In-vitro double-enzyme catalysis system for synthesizing beta-1, 3-glucan with specific polymerization degree from sucrose based on self-assembly system

Abstract

The invention relates to the technical field of biology, and discloses an in-vitro double-enzyme catalytic system for synthesizing beta-1, 3-glucan with a specific polymerization degree from sucrose based on a self-assembly system, and the in-vitro double-enzyme catalytic system comprises sucrose phosphorylase and beta-1, 3-glucan immobilized on the basis of a SpyTag / SpyCatcher system. The kit comprises a self-assembling enzyme of 1, 3-glucan phosphorylase, glucose, sucrose, a phosphate solution and a buffer solution. According to the in-vitro self-assembly double-enzyme system provided by the invention, the concentration of a local intermediate product can be increased by shortening the spatial distance of an enzyme activity center, and a substrate channel effect is realized, so that the catalysis speed is increased. The polymerization degree of beta-1, 3-glucan can be effectively customized by regulating and controlling the concentration ratio of glucose to cane sugar in the synthesis process; an economical, feasible and environment-friendly alternative scheme is provided for industrial production of the sucrose beta-1, 3-glucan, and a foundation is laid for wide application of the sucrose beta-1, 3-glucan.

Description

Technical Field

[0001] The present invention relates to the fields of synthetic biology and enzyme engineering technology, specifically to an enzymatic high-value technology with high conversion rate and high synthesis rate. More specifically, it relates to an in vitro dual-enzyme catalytic system for synthesizing β-1,3-glucan with a specific degree of polymerization from sucrose based on a self-assembly system. Background Art

[0002] β-1,3-glucan is a functional glucan connected by β-1,3-glycosidic bonds, widely distributed in the cell walls of bacteria, fungi, brown algae and plants. Its unique three-dimensional network structure endows it with significant biological activities and has important application values in the fields of immunomodulation, pathogen defense, anti-cancer and anti-infection. Currently, the preparation methods of β-1,3-glucan mainly include two major systems: chemical synthesis and enzymatic catalysis. However, the chemical extraction method is prone to destroying the natural structure of polysaccharides, restricting its medicinal development. Therefore, enzymatic synthesis has become a research hotspot in recent years due to its advantage of retaining biological activities.

[0003] In the field of biocatalytic engineering, the efficiency optimization of multi-enzyme cascade reactions has always been a research focus. In the traditional free enzyme system, due to the spatial dispersion of enzyme molecules, the diffusion loss of intermediate products often occurs, severely restricting the reaction efficiency. In recent years, the SpyTag / SpyCatcher system (protein covalent coupling technology) has provided an innovative solution to solve this problem.

[0004] This system is based on the spontaneous formation of an internal isopeptide bond between the side-chain carboxyl group of aspartic acid in SpyTag003 and the primary amine of lysine in SpyCatcher003 in this region, which can achieve the precise assembly of multiple enzymes. This "modular assembly" strategy not only effectively isolates the reaction interface and reduces substrate competition, but also significantly enhances the catalytic synergy through enzyme distance regulation and microenvironment optimization. It is worth noting that this protein interaction system mediated by non-covalent modification can, while maintaining enzyme activity, construct an artificial enzyme cascade reaction system through directional design. Existing research has shown that by preparing a cascade enzyme containing dockerin assembled based on a specific scaffold protein of SpyTag / SpyCatcher to form a multi-enzyme complex that can convert starch into inositol, its initial reaction rate is higher than that of a mixture of four enzymes. More importantly, it can still maintain the conformational stability of the enzyme complex under extreme conditions, which provides important technical support for industrial biocatalytic processes.

[0005] In the biosynthetic pathway, nature mainly relies on glycosyltransferases to catalyze the elongation of glucan chains. However, the cost of the required nucleotide-activated sugars (such as UDP-glucose) is high, restricting industrial applications. Glycoside phosphorylases have shown unique advantages in the field of β-1,3-oligosaccharide synthesis. These enzymes can directly use the glycosyl donor α-glucose-1-phosphate (G1P) for catalysis, providing a new idea for cost reduction and efficiency improvement. Sucrose, as an inexpensive and readily available natural disaccharide, the strategy of in-situ generating G1P through sucrose phosphorylase not only significantly reduces the raw material cost but also realizes the efficient utilization of resources, laying a foundation for large-scale production.

[0006] Therefore, the present invention proposes an in vitro dual-enzyme catalytic system for synthesizing β-1,3-glucan with a specific degree of polymerization from sucrose based on a self-assembly system. This catalytic system includes the substrate sucrose, phosphate buffer, glucose, and self-assembled enzymes (SpyCatcher-BaSP and SpyTag-TaβGP); mainly through sucrose phosphorylase (BaSP) and β-1,3-glucan phosphorylase (TaβGP), sucrose is phosphorolyzed into αG1P and fructose, and then β-1,3-glucan phosphorylase uses G1P as a glycosyl donor to synthesize β-1,3-glucan. Summary of the Invention

[0007] The purpose of the present invention is to provide an in vitro dual-enzyme catalytic system for synthesizing β-1,3-glucan with a specific degree of polymerization from sucrose based on a self-assembly system, so as to solve the technical problems that β-1,3-glucan is difficult to produce and has low purity at the present stage, and the structure and physicochemical properties of naturally extracted β-1,3-glucan are heterogeneous, resulting in limited wide applications.

