Preparation and application of immobilized hydantoinase

Abstract

The invention discloses immobilized hydantoin enzyme. The immobilized hydantoin enzyme comprises an immobilized carrier, a cross-linking agent and recombinant hydantoin enzyme, wherein the recombinant hydantoin enzyme is connected with the immobilized carrier through the cross-linking agent by a chemical bond. In addition, the immobilized hydantoin enzyme is cross-linked again through a bifunctional cross-linking agent, and the 3-isobutyl glutarimide can be converted into the (R)-(-)-3-(carbamyl methyl)-5-methylhexanoic acid by the re-cross-linked immobilized enzyme with higher stability.

Description

Technical Field

[0001] The present application relates to the field of bioengineering technology, and in particular to the preparation of an immobilized hydantoinase and its application in the catalytic synthesis of a pregabalin intermediate. Background Art

[0002] (R)-(-)-3-(Carbamoylmethyl)-5-methylhexanoic acid, also known as pregabalin intermediate, is an important raw material in the synthesis of pregabalin. There are two methods for synthesizing pregabalin intermediates: chemical method and enzymatic method. Among them, the pregabalin intermediate synthesized by chemical method is a mixture of two chiral configurations, R and S, and needs to be purified by a series of splitting methods. Enzymatic synthesis is widely known for its high chiral selectivity to synthesize target products, but it is usually difficult to be widely used due to the limitations of enzyme use conditions, especially the stability of the enzyme itself. Summary of the invention

[0003] In order to solve the problem of poor enzyme stability in the prior art, the present application prepares an immobilized hydantoinase, wherein the immobilized hydantoinase comprises an immobilized carrier, a cross-linking agent, and a recombinant hydantoinase chemically bonded to the immobilized carrier through the cross-linking agent. The immobilized hydantoinase described in the present application can convert 3-isobutylglutarimide into (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid under appropriate reaction conditions.

[0004] Furthermore, the immobilized hydantoinase described in the present application includes re-crosslinking of the immobilized enzyme, which is achieved by mixing and contacting the immobilized hydantoinase with a bifunctional crosslinker. The re-crosslinked immobilized enzyme can convert 3-isobutylglutarimide into (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid with higher stability, wherein the immobilized recombinant hydantoinase connects different monomeric hydantoinases with chemical covalent bonds through the bifunctional crosslinker, and the re-crosslinked immobilized hydantoinase can be stably recycled for at least 14 times in a reaction process with a 3-isobutylglutarimide loading of 200 g / L, and the time for each reaction to be completed is 9-20 hours.

[0005] The immobilized hydantoinase described in the present application includes a recombinant hydantoinase capable of converting 3-isobutylglutarimide into (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, a cross-linking agent and an immobilization carrier, wherein the recombinant hydantoinase forms a chemical covalent bond with the immobilization carrier through the cross-linking agent. In certain embodiments, the surface lysine side chains of the recombinant hydantoinase protein structure are chemically covalently bonded to the immobilization carrier through a cross-linking agent such as glutaraldehyde.

[0006] Optionally, the immobilized hydantoinase is cross-linked again by a bifunctional cross-linking agent, and the immobilized hydantoinase can be stored at 4° C. for at least one year with an activity loss of <10%.

[0007] In certain embodiments, the recombinant hydantoinase is selected from SEQ ID NOs: 1-10.

[0008] In some embodiments, the immobilization carrier comprises a resin. In some embodiments, the immobilization carrier is synthesized from methyl acrylate, wherein the monomer is methyl acrylate. In some embodiments, the immobilization carrier is LX-1000HA commercially available from Xi'an Lanxiao Technology New Materials Co., Ltd.

[0009] Secondly, the present application provides a method for preparing the immobilized hydantoinase, comprising the following steps:

[0010] 1) Enzyme solution preparation

[0011] 2) Cross-linker modification of immobilized carrier

[0012] 3) Enzyme immobilization

[0013] 4) Cross-linking of immobilized enzyme

[0014] 5) Immobilized enzyme cleaning

[0015] Finally, the present application also provides the application of the immobilized hydantoinase. The immobilized hydantoinase described in the present application can be used for batch reaction (or intermittent reaction), wherein the immobilized hydantoinase can be filtered out after the reaction is completed and used for the next reaction. Alternatively, the immobilized hydantoinase described in the present application can be used for continuous reaction, wherein the reaction substrate continuously passes through the immobilized hydantoinase and the product is separated.

[0016] The method for producing (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid described in the present application comprises the following steps:

[0017] 1) dissolving 3-isobutylglutarimide in a reaction system;

[0018] 2) adding the immobilized hydantoinase into a reaction system containing 3-isobutylglutarimide. The immobilized hydantoinase can convert 3-isobutylglutarimide into (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid under appropriate reaction conditions. DETAILED DESCRIPTION OF THE INVENTION

[0020] definition

[0021] Regarding the present disclosure, unless specifically defined otherwise, technical and scientific terms used in the specification herein have the meanings that are commonly understood by one of ordinary skill in the art.

