Recombinant immobilized enzyme, expression gene thereof and application of recombinant immobilized enzyme in production of macrocyclodextrin

Abstract

The invention belongs to the technical field of enzyme engineering and gene engineering, and particularly relates to a recombinant immobilized enzyme, an expression gene thereof and application of the recombinant immobilized enzyme in production of macrocyclodextrin. According to the preparation method, SpyTag and SpyCatcher peptides are fused to the N end of isoamylase and the N end of 4-alpha-glucanotransferase respectively, the 4-alpha-glucanotransferase modified by the SpyTag and the isoamylase modified by the SpyCatcher are obtained, and after spontaneous isopeptide bonds are formed between the SpyTag and the SpyCatcher, the self-assembled macrocyclodextrin production enzyme cluster is obtained. The obtained macrocyclodextrin production enzyme cluster forms an enzyme aggregate with a three-dimensional structure under the traditional glutaraldehyde-mediated catalytic action, namely the recombinant immobilized enzyme. The recombinant immobilized enzyme disclosed by the invention can be repeatedly utilized, is simple and convenient to operate, is easy to separate a converted product, and can be applied to industrial production of the macrocyclodextrin.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and genetic engineering technology. Specifically, it relates to a recombinant immobilized enzyme and its expression gene, and its application in the production of macrodextrin. Background Technology

[0002] Cyclodextrin (CD) is a general term for a class of polysaccharides that link different numbers of glucose units into a ring through α-1,4-glycosidic bonds. Large ring cyclodextrin (LRCD) is a general term for a series of cyclic dextrins with more than 8 glucose units. Compared with small ring CD, LRCD has a larger internal cavity size, higher water solubility and very low viscosity, is safe and non-toxic, and has unique encapsulation properties.

[0003] Macrocyclodextrins, due to their high water solubility, low viscosity, and non-retrogradation properties, can be widely used in the food, chemical, and pharmaceutical industries. Macrocyclodextrins are more soluble and stable in cold water than cyclodextrins. Furthermore, macrocyclodextrins can form inclusion complexes with various types of guest molecules and influence the properties of guest molecules through complex formation. Macrocyclodextrins have been used as effective artificial chaperones for protein refolding and as polysaccharide carriers in gene delivery systems. In addition, macrocyclodextrins improve the solubility and bioavailability of drug compounds. Despite the enormous potential of macrocyclodextrins as high-value-added biomaterials, their industrial application remains limited due to their high production costs.

[0004] Macrodextrin synthases mainly employ cyclodextrin glucosyltransferases and 4-α-glucantransferases. Cyclodextrin glucosyltransferase (EC2.4.1.19, CGTase), as a member of the amylase family, catalyzes the intramolecular glycosylation (cyclization) reaction between starch and linear α-1,4-glucose to form LRCD. The degree of polymerization of LRCD synthesized by CGTase ranges from 9 to 60. With prolonged reaction time, the number of macrocyclic CDs decreases, converting to CD6-CD8. Studies have found that several 4-α-glucantransferases (EC2.4.1.25), especially D-enzymes and maltose transglycosylation enzymes (Amylomaltase, AM), can act on starch to produce LRCD. D-enzymes catalyze the cyclization reaction of amylose, generating LRCDs with degrees of polymerization ranging from 17 to several hundred. AM catalyzes intramolecular and intermolecular transglycosylation reactions of starch substrates, producing LRCD and linear maltodextrin, respectively. However, compared to CGTase, the amount of LRCD with high polymerization degree produced by D-enzymes and AM decreases with prolonged reaction time due to the coupling reaction. In 2002, research found that glycogenolytic enzymes (GDE, EC2.4.1.25 / EC3.2.1.33) possess 4-α-glucosyltransferase and amylase-1,6-glucosidase activities, and can also produce LRCD mixtures. GDE derived from Saccharomyces cerevisiae can act on amylose, amylopectin, and DBS (short-chain starch) to prepare LRCD mixtures with a polymerization degree ranging from 11 to 40. Different enzyme preparations can be used to produce LRCD mixtures with different polymerization degrees.

