Alpha-amylase, alpha-amylase gene, high-yield alpha-amylase engineering bacterium and application thereof

Abstract

The invention relates to alpha-amylase, an alpha-amylase gene, an engineering bacterium for high yield of alpha-amylase and application of the engineering bacterium, and belongs to the technical field of biology and the technical field of enzyme engineering. According to the invention, an alpha-amylase gene is identified from a strain A8-1 with high starch hydrolysis activity, and the alpha-amylase coded by the alpha-amylase gene has the advantages of strong pH stability, wide temperature resistance range and strong resistance to organic solvents. Furthermore, by heterologous expression of the amylase gene in different hosts, a recombinant strain BAX-5 / PTamyy (A8-1) with the highest enzymatic activity is obtained, and the enzymatic activity of the recombinant strain is 156.1 U / mL. Finally, through signal peptide and promoter engineering, a recombinant strain BAX-5 / PTmy (A8-1) SP002 with the highest enzyme activity is obtained, and the enzyme activity reaches 205 U / mL.

Description

Technical Field

[0001] The invention relates to alpha-amylase, alpha-amylase gene, engineering bacteria with high alpha-amylase production and application thereof, belonging to the fields of biotechnology and enzyme engineering technology. Background Art

[0002] Amylase is an enzyme found in humans and microorganisms, and plays a fundamental role in the hydrolysis of starch and polysaccharides into simple sugars such as glucose and maltose. The wide application of amylase covers many industries: in food processing, it accelerates the hydrolysis of starch for products such as bread and beer; in pharmaceuticals, it helps to supplement digestive enzymes; in biotechnology, it promotes DNA extraction and enzymatic digestion reactions; in the textile industry, it enhances the softness of fabrics by removing starch residues. This versatile enzyme significantly improves production efficiency and product quality in these different fields.

[0003] According to the different positions of the hydrolysis catalyzed by amylase, there are three main types: α-amylase, β-amylase and γ-amylase. α-Amylase (EC 3.2.1.1) belongs to glycoside hydrolase family 13 (GH13), has a multidomain structure, and hydrolyzes internal α-1,4-glycosidic bonds in starch and related sugars to produce shorter oligosaccharides. In contrast, β-amylase (EC 3.2.1.2) belongs to glycoside hydrolase family 14 (GH14), is usually a monomeric enzyme, and specifically hydrolyzes the second α-1,4-glycosidic bond from the non-reducing end of the polyglucan chain to produce maltose. On the other hand, γ-amylase (EC 3.2.1.3) belongs to glycoside hydrolase family 15 (GH15), in addition to hydrolyzing the non-reducing α-1,4-glycosidic bonds of starch and amylopectin, it also hydrolyzes α-1,6-glycosidic bonds, unlike other forms of amylase, to produce glucose. Currently, α-amylase is more widely used than β-amylase and γ-amylase due to its advantages of broader substrate specificity, abundant sources in various organisms, wide pH range adaptability, and diversity in multiple industrial applications such as food processing, brewing, textiles, and biotechnology. In addition, the stability of α-amylase under various conditions and tolerance to high temperatures further enhance its practicality in industrial environments.

[0004] Alpha-amylase can be obtained from plants, animals and microorganisms. However, the activity and yield of alpha-amylase from plant and animal sources are low, which significantly limits its industrial development and application. With the progress and improvement of microbial fermentation technology, microbial fermentation has become the main method for industrial alpha-amylase production due to its advantages such as high efficiency, controllability, abundant sources, sustainability and high purity. At present, it is reported in the prior art that both fungi and bacteria can produce alpha-amylase, and most of the fungi that produce alpha-amylase are concentrated in heat-resistant fungi, especially Aspergillus (Aspergillus) and Penicillium (Penicillium) fungi, and specific culture conditions have been established, and excellent fungal strains have been selected for commercial-scale production. In addition, various bacteria including Bacillus (Bacillus) and certain halophilic bacteria such as Chromohalobacter sp. (Chromohalobacter) and Halobacillus sp. (Halobacillus) can also produce alpha-amylase. However, for commercial applications, α-amylases are mainly derived from Bacillus bacteria. Enzymes produced by species such as Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis, Bacillus megaterium and Bacillus circulans have shown potential application value in multiple industrial processes.

[0005] Compared with the complexity of molecular genetic manipulation in fungi and the endotoxin safety issues associated with Escherichia coli, the Bacillus expression system has the advantages of non-pathogenicity to the host, no endotoxin in the expressed protein, significant heat resistance, no significant codon preference, and efficient secretion signal peptides and molecular chaperone systems. In most cases, the recombinant protein secreted by Bacillus can maintain its native conformation and biological activity, thereby avoiding the formation of inclusion bodies. In addition, the Bacillus expression system has a short expression cycle and high protein yield. Therefore, the Bacillus expression system is widely used to efficiently express various enzymes. For example, Li et al. combined dual promoter engineering and promoter modification to enhance the secretion yield of myZ1 (an amylase gene) in B. subtilis (Bacillus subtilis), and the extracellular activity of AmyZ1 (an amylase) increased by 3.11 times. Chen et al. optimized the secretion efficiency of recombinant AmyM (an amylase) by overexpressing the molecular chaperone prsA (a molecular chaperone gene) gene, and its extracellular enzyme activity increased by 101.58%. Although considerable efforts have been made to increase production, the development of more efficient α-amylases remains of great importance due to the growing industrial demand. Summary of the invention

[0006] The first object of the present invention is to provide an alpha-amylase, which provides an alpha-amylase with strong pH stability, wide temperature resistance range and strong resistance to organic solvents.

[0007] The second purpose of the present invention is to provide an α-amylase gene, which provides a new α-amylase gene for the prior art.

[0008] The third object of the present invention is to provide an engineered bacterium with high α-amylase production, which provides an engineered bacterium with high-efficiency expression of α-amylase in the prior art.

[0009] The fourth object of the present invention is to provide an application of an engineered bacterium with high α-amylase production in α-amylase production to solve the problem of low α-amylase production efficiency of α-amylase producing bacteria in the prior art.

[0010] In order to achieve the above object, a technical solution of an α-amylase in the present invention is:

[0011] An alpha-amylase, the amino acid sequence of the alpha-amylase is shown as SEQ ID NO.1, SEQ ID NO.18 or SEQ ID NO.19.

