Crystallization-strengthened bioenzyme crosslinker, preparation method, application and continuous flow device thereof

Abstract

The present application relates to a kind of crystallization reinforced biological enzyme crosslinking body and its preparation method, application and continuous flow device, the biological enzyme crosslinking body is formed by the covalent assembly of amine dehydrogenase (BcAmDH) and formic acid dehydrogenase (AaFDH) Spatially ordered cascade structure;Wherein the nucleotide sequence of the amine dehydrogenase is as shown in SEQ ID No.1, the nucleotide sequence of the formic acid dehydrogenase is as shown in SEQ ID No.2.The present application is in situ crystallization auxiliary, constructs spatially ordered cascade enzyme crosslinking body by biological orthogonal covalent assembly, by continuous reaction device and spatially ordered cascade enzyme crosslinking body, high-efficiency catalysis phenylacetone asymmetric reduction amination synthesis substrate conversion rate ≥77.4%, ee ≥99.9% High enantiomeric selectivity R- ( + ) - alpha-methylbenzylamine;And spatially ordered cascade enzyme crosslinking body can be recycled, suitable for industrial amplification application.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a crystal-enhanced ordered-crosslinked-enzyme-assembly (OC-EAs) and its preparation method, application and continuous flow device. Background Technology

[0002] Nitrogen-containing compounds are among the most important structural elements in the pharmaceutical industry. Over the past decade, approximately 82% of FDA-approved small molecule drugs are nitrogen-containing molecules, and their chemical diversity continues to rise steadily. Therefore, developing green, readily available, and efficient biocatalysts for the asymmetric synthesis of chiral amines has become a core objective of contemporary drug synthesis research.

[0003] Compared to the transaminase route, asymmetric reductive amination catalyzed by amine dehydrogenase can directly convert prochiral ketones into highly enantioselective chiral amines. The reaction uses only inexpensive inorganic ammonia as the amino donor, and the theoretical byproduct is only water, which conforms to the principles of green and sustainable manufacturing.

[0004] However, amine dehydrogenases still face three major bottlenecks in practical applications:

[0005] (1) Economic issues of cofactors: Amine dehydrogenases are NAD(P)H-dependent oxidoreductases, and the stoichiometric consumption of expensive cofactors keeps the cost of large-scale synthesis high. Although a dual-enzyme cascade of "amine dehydrogenase-coenzyme regenerating enzyme" (such as coupling with glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase) has been established to realize the NAD(P)H cycle, the introduction of additional enzymes further amplifies the challenges of spatial organization and operational stability.

[0006] (2) Spatial organization defects: Traditional free or randomly cross-linked dual enzyme systems lack ordered spatial arrangement, resulting in large diffusion loss of intermediate (NAD(P)H), incoordination of local microenvironment pH and ionic strength, and a sharp drop in coenzyme channel efficiency when enzyme-enzyme distance is >10 nm. As a result, the overall reaction rate decreases by 30-70%, catalytic stability decreases, and productivity is difficult to meet industrial requirements.

[0007] (3) Poor operational stability: Free amine dehydrogenase and formate dehydrogenase are prone to subunit dissociation, active center oxidation or conformational drift in the presence of 30~40℃, high ionic strength or organic cosolvent. After 6 h of batch reaction, the activity often drops to below 50%; after 24 h of continuous flow operation, the inactivation is >80%, resulting in uncontrollable process and increased cost. Summary of the Invention

[0008] To address the problems of high economic cost and poor enzyme stability in the enzyme-catalyzed production of R-(+)-α-methylbenzylamine in existing technologies, this invention provides a crystallization-enhanced bio-enzyme cross-linker, its preparation method, application, and continuous flow apparatus. With in-situ crystallization assistance, a spatially ordered cascade enzyme cross-linker is constructed through bioorthogonal covalent assembly, enabling efficient catalysis of the asymmetric reductive amination of acetophenone to synthesize highly enantioselective R-(+)-α-methylbenzylamine.

[0009] A crystal-enhanced bioenzyme cross-linker, wherein the bioenzyme cross-linker is formed by the covalent assembly of amine dehydrogenase (BcAmDH) and formate dehydrogenase (AaFDH) into a spatially ordered cascade structure; wherein the nucleotide sequence of the amine dehydrogenase is shown in SEQ ID No. 1, and the nucleotide sequence of the formate dehydrogenase is shown in SEQ ID No. 2.

[0010] The crystal-enhanced bioenzyme crosslinking of this invention is a covalent assembly structure assisted by in-situ crystallization.

[0011] The amine dehydrogenase BcAmDH was obtained by introducing a five-point mutation (K70S / N263L / V293A / E116V / T136S) into the catalytic active site of leucine dehydrogenase (BcLeuDH) derived from Bacillus cereus as the parent enzyme.

[0012] The formate dehydrogenase AaFDH is derived from Bacillus bingmayongensis.

[0013] Preferably, the amine dehydrogenase and formate dehydrogenase are specifically and irreversibly linked through a SpyTag / SpyCatcher, QTag / KTag biorthogonal covalent cross-linking system to form a spatially ordered cascade enzyme cross-linker, wherein the nucleotide sequence of SpyTag is shown in SEQ ID No. 3, the nucleotide sequence of SpyCatcher is shown in SEQ ID No. 4, the nucleotide sequence of QTag is shown in SEQ ID No. 5, and the nucleotide sequence of KTag is shown in SEQ ID No. 6.

[0014] Among them, SpyTag / SpyCatcher is a protein self-assembly system derived from the CnaB2 domain of the fibronectin-binding protein FbaB from Streptococcus pyogenes. SpyTag is a short peptide tag containing specific aspartic acid residues, and SpyCatcher is a protein chaperone containing specific lysine residues. The two can spontaneously form stable heteropeptide bonds to achieve irreversible covalent linkage between proteins. QTag / KTag is a site-specific covalent cross-linking tag system for proteins mediated by microbial transglutaminase (MTG). It consists of a glutamine tag QTag and a lysine tag KTag. It can form stable heteropeptide bonds under the catalysis of transglutaminase MTG to achieve specific irreversible covalent linkage between proteins or peptides.

[0015] This invention also provides a method for preparing the above-mentioned crystal-enhanced bioenzyme cross-linked body, comprising the following steps:

[0016] (1) SpyCatcher-BcAmDH-QTag and KTag-AaFDH-SpyTag were co-expressed in the same E. coli;

[0017] (2) After cell disruption, transglutaminase MTG with a nucleotide sequence as shown in SEQ ID No. 7 is added for cross-linking of KTag and QTag;

[0018] (3) Collect the cross-linked particles by centrifugation, wash them, and you will get crystallized and reinforced bio-enzyme cross-linked bodies without the need for an additional carrier.

[0019] As a preferred embodiment, the construction and expression of SpyCatcher-BcAmDH-QTag in Escherichia coli are as follows:

[0020] (I) First round of polymerase chain reaction PCR: The amine dehydrogenase BcAmDH gene with nucleotide sequence SEQ ID No.1 was used as a template, and BcAmDH-QTag F1 and BcAmDH-QTag R1 were used as upstream and downstream primers. At the same time, SpyCatcher with nucleotide sequence SEQ ID No.4 was used as a template, and SpyCatcher-linker F and SpyCatcher-linker R were used as upstream and downstream primers. After PCR amplification, the purified BcAmDH-QTag' fragment and SpyCatcher-linker fragment were obtained by recovery.

[0021] (II) Second round of PCR: Using the BcAmDH-QTag' fragment obtained in the first round as a template, BcAmDH-QTag F2 and BcAmDH-QTag R2 were used as upstream and downstream primers, respectively. After PCR amplification, the purified BcAmDH-QTag fragment was obtained by recovery.

[0022] (III) Third round of PCR: Using the BcAmDH-QTag fragment obtained in the second round and SpyCatcher-linker as templates, SpyCatcher-linker F and BcAmDH-QTag R were used as upstream and downstream primers. After PCR amplification, the purified SpyCatcher-BcAmDH-QTag fragment was obtained by recovery.

[0023] (IV) Heterologous expression was performed in Escherichia coli Bl21. The SpyCatcher-BcAmDH-QTag gene fragment was inserted between NcoⅠ and XhoⅠ in pET-28a(+) by double digestion and ligation. An NdeⅠ restriction site was added between SpyCatcher-linker and BcAmDH-QTag. A 6×His tag was added to the C-terminus to construct the complete pET28a-SpyCatcher-BcAmDH-QTag-6His recombinant plasmid.

[0024] As a preferred embodiment, the construction and expression of KTag-AaFDH-SpyTag in Escherichia coli are as follows:

[0025] (i) First round of PCR: Using the AaFDH gene as a template, KTag-AaFDH-SpyTag F1 and KTag-AaFDH-SpyTag R1 were used as upstream and downstream primers. After PCR amplification, the purified KTag-AaFDH-SpyTag fragment was obtained by recovery.

[0026] (ii) Second round of PCR: Using the KTag-AaFDH-SpyTag' fragment obtained in the first round as a template, KTag-AaFDH-SpyTag F2 and KTag-AaFDH-SpyTag R2 were used as upstream and downstream primers. After PCR amplification, the purified KTag-AaFDH-SpyTag fragment was obtained by recovery.

[0027] (iii) Subsequently, heterologous expression was performed in E. coli Bl21. The KTag-AaFDH-SpyTag gene fragment was inserted between NcoⅠ and XhoⅠ of pET-28a(+) by double digestion and ligation, with a 6×His tag at the C-terminus, to construct the complete pET28a-KTag-AaFDH-SpyTag-6His recombinant plasmid.

[0028] The present invention also provides the application of the above-mentioned crystal-enhanced bioenzyme cross-linked polymer in the synthesis of R-(+)-α-methylbenzylamine, comprising the following steps:

[0029] (a) Using acetophenone as a substrate, ammonium formate (NH4COOH) as an amino donor, and NAD+ as a substrate. + As a coenzyme, it undergoes an asymmetric reductive amination reaction under the catalysis of a crystal-enhanced bioenzyme cross-linker.

[0030] (b) Adding in-situ crystallization additives to the reaction system to promote the reaction equilibrium constant and improve stereoselectivity;

[0031] (c) After the reaction was completed, the pH of the reaction solution was adjusted and extracted to obtain R-(+)-α-methylbenzamine with enantioselectivity (ee) ≥99.9% and conversion ≥77.4%.

[0032] Preferably, in step (a), the reaction conditions are controlled as follows: pH 8.0~9.0, reaction temperature 25~40 ℃, and stirring speed 100~300 rpm; in step (b), the in-situ crystallization additive is one of 3,3-diphenylpropionic acid (DPPA), diphenylacetic acid (DPAA), 3,4-dichlorobenzoic acid (34CA), and 4-chloro-3-naphthoic acid (43CNA), the amount of in-situ crystallization additive added is 1.0~4.0 equivalents, the reaction temperature is 25~35℃, and the reaction time is 6~12 h; in step (c), the pH of the reaction solution is adjusted to 8.5-10.0, and it is extracted with methyl tert-butyl ether.

[0033] As a preferred option, the crystal-enhanced bio-enzyme cross-linker can be recycled for more than 6 batches with a conversion rate of ≥60%.

[0034] The present invention also provides a continuous flow apparatus for implementing the above-described crystal-enhanced bioenzyme crosslinking, comprising:

[0035] Main apparatus: includes a reaction column, with a reaction chamber inside the reaction column for enzyme cross-linking catalysis; and a condensation circulation jacket outside the reaction column;

[0036] Retention components: Glass frit embedded in the reaction column;

[0037] Feeding components: The top of the reaction column is equipped with an injection port for continuous or intermittent feeding;

[0038] Slag discharge component: A glass valve is connected below the reaction column, and the opening / closing of the valve controls the discharge of the trapped solid crystals;

[0039] Circulation component: A peristaltic pump is connected to the bottom of the glass valve via a pipeline. The outlet of the peristaltic pump is connected to the upper part of the reaction column via a return pipeline, forming a reaction liquid circulation loop, which drives the reaction liquid to circulate continuously within the reaction column.

[0040] As a preferred option, the reaction conditions are controlled as follows:

[0041] Temperature control: A constant temperature water bath of 25~35℃ is introduced into the external condensation circulation jacket of the reaction column, and the medium flow rate in the jacket is maintained at 5~8mL / min by the circulation pump to ensure the temperature of the reaction system is stable;

[0042] Circulation control: Start the peristaltic pump and adjust the pump speed to 0.1~0.5 mL / min to make the reaction solution continuously circulate in the reaction column through the reflux pipeline. During the circulation process, the glass frit core traps the solid ammonium salt crystals generated in the reaction.

[0043] Feed control: During the reaction, the consumed substrates acetophenone and NAD are intermittently replenished through the top injection port. + and NH4COOH;

[0044] Slag discharge control: After the reaction has proceeded for 6 to 10 hours, turn off the peristaltic pump, slowly open the glass valve below the glass reaction column to discharge the trapped solid crystals, close the valve after slag discharge, restart the peristaltic pump to continue the reaction, and the total reaction time is 20 to 28 hours.

[0045] This invention uses leucine dehydrogenase (BcLeuDH) derived from Bacillus cereus as the parent enzyme, and introduces a five-point mutation (K70S / N263L / V293A / E116V / T136S) into its catalytic active site to obtain amine dehydrogenase. The amine dehydrogenase is then covalently assembled with formate dehydrogenase AaFDH derived from Bacillus bingmayongensis through a SpyTag / SpyCatcher and QTag / KTag biorthogonal covalent cross-linking system to obtain spatially ordered cascade enzyme cross-linkers OC-EAs.

[0046] SpyTag / SpyCatcher is a protein self-assembly system derived from the CnaB2 domain of the fibronectin-binding protein FbaB from Streptococcus pyogenes. SpyTag is a short peptide tag containing a specific aspartic acid (Asp) residue, and SpyCatcher is a protein chaperone containing a specific lysine (Lys) residue. The two can spontaneously form stable heteropeptide covalent bonds under various conditions to achieve irreversible protein linkage. QTag / KTag is a protein site-specific covalent cross-linking tag system based on microbial transglutaminase (MTG). It consists of a glutamine tag QTag and a lysine tag KTag. It can form stable heteropeptide covalent bonds under MTG catalysis to achieve specific irreversible linkage between proteins or peptides.

[0047] By covalently assembling amine dehydrogenase (BcAmDH) and formate dehydrogenase (AaFDH) to construct a spatially ordered-crosslinked-enzyme-assembly (OC-EAs), and utilizing its efficient catalysis of the asymmetric reductive amination of acetophenone, highly enantioselective R-(+)-α-methylbenzylamine was prepared.

[0048] The beneficial effects of this invention are as follows:

[0049] (1) With the assistance of in-situ crystallization, an ordered-crosslinked-enzyme-assembly (OC-EAs) is formed by covalently assembling amine dehydrogenase (BcAmDH) and formate dehydrogenase (AaFDH); this solves the problems of high synthesis cost and poor enzyme stability in the process of synthesizing R-(+)-α-methylbenzylamine by enzyme-catalyzed asymmetric reduction amination of acetophenone.

[0050] (2) The asymmetric reductive amination of acetophenone via spatially ordered cascade enzyme cross-linkers (OC-EAs) achieves a substrate conversion rate of ≥77.4% and an ee of ≥99.9% for the synthesis of R-(+)-α-methylbenzylamine; and OC-EAs are recyclable, with a conversion rate of ≥60% for six consecutive batches.

[0051] (3) By using a continuous reaction device, the solid crystals are retained by glass frit and the reaction liquid is circulated by a peristaltic pump, which realizes the continuous conversion of the substrate and the efficient separation of the product. The condensation jacket effectively controls the reaction temperature and avoids enzyme activity loss. The glass valve facilitates the discharge of solid impurities, ensuring that the reaction proceeds continuously and stably. Finally, R-(+)-α-methylbenzylamine product with high enantioselectivity and high purity is obtained, which is suitable for industrial scale-up applications. Attached Figure Description

[0052] Figure 1 This is a MALDI-TOF mass spectrometry and protein expression analysis diagram of pET28a-SpyCatcher-BcAmDH-QTag-6His in an embodiment of the present invention.

[0053] in, Figure 1 The left image in the image is a MALDI-TOF mass spectrometry analysis of the fusion enzyme; Figure 1 The right side of the image shows a protein expression electrophoresis diagram. Channels 1-3 in the protein expression electrophoresis diagram represent the electrophoresis diagrams of soluble fractionation, insoluble fractionation, and purified protein, respectively.

[0054] Figure 2 This is a mass spectrometry and protein expression analysis diagram of pET28a-KTag-AaFDH-SpyTag-6His in an embodiment of the present invention;

[0055] in, Figure 2 The left image in the image is a MALDI-TOF mass spectrometry analysis of the fusion enzyme; Figure 2 The right side of the image shows a protein expression electrophoresis diagram. Channels 1-3 in the protein expression electrophoresis diagram represent the electrophoresis diagrams of soluble fractionation, insoluble fractionation, and purified protein, respectively.

[0056] Figure 3 This is a MALDI-TOF mass spectrometry and protein expression analysis diagram of BcAmDH in this embodiment of the invention;

[0057] in, Figure 3 The left image in the image is a MALDI-TOF mass spectrometry analysis of the fusion enzyme; Figure 3 The right side of the image shows a protein expression electrophoresis diagram. Channels 1-3 in the protein expression electrophoresis diagram represent the electrophoresis diagrams of soluble fractionation, insoluble fractionation, and purified protein, respectively.

[0058] Figure 4 This is a MALDI-TOF mass spectrometry and protein expression analysis diagram of AaFDH in an embodiment of the present invention;

[0059] in, Figure 4 The left image in the image is a MALDI-TOF mass spectrometry analysis of the fusion enzyme; Figure 4 The right side of the image shows a protein expression electrophoresis diagram. Channels 1-3 in the protein expression electrophoresis diagram represent the electrophoresis diagrams of soluble fractionation, insoluble fractionation, and purified protein, respectively.

[0060] Figure 5 A schematic diagram illustrating the synthesis and catalytic function of KSC@AaFDH-BcAmDH OC-EAs;

[0061] in, Figure 5Figure a shows the stepwise construction process of OC-EAs; Figure b shows the directional immobilization strategy of enzymes and vectors; Figure c shows the catalytic function and coenzyme cycling mechanism of OC-EAs.

[0062] Figure 6 Fluorescence microscopy images of KSC@AaFDH-BcAmDH OC-EAs under different fluorescence channels;

[0063] Figure 7 SEM images of the KSC protein scaffold taken at different magnifications;

[0064] Figure 7a shows a high-magnification image of the KSC protein scaffold (scale bar 1 μm); Figure 7b shows a low-magnification image of the KSC protein scaffold (scale bar 10 μm).

[0065] Figure 8 SEM images of KSC@AaFDH-BcAmDH OC-EAs obtained at different magnifications;

[0066] In Figure 8, image c is a high-magnification image of KSC@AaFDH-BcAmDH OC-EAs (scale bar 1 μm); image d is a low-magnification image of KSC@AaFDH-BcAmDH OC-EAs (scale bar 10 μm).

