Preferably, the pH is 3-5, which may be, for example, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8 or 5, preferably 3.5-4.5.

Preferably, the temperature of the stirring on the magnetic stirrer is 60 to 80 ℃, for example, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃ or 80 ℃, preferably 65 to 75 ℃.

Preferably, the stirring time on the magnetic stirrer is 0.5-3h, for example, 0.5h, 0.6h, 0.8h, 0.9h, 1h, 1.2h, 1.3h, 1.5h, 1.6h, 1.8h, 2h, 2.2h, 2.3h, 2.5h, 2.8h or 3h, preferably 1-2h.

Preferably, the period of time during which the APTES is added and stirring is continued is 10 to 15 hours, for example, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours or 15 hours, preferably 11 to 13 hours.

According to the invention, the step (2) further comprises the step of crosslinking and fixing by using a crosslinking agent.

Preferably, the temperature of the crosslinking fixation is 15 to 25℃and may be, for example, 15℃16℃17℃18℃19℃20℃21℃22℃23℃24℃or 25℃and preferably 18 to 23 ℃.

Preferably, the cross-linked fixed pH is 4-6, which may be, for example, 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8 or 6, preferably 4.5-5.5.

As a preferred technical scheme, the preparation method of the immobilized enzyme comprises the following steps:

(1) Mixing chitin with mass concentration of 8-15g/L, molar concentration of 0.5-3mM TEMPO, molar concentration of 5-15mM sodium bromide and molar concentration of 5-15mM sodium hypochlorite, stirring for reaction, and regulating pH to be 9-11 all the time during the reaction;

(2) Centrifuging the reacted mixture at 6000-15000rpm for 5-15min, collecting precipitate, purifying, and performing ultrasonic crushing at 200-500W for 20-40min;

(3) Activating the crushed chitin nano fiber for 1-3h at the temperature of 15-25 ℃ through EDC with the mass concentration of 5-15mg/mL and NHS with the mass concentration of 40-60mg/mL;

(4) Amination is carried out on the magnetic nano material, and the aminated magnetic nano material is added into the activated chitin nano fiber to carry out magnetic modification;

wherein the amination comprises the following steps: adding an ethanol solution into the magnetic nano material reaction system, adjusting the pH to 3-5, placing the mixture on a magnetic stirrer under the protection of nitrogen, stirring the mixture at 60-80 ℃ for reaction for 0.5-3h, adding APTES, continuously stirring the mixture for reaction for 10-15h, and washing the mixture after magnetic separation to obtain the aminated magnetic nano material;

(5) Adding enzyme to react with the chitin nanofiber magnetically modified in the step (4) for fixation;

(6) Adding a cross-linking agent into the immobilized mixed solution, and carrying out cross-linking immobilization at the temperature of 15-25 ℃ and the pH value of 4-6 to obtain the immobilized enzyme.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) According to the invention, the hydroxyl groups of the chitin nanofibers are oxidized into carboxylic acid groups, so that enzymes can be covalently connected through the carboxylic acid groups and fixed on a carrier, the enzymes are further fixed by the aid of the cross-linking agent, and the two methods are matched, so that the fixed amount of the enzymes is further increased, the stability of the enzymes is improved, and the catalytic efficiency of the enzymes is also improved;

(2) According to the invention, the length of the chitin nanofiber is adjusted, and the magnetic nanomaterial is modified on the carrier, so that the enzyme can repeatedly react for a plurality of times, can be effectively separated from a reaction product, and avoids the pollution of the enzyme to the product;

(3) The method has simple process and easy operation, and the enzyme can be repeatedly utilized, so that the method can be used for industrialized mass production.

Detailed Description

To facilitate understanding of the present invention, examples are set forth below. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

Example 1

The embodiment provides a preparation method of immobilized enzyme, which comprises the following specific steps:

(1) Mixing chitin with the mass concentration of 8g/L, TEMPO with the molar concentration of 0.5mM, sodium bromide with the molar concentration of 5mM and sodium hypochlorite with the molar concentration of 5mM, stirring for reaction, and regulating the pH to be kept at 9 all the time during the reaction;

(2) Centrifuging the reacted mixture at 6000rpm for 15min, collecting precipitate, purifying, and performing ultrasonic crushing at 200W for 40min;

(3) Activating the crushed chitin nano fiber for 3 hours at 15 ℃ through EDC with the mass concentration of 5mg/mL and NHS with the mass concentration of 40 mg/mL;

wherein the length of the chitin nanofiber is 30-100nm;

(4) The magnetic nano material is aminated, and aminated ferroferric oxide is added into activated chitin nano fiber to carry out magnetic modification;

wherein the amination comprises the following steps: adding an ethanol solution into the magnetic nano material reaction system, adjusting the pH to 3, placing the mixture on a magnetic stirrer under the protection of nitrogen, stirring the mixture at 60 ℃ for reaction for 3 hours, adding 3mL of APTES, continuously stirring the mixture for reaction for 15 hours, and cleaning the mixture after magnetic separation to obtain the aminated magnetic nano material;

(5) And (3) adding chymotrypsin to react with the chitin nanofiber subjected to the magnetic modification in the step (4) for fixation.