[0008] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0009] The present invention provides an in vitro dual-enzyme catalytic system for synthesizing β-1,3-glucan with a specific degree of polymerization from sucrose based on a self-assembly system. The in vitro dual-enzyme catalytic system includes self-assembled enzymes immobilizing sucrose phosphorylase and β-1,3-glucan phosphorylase based on the SpyTag / SpyCatcher system, glucose, sucrose, phosphate solution, and buffer solution.

[0010] Preferably, the self-assembled enzyme is obtained by fully mixing the aqueous solutions of the recombinant protein SpyCatcher-BaSP with the amino acid sequence shown in SEQ ID NO.5 and the recombinant protein SpyTag-TaβGP with the amino acid sequence shown in SEQ ID NO.6, and incubating at 25±5°C for more than 20 minutes.

[0011] Preferably, the recombinant protein SpyCatcher-BaSP is obtained by homologous recombination of the SpyCatcher gene with homologous arms and pET-28a-BaSP, and then transferred into a host cell for expression and purification; the recombinant protein SpyTag-TaβGP is obtained by homologous recombination of the TaβGP gene with homologous arms and pET-28a-SpyTag, and then transferred into a host cell for expression and purification.

[0012] Preferably, the PCR amplification primer sequences of the SpyCatcher gene with homologous arms are shown in SEQ ID NO.1 and SEQ ID NO.2, and the PCR amplification primer sequences of the TaβGP gene with homologous arms are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0013] The present invention also provides the application of the above in vitro dual-enzyme catalytic system in the synthesis of β-1,3-glucan with a specific degree of polymerization from sucrose.

[0014] Preferably, the application is as follows: using the self-assembled enzyme as a catalyst, sucrose as a donor substrate, glucose as a primer substrate, and a phosphate solution and a buffer with a pH of 6-8 to form an in vitro dual-enzyme catalytic system, and reacting at 40-60 °C for 6-36 h.

[0015] Preferably, the concentration of the self-assembled enzyme is 2 U / mL, the concentration of sucrose is 100-600 mM, the concentration of glucose is 1-150 mM, and the concentration of phosphate is 50 mM.

[0016] Preferably, when the concentration of the self-assembled enzyme is 2 U / mL, the concentration of phosphate is 50 mM, the concentration of sucrose is 400-600 mM, and the concentration of glucose is 1-50 mM, the average degree of polymerization of the obtained β-1,3-glucan product is 27-31, and the degree of polymerization distribution is 22-35.

[0017] Preferably, when the concentration of the self-assembled enzyme is 2 U / mL, the concentration of phosphate is 50 mM, and the concentration of sucrose is 400-600 mM, the average degree of polymerization of the obtained β-1,3-glucan product is 13-18, and the degree of polymerization distribution is 5-27.

[0018] Preferably, when the concentration of the self-assembled enzyme is 2 U / mL, the concentration of phosphate is 50 mM, the concentration of sucrose is 100-200 mM, and the concentration of glucose is 25-75 mM, the average degree of polymerization of the obtained β-1,3-glucan product is 12-14, and the degree of polymerization distribution is 7-26.

[0019] The sequences involved in this patent are as follows:

[0020] >SpyCatcher-BaSP(SEQ ID NO.5)

[0021] MGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATM

[0022] ELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTV

[0023] NEDGQVTVDGEATEGDAHTGSSGSGSSGSG KNKVQLITYADRLGDGTIK

[0024] SMTDILRTRFDGVYDGVHILPFFTPFDGADAGFDPIDHTKVDERLGSWD

[0025] DVAELSKTHNIMVDAIVNHMSWESKQFQDVLAKGEESEYYPMFLTMSS

[0026] VFPNGATEEDLAGIYRPRPGLPFTHYKFAGKTRLVWVSFTPQQVDIDTDS

[0027] DKGWEYLMSIFDQMAASHVSYIRLDAVGYGAKEAGTSCFMTPKTFKLI

[0028] SRLREEGVKRGLEILIEVHSYYKKQVEIASKVDRVYDFALPPLLLHALST

[0029] GHVEPVAHWTDIRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDED

[0030] VDNLVNTIHANTHGESQAATGAAASNLDLYQVNSTYYSALGCNDQHYI

[0031] AARAVQFFLPGVPQVYYVGALAGKNDMELLRKTNNGRDINRHYYSTA

[0032] EIDENLKRPVVKALNALAKFRNELDAFDGTFSYTTDDDTSISFTWRGET

[0033] SQATLTFEPKRGLGVDNTTPVAMLEWEDSAGDHRSDDLIANPPVVALEHHHHHH;

[0034] >SpyTag-TaβGP(SEQ ID NO.6)