[0067] Figure 9 This is a liquid chromatography detection image of the preparation of R-(+)-α-methylbenzamide using OC-EAs and a free dual-enzyme system in an embodiment of the present invention;

[0068] Figure 10 The figure shows the operational stability results of OC-EAs in repeated catalytic cycles;

[0069] Figure 11 This is a schematic diagram of the continuous reaction apparatus for the catalytic synthesis of highly enantioselective R-(+)-α-methylbenzylamine in an embodiment of the present invention. Detailed Implementation

[0070] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0071] Unless otherwise specified, all methods used in the embodiments of this invention are conventional methods, and all reagents used are commercially available. The substrate acetophenone, and the products (R)-(+)-α-methylbenzylamine and (S)-(-)-α-methylbenzylamine, as well as other chemical standards, are all derived from Sigma-Aldrich.

[0072] The culture medium and solution preparation required:

[0073] (1) LB medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract.

[0074] (2) LB solid plate medium: peptone 10 g / L, sodium chloride 10 g / L, yeast extract 5 g / L, agar 20 g / L.

[0075] (3) Kanamycin (Kan, Kanamycin resistance) stock solution: Weigh 0.5 g of kanamycin and dissolve it in 10 mL of sterile water. Sterilize by passing the solution through a 0.22 μm filter membrane to obtain a final concentration of 50 g / L.

[0076] (4) IPTG solution: Weigh 0.476 g of IPTG and dissolve it in 10 mL of sterile water to prepare a 0.2 mol / L IPTG solution (working concentration is 0.2 mM). Use a 0.22 μm sterile filter membrane for filtration and sterilization.

[0077] (5) 50% glycerol: Measure 50 mL of glycerol and add sterile water to make up to 100 mL.

[0078] (6) 2 M ammonium formate (NH4COOH) buffer (pH 8.0-9.0): Weigh 21.02 g of NH4Cl, dissolve it in an appropriate amount of deionized water, and then add 25% ammonia dropwise while measuring the pH until the target value of 8.0–9.0 (room temperature 25 ℃) is reached. Store at room temperature for later use.

[0079] In the process of constructing the production strains pET28a-SpyCatcher-BcAmDH-QTag-6His and pET28a-KTag-AaFDH-SpyTag-6His, this invention further designed and incorporated GFP (SEQ ID No. 8) and mCherry (SEQ ID No. 9) fluorescent markers, generating pET28a-KTag-AaFDH-GFP-SpyTag-6His (SEQ ID No. 10) and ET28a-SpyCatcher-BcAmDH-mCherry-QTag-6His (SEQ ID No. 11), respectively. These fusion plasmids enabled the visualization of enzyme cross-linking and spatial localization. To verify the precise cross-linking of the BcAmDH and AaFDH dual-enzyme system, the resulting enzyme cross-links (OC-EAs) were characterized using multiple analytical techniques, including SDS-PAGE, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM).

[0080] Example 1

[0081] Construction of BcAmDH / AaFDH recombinant plasmid

[0082] 1. Construction of recombinant plasmid pET28a-SpyCatcher-BcAmDH-QTag-6His

[0083] The first round of PCR used the BcAmDH gene (SEQ ID No. 1) as a template, with BcAmDH-QTag F1 and BcAmDH-QTag R1 as upstream and downstream primers, respectively. Simultaneously, SpyCatcher (SEQ ID No. 4) was used as a template, with SpyCatcher-linker F and SpyCatcher-linker R as upstream and downstream primers, respectively. After PCR amplification, the purified BcAmDH-QTag' and SpyCatcher-linker fragments were recovered by 1% agarose gel electrophoresis. The second round of PCR used the BcAmDH-QTag' fragment obtained in the first round as a template, with BcAmDH-QTag F2 and BcAmDH-QTag R2 as upstream and downstream primers, respectively. After PCR amplification, the purified BcAmDH-QTag fragment was recovered by 1% agarose gel electrophoresis. The third round of PCR used the BcAmDH-QTag fragment obtained in the second round as a template, along with SpyCatcher-linker. SpyCatcher-linker F and BcAmDH-QTag R were used as upstream and downstream primers, respectively. After PCR amplification, the purified SpyCatcher-BcAmDH-QTag fragment was recovered by 1% agarose gel electrophoresis. Subsequently, heterologous expression was performed in E. coli Bl21. The SpyCatcher-BcAmDH-QTag gene fragment was inserted between Nco I and Xho I in pET-28a(+) via double digestion and ligation. An Nde I restriction site was added between SpyCatcher-linker and BcAmDH-QTag. A 6×His tag was added to the C-terminus, constructing the complete pET28a-SpyCatcher-BcAmDH-QTag-6His recombinant plasmid. Figure 1 The protein expression map of pET28a-SpyCatcher-BcAmDH-QTag-6His constructed in this embodiment and the MALDI-TOF mass spectrometry analysis of the fusion enzyme were verified by dual analysis of SDS-PAGE protein expression electrophoresis and MALDI-TOF mass spectrometry. This confirmed that the constructed fusion enzyme was correctly expressed, and its molecular weight and amino acid sequence were completely consistent with the design, providing structural and purity assurance for subsequent cross-linking assembly and catalytic applications. The primer sequences designed for constructing the recombinant plasmid pET28a-SpyCatcher-BcAmDH-QTag-6His are shown in Table 1.

[0084] Table 1. Primers designed for constructing the recombinant plasmid pET28a-SpyCatcher-BcAmDH-QTag-6His 2.

[0086] 3. Construction of recombinant plasmid pET28a-KTag-AaFDH-SpyTag-6His

[0087] The first round of PCR used the AaFDH gene (SEQ ID No. 2) as a template, with KTag-AaFDH-SpyTag F1 and KTag-AaFDH-SpyTag R1 as upstream and downstream primers, respectively. After PCR amplification, the purified KTag-AaFDH-SpyTag fragment was recovered by 1% agarose gel electrophoresis. The second round of PCR used the KTag-AaFDH-SpyTag' fragment obtained in the first round as a template, with KTag-AaFDH-SpyTag F2 and KTag-AaFDH-SpyTag R2 as upstream and downstream primers, respectively. After PCR amplification, the purified KTag-AaFDH-SpyTag fragment was recovered by 1% agarose gel electrophoresis. Subsequently, heterologous expression was performed in E. coli Bl21. The KTag-AaFDH-SpyTag gene fragment was inserted between Nco I and Xho I of pET-28a(+) by double digestion and ligation, with a 6×His tag at the C-terminus, to construct the complete pET28a-KTag-AaFDH-SpyTag-6His recombinant plasmid. Figure 2 The protein expression map of pET28a-KTag-AaFDH-SpyTag-6His constructed in this embodiment and the MALDI-TOF mass spectrometry analysis of the fusion enzyme are shown. Dual verification by SDS-PAGE protein expression electrophoresis and MALDI-TOF mass spectrometry confirmed that the constructed fusion enzyme was correctly expressed, and its molecular weight and amino acid sequence were completely consistent with the design, providing structural and purity assurance for subsequent cross-linking assembly and catalytic applications. The primer sequences designed for constructing the recombinant plasmid pET28a-KTag-AaFDH-SpyTag-6His are shown in Table 2.