[0022] "Hydantoinase" is used in this application to refer to a polypeptide having the enzymatic ability to selectively convert a cyclic hydantoin compound (1) into an amino acid derivative (2) or (3) of high chiral purity:

[0023]

[0024] Regarding the immobilized hydantoinase described in the present application, the hydantoinase polypeptide is capable of converting (1a) 3-isobutylglutarimide (substrate) to (2a) (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid (product) under appropriate reaction conditions, such as Figure 1 shown.

[0025] "Protein," "enzyme," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). This definition includes both D- and L-amino acids, as well as mixtures of D- and L-amino acids.

[0026] "Amino acid difference" or "residue difference" refers to the difference in amino acid residues at one position of a polypeptide sequence relative to the amino acid residues at the corresponding position in a reference sequence. The position of the amino acid difference is generally referred to as "Xn" herein, where n refers to the corresponding position in the reference sequence on which the residue difference is based. In most examples herein, a specific amino acid residue difference at one position is represented as "XnY", where "Xn" refers to the corresponding position as described above, and "Y" is a one-letter identifier for the amino acid found in the engineered polypeptide (i.e., a different residue from the reference polypeptide). The present disclosure also provides specific amino acid differences represented by the conventional symbol "AnB", where A is a one-letter identifier for a residue in a reference sequence, "n" is the number of the residue position in the reference sequence, and B is a single-letter identifier for a residue substitution in the sequence of the engineered polypeptide. In some examples, a polypeptide of the present disclosure may include one or more amino acid residue differences relative to a reference sequence, which is represented by a list of specific positions at which residue differences exist relative to the reference sequence.

[0027] "Stereoselectivity" refers to the preferential formation of one stereoisomer over one or more other isomers in a chemical or enzymatic reaction. Stereoselectivity can be partial, in which one stereoisomer is formed in preference to the other, or complete, in which only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is called enantioselectivity, and the fractional excess of one enantiomer in a mixture of two enantiomers (usually reported as a percentage) is often alternatively reported as "enantiomeric excess" (ee). When the stereoisomers are diastereoisomers, the stereoselectivity is called diastereoselectivity, that is, the excess fraction (usually reported as a percentage) of one diastereomer in a mixture of two diastereomers, often alternatively reported as "diastereoisomeric excess" (de). In the art, this fraction (typically a percentage) is often alternatively reported as enantiomeric excess (ee) calculated therefrom according to the following formula: {major enantiomer concentration - minor enantiomer concentration} / {major enantiomer concentration + minor enantiomer concentration}.

[0028] "Stereoisomers", "stereoisomeric forms" and similar expressions are used interchangeably herein and refer to all isomers of molecules that differ only in the orientation of their atoms in space. This includes enantiomers and isomers of compounds with more than one chiral center that are not mirror images of each other (i.e., "diastereomers").

[0029] "Reaction yield" refers to the molar percentage of the product generated in the reaction system to the initial feed substrate under specified reaction conditions and within a specified reaction time. Therefore, the "enzyme activity" or "activity" of hydantoin or engineered polypeptides can be expressed as "reaction yield". The reaction is generally calculated by sampling to determine the molar concentration of the product in the reaction system and the molar concentration of the initial feed substrate: [molar concentration of product] / [molar concentration of the initial feed substrate].

[0030] "Stable" refers to the ability of the immobilized hydantoinase described in the present application to maintain their structural conformation and / or activity after multiple cycles of use under high substrate concentration reaction conditions. In certain embodiments, the stable immobilized hydantoinase refers to a loss of up to 10% of activity after being used once in a high substrate concentration cycle. Preferably, the stable immobilized hydantoinase described in the present application refers to a loss of up to 9% of activity after being used once in a high substrate concentration cycle. Preferably, the stable immobilized hydantoinase described in the present application refers to a loss of up to 8% of activity after being used once in a high substrate concentration cycle. Preferably, the stable immobilized hydantoinase described in the present application refers to a loss of up to 7% of activity after being used once in a high substrate concentration cycle. Preferably, the stable immobilized hydantoinase described in the present application refers to a loss of up to 6% of activity after being used once in a high substrate concentration cycle. Preferably, the stable immobilized hydantoinase described in the present application refers to a loss of up to 5% of activity after being used once in a high substrate concentration cycle. Preferably, the stable immobilized hydantoinase described in the present application refers to a loss of up to 4% of activity after being used once in a high substrate concentration cycle. Preferably, the stable immobilized hydantoinase described in the present application means that it loses at most 3% of its activity after being recycled once at a high substrate concentration. Preferably, the stable immobilized hydantoinase described in the present application means that it loses at most 2% of its activity after being recycled once at a high substrate concentration. Preferably, the stable immobilized hydantoinase described in the present application means that it loses at most 1% of its activity after being recycled once at a high substrate concentration.