[0035] MGRGVPHIVMVDAYKRYKGSSGSGS KKFDFVIENYSKQKLFSSFLPGIA

[0036] GKNGIPLWVFYVNRGQCIASFGIENKANSILEFKPAGQSHTDTPLKGFRT

[0037] FIKVDGRYYEPFSELTNFKREMRINKNSLEIEERNNELGLKVKVIYFVLP

[0038] NEDFAALVRRVEIENEDKYEKHIEIIDGLPEVIPYGVSNGLYKEMGYTAR

[0039] AWMHVYNYEKKVPFYSVRTTIGDLEIVEEINNGYFYFASSGDELLEVIY

[0040] DKNVLFGNSTSLQVPLVFKELGIKEVLRKEQYDENLLPSAFGVLERKLK

[0041] DKVVINSMIGFSKEKGLINNNINTLKKDEYIISKKEEADLIVEELVSEIKT

[0042] KTSNKLFDKYCEQNYLDNVLRGGYPLVFENKDGKVVYHIYSRKHGDLE

[0043] RDYNFFVLEAKKYSQGNGNFRDVAQNRRNDVIFHPEIEDFNLSMFVNLI

[0044] QADGNNPLVVKGTRFKFEGDPSILDGVNEELKEFILNNYFTPGEILEKLK

[0045] DKNVNEDEFVSKILYHSSQHEQAEFGEGYWIDHWTYLMDLVDTYKEIY

[0046] PDKLQKTLFEKFEYKIYDSHAYVKPRKEKYKLYKGKVRQIAAVGESHEK

[0047] LKIIQERGHNYLTDRNGNIYKTNMFEKLLLLAVNKFATLDPYGMGLEME

[0048] ANKPGWNDALNGLPALFGSGMSETFELKRLINFMYEELKKYNKDIEVF

[0049] EELQEFIQKIKVELENYFNDNDQFIYWDNVSNAKEEYRSKVFYSITGNK

[0050] KKISKVELLAILKKMIKKLDEGIERAKNYGKGIYPTFFTYELVDYEVIDG

[0051] VIIPKKFEVNVLPYFLEGIVRAFKVIDKDEKKKLYDFVKNSNIYDKKLKM

[0052] YKTSESILNQPYSIGRIRAFTPGWLENESVFMHMEFKYLLELIKSDMLEE

[0053] FYEDIKTALPPYMDYKVYGRSILENSSFIVSSANSNPNLHGQGFYARLSG

[0054] STAEFLSMWKYMFIGDKLFTLENNELTFTFEPKINKEFFENGVIEFKLFSK

[0055] TKVKYVNPQLKEKIGRIEVFVDGKKFEIHGNKIKGELAHKLRNKKIDEVICYFELEHHHHHH。

[0056] In summary, compared with the prior art, the solution of the present invention has the following beneficial effects:

[0057] 1. By introducing the gene-encoded click chemistry - Spy chemistry technology, the present invention constructs an in vitro self-assembled dual-enzyme system composed of heat-resistant BaSP and TaβGP, and successfully realizes the sustainable production of β-1,3-glucan from bulk renewable substrates sucrose and glucose; moreover, the in vitro self-assembled dual-enzyme system provided by the present invention can improve the local intermediate concentration by shortening the spatial distance between the enzyme active centers, realizing the substrate channel effect, so as to enhance the catalytic speed.

[0058] 2. The self-assembled dual-enzyme catalytic system provided by the present invention achieves nearly complete conversion of 500 mM sucrose within 6 hours. Its reaction rate is four times higher than that of the free enzyme system, and it also exhibits a remarkable production efficiency of 13.2 g / L / h. Moreover, by regulating the concentration ratio of glucose to sucrose during the synthesis process, β-1,3-glucans with different average degrees of polymerization can be precisely synthesized, realizing the customized production of the degree of polymerization (DP) of β-1,3-glucan, thereby expanding the applications of β-1,3-glucans with different molecular weights.

[0059] 3. The self-assembled dual-enzyme catalytic system provided by the present invention has excellent scale-up potential, demonstrating the application prospects of industrial production. It provides an economically viable and environmentally friendly alternative for the industrial production of sucrose-based β-1,3-glucan, laying a foundation for its wide application. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 is a schematic diagram of the construction of the recombinant plasmids pET-28a-SpyCatcher-BaSP and pET-28a-SpyTag-TaβGP in Example 1;

[0061] Figure 2 is the amplification electrophoresis pattern of the SpyCatcher and TaβGP genes in Example 1 (M: DNA Marker; Lane 1: amplification product of the SpyCatcher gene; Lane 2: amplification product of the TaβGP gene);

[0062] Figure 3 is the linearization of the plasmid vectors pET-28a-BaSP and pET-28a-SpyTag in Example 1 (M: DNA Marker; Lane 1: single digestion product of pET-28a-BaSP; Lane 2: double digestion product of the pET-28a-SpyTag plasmid);

[0063] Figure 4 is the colony PCR verification of the recombinant vectors in Example 1 (M: DNA Marker; Lane 1: negative colony PCR of SpyCatcher-BaSP; Lane 2: positive colony PCR of SpyCatcher-BaSP; Lane 3: negative colony PCR of SpyTag-TaβGP; Lane 4: positive colony PCR of SpyTag-TaβGP);

[0064] Figure 5SDS-PAGE analysis results of BaSP, SpyCatcher-BaSP, TaβGP, and SpyTag-TaβGP in Example 2 (M: protein Marker; Lane 1: BaSP; Lane 2: SpyCatcher-BaSP; Lane 3: TaβGP; Lane 4: SpyTag-TaβGP);

[0065] Figure 6 Comparison and analysis of specific enzyme activities before and after modification with the two glycosylphosphorylase SpyTag / SpyCatcher tags in Example 2;

[0066] Figure 7 Schematic diagram of the assembly of SpyCatcher-BaSP and SpyTag-TaβGP in Example 3;

[0067] Figure 8 SDS-PAGE analysis of the in vitro self-assembly of SpyCatcher-BaSP and SpyTag-TaβGP in Example 3 (M: protein Marker; Lane 1: SpyCatcher-BaSP; Lane 2: SpyTag-TaβGP; Lane 3: self-assembled complex of SpyCatcher-BaSP and SpyTag-TaβGP);

[0068] Figure 9 Dynamic analysis of substrate phosphorolysis and G1P conversion in a reaction system composed of different substrate concentrations over a period of time in Example 4 (A and C are dynamic analyses of substrate phosphorolysis, B and D are dynamic analyses of G1P conversion; where the substrate concentration in A and B is 150 mM sucrose and the reaction time is 6 h; the substrate concentration in C and D is 500 mM sucrose and the reaction time is 36 h; Free represents the free enzyme system, self-assembly represents the self-assembled enzyme system, F represents the fructose content, and S represents the sucrose content);

[0069] Figure 10 Effect of adding different concentrations of glucose on the degree of polymerization of the synthesized β-1,3-glucan product in a reaction system with different sucrose concentrations in Example 5 (A - D represent 500 mM sucrose, and the added glucose concentrations are 1 mM, 50 mM, 100 mM, and 150 mM respectively; E - F represent 150 mM sucrose, and the added glucose concentrations are 1 mM and 50 mM respectively). Detailed implementation mode

[0070] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments and accompanying drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0071] The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

[0072] The experimental methods of the present invention are all conventional methods unless otherwise specified.

[0073] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0074] Example 1 Construction of Recombinant Plasmids pET-28a-SpyCatcher-BaSP and pET-28a-SpyTag-TaβGP

[0075] 1 Experimental Method

[0076] The gene encoding BaSP (AAO33821.1) was codon-optimized, synthesized, and cloned into the pET-28a(+) vector using Nco I and Xho I restriction enzyme cleavage sites, thereby obtaining the recombinant plasmid pET-28a-BaSP. The recombinant plasmids pET-21a-TaβGP, pET-28a-SpyTag, pET-28a-SpyCatcher, Escherichia coli DH5α and BL21(DE3) are all preserved in the Energy Microorganism Laboratory of the College of Life Sciences, Henan Agricultural University.

[0077] Schematic diagrams of the recombinant plasmids pET-28a-SpyCatcher-BaSP and pET-28a-SpyTag-TaβGP are as Figure 1 shown.

[0078] The construction principle of the pET-28a-SpyCatcher-BaSP plasmid is as follows: relying on the homologous arms carried at both ends of the SpyCatcher gene to recognize and recombine with the corresponding sequences on pET-28a-BaSP. To prevent the influence of the SpyCatcher gene on the three-dimensional structure of BaSP, a flexible Linker composed of (GSSGS)2 was added between the enzyme and the SpyCatcher protein to achieve the effective connection of the SpyCatcher gene with the vector containing the BaSP gene. Recombining the TaβGP gene with homologous arms with pET-28a-SpyTag can construct the pET-28a-SpyTag-TaβGP recombinant plasmid. The specific operations are as follows:

[0079] Using pET-28a-SpyCatcher and pET-21a-TaβGP as templates respectively for PCR amplification. The primers for PCR amplification are shown in Table 1. The reaction system includes 1 μL of DNA template, 1 μL of upstream primer, 1 μL of downstream primer, 20 μL of 2×i5 High-Fidelity DNA Polymerase PCR mix, and ddH2O to make up 40 μL. The reaction program is 98°C for 300 s, 98°C for 30 s, 58°C for 30 s, 72°C for 5 / 35 s, 72°C for 600 s, with 25 cycles. Among them, the extension time for SpyCatcher is 5 s and for TaβGP is 35 s. After the PCR reaction, use 1% agarose gel electrophoresis to analyze the reaction products to determine whether the target gene has been successfully amplified. Use the column-concentrated DNA gel recovery kit to recover the bands with the expected size in the electrophoresis results to obtain the target genes SpyCatcher and TaβGP.

[0080] Table 1 Primer sequences of SpyCatcher-BaSP and SpyTag-TaβGP

[0081]

[0082]

[0083] Use the restriction enzyme NcoⅠ to digest the vector pET-28a-BaSP at 37°C; use the restriction enzymes Bam HⅠ and XhoⅠ to digest the vector plasmid of pET-28a-SpyTag for 4 h to linearize it. The template digestion reaction system is 20 μL of plasmid, 1 μL of NcoⅠ, 4 μL of 10×Buffer, and ddH2O to make up 40 μL.

[0084] The linearized plasmids pET-28a-SpyTag and pET-28a-BaSP were recovered using a microcolumn-concentrated DNA gel recovery kit, and the purified products of the target genes SpyCatcher and TaβGP with homologous arms were recombined with the vector in an ice-water bath according to the homologous recombination reaction system configured in Table 2. In this reaction system, the amount of the vector was set to 120 ng, and the amount of the target fragment was 50 ng. Subsequently, the reaction system was placed at 50 °C for 45 min.