Example 2

(1) Mixing chitin with the mass concentration of 15g/L, TEMPO with the molar concentration of 3mM, sodium bromide with the molar concentration of 15mM and sodium hypochlorite with the molar concentration of 15mM, stirring for reaction, and regulating the pH to be kept at 11 all the time during the reaction;

(2) Centrifuging the reacted mixture at 15000rpm for 5min, collecting precipitate, purifying, and performing ultrasonic crushing with power of 500W for 20min;

(3) Activating the crushed chitin nano fiber for 3 hours at the temperature of 25 ℃ through EDC with the mass concentration of 15mg/mL and NHS with the mass concentration of 60mg/mL;

wherein the length of the chitin nanofiber is 150-300nm;

wherein the amination comprises the following steps: adding an ethanol solution into the magnetic nano material reaction system, adjusting the pH to 5, placing the mixture on a magnetic stirrer under the protection of nitrogen, stirring the mixture at 80 ℃ for reaction for 0.5h, adding 3mL of APTES, continuously stirring the mixture for reaction for 10h, and cleaning the mixture after magnetic separation to obtain the aminated magnetic nano material;

(5) Adding chymotrypsin to react with the chitin nanofiber magnetically modified in the step (4) for fixation;

(6) And adding a cross-linking agent into the immobilized mixed solution, and carrying out cross-linking immobilization at the temperature of 25 ℃ and the pH value of 6 to obtain the immobilized enzyme.

Example 3

(1) Mixing chitin with the mass concentration of 9g/L, TEMPO with the molar concentration of 0.8mM, sodium bromide with the molar concentration of 8mM and sodium hypochlorite with the molar concentration of 12mM, stirring for reaction, and regulating the pH to be 10 all the time during the reaction;

(2) Centrifuging the reacted mixture at 12000rpm for 10min, collecting precipitate, purifying, and performing ultrasonic crushing at power of 300W for 25min;

(3) Activating the crushed chitin nano fiber for 3 hours at the temperature of 20 ℃ through EDC with the mass concentration of 12mg/mL and NHS with the mass concentration of 50 mg/mL;

wherein the length of the chitin nanofiber is 50-200nm;

wherein the amination comprises the following steps: adding an ethanol solution into the magnetic nano material reaction system, adjusting the pH to 4, placing the mixture on a magnetic stirrer under the protection of nitrogen, stirring the mixture at 70 ℃ for reaction for 2 hours, adding 3mL of APTES, continuously stirring the mixture for reaction for 12 hours, and cleaning the mixture after magnetic separation to obtain the aminated magnetic nano material;

(5) Adding alpha agarase to react with the chitin nanofiber magnetically modified in the step (4) for fixation.

Example 4

This example provides a method for preparing immobilized enzyme, which, compared with example 3, only comprises the step of crosslinking glutaraldehyde as a crosslinking agent, specifically comprising the following steps:

(2) Centrifuging the reacted mixture at 12000rpm for 10min, collecting precipitate, purifying, and performing ultrasonic crushing with power of 300W for 35min;

wherein the length of the chitin nanofiber is 100-300nm;

(5) Adding alpha agarase to react with the chitin nanofiber subjected to the magnetic modification in the step (4) for fixation;

Example 5

The embodiment provides a preparation method of immobilized enzyme, which specifically comprises the following steps:

(1) Mixing chitin with the mass concentration of 12g/L, TEMPO with the molar concentration of 2mM, sodium bromide with the molar concentration of 10mM and sodium hypochlorite with the molar concentration of 10mM, stirring for reaction, and regulating the pH to be 10 all the time during the reaction;

(2) Centrifuging the reacted mixture at 14000rpm for 8min, collecting precipitate, and performing ultrasonic crushing at 400W for 23min after purification;

(3) Activating the crushed chitin nano fiber for 3 hours at the temperature of 20 ℃ through EDC with the mass concentration of 10mg/mL and NHS with the mass concentration of 50 mg/mL;

wherein the length of the chitin nanofiber is 150-300nm;

(5) Adding phospholipase D to react with the chitin nanofiber magnetically modified in the step (4) for fixation;

(6) And adding a cross-linking agent into the immobilized mixed solution, and carrying out cross-linking immobilization at the temperature of 20 ℃ and the pH value of 5 to obtain the immobilized enzyme.