[0031] "Suitable reaction conditions" refer to ranges of, for example, enzyme loading, substrate loading, cofactor loading, temperature, pH, buffer, cosolvents, etc., in a biocatalytic reaction solution under which the engineered polypeptide of the present application can convert a substrate into a desired product compound. Exemplary "suitable reaction conditions" are provided in this disclosure and exemplified by the Examples. Compounds may be identified by their chemical structure and / or chemical name. When the chemical structure conflicts with the chemical name, the chemical structure determines the identity of the compound.

[0032] "Crosslinking" refers to the chemical bonding and molecular connection caused by the interaction between the active groups of molecules. In the present application, it refers to the mutual bonding between enzyme-enzyme and enzyme-carrier to form a network structure through a crosslinking agent to improve the performance of immobilized enzyme strength, heat resistance, solvent resistance, etc. The crosslinking described in the present application can be crosslinking between enzyme and carrier to fix the enzyme on the carrier, or crosslinking between enzymes fixed on the carrier to improve stability, or crosslinking between each monomer of polymeric enzyme to improve stability, such as forming chemical covalent bonds by glutaraldehyde between subunits inside the immobilized hydantoin enzyme molecule and between tetramers outside the molecule.

[0033] Hydantoin

[0034] Hydantoinase is a metalloenzyme that catalyzes the hydrolysis of cyclic hydantoin compounds into D-amino acids or L-amino acid intermediate compounds. These chiral intermediates catalyzed by hydantoinase are widely used in semi-synthetic antibiotics, peptide hormones, pyrethroids and pesticides. Examples of hydantoinase-catalyzed reactions are as follows:

[0035]

[0036] When R is a hydrogen atom, compound (1) is not chiral. When R is not a hydrogen atom, compound (1) has a chiral structure. At this time, it is a mixed state of two enantiomeric compounds. Hydantoinase will hydrolyze one of the chiral compounds with high stereoselectivity to generate a single chiral product (2) or (3).

[0037] The immobilized hydantoinase described in the present application comprises a recombinant hydantoinase, wherein the recombinant hydantoinase is capable of converting 3-isobutylglutarimide (substrate) into (2a)(R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid (product).

[0038]

[0039] In certain embodiments, the hydantoinase described herein is capable of converting (1a) into (2a) under appropriate reaction conditions, and the hydantoinase comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1-10.

[0040] In certain embodiments, at least one of the amino acids at residue positions n=407 and n=478 of the hydantoinase is lysine.

[0041] In certain embodiments, at least one of the amino acids at the two positions n=407 and n=478 of the hydantoinase corresponding to the residue positions of SEQ ID NO: 1-10 is lysine, and the spatial positions of K407 and K478 are as follows: Figure 2 As shown, the common feature of these two sites is that they are far away from the active pocket and on the protein surface.

[0042] Immobilized carrier

[0043] The immobilized hydantoin enzyme described in the present application comprises the immobilized hydantoin enzyme connected with the immobilized carrier by glutaraldehyde with chemical covalent bond, wherein the material of the immobilized carrier can include biological, non-biological, organic, inorganic or any combination of these. For example, the material of the immobilized carrier can be any one of many kinds of gels or polymers, such as (poly) tetrafluoroethylene, (poly) vinylidene fluoride, polystyrene, cross-linked polystyrene, polyacrylic acid, polylactic acid, polyglycolic acid, poly (lactide-co-glycolide), polyanhydride, poly (methyl methacrylate), ethylene-vinyl acetate copolymer, polysiloxane, polymerized silica, latex, dextran polymer, epoxy resin, polycarbonate or its combination. It can also be an inorganic carrier material, mesoporous material, non-metallic mineral, metal oxide and salt, nanotube etc. It can also be a fiber material, natural polysaccharide etc.

[0044] The material of the immobilization support can be taken in any form or shape, and is generally a plate, bead, pill, disk, particle, wire, precipitate, membrane, optionally a porous gel. It may contain raised or recessed or cracked areas, where the capture probe is located. The immobilization support can be etched using well-known techniques to provide desired surface features, such as grooves, v-grooves, terrace structures, etc. to the surface of the material of the immobilization support.

[0045] About the immobilized hydantoinase of being connected to hydantoinase by chemical covalent bond with immobilized carrier described in the present application by crosslinking agent, wherein immobilized carrier is resin.Described resin comprises styrene / divinylbenzene (DVB) and polymethacrylate copolymer with epoxy functional group, styrene / divinylbenzene (DVB) and polymethacrylate copolymer with aminoepoxy functional group, styrene / divinylbenzene (DVB) and polymethacrylate copolymer with octadecyl functional group.Exemplary immobilized carrier includes but is not limited to LX-1000EP, LX-1000EPF, LX-1000EPB, LX-103B, EP200, LX-1000HA (A), SEPABEAD:EC-EP, EC-HFA / S, EXA252, EXE119 and EXE120.