[0012] The beneficial effect of the above technical scheme is that: an α-amylase of the present invention is a pioneering invention. The present invention obtains a natural strain A8-1 with strong starch degradation ability by screening, and analyzes the enzymatic characteristics of the α-amylase produced by A8-1 fermentation. It is found that the α-amylase has the maximum enzyme activity at 50°C, but can maintain a high enzyme activity in the temperature range of 30-70°C, and when the temperature is 80°C, it can still maintain more than 50% enzyme activity; at the same time, the α-amylase has the maximum enzyme activity at pH 7, but can maintain a high enzyme activity in the range of pH 4-8; and organic solvents such as ethanol, glycerol, and propylene glycol have little effect on the enzyme activity of the α-amylase. This shows that the α-amylase provided by the present invention having an amino acid sequence as shown in SEQ ID NO.1 has the advantages of strong pH stability, a wide temperature resistance range, and strong resistance to organic solvents.

[0013] Furthermore, the present invention determines the signal peptide in the α-amylase with the amino acid sequence shown in SEQ ID NO.1 through bioinformatics analysis, and replaces the original signal peptide with tag1 (signal peptide SP001) derived from lactic acid bacteria Lactobacillus plantarum and bglS (signal peptide SP002) derived from Geobacillus stearothermophilus through genetic engineering to obtain α-amylase with the amino acid sequence shown in SEQ ID NO.18 and SEQ ID NO.19.

[0014] In order to achieve the above object, a technical solution of an α-amylase gene in the present invention is:

[0015] An alpha-amylase gene, the nucleotide sequence of the alpha-amylase gene is shown as SEQ ID NO.2, SEQ ID NO.16 or SEQ ID NO.17.

[0016] The beneficial effect of the above technical solution is that: the present invention analyzes the genome of the natural strain A8-1 with strong starch degradation ability and finds that it has an α-amylase gene amy(A8-1) with a nucleotide sequence as shown in SEQ ID NO.2, and the α-amylase encoded by it has the advantages of strong pH stability, wide temperature resistance range and strong resistance to organic solvents. The present invention provides a new α-amylase gene derived from Bacillus velez for the prior art.

[0017] Furthermore, the present invention replaces the nucleotide sequence encoding the signal peptide portion in the α-amylase gene amy (A8-1) through genetic engineering technology to obtain α-amylase genes having nucleotide sequences as shown in SEQ ID NO.16 and SEQ ID NO.17.

[0018] In order to achieve the above object, the technical scheme of an engineering bacterium for high-yield α-amylase in the present invention is:

[0019] An engineered bacterium with high alpha-amylase production, wherein the engineered bacterium is started from Bacillus and overexpresses an alpha-amylase gene having a nucleotide sequence as shown in SEQ ID NO.2, SEQ ID NO.16 or SEQ ID NO.17; the Bacillus is Bacillus amyloliquefaciens, Bacillus subtilis or Bacillus licheniformis.

[0020] The beneficial effect of the above technical solution is that the present invention uses genetic engineering technology to construct a recombinant expression vector expressing the α-amylase gene amy (A8-1), transforms it into different Bacillus hosts, overexpresses the α-amylase gene amy (A8-1) in different Bacillus hosts, and obtains an engineered bacterium with high α-amylase production. Analysis of the fermentation activity of α-amylase shows that the α-amylase gene amy (A8-1) can be efficiently expressed in Bacillus amyloliquefaciens, Bacillus subtilis or Bacillus licheniformis.

[0021] As a further improvement, the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens HZ12 or Bacillus amyloliquefaciens BAX-5; the Bacillus subtilis is Bacillus subtilis WB800 or Bacillus subtilis 168; and the Bacillus licheniformis is Bacillus licheniformis BL10.

[0022] The beneficial effect of the above technical solution is that: the present invention conducts α-amylase fermentation activity analysis on multiple engineered bacteria constructed, and the results show that the α-amylase gene amy (A8-1) can be expressed in the above host bacteria, among which BAX-5 is the best host.

[0023] As a further improvement, the method for constructing the engineered bacteria comprises the following steps: constructing a recombinant expression vector for expressing the α-amylase gene, and transforming the vector into Bacillus to overexpress the α-amylase.

[0024] As a further improvement, the starting vector of the recombinant expression vector is pT-17.

[0025] In order to achieve the above object, the technical scheme of the application of an engineering bacterium with high α-amylase production in the present invention is:

[0026] The invention discloses an application of an engineering bacterium with high alpha-amylase production in the production of alpha-amylase.

[0027] The beneficial effect of the above technical solution is that the present invention further optimizes the fermentation conditions in order to further improve the activity of α-amylase. Under the optimal conditions, the enzyme activity of BAX-5 / PTamy (A8-1) SP002 reaches 1440U / mL, which is 9.2 times higher than that of the original wild-type strain A8-1, and the viscosity problem of the fermentation liquid is alleviated, which has obvious advantages in the production of α-amylase. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The amino acid comparison result of α-amylase of strain A8-1 and strain Bacillus velezensis UCMB5033 in Example 1 of the present invention (in the figure, the upper row represents strain A8-1, and the lower row represents Bacillus velezensis UCMB5033);

[0029] Figure 2 The nucleotide comparison result of α-amylase of strain A8-1 and strain Bacillus velezensis UCMB5033 in Example 2 of the present invention (in the figure, the upper row represents strain A8-1, and the lower row represents Bacillus velezensis UCMB5033);

[0030] Figure 3 Screening of high-yield amylase strains in Experimental Example 1 (in the figure, (A) is the α-amylase activity of 7 wild-type strains; (B) is the Gram staining of strains CK3-5 and A8-1);

[0031] Figure 4 A further identification diagram of strains CK3-5 and A8-1 in Experimental Example 1 of the present invention (in the figure, (A) is the non-redundant protein NR annotation result of CK3-5 and A8-1; (B) is a phylogenetic tree with 8 representative Bacillus strains, the horizontal coordinate represents the first 20 species, and the vertical coordinate represents the number of matched genes);

[0032] Figure 5 is the relative enzyme activity of α-amylase in Experimental Example 3 of the present invention under different pH environments;

[0033] Figure 6 is the relative enzyme activity of α-amylase in Experimental Example 3 of the present invention under different temperature environments;

[0034] Figure 7 The effect of metal ions on α-amylase activity in Experimental Example 3 of the present invention (NC in the figure represents a negative control, without adding metal ions);

[0035] Figure 8 The relative enzyme activity of α-amylase in Experimental Example 3 of the present invention under different organic reagent environments;

[0036] Fig. 9 The heterologous expression and molecular docking of different α-amylase genes in different hosts in Experimental Example 4 of the present invention (in the figure, (A) is a schematic diagram of the construction of recombinant strains and molecular docking; (B) is a comparison of α-amylase activities of recombinant strains with different α-amylase genes, * represents P≤0.05, ** represents P≤0.01; (C) is a comparison of α-amylase activities of recombinant strains of different hosts; (D) is a heat map of α-amylase activities of recombinant strains, purple squares represent low enzyme activity, and yellow squares represent high enzyme activity; (E) is the molecular docking of different amylases with maltotriose, different secondary structures of proteins are displayed in different colors, and the ligand is highlighted in yellow);

