Glucoamylase mutant and application thereof

Abstract

The invention provides a glucamylase mutant and application thereof, and belongs to the technical field of bioengineering. The method comprises the following steps: training an amino acid sequence and three-dimensional structure data of GLL by utilizing a machine learning algorithm, and selecting specific amino acid sites according to a prediction result of a machine learning model to carry out site-specific mutagenesis of six amino acids to obtain a glucamylase mutant; the amino acid sequence of the glucamylase mutant is as shown in SEQ ID NO: 5, and the nucleotide sequence for coding the glucamylase mutant is as shown in SEQ ID NO: 6. Compared with a wild type, the glucamylase mutant has higher hydrolytic activity and thermal stability, has important significance in improving the production efficiency and reducing the cost, and has good practicability.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a glucose amylase mutant and application thereof. Background Art

[0002] Glucoamylase (GLL) is an important industrial enzyme that can hydrolyze starch into glucose syrup without producing intermediate oligosaccharides. GLL has good applications in the food industry, biofuels, and bioplastics. For example, GLL is used in the food industry to produce high fructose syrup, which is an important sweetener that can be used to replace sucrose; in the production of biofuels, GLL can convert starch into glucose, which is then fermented to produce ethanol; in the production of bioplastics, GLL can produce raw materials for making bioplastics by hydrolyzing starch.

[0003] GLL has the defects of low enzyme activity and insufficient thermal stability, which seriously affect its widespread use in industrial production. Low enzyme activity means that GLL is not efficient in catalyzing the hydrolysis of starch into glucose, which limits its application in food processing, biofuel production and other fields. Especially under high temperature conditions, the activity of GLL decreases rapidly, resulting in a significant reduction in its efficiency and stability in industrial processes. These limitations require more enzymes or more frequent enzyme replacements to maintain the production process, which not only affects production efficiency but also increases production costs.

[0004] In order to solve the above problems, researchers have adopted a variety of strategies to improve the thermal stability and enzyme activity of GLL. These strategies include directed evolution, rational design and semi-rational design, which enhance the performance of GLL under industrial conditions by molecular modification; through rational design, on the basis of understanding the three-dimensional structure, catalytic mechanism and thermal stability mechanism of GLL, site-directed mutations are performed in key areas to improve its thermal stability and activity. In addition, the physicochemical properties of the enzyme can also be improved by immobilization technology, so that it can adapt to a wider range of industrial applications. Although the above methods can improve the industrial application value of GLL, reduce production costs, improve production efficiency, and promote the application of GLL in more industrial fields, they still have the shortcomings of low enzyme activity and poor thermal stability. Therefore, it is necessary to develop a new strategy to improve the thermal stability and enzyme activity of GLL. Summary of the invention

[0005] In view of some deficiencies in the prior art, the present invention provides a glucoamylase mutant and an application thereof; the present invention uses a machine learning algorithm to train the amino acid sequence and three-dimensional structure data of GLL, selects specific amino acid sites for site-directed mutation of six amino acids according to the prediction results of the machine learning model, and obtains a glucoamylase mutant; the amino acid sequence of the glucoamylase mutant is shown in SEQ ID NO:5, and the nucleotide sequence encoding the glucoamylase mutant is shown in SEQ ID NO:6; the glucoamylase mutant has higher hydrolysis activity and thermal stability than the wild type, which is of great significance for improving production efficiency and reducing costs, and has good practicality.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0007] The present invention first provides a glucoamylase mutant. The glucoamylase mutant is subjected to site-directed mutation of six amino acids based on a wild-type glucoamylase, wherein the mutation sites include: I73L, T130V, N212V, D238G, N327M and S332P.

[0008] Preferably, the amino acid sequence of the glucoamylase mutant is shown in SEQ ID NO:5.

[0009] The present invention also provides a nucleotide encoding a glucoamylase mutant, wherein the sequence of the nucleotide is shown in SEQ ID NO:6.

[0010] The present invention also provides a recombinant vector, which comprises the nucleotide encoding the glucoamylase mutant.