[0046] In certain embodiments of the immobilized hydantoinase described herein, the resin comprises methyl acrylate. Examples of suitable resins include, but are not limited to, LX-1000HA.

[0047] In certain embodiments of the immobilized hydantoinase described in the invention, the immobilized hydantoinase comprises a resin chemically covalently linked to the hydantoinase via a cross-linking agent. Suitable resins include methyl acrylate modified with a short chain (C2-C6) amino group. Examples include, but are not limited to, LX-1000HA.

[0048] In certain embodiments of the immobilized hydantoinase described in the present application, the immobilized hydantoinase comprises a cross-linking agent that connects the resin LX-1000HA and the hydantoinase with a chemical covalent bond. Suitable cross-linking agents include soluble dialdehyde compounds, NHS esters, imidates, hydroxymethylphosphine, etc. Examples include, but are not limited to, glutaraldehyde.

[0049] In certain embodiments of the immobilized hydantoinase described in the invention, the immobilized hydantoinase comprises hydantoinase SEQ ID NO: 1-10, wherein at least one of the amino acids at residue positions n=407 and n=478 is lysine, which is chemically covalently bonded to the resin LX-1000HA via a crosslinking agent glutaraldehyde.

[0050] Preparation of immobilized hydantoinase

[0051] The present application also provides a method for preparing immobilized hydantoinase. In certain embodiments of the method for preparing immobilized hydantoinase described in the present application, the method begins by dissolving hydantoinase in water or a buffer to prepare an enzyme solution.

[0052] In certain embodiments of the method for preparing immobilized hydantoinase, the pH of the hydantoinase solution is pH 5.0-9.0. In certain embodiments, the reaction conditions of the method are pH about 7.0-about 8.5. In other embodiments, the reaction conditions of the method are pH about 8.0.

[0053] The method also includes cross-linking agent modification of the immobilized carrier. This can be achieved by adding the immobilized carrier resin and the cross-linking agent to a buffer. The solution is then shaken for a certain period of time, such as shaking on a shaker for 2 hours. After removing the liquid, the solution is repeatedly washed with clean water several times.

[0054] The method further comprises mixing and contacting the hydantoinase and the immobilized support modified by the crosslinking agent. This can be achieved by adding the modified resin to the enzyme solution. The mixture is then shaken for a certain period of time, such as shaking on a shaking table for 20-24 hours.

[0055] In certain embodiments, the reaction conditions for carrying out the method may include a temperature of about 5° C. to about 70° C. In certain embodiments, the reaction conditions are 40° C.

[0056] The method also includes a process of re-crosslinking the immobilized enzyme, which is achieved by mixing and contacting the hydantoinase already fixed on the carrier with a bifunctional crosslinking agent. The crosslinking agent includes a soluble dialdehyde compound, NHS ester, imidate, hydroxymethylphosphine, etc. In certain embodiments, the immobilized hydantoinase is added to a 0.001-5% preferably 0.008%-0.8% glutaraldehyde solution. In certain embodiments, the process is carried out at a temperature of 10-50° C., and the shaking or stirring lasts for 1-24 hours.

[0057] The method further comprises washing the re-crosslinked immobilized hydantoinase prepared in the solution. The re-crosslinked immobilized hydantoinase can be filtered to remove the liquid, and then washed with phosphate buffer, and this process needs to be repeated several times to remove some proteins that are easy to fall off.

[0058] Application of immobilized hydantoinase

[0059] Suitable reaction conditions include a combination of reaction parameters that catalyze the conversion of a substrate compound into its corresponding product compound. Accordingly, in some embodiments of the method, the combination of reaction parameters includes: (a) a substrate loading of about 10 g / L to 200 g / L; (b) an immobilized hydantoinase loading of about 1 g / L to 50 g / L; (c) a pH of about 5.0 to 9.0; (d) a temperature of about 10° C. to 65° C. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 . Preparation of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid by asymmetric hydrolysis of 3-isobutylglutarimide catalyzed by hydantoinase

[0061] Figure 2 . Spatial distribution of amino acids K407 and K478 in protein structure

[0062] Figure 3 .Stability of immobilized hydantoinase in high salt concentration

[0063] Figure 4 .Protein shedding of immobilized hydantoinase after high concentration salt treatment

[0064] Figure 5 .Reaction results of hydantoinase immobilized by cross-linking with glutaraldehyde at different concentrations (GD is the abbreviation of glutaraldehyde) Specific embodiments