[0088] Table 2. Primers designed for constructing the recombinant plasmid pET28a-KTag-AaFDH-SpyTag-6His

[0089]

[0090] Example 2

[0091] Co-expression and self-crosslinking of recombinant proteins

[0092] Co-expression of SpyCatcher-BcAmDH-QTag and KTag-AaFDH-SpyTag

[0093] First, the co-expression conditions of the SpyCatcher-BcAmDH-QTag and KTag-AaFDH-SpyTag fusion proteins were optimized: 10 μL of engineered E. coli culture carrying the bicistronic recombinant expression plasmid was inoculated into 10 mL of LB broth containing kanamycin resistance (final concentration 50 μg / mL) and pre-cultured overnight at 37℃ and 220 rpm; subsequently, 0.5 mL of the pre-cultured culture was inoculated into 50 mL of LB broth containing kanamycin resistance for expansion culture, and cultured at 37℃ and 220 rpm with shaking until the culture reached OD. 600 When the value reached 0.6–0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.2 mM, and expression was induced at 16℃–23℃ for 18 h to achieve co-expression of SpyCatcher-BcAmDH-QTag and KTag-AaFDH-SpyTag in the same Escherichia coli.

[0094] Self-crosslinking of SpyCatcher-BcAmDH-QTag and KTag-AaFDH-SpyTag

[0095] After induction, bacterial cells were collected by centrifugation and sonicated to obtain cell lysates containing the target fusion protein. Transglutaminase (MTG) with the nucleotide sequence shown in SEQ ID No. 7 was then added to the lysates, and the mixture was incubated at 30°C for 4 h. This catalyzed a specific cross-linking reaction between the KTag and QTag tags, inducing the self-assembly of the two enzyme molecules into ordered aggregates. After incubation, the cross-linked particles were collected by centrifugation at 12000 rpm for 10 min and washed three times with phosphate buffer (pH 7.5) to remove unreacted proteins and impurities, yielding crystallized and reinforced bio-enzyme cross-links. This process required no additional carrier.

[0096] In this embodiment, the protein expression analysis diagrams of BcAmDH and AaFDH and the MALDI-TOF mass spectrometry analysis diagrams are shown in Figures 3 and 4, respectively. The results show that the SpyCatcher-BcAmDH-QTag and KTag-AaFDH-SpyTag fusion proteins were correctly expressed, and their molecular weight and amino acid sequence were completely consistent with the design. Subsequent SDS-PAGE and dynamic light scattering analysis confirmed that after MTG cross-linking, the two enzyme molecules formed a stable and ordered aggregate, and the carrier-free self-crosslinking enzyme aggregate was successfully constructed.

[0097] Example 3

[0098] Preparation and assembly of the K4-SpyCatcher-6His (KSC) protein backbone

[0099] By inserting four lysine residues into the N-terminus of the CnaB2 domain SpyCatcher derived from Streptococcus pyogenes, the K4-SpyCatcher structure was obtained. By increasing the number of N-terminal lysine residues, glutaraldehyde is more likely to undergo cross-linking reactions in the N-terminal region of the K4-SpyCatcher, exposing the domain to the outside and thus forming a multi-level spatial cross-linked structure.

[0100] Based on the K4 domain and the SpyCatcher gene sequence, upstream and downstream primers were designed, with appropriate restriction endonuclease sites introduced at both ends of the primers. The recombinant plasmid K4-SpyCatcher-6His was constructed using primers listed in Table 3. Expression was induced at 23℃ for 20 h using IPTG at a final concentration of 0.1 mM to obtain the recombinant K4-SpyCatcher-6His protein. After purification and concentration, the protein concentration was adjusted to 10 mg / mL for protein backbone preparation. Take 4 mg of K4-SpyCatcher-6His protein solution, add twice the volume of saturated ammonium sulfate solution, and precipitate at 4℃ for 1 h. Mix the ammonium sulfate-precipitated protein solution with glutaraldehyde at four volume ratios of 1:10, 1:20, 1:30, and 1:40, and vortex at 4℃ and 100 rpm to complete cross-linking. After cross-linking, wash three times each with 2 M NaCl solution and PBS buffer to obtain the K4-SpyCatcher-6His protein backbone, denoted as KSC protein backbone, with the nucleotide sequence shown in SEQ ID No. 12. The cross-linking rate of KSC protein is calculated by measuring the protein concentration in the supernatant. The cross-linking rate calculation formula is as follows:

[0101] KSC protein cross-linking rate = (Total protein content - Supernatant protein content) / Total protein content × 100%

[0102] Table 3. Primers designed for the construction of recombinant plasmid K4-SpyCatcher-6His

[0103]

[0104] Take 5 mg of the KSC protein backbone prepared from K4-SpyCatcher-6His protein, add it to the lysed pET28a-KTag-AaFDH-SpyTag-GFP-6His crude enzyme solution, and vortex at 4℃ and 200 rpm for 2 h to crosslink, allowing the SpyTag on the enzyme molecule to specifically covalently bind to the SpyCatcher on the KSC backbone. After the reaction, centrifuge at 8000 rpm for 2 min, collect the enzyme aggregates, and wash three times each with 2 M NaCl solution and PBS buffer (20 mM, pH 8.5) to remove physically adsorbed contaminating proteins. Subsequently, the lysed pET28a-SpyCatcher-BcAmDH-QTag-mCherry-6His crude enzyme solution was added to the above complex, along with transglutaminase MTG with the nucleotide sequence shown in SEQ ID No. 7. This catalyzed the covalent cross-linking of QTag on BcAmDH with KTag on AaFDH, while simultaneously further covalently linking SpyCatcher on BcAmDH with SpyTag on AaFDH. After the reaction was complete, the enzyme aggregates were collected by centrifugation and washed to obtain KSC@AaFDH-BcAmDH OC-EAs, which were then immersed in PBS buffer and stored at 4°C for later use. Figure 5 This diagram illustrates the synthesis and catalytic function of KSC@AaFDH-BcAmDH OC-EAs, including the construction of its unit enzyme cross-linkers, a comparison of the protein covalent coupling mechanisms mediated by SpyTag / SpyCatcher and QTag / KTag, and the reaction mechanism of the cascade catalytic synthesis of R-(+)-α-methylbenzylamine by BcAmDH and AaFDH. Figure 6 Multichannel fluorescence microscopy images of the two enzymes assembled: the green channel shows the distribution of BcAmDH, the red channel shows the distribution of AaFDH, the yellow superimposed channel confirms the co-localization of the two enzymes on the carrier, and the bright-field image shows the morphology of the carrier; the results show that the two enzymes achieved ordered assembly and spatial proximity distribution on the protein backbone through biorthogonal covalent cross-linking, providing a structural basis for the efficient cascade catalysis.