[0011] Preferably, the recombinant vector is a recombinant prokaryotic vector.

[0012] Preferably, the prokaryotic vector comprises a pET(+) plasmid or a pGEX vector.

[0013] The present invention also provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium comprises the recombinant vector or the nucleotide encoding the glucoamylase mutant.

[0014] Preferably, the host bacteria of the recombinant engineered bacteria include Escherichia coli, preferably Escherichia coli BL21 (DE3).

[0015] The present invention also provides a method for preparing the above-mentioned glucoamylase mutant, the method comprising:

[0016] The above recombinant engineering bacteria are inoculated into a fermentation medium, fermented and cultured, and then centrifuged to obtain a precipitate to obtain a crude product of the glucoamylase mutant.

[0017] Preferably, the fermentation medium comprises LB medium; the fermentation culture is performed until OD600 is 0.4-0.8.

[0018] The present invention also provides the use of the above-mentioned glucoamylase mutant, nucleotides encoding the glucoamylase mutant, recombinant vectors or recombinant engineering bacteria in food processing, biofuel production, and bioplastic production.

[0019] Preferably, the food processing comprises producing high fructose corn syrup;

[0020] The biofuel production includes converting starch into glucose, which is then fermented to produce ethanol;

[0021] The bioplastic production includes hydrolyzing starch to produce raw materials for making bioplastics.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention combines machine learning and genetic engineering technology, utilizes machine learning technology, quickly and accurately predicts the mutation site that improves enzyme activity, and then carries out site-directed mutation of six amino acids on the basis of wild-type GLL, thereby constructs glucoamylase mutant. The glucoamylase mutant has higher hydrolysis activity and better thermal stability than the wild-type, which is of great significance for improving production efficiency and reducing costs. The glucoamylase mutant of the present invention can not only broaden the scope of application of GLL, but also provide more efficient and stable biocatalysts for industrial production, thereby being expected to be more widely used in multiple fields such as food processing and biofuel production.

[0024] The present invention adopts an Escherichia coli expression system to express a glucoamylase mutant. The Escherichia coli cells have strong metabolic capacity and rich cell mechanisms, which enable them to efficiently synthesize and fold proteins. Compared with other expression systems, the Escherichia coli expression system is simple to operate and low in cost. The recombinant engineered bacteria culture and protein expression cycle of the present invention are short, and are suitable for rapid large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the X-ray crystal structure modeled for PyMOL version 4.4; in the figure, the target amino acid in 2Fo-Fc electron density (represented by blue mesh, contoured at 1.5σ) is superimposed on the licorice model of the reference and mutant amino acids; the original (orange) and replaced (light green) amino acids are represented by the licorice model; the active site (D207 and E210) is shown in magenta; the substrate binding site (W139) is shown in yellow.

[0026] Figure 2The figures are the expression (A) and purification (B) of GLL and its mutants; in Figure A, M is the protein marker; Ctrl is the control Escherichia coli containing the empty pET28a(+) plasmid; Lys is the whole cell lysate with expressed enzymes (GLL, GLL-4, GLL-6, GLL-10); Sup is the protein in the supernatant after centrifugation at 4°C; Pel is the precipitate or insoluble protein component after centrifugation at 4°C; in Figure B, Lys is the whole cell lysate with expressed enzymes (GLL, GLL-4, GLL-6, GLL-10), and Pure is the GLL, GLL-4, GLL-6, GLL-10 protein obtained after purification.

[0027] Figure 3 A diagram of the GLL polar interaction and the binding of the target sequence (before and after substitution) to any atom in its direct microenvironment; in the figure, A is before amino acid substitution and B is after amino acid substitution.

[0028] Figure 4 The structure of GLL (A) and the polar interactions and intermolecular bonding of GLN-25 (B) and GLU-25 (C); and the polar interactions of ASP-207 (D) and ASN-212 (E) before substitution.

[0029] Figure 5 Diagram of the starch hydrolysis mechanism of GLL with GLU-210 and ASP-207 as catalytic sites.

