Fusion enzyme protein, immobilized enzyme of fusion enzyme protein and application of fusion enzyme protein in catalytic synthesis of L-carnitine precursor

Abstract

The invention relates to a fusion enzyme protein, an immobilized enzyme of the fusion enzyme protein and application of the fusion enzyme protein in catalytic synthesis of an L-carnitine precursor, and belongs to the technical field of bioengineering. The fusion enzyme protein is constructed by connecting the C end of carbonyl reductase CgKR with the N end of glucose dehydrogenase BmGDH through a connecting peptide, the fusion enzyme protein simultaneously has carbonyl reduction activity and glucose dehydrogenase activity, the amino acid sequence of the carbonyl reductase CgKR is as shown in SEQ ID No. 1, and the amino acid sequence of the glucose dehydrogenase BmGDH is as shown in SEQ ID No. 2. The invention also provides an application of catalyzing the reduction of the ethyl 4-chloro-3-oxobutyrate to generate the ethyl (R)-4-chloro-3-hydroxybutyrate. Through double-enzyme fusion expression, compared with a free enzyme, the catalytic efficiency of the fusion enzyme protein CgKR-GSTGS-BmGDH is improved by 30%. When the immobilized fusion enzyme protein CgKR-GSTGS-BmGDH-coated LXHFA-001 disclosed by the invention is used for catalytically preparing ethyl (R)-4-chloro-3-hydroxybutyrate, the catalyst stability is good, and the application cost is low; the method has the remarkable advantages of good reusability, simple and convenient product separation and the like, and has a good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a fusion enzyme protein, an immobilized enzyme thereof and application in catalyzing the synthesis of a L-carnitine precursor. Background Art

[0002] L-Carnitine is a naturally occurring compound found throughout the body and possesses important physiological functions. It is a key player in the entry of fatty acids into mitochondria for β-oxidation, promoting fat metabolism and thereby increasing energy levels. It also improves fatty acid utilization in muscle tissue while reducing lactic acid accumulation, alleviating muscle fatigue and enhancing performance during endurance exercise. L-Carnitine also possesses antioxidant properties, helping to mitigate post-exercise oxidative stress damage to muscles and potentially delaying aging. L-Carnitine can also lower serum total cholesterol and triglycerides and increase serum high-density lipoprotein, helping to prevent cardiovascular disease.

[0003] Due to its important physiological functions, L-carnitine plays an important role in the nutritional health products, sports nutrition supplements, feed and pharmaceutical industries. As people pay more attention to health and fitness, the market prospects of L-carnitine are very broad.

[0004] Currently, a variety of methods for synthesizing L-carnitine have been developed. Among them, the enzymatic asymmetric reduction of the potentially chiral substrate 4-chloro-3-carbonylbutyric acid ethyl ester (COBE) to produce optically active (R)-4-chloro-3-hydroxybutyric acid ethyl ester [(R)-CHBE], followed by chemical amination to L-carnitine, has become an important method for L-carnitine synthesis. This process offers significant advantages, such as a short route and high optical purity, and has garnered widespread attention in recent years.

[0005] It has been reported that various carbonyl reductases can catalyze the asymmetric reduction of ethyl 4-chloro-3-carbonylbutyrate to obtain (R)-ethyl 4-chloro-3-hydroxybutyrate with high optical purity.

[0006] Chinese patent CN106164260B discloses a carbonyl reductase from Candida parapsilosis and its expression, the enzyme's encoding gene and amino acid sequence, as well as a recombinant expression vector and recombinant expression transformant containing the encoding gene, and the use of the carbonyl reductase or recombinant expression transformant as a catalyst in the asymmetric reduction of potential chiral carbonyl compounds. The inventors of this application also previously molecularly modified the carbonyl reductase CgKR from Candida parapsilosis to obtain a highly active and stereoselective reductase mutant CgKR. M10(Hereinafter referred to as CgKR). Carbonyl reductase CgKR is an NADPH-dependent enzyme that catalyzes the asymmetric reduction of ethyl 4-chloro-3-carbonylbutyrate, oxidizing the coenzyme NADPH to generate the oxidized coenzyme NADP. + Since coenzymes are expensive, the substrate hydrogenation reduction reaction catalyzed by carbonyl reductase CgKR is coupled with the glucose dehydrogenation and oxidation reaction catalyzed by glucose dehydrogenase (BmGDH) to achieve in situ regeneration of the coenzyme, which can efficiently realize the asymmetric reduction of COBE.