[0065] Example 1 Expression of hydantoin polypeptide

[0066] A single microbial colony of Escherichia coli BL21 (DE3) containing a plasmid expressing the hydantoin polypeptide was inoculated into a 250 mL conical flask containing 50 mL LB medium (containing 30 μg / mL chloramphenicol) and cultured overnight in a shaking incubator at 30°C. When the OD of the culture solution reached 600 When the OD value of the culture medium reached 2, the inoculum was inoculated into a 1000 mL conical flask containing 250 mL TB medium at a volume of 5% (v / v) and placed in a shaking incubator at 30°C for culture. 600When the pH reaches 0.6, IPTG with a final concentration of 1 mM is added to induce the expression of hydantoin. After culturing for 20 hours, the culture solution is centrifuged (8000 rpm, 10 minutes), the supernatant is discarded after centrifugation, and the cells are collected to obtain wet cells. The wet cells are directly used to prepare the enzyme solution, or can be frozen and stored at -20°C until use.

[0067] Example 2 Preparation of immobilized enzyme

[0068] Weigh the required mass of wet cells containing SEQ ID NO: 1-10, resuspend the wet cells in 5 volumes of phosphate buffer (0.02M, pH 8.0) and mix thoroughly, use a homogenizer 850Bar to homogenize twice at low temperature (cooling water 10-15°C), put the homogenized enzyme solution into a glass container, place it in a preheated 50°C water bath, mechanically stir at 200rpm, start timing after the enzyme solution temperature rises to 50°C, and heat-treat in a water bath for 4h. Cool the enzyme solution to less than 20°C in an ice water bath, centrifuge the enzyme solution at 8000rpm for 20min, and take the supernatant to obtain the enzyme solution.

[0069] Weigh 10g of LX-1000HA immobilized carrier, add 30mL of phosphate buffer (0.02M, pH 7.4) to completely immerse the carrier, add 1mL of 50% glutaraldehyde, and place on a shaker at 37°C and 200rpm for 2 hours. After sufficient reaction, remove the liquid by suction with a double-layer neutral filter paper. Then add 30-50mL of pure water, shake on a shaker at 37°C and 200rpm for 20min, remove the liquid by suction, and repeat the washing 3 times to obtain the modified LX-1000HA immobilized carrier.

[0070] Take 5 g of the modified LX-1000HA immobilized carrier into a 50 mL centrifuge tube, add 25 mL of the prepared enzyme solution, put the centrifuge tube into a shaker at 20°C and 250 rpm to react for 20-24 hours, and after sufficient reaction, the immobilized hydantoinase crude product is obtained.

[0071] The prepared immobilized hydantoinase crude product was taken out, and the excess liquid was removed by filtration. Then 25 mL of phosphate buffer (0.02 M, pH 8.0, 1 M NaCl) was added, and the mixture was shaken in a shaker at 30° C. and 250 rpm for 30 min. The liquid was removed by filtration, but the immobilized hydantoinase was not filtered to complete dryness. The washing was repeated for more than 3 times.

[0072] The immobilized hydantoinase crude product after washing was taken out, and 25 mL of phosphate buffer (0.02 M, pH 8.0) was added, and 4 μL of 50% glutaraldehyde was added for re-crosslinking, and the product was placed in a shaker at 30° C. and 250 rpm for 20-24 h. The liquid was removed by filtration, but the immobilized hydantoinase was not filtered to complete dryness, and then the washing was repeated 3 times to obtain the immobilized enzyme described in the present application.

[0073] Example 3 Enzyme liquid reaction

[0074] In a reaction bottle with a total volume of 250mL, 6mL of enzyme solution containing SEQ ID NO:9 was added, 24g of 3-(2-methylpropyl)glutarimide was added, and finally 114mL of phosphate buffer (0.05M, pH 7.6) was added to make the concentration of each component in the reaction system 5% (v / v) enzyme solution, 200g / L of 3-(2-methylpropyl)glutarimide. The reaction bottle was placed in a 40°C water bath, and the mechanical stirring paddle speed was set to 300rpm to start the reaction. During the reaction, the generation of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid gradually reduced the pH of the reaction system, so 10-15% ammonia water was continuously added to maintain a constant pH of 8.0±0.2 until the reaction was completed. After 10.5h of reaction, ammonia water was no longer added and the reaction was completed. 1 mL of sample was taken and 9 mL of acetonitrile was added for quenching for 30 min. After centrifugation (13000 rpm, 3 min), the supernatant was diluted with 50% acetonitrile and the sample was tested by HPLC. The conversion rate was 98.4% and the product ee was 99.8%.

