A glucoamylase mutant and use thereof

Abstract

The application provides a glucose amylase mutant and application thereof, and belongs to the technical field of bioengineering; the amino acid sequence and three-dimensional structure data of GLL are trained by using a machine learning algorithm, specific amino acid sites are selected according to the prediction result of the machine learning model to perform site-directed mutation of six amino acids, and a glucose amylase mutant is obtained; the amino acid sequence of the glucose amylase mutant is shown as SEQ ID NO: 5, and the nucleotide sequence for coding the glucose amylase mutant is shown as SEQ ID NO: 6; compared with a wild type, the glucose amylase mutant has higher hydrolysis activity and thermal stability, has important significance for improving production efficiency and reducing cost, and has good practicability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a glucose amylase mutant and application thereof. BACKGROUND

[0002] Glucose amylase (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, etc. For example, GLL is used in the food industry to produce high fructose syrup, which is an important sweetener and can be used to replace sucrose; in the production of biofuels, GLL can convert starch into glucose for fermentation to produce ethanol; in the production of bioplastics, GLL can produce raw materials for manufacturing bioplastics by hydrolyzing starch.

[0003] GLL has the defects of low enzyme activity and poor 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. In particular, 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 replacement 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 various strategies to improve the thermal stability and enzyme activity of GLL. These strategies include directed evolution, rational design, and semi-rational design, which modify GLL molecules to enhance its performance under industrial conditions; through rational design, based on the understanding of the stereostructure, catalytic mechanism, and thermal stability mechanism of GLL, key regions are subjected to site-directed mutagenesis to improve its thermal stability and activity. In addition, immobilization technology can also improve the physicochemical properties of enzymes, making them suitable for 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, there are still deficiencies in 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

[0005] In view of some deficiencies in the prior art, the present application provides a glucoamylase mutant and application thereof; the present application trains amino acid sequences and three-dimensional structure data of GLL by using a machine learning algorithm, selects specific amino acid sites for six amino acid site-directed mutagenesis according to a prediction result of the machine learning model, and obtains a glucoamylase mutant; the amino acid sequence of the glucoamylase mutant is shown as SEQ ID NO: 5, and the nucleotide sequence for encoding the glucoamylase mutant is shown as SEQ ID NO: 6; the glucoamylase mutant has higher hydrolysis activity and thermal stability compared with a wild type, has important significance for improving production efficiency and reducing cost, and has good practicability.

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

[0007] The present application first provides a glucoamylase mutant, which is subjected to six amino acid site-directed mutagenesis on the basis of a wild type glucoamylase, and the mutation sites include I73L, T130V, N212V, D238G, N327M and S332P.

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

[0009] The present application also provides a nucleotide for encoding the glucoamylase mutant, and the sequence of the nucleotide is shown as SEQ ID NO: 6.

[0010] The present application also provides a recombinant vector, which comprises the above-mentioned nucleotide for encoding the glucoamylase mutant.

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

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

[0013] The present application also provides a recombinant engineering bacterium, which comprises the above-mentioned recombinant vector or the above-mentioned nucleotide for encoding the glucoamylase mutant.

[0014] Preferably, the host bacterium of the recombinant engineering bacterium includes Escherichia coli, preferably Escherichia coli BL21(DE3).

[0015] The present application also provides a method for preparing the above-mentioned glucoamylase mutant, and the method comprises:

[0016] The above-mentioned recombinant engineering bacterium is inoculated into a fermentation medium, is subjected to fermentation culture, is then centrifuged to obtain a precipitate, and a crude product of the glucoamylase mutant is obtained.

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

[0018] The application also provides the application of the above-mentioned glucoamylase mutant, the nucleotide encoding the glucoamylase mutant, the recombinant vector or the recombinant engineering bacteria in food processing, biofuel production and bioplastic production.

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

[0020] The biofuel production comprises converting starch into glucose, and then fermenting to produce ethanol;

[0021] The bioplastic production comprises hydrolyzing starch to produce raw materials for manufacturing bioplastics.

[0022] Compared with the prior art, the application has the beneficial effects that:

[0023] The application combines machine learning and genetic engineering technology, uses machine learning technology to quickly and accurately predict mutation sites that can improve enzyme activity, and then performs site-directed mutagenesis of six amino acids based on the wild type GLL, thereby constructing a 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 cost. The glucoamylase mutant of the application can not only broaden the application range of GLL, but also provide more efficient and stable biological catalysts for industrial production, thereby being expected to achieve more extensive application in food processing, biofuel production and other fields.