[0007] A dual-enzyme coupled coenzyme regeneration system is a common system for biological asymmetric reduction. However, this system requires the synergistic action of two enzymes. The expression of each enzyme individually is time-consuming and labor-intensive, and the shuttling and regeneration of the coenzyme between the two enzymes is inefficient. To achieve enzyme reuse, both enzymes must be immobilized separately, which is costly. Summary of the Invention

[0008] Based on the problems in the prior art that the expression of two enzymes separately is time-consuming and labor-intensive, and the shuttling and regeneration efficiency of the coenzyme between the two enzymes is low, the present invention provides a fusion enzyme protein, the preparation of an immobilized fusion enzyme protein, and its application in the catalytic synthesis of L-carnitine precursors.

[0009] Specifically, the present invention connects carbonyl reductase CgKR and glucose dehydrogenase BmGDH through a connecting peptide at the genetic level to construct a fusion enzyme protein, thereby realizing substrate-coupled coenzyme regeneration; the fusion enzyme protein is further immobilized on a resin surface to prepare an immobilized enzyme, and the prepared immobilized enzyme is used to efficiently catalyze the asymmetric reduction of 4-chloro-3-carbonylbutyric acid ethyl ester to prepare the carnitine precursor (R)-4-chloro-3-hydroxybutyric acid ethyl ester, with excellent reusability.

[0010] The constructed fusion enzyme protein provided by the present invention can realize the simultaneous catalysis of the asymmetric reduction of the substrate COBE and the oxidative dehydrogenation of glucose by a single enzyme, and mediate the efficient cyclic regeneration of the coenzyme NADPH in situ.

[0011] The immobilized enzyme provided by the present invention has the advantages of being single, efficient, stable and reusable, and is of great significance for the efficient, stable and continuous production of chiral alcohols.

[0012] The purpose of the present invention can be achieved by the following technical solutions:

[0013] One of the technical solutions adopted by the present invention is to provide a fusion enzyme protein, which is constructed by connecting the C-terminus of carbonyl reductase CgKR to the N-terminus of glucose dehydrogenase BmGDH through a connecting peptide, to obtain a fusion enzyme protein having both carbonyl reduction activity and glucose dehydrogenase activity, wherein the amino acid sequence of the carbonyl reductase CgKR is shown in SEQ ID No. 1, and the amino acid sequence of the glucose dehydrogenase BmGDH is shown in SEQ ID No. 2.

[0014] In one embodiment of the present invention, the connecting peptide can be any of the currently reported peptide chains with different lengths, flexibility or rigidity, as long as the constructed fusion enzyme protein retains both high carbonyl reduction activity and glucose dehydrogenase activity.

[0015] Preferably, the connecting peptide is selected from one of AEAAAKEAAAKA, GGGGS, (GGGGS)3, (GSG)3, or GSTGS.

[0016] The second technical solution adopted by the present invention is to provide an isolated nucleic acid, wherein the nucleic acid is a nucleic acid molecule encoding the fusion enzyme protein as described in the first technical solution.

[0017] The third technical solution adopted by the present invention provides a recombinant expression vector containing the fusion enzyme protein nucleic acid.

[0018] The recombinant expression vector is obtained by cloning the nucleic acid of the fusion enzyme protein into various expression vectors using conventional methods in the art. The expression vector includes various conventional vectors in the art, such as commercially available plasmids, phages, or viral vectors, and the preferred vector is plasmid pET28a.