[0005] Traditionally, macrodextrins are produced from amylose or intact starch polymers via intramolecular transglycosylation catalyzed by 4-α-glucan transferases. Synthetically produced potato amylose, primarily composed of α-1,4-linked amylose, has long been the primary substrate for macrodextrin production. However, utilizing isolated amylose is extremely expensive and time-consuming, limiting the large-scale production and industrial application of macrodextrins. Furthermore, using intact starch polymers containing both amylose and highly amylopectin as substrates results in lower macrodextrin yields.

[0006] There are few reports on macrocyclodextrin yield studies both domestically and internationally. In 2014, Yan Xu et al. first reported a biotransformation process for producing macrocyclodextrins (LRCDs) using the combined action of isoamylase and TA 4GTase. This process uses natural starch substrates containing a large amount of amylopectin, and the conversion rate of the debranched LRCD product is high. Compared with amylopectin from high-amylose corn, debranched high-amylose corn amylopectin is a more suitable substrate for LRCD production. The maximum conversion rate of LRCD products from high-amylose corn was 24.55%, while after debranching, the conversion rate reached 45.58% in an aqueous reaction system. Regardless of the reaction conditions, the minimum yield (DP) of LRCD treated with TA 4GTase was 5, and the number of smaller cyclic dextran decreased as the reaction proceeded. In 2016, Sun Chu et al. used enzymatic hydrolysis to debranch starch, and then 4-α-GTase used these amylose as intermediate substrates for cyclization, achieving a maximum yield of 48.56%, which is the highest production efficiency obtained from starch to date. In 2018, Zhu Rong et al. used ordinary potato starch as a substrate and coupled isoamylase and 4-α-GTase, achieving a maximum yield of 24.8%.

[0007] Therefore, there is an urgent need for a method for preparing macrocyclodextrins that utilizes inexpensive substrates, is easy to operate, and has the potential for large-scale industrial production. Summary of the Invention

[0008] The purpose of this invention is to provide a recombinant immobilized enzyme and its expression gene, and its application in the production of macrodextrin, so as to realize the large-scale industrial production of macrodextrin.

[0009] Therefore, the present invention provides the following technical solution.

[0010] A first aspect of the present invention provides a recombinant immobilized enzyme, the recombinant immobilized enzyme comprising: (1) 4-α-glucan transferase; (2) Isoamylase; and (3) Bridging intermediates; The bridging intermediate is SpyCatcher-SpyTag, which includes SpyCatcher and SpyTag; wherein, SpyCatcher is modified on isoamylase, and SpyTag is modified on 4-α-glucantransferase, and SpyCatcher and SpyTag react via an amide bond to generate the bridging intermediate SpyCatcher-SpyTag that couples isoamylase and 4-α-glucantransferase.

[0011] In a preferred embodiment of the present invention, the recombinant immobilized enzyme is selected from: (1) An amino acid sequence consisting of SpyTag-4αG having the amino acid sequence shown in SEQ ID NO. 10 and SpyCatcher-IA having the amino acid sequence shown in SEQ ID NO. 11; or (2) A protein whose amino acid sequence is homologous to or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the combination of SpyTag-4αG (amino acid sequence shown in SEQ ID NO. 10) and SpyCatcher-IA (amino acid sequence shown in SEQ ID NO. 11); or (3) Recombinant immobilized enzymes derived from (1) or (2) whose amino acid sequences of (1) or (2) have been substituted, deleted or added with one or more amino acids.

[0012] A second aspect of the present invention provides a nucleic acid molecule comprising a sequence selected from the following: (i) Encodes the recombinant immobilized enzyme as described above; (ii) A variant having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with (i); or (iii) Complementary sequences of (i) or (ii).

[0013] A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid molecules as described above; Preferably, the recombinant expression vector includes one or a combination of retroviral vectors, DNA vectors, plasmids, RNA vectors, adenovirus vectors, adenovirus-associated vectors, and lentiviral vectors. Preferably, the recombinant expression vector is pRB1k and / or pBAD-HisB.

[0014] A fourth aspect of the present invention provides a recombinant bacterium comprising the recombinant expression vector as described above.