[0024] The application uses an E. coli expression system to express the glucoamylase mutant. The E. coli cell has strong metabolic capacity and rich cell mechanisms, so that it can efficiently synthesize and fold proteins. Compared with other expression systems, the E. coli expression system is simple to operate and low in cost. The recombinant engineering bacteria of the application have a relatively short culture and protein expression period, and are suitable for rapid large-scale production. BRIEF DESCRIPTION OF DRAWINGS

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

[0026] Figure 2Figure A, M is protein Marker; Ctrl is control E. coli containing empty pET28a(+) plasmid; Lys is whole cell lysate with expressed enzyme (GLL, GLL-4, GLL-6, GLL-10); Sup is protein in supernatant after centrifugation at 4°C; Pel is precipitate or insoluble protein fraction after centrifugation at 4°C; Figure B, Lys is whole cell lysate with expressed enzyme (GLL, GLL-4, GLL-6, GLL-10), Pure is GLL, GLL-4, GLL-6, GLL-10 protein after purification.

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

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

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

[0030] Figure 6 Figure for comparison of enzyme activity of GLL, GLL-4M, GLL-6M and GLL-10M.

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

[0032] The present application will be further described with reference to the following figures and embodiments, but the scope of the present application is not limited thereto. In the following examples, the specific conditions not specified are carried out according to the conventional known conditions or the conditions recommended by the manufacturer. The reagents or instruments used are conventional products that can be obtained by commercial purchase, unless otherwise specified. Unless otherwise specified, the present application employs the existing technology in the field.

[0033] Example 1: Prediction and obtaining of glucose amylase mutants

[0034] Machine learning assisted computational methods use bioinformatics data from multiple protein sequences to predict one or more amino acid sequences that can be replaced according to ancestral or consensus homology to produce stable mutants that are experimentally attractive in enzyme engineering studies. This embodiment uses machine learning assisted computational methods to predict and design glucose amylase mutants of wild-type glucose amylase (Saccharomycopsis fibuligera Glucoamylase Accession Number: ADZ30931.1) in order to obtain glucose amylase mutants with higher hydrolytic activity and stability. The amino acid sequence of the wild-type glucose amylase is shown as SEQ ID NO: 1, and the nucleotide sequence encoding the glucose amylase is shown as SEQ ID NO: 2.

[0035] The specific steps are as follows:

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

[0037] First, the web server for automatic design of thermostable proteins (http: / / loschmidt.chemi.muni.cz / fireprot) was used to determine the possible amino acids that can be replaced to produce mutants with improved thermal stability, and the consensus homology of this program predicted 23 substitutions. The predicted Fireprot substitutions were used to study the effect of induced multiple point mutations on GLL expression and performance, and the specific substitutions are shown in Table 1.

[0038] Table 1. Thermostability values of different glucose amylase mutants

[0039]

[0040]

[0041] As can be seen from Table 1, different amino acid mutations result in different SCMTTPs, and the higher the value, the better the thermostability.

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

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

[0044] Table 2. Predicted thermal stability values for experimentally determined thermophilic 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, the values for all proteins are above the thermal stability threshold of the procedure, 418, indicating that the higher the TPPValue, the better the thermal stability, and also that the SCMTPP prediction of stability is completely feasible. This indicates that the thermal stability values for the wild-type glucoamylase (WT-GLL) and the 23 mutants predicted are all below the required threshold, and therefore, mutants with SCMTTP values lower than the WT glucoamylase (368.39) were eliminated.

[0047] To improve the thermal stability of the mutants, 10 amino acid substitutions were selected in total, and three new GLL mutants were designed, including GLL-4M, GLL-6M (GLLI73L / T130V / N212V / D238G / N327M / S332P), and GLL-10M (GLLQ25E / S29A / I73L / T130V / N144R / N212V / D238G / N327M / K328R / S332P). Among them, the mutation sites of GLL-4M are GLLQ25E / S29A / N144R / K328R, the amino acid sequence thereof is SEQ ID NO: 3, and the nucleotide sequence thereof is SEQ ID NO: 4; the mutation sites of GLL-6M are GLLI73L / T130V / N212V / D238G / N327M / S332P, the amino acid sequence thereof is SEQ ID NO: 5, and the nucleotide sequence thereof is SEQ ID NO: 6; the mutation sites of GLL-10M are GLLQ25E / S29A / I73L / T130V / N144R / N212V / D238G / N327M / K328R / S332P, the amino acid sequence thereof is SEQ ID NO: 7, and the nucleotide sequence thereof 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 three mutations of GLL-4M, GLL-6M and GLL-10M are designed, the TS of the three mutations are higher than that of GLL, and the lowest ΔΔGfold Fold X of GLL-6M indicates that it is likely to be more stable.