[0105] Characterization analysis of the two-enzyme immobilization system

[0106] Following the directed assembly of the K4-SpyCatcher-6His (KSC) protein backbone, AaFDH, and BcAmDH, the successful construction and structural integrity of the ordered cascade enzyme assembly (OC-EAs) were verified. First, the structural and morphological changes of the conjugated protein scaffold were investigated using scanning electron microscopy (SEM). Figure 7 , Figure 8The images are SEM morphology images, using 10 μm and 1 μm scale bars to display the microstructure and surface morphology of the protein backbone and cascade enzyme assemblies. The results show that the KSC protein scaffold possesses excellent multi-enzyme covalent immobilization adaptability, and the dense enzyme network formed within the scaffold can generate a macromolecular crowding effect, further enhancing enzymatic catalytic efficiency.

[0107] Secondly, to demonstrate the multilayer immobilization of the enzyme protein, mCherry (red fluorescent protein, emission excitation wavelength 610nm-587nm) and GFP (green fluorescent protein, emission excitation wavelength 507nm-484nm) were fused to the C-terminus of pET28a-SpyCatcher-BcAmDH-QTag-6His and pET28a-KTag-AaFDH-SpyTag-6His, respectively, according to Table 4, by PCR digestion and enzyme ligation. Following the double-layer immobilization method, the cross-linking order was pET28a-KTag-AaFDH-SpyTag-GFP-6His and pET28a-SpyCatcher-BcAmDH-QTag-mCherry-6His. After lysing both cell types, transglutaminase MTG was added for cross-linking. The cross-linked particles were collected by centrifugation, washed, and KSC@BcAmDH-AaFDH was prepared. The synthesis and catalytic function of KSC@AaFDH-BcAmDH are described in the following figures. Figure 5 As shown, Figure 6 The images show fluorescence microscopy images of KSC@AaFDH-BcAmDH (OC-EAs) under different fluorescence channels. The results show that the double enzyme layer is spatially ordered and fixed on the KSC protein backbone.

[0108] Table 4. Primers designed for the construction of BcAmDH / AaFDH series protein fusion fluorescent proteins

[0109]

[0110] Example 4

[0111] Catalytic Analysis of Enzyme Crosslinkers OC-EAs

[0112] R-(+)-α-methylbenzylamine was synthesized using acetophenone as a substrate, catalyzed by two multilayer immobilized enzymes, KSC@BcAmDH-AaFDH. The KSC@BcAmDH-AaFDH reaction system consisted of 2 mL of acetophenone containing 5 g / L, 20% DMSO, and 1 mM NAD. +KSC@BcAmDH-AaFDH with 1 g / L immobilized enzyme (BcAmDH protease content) was used. The final volume was 2 M ammonium formate (pH 8.0-9.0), and the reaction was carried out at 30℃ with shaking at 200 rpm / min for 12 h. After the reaction, NaOH solution was added to adjust the pH to 9.0-10.0. 1 mL of the reaction solution was added to an equal volume of acetonitrile, and the yield was determined by high-performance liquid chromatography (HPLC). The reaction solution was filtered through a 0.22 μm organic filter and detected by chiral HPLC (Shimadzu LC-6). An OD-H column (4.6 mm × 250 mm, 5 μm) was used, with a mobile phase of hexane / isopropanol (80:20, v / v) pumped at a flow rate of 1 mL / min. The injection volume was 10 μL, the column temperature was set at 30°C, and the detection wavelength was 210 nm.

[0113] In the asymmetric synthesis system of R-(+)-α-methylbenzylamine, at least one of DPPA, DPAA, 34CA, and 43CNA was added in 1.0-4.0 equivalents to induce crystallization of the target product R-(+)-α-methylbenzylamine, thereby shifting the reaction equilibrium towards product formation. The resulting crystals were collected by vacuum filtration and then thoroughly washed with methyl tert-butyl ether (MTBE). A 2 M sodium hydroxide solution was then introduced, and the crystalline salt was dissolved by vigorous vortex mixing. Subsequently, 400 μL of dichloromethane was added to the aqueous phase to promote the extraction of the final product, followed by high-performance liquid chromatography (HPLC) analysis according to the above protocol. The ee value was calculated using the following formula.

[0114] R-α-methylbenzylamine ee value = (R configuration product content - S configuration product content) / (R configuration product content + S configuration product content) × 100%.

[0115] Liquid phase detection results as follows Figure 9 As shown. Figure 9 To prepare R-(+)-α-methylbenzylamine by HPLC using OC-EA and a free two-enzyme system. Peak A represents the substrate acetophenone, and peak B represents the product R-(+)-α-phenylethylamine. Results showed that without the addition of an in-situ crystallization additive, the substrate conversion rate of the free two-enzyme system was only 25.6%, while the OC-EA system increased the R-(+)-α-methylbenzylamine conversion to 49.7%. After introducing the in-situ crystallization additive into the OC-EA system, the R-(+)-α-methylbenzylamine conversion reached 77.4%, approximately 3.02 times that of the free two-enzyme system, with an enantiomeric excess (ee) greater than 99.99%. Adding the in-situ crystallization additive to the free two-enzyme system, however, led to a significant decrease in substrate conversion.

[0116] R-(+)-α-methylbenzylamine was reduced at 30 °C for 10 hours. After each cycle, the immobilized enzyme was recovered by centrifugation and reused in subsequent reactions. The operational stability of OC-EAs in repeated catalytic cycles is shown in the figure. Figure 10 As shown, after six consecutive cycles and 60 hours, KSC@FDH-AmDH retained approximately 70% of its initial activity.

[0117] Example 5

[0118] Recovery of in-situ crystallization additives

[0119] Deionized water was added to the collected DPPA-amine crystal mixture at a liquid-to-solid volume ratio of 5:1 (v / w). Then, 10 mol / L NaOH aqueous solution was added dropwise while magnetically stirring at 300–400 r / min to adjust the pH to >13. Stirring continued for 20–30 min until the DPPA-amine crystal mixture was completely dissolved, allowing both the amine product and DPPA to enter the aqueous phase, eliminating the solid-phase crystal morphology and yielding a homogeneous aqueous system. Methyl tert-butyl ether (MTBE) was added to the homogeneous aqueous system, maintaining an aqueous-to-MTBE ratio of 1:1 (v / v). Extraction was performed at 25–30 °C with magnetic stirring at 300 r / min for 15–20 min. The mixture was then allowed to stand for 15–20 min to separate the phases. The upper organic phase (containing highly enantioselective R-(+)-α-methylbenzylamine) was collected. This organic phase was then subjected to vacuum distillation to remove MTBE, yielding the purified product.