[0037] Fig.10 This is a molecular docking diagram of α-amylase of Bacillus velezensis UCMB5033 strain and maltotriose in Experimental Example 4 of the present invention (in the figure, different secondary structures of the protein are displayed in different colors, and the ligand is highlighted in yellow);

[0038] Fig.11 The bioinformatics signal peptide sequence analysis of α-amylase AMY (A8-1) in Experimental Example 5 of the present invention;

[0039] Fig.12 The effects of different elements in Experimental Example 5 of the present invention on the enzymatic activity of α-amylase AMY (A8-1) (in the figure, (A) is a signal peptide; (B) is a promoter, ns represents no significant difference, * represents P≤0.05, ** represents: P≤0.01);

[0040] Fig.13 This is the enzyme activity during the fermentation process in Experimental Example 6 of the present invention (the orange column in the figure represents the recombinant strain BAX-5 / PTamy (A8-1) SP002, and the blue column represents the wild-type strain A8-1). DETAILED DESCRIPTION

[0041] α-amylase is one of the main enzymes widely used in industry. With the progress of industrialization, the global demand for α-amylase is increasing. Therefore, people have shown significant interest in isolating strains with higher α-amylase production performance. In the early stage of the present invention, a strain with high starch hydrolysis activity was isolated from tobacco leaves through a large number of separation and screening, and was named A8-1. Through whole genome sequencing, genome alignment and gene annotation analysis, A8-1 was confirmed to be Bacillus velezensis, and the α-amylase gene amy (A8-1) was identified. The nucleotide sequence of the α-amylase gene amy (A8-1) is shown in SEQ ID NO.1. The enzymatic characteristics of the α-amylase produced by A8-1 fermentation were analyzed, and it was found that the α-amylase had the highest enzyme activity at 50°C, but it could maintain a high enzyme activity in the temperature range of 30-70°C, and when the temperature was 80°C, it could still maintain more than 50% of the enzyme activity; at the same time, the α-amylase had the highest enzyme activity at pH 7, but it could maintain a high enzyme activity in the pH range of 4-8; and organic solvents such as ethanol, glycerol, and propylene glycol had little effect on the enzyme activity of the α-amylase. This shows that the α-amylase encoded and expressed by the α-amylase gene amy (A8-1) has the advantages of strong pH stability, wide temperature resistance, and strong resistance to organic solvents. Moreover, A8-1 was isolated from tobacco leaves, and the expressed α-amylase was more advantageous in degrading starch in tobacco than α-amylases from other sources.

[0042] Furthermore, the present invention uses genetic engineering technology to construct a recombinant expression vector expressing the α-amylase gene amy (A8-1), transforms it into different Bacillus hosts, overexpresses the α-amylase gene amy (A8-1) in different Bacillus hosts, and obtains an engineered bacterium with high α-amylase production. Moreover, in order to further improve the enzymatic activity of the engineered bacteria producing α-amylase, the present invention also screens and verifies the promoter and signal peptide, and finds that compared with the signal peptide in the α-amylase gene amy (A8-1) gene, tag1 (signal peptide SP001) from lactic acid bacteria Lactobacillus plantarum and bglS (signal peptide SP002) from Geobacillus stearothermophilus show higher α-amylase activity; the recombinant strain with the promoter P43 from Bacillus subtilis shows the highest α-amylase activity.

[0043] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto; unless otherwise specified, the various reagents, instruments, etc. used in the embodiments are commercially available products. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0044] Whole genome sequencing and bioinformatics analysis in the following examples of the present invention: Whole genome sequencing was performed by PacBio sequencing and Illumina PE150 sequencing platforms (Beijing Novo Gene Technology Co., Ltd., Beijing, China). Whole genome alignment analysis was performed using CLCworkbench, COG, KEGG, GO and EC annotations. Signal peptides were analyzed using SignalP 5.0.

[0045] Statistical analysis in the following examples of the present invention: Three independent replicates were designed for each group of experiments. SPSS 26.0 was used for statistical analysis, including calculation of mean and deviation values. Graphpad prism 9 was used for data processing and preparation of histograms.

[0046] The Bacillus used in the following examples of the present invention are Bacillus amyloliquefaciens HZ12, Bacillus amyloliquefaciens BAX-5, Bacillus subtilis WB800, Bacillus subtilis 168, and Bacillus licheniformis BL10.

[0047] Among the above-mentioned Bacillus, Bacillus amyloliquefaciens HZ12 and Bacillus subtilis 168 are mature chassis cells in synthetic biology and have been widely studied and applied in metabolic engineering. [1-4] Bacillus amyloliquefaciens BAX-5 was derived from HZ12 by deleting five extracellular protease genes, including epr, nprE, aprE, aprX, and mpr [5] Bacillus subtilis strain WB800 lacks eight extracellular proteases, including nprE, aprE, epr, bpr, mpr, nprB, vpr, and wprAP [6] Bacillus licheniformis BL10 was obtained by deleting 10 genes encoding extracellular proteases, flagellin, and α-amylase (mpr, vpr, aprX, epr, bpr, wprA, aprE, bprA, hag, and amyl). [7] .

[0048] References are as follows:

[0049] [1]Cai,D.;Liu,M.;Wei,X.;Li,X.;Wang,Q.;Nomura,C.T.;Chen,S.,Use ofBacillus amyloliquefaciens HZ-12for high-level production of the bloodglucose lowering compound,1-deoxynojirimycin(DNJ),and nutraceutical enrichedsoybeans via fermentation.Applied biochemistry and biotechnology 2017,181,1108-1122.

[0050] [2]Xu,Y.;Cai,D.;Zhang,H.;Gao,L.;Yang,Y.;Gao,J.;Li,Y.;Yang,C.;Ji,Z.;Yu,J.,Enhanced production of iturin A in Bacillus amyloliquefaciens bygenetic engineering and medium optimization.Process biochemistry 2020,90,50-57.

[0051] [3]Liu,Y.;Su,A.;Tian,R.;Li,J.;Liu,L.;Du,G.,Developing rapid growingBacillus subtilis for improved biochemical and recombinant proteinproduction.Metabolic Engineering Communications 2020,11,e00141.

[0052] [4]Zhang,X.;Lu,R.;Wang,Q.;Hu,M.;Li,Z.;Xu,M.;Yang,T.;Zhang,R.;Rao,Z.,Production of d-tagatose by whole-cell conversion of recombinant Bacillussubtilis in the absence of antibiotics.Biology 2021,10(12),1343.

[0053] [5] Chen, W.; Li, L.; Ye, C.; Zhao, Z.; Huang, K.; Zou, D.; Wei, X., Efficient production of extracellular alkaline protease in Bacillus amyloliquefaciens by host strain construction. LWT 2022, 163, 113620.

[0054] [6] Wu, S.C.; Yeung, J.C.; Duan, Y.; Ye, R.; Szarka, S.J.; Habibi, H.R.; Wong, S.L., Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production. Appl Environ Microbiol 2002, 68(7), 3261-9.

[0055] [7] Wei, X.; Zhou, Y.; Chen, J.; Cai, D.; Wang, D.; Qi, G.; Chen, S., Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization. Journal of Industrial Microbiology and Biotechnology 2015, 42(2), 287-295.

[0056] The preparation processes of the culture media and reagents used in the present invention are as follows:

[0057] Preparation of soluble starch solution (2g / L): weigh 1.000g (accurate to 0.001g) soluble starch (on an absolute dry basis) in a beaker, add a small amount of water to make a slurry, slowly add it into 200mL boiling water while stirring, then rinse the beaker containing starch with water several times, pour the washing liquid into it, stir and heat until completely transparent, cool and dilute to 500mL, and use the solution immediately after preparation. Note: Soluble starch should be starch specially used for enzyme analysis.

[0058] Original iodine solution: Weigh 11.0g iodine and 22.0g potassium iodide, use a small amount of water to completely dissolve the iodine, make up to 500mL, and store in a brown bottle.

[0059] Dilute iodine solution: Take 2.00 mL of the original iodine solution, add 20.0 g of potassium iodide, dissolve it in water and make up to 500 mL, and store it in a brown bottle.

[0060] Phosphate buffer (pH 6.0): Weigh 45.23 g of disodium hydrogen phosphate (Na2HPO4·12H2O) and 8.07 g of citric acid (C6H8O7·H2O), dissolve them in water and make up to 1000 mL, and use after calibration with a pH meter.

[0061] LB solid medium: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L, agar powder 15g / L, adjust pH to 7.0, sterilize at 121℃ for 30min.

[0062] LB liquid culture medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, adjust pH to 7.0, and sterilize at 121°C for 30 min.

[0063] Liquid enzyme production medium: soybean meal 45g / L, corn flour 95g / L, disodium hydrogen phosphate 8g / L, ammonium sulfate 4g / L, ammonium chloride 1.3g / L, calcium chloride 2g / L, natural pH, liquid volume 50mL / 250mL triangular flask, sterilization at 121℃ for 30min. Liquefaction process: liquefaction temperature 85-90℃, time 20min.

[0064] The specific process of α-amylase activity detection in the following embodiments of the present invention is as follows:

[0065] The method for determining α-amylase activity in the national standard GB1886.174-2016 was modified. The main modification was to prepare the standard solution, specify the standard curve, and obtain the concentration-absorbance equation to obtain the amylase activity. The specific improvement method is as follows:

[0066] (1) Drawing of standard curve

[0067] The standard curve is drawn as follows:

[0068] Soluble starch standard solution: prepare according to Table 1.

[0069] Table 1 Soluble starch standard solution

[0070]

[0071] 1.00 mL of each of the above solutions was taken (three parallel tests) and added to centrifuge tubes containing 0.5 mL of 0.1 mol / L hydrochloric acid solution and 5.00 mL of dilute iodine solution, respectively. The tubes were shaken thoroughly, and the absorbance (A) was quickly determined at a wavelength of 660 nm using a spectrophotometer with 0.5 mL of 0.1 mol / L hydrochloric acid solution and 5.00 mL of dilute iodine solution as blanks. The standard curve was plotted with the absorbance (A) as the abscissa and the concentration of the soluble starch standard solution as the ordinate. 2 The closer it is to 1, the more effective it is, and the slope K is obtained.

[0072] (2) Preparation of enzyme solution to be tested

[0073] Pick a single colony and inoculate it into 50mL LB liquid medium, and culture it at 37℃, 180r / min for 9h. Inoculate it into 50mL fermentation medium with 3% (v / v) inoculation, and culture it at 37℃, 180r / min for 60h. Take the fermented bacterial liquid, centrifuge it at 10000r for 10min, and filter it with a 0.22μm aqueous filter membrane to obtain the crude enzyme liquid. Take 1mL of the crude enzyme liquid and dilute it to the appropriate concentration with phosphate buffer at pH 6.0.

[0074] (3) Determination

[0075] Pipette 20.0 mL of soluble starch solution (2 mg / mL) into a 50 mL centrifuge tube, add 5.00 mL of pH 6.0 phosphate buffer, shake well, and preheat in a 70±0.2°C constant temperature water bath for 8 min.

[0076] Add 1.00mL of the enzyme solution to be tested, start the time immediately, shake well, and maintain a constant temperature of 70±0.2℃ for accurate reaction for 10 minutes; immediately use an automatic pipette to draw 1.00mL of the reaction solution, add it to a centrifuge tube pre-filled with 0.5mL of 0.1mol / L hydrochloric acid solution and 5.00mL of dilute iodine solution, shake well, and use 0.5mL of hydrochloric acid solution and 5.00mL of dilute iodine solution as blanks, and quickly measure its absorbance (A) at a wavelength of 660nm using a spectrophotometer.

[0077] (4) Calculation of results

[0078] Enzyme activity of thermostable α-amylase

[0079] The enzyme activity X of thermostable α-amylase, expressed in U / mL or U / g, is calculated as follows:

[0080] X = (2 × 20-26 × AK) × n / 10;

[0081] Where:

[0082] 2—initial concentration of soluble starch solution;

[0083] 20—volume of soluble starch solution during measurement;

[0084] 26—the sum of the volume of the enzyme solution to be tested, the volume of the pH 6.0 phosphate buffer and the volume of the soluble starch solution during the test;

[0085] A—absorbance value of the enzyme solution measured;

[0086] K—the slope of the obtained standard curve;

[0087] n—dilution multiple of enzyme solution;

[0088] 10—Reaction time of enzyme solution during determination.

[0089] The test result shall be based on the arithmetic mean of the parallel measurement results. The relative error of two independent measurement results obtained under repeatability conditions shall not exceed 5%.

[0090] Example 1 A kind of α-amylase

[0091] The α-amylase of this embodiment is derived from Bacillus velezensis A8-1, which has been deposited in the China Center for Type Culture Collection (CCTCC) on December 22, 2023, with a deposit number of CCTCC NO: M20232632. The amino acid sequence of the α-amylase is shown in SEQ ID NO.1.