[0015] A fifth aspect of the present invention provides a method for constructing the recombinant bacteria as described above, the method comprising the following steps: The gene fragments encoding 4-α-glucan transferase and SpyTag were ligated into the pRB1k vector to obtain the recombinant plasmid pRB1k-SpyTag-4αG; The gene fragments encoding isoamylase and SpyCatcher were ligated into the pBAD-HisB vector to obtain the recombinant plasmid pBAD-SpyCatcher-IA. The two recombinant plasmids obtained above were transformed into host cells to obtain the recombinant bacteria BW / pRB1k-SpyTag-4αG+pBAD-SpyCatcher-IA.

[0016] Preferably, the host cell is Escherichia coli BW25113.

[0017] A sixth aspect of the present invention provides a method for preparing the recombinant immobilized enzyme as described above, the method comprising the following steps: (1) After the recombinant bacteria described above are induced to express, the bacterial cells are collected by centrifugation, the bacterial cells are broken up and the supernatant is taken to obtain crude enzyme solution containing recombinant immobilized enzyme. (2) The crude enzyme solution was concentrated, shaken and centrifuged to obtain recombinant immobilized enzyme spheroids; (3) Cross-link the obtained recombinant immobilized enzyme spheres to obtain recombinant immobilized enzyme aggregates.

[0018] In a preferred embodiment of the present invention, in step (1), the induction conditions are: 1-2 wt% arabinose is used to induce enzyme production, the induction temperature is 25-30°C, the induction speed is 180-220 rpm, and the induction time is 14-18 h. The centrifugation conditions are: rotation speed of 6000-8000 rpm and time of 5-10 min.

[0019] In a preferred embodiment of the present invention, in step (2), the oscillation conditions are: temperature of 4-18℃, rotation speed of 180-220 rpm, and time of 3h-10h; The centrifugation conditions are: rotation speed of 8000-12000×g, and time of 5-15min.

[0020] In a preferred embodiment of the present invention, in step (3), the crosslinking is performed using glutaraldehyde as a crosslinking agent; The cross-linking process is as follows: the recombinant immobilized enzyme spheres are shaken with 0.6-1.2 wt% glutaraldehyde solution at 4℃-10℃ and 200-500 rpm for 10-30 min.

[0021] A seventh aspect of the present invention provides the use of the recombinant immobilized enzyme, nucleic acid molecule, recombinant expression vector or recombinant bacteria as described above in the production of macrodextrin.

[0022] In a preferred embodiment of the present invention, the following steps are included: Recombinant immobilized enzymes were added to a starch solution for reaction, followed by the addition of saccharifying enzymes to hydrolyze any unreacted starch. The obtained product was centrifuged, precipitated, and centrifuged again. The precipitate was then dried to obtain macrocyclodextrin. The reaction conditions are: temperature 30-70℃, rotation speed 180-220rpm, and time 6-24h.

[0023] By employing the above technical solution, the present invention has at least the following advantages: This invention involves fusing SpyTag and SpyCatcher peptides to the N-terminus of isoamylase and 4-α-glucantransferase, respectively, to obtain SpyTag-modified 4-α-glucantransferase and SpyCatcher-modified isoamylase. After spontaneous isopeptide bonds are formed between SpyTag and SpyCatcher, a self-assembled macrodextrin-producing enzyme cluster is obtained. The resulting macrodextrin-producing enzyme cluster forms a three-dimensional enzyme aggregate, i.e., a recombinant immobilized enzyme, under conventional glutaraldehyde-mediated catalysis. The recombinant immobilized enzyme of this invention exhibits good reusability and can catalyze high-yield macrodextrin production from starch. The recombinant immobilized enzyme of this invention is reusable, easy to operate, and its conversion products are easily separated, making it applicable to the industrial production of macrodextrin.

[0024] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0025] Figure 1 The spectrum of the recombinant plasmid pRB1k-SpyTag-4αG is shown; Figure 2 The map of the recombinant plasmid pBAD-SpyCatcher-IA is shown; Figure 3 The gel electrophoresis diagram of the recombinant immobilized enzyme BW / pRB1k-SpyTag-4αG + pBAD-SpyCatcher-IA is shown. Figure 4 The gel electrophoresis diagram of the complex enzyme BW / pRB1k-4αG + pBAD-IA is shown. Detailed Implementation