[0075] Example 4 Immobilized enzyme reaction

[0076] 24g 3-(2-methylpropyl)glutarimide was added to a three-necked flask with a container volume of 250mL, 114mL phosphate buffer (0.05M, pH 7.6) was added to make the final substrate concentration 200g / L, and then 6g SEQ ID NO:9 immobilized hydantoinase was added, and the reaction system was placed at 40°C, and the reaction continued for 8-24h under the conditions of 450rpm. During the reaction, the generation of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid gradually reduced the pH of the reaction system, so 10-25% ammonia was used to control the reaction pH between 7.9-8.1 in real time until the reaction was completed. 1mL of the sample was taken, 9mL of acetonitrile was added for quenching, and the quenching time was 30min. After centrifugation (13000rpm, 3min), the supernatant was diluted with 50% acetonitrile, and the sample was detected by HPLC, and the conversion rate was 97.5%, and the product ee was 99.8%.

[0077] Example 5 Recovery and application of immobilized hydantoinase

[0078] After the reaction of Example 4 was completed, the reaction liquid was removed by suction filtration, and the immobilized hydantoinase was collected. The collected immobilized hydantoinase was rinsed with 0.01-0.1 M phosphate buffer (pH 6.5-8.0).

[0079] 24g 3-(2-methylpropyl)glutarimide was added to a clean reaction bottle, and 114mL phosphate buffer (0.05M, pH 7.6) was added to make the final substrate concentration 200g / L, and then the recovered immobilized hydantoinase was added to the reaction system, and the reaction system was placed at 40°C and 450rpm for continuous reaction for 8-24h. During the reaction, the generation of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid gradually reduced the pH of the reaction system, so 10-25% ammonia water was used to control the reaction pH in real time between 7.9-8.1. Until the reaction was completed.

[0080]

[0081] Reaction time: The reaction time when the conversion rate exceeds 98%

[0082] Repeating the above steps is the cyclic use of the immobilized hydantoinase. In this embodiment, the immobilized hydantoinase reaction was repeated 23 times, and the enzyme activity was retained> 60%. The details are shown in Table 1.

[0083] Table 1. Results of the cyclic reaction of immobilized hydantoinase

[0084]

[0085] Note: Considering the industrial production cost, the immobilized hydantoinase will not be used after the reaction time exceeds 24h. There is a certain error in the reaction time.

[0086] Therefore, the calculated enzyme activity retention allows a certain range of error. The re-crosslinked immobilized enzyme can actually continue to be recycled, but in this experiment

[0087] Terminate after 23 cycles.

[0088] Example 6 Stability test of re-crosslinked immobilized hydantoinase in high salt concentration environment

[0089] The immobilized hydantoinase of SEQ ID NO: 9 described in the present application was placed in a 1.6M sodium chloride, 1.0-2.0M (R)-(-)-3-(carbamoylmethyl)-5-methylacetic acid ammonium salt system for stability testing. Compared with the immobilized hydantoinase prepared by the non-recrosslinking method ( Figure 3 Control), immobilized hydantoinase prepared by the re-crosslinking method of the present application ( Figure 3 Cross-linking) has higher activity and stability, especially after multiple cycles of use, the immobilized hydantoinase described in the present application can better maintain its catalytic activity.

[0090] Example 7 Detection of protein shedding in high-concentration salt environment of re-crosslinked immobilized hydantoinase

[0091] The immobilized hydantoinase of SEQ ID NO: 9 described in the present application was placed in a system of 1.6 M sodium chloride and 1.0-2.0 M (R)-(-)-3-(carbamoylmethyl)-5-methylacetic acid ammonium salt (ie, product salt) for protein shedding detection.

[0092] Weigh 3 portions of 0.1g of re-crosslinked immobilized hydantoinase and control immobilized hydantoinase, add 1mL of 100g / L, 160g / L, and 200g / L product salt solution to EP tubes, incubate in a shaker at 40℃, 200rpm for 24h, centrifuge at 5000rpm for 5min, then take 20μL of the centrifuged supernatant solution, add 5μL of 5× loading buffer, denature at 100℃ for 10min. Take 5μL and load SDS-PAGE precast gel, electrophoresis conditions 200V, 45min.

[0093] Compared with the control conventional immobilized hydantoinase, less protein shedding was detected after pretreatment in the reaction system with three different product salt concentrations of 100 g / L, 160 g / L, and 200 g / L (results are shown in Figure 4 This indicates that the immobilized hydantoinase method described in the present application can better maintain the structural stability of the immobilized enzyme.

[0094] Example 8 Stability test of immobilized hydantoinase after cross-linking with glutaraldehyde

[0095] Weigh the required mass of wet cells containing SEQ ID NO: 9, resuspend in 5 volumes of phosphate buffer (0.02M, pH 8.0), mix thoroughly, homogenize twice at low temperature with a homogenizer at 850Bar (cooling water 10-15°C), put the homogenized enzyme solution in a glass container, put it in a preheated 50°C water bath, stir mechanically at 200rpm, start the timing when the enzyme solution temperature rises to 50°C, and heat-treat in a water bath for 4h. Cool the enzyme solution to less than 20°C in an ice water bath, centrifuge the enzyme solution at 8000rpm for 20min, and take the supernatant to obtain the enzyme solution.