[0120] A 2M hydrochloric acid aqueous solution was slowly added dropwise to the raffinate aqueous phase under magnetic stirring (200~300 r / min). The pH of the system was monitored in real time and adjusted to <4. After the addition was completed, stirring was continued at 4℃ for 10~15 min to allow DPPA to quantitatively and directionally precipitate from the aqueous phase, forming a white suspension. Solid DPPA was recovered by vacuum filtration. High-performance liquid chromatography (HPLC) analysis showed that the purity of the recovered in-situ crystallization additive solid was ≥95%.

[0121] Example 6

[0122] Continuous fixed-bed reactor for the highly enantioselective synthesis of R-(+)-α-methylbenzylamine

[0123] 1. Reaction apparatus

[0124] Reference Figure 11 A continuous flow apparatus for implementing the above-mentioned crystallization-enhanced bio-enzyme crosslinking body includes:

[0125] Main apparatus: 500mL glass reaction column 1 (effective volume 500mL), with a condensation circulation jacket 2 on the outside of the reaction column for circulating cooling medium to maintain the reaction temperature;

[0126] Retention component: A glass frit 3 (pore size 10-20μm) is built into the glass reaction column at 1 / 3 of its height to retain solid crystals formed by one of DPPA, DPAA, 34CA, and 43CNA in the reaction solution with amines;

[0127] Feeding components: The top of the reaction column is equipped with an injection port 4 for continuous or intermittent replenishment of substrate and NAD. + and NH4COOH;

[0128] Slag discharge component: Glass valve 5 is connected below the glass reaction column, and the discharge of the trapped solid crystals is controlled by opening / closing the valve;

[0129] Circulation component: A peristaltic pump (model BT100-1F) is connected to the bottom of the glass valve via a pipeline. The outlet of the peristaltic pump is connected to the upper part of the glass reaction column via a return pipeline, forming a reaction liquid circulation loop, which drives the reaction liquid to circulate continuously within the reaction column.

[0130] 2. Preparation of the reaction system

[0131] Prepare a 500mL reaction system according to the following proportions, with the final concentrations of each component as follows:

[0132] Substrate: Acetophenone 5 g / L;

[0133] Coenzyme: NAD + 1mM;

[0134] Immobilized enzyme: BcAmDH (amine dehydrogenase) 1g / L (enzyme activity ≥100U / mg based on protein content).

[0135] Solvent system: 20% (v / v) DMSO mixed with 80% (v / v) buffer;

[0136] Buffer solution: 2M NH4COOH buffer, adjusted to pH 8.5 with 2M ammonia (NH4OH);

[0137] The above components are added to the glass reaction column through the injection port at the top of the reaction column. The complete device is assembled and the tubing is checked for tightness.

[0138] 3. Control of reaction conditions

[0139] Temperature control: A 30℃ constant temperature water bath is introduced into the external condensation circulation jacket of the reaction column, and the medium flow rate in the jacket is maintained at 5-8 mL / min by the circulation pump to ensure that the temperature of the reaction system is stable at 30±0.5℃;

[0140] Circulation control: Start the peristaltic pump and adjust the pump speed to 0.1-0.5 mL / min to allow the reaction solution to circulate continuously in the reaction column through the reflux pipeline. During the circulation process, the glass frit core retains the DPPA / DPAA / 34CA / 43CNA-amine solid crystals generated in the reaction.

[0141] Feed control: During the reaction, the consumed substrates acetophenone and NAD are intermittently replenished through the top injection port. + And NH4COOH, each time the amount added is 10% of the initial amount, and the addition interval is 4 hours, to maintain the stability of the component concentration in the reaction system;

[0142] Slag discharge control: After the reaction has proceeded for 8 hours, the peristaltic pump is turned off, and the glass valve below the glass reaction column is slowly opened to discharge the trapped solid crystals. After the slag discharge is completed, the valve is closed, the peristaltic pump is restarted, and the reaction continues. The total reaction time is 24 hours.

[0143] 4. Post-processing and product testing

[0144] After the reaction is complete, stop the condensation circulation and peristaltic pump, and drain the reaction solution from the bottom of the reaction column. Adjust the pH of the reaction solution to 9-10 with 40% (w / v) NaOH solution and stir for 10 minutes.

[0145] Acetonitrile extraction (extraction ratio 1:1, v / v) was performed three times. The organic phases were combined, dried over anhydrous sodium sulfate, and then distilled under reduced pressure (vacuum degree 0.08 MPa, distillation temperature 65-70℃) to obtain R-(+)-α-methylbenzylamine product.

[0146] Product detection: The enantioselectivity of the product was detected by high performance liquid chromatography (HPLC, chiral column OD-H), and the ee value was measured to be ≥99.9%.

[0147] 5. Advantages of the device

[0148] This embodiment utilizes a continuous circulating reaction device with condensation circulation. By using a glass frit core to trap solid crystals and a peristaltic pump to drive the circulation of the reaction liquid, it achieves continuous substrate transformation and efficient product separation. The condensation jacket effectively controls the reaction temperature, avoiding enzyme activity loss. The glass valve facilitates the discharge of solid impurities, ensuring the continuous and stable progress of the reaction. Ultimately, a highly enantioselective and high-purity R-(+)-α-methylbenzylamine product is obtained, suitable for industrial scale-up applications.

[0149] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A crystal-reinforced bio-enzyme cross-linker, characterized in that, The bioenzyme cross-linker is a spatially ordered cascade structure formed by the covalent assembly of amine dehydrogenase and formate dehydrogenase; wherein the nucleotide sequence of the amine dehydrogenase is shown in SEQ ID No. 1, and the nucleotide sequence of the formate dehydrogenase is shown in SEQ ID No.

2.

2. The crystal-strengthened bio-enzyme crosslinker according to claim 1, characterized in that, The amine dehydrogenase and formate dehydrogenase are specifically and irreversibly linked through a SpyTag / SpyCatcher, QTag / KTag biorthogonal covalent cross-linking system to form a spatially ordered cascade enzyme cross-linker. The nucleotide sequence of SpyTag is shown in SEQ ID No. 3, the nucleotide sequence of SpyCatcher is shown in SEQ ID No. 4, the nucleotide sequence of QTag is shown in SEQ ID No. 5, and the nucleotide sequence of KTag is shown in SEQ ID No.