[0026] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] As used in this paper, SpyCatcher-SpyTag, derived from the CnaB2 domain of Streptococcus pyogenes mucin FbaB, can form stable and specific amide bonds, providing a basis for constructing modular proteins. The CnaB2 domain is divided into two parts: one is the immunoglobulin-like domain SpyCatcher, composed of 138 residues (15 kDa), and the other is the short peptide SpyTag, composed of 13 residues. When they meet, they can covalently bind to form a complete protein within minutes through the formation of an amide bond between the aspartic acid residue of SpyTag and the lysine residue of SpyCatcher. Compared with traditional methods such as chemical coupling and enzyme linkage, the SpyCatcher-SpyTag protein linking system has advantages such as simple reaction conditions, high specificity, high efficiency, large yield, and stable reaction products. In this invention, SpyTag is fused to the N-terminus of 4-αG and SpyCatcher is fused to the N-terminus of IA using PCR technology and protein engineering. The SpyTag-4-αG and SpyCatcher-IA structures were then effectively expressed in E. coli. Furthermore, through in vitro reactions, 4-α-glucan transferase 4-αG and isoamylase IA were linked via SpyCatcher-SpyTag to form a recombinant enzyme with a novel structure.

[0028] Unless otherwise specified, the 2YT liquid culture medium in this embodiment of the invention comprises: 10 g / L molecular-grade peptone, 5 g / L molecular-grade yeast extract, 5 g / L NaCl, 50 μg / mL kanamycin, 100 μg / mL ampicillin, and pH 7.0. The 2YT solid culture medium is prepared by adding 1.5 g / L agar powder to the 2YT liquid culture medium.

[0029] Example 1: Construction of Recombinant Vector 1. Construction of 4-α-glucantransferase recombinant plasmid Using the synthesized 4-α-glucan transferase (4αG) plasmid with the nucleotide sequence shown in SEQ ID NO. 1 as a template, PCR amplification was performed using primer pairs 4αG-F and 4αG-R, ST-4αG-F and 4αG-R, to obtain two target gene fragments, 4αG and SpyTag-4αG, respectively. The obtained 4αG and SpyTag-4αG gene fragments were ligated into the pRB1k vector, respectively, to obtain recombinant plasmids pRB1k-4αG and pRB1k-SpyTag-4αG. The obtained recombinant plasmids were transformed into *E. coli* Fast-T1 competent cells, and positive clones were screened on LB plates containing kanamycin. Sequencing with primer pBAD-F confirmed the correct sequencing of the recombinant plasmids pRB1k-4αG and pRB1k-SpyTag-4αG (plasmid maps shown in SEQ ID NO. 1). Figure 1 (As shown).

[0030] SEQ ID NO. 1 The sequence of 4αG-F is shown in SEQ ID NO. 2: 5'-TAAGGATCCGAGCTCGAGATGCAGATTCGCCGTAGC-3' The sequence of 4αG-R is shown in SEQ ID NO. 3: 5'-cagccaagcttcgaattcTTACAGGCGCTGATGTGC-3' The sequence of ST-4αG-F is shown in SEQ ID NO. 4: 5'-TAAGGATCCGAGCTCGAGATGGCACATATTGTTATGGTTGATGCCTATAAACCGACCAAACAGATTCGCCGTAGCTTT-3' The sequence of pBAD-F is shown in SEQ ID NO. 5: 5'-aggaggaattaaccatgggg-3' 2. Construction of recombinant plasmids containing isoamylase Using plasmid A0A147KK56 / pBAD HisB (details of which can be found in application number 201910903125.8, which is incorporated herein by reference) and the synthetic plasmid pUC57-SpyCatcher as templates, PCR amplification was performed using primer pairs SpyCatcher-F and SpyCatcher-R, SC-IA-F and SC-IA-R, yielding two target gene fragments, SpyCatcher and isoamylase (IA). The obtained SpyCatcher and IA gene fragments were ligated into the pBAD-HisB vector to obtain the recombinant plasmid pBAD-SpyCatcher-IA. The recombinant plasmid was transformed into *E. coli* Fast-T1 competent cells, and positive clones were screened on ampicillin-treated LB plates. Sequencing with primer PBAD-F confirmed the correct sequence of the recombinant plasmid pBAD-SpyCatcher-IA (plasmid map shown below). Figure 2 (As shown).