[0019] Preferably, the recombinant expression vector can be obtained by the following example method: plasmid pET28a-CgKR is PCR amplified using primers and linearized; then it is connected with the BmGDH fragment obtained by PCR amplification through a one-step cloning indirect technology to construct a recombinant expression vector containing the fusion enzyme protein gene for Escherichia coli expression.

[0020] The fourth technical solution adopted by the present invention is to provide a recombinant expression transformant comprising the fusion enzyme protein gene of the present invention.

[0021] The recombinant expression vector containing the nucleic acid sequence of the fusion enzyme protein of the present invention is transformed into a host cell to obtain the recombinant expression transformant.

[0022] The recombinant expression vector is obtained by linking the nucleic acid sequence of the fusion enzyme protein of the present invention to various expression vectors; the expression vectors include various conventional vectors in the art, preferably plasmid pET-28a(+). The host cell can be any conventional host cell in the art that can stably replicate the recombinant expression vector and effectively express the fusion enzyme protein gene, preferably Escherichia coli BL21(DE3).

[0023] The fifth technical solution adopted by the present invention is to provide a method for preparing the fusion enzyme protein as described in technical solution 1. The method for preparing the fusion enzyme protein of the present invention is preferably as follows: culturing the recombinant expression transformant as described above, and isolating the recombinantly expressed fusion enzyme protein.

[0024] Wherein cultivating the used substratum of recombinant expression transformant can be any substratum that can make transformant grow and produce recombinant fusion enzyme protein of the present invention in this area.Cultural method and culture condition do not have special restrictions, can be according to the difference of factors such as host cell type and cultural method, carry out suitable selection by this area routine knowledge, as long as transformant can be grown and produce described recombinant fusion enzyme protein.The concrete operation that the recombinant expression transformant is cultivated can be carried out by this area routine operation.

[0025] For example, 20 μL of glycerol culture of recombinant fusion enzyme protein was inoculated into a 4 mL LB liquid culture medium test tube containing 50 μg / mL kanamycin, and cultured in a shaker at 37°C, 200 rpm for 12 hours. 1 mL of the test tube culture was inoculated into a 500 mL shake flask containing 100 mL LB liquid culture medium, and cultured in a shaker at 37°C, 200 rpm for 3-4 hours until the OD 600 When the pH reaches 0.5-1.0, add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1-1.0 mM (preferably 0.2 mM) to induce enzyme production, and continue to culture at 16°C for 24 hours. Use a centrifuge to collect the bacteria. Use 10 mL of potassium phosphate buffer (100 mM, pH 6.0) to resuspend the bacteria, and ultrasonically disrupt them in an ice water bath for 15 minutes to obtain a crude enzyme solution. The LB liquid medium formula is: 10 g / L peptone, 5 g / L yeast powder, and 10 g / L sodium chloride.

[0026] The sixth technical solution adopted by the present invention is to provide a fusion enzyme protein catalyst in one of the following forms:

[0027] (1) culturing the recombinant expression transformant and isolating transformant cells containing the fusion enzyme protein;

[0028] (2) disrupting the transformant cells as described in form (1) to obtain a cell disrupted liquid containing the fusion enzyme protein;

[0029] The seventh technical solution adopted by the present invention is to provide an immobilized enzyme. The preparation method of the immobilized enzyme is to immobilize the fusion enzyme protein described in the first technical solution by a resin surface binding method.

[0030] The resin may be any commercially available resin carrier, preferably an amino resin or an epoxy resin.

[0031] The amino resin is one of LX-1000NH, ESR-2, ESR-3, LX-701, LX-703 and LX-705, and needs to be activated by reaction with glutaraldehyde before immobilization;

[0032] The epoxy resin is one of LXHFA-001, LX-604, XJGD-89, LX-609, ES-103B or LX-600. More preferably, the epoxy resin is LXHFA-001.