[0092] Through comparison with the Uniprot database, the α-amylase from strain A8-1 of the present invention has the highest similarity to the α-amylase from Bacillus velezensis UCMB5033. The amino acid sequences of the two are different. Figure 1 It can be seen that among the full-length 659 amino acids encoded, the consistency is 96.2%, there are 25 amino acid mutations, and the mutation rate is 3.8%.

[0093] Example 2 An α-amylase gene

[0094] The α-amylase gene of this example is derived from Bacillus velezensis A8-1, which has been deposited in the China Center for Type Culture Collection (CCTCC) on December 22, 2023, with a deposit number of CCTCC NO: M20232632. The nucleotide sequence of the α-amylase gene is shown in SEQ ID NO.2.

[0095] Depend on Figure 2 It can be seen that the nucleotide sequence of the α-amylase gene derived from the A8-1 strain of the present invention is different from that of the α-amylase gene derived from Bacillus velezensis UCMB5033. Figure 2 It can be seen that in the gene fragment with a total length of 1980bp, the consistency is 97%, there are 66 mutant nucleotides, and the mutation rate is 3%.

[0096] Example 3 An engineered bacterium that produces high α-amylase

[0097] The high-yield α-amylase engineering bacteria of this embodiment uses BAX-5 as the starting bacteria, overexpresses the α-amylase gene with a nucleotide sequence as shown in SEQ ID NO.2, and the construction method of the high-yield α-amylase engineering bacteria is as follows:

[0098] (1) Obtain an α-amylase gene and clone it into the starting vector pT-17 to obtain a recombinant expression vector; the nucleotide sequence of the α-amylase gene is shown in SEQ ID NO.2.

[0099] (2) The recombinant vector was transformed into BAX-5 and screened for verification.

[0100] Screening of Experimental Example 1A8-1

[0101] In this experimental example, a strain A8-1-1 that can efficiently degrade tobacco starch was screened out from the aged tobacco leaf samples. After physiological, biochemical and molecular biological identification, the bacterium was identified as Bacillus velezensis, which was deposited in the China Center for Type Culture Collection (CCTCC) on December 22, 2023, with the deposit number CCTCC NO: M20232632.

[0102] The specific operations are as follows:

[0103] 1. Screening of strains

[0104] 5 g of tobacco sample was chopped and mixed with 45 mL of sterile phosphate buffer and stirred for 10 minutes. Then heated in a water bath at 80 ° C for 15 minutes. Subsequently, 1 mL of sample was added to 50 mL of starch enrichment medium (tobacco starch 1 g / L, sodium chloride 5 g / L, yeast powder 2 g / L, pH 7.0), and cultured at 37 ° C with shaking at 180 rpm for 48 h for target enrichment.

[0105] The enriched culture solution was diluted in gradients and evenly coated on starch screening medium (tobacco starch 1g / L, sodium chloride 5g / L, yeast powder 2g / L, agar powder 15g / L, pH 7.0), and incubated at 37°C for 48h. After adding iodine solution, colonies with large clear zones were selected for marking and purification. The selected colonies were inoculated into 50 ml of LB liquid medium (yeast powder 5g / L, starch peptone 10g / L, NaCl 10g / L, pH 7.2-7.4), and cultured at 37°C with shaking at 180rpm for 48h. The culture was centrifuged at 10000rpm for 10 minutes, and the supernatant was filtered through a 0.22μm membrane filter. The supernatant was added to the starch screening medium containing 6mm diameter holes, and iodine solution was added after incubation at 37°C for 24 hours. The diameter of the clear zone was measured with a vernier caliper, and the strain with a larger clear zone was selected for further amylase fermentation analysis. Then, the amylase activity of the isolated strain was determined according to the α-amylase activity determination method in the national standard GB1886.174-2016. The specific results are shown in Figure 3 shown.

[0106] The present invention uses tobacco starch as the sole carbon source to enrich the medium to screen the target strains, and successfully isolates seven strains that produce α-amylase. The iodine staining method (modified according to the α-amylase activity determination method in the national standard GB1886.174-2016) was used to determine the α-amylase activity of the isolated strains ( Figure 3 A), of which CK3-5 and A8-1 strains showed higher enzyme activities, 158.5U / mL and 138.7U / mL, respectively. Morphological observation and Gram staining were then performed ( Figure 3 B) The results showed that both strains were Gram-positive rod-shaped bacteria.

[0107] Furthermore, molecular biological techniques were used to further identify these two high-yield amylase strains (e.g. Figure 4 ). The species classification was performed on the non-redundant protein database (NR) through whole genome sequencing, as shown in Figure 4As shown in A, the genes of CK3-5 were mainly annotated as B. velezensis, followed by B. amyloliquefaciens; while the genes of A8-1 were mainly annotated as B. amyloliquefaciens, followed by B. velezensis. In order to further verify the genetic relationship between CK3-5 and A8-1 strains, we selected eight representative Bacillus strains to construct a phylogenetic tree. Figure 4 As shown in B, strain A8-1 has the highest affinity with B. velezensis. Strain CK3-5 also has the highest affinity with B. velezensis, but is slightly less related than A8-1. In the classification of A8-1, the inconsistency between the phylogenetic tree analysis results and the gene NR annotation may be due to the previous classification confusion of B. velezensis. A8-1 was deposited in the China Center for Type Culture Collection (CCTCC) on December 22, 2023, with the deposit number CCTCC NO: M 20232632.

[0108] Experimental Example 2 Alpha-amylase and gene analysis of CK3-5 and A8-1 strains

[0109] This experimental example analyzed the α-amylase and genes of the CK3-5 and A8-1 strains. The specific process is as follows:

[0110] After the genomes of CK3-5 and A8-1 strains were annotated with Gene Ontology (GO) and Enzyme Commission (EC) in the early stage of the present invention, the genes encoding α-amylase (EC3.2.1.1) in B. velezensis CK3-5 and A8-1 strains were analyzed in this experimental example, and compared with the gene sequences in the UniProt database (see Table 2). The results showed that both strains contained α-amylase derived from B. velezensis, which consisted of 659 amino acids and had a molecular weight of 72.4 kDa, and were named AMY (CK3-5) and AMY (A8-1), respectively. Specifically, the genes encoding α-amylase in CK3-5 and A8-1 strains were located at positions 279359-281338 and 279363-281342 of their genomes, respectively, and were referred to as amy (CK3-5) and amy (A8-1). By comparing with the α-amylase from Bacillus velezensis UCMB5033 in the Uniprot database, 22 and 25 amino acids were mutated in these 659 amino acids, and the mutation rates (total number of mutated amino acids / total number of amino acids) reached 3.3% and 3.8%, respectively (the comparison results of CK3-5 are omitted here). These amino acid mutations may lead to differences in the properties of the expressed α-amylase. In addition, this experimental example analyzed the similarity and homology of the α-amylase genes and their encoded amino acids in different strains. The results showed that the similarity between amy (CK3-5) and amy (A8-1) was 97.57%, and the homology exceeded 95.45%.

[0111] Table 2 Analysis results of α-amylase and genes

[0112]

[0113] Experimental Example 3 Determination of enzymatic properties of α-amylase

[0114] This experiment controls the reaction of α-amylase in different temperatures, pH, metal ions and organic solvents, measures its enzyme activity, determines the optimal temperature and temperature stability of α-amylase, the optimal pH and pH stability, the influence of metal ions and organic solvents on the activity of amylase, and determines the conditions for exerting the maximum enzyme activity of α-amylase. The specific operation is as follows:

[0115] 1. Preparation of crude enzyme solution

[0116] Take 50 mL of fermentation broth and centrifuge at 8000 r / min for 10 min. Repeat the centrifugation twice, take the supernatant into a centrifuge tube and put it in a 4°C refrigerator for later use.

[0117] 2. Preparation of purified enzyme solution

[0118] Take 5 mL of crude enzyme solution into an ultrafiltration tube (the ultrafiltration tube was rinsed with pure water by centrifugation in advance), centrifuge at 2000g, 4°C for 30 min, retain the supernatant in the ultrafiltration tube (i.e., the refined enzyme solution described in the following steps), and measure the amylase activity.

[0119] 3. Optimal reaction temperature and thermal stability of α-amylase

[0120] The 100-fold diluted refined enzyme solution was reacted in a water bath at 30, 40, 50, 60, 70, 80, and 90°C for 30 minutes, and the amylase activity was measured to determine the temperature stability. At the same time, the diluted refined enzyme solution was placed at 30, 40, 50, 60, 70, and 80°C for enzyme activity determination. The amylase activity was measured, and the highest enzyme activity was taken as 100%. The relative enzyme activity at different reaction temperatures was calculated using the following formula:

[0121] Relative enzyme activity (%) = A / A0×100%

[0122] Wherein: A represents the enzyme activity of each experimental group; A0 represents the highest enzyme activity in the experimental group.

[0123] Test results such as Figure 5 As shown in the figure, it can be seen that the enzyme activity of α-amylase is maximum at 50°C, but it can maintain a high enzyme activity in the temperature range of 30-70°C, and when the temperature is 80°C, it can still maintain more than 50% of the enzyme activity.

[0124] 4. Optimal reaction pH and pH stability of α-amylase

[0125] The refined enzyme solution was diluted 100 times in phosphate buffer solutions with pH values ​​of 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, respectively, and reacted in a 40°C water bath for 30 minutes. The enzyme activity of the enzyme solution in different pH buffer solutions was measured at 50°C to determine the pH stability of the enzyme solution. The enzyme solution diluted 100 times was measured in phosphate buffer solutions with pH values ​​of 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 at a temperature of 50°C to determine the amylase activity, calculate the relative enzyme activity under different pH conditions, and determine the optimal reaction pH.

[0126] Test results such as Figure 6 As shown in the figure, it can be seen that the enzyme activity of α-amylase is maximum at pH 7, but it can maintain a high enzyme activity in the pH range of 4 to 8.

[0127] 5. Effect of metal ions on α-amylase activity

[0128] The refined enzyme solution was placed in a phosphate buffer solution of pH 7 containing FeCl2, CaCl2, NaCl, MgCl2, KCl, CuCl2 with a metal ion concentration of 5 mmol / L, and the amylase activity was measured at 50°C. The enzyme activity without adding metal ions was taken as 100%, and the relative enzyme activity with different metal ions added was calculated.

[0129] Test results such as Figure 7 As shown in the figure, Fe 2+ , Ca 2+ 、Na + Mg 2+ Ions have little effect on the activity of α-amylase. + and Cu 2+ Ions can slightly increase the enzymatic activity of α-amylase.

[0130] 6. Effect of organic solvents on α-amylase activity

[0131] The enzyme solution was placed in an organic solvent such as ethanol, glycerol, propylene glycol with a volume fraction of 5% (phosphate buffer solution with a pH of 7), and the amylase activity under this condition was measured at 50°C. The enzyme activity without adding organic solvents was taken as 100%, and the relative enzyme activity with the addition of different organic solvents was calculated.

[0132] Test results such as Figure 8 As shown in the figure, it can be seen that organic solvents such as ethanol, glycerol, and propylene glycol have little effect on the activity of the α-amylase.

[0133] From the above results, it can be seen that the α-amylase secreted by A8-1 has the advantages of strong pH stability, wide temperature resistance range and strong resistance to organic solvents.

[0134] Experimental Example 4 Construction of an engineered bacterium that produces high α-amylase

[0135] In this experimental example, two α-amylase genes amy(CK3-5) (nucleotide sequence such as SEQ ID NO.3) and amy(A8-1) (nucleotide sequence such as SEQ ID NO.1) of B. velezensis and one α-amylase gene amy(FYZ1-3) (nucleotide sequence such as SEQ ID NO.4) of B. subtilis were selected for recombinant expression, and their expression in different Bacillus hosts was compared. The specific implementation operation is as follows:

[0136] 1. Construction of recombinant expression vector

[0137] The construction process of the recombinant expression vector is described by taking the recombinant vector overexpressing the α-amylase gene amy (A8-1) as an example:

[0138] Primers amy-F and amy-R were designed according to the α-amylase gene sequence of A8-1, and XbaI and BamHI restriction enzyme sites were added to their 5' ends. The amy(A8-1) gene was amplified by PCR using the genomic DNA of B. velezensis A8-1 as a template. After the PCR product and pT-17 vector digested with BamHI and XbaI were purified, they were connected using T4 DNA ligase at 25°C for 10-20 minutes. The ligated product was then transformed into E. coli DH5α competent cells. The pT-17amy(A8-1) recombinant expression vector was successfully constructed by PCR amplification and gene sequencing.

[0139] The construction method of the recombinant expression vectors pT-17amy(CK3-5) and pT-17amy(FYZ1-3) for overexpressing the α-amylase gene amy(CK3-5) or amy(FYZ1-3) is the same as above.

[0140] 2. Construction of engineered bacteria with high α-amylase production

[0141] The construction process of engineering bacteria is explained by taking the recipient bacteria BAX-5 and the recombinant expression vector pT-17amy (A8-1) as examples:

[0142] The pT-17amy(A8-1) plasmid identified correctly in step 1 was electrotransformed into B.amyloliquefaciens BAX-5 competent cells. Positive transformants were verified by PCR amplification and gene sequencing. The construction of other recombinant expression strains was carried out in the same way. All strains and plasmids used and constructed in this experimental example are shown in Table 3.

[0143] Table 3 Constructed strains and plasmids

[0144]

[0145]

[0146] In summary, after successfully constructing the recombinant expression vectors expressing the α-amylase gene amy(CK3-5), amy(A8-1) or amy(FYZ1-3), they were introduced into five different hosts, including HZ1227, BAX-5, 168, WB800 and BL10 ( Fig. 9A), the resulting recombinant strains included HZ12 / PT17, HZ12 / PT17amy(FYZ1-3), HZ12 / PT17amy(CK3-5), HZ12 / PT17amy(A8-1), BAX-5 / PT17, BAX-5 / PT17amy(FYZ1-3), BAX-5 / PT17amy(CK3-5), BAX-5 / PT17amy(A8-1), 168 / PT17, 168 / PT17amy(FYZ1-3), BAX-5 / PT17amy(CK3-5), BAX-5 / PT17amy(A8-1), 3), 168 / PT17amy(CK3-5), 168 / PT17amy(A8-1), WB800 / PT17, WB800 / PT17amy(FYZ1-3), WB800 / PT17amy(CK3-5), WB800 / PT17amy(A8-1), BL10 / PT17, BL10 / PT17amy(FYZ1-3), BL10 / PT17amy(CK3-5) and BL10 / PT17amy(A8-1).

[0147] 3. Analysis of α-amylase activity of recombinant strains

[0148] Further fermentation analysis showed the α-amylase activity of the recombinant strain, e.g. Fig. 9 As shown. Genes amy(CK3-5), amy(A8-1) or amy(FYZ1-3) all successfully expressed α-amylase ( Fig. 9 B), the enzyme activity ranking is AMY(A8-1)>AMY(CK3-5)>AMY(FYZ1-3). Expression results in different hosts ( Fig. 9 C) showed that the host BAX-5 exhibited the highest α-amylase activity, which may be attributed to its lack of extracellular protease genes (epr, nprE, aprE, aprX, and mpr), thereby reducing the expression burden of redundant proteins. Therefore, among all recombinant strains, BAX-5 / PT17amy (A8-1) exhibited the highest enzyme activity, reaching 156.1 U / mL ( Fig. 9 D).

[0149] 4. Analysis of the interaction between α-amylase and maltotriose

[0150] In addition, we also explored the interaction between α-amylase AMY(FYZ1-3), AMY(CK3-5) and AMY(A8-1) and maltotriose, using molecular docking analysis ( Fig. 9E). The calculated -CDOCKER interaction energies were 43, 43.3, and 46.5 kcal / mol, respectively. A higher -CDOCKER interaction energy indicates a stronger binding affinity between the protein and the ligand. This suggests that the α-amylases expressed by B. subtilis and B. velezensis have similar binding affinities for maltotriose. Among them, AMY(A8-1) exhibited the highest -CDOCKER interaction energy, indicating that it had the strongest binding affinity to maltotriose, suggesting better stability of the enzyme-substrate complex. This higher affinity may contribute to its enhanced enzymatic activity in hydrolyzing the α-1,4-glycosidic bonds of maltotriose. The results of the interaction analysis were consistent with the results of the above enzyme activity assay, and the amy(A8-1) gene exhibited the highest enzyme activity in the recombinant strain ( Fig. 9 B).

[0151] In addition, we conducted molecular docking between the α-amylase from the Bacillus velezensis UCMB5033 strain in the database and maltotriose, and the results were as follows: Fig.10 As shown. The calculated -CDOCKER interaction energy is 43.1 kcal / mol. It is speculated that the 25 mutant amino acids present in AMY(A8-1) compared with the α-amylase of Bacillus velezensis UCMB5033 strain have changed the secondary decomposition of the protein, thus causing a higher -CDOCKER interaction and stronger binding affinity.

[0152] Experimental Example 5 Further optimization of expression elements of engineering bacteria with high α-amylase production

[0153] In this experiment, the α-amylase gene amy (A8-1) was subjected to bioinformatics analysis to determine the signal peptide of α-amylase AMY (A8-1), and signal peptides from other sources were selected for comparison to select a more suitable signal peptide. At the same time, this experiment also compared the effects of different promoters on the activity of engineered bacteria α-amylase. The specific process is as follows:

[0154] 1. Signal peptide analysis of α-amylase AMY (A8-1)

[0155] Based on the above results, the amy (A8-1) gene of A8-1 plays the most important role in the expression of α-amylase. In order to further clarify the function of the amy (A8-1) gene, the gene was translated into 659 amino acids using BioEdit software, and the amino acid sequence was further analyzed. The results showed that α-amylase AMY (A8-1) has a SP (Sec / SPI) type signal peptide consisting of 33 amino acids, with a cleavage site of 33-34, and its sequence is: MFKKRFKTSLLPLFAGFLLLFHLVLSGPAAVNA( Fig.11 , the amino acid sequence is shown in SEQ ID NO.5).

[0156] 2. Effects of different signal peptides on the activity of α-amylase AMY (A8-1)

[0157] The signal peptide consists of 5-30 amino acids at the N-terminus of the protein and plays a key role in the extracellular secretion of the target protein. Changes in signal peptides from different strains usually lead to different expression levels of the target protein, so signal peptide replacement is an important optimization strategy to enhance protein expression. Given that α-amylase is an extracellular protein, the signal peptide plays a key role in its secretion process.

[0158] According to the prediction of the signal peptide database SPSED (http: / / www.spsed.com), two signal peptides with significant potential to enhance the expression of α-amylase genes were identified. These signal peptides are derived from specific genes: tag1 (signal peptide SP001) of lactic acid bacteria Lactobacillus plantarum and bglS of Geobacillus stearothermophilus. In this study, these signal peptides were named SP001 (amino acid sequence as shown in SEQ ID NO.6) and SP002 (amino acid sequence as shown in SEQ ID NO.7), respectively, consisting of 27 and 28 amino acid residues, and the specific sequences are as follows:

[0159] SP001: VKEVRFWGLLLGLFVCLGAVIPLVSKA (as shown in SEQ ID NO. 6);

[0160] SP002: MPYLKRVLLLLVTGLFMSLFAVTATASA (as shown in SEQ ID NO. 7).

[0161] In this experiment, the signal peptide in the amy(A8-1) gene was replaced with SP001 and SP002 to study the effect of different signal peptides on the enzymatic activity of α-amylase AMY(A8-1) ( Fig.12A). SP001 and SP002 sequences were amplified by PCR using specific primers, and the non-signal peptide region of amy(A8-1) was amplified using primers. The specific primer sequences are shown in Table 4. The signal peptides SP001 and SP002 were connected to the non-signal peptide region of amy(A8-1) using SOE technology to form a new fusion fragment (the nucleotide sequence of the amy(A8-1)SP001 fusion fragment is shown in SEQ ID NO.16, the nucleotide sequence of the amy(A8-1)SP002 fusion fragment is shown in SEQ ID NO.17, the amino acid sequence of the α-amylase with SP001 is shown in SEQ ID NO.18, and the amino acid sequence of the α-amylase with SP002 is shown in SEQ ID NO.19). The two ends of the fusion fragment include restriction sites and protective bases. After restriction endonuclease digestion and ligase treatment, they are connected to the expression vector PT17, and finally two free expression recombinant vectors carrying different signal peptides are obtained. Finally, the recombinant expression vectors are transferred into the recipient bacteria BAX-5 to obtain the recombinant strain.

[0162] The results showed that the recombinant strain BAX-5 / PTamy(A8-1)SP002, which had the signal peptide SP002 from the α-amylase gene of Geobacillus stearothermophilus, exhibited the highest α-amylase activity, reaching 205 U / mL.

[0163] Table 4. Signal peptide related primers

[0164]

[0165]

[0166] 3. Effects of different promoters on the activity of α-amylase AMY (A8-1)

[0167] Since the promoter is one of the most critical factors in gene expression regulation, its engineering is also an important strategy to improve microbial gene expression and metabolite production. This experiment further analyzed the effects of different promoters on AMY (A8-1) enzyme activity ( Fig.12 B). The results showed that the recombinant strain with the promoter P43 from Bacillus subtilis exhibited the highest α-amylase activity.

[0168] Note: The engineered bacteria and plasmids constructed in this experimental example are shown in Table 5, and the construction method is as described in Experimental Example 4.

[0169] Table 5 Constructed strains and plasmids

[0170]

[0171] Note: The signal peptide of amy(A8-1) gene itself was used for comparison of different promoter activities, while the P43 promoter was used for comparison of promoter activities.

[0172] Experimental Example 6 Fermentation process optimization and α-amylase production process

[0173] This experimental example uses the recombinant strain BAX-5 / PTamy (A8-1) SP002 constructed in Experimental Example 5 to optimize the fermentation process. The specific process is as follows:

[0174] In order to further improve the α-amylase production of the recombinant strain BAX-5 / PTamy (A8-1) SP002 in industrial applications, the present invention performs a large amount of optimization of the culture medium and culture conditions.

[0175] The optimal culture medium of the recombinant strain BAX-5 / PTamy(A8-1)SP002 is: corn flour 65g / L, soybean cake powder 70g / L, NaCl 10g / L, CaCl2 3g / L, and the optimal fermentation conditions are: temperature 40℃, initial pH 6, initial inoculation amount 3%, and liquid volume 50mL.

[0176] The best culture medium for the wild-type strain A8-1 is: 20 g / L corn flour, 80 g / L soybean cake powder, 10 g / L K2HPO4, and the best fermentation conditions are: temperature 37°C, pH 7, inoculation volume 3%, and liquid volume 50 mL.

[0177] The recombinant strain BAX-5 / PTamy(A8-1)SP002 and the wild-type strain A8-1 were fermented under the optimal conditions. During the fermentation process, the α-amylase activity was measured at different time points. Fig.13 It was shown that the enzyme activities of A8-1 and BAX-5 / PTamy (A8-1) SP002 continued to increase with the extension of fermentation time. After 40 hours of fermentation, the enzyme activity of A8-1 reached 156U / mL, while the enzyme activity of BAX-5 / PTamy (A8-1) SP002 reached 1440U / mL, which was 9.2 times that of the wild-type strain. In addition, since the metabolites produced by Bacillus subtilis during the fermentation process are viscous, it significantly affects industrial applications. Overall, heterologous expression of the α-amylase gene of Bacillus subtilis not only alleviates the viscosity problem of the fermentation broth, facilitates industrial application, but also significantly improves the activity of α-amylase and strain performance.

[0178] Finally, the present invention sprays the α-amylase AMY (A8-1) expressed by the recombinant strain BAX-5 / PTamy (A8-1) SP002 on the tobacco leaves. After 48 hours of enzyme treatment (temperature 45°C, humidity 75%), the starch content in the tobacco leaves is detected by near-infrared spectroscopy. Compared with the control group, the results show that the starch content in the enzyme-treated tobacco leaves is reduced by 6%, which is significantly different from the untreated control samples. Afterwards, the above tobacco leaves are processed into cigarette samples, which are evaluated and scored by 7 experts with qualification certificates for cigarette sensory quality evaluation. According to industry requirements, the scoring is based on 9 items including aroma quality, aroma quantity, miscellaneous odor, irritation, concentration, strength, sweetness, aftertaste and softness, and each item has a full score of 9 points. The average total score of the AMY (A8-1) amylase-treated group was 43.1 points, and that of the untreated control group was 41.07 points. According to the sensory deviation reference method, if the total score deviation is greater than 1 point, it means that the sensory quality has been improved, and if the total score difference is greater than 2 points, it means that the sensory quality has been significantly improved. Therefore, the above results show that the sensory quality of cigarette products treated with AMY (A8-1) amylase is significantly improved during smoking.

[0179] Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An alpha-amylase, characterized in that: The amino acid sequence of the α-amylase is shown in SEQ ID NO.1, SEQ ID NO.18 or SEQ ID NO.

19.

2. An α-amylase gene, characterized in that: The nucleotide sequence of the α-amylase gene is shown as SEQ ID NO.2, SEQ ID NO.16 or SEQ ID NO.

17.

3. An engineered bacterium with high α-amylase production, characterized in that: The engineering bacteria uses Bacillus as a starting bacterium and overexpresses an alpha-amylase gene having a nucleotide sequence as shown in SEQ ID NO.2, SEQ ID NO.16 or SEQ ID NO.17; the Bacillus is Bacillus amyloliquefaciens, Bacillus subtilis or Bacillus licheniformis.

4. The engineered bacteria for high α-amylase production according to claim 3, characterized in that: The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens HZ12 or Bacillus amyloliquefaciens BAX-5; the Bacillus subtilis is Bacillus subtilis WB800 or Bacillus subtilis 168; and the Bacillus licheniformis is Bacillus licheniformis BL10.

5. The engineered bacteria for high α-amylase production according to claim 3 or 4, characterized in that: The construction method of the engineering bacteria comprises the following steps: constructing a recombinant expression vector for expressing the alpha-amylase gene, and transforming the vector into Bacillus to over-express the alpha-amylase.

6. The engineered bacteria for high α-amylase production according to claim 5, characterized in that: The starting vector of the recombinant expression vector is pT-17.

7. Use of the engineered bacteria with high α-amylase production according to any one of claims 3 to 6 in the production of α-amylase.

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