[0031] The sequence of SpyCatcher-F is shown in SEQ ID NO. 6: 5'-AGGATCCGAGCTCGAGGCTATGGTTGATACCCTGAGC-3' The sequence of SpyCatcher-R is shown in SEQ ID NO. 7: 5'-TTAAATATGTGCATCACCTT-3' The sequence of SC-IA-F is shown in SEQ ID NO. 8: 5'-AAGGTGATGCACATATTTAAatggtggaagtctggccc-3' The sequence of SC-IA-R is shown in SEQ ID NO. 9: 5'-cagccaagcttcgaattctcagtcccccgaagcctc-3' In the plasmid construction process described above, the PCR amplification system consisted of: 10 μL of 5 × SF Buffer, 1 μL of dNTP Mix (10 mM each), 20 ng of template, 2 μL each of primers (10 μM), 1 μL of Phanta Super-Fidelity DNA Polymerase (Nanjing Novizan Biotechnology Co., Ltd., product catalog P501), and 34 μL of distilled water, for a total volume of 50 μL. The PCR amplification conditions were: 95 ℃ pre-denaturation for 2 minutes (1 cycle); 95 ℃ denaturation for 10 seconds, 55 ℃ annealing for 20 seconds, and 72 ℃ extension for 1.5 minutes (30 cycles); and 72 ℃ extension for 10 minutes (1 cycle).

[0032] The specific steps for transforming recombinant plasmids into E. coli Fast-T1 competent cells during the above plasmid verification process are as follows: (1) Take a tube of Fast-T1 competent cells stored at -80℃ containing 100 μL; (2) Add the ligation product to the thawed competent cells, gently tapping them with your fingers during the addition process; (3) Place the product obtained in step (2) on ice for 20-30 min, then place it in a 42℃ water bath for 45 s for heat shock, and immediately remove it and place it back on ice for 2-5 min after the heat shock is completed; (4) Add 800 μL of 2YT liquid medium and incubate in a 37℃ shaking incubator at a shaking frequency of 200 rpm for 40 min to obtain the transformed bacterial solution; (5) Centrifuge the transformed bacterial solution from step (4) at 4000 rpm for 2 min and spread it on a 2YT solid medium plate, and incubate at 37℃ for 10-12 h; (6) Select single colonies to contain 100 μL of the product. The assay was performed in μg / mL ampicillin-resistant liquid 2YT medium at 37°C for 4–6 h.

[0033] Example 2: Construction of recombinant bacteria The recombinant plasmid pRB1k-4αG constructed in Example 1 was transformed into Escherichia coli host cell BW25113 to obtain the genetically engineered bacterium BW / pRB1k-4αG.

[0034] The recombinant plasmids pRB1k-SpyTag-4αG and pBAD-SpyCatcher-IA constructed in Example 1 were co-transformed into Escherichia coli host cells BW25113 to obtain the genetically engineered bacteria BW / pRB1k-SpyTag-4αG+pBAD-SpyCatcher-IA, with a recombinant immobilized enzyme molecular weight of approximately 148 kDa.

[0035] The amino acid sequence of SpyTag-4αG is shown in SEQ ID NO. 10: MAHIVMVDAYKPTKMQIRRSFGVLLHPTSFPGRWGIGALGLEAERFLDWLAQAGARWWQVLPLGPTSYGDSPYQSFSAFAGNPYLIDPERLFAQGWLEEEAPPNYPPDFVDYGQLYQTRWPLLRRAFA GFEARASRSREALAAFVQAERAWLEDYALFMALKNRFGGRPWNQWSPELRDREPAALAKAREELAYEVALHEWVQWQFYTEGRIKAYAEARGIQIIGDMPIFVAFDSADVWAHPEYFYLDESGQPTVV AGVPPDYFSETGQLWGNPLYRWEVMEADGFSWWVARIRQALKQAHLVRIDHFRGFEAYWEIPFGRPDAVEGRWVKAPGEKLFQAVRQALGDAPIIAEDLGVITPEVEALRDAFGFPGMKILQFAFSDE SNPFLPHYPEHGHVVVYSGTHDNDTTLGWFRTAPESERAFMRAYLARQGIRCLSEYEAAGALIELAFQSRAKLAVVPLQDVLGLGPEARMNYPGRLGGNWAWRYREGDLEPGLAQSLRALAKAHQRL* The amino acid sequence of SpyCatcher-IA is shown in SEQ ID NO. 11: * The recombinant plasmids pRB1k-4αG and A0A147KK56 / pBAD HisB constructed in Example 1 were co-transformed into Escherichia coli host cells BW25113 (as described in application number 201910903125.8) to obtain the genetically engineered bacteria BW / pRB1k-4αG+pBAD-IA.