[0085] Table 2 Ligation system of target gene and vector

[0086]

[0087] The recombinant reaction solution was transferred into Escherichia coli competent cells DH5α and evenly spread on a solid LB plate containing 50 μg / mL Kana antibiotic in a laminar flow hood. After sealing with a sealing film, it was incubated overnight in an inverted position in a 37 °C incubator. Single colonies on the plate were picked and inoculated into a liquid LB medium containing 50 μg / mL Kana antibiotic, and cultured with shaking at 37 °C until the logarithmic growth phase (OD = 0.6); PCR amplification was performed. The colony PCR reaction system was 1 μL T7, 1 μL T7-ter, 25 μL 2×Green Taq mix, and ddH2O was added to make up 50 μL. The colony PCR program was 95 °C for 180 s, 95 °C for 10 s, 55 °C for 10 s, 72 °C for 120 / 190 s, 72 °C for 600 s, with 25 cycles. Among them, the extension time for SpyCatcher-BaSP was 120 s, and for SpyTag-TaβGP was 190 s.

[0088] After the reaction, an appropriate amount of the PCR product was subjected to 1% agarose gel electrophoresis, and whether the target fragment was successfully amplified was judged according to the presence, absence, and size of the bands; 10 mL of the bacterial liquid with a positive electrophoresis result was taken to extract the plasmid, and the plasmid sample was sequenced. The sequencing primer used was the T7 promoter-specific primer. The sequencing results were aligned with the target gene using Snapgene software to judge whether the plasmid was successfully constructed.

[0089] 2 Experimental results

[0090] 2.1 Obtaining the target genes with homologous arms

[0091] The target genes were amplified using pET-28a-SpyCatcher and pET-21a-TaβGP as templates. The results were as Figure 2As shown, after PCR amplification, we can clearly observe obvious DNA bands in the range of 1000 - 2000 bp from agarose gel electrophoresis, which are consistent with the theoretical DNA molecular weights of SpyCatcher and TaβGP genes (381 bp and 3057 bp).

[0092] 2.2 Successful construction of recombinant expression vectors

[0093] After digesting the templates pET - 28a - BaSP and pET - 28a - SpyTag with restriction enzymes to open the circular plasmids (see Figure 3 ), homologous recombination was carried out with the target gene with homologous arms at appropriate positions, and the recombinant reaction solution was transferred into Escherichia coli DH5α. Colony PCR verification showed that the size of the target band was consistent with the expectation (see Figure 4 ). At the same time, the comparison result between the plasmid sequencing result and the target sequence was accurate, indicating the successful construction of the recombinant expression vector.

[0094] Example 2 Recombinant expression and purification of SpyCatcher - BaSP and SpyTag - TaβGP

[0095] 1 Experimental method

[0096] The recombinant plasmid containing the gene encoding the target protein in Example 1 was transformed into competent cells of Escherichia coli BL21(DE3), inoculated onto a solid LB plate containing 50 μg / mL kanamycin (kana), and cultured overnight at 37°C in an inverted position. After obtaining positive clones, single colonies were picked and amplified in LB medium (220 rpm at 37°C). After adding IPTG for induction expression for 16 h, the cells were lysed by an ultra - high - pressure cell disruptor under ice - bath conditions, and the supernatant was collected after centrifugation and heat - treated at 50°C for 20 min. The target proteins were purified by nickel - medium affinity chromatography: first, washed with 5 mM imidazole to remove impurities, and then further treated with 1 mM imidazole to remove non - specifically bound proteins; finally, the target proteins were eluted with 500 mM imidazole, and the eluate was collected. The eluate was concentrated to an appropriate volume through 50 and 120 kDa ultrafiltration centrifugal tubes to obtain pure enzymes SpyCatcher - BaSP and SpyTag - TaβGP. The protein concentration was measured according to the Bradford method. After diluting the pure enzyme by a certain multiple, it was mixed with Coomassie Brilliant Blue G250, the absorbance value was measured, and the protein concentration was calculated by substituting into the standard curve.

[0097] The enzymatic activities of the purified SpyCatcher-BaSP (amino acid sequence shown in SEQ ID NO.5) and SpyTag-TaβGP (amino acid sequence shown in SEQ ID NO.6) were determined. Since the self-assembled enzyme reaction process is a cascade reaction and the method for measuring enzyme activity is related to the intermediate products of their reactions, it is impossible to directly calculate the specific enzyme activities of the assembled enzymes separately. Only the enzymes of SpyCatcher-BaSP and SpyTag-TaβGP before assembly can be assayed for their activities.

[0098] Method for determining the enzymatic activity of SpyCatcher-BaSP: Sucrose and phosphate are catalyzed by BaSP to produce fructose and G1P, and the BaSP enzyme activity is calculated based on the fructose content in the product. The assay system is 100 μL, containing 150 mM Sucrose, 50 mM phosphate buffer (pH 7.0) and diluted BaSP. After reacting in a 50 °C water bath for 10 min, 150 μL of DNS reagent is added, boiled for 7 min and then ice-bathed for 2 min. The absorbance at 540 nm is detected by an enzyme-linked immunosorbent assay (ELISA) reader, and a standard curve is plotted using fructose as the standard. One unit (U) of BaSP enzyme activity is defined as the amount of enzyme required to hydrolyze sucrose to produce 1 μmol of fructose per minute.