[0033] The epoxy resin immobilization method of the present invention is preferably as follows: mixing the epoxy resin with the enzyme solution of the fusion enzyme protein, wherein the ratio of the fusion enzyme protein to the epoxy resin is 30-180 mg 酶蛋白 / g 树脂 The pH of the enzyme solution is 5.0-8.0. It is shaken at 20-40°C for 4-20 hours, filtered, and washed to obtain the epoxy resin-immobilized fusion enzyme protein.

[0034] The eighth technical solution adopted by the present invention is:

[0035] As described in Technical Solution 7, the immobilized enzyme is used to catalyze the asymmetric reduction of the substrate 4-chloro-3-carbonylbutyric acid ethyl ester to prepare 4-chloro-3-hydroxybutyric acid ethyl ester.

[0036] In one embodiment of the present invention, the enzymatic reaction is carried out in a toluene-water two-phase reaction system, the loading amount of the substrate COBE in the catalytic reaction system is 10-100 g / L, the molar ratio of glucose to COBE is 1-1.5:1, and NADP + 0.2-0.5mM. The reaction temperature is 25-35°C, pH 5.0-8.0. Samples are taken intermittently during the reaction, and the amount of product generated is analyzed by liquid chromatography. After each batch of reaction, the immobilized fusion enzyme protein is separated by filtration. The filtrate is allowed to stand for stratification, and the organic phase is collected and the target product (R)-CHBE is isolated and extracted. The isolated immobilized fusion enzyme protein is added to a fresh reaction system and reused.

[0037] The analytical conditions were as follows: the chromatographic column was CP-Chirasil-Dex CB, and the detection conditions were: inlet temperature 280°C, detector temperature 280°C, column temperature initially 60°C, held for 5 minutes, then increased at 10°C / min to 140°C, and held for 5 minutes. The elution time of the internal standard p-methylanisole was 10.4 minutes, the retention time of the substrate COBE was 13.3 minutes, and the retention time of the product (R)-CHBE was 15.1 minutes.

[0038] Compared with the prior art, the present invention has the following advantages and significant advantages:

[0039] The present invention fuses carbonyl reductase CgKR and glucose dehydrogenase BmGDH to construct a fusion enzyme protein that simultaneously catalyzes COBE asymmetric reduction and glucose dehydrogenation oxidation, and can efficiently achieve substrate-coupled and efficient regeneration of the coenzyme NADPH. Compared with the free enzyme, the catalytic efficiency of the fusion enzyme protein is improved by 30%. The immobilized enzyme prepared by immobilizing the fusion enzyme protein has high stability and retains good activity after multiple rounds of repeated use, greatly reducing the application cost of the catalyst. By immobilizing the fusion enzyme protein, after the reaction is completed, the catalyst is separated by filtration, and the two-phase reaction liquid can be quickly separated, greatly simplifying downstream extraction. It has good industrial application prospects in the enzymatic synthesis of L-carnitine intermediates. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 : Immobilized enzyme is repeatedly used to catalyze the COBE asymmetric reduction process. DETAILED DESCRIPTION

[0041] The various reaction or detection conditions described in the present invention may be combined or modified according to common knowledge in the art and may be verified by experiments. The technical solutions and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments. However, the scope of protection of the present invention is not limited to these embodiments, and any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the scope of protection of the present invention.

[0042] The content of the present invention is further described below through specific examples.

[0043] The sources of materials in the following examples are:

[0044] The expression plasmid pET-28a(+) was purchased from Novagen, and the plasmids pET-28a(+)-CgKR and pET-28a(+)-BmGDH were constructed and prepared using conventional biotechnology.

[0045] Escherichia coli BL21 (DE3) competent cells and agarose gel DNA recovery kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0046] The restriction endonuclease Dpn I was a commercial product from New England Biolabs (NEB).

[0047] Rapid PCR polymerase PrimeSTAR MAX was purchased from Takara.

[0048] Unless otherwise stated, the specific experiments in the following examples were performed according to conventional methods and conditions in the art, or in accordance with the commercial instructions of the kits.