[0036] The specific steps for transforming host cells with the above recombinant plasmid are as follows: (1) Take a vial of E. coli BW25113 competent cells stored at -80℃ containing 50~200 μL and thaw it on ice for 20 min; (2) Take 2~3 μL of the verified correct recombinant plasmid solution and add it to the thawed competent cells, gently flicking it with your fingers during the addition process; (3) Place the product of step (2) on ice for 20~30 min, and then place it in a 42℃ water bath for 90 s for heat shock. After the heat shock is completed, immediately remove it and place it back on ice to stand for 2~5 min; (4) Add 800 μL of 2YT liquid culture medium and culture it in a 37℃ shaking incubator with a shaking frequency of 200 rpm for 40 min to obtain the transformation bacterial solution; (5) Take 50 μL of the transformation bacterial solution from step (4) and spread it on a 2YT solid culture medium plate, and culture it at 37℃ for 10~12 minutes. h; (6) Select a single colony into liquid 2YT medium containing the corresponding antibiotic and incubate at 37℃ for 6~8 h; (7) Take an equal volume of bacterial solution and mix it with an equal volume of sterile 80% (volume) glycerol solution and place it in a preservation tube and store it in a -80℃ refrigerator for later use.

[0037] Example 3: Preparation of recombinant immobilized enzyme 1. Preparation of recombinant immobilized enzymes The genetically engineered bacteria BW / pRB1k-SpyTag-4αG+pBAD-SpyCatcher-IA constructed in Example 2 above were inoculated into 2YT liquid medium and cultured at 37℃ for 4-6 h. Then, the resulting culture was inoculated into shake flasks at an inoculation rate of 1-2% and cultured at 37℃ and 200 rpm for 2-3 h. Enzyme production was then induced with arabinose at a final concentration of 1-2 wt% at an induction temperature of 25-30℃, an induction speed of 220 rpm, and an induction time of 14-18 h. After induction, the bacterial culture was centrifuged at 8000 rpm for 5 minutes to collect the cell pellet. The cell pellet was resuspended in 50 mM PBS buffer (pH 7) to obtain a cell suspension. The cell suspension was lysed by sonication (25 kHz, 315 W, 10 min), centrifuged, and the supernatant and pellet were used to prepare protein samples for electrophoresis. The gel electrophoresis images of each protein are shown in [Figure number missing]. Figure 3 .like Figure 3 As shown, the molecular weight of the 4αG protein is approximately 57 kDa, the molecular weight of the IA protein is approximately 79 kDa, the molecular weight of the SpyCatcher is approximately 12 kDa, and the molecular weight of the resulting self-assembled fusion protein recombinant immobilized enzyme is approximately 148 kDa.

[0038] The supernatant after sonication was concentrated to 2 mL, and then shaken at 220 rpm for 3 h at 18 °C to produce recombinant immobilized enzyme spheroids. The shaken product was centrifuged at 12,000 × g for 15 min. After centrifugation, the obtained recombinant immobilized enzyme spheroids were washed three times with PBS buffer (pH 7). Then, glutaraldehyde was used as a cross-linking agent, and the obtained recombinant immobilized enzyme spheroids were shaken with 1.2 wt% glutaraldehyde solution at 220 rpm for 30 min at 4 °C to prepare recombinant immobilized enzyme aggregates, i.e., recombinant immobilized enzyme.

[0039] 2. Preparation of the complex enzyme 4αG+IA The genetically engineered bacteria BW / pRB1k-4αG+pBAD-IA obtained in Example 2 were inoculated into 2YT liquid medium and cultured at 37°C for 4-6 h. Then, the resulting culture was inoculated into shake flasks at an inoculation rate of 1-2% and cultured at 37°C and 200 rpm for 2-3 h. Enzyme production was then induced with arabinose at a final concentration of 1-2 wt% at an induction temperature of 25-30°C, an induction speed of 220 rpm, and an induction time of 14-18 h. After induction, the resulting culture was centrifuged at 8000 rpm for 5 minutes to collect the bacterial cell pellet, which was then frozen for later use.