[0051] The crystal structure of GLL (PDB ID: 1AYX) is constructed by PyMol, in which the target replacement amino acids (before and after replacement) and the active site are in a network. The online program of protein surface topography computer atlas (CASTp) (http: / / sts.bioe.uic.edu / castp / ) determines the enzyme active site ( Figure 1 ). As can be seen from the figure, glucoamylase is a typical hydrolase, and the active site is acid / base and nucleophilic amino acid, GLU, E-210 acts as a proton donor responsible for initiating the hydrolysis process, and ASP, D-207 acts as a proton acceptor.

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

[0053] Example 2. Construction of recombinant vector

[0054] The GLL, GLL-4M, GLL-6M and GLL-10M obtained in Example 1 are synthesized by Nanjing Kingsriver Company, and the 5' and 3' of the GLL, GLL-4M, GLL-6M and GLL-10M sequences are respectively provided with BamH I and EcoR I restriction enzyme sites, to obtain GLL, GLL-4M, GLL-6M and GLL-10M nucleotide sequences with BamH I and EcoR I restriction enzyme sites, and then the GLL, GLL-4M, GLL-6M and GLL-10M nucleotide sequences with BamH I and EcoR I restriction enzyme sites are constructed into pET28a(+) plasmid, which is denoted as pET28a(+)-GLL, pET28a(+)-GLL-4M, pET28a(+)-GLL-6M and pET28a(+)-GLL-10M.

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

[0056] The BL21 (DE3) competent cells were taken out from the -80 °C refrigerator, placed on ice to thaw, 5 μL of pET28a (+) -GLL, pET28a (+) -GLL-4M, pET28a (+) -GLL-6M and pET28a (+) -GLL-10M were added respectively, and the EP tube bottom was gently tapped with hands to mix, placed in ice for 30 minutes, 42 °C water bath for 45 seconds, quickly placed on ice for two minutes. After standing, 700 μL of preheated LB medium was added in the clean bench, mixed and then shaken in the shaking table at 37 °C, 200 rpm for 2 h to recover the cells, then centrifuged at room temperature 10000 rpm for one minute to collect the bacteria, 600 μL of supernatant was discarded, and the remaining supernatant and bacteria were mixed and then coated on the LB solid medium containing kanamycin, and cultured in the 37 °C constant temperature incubator overnight to obtain recombinant E. coli transformed with pET28a (+) -GLL, pET28a (+) -GLL-4M, pET28a (+) -GLL-6M and pET28a (+) -GLL-10M, namely recombinant E. coli expressing GLL, GLL-4M, GLL-6M and GLL-10M.

[0057] Example 3. Scale-up culture of E. 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 was transferred to solid LB medium containing 50 μg / mL kanamycin, and cultured overnight at 37 °C. After the culture, a single colony in the medium was transferred to 5 mL of liquid LB medium containing 50 μg / mL kanamycin, and then cultured overnight in a 37 °C, 200 rpm orbital shaker. After the culture, 3 mL of recombinant E. coli was inoculated into 300 mL of liquid LB medium containing 50 μg / mL kanamycin and grown at 37 °C, 200 rpm for about 3 h until the OD600 of the bacteria reached between 0.4-0.6.

[0059] (2) The liquid LB medium after step (1) was placed on ice for 20 minutes, and isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM, and GLL, GLL-4M, GLL-6M and GLL-10M expression was induced by shaking at 25 °C, 180 rpm for 16-20 h (overnight). After the induction, the liquid medium was centrifuged at 3000 rpm, 4 °C for 20 minutes to discard the supernatant, and the cell precipitate cultured at 25 °C was obtained and stored at -80 °C for standby.