[0030] Figure 6 This is a comparison chart of the enzyme activities of GLL, GLL-4M, GLL-6M and GLL-10M.

[0031] Figure 7 The thermal stability and storage stability of GLL and GLL-6 are shown in the figure; in the figure, A is heat treatment for 30 minutes, B is heat treatment for 60 minutes, C is heat treatment for 120 minutes, and D is the stability after storage at 25°C for 30 days. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. In the following examples, if specific conditions are not specified, they are all carried out according to conventional known conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially. Unless otherwise specified, the present invention adopts the prior art in this field.

[0033] Example 1: Prediction and acquisition of glucoamylase mutants

[0034] Machine learning assisted calculation method uses the bioinformatics data of multiple protein sequences to predict one or more amino acid sequences, which can be replaced according to ancestor or consensus homology to produce stable mutants, which have experimental appeal in enzyme engineering research. The present embodiment predicts and designs glucoamylase mutants to wild-type glucoamylase (Saccharomycopsis fibuligera Glucoamylase accession number: ADZ30931.1) by machine learning assisted calculation method, in order to obtain glucoamylase mutants with higher hydrolytic activity and stability. Wherein, the amino acid sequence of the wild-type glucoamylase is as shown in SEQ ID NO:1, and the nucleotide sequence encoding the glucoamylase is as shown in SEQ ID NO:2.

[0035] The specific steps are as follows:

[0036] To improve the hydrolytic activity and stability of GLL, machine-assisted computational methods using FireProt (a web server for automated design of thermostable proteins, http: / / loschmidt.chemi.muni.cz / fireprot) and a web server (http: / / pmlabstack.pythonanywhere.com / SCMTPP) were used to predict and design glucoamylase mutants with potential for improved activity and stability, respectively.

[0037] First, a web server for the automated design of thermostable proteins (http: / / loschmidt.chemi.muni.cz / fireprot) was used to identify possible amino acids whose substitutions could produce mutants with improved thermostability. The consensus homology of this program predicted 23 substitutions. The predicted Fireprot substitutions were used to study the effects of induced multi-point mutations on GLL expression and performance. The specific substitutions are shown in Table 1.

[0038] Table 1. Thermostability values ​​of different glucoamylase mutants

[0039]

[0040]

[0041] As can be seen from Table 1, different amino acid mutations lead to different SCMTTPs, and the higher the value, the better the heat resistance.

[0042] This example also uses a second web server (http: / / pmlabstack.pythonanywhere.com / SCMTPP) to predict the thermal stability of proteins, which is then used to analyze the potential impact of each substitution on the stability of the resulting mutant GLL.

[0043] To confirm the accuracy of the program, 10 known experimentally characterized thermophilic proteins (TTPs with thermal stability between 80-100°C) were analyzed and the results are shown in Table 2.

[0044] Table 2. Predicted thermostability values ​​of experimentally determined thermostable proteins

[0045] Protein name Accession No. Thermal stability(℃) TPP Value Glutamate dehydrogenase 1BVU 109 432.64 β-xylosidase ACK42133.1 98 436.63 Cellulase ADA67783.1 90 436.99 Xylanase B QJQ82418.1 100 442.9 β-glucosidase WP_101510358.1 90 420.5 β-galactosidase ADA66698.1 95 425.4 β-glycosidase AAF36392.1 90 420.07 β-galactosidase ABI35983.1 85 430.58 Xylose isomerase AKE27551.1 95 425.01 α-galactosidase AAS82402.1 90 424.18

[0046] As can be seen from Table 2, all proteins have values ​​higher than the program's thermostability threshold of 418, indicating that higher TPPValue values ​​have better thermostability, and that SCMTPP prediction of stability is completely feasible. This indicates that the thermostability values ​​of the wild-type glucoamylase (WT-GLL) and the predicted 23 mutants are all below the required threshold, and therefore, mutants with SCMTTP values ​​lower than WT glucoamylase (368.39) were eliminated.