[0040] The obtained products were subjected to gel electrophoresis, and the results are shown in the figure. Figure 4 .like Figure 4 As shown, the molecular weight of 4-α-glucan transferase 4αG protein is 57 kDa, and the molecular weight of isoamylase IA protein is 79 kDa.

[0041] 3. Preparation of 4-α-glucantransferase 4αG The genetically engineered bacteria BW / pRB1k-4αG obtained in Example 1 was inoculated into 2YT liquid medium and cultured at 37°C for 4-6 h. Then, the resulting culture was inoculated into shake flasks at an inoculation rate of 1-2% and cultured at 37°C and 200 rpm for 2-3 h. Enzyme production was then induced with arabinose at a final concentration of 1-2 wt% at an induction temperature of 25-30°C, an induction speed of 220 rpm, and an induction time of 14-18 h. After induction, the resulting culture was centrifuged at 8000 rpm for 5 minutes to collect the bacterial cell pellet, which was then frozen for later use.

[0042] Example 4: Preparation of macrocyclodextrin Weigh 10g of cassava starch and dissolve it in 100mL of water to obtain a cassava starch solution. Add 2g of the compound enzyme 4αG+IA prepared in Example 3, or 2g of the 4-α-glucan transferase 4αG prepared in Example 3, or 2g of the recombinant immobilized enzyme prepared in Example 3 to the cassava starch solution. React at 50℃ and 220rpm for 16h, then add 0.5mL of saccharifying enzyme to hydrolyze the unreacted starch. Centrifuge the obtained product and collect the supernatant. Add 90% ethanol to the supernatant to precipitate the product, centrifuge, and remove the supernatant. Dry the precipitate to obtain the macrocyclodextrin sample. The yield statistics of macrocyclodextrin prepared by different enzymes are shown in Table 1.

[0043] Table 1. Production of macrocyclodextrin ; As shown in Table 1, 3.4 g of macrodextrin sample can be obtained by catalyzing starch with 4-α-glucan transferase 4αG, 6.6 g of macrodextrin sample can be obtained by catalyzing starch with the complex enzyme 4αG + IA, and 8.4 g of macrodextrin sample can be obtained by catalyzing starch with recombinant immobilized enzyme.

[0044] In addition, the recombinant immobilized enzyme of the present invention can be recycled back into the reaction, as detailed in Example 5 below.

[0045] Example 5: Preparation of macrodextrin from the recovery of immobilized enzymes Weigh 10g of cassava starch and dissolve it in 100mL of water to obtain a cassava starch solution. Add 2g of the recombinant immobilized enzyme aggregate recovered in Example 5 to the cassava starch solution, react at 50℃ and 220rpm for 16h, and then add saccharifying enzyme and liquefying enzyme to hydrolyze the unreacted starch. Centrifuge the obtained product and collect the supernatant. Add 90% ethanol to the obtained supernatant to precipitate and dry it. The enzyme aggregates in the precipitate can be recovered and reacted again.

[0046] Calculations show that the enzyme aggregate reaction after one recovery yielded 8g of macrodextrin, representing 95% of the first reaction. The enzyme aggregate reaction after two recovery yielded 7.6g of macrodextrin, representing 90% of the first reaction. The recombinant immobilized enzyme of this invention can be utilized at least three times, with a conversion rate (product to substrate) of over 70%.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A recombinant immobilized enzyme, characterized in that, The recombinant immobilized enzyme comprises: (1) 4-alpha-glucanotransferase; (2) isoamylase; and (3) a bridging intermediate; The bridging intermediate is SpyCatcher-SpyTag, which comprises SpyCatcher and SpyTag; wherein the SpyCatcher is modified on the isoamylase, the SpyTag is modified on the 4-alpha-glucanotransferase, and the SpyCatcher and SpyTag react through an amide bond to generate the bridging intermediate SpyCatcher-SpyTag coupling the isoamylase and the 4-alpha-glucanotransferase.