[0099] Method for determining the enzymatic activity of SpyTag-TaβGP: TaβGP uses the phosphomolybdate colorimetric method to detect the phosphate released from αG1P. The system is 100 μL, containing 50 mM Tris-HCl buffer (pH 7.0), 20 mM G1P, 1 mM Glucose and diluted TaβGP. After reacting in a 50 °C water bath for 10 min, 1 mL of P i The assay working solution (ammonium molybdate-ascorbic acid solution) is added to terminate the reaction. After incubating at 30 °C for 15 minutes, the absorbance at 850 nm is detected by an ELISA reader, and a standard curve is plotted using G1P as the standard. One unit of enzyme activity (U) of TaβGP activity is defined as: under the given conditions, when TaβGP enzyme catalyzes the utilization of G1P to produce dextran, the amount of TaβGP enzyme that releases 1 μmol of phosphate per minute.

[0100] 2 Experimental results

[0101] 2.1 Results of the determination of the recombinantly expressed and purified proteins

[0102] Analysis by SDS-PAGE showed that the N-terminals of BaSP and TaβGP were respectively inserted with the SpyCatcher and SpyTag interacting protein tags. The purified SpyCatcher-BaSP and SpyTag-TaβGP had high purity, low content of heteroproteins, and could still be highly soluble expressed in Escherichia coli after site-directed mutagenesis of the gene sequence (see Figure 5), indicating that its folding is not affected by SpyTag / SpyCatcher, and the two recombinant proteins have been correctly folded according to the protein sequences involved, providing a reliable experimental basis for subsequent self-assembly research.

[0103] 2.2 Activity analysis of SpyCatcher-BaSP and SpyTag-TaβGP

[0104] The results showed that the activities of SpyCatcher-BaSP and SpyTag-TaβGP were respectively close to those of the original enzymes (unmodified), and their interaction had no significant effect on the catalytic efficiency of each other (see Figure 6 ). The results indicated that the self-assembly strategy mediated by SpyCatcher-SpyTag did not introduce obvious structural perturbations or activity losses, providing a feasibility basis for the in vitro directed self-assembly catalytic system based on dual enzymes.

[0105] Example 3 In vitro self-assembly of SpyCatcher-BaSP and SpyTag-TaβGP

[0106] Dilute the two purified enzyme solutions in Example 2 to an appropriate multiple and incubate them at room temperature for 20 minutes to allow sufficient reaction. Based on the unique covalent binding characteristics of the SpyTag / SpyCatcher system, by fusing SpyTag containing aspartic acid residue D117 and SpyCatcher containing lysine residue K31 to the N-terminus of BaSP and TaβGP respectively (see Figure 7 ), after incubation at room temperature at appropriate concentrations in vitro, these two recombinant proteins spontaneously formed stable isopeptide bond linkages; thus enabling the two proteins to form a stable assembled complex in SDS-PAGE, and successfully constructing a self-assembled dual-enzyme complex system. To verify the protein assembly effect, mix the reacted sample with protein loading buffer, boil for 10 minutes to fully denature, and then perform SDS-PAGE analysis. By observing the electrophoresis bands, the specific assembly of SpyTag / SpyCatcher proteins can be visually verified.

[0107] The results of SDS-PAGE analysis (see Figure 8)It was shown that in the β-1,3-glucan synthesis reaction system, SpyCatcher-BaSP (70 kDa) and SpyTag-TaβGP (123 kDa) successfully assembled to form a complex with a molecular weight of 193 kDa, which was exactly the same as the sum of the theoretical molecular weights of the two enzymes, confirming the formation of a covalently bound-mediated self-assembled dual-enzyme complex. Notably, some unassembled SpyTag-TaβGP was still observed in the electrophoresis pattern, which was due to the imbalance in the molar ratio of enzyme molecules in the reaction system. This result not only verified the reliability of the SpyTag / SpyCatcher system in constructing multi-enzyme complexes but also provided an important reference for subsequent optimization of the assembly efficiency.

[0108] Example 4 Synthesis of β-1,3-glucan using self-assembled enzymes

[0109] 1 Experimental method

[0110] 1.1 Synthesis of β-1,3-glucan

[0111] The reaction systems were all prepared on an ice-water bath. Before preparing the reaction systems, calculate the amount of enzymes to be added to the systems for SpyCatcher-BaSP and SpyTag-TaβGP so that the volume can reach 2 U / mL, and incubate them in 1.5 mL EP tubes at room temperature for 20 minutes. Incubate the free enzymes in the same way, and at the same time add them to the reaction systems prepared in the ice-water bath, aiming to make the free enzymes and self-assembled enzymes start reacting simultaneously. All other reaction conditions were the same as those of the free dual-enzyme system.