[0049] Example 1 Construction of fusion enzyme protein CgCR-linker-BmGDH

[0050] Primer pairs CgCR-F and CgCR-linker-R were designed to linearize the plasmid pET28a-CgKR by plasmid PCR. Primer pairs linker-BmGDH-F and BmGDH-R were designed to amplify the BmGDH sequence by PCR. Primers CgCR-linker-R and linker-BmGDH-F contain the linker sequence. The linearized fragments were separated and recovered by nucleic acid electrophoresis. The two fragments were mixed in equal proportions and integrated into a recombinant plasmid expressing the fusion enzyme protein CgCR-Linker-BmGDH using a one-step cloning system. The plasmid was then transformed into E. coli BL21(DE3) to construct a recombinant E. coli expression strain.

[0051] The linker peptides used in the constructed fusion enzyme protein CgCR-Linker-BmGDH are: AEAAAKEAAAKA, GSTGS, GGGGS, (GSG)3 and (GSG)3. The primers for constructing the corresponding fusion enzyme proteins are shown in Table 1.

[0052] Table 1 Primers required for constructing the fusion enzyme protein CgKR-linker-BmGDH

[0053]

[0054]

[0055] Among them, the sequences of primers CgKR-F, BmGDH-R, GGGGS-BmGDH-F, CgKR-GGGGS-R, GSTGS-BmGDH-F, CgKR-GSTGS-R, GSGGSGGSG-BmGDH-F, CgKR-GSGGSGGSG-R, AEAAAKEAAAKA-BmGDH-F, CgKR-AEAAAKEAAAKA-R, (GGGGS)3-BmGDH-F, and CgKR-(GGGGS)3-R are shown in SEQ ID No.3-14, respectively.

[0056] Example 2 Inducible expression of fusion enzyme protein CgKR-linker-BmGDH and preparation of crude enzyme solution

[0057] The recombinant E. coli obtained in Example 1 was inoculated into a test tube containing 4 mL of LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C and 200 rpm with shaking for 12 h to prepare a seed solution. The seed solution was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of LB medium (containing 50 μg / mL kanamycin) at a 1% (v / v) inoculum and cultured at 37°C and 200 rpm with shaking until the OD value of the culture solution reached 0. 600 When the p-value reached approximately 0.6, IPTG was added to a final concentration of 0.2 mM to induce expression of the target protein. The cells were cultured at 16°C and 200 rpm for 24 h. The cells were harvested by centrifugation at 8,000 × g at 4°C and washed twice with saline.

[0058] Weigh 1.0 g of wet cells, add 10 mL of potassium phosphate buffer (10 mM, pH 7.0), shake to resuspend the cells, and ultrasonicate (power 400 W, ultrasonication 4 s, rest 6 s, repeated 99 times). Centrifuge at 4°C (8000 rpm, 20 min) to remove cell debris to obtain the crude enzyme solution of the recombinant fusion enzyme protein CgKR-linker-BmGDH.

[0059] Example 3 Purification and Activity Characterization of Fusion Enzyme Protein CgKR-linker-BmGDH

[0060] The crude enzyme solution of the recombinant fusion enzyme protein CgKR-linker-BmGDH obtained in Example 2 was loaded onto a nickel chromatography column, and the flow rate was controlled to ensure that the target protein was fully bound. After loading, non-specific binding proteins were rinsed with 2 times the column volume of Buffer A, and weakly bound miscellaneous proteins were eluted with 2 times the column volume of 10% Buffer B (containing 50mM imidazole). Finally, the target protein was eluted with 30% Buffer B (containing 150mM imidazole), the effluent was collected, and concentrated with an ultrafiltration tube with a 30kDa molecular weight cutoff: centrifuged at 4 ° C, 3300 rpm to a volume of less than 500 μL (about 30 minutes), an equal volume of Buffer C was added, and the concentration was repeated twice to obtain the target purified protein solution.

[0061] Buffer A: 1.21 g K2HPO4, 0.414 g KH2PO4, 14.71 g NaCl, 91.5 μL β-mercaptoethanol, and 680.1 mg imidazole were dissolved in deionized water, diluted to 500 mL, and filtered through a 0.22 μm filter membrane.