2. The recombinant immobilized enzyme according to claim 1, characterized in that, The recombinant immobilized enzyme is selected from: (1) an amino acid sequence composed of SpyTag-4αG having the amino acid sequence shown in SEQ ID NO. 10 and SpyCatcher-IA having the amino acid sequence shown in SEQ ID NO. 11; or (2) a protein homologous to or having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence composed of SpyTag-4αG having the amino acid sequence shown in SEQ ID NO. 10 and SpyCatcher-IA having the amino acid sequence shown in SEQ ID NO. 11; or (3) a recombinant immobilized enzyme derived from (1) or (2) with one or several amino acids substituted, deleted, or added in the amino acid sequence of (1) or (2).

3. A nucleic acid molecule, characterized in that, comprises a sequence selected from: (i) encoding the recombinant immobilized enzyme of claim 1 or 2; (ii) a variant having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to (i); or (iii) a complementary sequence of (i) or (ii).

4. A recombinant expression vector, characterized in that, The vector comprises the nucleic acid molecule of claim 3; Preferably, the recombinant expression vector comprises one or a combination of a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-associated vector, a lentiviral vector; Preferably, the recombinant expression vector is pRB1k and / or pBAD-HisB.

5. A recombinant bacterium, characterized in that, comprises the recombinant expression vector of claim 4.

6. A method for constructing the recombinant bacteria of claim 5, wherein, The method comprises the following steps: connecting a gene fragment encoding 4-alpha-glucanotransferase and SpyTag to a pRB1k vector to obtain a recombinant plasmid pRB1k-SpyTag-4αG; connecting a gene fragment encoding isoamylase and SpyCatcher to a pBAD-HisB vector to obtain a recombinant plasmid pBAD-SpyCatcher-IA; transforming the above two recombinant plasmids into a host cell to obtain a recombinant bacteria BW / pRB1k-SpyTag-4αG+ pBAD-SpyCatcher-IA; Preferably, the host cell is Escherichia coli BW25113.

7. The method of producing a recombinant immobilized enzyme according to claim 1 or 2, wherein The preparation method comprises the following steps: (1) centrifuging the recombinant bacteria of claim 5 after induction expression, collecting the bacterial bodies, crushing the bacterial bodies to obtain supernatant, and obtaining a crude enzyme solution containing the recombinant immobilized enzyme; (2) concentrating, oscillating and centrifuging the obtained crude enzyme solution to obtain a recombinant immobilized enzyme spheroplast; (3) cross-linking the obtained recombinant immobilized enzyme spheroplast to obtain a recombinant immobilized enzyme aggregate.

8. The preparation method according to claim 7, characterized in that, In step (1), the induction conditions are as follows: the enzyme is induced by using arabinose with a final concentration of 1-2 wt%, the induction temperature is 25-30℃, the induction rotation speed is 180-220 rpm, and the induction time is 14-18 h. In step (1), the centrifugation conditions are as follows: the rotation speed is 6000-8000 rpm, and the time is 5-10 min.

9. The preparation method according to claim 7, characterized in that, In step (2), the oscillation conditions are as follows: the temperature is 4-18℃, the rotation speed is 180-220 rpm, and the time is 3-10 h. In step (2), the centrifugation conditions are as follows: the rotation speed is 8000-12000×g, and the time is 5-15 min. In step (3), the cross-linking uses glutaraldehyde as a cross-linking agent, and the cross-linking process is as follows: oscillating the recombinant immobilized enzyme spheroplast with a 0.6-1.2 wt% glutaraldehyde solution at 4-10℃ and 200-500 rpm for 10-30 min.

10. Use of the recombinant immobilized enzyme according to claim 1 or 2, the nucleic acid molecule according to claim 3, the recombinant expression vector according to claim 4 or the recombinant bacterium according to claim 5 in the production of a macrocyclic cyclodextrin, characterized in that, The method comprises the following steps: adding the recombinant immobilized enzyme to the starch solution for reaction, and then adding saccharifying enzyme to hydrolyze the unreacted starch; centrifuging, precipitating, and centrifuging again, taking the precipitate, drying, and obtaining the macrocyclic cyclodextrin; The reaction conditions are as follows: the temperature is 30-70℃, the rotation speed is 180-220 rpm, and the time is 6-24 h.

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