[0112] The reaction system with 150 mM sucrose as the substrate contained 50 mM phosphate, 1 mM glucose, and reacted at pH 7 and 50 °C for 6 h. Samples were taken every hour, and the contents of sucrose, fructose, and G1P in the system were analyzed by high-performance liquid chromatography, and the differences between the self-assembled enzymes and free enzymes were calculated;

[0113] The reaction system with 500 mM sucrose as the substrate contained 50 mM phosphate, 1 mM glucose, and reacted at pH 7.0 and 50 °C for 36 h, and samples were taken every 6 h within this period. Analyze the contents of sucrose, fructose, and G1P in the system in the same way, and calculate the differences between the self-assembled enzymes and free enzymes.

[0114] In this example, since the phosphate groups were recycled continuously in the dual-enzyme reaction system, the phosphate groups were considered sufficient; therefore, it was assumed that 100% of the sucrose was phosphorolyzed by BaSP into one molecule of fructose and one molecule of G1P, and the total content of fructose detected by liquid phase could be used to represent the total production amount of G1P.

[0115] The contents of fructose and sucrose were determined by high-performance liquid chromatography, and the concentration of G1P was determined by the dual-enzyme coupling method.

[0116] The yield calculation formula of the dual-enzyme catalysis system is as follows:

[0117] Amount of G1P converted (mM) = Amount of fructose produced - Amount of G1P remaining (1)

[0118] Substrate conversion rate (mol%) = Amount of G1P converted / Initial sucrose concentration (2).

[0119] 2 Experimental results

[0120] 2.1 Effect of self-assembly on the synthesis rate of β-1,3-glucan

[0121] As Figure 9 shown, when the substrate is 150 mM sucrose and the reaction time is 6 h: at the same enzyme dosage, the catalytic efficiency of the self-assembly system is 2 times faster than that of the free enzyme, and the conversion rate of G1P increases by 1.5 times (see Figure 9 A). However, compared with the free enzyme reaction system, the self-assembly system does not increase the utilization amount of G1P (142.11 mM vs 142.64 mM) (see Figure 9 B). When the substrate concentration in the system is increased to 500 mM and the enzyme amount remains unchanged, and the reaction time is extended: sucrose completes the phosphorolysis reaction within 6 h under the catalysis of the self-assembled enzyme (see Figure 9 C); and the utilization of G1P is also completed during the same period and reaches the reaction equilibrium. On the contrary, the free enzyme reaches the reaction equilibrium after 24 h. And when the sucrose substrate concentration is different, the conversion amount of G1P is the same, indicating that self-assembly does not increase the yield, but the reaction rate is increased (see Figure 9 D). Although there is no significant difference in the substrate conversion rate between the two assembly methods at the same substrate concentration level, however, at different concentration levels, taking the free enzyme as an example, when 500 mM sucrose is used as the substrate, the substrate conversion rate is as high as 96.4% within 36 h, which can be said that 100% of the BaSP phosphorolysis product is converted, while at the 150 mM substrate concentration level, the highest substrate conversion rate is 92.4% (see Figure 9 D). In short, under the self-assembly system, 92% of the 150 mM substrate can be converted within 2 h, and 97.4% of the 500 mM substrate can be converted within 4 h. Thus, it can be seen that the self-assembly system has great advantages.

[0122] In summary, the G1P conversion rate of the self-assembled enzyme system is increased by four times compared with the free enzyme. In addition, under high-concentration conditions, the self-assembly system achieves nearly 100% conversion of the substrate G1P, further optimizing the existing high-conversion free enzyme system. Significantly demonstrates the great potential of the self-assembly reaction system in the application of β-1,3-glucan synthesis.

[0123] Example 5 Regulation of the degree of polymerization of β-1,3-glucan

[0124] 1. Experimental methods

[0125] After the β-1,3-glucan biosynthesis in Example 4 was completed, the soluble and insoluble products in the reaction system were separated. The reaction mixture containing the insoluble product was boiled for 10 minutes, cooled to room temperature at 4°C, and centrifuged at 12,000 rpm for 5 minutes to collect the bottom precipitate. Molecular weight analysis was performed using Maldi-TofMas.

[0126] Based on the obtained mass spectrometry data, the average degree of polymerization (DPn), number-average molecular weight weight-average molecular weight and polydispersity index (PDI) of the synthesized β-1,3-glucan were calculated using formulas to evaluate its molecular characteristics.

[0127] The degree of polymerization (DP) is defined as the average number of monomer units contained in each polymer chain, and the calculation formula is as follows:

[0128]

[0129] In the formula: is the number-average molecular weight of β-1,3-glucan, and Mo is the molecular weight of the repeating unit (β-1,3-glucose losing one water molecule, molecular weight 162.14 g / mol).

[0130] Number-average molecular weight represents the number-weighted average molecular weight of all β-1,3-glucan molecular chains, and the calculation formula is as follows:

[0131]

[0132] In the formula: Ni represents the peak area of the i-th β-1,3-glucan component, and Mi represents the molar mass represented by this peak.

[0133] Weight-average molecular weight represents the mass-weighted average molecular weight of all β-1,3-glucan molecular chains, and the calculation formula is as follows:

[0134]

[0135] The polydispersity index (PDI) is defined as the ratio of the weight-average molecular weight to the number-average molecular weight of β-1,3-glucan molecular chains, which is a key indicator characterizing the molecular weight homogeneity, and its calculation formula is:

[0136]

[0137] This example explored the regulation of the synthesized β-1,3-glucan with adjustable degree of polymerization (see Table 3 and Figure 10 ).