[0047] In order to improve the thermal stability of the mutants, a total of 10 amino acid substitutions were selected in this example to design three new GLL mutants, including GLL-4M, GLL-6M (GLL I73L / T130V / N212V / D238G / N327M / S332P) and GLL-10M (GLLQ25E / S29A / I73L / T130V / N144R / N212V / D238G / N327M / K328R / S332P). Among them, the mutation site of GLL-4M is GLLQ25E / S29A / N144R / K328R, its amino acid sequence is SEQ ID NO:3, and its nucleotide sequence is SEQ ID NO:4; the mutation site of GLL-6M is GLL I73L / T130V / N212V / D238G / N327M / S332P, its amino acid sequence is SEQ ID NO:5, and its nucleotide sequence is SEQ ID NO:6; the mutation site of GLL-10M is GLLQ25E / S29A / I73L / T130V / N144R / N212V / D238G / N327M / K328R / S332P, its amino acid sequence is SEQ ID NO:7, and its nucleotide sequence is SEQ ID NO:8. The prediction results are shown in Table 3.

[0048] Table 3. Designed mutants and their predicted thermal stability (TS) values

[0049]

[0050] As can be seen from Table 3, the TS of the three designed mutations of GLL-4M, GLL-6M and GLL-10M are all higher than that of GLL, and the ΔΔGfold Fold X of GLL-6M is the lowest, indicating that it may be more stable.

[0051] The crystal structure of GLL (PDB ID: 1AYX) was constructed using PyMol, in which the target amino acid substitution (before and after substitution) and the active site are in a network shape. The protein surface topography computer map (CASTp) online program (http: / / sts.bioe.uic.edu / castp / ) was used to determine the enzyme active site ( Figure 1 ). As can be seen from the figure, glucoamylase is a typical hydrolase with an acid / base and nucleophilic amino acid as active sites, GLU, E-210 acting as proton donors responsible for initiating the hydrolysis process, while ASP, D-207 acting as proton acceptors.

[0052] In summary, in order to obtain GLL with high enzyme activity and high stability, GLL-4M, GLL-6M and GLL-10M mutants were constructed according to the above analysis, and the solubility, enzyme activity and thermal stability of these three mutations were further studied subsequently.

[0053] Example 2. Construction of recombinant vector

[0054] The GLL, GLL-4M, GLL-6M, and GLL-10M obtained in Example 1 of the full gene synthesis of Nanjing GenScript Company were added with BamH I and EcoR I restriction sites at the 5' and 3' of the GLL, GLL-4M, GLL-6M, and GLL-10M sequences, respectively, to obtain GLL, GLL-4M, GLL-6M, and GLL-10M nucleotide sequences with BamH I and EcoR I restriction sites, and then the GLL, GLL-4M, GLL-6M, and GLL-10M nucleotide sequences with BamH I and EcoR I restriction sites were constructed into the pET28a(+) plasmid, and recorded as pET28a(+)-GLL, pET28a(+)-GLL-4M, pET28a(+)-GLL-6M, and pET28a(+)-GLL-10M.

[0055] E. coli was transformed with pET28a(+)-GLL, pET28a(+)-GLL-4M, pET28a(+)-GLL-6M and pET28a(+)-GLL-10M, respectively. The specific steps are as follows:

[0056] Take out the BL21(DE3) competent cell from the -80℃ refrigerator, place it on ice to melt, add 5μL of pET28a(+)-GLL, pET28a(+)-GLL-4M, pET28a(+)-GLL-6M and pET28a(+)-GLL-10M respectively, and gently tap the bottom of the EP tube with your hand to mix it. Place it in ice for 30 minutes, bathe in 42℃ water for 45 seconds, and quickly place it on ice for two minutes. After standing, 700 μL of preheated LB medium was added to the clean bench, mixed and shaken at 37°C and 200 rpm for 2 hours to revive the cells, then centrifuged at 10,000 rpm at room temperature for one minute to collect the bacteria, discarded 600 μL of supernatant, and the remaining supernatant was mixed with the bacteria, spread on LB solid medium containing kanamycin, and cultured in a constant temperature incubator at 37°C overnight to obtain recombinant Escherichia coli transformed with pET28a(+)-GLL, pET28a(+)-GLL-4M, pET28a(+)-GLL-6M and pET28a(+)-GLL-10M, i.e., recombinant Escherichia coli expressing GLL, GLL-4M, GLL-6M and GLL-10M.