[0062] Protein purification Buffer B: 1.21 g K2HPO4, 0.414 g KH2PO4, 14.71 g NaCl, 91.5 μL β-mercaptoethanol, and 34.02 g imidazole were dissolved in deionized water, diluted to 500 mL, and filtered through a 0.22 μm filter.

[0063] Protein purification Buffer C: 1.21 g K2HPO4, 0.414 g KH2PO4, 14.71 g NaCl, 50 mL glycerol, 154 mg DTT, dissolved in deionized water, dilute to 500 mL, and filter through a 0.22 μm filter.

[0064] The recombinantly expressed fusion enzyme proteins were characterized for activity, with the results shown in Table 2. The fusion enzyme CgKR-AEAAAKEAAAKA-BmGDH exhibited the highest carbonyl reduction activity, while the fusion enzyme CgKR-GSTGS-BmGDH exhibited the highest glucose oxidation activity. Considering that the activity of glucose dehydrogenase is generally lower than that of carbonyl reductase among the constructed fusion enzymes, and that glucose dehydrogenase activity is the rate-limiting factor in the overall activity of the fusion enzymes, the fusion enzyme CgKR-GSTGS-BmGDH was selected for subsequent immobilization studies.

[0065] Table 2 Fusion enzyme protein specific activity

[0066]

[0067] Example 4 Determination of kinetic parameters of fusion enzyme protein

[0068] The kinetic parameters of the fusion enzyme protein CgKR-GGGGS-BmGDH and the single enzymes CgKR and BmGDH were characterized to investigate their catalytic performance for substrates COBE and glucose. The results are shown in Table 3. Compared with the single enzyme, the catalytic constant k of the fusion enzyme protein catalyzing the COBE reduction reaction is cat Compared with the single enzyme CgKR, the k cat The activity of the enzyme decreased by 10% compared with that of the single enzyme BmGDH. The results showed that the fusion process had a certain impact on the activity of the dual enzyme. The bifunctional characteristics of the fusion enzyme protein enabled it to simultaneously catalyze COBE reduction and NADPH regeneration, achieving substrate-coupled coenzyme regeneration.

[0069] Table 3 Kinetic parameter characterization of CgKR-GGGGS-BmGDH fusion protein and single enzyme

[0070]

[0071] Example 5 Screening of immobilized carriers of fusion enzyme protein CgKR-GSTGS-BmGDH

[0072] Preactivation of amino resin: Weigh 1 g of resin and mix with 10 mL of potassium phosphate buffer (100 mM, pH 7.0) containing 2% (w / v) glutaraldehyde. Oscillate at 30°C and 200 rpm for 2 h. Wash four times with potassium phosphate buffer (100 mM, pH 7.0) to remove any residual glutaraldehyde solution. Immobilize the resin within 12 h.

[0073] The epoxy resin did not require pretreatment and was directly washed four times with potassium phosphate buffer (100 mM, pH 7.0), filtered, and stored at 4°C for later use.

[0074] 100 mg of resins of varying origins and properties were mixed with 1 mL of buffer containing 2 mg / mL of the target protein. The mixture was shaken at 30°C and 1000 rpm for 15 hours, filtered, and rinsed four times with an equal volume of potassium phosphate buffer. The filtrate and washes were combined. The residual enzyme activity of the free enzyme before immobilization and in the filtrate washes, as well as the specific activity of the immobilized enzyme, were measured. The activity recovery during the immobilization process was calculated to evaluate the performance of the immobilized resins. As shown in Table 4, both epoxy and amino-derivatized resins effectively immobilized the fusion enzyme CgKR-GSTGS-BmGDH. The epoxy resin-immobilized enzyme CgKR-GSTGS-BmGDH@LXHFA-001 exhibited the highest specific activity and activity recovery.