[0138] Table 3 Regulation of the degree of polymerization of β-1,3-glucan

[0139]

[0140] As can be seen from Table 3, under the condition that the sucrose concentration was 500 mM, when the glucose concentration was gradually increased from 1 mM to 150 mM, significant changes occurred in the product. When the glucose concentration was 1 mM and 50 mM, the degree of polymerization DP of the product β-1,3-glucan n was 31 and 27 respectively; while when the glucose concentration was increased to 100 mM and 150 mM, the degree of polymerization DP of the product β-1,3-glucan n was 18 and 13 respectively. Similarly, when the glucose concentration was maintained at 50 mM and the sucrose concentration was reduced from 500 mM to 150 mM, the degree of polymerization DP of the formed β-1,3-glucan n was 13 (see Figure 10 ). The above results indicate that by adjusting the ratio between sucrose as the donor substrate (source of G1P) and glucose as the primer substrate, the degree of polymerization of the synthesized β-1,3-glucan can be effectively controlled.

[0141] The β-1,3-glucan synthesized in the present invention (covering soluble and insoluble products) has an estimated polydispersity index (PDI) between 1.01 and 1.12, indicating that the molecular weight distribution of this product is highly uniform. Compared with other β-1,3-glucans synthesized from sucrose, the degree of polymerization distribution of the product obtained in the present invention is significantly narrower.

[0142] In summary, the self-assembled dual-enzyme system can synthesize β-1,3-glucan with a highly uniform molecular weight distribution and adjustable degree of polymerization by regulating the concentrations of sucrose and glucose.

[0143] The above-described embodiments only represent the preferred embodiments of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation to the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. An in vitro dual enzyme catalytic system for synthesizing β-1,3-glucan with a specific degree of polymerization from sucrose based on a self-assembly system, characterized in that: The in vitro dual-enzyme catalytic system comprises a self-assembling enzyme based on a SpyTag / SpyCatcher system to immobilize sucrose phosphorylase and beta-1,3-glucan phosphorylase, glucose, sucrose, a phosphate solution and a buffer solution.

2. The in vitro dual enzyme catalytic system according to claim 1, characterized in that: The self-assembling enzyme is obtained by fully mixing aqueous solutions of the recombinant protein SpyCatcher-BaSP with an amino acid sequence as shown in SEQ ID NO.5 and the recombinant protein SpyTag-TaβGP with an amino acid sequence as shown in SEQ ID NO.6, and then incubating at 25±5°C for more than 20 minutes.

3. The in vitro dual enzyme catalytic system according to claim 2, characterized in that: The recombinant protein SpyCatcher-BaSP is obtained by homologous recombination between the SpyCatcher gene with homology arms and pET-28a-BaSP, and then transferred into host cells for expression and purification; the recombinant protein SpyTag-TaβGP is obtained by homologous recombination between the TaβGP gene with homology arms and pET-28a-SpyTag, and then transferred into host cells for expression and purification.

4. The in vitro dual enzyme catalytic system according to claim 3, characterized in that: The PCR amplification primer sequences of the SpyCatcher gene with homology arms are shown in SEQ ID NO.1 and SEQ ID NO.2, and the PCR amplification primer sequences of the TaβGP gene with homology arms are shown in SEQ ID NO.3 and SEQ ID NO.

4.

5. Use of the in vitro dual enzyme catalytic system according to any one of claims 1 to 4 in the synthesis of β-1,3-glucan with a specific degree of polymerization from sucrose.

6. The use according to claim 4, characterized in that: The application is: using the self-assembling enzyme as a catalyst, sucrose as a donor substrate, glucose as a primer substrate, a phosphate solution and a buffer solution with a pH of 6 to 8 to form an in vitro double enzyme catalytic system, and reacting at 40 to 60°C for 6 to 36 hours.

7. The use according to claim 5, characterized in that: The concentration of the self-assembly enzyme is 2 U / mL, the concentration of sucrose is 100-600 mM, the concentration of glucose is 1-150 mM, and the concentration of phosphate is 50 mM.

8. The use according to claim 5, characterized in that: When the concentration of the self-assembly enzyme is 2 U / mL, the concentration of the phosphate is 50 mM, the concentration of the sucrose is 400-600 mM, and the concentration of the glucose is 1-50 mM, the average polymerization degree of the obtained β-1,3-glucan product is 27-31, and the polymerization degree distribution is 22-35.

9. The use according to claim 5, characterized in that: When the concentration of the self-assembly enzyme is 2 U / mL, the concentration of the phosphate is 50 mM, the concentration of the sucrose is 400-600 mM, and the concentration of the glucose is 75-150 mM, the average polymerization degree of the obtained β-1,3-glucan product is 13-18, and the polymerization degree distribution is 5-27.

10. The use according to claim 6, characterized in that: When the concentration of the self-assembly enzyme is 2 U / mL, the concentration of the phosphate is 50 mM, the concentration of the sucrose is 100-200 mM, and the concentration of the glucose is 25-75 mM, the average polymerization degree of the obtained β-1,3-glucan product is 12-14, and the polymerization degree distribution is 7-26.

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