[0057] Example 3. Expanded culture of Escherichia coli expressing GLL, GLL-4M, GLL-6M and GLL-10M

[0058] (1) The recombinant E. coli expressing GLL, GLL-4M, GLL-6M and GLL-10M obtained in Example 2 were transferred to solid LB medium containing 50 μg / mL kanamycin, respectively, and cultured overnight at 37°C. After the culture, the single colony in the medium was transferred to 5 mL of liquid LB medium containing 50 μg / mL kanamycin, and then cultured overnight in an orbital shaker at 37°C and 200 rpm. After the culture, 3 mL of the recombinant E. coli was inoculated into 300 mL of liquid LB medium containing 50 μg / mL kanamycin and grown at 37°C and 200 rpm for about 3 h until the OD600 of the bacteria reached between 0.4 and 0.6.

[0059] (2) After the cultivation in step (1), the liquid LB culture medium was placed on ice for 20 minutes, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added thereto at a final concentration of 0.4 mM, and the culture was shaken at 180 rpm for 16-20 h (overnight) at 25°C to induce the expression of GLL, GLL-4M, GLL-6M and GLL-10M. After the induction, the liquid culture medium was centrifuged at 3000 rpm and 4°C for 20 minutes, and the supernatant was discarded to obtain a cell pellet cultured at 25°C, which was stored at -80°C for later use.

[0060] (3) The cell pellets obtained in step (2) were thawed and resuspended in 10 mL of Tris-HCl (50 mM, pH 8.0), and then centrifuged at 3000 rpm and 4°C for 20 minutes, the supernatant was discarded, and the cells were collected and washed twice with 50 mM Tris-HCl (pH 8.0).

[0061] The cells were resuspended in 20 mL Tris-HCl buffer containing 1 mM PMSF (200 μL), and ultrasonically lysed on ice for 30 minutes using an ultrasonic cell disruptor, followed by alternating ultrasonic treatment for 6 seconds and intermittent cooling for 6 seconds to obtain a solution containing cell precipitates (total protein). The total protein was centrifuged at 13,000 rpm for 30 minutes at 4°C to obtain a supernatant and a precipitate. The supernatant was transferred to a new EP tube, and the precipitate was resuspended with 50 mM Tris-HCl, pH 8.0. The total protein, supernatant, and precipitate were subjected to SDS-PAGE to analyze the expression and solubility of the recombinant protein. The analysis results are shown in the figure. Figure 2 shown.

[0062] Figure 2 A shows that under the induction condition of 25℃, GLL, GLL-4M, GLL-6M and GLL-10M cell lysates (total protein) all showed significant bands at 58kDa, indicating that the above four recombinant proteins were successfully expressed; among them, GLL and GLL-4M expressed a lot in the supernatant, indicating that they were soluble, while GLL-4M and GLL-10M expressed very little in the supernatant, most of which were in the precipitate, indicating that they were expressed in the form of insoluble inclusion bodies. The insolubility and loss of activity of GLL-4M and GLL-6M can be attributed to an amino acid, whose substitution may have a reproductive effect on the intermolecular, intramolecular and polar bonds of the enzyme, thereby affecting the 3D conformational stability of the enzyme.

[0063] The homology model of the GLL mutant based on the X-ray crystal structure of WT-GLL (PDB ID: 1AYX) was checked using the SWISS-MODEL protein modeling server (https: / / swissmodel.expasy.org / interactive), and the results showed that GLL and mutant GLL were similar in structure. The original ( Figure 3 A) and replace ( Figure 3 B) All possible polar bonding of an amino acid to any atom within the sequence and its immediate microenvironment, revealing the impact of some substitutions on polar contacts, such as Q25E. However, most of the substituted amino acids made nearly identical polar contacts to neighboring atoms as the original amino acid (I73L, T130V, D238G, and S332P), with no significant effect on enzyme function.

[0064] The insolubility and loss of activity of GLL-4M and GLL-10M may be caused by a substitution in GLL-4Ms, as GLL-10 contains all the target substitutions for GLL-4M and GLL-6M. Looking at the FoldX values ​​provided by Fireprot, one can theoretically understand specific substitutions that may lead to insolubility and subsequent loss of activity. Although the 23 sequences generated by Fireprot are predicted to theoretically improve the stability of GLL, any amino acid with a positive FoldX energy value indicates that there is a problem with the 3D structure of the enzyme and may not be suitable for improving the solubility and function of the enzyme. These FoldX values ​​estimate the energy contribution of each amino acid in the protein, thereby providing an empirical understanding of their effects on maintaining protein stability. As shown in Table 3 in Example 1, Q25E has the highest positive FoldX energy value of +0.47 compared to other substituted amino acids, which may cause the enzyme to be not only insoluble but also inactive. Polar bonding analysis further evaluates changes in the polar interactions of Q25E with neighboring atoms in its direct microenvironment, as the stability of an enzyme depends on its ability to maintain its active structural conformation under changing conditions. While the polar neutral amino acid GLN-25 forms polar contacts with TYR-11, PHE-21, SER-29, and GLY-452, when it is replaced by the hydrophilic GLU-25, no interactions are formed with any amino acid except for contacts with a single water molecule. The lack of strong polar interactions at this site may lead to the disruption of ionic cohesion within the enzyme structure, thereby initiating and perpetuating the misfolding process through a self-reinforcing cascade ( Figure 4 A, B, C).

[0065] In terms of increasing GLL activity, replacing ASN-212 with VAL-212 had the greatest effect on GLL-6M function. Figure 4 The crystal structure of 1AYX, shown in D, E, shows that ASN-212 forms ∼5 polar interactions with surrounding atoms in its immediate environment, including 3 bonds to the nucleophilic ASP-207 residue and 2 water molecules. However, upon replacement with VAL-212, the bonding with ASP-207 was reduced to 1. This reduced binding may allow greater flexibility around the enzyme active site, thereby improving substrate accessibility to the catalytic site and thus enhancing hydrolysis.

[0066] Glucoamylase utilizes six conserved amino acid residues [Arg-69 (54), Asp-70 (55), Leu-208 (177), Try-209 (178), Glu-211 (180), and Arg-330 (305)] to bind and coordinate substrates at the catalytic site where two catalytic residues perform glycosidic bond hydrolysis. Structural analysis of glucoamylase studies has shown that the conserved catalytic base Glu-435 (400) and catalytic acid Glu-210 (179) are suitable catalytic residues. Based on predictions from the website (http: / / sts.bioe.uic.edu / castp / ), we hypothesize that Asp-207 and Glu-210 are the main catalytic residues responsible for catalyzing starch hydrolysis. In addition, the reaction mechanism of glucoamylase has been characterized for starch hydrolysis, which has been shown to occur through the simultaneous action of nucleophilic residues and acid / base residues. Glucoamylase-mediated starch hydrolysis involves a two-step double displacement hydrolysis reaction, in which the first step involves a nucleophilic attack of the carboxyl group of GLU-210 on the C1 carbon of the non-reducing end of starch and the cleavage of the glycosidic bond C1 Ogly, resulting in the formation of an intermediate covalently bound enzyme: substrate complex. The glycosyl intermediate (glucose) bound to an acid / base residue (ASP-207) is then transferred to a water donor to complete the hydrolysis process ( Figure 5 ).

[0067] In summary, although GLL-4M and GLL-10M can increase thermal stability, they are insoluble, and GLL-6M does not affect the solubility of the enzyme and increases the enzyme activity and thermal stability.

[0068] Example 4. Ni-NTA nickel column purification of GLL, GLL-4M, GLL-6M and GLL-10M proteins

[0069] The supernatant containing GLL, GLL-4M, GLL-6M and GLL-10M obtained in Example 3 was purified by Ni-NTA column, and the specific method and steps are as follows:

[0070] Add 10 mL of nickel removal buffer to the column, rinse twice until the column turns from blue to white, rinse the column twice with 10 mL of deionized water, and then slowly add 10 mL of NiSO 4 Buffer, cover the upper and lower lids, and incubate in a 4°C chromatography cabinet with rotation for 30 min. Open the lower lid to allow excess NiSO to 4 Drain and then rinse once with 10 mL of deionized water and once with 10 mL of PBS.

[0071] Add 4 mL of supernatant solution sample of GLL, GLL-4M, GLL-6M and GLL-10M after cell lysis to the column and rotate and incubate in a 4°C chromatography cabinet for 2 hours. Open the bottom cover to allow the protein sample that is not bound to the column to flow out, and rinse again with 15 mL of PBS buffer. After the rinse, elute with 2 mL of 150 mM, 250 mM, and 450 mM imidazole buffer respectively. Prepare protein loading samples and judge the purification results of GLL, GLL-4M, GLL-6M and GLL-10M by SDS-PAGE. The results are as follows: Figure 2 As shown in B.

[0072] As can be seen from the figure, GLL and GLL-6M have significant bands at 58kDa, and there are relatively few impurities, indicating that soluble GLL and GLL-6M were successfully purified. GLL-4M and GLL-10M have no significant bands, and the purified amount is very small. The main reason is that the content of GLL-4M and GLL-10M in the supernatant is very low, and they are mainly expressed in the form of insoluble inclusion bodies.

[0073] Example 5: GLL, GLL-4M, GLL-6M and GLL-10M enzyme activity detection

[0074] In this example, soluble starch was used as the substrate to determine the enzymatic activities of GLL, GLL-4M, GLL-6M and GLL-10M by reconstituting 5% (w / v) starch in distilled water and carefully heating at 80°C until the starch dissolved.

[0075] The reaction system for determining enzyme activity includes: 340 μL sodium phosphate buffer (pH 7.4), 50 μL starch and 15 μL enzyme (0.6 mg / mL), the reaction system is hydrolyzed at 40°C for 10 minutes, 100 μL 3,5-dinitrosalicylic acid (DNSA) reagent is added to the reaction system after the reaction is completed, boiled for 5 minutes, and then cooled on ice, 500 μL distilled water is added after cooling, the amount of reducing sugar released is measured at 540 nm, and the absorbance is used to determine the reducing sugar concentration relative to the glucose standard. As shown in the following formula, one unit (U) of glucoamylase activity is defined as the amount of enzyme required to release 1 mM reducing sugar (glucose) per minute at 40°C.

[0076] Where A is the change in absorbance per minute, ε is the molar extinction coefficient of DNSA and glucose standard, GLL is the enzyme concentration, and V is the total volume of the reaction. In this reaction system, the molar extinction coefficient of DNSA and glucose standard is 105.88 L mol -1 cm -1 , measured at 540nm, the results are as follows Figure 6 shown.

[0077] As can be seen from the figure, the enzyme activities of GLL, GLL-4M, GLL-6M and GLL-6M are 0.32, 0.07, 1.1 and 0.11 U / mg, respectively. The substitution of a single amino acid (most likely Q25E) in GLL-4M and GLL-10M may be the reason for the insolubility and inactivation of the enzyme due to its high FoldX energy value. Compared with the wild-type GLL, the activity of GLL-6M increased by 2.53 times. This indicates that the mutation of six amino acids in GLL-6M significantly increased the enzyme activity mutation and the catalytic activity of the enzyme was enhanced.

[0078] Example 6: Thermal and Storage Stability of GLL and GLL-6M

[0079] In this example, the thermal stability and storage stability of GLL and GLL-6M were investigated respectively to evaluate the stability of the glucoamylase mutants, as shown below.

[0080] The purified GLL and GLL-6M were placed at 4, 40, 45, 50, 55, 60 and 70°C for 30 minutes, 60 minutes and 120 minutes respectively to test the heat resistance of GLL and GLL-6M. The sample placed at 4°C was set as the reference point (100%) to evaluate the residual activity, and the curve of relative activity at different temperatures was drawn. The test results are shown in Figure 7 As shown in A, B, and C.

[0081] As can be seen from the figure, GLL and GLL-6M completely lost their activity after 30 min at 55 °C, but the residual activity of GLL-6M within 120 min at 45 °C and 50 °C was still relatively high, indicating that the amino acid substitution in GLL-6M improved the stability of the enzyme by inducing partial structural stability within the tolerable temperature point of the enzyme.

[0082] In this example, the purified GLL-6M and GLL were stored at 4°C and 40°C for 30 days, and the residual enzyme activity was detected every 5 days to investigate the storage stability of GLL-6M and GLL. The results are as follows: Figure 7 As shown in D.

[0083] As can be seen from the figure, with the extension of storage time, the activities of GLL-6M and GLL both showed a downward trend, but the downward trend of GLL was more obvious. On the 20th day, the activity of GLL decreased by about 16%, and on the 30th day it decreased by about 37%. The residual activity of GLL-6M after 20 days and 30 days was 95% and 83%, respectively. The results showed that the storage stability of the GLL-6M mutant was better than that of GLL, so the mutation of amino acids increased the storage stability of GLL-6M.

[0084] In summary, the present invention uses a machine learning algorithm to train the amino acid sequence and three-dimensional structure data of GLL, selects specific amino acid sites for site-directed mutation of six amino acids according to the prediction results of the machine learning model, and obtains a glucoamylase mutant; the amino acid sequence of the glucoamylase mutant is shown in SEQ ID NO:5, and the nucleotide sequence encoding the glucoamylase mutant is shown in SEQ ID NO:6; the glucoamylase mutant has higher hydrolysis activity and thermal stability than the wild type, which is of great significance to improving production efficiency and reducing costs, and has good practicality.

[0085] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A glucoamylase mutant, characterized in that The glucoamylase mutant is subjected to site-directed mutation of six amino acids based on the wild-type glucoamylase, and the mutation sites include: I73L, T130V, N212V, D238G, N327M and S332P.

2. The glucoamylase mutant according to claim 1, characterized in that The amino acid sequence of the glucoamylase mutant is shown in SEQ ID NO:

5.

3. A nucleotide encoding the glucoamylase mutant according to any one of claims 1 or 2, characterized in that: The nucleotide sequence is shown in SEQ ID NO:

6.

4. A recombinant vector, characterized in that: The recombinant vector comprises the nucleotide encoding the glucoamylase mutant according to claim 3.

5. The recombinant vector according to claim 4, characterized in that The recombinant vector is a recombinant prokaryotic vector.

6. The recombinant vector according to claim 5, characterized in that In the recombinant prokaryotic vector, the prokaryotic vector includes a pET(+) plasmid or a pGEX vector.

7. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria comprises the recombinant vector according to any one of claims 4 to 6 or the nucleotide encoding the glucoamylase mutant according to claim 3.

8. The recombinant engineered bacterium according to claim 7, characterized in that The host bacteria of the recombinant engineering bacteria include Escherichia coli.

9. A method for preparing the glucoamylase mutant according to claim 1 or 2, characterized in that: The method comprises: The recombinant engineered bacteria according to claim 7 or 8 are inoculated into a fermentation medium, fermented and cultured, and then centrifuged to obtain a precipitate to obtain a crude product of the glucoamylase mutant.

10. Use of the glucoamylase mutant according to claim 1 or 2, or the nucleotide according to claim 3, or the recombinant vector according to any one of claims 4 to 6, or the recombinant engineered bacteria according to any one of claims 7 to 8 in food processing, biofuel production, and bioplastic production.

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