6.

3. The method for preparing the crystal-reinforced bio-enzyme crosslinker according to any one of claims 1-2, characterized in that, Includes the following steps: (1) SpyCatcher-BcAmDH-QTag and KTag-AaFDH-SpyTag were co-expressed in the same E. coli; (2) After cell lysis, transglutaminase MTG with the nucleotide sequence shown in SEQ ID No.7 was added for cross-linking of KTag and QTag; (3) The cross-linked particles were collected by centrifugation, washed, and crystal-enhanced bioenzyme cross-linked bodies were obtained without the need for additional carriers.

4. The preparation method according to claim 3, characterized in that, The construction and expression of SpyCatcher-BcAmDH-Qtag in E. coli are as follows: (I) First round of polymerase chain reaction PCR: Using the BcAmDH gene as a template, BcAmDH-QTag F1 and BcAmDH-QTag R1 were used as upstream and downstream primers, while SpyCatcher was used as a template, and SpyCatcher-linker F and SpyCatcher-linker R were used as upstream and downstream primers. After PCR amplification, the purified BcAmDH-QTag' fragment and SpyCatcher-linker fragment were obtained by recovery. (II) Second round of PCR: Using the BcAmDH-QTag' fragment obtained in the first round as a template, BcAmDH-QTag F2 and BcAmDH-QTag R2 were used as upstream and downstream primers, respectively. After PCR amplification, the purified BcAmDH-QTag fragment was obtained by recovery. (III) Third round of PCR: Using the BcAmDH-QTag fragment obtained in the second round and SpyCatcher-linker as templates, SpyCatcher-linker F and BcAmDH-QTag R were used as upstream and downstream primers. After PCR amplification, the purified SpyCatcher-BcAmDH-QTag fragment was obtained by recovery. (IV) Heterologous expression was performed in Escherichia coli Bl21. The SpyCatcher-BcAmDH-QTag gene fragment was inserted between NcoⅠ and XhoⅠ in pET-28a(+) by double digestion and ligation. An NdeⅠ restriction site was added between SpyCatcher-linker and BcAmDH-QTag. A 6×His tag was added to the C-terminus to construct the complete pET28a-SpyCatcher-BcAmDH-QTag-6His recombinant plasmid.

5. The preparation method according to claim 3, characterized in that, The construction and expression of KTag-AaFDH-SpyTag in E. coli are as follows: (i) First round of PCR: Using the formate dehydrogenase AaFDH gene as a template, KTag-AaFDH-SpyTag F1 and KTag-AaFDH-SpyTag R1 were used as upstream and downstream primers. After PCR amplification, the purified KTag-AaFDH-SpyTag' fragment was obtained by recovery. (ii) Second round of PCR: Using the KTag-AaFDH-SpyTag' fragment obtained in the first round as a template, KTag-AaFDH-SpyTag F2 and KTag-AaFDH-SpyTag R2 were used as upstream and downstream primers. After PCR amplification, the purified KTag-AaFDH-SpyTag fragment was obtained by recovery. (iii) Subsequently, heterologous expression was performed in E. coli Bl21. The KTag-AaFDH-SpyTag gene fragment was inserted between NcoⅠ and XhoⅠ of pET-28a(+) by double digestion and ligation, with a 6×His tag at the C-terminus, to construct the complete pET28a-KTag-AaFDH-SpyTag-6His recombinant plasmid.

6. The use of the crystal-reinforced bioenzyme cross-linker of claim 1 in the synthesis of R-(+)-α-methylbenzylamine, characterized in that... Includes the following steps: (a) Using acetophenone as a substrate, ammonium formate as an amino donor, and NAD+ as a substrate. + (a) Asymmetric reduction amination reaction was carried out under the catalysis of a crystallization-enhanced bioenzyme cross-linker as a coenzyme; (b) an in-situ crystallization additive was added to the reaction system; (c) after the reaction was completed, the pH of the reaction solution was adjusted, and R-(+)-α-methylbenzylamine with enantioselectivity ≥99.9% and conversion ≥77.4% was obtained by extraction.

7. The application according to claim 6, characterized in that, In step (a), the reaction conditions are controlled as follows: pH 8.0~9.0, reaction temperature 25~40 ℃, and stirring speed 100~300 rpm; in step (b), the in-situ crystallization additive is one of 3,3-diphenylpropionic acid, diphenylacetic acid, 3,4-dichlorobenzoic acid, and 4-chloro-3-naphthoic acid, the amount of in-situ crystallization additive added is 1.0~4.0 equivalents, the reaction temperature is 25~35℃, and the reaction time is 6~12 h.

8. The application according to claim 6, characterized in that, The crystallization-enhanced bio-enzyme cross-linker can be recycled for more than 6 batches, and the conversion rate is still ≥60%.

9. A continuous flow apparatus for implementing the crystal-strengthened bioenzyme crosslinker of claim 1, characterized in that, include: Main apparatus: includes a reaction column, with a reaction chamber inside the reaction column for enzyme cross-linking catalysis; and a condensation circulation jacket outside the reaction column; Retention components: Glass frit embedded in the reaction column; Feeding components: The top of the reaction column is equipped with an injection port for continuous or intermittent feeding; Slag discharge component: A glass valve is connected below the reaction column, and the opening / closing of the valve controls the discharge of the trapped solid crystals; Circulation component: A peristaltic pump is connected to the bottom of the glass valve via a pipeline. The outlet of the peristaltic pump is connected to the upper part of the reaction column via a return pipeline, forming a reaction liquid circulation loop, which drives the reaction liquid to circulate continuously within the reaction column.

10. The apparatus according to claim 9, characterized in that, The reaction conditions are controlled as follows: Temperature control: A constant temperature water bath of 25~35℃ is introduced into the external condensation circulation jacket of the reaction column, and the medium flow rate in the jacket is maintained at 5~8mL / min by the circulation pump; Circulation control: Start the peristaltic pump and adjust the pump speed to 0.1~0.5 mL / min to make the reaction solution continuously circulate in the reaction column through the reflux pipeline. During the circulation process, the glass frit core traps the solid ammonium salt crystals generated in the reaction. Feed control: During the reaction, the consumed substrates acetophenone and NAD are intermittently replenished through the top injection port. + and ammonium formate; Slag discharge control: After the reaction has proceeded for 6 to 10 hours, turn off the peristaltic pump, open the glass valve below the reaction column to discharge the trapped solid crystals, close the valve after slag discharge, restart the peristaltic pump to continue the reaction, and the total reaction time is 20 to 28 hours.

Images