[0096] Then weigh 30g of LX-1000HA immobilized carrier, add 90mL of phosphate buffer (0.02M, pH 7.4) to completely immerse the carrier, add 3mL of 50% glutaraldehyde, and place it on a shaker at 37°C and 200rpm for 2 hours. After sufficient reaction, use double-layer neutral filter paper to filter out the liquid. Then add 30-50mL of pure water, place it on a shaker at 37°C and 200rpm for 20min, filter out the liquid, and repeat the washing 3 times to obtain the modified LX-1000HA immobilized carrier.

[0097] Then, 25 g of the modified LX-1000HA immobilized carrier was placed in a 250 mL centrifuge cup, 125 mL of the prepared enzyme solution was added, and the centrifuge tube was placed in a shaker at 20°C and 250 rpm to react for 20-24 hours. After sufficient reaction, the excess liquid was removed by filtration to obtain the crude immobilized hydantoinase.

[0098] After the crude immobilized enzyme is obtained, 5 clean 50 mL centrifuge tubes are prepared, 5 g of the crude immobilized hydantoinase is weighed respectively, and then 25 mL of phosphate buffer (0.02 M, pH 8.0) is added, and 4 μL, 50 μL, 200 μL, and 400 μL of 50% glutaraldehyde are added respectively. The control group is added with 200 μl of phosphate buffer, and the mixture is placed in a shaker at 30° C. and 250 rpm for 16-24 h, and the liquid is removed by suction but not filtered to complete dryness, to obtain crude immobilized hydantoinase cross-linked with glutaraldehyde of different concentrations. 25 mL of phosphate buffer (0.02 M, pH 8.0, 1 M sodium chloride) is added to the crude product, and the mixture is placed in a shaker at 30° C. and 250 rpm for 30 min, and the liquid is removed by suction but the immobilized hydantoinase is not filtered to dryness, and the washing is repeated 3 times to obtain the immobilized hydantoinase described in the present application.

[0099] Weigh 0.1g of each immobilized enzyme cross-linked with glutaraldehyde of different concentrations, and soak it in 1.0M, 1.6M, and 2.0M (R)-(-)-3-(carbamoylmethyl)-5-methylacetic acid ammonium salt at 40℃ for 24h. Remove the soaking solution, add 1mL pH 7.5 phosphate buffer to wash the immobilized hydantoinase, remove the washing solution, add 10g / L 3-2-(methylpropyl) glutarimide, and react at 40℃ and 200rpm on a shaker for 1h. Remove the reaction solution, wash it with 1mL pH 7.5 phosphate buffer, and then add 10g / L 3-(2-methylpropyl) glutarimide and repeat the reaction once. The reaction results are shown in the figure. Figure 5 As shown, the immobilized hydantoinase re-crosslinked with different concentrations (0.008%-0.8%) of glutaraldehyde showed better high-concentration salt stability than the immobilized hydantoinase without re-crosslinking.

[0100] Depend on Figure 5It can be seen that after soaking in high concentration ammonium salt, the activity of the immobilized enzyme after re-crosslinking with 50-400 μL glutaraldehyde is very stable. Compared with the result of re-crosslinking with high concentration glutaraldehyde, the activity of the immobilized enzyme after re-crosslinking with 4 μL glutaraldehyde decreases significantly, so it is speculated that the crosslinking with 4 μL glutaraldehyde is not particularly sufficient. However, by comparing the number of cycles of the conventional immobilized enzyme and the immobilized enzyme re-crosslinked with 4 μL glutaraldehyde, it can be seen that the stability of the re-crosslinked immobilized enzyme is significantly improved. The reason may be that the conventional immobilized enzyme is quickly inactivated under the action of the product ammonium salt, and the part of the enzyme that is not fully crosslinked in the re-crosslinked immobilized enzyme is also quickly inactivated, but the part of the enzyme that is fully crosslinked in the re-crosslinked immobilized enzyme will maintain activity. Therefore, although the immobilized enzyme after re-crosslinking with 4 μL glutaraldehyde is not completely crosslinked, because the part of the enzyme that is fully crosslinked will maintain activity, its number of cycles can be greatly increased compared with the conventional immobilized enzyme.