[0075] Table 4 Screening of fusion enzyme protein immobilized resin carriers

[0076]

[0077]

[0078] Example 6 Optimization of the fusion enzyme protein CgKR-GSTGS-BmGDH immobilization conditions

[0079] 1g of epoxy resin LXHFA-001 was mixed with 10mL of potassium phosphate buffer (100mM, pH 6.0) containing 120mg of the target protein. The mixture was shaken at 200rpm at 25°C for 8 hours, filtered, and rinsed four times with an equal volume of potassium phosphate buffer. The filtered washes were combined. The residual enzyme activity of the free enzyme before immobilization and in the filtered washes, as well as the specific activity of the immobilized enzyme, were measured, and the activity recovery rate of the immobilization process was calculated. Under optimized conditions, the activity recovery rate of the immobilized enzyme CgKR-GSTGS-BmGDH@LXHFA-001 increased to 40.1%, and the specific activity reached 90.1U / g.

[0080] Example 7 Immobilized enzyme CgKR-GSTGS-BmGDH@LXHFA-001 catalytic reduction of COBE

[0081] The reaction was carried out in a 250 mL three-necked flask in a two-phase system with a toluene / water ratio of 1:1. The total volume of the reaction system was 100 mL. The toluene organic phase contained 10 g COBE, and the aqueous phase was 6.0% potassium phosphate (100 mM, pH 6.0), 15 g glucose, 5 g immobilized enzyme prepared as in Example 5, and 0.4 mM NADP. + The reaction was carried out at 30°C and 200 rpm with mechanical stirring. 2M Na2CO3 was added to control the pH of the reaction solution to a constant 6.0. Samples were taken intermittently to monitor the reaction conversion rate. After the reaction was complete and the substrate conversion rate exceeded 99%, the immobilized enzyme was recovered by filtration, washed three times with 50 mL of potassium phosphate buffer (20 mM, pH 6.0), and then added to fresh reaction solution for reuse. The results are shown in Figure 2. Figure 1 As shown in the figure, the immobilized enzyme still has high activity after being reused 13 times with a half-life of 136.6 h.

[0082] Comparative Example 1 Construction and Characterization of Fusion Enzyme Protein BmGDH-linker-CgCR

[0083] Referring to the methods of Examples 1-3, a series of fusion enzyme proteins BmGDH-linker-CgCR with CgCR at the N-terminus and BmGDH at the C-terminus were constructed and characterized for catalytic activity. The results are shown in Table 5. Compared with the CgCR-linker-BmGDH series fusion enzyme proteins, the BmGDH-linker-CgCR series fusion enzyme proteins generally have very low glucose dehydrogenation activity, which severely limits the overall activity of the fusion enzyme protein.

[0084] Table 5 Fusion enzyme protein specific activity

[0085]

[0086] Comparative Example 2 Comparison of catalytic reactions between fusion enzyme protein and free enzyme BmGDH & CgKR

[0087] The fusion enzyme protein CgKR-GGGGS-BmGDH (50 mg / L) and the mixed enzyme protein of equimolar amounts of the free enzymes CgKR & BmGDH were used to catalyze the reduction of 100 mM COBE. The pH was maintained constant by titrating a 2M sodium carbonate solution, and the catalytic efficiency of the two enzyme preparations during the reaction was compared. The results showed that the initial rate of the reaction catalyzed by the fusion enzyme protein was over 30% higher than that of the free enzyme. The reaction catalyzed by the fusion enzyme protein achieved a conversion rate of over 99% after 12 hours, while the conversion rate of the reaction catalyzed by the mixed free enzyme was only 91%. These results clearly demonstrate that the construction of the fusion enzyme protein achieves dual enzyme synergy between the carbonyl reductase CgKR and the glucose dehydrogenase BmGDH, significantly improving the catalytic efficiency.