Example 6

(1) Mixing chitin with the mass concentration of 10g/L, TEMPO with the molar concentration of 1mM, sodium bromide with the molar concentration of 10mM and sodium hypochlorite with the molar concentration of 10mM, stirring for reaction, and regulating the pH to be 10 all the time during the reaction;

(2) Centrifuging the reacted mixture at 10000rpm for 10min, collecting precipitate, purifying, and performing ultrasonic crushing with power of 360W for 30min;

wherein the length of the chitin nanofiber is 50-200nm;

wherein the amination comprises the following steps: adding 100mL of 50% ethanol solution into the ferroferric oxide reaction system, adjusting the pH to 4, placing the ferroferric oxide reaction system on a magnetic stirrer under the protection of nitrogen, stirring and reacting for 1h at 70 ℃, adding 3mL of APTES, continuously stirring and reacting for 12h, and cleaning after magnetic separation to obtain the aminated magnetic nano material;

(5) Adding lipase to react with the chitin nanofiber magnetically modified in the step (4) for fixation;

(6) And adding glutaraldehyde as a crosslinking agent into the immobilized mixed solution, and performing crosslinking immobilization at the temperature of 20 ℃ and the pH value of 5 to obtain the immobilized enzyme.

Example 7

wherein the length of the chitin nanofiber is 50-200nm;

(5) Adding astaxanthin esterase to react with the chitin nanofiber magnetically modified in the step (4) for fixation;

Example 8

The length of the chitin nanofiber was 10-20nm compared with example 7, and otherwise the same as in example 7.

Example 9

The length of the chitin nanofiber was 300-500nm as compared with example 7, and otherwise the same as in example 7.

Example 10

In contrast to example 7, the magnetic nanomaterial was not modified on the support, and the other steps were the same as in example 7.

Example 11

The oxidation of the C3 primary hydroxyl group of the chitin nanofiber to a carboxylic acid group was performed in comparison with example 7, except that the same was performed in example 7.

Comparative example 1

In comparison with example 7, only chitin was used, the preparation of chitin fiber was not performed, oxidation was not performed, and glutaraldehyde as a crosslinking agent was directly used for crosslinking, otherwise the same as in example 7.

Enzyme activity detection

The results of the enzyme activity tests performed in comparative example 1 and examples 1 to 11 are shown in Table 1 below.

TABLE 1

	Enzyme immobilization amount (mg/g)	Enzyme activity
			Example 1	90	9000U
Example 2	320	25000U
			Example 3	120	4000U
Example 4	430	15000U
			Example 5	440	600U
Example 6	490	27000U
			Example 7	500	4800U
Example 8	60	2600U
			Example 9	450	-
Example 10	460	4300U
			Example 11	320	3200U
Comparative example 1	50	300U

From the comparison of examples 1-11 and comparative example 1, it can be seen that the method of the present application can be used for immobilization of chymotrypsin, alpha agarase, beta agarase, phospholipase D, lipase and astaxanthin esterase, and is equally effective for various enzymes, and from the comparison of examples 1-4, the enzyme immobilization is increased by further glutaraldehyde crosslinking, and covalent linkage and crosslinking are mutually promoted, so that the enzyme stability is increased and the enzyme activity is increased; as can be seen from comparison of examples 7 and examples 8 to 9, the too short loading amount of the chitin nanofiber is low, and the too long chitin nanofiber has poor dispersibility in the solution and cannot be catalyzed; as can be seen from comparison of examples 7 and examples 10 to 11, the immobilized enzyme cannot be reused without modifying the magnetic nanomaterial on the carrier, and the enzyme activity is also reduced, the chitin nanofiber is covalently connected on C3, the enzyme load is obviously reduced, and the enzyme activity is also obviously reduced.

As can be seen from the examples and comparative examples, the enzyme loading was only 50mg/g and the enzyme activity was also significantly reduced after crosslinking directly with glutaraldehyde without covalent bonding.

In summary, the hydroxyl groups of the chitin nanofibers are oxidized to carboxylic acid groups, so that enzymes can be covalently connected through the carboxylic acid groups and fixed on a carrier, the enzymes are further fixed by the aid of the crosslinking agent, and the two methods are matched, so that the fixed amount of the enzymes is further increased, the stability of the enzymes is improved, and the catalytic efficiency of the enzymes is also improved; moreover, by adjusting the length of the chitin nanofiber and modifying the magnetic nanomaterial on the carrier, the enzyme can repeatedly react for a plurality of times and can be effectively separated from a reaction product, so that the pollution of the enzyme to the product is avoided.