[0101] Example 9: Enzyme activity retention test of immobilized hydantoinase containing the same characteristic lysine

[0102] 24g 3-(2-methylpropyl)glutarimide was added to a three-necked flask with a container volume of 250mL, 114mL phosphate buffer (0.05M, pH 7.6) was added to make the final substrate concentration 200g / L, and then 6g SEQ ID NO:1-10 immobilized hydantoinase was added, and the reaction system was placed at 40°C and continued to react for 8-24h under the conditions of 450rpm. During the reaction, the generation of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid gradually reduced the pH of the reaction system, so 10-25% ammonia water was used to control the reaction pH between 7.9-8.1 in real time until the reaction was completed. 1mL of the sample was taken, 9mL of acetonitrile was added for quenching, and the quenching time was 30min. After centrifugation (13000rpm, 3min), the supernatant was diluted with 50% acetonitrile and the sample was detected by HPLC. After the reaction was completed, the reaction liquid was removed by suction filtration, and the immobilized hydantoinase was collected. The collected immobilized hydantoinase was eluted with 0.01-0.1 M phosphate buffer (pH 6.5-8.0).

[0103] 24g 3-(2-methylpropyl)glutarimide was added to a clean reaction bottle, and 114mL phosphate buffer (0.05M, pH 7.6) was added to make the final substrate concentration 200g / L, and then the recovered immobilized hydantoinase was added to the reaction system, and the reaction system was placed at 40°C and 450rpm for continuous reaction for 8-24h. During the reaction, the generation of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid gradually reduced the pH of the reaction system, so 10-25% ammonia water was used to control the reaction pH in real time between 7.9-8.1. Until the reaction was completed.

[0104]

[0105] Reaction time: The reaction time when the conversion rate exceeds 98%

[0106] Repeating the above steps is the cyclic application of immobilized hydantoinase. The enzyme activity retention results of SEQ ID NO: 1-10 are shown in Table 2:

[0107] Table 2. Cyclic application of re-crosslinked immobilized hydantoinase

[0108] SEQ ID NO: First reaction time (h) Number of applications Average enzyme activity loss / time 1 12.5 17 1.82％ 2 13.4 15 2.08％ 3 14.1 14 2.26％ 4 13.7 15 2.23％ 5 11.4 20 1.68％ 6 13.2 16 2.05％ 7 12.4 18 1.78％ 8 11.1 20 1.53％ 9 10.9 23 1.48％ 10 10.2 25 1.17％

[0109] Note: In this experiment, when the enzyme activity loss is about 35%, the cycle will no longer be carried out and the experiment will be terminated.

Claims

1. An immobilized hydantoinase, comprising: The recombinant hydantoinase comprises a resin of methyl acrylate and a cross-linking agent; the recombinant hydantoinase is immobilized by forming a chemical covalent bond with a resin carrier through the cross-linking agent, and further the cross-linking agent is used to form a chemical covalent bond between hydantoinase molecules to generate a stable immobilized structure.

2. The immobilized hydantoinase according to claim 1, wherein the recombinant hydantoinase sequence is as shown in SEQ ID NO: 1-10.

3. The immobilized hydantoin enzyme according to claim 1, wherein the subunits inside the immobilized hydantoin enzyme molecules form chemical covalent bonds through a cross-linking agent, and / or the tetramers outside the immobilized hydantoin enzyme molecules form chemical covalent bonds through a cross-linking agent.

4. The immobilized hydantoinase according to claim 1-2, wherein the resin is synthesized from methyl acrylate, preferably LX1000HA commercially available from Xi'an Lanxiao Technology New Materials Co., Ltd.

5. The immobilized hydantoinase according to claims 1-2, wherein the cross-linking agent is selected from the group consisting of soluble dialdehyde compounds, NHS esters, imidates, and hydroxymethylphosphines.

6. The immobilized hydantoinase according to claims 1-2, wherein the immobilized hydantoinase can be stably circulated in a reaction system comprising 200 g / L 3-isobutylglutarimide converted into (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

7. An immobilized recombinant hydantoinase sequence, wherein at least one of the amino acids at residue positions 407 and 478 of the hydantoinase is lysine, and the sequence is preferably SEQ ID NO: 1-10.

8. A method for preparing immobilized hydantoinase, comprising the following steps: (1) Preparation of enzyme solution of hydantoinase with sequence as shown in SEQ ID NO: 1-10; (2) Cross-linker modification of immobilized carriers; (3) incubating the immobilized carrier with hydantoinase to immobilize the enzyme; (4) re-crosslinking of immobilized hydantoinase; (5) Cleaning of immobilized hydantoinase.

9. The method of claim 8, wherein the cross-linking agent is selected from the group consisting of soluble dialdehyde compounds, NHS esters, imidates, and hydroxymethylphosphines.

10. A method for preparing (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, comprising the following steps: (1) dissolving 3-isobutylglutarimide in a reaction system; (2) placing the immobilized hydantoinase in a reaction system containing 3-isobutylglutarimide for incubation; in, The immobilized hydantoinase is selected from the sequences shown in SEQ ID NOs: 1-10.

Images