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

[0061] The bacterial cells were resuspended in 20 mL Tris-HCl buffer containing 1 mM PMSF (200 μL) and lysed using an ultrasonic cell disruptor for 30 min on ice, followed by 6 s of alternating sonication and 6 s of intermittent cooling, to obtain a solution containing cell pellets (total protein). The total protein was centrifuged at 13000 rpm for 30 min at 4 °C to obtain the supernatant and the precipitate. The supernatant was transferred to a new EP tube, while the precipitate was resuspended with 50 mM Tris-HCl (pH 8.0). SDS-PAGE was performed on the total protein, supernatant, and precipitate to analyze the expression and solubility of the recombinant proteins, and the results are shown in Figure 2

[0062] Figure 2 A shows that under the induction conditions at 25 °C, the cell lysates (total protein) of GLL, GLL-4M, GLL-6M, and GLL-10M all have a significant band at 58 kDa, indicating that the above four recombinant proteins are successfully expressed; among them, the expression amount in the supernatant of GLL and GLL-4M is large, indicating that it is soluble, while the expression amount in the supernatant of GLL-4M and GLL-10M is small, most of which is in the precipitate, indicating that it is 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, the substitution of which may have a proliferative effect on the intermolecular, intramolecular, and polar bonds of the enzyme, thereby affecting the 3D conformational stability of the enzyme.

[0063] The homology models of GLL mutants based on the WT-GLL (PDB ID: 1AYX) X-ray crystal structure were checked using the SWISS-MODEL protein modeling server (https: / / swissmodel.expasy.org / interactive), and the results showed that GLL and mutant GLL were structurally similar. Then the original ( Figure 3 A) and the substituted ( Figure 3 B) amino acids were checked for all possible polar bonds with any atom within the sequence and its immediate microenvironment using PyMol software, which revealed that certain substitutions had an impact on polar contacts, such as Q25E. However, the polar contacts of most substituted amino acids with adjacent atoms were almost the same as the original amino acids (I73L, T130V, D238G, and S332P), which had 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 one of the GLL-4Ms, as GLL-10 contains all the target substitutions of GLL-4M and GLL-6M. Examining the FoldX values ​​provided by Fireprot allows for a theoretical understanding of the specific substitutions that may lead to insolubility and subsequent loss of activity. Although the 23 sequences predicted by Fireprot are theoretically likely to improve GLL stability, any amino acid with a positive FoldX energy value indicates a problem with the enzyme's 3D structure and may not be suitable for improving enzyme solubility and function. These FoldX values ​​estimate the energy contribution of each amino acid in the protein, thus providing an empirical understanding of their impact on maintaining protein stability. As shown in Table 3 of Example 1, Q25E has the highest positive FoldX energy value of +0.47 compared to other substitution amino acids, which may lead to not only insolubility but also inactivation of the enzyme. Polar bonding analysis further assesses variations in the polar interactions of Q25E with neighboring atoms in its immediate microenvironment, as enzyme stability depends on its ability to maintain its active structural conformation under varying conditions. Although 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, it does not interact with any amino acid except for contact with a single water molecule. This lack of strong polar interaction at this site may lead to the disruption of ionic cohesion within the enzyme structure, thereby initiating and prolonging misfolding processes through a self-reinforcing cascade. Figure 4 (A, B, C)

[0065] In terms of improving GLL activity, replacing ASN-212 with VAL-212 has the greatest impact on GLL-6M function. For example... Figure 4 As shown in D and E, the crystal structure of 1AYX indicates that ASN-212 forms ~5 polar interactions with surrounding atoms in its immediate environment, including 3 bonds with nucleophilic ASP-207 residues and 2 water molecules. However, after replacing VAL-212 with ASP-207, the binding is reduced to 1. This reduced binding may allow for greater flexibility around the enzyme's active site, thereby improving substrate accessibility to the catalytic site and enhancing hydrolysis.

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

[0067] In summary, GLL-4M and GLL-10M, while increasing thermal stability, 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 subjected to Ni-NTA column purification, the method steps are described as follows:

[0070] 10 mL of Ni removal buffer was added to the column, washed twice until the column changed from blue to white, the column was washed twice with 10 mL of deionized water, then 10 mL of NiSO4buffer was slowly added to the column, covered with the upper and lower covers, and incubated at 4°C in the chromatography cabinet for 30 min. Open the lower cover, let the excess NiSO4flow out, then wash with 10 mL of deionized water and 10 mL of PBS respectively.

[0071] ​The column was added with 4 mL supernatant sample of GLL, GLL-4M, GLL-6M and GLL-10M after centrifugation after cell lysis, and incubated at 4°C in the rotating incubator for 2 h. The lower cover was opened to allow the protein sample not bound to the column to flow out. Then, 15 mL PBS buffer was used for flushing once again. After flushing, 2 mL 150 mM, 250 mM and 450 mM imidazole buffer was used for elution, respectively. The protein sample was prepared, and the purification results of GLL, GLL-4M, GLL-6M and GLL-10M were determined by SDS-PAGE, and the results are shown in Figure 2 B.

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

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

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

[0075] The reaction system for determining the 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 was hydrolyzed at 40°C for 10 min. After the reaction was completed, 100 μL 3,5-dinitrosalicylic acid (DNSA) reagent was added to the reaction system, boiled for 5 min, then cooled on ice, and after cooling, 500 μL distilled water was added. The amount of reducing sugar released was measured at 540 nm, and the absorbance was used to determine the concentration of reducing sugar 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 of reducing sugar (glucose) per minute at 40°C.

[0076] Wherein A is the change of absorbance per minute, ε is the molar extinction coefficient of DNSA with 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 with glucose standard is 105.88 L mol -1 cm -1 at 540 nm. The results are shown in Figure 6 .

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

[0078] Example 6: Thermal stability and storage stability of GLL and GLL-6M

[0079] This example investigates the thermal stability and storage stability of GLL and GLL-6M, respectively, to evaluate the stability of the glucose amylase mutant, 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 to detect the heat resistance of GLL and GLL-6M, and the samples placed at 4°C were set as the reference point (100%) to evaluate the residual activity, and the curves of different temperatures versus relative activity were drawn, and the test results are shown in Figure 7 A, B and C.

[0081] As can be seen from the figure, GLL and GLL-6M completely lose activity after 30 minutes at 55°C, but the residual activity of GLL-6M is still relatively high within 120 minutes at 45°C and 50°C, which indicates that the amino acid substitution in GLL-6M improves 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 also stored at 4°C and 40°C for 30 days, respectively, and the residual enzyme activity was detected every 5 days to investigate the storage stability of GLL-6M and GLL, and the investigation results are shown in Figure 7 D.

[0083] As can be seen from the figure, the activities of GLL-6M and GLL both show a downward trend as the storage time is prolonged, but the downward trend of GLL is 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 activities of GLL-6M after 20 days and 30 days are 95% and 83%, respectively. The results show that the storage stability of GLL-6M mutant is better than that of GLL, and therefore the mutation of amino acids increases the storage stability of GLL-6M.

[0084] To sum up, the amino acid sequence and three-dimensional structure data of GLL are trained by using a machine learning algorithm, a specific amino acid site is selected for six amino acid site-directed mutation according to the prediction result of the machine learning model, and a glucose amylase mutant is obtained; the amino acid sequence of the glucose amylase mutant is shown as SEQ ID NO: 5, the nucleotide sequence encoding the glucose amylase mutant is shown as SEQ ID NO: 6; the glucose amylase mutant has higher hydrolysis activity and thermal stability compared with the wild type, has important significance for improving production efficiency and reducing cost, and has good practicability.

[0085] The embodiments are preferred embodiments of the present application, but the present application is not limited to the above embodiments, and any obvious improvements, replacements or modifications made by those skilled in the art without departing from the essential content of the present application shall fall within the protection scope of the present application.

Claims

1. A glucoamylase mutant, characterized in that, The glucoamylase mutant is based on the wild-type glucoamylase and has six site-directed mutations of amino acids, and the mutation sites include I73L, T130V, N212V, D238G, N327M and S332P; the amino acid sequence of the glucoamylase mutant is shown in SEQ ID NO:

5.

2. Nucleic acid encoding the glucoamylase mutant of claim 1, characterized in that, The sequence of the nucleotide is shown in SEQ ID NO:

6.

3. A recombinant vector, characterized in that, The recombinant vector comprises the nucleotide encoding the glucoamylase mutant according to claim 2.

4. The recombinant vector of claim 3, wherein, The recombinant vector is a recombinant prokaryotic vector.

5. The recombinant vector of claim 4, wherein, In the recombinant prokaryotic vector, the prokaryotic vector includes a pET (+) plasmid or a pGEX vector.

6. A recombinant engineered bacterium, characterized in that, The recombinant engineering bacteria comprise the recombinant vector according to any one of claims 3-5 or the nucleotide encoding the glucoamylase mutant according to claim 2.

7. The recombineering bacteria of claim 6, wherein, The host bacteria of the recombinant engineering bacteria include Escherichia coli.

8. A method of producing the glucoamylase mutant of claim 1, comprising, The method comprises: Inoculating the recombinant engineering bacteria according to claim 6 or 7 into a fermentation medium, fermenting, then centrifuging to obtain the crude product of the glucoamylase mutant.

9. The glucoamylase mutant according to claim 1, or the nucleotide according to claim 2, or the recombinant vector according to any one of claims 3-5, or the recombinant engineering bacteria according to any one of claims 6-7 are applied in food processing, biofuel production and bioplastic production.

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