[0088] The sequence information involved in the present invention is as follows:

[0089] SEQ ID No. 1:

[0090] Amino acid sequence of carbonyl reductase CgKR

[0091] MASDNSNTTVFVSGATGFIAQHVVRQLLDQNYKVIGSVRSAEKGDHLKNVIFKGGDFNYEIVKDISDPTAFDHVFEKHGKDIKVVLHTASPLHFNTTDYEKDLLIPAVNGTKGILESIKKYAAQTVERVVVTSSFAANSSTVDMFYAKDSSKTITEESWNQDTWESCQSDPIRAYC GSKKFAEKAAWDFYNANKDSVKFKLSIINPVYVFGPQNYVEPGKKILNTSSEVINSLVHLKKDDPLPEFAGGHIDVRDVAKAHILAFQKDELIEQRLMLHAGLFTTQTLLDIINEQFPELKGKIPAGKPGTGNPDDALTPVDNSKTKKLLGFEFIDLKKDLYDTISQILEAEKNSN

[0092] SEQ ID No. 2:

[0093] Amino acid sequence of glucose dehydrogenase BmGDH

[0094] MMYKDLEGKVVVITGSSTGLGKSMAIRFATEKAKVVVNYRSKEDEANSVLEEIKRVGGEAIAVKGDVTVESDIINLVQSAIKEFGKLDVMINNAGLENPVPSHEMSLSDWNKVIDTNLTGAFLGSREAIKY FVENDIRGTVINMSSVHEKIPWPLFVHYAASKGGMRLMTKTLALEYAPKGIRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTLYPSFQAGRG

[0095] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A fusion enzyme protein, characterized in that The C-terminus of carbonyl reductase CgKR is connected to the N-terminus of glucose dehydrogenase BmGDH via a connecting peptide to construct a fusion enzyme protein having both carbonyl reduction activity and glucose dehydrogenase activity, wherein the amino acid sequence of carbonyl reductase CgKR is shown in SEQ ID No. 1, and the amino acid sequence of glucose dehydrogenase BmGDH is shown in SEQ ID No.

2.

2. The fusion enzyme protein according to claim 1, characterized in that The connecting peptide is selected from one of the following amino acid sequences: AEAAAKEAAAKA, GGGGS, (GGGGS)3, (GSG)3 or GSTGS.

3. An isolated nucleic acid, characterized in that The nucleic acid is a nucleic acid molecule encoding the fusion enzyme protein according to claim 1 or 2.

4. A recombinant expression vector, characterized in that: Comprising the nucleic acid according to claim 3.

5. A recombinant expression transformant, characterized in that: Comprising the recombinant expression vector according to claim 4.

6. A fusion enzyme protein catalyst, characterized in that Is one of the following forms: (1) culturing the recombinant expression transformant according to claim 5, and isolating transformant cells containing the fusion enzyme protein according to claim 1 or 2; (2) The transformant cells according to embodiment (1) are disrupted to obtain a cell disrupted liquid containing the fusion enzyme protein according to claim 1 or 2.

7. An immobilized enzyme, characterized in that The fusion enzyme protein as claimed in claim 1 or 2 is immobilized on the surface of an amino resin or an epoxy resin by a covalent bonding method.

8. An immobilized enzyme according to claim 7, characterized in that: The amino resin is selected from one of LX-1000NH, ESR-2, ESR-3, LX-701, LX-703 or LX-705, and the amino resin needs to be activated by reaction with glutaraldehyde before immobilization; The epoxy resin is selected from one of LXHFA-001, LX-604, XJGD-89, LX-609, ES-103B or LX-600.

9. Use of the fusion enzyme protein according to claim 1 or 2, the fusion enzyme protein catalyst according to claim 6, or the immobilized enzyme according to claim 7 or 8 in catalyzing the reduction of ethyl 4-chloro-3-carbonylbutyrate to produce (R)-4-chloro-3-hydroxybutyrate.

10. The use according to claim 9, characterized in that The reaction was carried out in a toluene-water two-phase reaction system. The loading amount of substrate COBE in the catalytic reaction system was 10–100 g / L, the molar ratio of glucose to COBE was 1-1.5:1, and NADP + 0.2-0.5mM, reaction temperature is 25-35℃, pH 5.0-8.0.

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