The applicant states that the invention is illustrated by the above examples for products and methods of preparation, but the invention is not limited to the above detailed products and methods of preparation, i.e. it is not meant that the invention must be practiced in dependence upon the above products and methods of preparation. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims

1. An immobilized enzyme is characterized in that chitin nanofibers are used as a carrier, and the enzyme is immobilized on the carrier through covalent connection;

wherein the hydroxyl groups of the chitin nanofibers are oxidized to carboxylic acid groups for covalent attachment of enzymes to the chitin nanofibers;

oxidizing a primary hydroxyl group on C6 of the chitin nanofiber into a carboxylic acid group;

the length of the chitin nanofiber is 30-300nm;

the carrier is also modified with an aminated magnetic nano material;

the magnetic nano material is any one or the combination of at least two of amino-functionalized ferroferric oxide, silicon oxide or aluminum oxide;

the enzyme is also connected with the chitin nanofiber carrier through a crosslinking effect;

the crosslinking is achieved by the crosslinking agent glutaraldehyde.

2. The immobilized enzyme according to claim 1, wherein,

the length of the chitin nanofiber is 50-200nm;

the magnetic nano material is ferroferric oxide.

3. The immobilized enzyme according to claim 1 or 2, wherein,

the enzyme is any one or the combination of at least two of chymotrypsin, alpha agarase, beta agarase, phospholipase D, lipase or astaxanthin esterase.

4. A method for preparing the immobilized enzyme of any one of claims 1 to 3, comprising the steps of:

(1) Preparing chitin nano-fiber, oxidizing hydroxyl, and performing activation and magnetic modification;

5. The method according to claim 4, wherein the step (1) comprises the steps of:

(1') mixing chitin, TEMPO, sodium bromide and sodium hypochlorite, and stirring for reaction;

(3') activating the crushed chitin nano fibers through EDC/NHS reaction.

6. The preparation method according to claim 5, wherein the mass concentration of the chitin is 8-15g/L;

the molar concentration of TEMPO is 0.5-3mM;

the molar concentration of the sodium bromide is 5-15mM;

the molar concentration of the sodium hypochlorite is 5-15mM;

the pH of the reaction is 10-11.

7. The preparation method according to claim 6, wherein the mass concentration of the chitin is 9-12g/L;

the molar concentration of TEMPO is 0.8-2mM;

the molar concentration of the sodium bromide is 8-12mM;

the molar concentration of the sodium hypochlorite is 8-12mM;

the pH of the reaction is 10-10.5.

8. The method according to claim 6, wherein the rotational speed of centrifugation in step (2') is 6000 to 15000rpm;

the centrifugation time in the step (2') is 5-15min;

the crushing in the step (2') adopts ultrasonic crushing;

the power of the ultrasonic crushing in the step (2') is 200-500W;

the ultrasonic crushing time in the step (2') is 20-40min;

the mass concentration of the EDC is 5-15mg/mL;

the mass concentration of NHS is 40-60mg/mL;

the activation temperature is 15-25 ℃;

the activation time is 0.5-5h.

9. The method according to claim 8, wherein the rotational speed of centrifugation in step (2') is 8000 to 12000rpm;

the centrifugation time in the step (2') is 8-12min;

the power of the ultrasonic crushing in the step (2') is 300-400W;

the ultrasonic crushing time in the step (2') is 25-35min;

the mass concentration of the EDC is 8-12mg/mL;

the mass concentration of NHS is 45-55mg/mL;

the activation temperature is 18-23 ℃;

the activation time is 1-3h.

10. The method of any one of claims 4-9, further comprising a step of magnetic modification prior to step (2);

the magnetic modification is carried out by adopting an aminated magnetic nano material;

the preparation method of the aminated magnetic nano material comprises the following steps: preparing a magnetic nanomaterial, and amination the magnetic nanomaterial;

the amination of the magnetic nanomaterial specifically comprises the following steps: adding an ethanol solution into the magnetic nano material reaction system, adjusting the pH, placing the mixture on a magnetic stirrer for stirring under the protection of protective gas, adding APTES, continuing stirring, and cleaning after magnetic separation to obtain the aminated magnetic nano material;

the pH is 3-5;

the temperature of the stirring on the magnetic stirrer is 60-80 ℃;

the stirring time on the magnetic stirrer is 0.5-3h;

and the continuous stirring time of adding APTES is 10-15h.

11. The method of claim 10, wherein the pH is 3.5-4.5;

the temperature of the stirring on the magnetic stirrer is 65-75 ℃;

the stirring time on the magnetic stirrer is 1-2h;

the period of adding APTES and continuing stirring is 11-13h.

12. The method according to any one of claims 4 to 9, wherein the step (2) further comprises a step of crosslinking fixation with a crosslinking agent;

the temperature for crosslinking and fixing is 15-25 ℃;

the pH of the crosslinking fixation is 4-6.

13. The method according to claim 12, wherein,

the temperature for crosslinking and fixing is 18-23 ℃;

the pH of the crosslinking fixation is 4.5-5.5.

14. The preparation method according to any one of claims 4 to 9, comprising the steps of: