Preparation method of a halophilic archaea-derived amylase and application thereof

Abstract

This invention belongs to the field of genetic engineering and relates to a method for preparing amylase derived from halophilic archaea and its application; based on halophilic archaea... Haloarcula sp. ZJK10-12、 Haladaptatus The genes for α-amylase, maltose amylase, and glucoamylase were obtained from the whole genome sequence of sp. ZJK29-3. Recombinant plasmids were constructed using primer design and molecular cloning, and transformed into prokaryotic host bacteria to obtain the corresponding recombinant strains. The bacteria were enriched and cultured in Hv-YPC liquid medium, and the cells were collected and lysed by sonication. α-amylase, maltose amylase, and glucoamylase were obtained using nickel column affinity chromatography. The amylases exhibit excellent enzymatic properties and outstanding salt tolerance. Their application in the fermentation of high-salt, dilute soy sauce can improve raw material utilization and product quality, demonstrating good industrial applicability and promotional value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and discloses a method for preparing amylase from halophilic archaea and its application; specifically, it relates to a method for preparing a novel α-amylase, maltose amylase and glucoamylase from halophilic archaea and their combined application in the soy sauce brewing process. Background Technology

[0002] α-Amylase (EC 3.2.1.1) is an endoamylase belonging to the GH13 family of glycoside hydrolases. It acts on the α-1,4-glycosidic bonds within starch molecules but cannot cleave the α-1,6-glycosidic bonds. Its hydrolysis products are mainly dextrins and oligosaccharides. Maltose amylase (EC 3.2.1.133) hydrolyzes the α-1,4-glycosidic and α-1,6-glycosidic bonds in starch and related polysaccharide molecules, producing maltose and oligosaccharides. It belongs to the 20th subfamily of the GH13 family of glycoside hydrolases (GH13_20, also known as the neopulllanase subfamily). Glucoamylase (EC 3.2.1.3), also known as γ-amylase or saccharifying enzyme, is a typical exoglycosidase that sequentially hydrolyzes the α-1,4-glycosidic bonds at the non-reducing ends of starch molecules and the α-1,6-glycosidic bonds at the branching points, thereby completely hydrolyzing the starch molecule into glucose. These three types of enzymes are widely used in industrial production and are of significant value in the food, pharmaceutical, feed, and fermentation industries.

[0003] In the production of soy sauce, seafood seasonings, and other products, the starch in the raw materials is hydrolyzed into sugars. These hydrolysis products can serve as precursors to promote the formation of flavor compounds or as carbon source substrates for microbial fermentation. Currently, *Aspergillus oryzae* is often used as a single strain in industry, primarily producing neutral proteases. However, the activity of amylases (such as α-amylase and saccharifying enzymes) is limited by high-salt environments, resulting in the ineffective utilization of starchy raw materials. Therefore, adding multiple high-salt-tolerant amylases to the soy sauce production process allows for the efficient hydrolysis of starch substrates through the combined action of different types of amylases. This completely converts the raw starch into maltose and glucose, thereby promoting the enrichment of reducing sugars, enhancing the formation of soy sauce flavor compounds, and improving raw material utilization.

[0004] Halophilic archaea are mostly distributed in coastal mudflats, salt lakes, saline-alkali lands, and environments where high-salt preserved foods are processed. Their optimal growth salt concentration is usually no less than 15% (2.5 M NaCl), and some strains can survive under saturated salt conditions. Halophilic archaea excrete Na+ from their cells. + Simultaneously, intracellular absorption and enrichment of K +This helps maintain osmotic pressure balance inside and outside the cell. The enzymes they produce mainly form a negatively charged shield by increasing the number of acidic amino acid residues on the protein surface, creating a stable hydration layer on the protein surface, thus ensuring the structural stability of enzyme molecules in high-salt, low-water-activity environments. Furthermore, these enzymes generally possess excellent environmental tolerance, exhibiting excellent properties such as resistance to salt and alkali, high temperatures, and organic solvents. The safety of halophilic archaea has been certified as "practically non-toxic," making them an ideal source for screening and preparing high-salt-tolerant amylases. Currently, research on α-amylases from halophilic archaeas is relatively limited both domestically and internationally, and no relevant research has been found on maltose amylase and glucoamylase from halophilic archaeas. Therefore, using halophilic archaeas as starting strains to explore and obtain amylase resources that can tolerate high-salt environments has sufficient theoretical basis and practical feasibility. Summary of the Invention

[0005] This invention aims to provide a method for preparing α-amylase, maltose amylase, and glucoamylase from halophilic archaea, and their combined application in soy sauce brewing. The three types of halophilic archaea amylase maintain high catalytic activity and stability over a wide range of temperature, salinity, and pH, and exhibit significant differences in tolerance to metal ions, organic solvents, and surfactants. Without modifying existing production processes, α-amylase, maltose amylase, and glucoamylase are added to the soy sauce fermentation process at amounts of 0.02%, 0.01%, and 0.01% of the total fermentation volume, respectively. Measurements of reducing sugar content, color, and total free amino acids revealed that the combined use of these three enzymes increased the reducing sugar content by 32%, the yellow and red indices by 4.0% and 3.4%, respectively, and the total free amino acid content by 6.4%, demonstrating excellent application potential. Research on the purification process, enzymatic properties, and application of amylase in high-salt dilute soy sauce can provide theoretical and technical support for the practical application of halophilic archaea amylase, and at the same time provide new enzyme preparations for the fermented food industry, especially in the field of high-salt fermented foods.

[0006] The halophilic archaea used in this invention Haloarcula sp. ZJK10-12 was isolated from Nitan, Dongwan City, Guangdong Province. Haladaptatus sp. ZJK29-3 was isolated from Nitan, Nansha District, Guangzhou. All strains are conventionally published strains and are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC 1.62770 and CGMCC 1.62772, respectively.

[0007] A halophilic archaea-derived amylase, specifically comprising α-amylase, maltose amylase, and glucoamylase; based on halophilic archaea. Haloarcula A halophilic archaea α-amylase gene was obtained from the whole genome of sp. ZJK10-12. α-amy Har The gene is 1179 bp in size, and its nucleotide sequence is shown in SEQ ID No. 1. The encoded α-amylase is named α-Amy. Har The protein has a size of 392 amino acid residues, and its amino acid sequence is shown in SEQ ID No. 2; Based on halophilic archaea Haladaptatus A halophilic archaea maltose amylase gene was obtained from the whole genome of sp. ZJK29-3. maa Hap A halophilic archaea glucoamylase gene ga Hap , maa Hap The gene is 2109 bp in size, and its nucleotide sequence is shown in SEQ ID No. 3. The encoded maltose amylase is named MAA. Hap The protein has a size of 702 amino acid residues, and its amino acid sequence is shown in SEQ ID No. 4; ga Hap The gene is 2124 bp in size, and its nucleotide sequence is shown in SEQ ID No. 5. The encoded glucosylase is named GA. Hap The protein has a size of 707 amino acid residues, and its amino acid sequence is shown in SEQ ID No. 6.

[0008] This invention also provides a method for preparing amylases derived from halophilic archaea, specifically involving the preparation of halophilic archaea α-amylase, maltose amylase, and glucoamylase, which includes the following steps: (1) Construction of recombinant plasmid pTA04- α-amy Har pTA04- maa Hap and pTA04- ga Hap ; Based on halophilic archaea Haloarcula The whole genome sequence of sp. ZJK10-12 was used to obtain an α-amylase gene through pairwise alignment on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). α-amy Har Its nucleotide sequence is shown in SEQ ID No. 1; based on halophilic archaea. Haladaptatus The complete genome sequence of sp. ZJK29-3 was obtained, and a maltose amylase gene was derived. maa Hap and a glucosidase gene ga HapThe nucleotide sequences are shown in SEQ ID No. 3 and SEQ ID No. 5, respectively. Primers for the target gene were designed, and the gene fragments of the three amylases were amplified using PCR technology. The target gene was then cloned using molecular cloning technology. α-amy Har , maa Hap , ga Hap The recombinant plasmid pTA04- was ligated to the pTA04 vector to construct the recombinant plasmid pTA04- α-amy Har pTA04- maa Hap and pTA04- ga Hap .

[0009] (2) PEG-mediated plasmid transformation and efficient expression; The recombinant plasmid pTA04- constructed in step (1) α-amy Har pTA04- maa Hap and pTA04- ga Hap Transformation into prokaryotic host bacteria using the PEG-mediated method. Haloferax volcanii The corresponding recombinant strain was obtained from H1424. The recombinant strain was inoculated into Hv-YPC liquid medium and cultured on a shaker. After reaching the stationary phase, the bacterial cells were collected by centrifugation. (3) Purification of halophilic archaea α-amylase, maltose amylase and glucoamylase; The bacterial cells collected in step (2) were resuspended in cell lysis buffer, and the cells were sonicated for 30 min under ice bath conditions. After centrifugation for 10 min, the supernatant was collected, and the proteins were purified by nickel column affinity chromatography to obtain high-purity halophilic archaea α-amylase, maltose amylase, and glucoamylase, which were denoted as α-Amy. Har MAA Hap and GA Hap Their amino acid sequences are SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6, respectively.

[0010] Preferably, the halophilic archaea described in step (1) Haloarcula sp. ZJK10-12 was obtained from the China General Microbiological Culture Collection Center, accession number CGMCC 1.62770; the halophilic archaea described Haladaptatus sp. ZJK29-3 is from the China General Microbiological Culture Collection Center, accession number CGMCC 1.62772; Preferably, the prokaryotic host in step (2) is based on a bacterial strain. Haloferax volcanii The constructed uracil-thymidine auxotrophic mutant strain was named Haloferax volcanii H1424. Wild type Haloferax volcanii Purchased from the Japan Culture Collection (JCM 8879), the strain was constructed using the method described in: Allers, T., Ngo, HP, Mevarech, M., et al. Development of additional selectable markers for the halophilic archaeon. Haloferax volcanii based on the leuB and trpA genes[J]. Applied and environmental microbiology, 2004, 70(2):943-953. The gene α-amy Har The F-terminal primer is 5'-CGCGAATTCGTCGGGTCCAGTGCGGTG-3', where GAATTC is... Eco The restriction enzyme site for RI is 5'-ATAGGATCCCGGCGCGTAGCAGACCCA-3', and GGATCC is... Bam HI restriction enzyme site; maa Hap The F-terminal primer is 5'-ATAGGTACCCACACTGCTGATCCCCCG-3', and the GGTACC is... Kpn The restriction enzyme site for I, the R-terminal primer is 5'-ATAGCATGCTTTACTTTCCTCGGTGGT-3', and GCATGC is... Sph I restriction enzyme site; ga Hap The F-terminal primer is 5'-CGCAGATCTAGAATCTGTGAAGGATTA-3', where AGATCT is... Bgl The restriction enzyme site for II has an R-terminal primer of 5'-ATAGGATCCGTCCAACGACGGTTTCAC-3', and GGATCC is... Bam HI restriction enzyme site.

[0011] Preferably, the Hv-YPC culture medium in step (2) consists of: 5.0 g yeast extract, 1.0 g soybean peptone, 1.0 g acid-hydrolyzed casein, 4.2 g KCl, 33.0 g MgSO4·7H2O, 30.0 g MgCl2·6H2O, 144.0 g NaCl, 12.0 mL 1 M Tris-HCl (pH 7.5), and 0.33 g CaCl2 (pre-dissolved in a small amount of water and added last after the other components have dissolved). All components are dissolved in distilled water, mixed and brought to a final volume of 1 L, pH adjusted to 7.2-7.5, and sterilized at 115 °C for 30 min.

[0012] Preferably, the culture conditions in step (2) are 37 ℃, 200 rpm, and a culture time of 3-4 days; the centrifugation conditions are 4 ℃, 8000 rpm, and a centrifugation time of 10-15 min.

[0013] Preferably, the cell lysis buffer in step (3) consists of 2 M NaCl, 40 mM Tris-HCl, and pH 8.0.

[0014] Preferably, the conditions for ultrasonic cell disruption in step (3) are: ultrasonic time 3 s, interval 5 s, power 180 W, and total time 30 min.

[0015] Preferably, the nickel column affinity chromatography steps in step (3) are as follows: drain the 20% ethanol preservation solution from the nickel column, then rinse the chromatography column with 10 column bed volumes of ddH2O, and then fully equilibrate the nickel column with 10 column bed volumes of cell lysis buffer; slowly load the supernatant obtained after cell disruption and centrifugation onto the equilibrated nickel column to ensure that the protein is fully bound to the nickel column packing material; then first elute with 10 column bed volumes of buffer I to remove impurities, and then elute with 5 column bed volumes of buffer II to collect the target protein.

[0016] Preferably, the buffer solution I consists of 40 mM imidazole, 2 M NaCl, 40 mM Tris-HCl, and pH 8.0.

[0017] Preferably, the buffer solution II consists of 100 mM imidazole, 2 M NaCl, 40 mM Tris-HCl, and pH 8.0.

[0018] The present invention also provides an application of soy sauce reprocessing based on halophilic archaea amylase, which is used to improve the reducing sugar content, color and total free amino acid content of soy sauce; After mixing the soy sauce starter with a brine solution, the resulting halophilic archaea α-amylase (α-Amy) is added. Har ), maltose amylase (MAA)Hap ) and glucoamylase (GA) Hap Fermentation improves the reducing sugar content, color, and total free amino acid content of soy sauce.

[0019] The ratio of soy sauce starter to brine is 1g:4ml, the brine concentration is 18%, and the amounts of α-amylase, maltose amylase, and glucoamylase added are 0.02%, 0.01%, and 0.01% of the total fermentation volume, respectively. The fermentation temperature is 30-40℃, and the fermentation time is 25-30 days.

[0020] (1) Halophilic archaea α-amylase (α-Amy) Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap Determination of optimal reaction conditions and environmental tolerance; α-Amylase (α-Amylase) was determined using a 1% soluble starch solution as a substrate. Har ) and maltose amylase (MAA) Hap The enzymatic properties of glucoamylase (GA) were characterized using a 1% dextrin solution as a substrate. Hap The enzymatic properties of the enzyme, specifically including: the optimal temperature for enzyme-catalyzed reaction, the optimal NaCl concentration, the optimal pH, the effects of different metal ions on enzyme activity, the effects of different organic solvents and surfactants on enzyme activity, and the determination of enzyme kinetic parameters; wherein the different metal ions are: Ni 2+ Cu 2+ Ca 2+ 、Sr 2+ Mg 2+ Mn 2+ Zn 2+ Fe 3+ Ba 2+ K + and Co 2+ The different organic solvents and surfactants are: DMSO, glycerol, methanol, ethanol, acetone, DMF, isopropanol, Tween 20, Triton X-100, SDS, Tween 80 and PEG600.

[0021] (2) Halophilic archaea α-amylase (α-Amy) Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap Stability analysis of ) Based on the optimal reaction conditions of the three enzymes, the enzyme solutions were incubated at different temperatures, NaCl concentrations, and pH levels for 0 min, 30 min, 60 min, and 90 min, respectively. The residual enzyme activity was then measured under each optimal reaction condition. Among them, α-amylase (α-Amylase)... Har The incubation conditions were as follows: temperatures of 20 ℃, 40 ℃, and 50 ℃; NaCl concentrations of 0.1 M, 0.5 M, 1.0 M, 1.5 M, 3.0 M, and 4.5 M; and pH values ​​of 5.0, 5.5, 6.0, 7.5, and 9.5; maltose amylase (MAA) was used. Hap The incubation conditions were as follows: temperatures of 20 ℃, 40 ℃, 45 ℃, 50 ℃, and 60 ℃; NaCl concentrations of 0.5 M, 0.7 M, 0.8 M, 1.0 M, 1.5 M, 3.0 M, and 4.5 M; and pH values ​​of 5.5, 6.0, 7.0, and 8.0; glucoamylase (GA) was used. Hap The incubation conditions were as follows: temperatures of 20 ℃, 40 ℃, 45 ℃, and 50 ℃; NaCl concentrations of 0.3 M, 1.0 M, 1.5 M, 3.0 M, and 4.5 M; and pH values ​​of 5.0, 5.5, 6.0, and 7.0.

[0022] (3) Halophilic archaea α-amylase (α-Amy) Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap The combined application of ) on the quality of soy sauce; The reducing sugar content, yellow index, red index, and total free amino acid content of soy sauce were determined during the fermentation process. Specifically, the reducing sugar content of soy sauce during fermentation was determined using the DNS method; the yellow index and red index were determined using formulas (a) and (b); and the content of free amino acids was determined using a fully automated amino acid analyzer.

[0023] The DNS method involves mixing the diluted soy sauce supernatant with an equal volume of DNS solution (3,5-dinitrosalicylic acid), incubating in a boiling water bath for 5 minutes for color development, then rapidly cooling in an ice-water mixture, and measuring the absorbance (OD) using a microplate reader. 540 Preparation of DNS solution: Dissolve 1.6 g NaOH solution in water, add 1.0 g 3,5-dinitrosalicylic acid, stir to dissolve in the dark, then add 30 g potassium sodium tartrate, stir and make up to 100 mL, store in the dark, and use after one week of stability;

[0024] In the formula: A460, A510, and A610 represent the absorbance values ​​at 460 nm, 510 nm, and 610 nm, respectively; the free amino acid content was determined by the Modern Analysis and Testing Center of Nanjing Forestry University.

[0025] (1) This invention discovered genes for α-amylase, maltose amylase, and glucoamylase from halophilic archaeas, expressing the corresponding halophilic archaea amylases. A method for preparing high-salt-tolerant halophilic archaea amylases was proposed. This method is simple to operate, and the resulting amylases have high purity, excellent salt tolerance, and good stability. It overcomes the defect of traditional Aspergillus oryzae amylase having inhibited activity under high-salt conditions, indicating that enzymes from halophilic archaeas are more adaptable to complex high-salt fermentation environments. By combining multiple amylases (α-amylase, maltose amylase, and glucoamylase) to hydrolyze starch in fermented bean paste, the utilization rate of raw materials and the quality of products are improved, demonstrating strong industrial applicability and promotional value.

[0026] (2) The halophilic archaea α-amylase (α-Amy) prepared in this invention Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap All of them possess excellent enzymatic properties, exhibiting superior adaptability and stability in terms of temperature, salinity, and pH. Among them, α-amylase (α-Amylase) Har It maintains high activity within the range of 35-60 ℃, 1.0-4.8 M NaCl, and pH 5.0-8.5; maltose amylase (MAA) Hap It maintains high activity within the range of 30-50 ℃, 1.0-4.8 M NaCl, and pH 5.5-7.0; glucoamylase (GA) Hap The three enzymes exhibit strong activity within the range of 25-55 °C, 1.0-2.0 M NaCl, and pH 5.5-6.0. Furthermore, they show varying degrees of tolerance to different metal ions, organic solvents, and surfactants.

[0027] (3) Without modifying the existing production process, halophilic archaea α-amylase (α-Amy) is introduced. Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap This halophilic archaea amylase, added directly to the early stages of soy sauce brewing, fully hydrolyzes the starch substrate with amylase. After fermentation, the reducing sugar content increases by 33.82%, the yellow index by 3.98%, the red index by 3.39%, and the total free amino acid content by 6.42%. This amylase possesses excellent properties, adapting to complex fermentation environments. It can be directly added to the fermentation process of high-salt, low-concentration soy sauce to improve the utilization rate of starch in the raw materials and enhance the flavor and color of the soy sauce. Attached Figure Description

[0028] Figure 1 Image for double enzyme digestion verification of recombinant plasmid (a: pTA04-) α-amy Har b: pTA04- maa Hap c: pTA04- ga Hap ).

[0029] Figure 2 Transformation of recombinant plasmids Haloferax volcanii H1424 transformant verification diagram (a: α-amy Har b: maa Hap c: ga Hap ).

[0030] Figure 3 SDS-PAGE gel image for purification of halophilic archaeal amylase by nickel column affinity chromatography (a: α-Amy) Har b: MAA Hap c:GA Hap ).

[0031] Figure 4 Figure a shows the effect of temperature on enzyme catalytic activity (a: α-Amy). Har b: MAA Hap c:GA Hap ).

[0032] Figure 5 Figure a shows the effect of NaCl concentration on enzyme catalytic activity (a: α-Amy). Har b: MAA Hap c:GA Hap ).

[0033] Figure 6 Figure a shows the effect of pH on enzyme catalytic activity (a: α-Amy). Har b: MAA Hap c:GA Hap ).

[0034] Figure 7 Figure 1 shows the results of enzyme thermostability analysis (a: α-Amy) Har b: MAA Hap c:GA Hap ).

[0035] Figure 8 Figure 1 shows the results of NaCl stability analysis of the enzyme (a: α-Amy). Har b: MAA Hap c:GAHap ).

[0036] Figure 9 Figure 1 shows the results of the enzyme's pH stability analysis (a: α-Amy). Har b: MAA Hap c:GA Hap ).

[0037] Figure 10 Figure a shows the effect of different metal ions on enzyme catalytic activity (a: α-Amy). Har b: MAA Hap c:GA Hap ).

[0038] Figure 11 Figure a shows the effect of different organic solvents and surfactants on enzyme catalytic activity (a: α-Amy). Har b: MAA Hap c:GA Hap ).

[0039] Figure 12 Enzyme kinetic curve (a: α-Amy) Har b: MAA Hap c:GA Hap ).

[0040] Figure 13 This is a graph showing the change in reducing sugar content in soy sauce during fermentation.

[0041] Figure 14 This is a graph showing the changes in the yellow and red indices of soy sauce during fermentation.

[0042] Figure 15 This is a heatmap showing the content of various free amino acids in soy sauce after fermentation. Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and specific implementation examples.

[0044] The halophilic archaea of ​​the present invention Haloarcula sp. ZJK10-12 and Haladaptatus sp. ZJK29-3 is a publicly available strain, obtained from the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC1.62770 and CGMCC1.62772, respectively.

[0045] Prokaryotic expression hosts are Haloferax volcanii Purchased from the Japan Culture Collection, strain accession number JCM 8879. Regarding the strain... Haloferax volcanii Mutation was performed to obtain a uracil-thymidine auxotrophic mutant strain, denoted as... Haloferax volcanii H1424; its construction method is well-known, and can be found in: Allers, T., Ngo, HP, Mevarech, M., et al. Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes[J]. Applied and environmental microbiology, 2004, 70(2):943-953. Daqu (a type of Chinese liquor) was purchased from Jiangsu Hengshun Group Co., Ltd., and the saline solution was sterilized at 121 ℃ for 15 min and used after cooling. Other reagents or materials can be obtained through regular commercial channels unless otherwise specified. Example 1:

[0046] Construction of recombinant plasmid pTA04- α-amy Har pTA04- maa Hap and pTA04- ga Hap (1) Amplification of the target gene One loopful of bacterial cells was picked and placed into 50 µL ddH2O. The cells were lysed by boiling at 100 °C for 10 min. Using this as a DNA template, the following primers were used for amplification of the target gene. The PCR product was detected by 1% agarose gel electrophoresis and then purified using a DNA gel recovery kit.

[0047]

[0048] PCR amplification system (25 µL): PCR Master mix (12.5 µL); DNA template (2 µL); F-terminal primer (10 µM) (1 µL); R-terminal primer (10 µM) (1 µL); ddH2O (8.5 µL).

[0049] PCR amplification program: Pre-determination: 95 ℃, 5 min; Determination: 95 ℃, 30 s; Annealing: 65 ℃, 30 s; Extension: 72 ℃, 2.5 min; 30 cycles; Post-extension: 72 ℃, 10 min.

[0050] (2) Enzyme digestion of the target gene and vector Using the corresponding two restriction endonucleases (QuickCut) TM Ⅰ. QuickCutTM II) The target gene fragment and pTA04 vector were double-digested with restriction endonucleases according to the instructions (37 °C for 30 min). The digestion products were purified using a DNA gel extraction kit.

[0051] Enzyme digestion system (100 µL):

[0052] (3) The target fragment is ligated to the pTA04 vector. The purified target gene from step (2) was ligated with the pTA04 vector at 16 °C for 2 h.

[0053] Connection system (10 µL):

[0054] (4) Recombinant plasmid transformation of cloning host E . coli DH5α After the target gene is ligated with the pTA04 vector, add the entire ligation system to 100 µL. E . coli In DH5α competent cells, after being thoroughly mixed by pipetting, the cells were placed in an ice bath for 30 min, then heat-shocked at 42 ℃ for 90 s. After being removed, the cells were placed in an ice-water mixture for 2 min. Finally, 600 µL of LB liquid medium was added and the cells were shaken and cultured in a shaker for 1 h (37 ℃, 200 rpm). 200 µL of the bacterial culture was spread on LB agar plates (containing 0.1 mg / mL ampicillin) and cultured overnight at 37 ℃ (12-15 h). Single colonies were then selected for PCR verification. The verification system is shown in (1). Subsequently, 1% agarose gel electrophoresis was performed to identify positive transformants.

[0055] (5) Plasmid extraction and validation The positive transformants from step (4) were inoculated into 3 mL of LB liquid medium (containing 0.1 mg / mL ampicillin) and cultured overnight on a shaker (12-15 h, 37 ℃, 200 rpm). The plasmid was extracted using a plasmid DNA mini-extraction kit and verified by double enzyme digestion. The enzyme digestion conditions were the same as in (2).

[0056]

[0057] The enzyme digestion products were detected by 1% agarose gel electrophoresis, such as... Figure 1 As shown, this is the recombinant plasmid pTA04- α-amy Har (a) pTA04- maa Hap(b) and pTA04- ga Hap (c) Double enzyme digestion verification diagram; lane M in the diagram represents the DNA marker, lane 1 represents the result of double enzyme digestion of plasmid. The successfully constructed plasmid was stored at -20 °C and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The recombinant plasmid with correct base sequence was used for subsequent experiments. Example 2:

[0058] halophilic archaea α-amylase (α-Amy) Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap Heterologous expression in prokaryotes mutant strain Haloferax volcanii H1424 was inoculated into Hv-YPC liquid medium (with thymine added to a final concentration of 40 μg / mL) and cultured on a shaker (37 ℃, 200 rpm). After reaching the exponential phase, it was converted into protoplasts. The recombinant plasmid was transformed into the protoplasts using PEG-mediated transformation and cultured overnight. 200 µL of bacterial culture was spread onto Hv-YPC agar plates and cultured at 37 ℃ for about one week. Single colonies were picked for PCR verification under the same conditions as in Example 1 (1). Finally, positive transformants containing the recombinant plasmid were obtained.

[0059] like Figure 2 As shown, it contains recombinant plasmid pTA04- α-amy Har (a) pTA04- maa Hap (b) and pTA04- ga Hap (c) Transformant verification diagram; lane M in the diagram represents DNA marker, lane 1 represents the transformation verification result. Successfully verified transformants are inoculated into 3 mL of Hv-YPC liquid medium and cultured at 37 °C and 200 rpm for 3-4 days. Then, they are inoculated into 300 mL of Hv-YPC liquid medium at a 1% (v / v) inoculation rate for enrichment culture. After the cells grow to the stationary phase, they are centrifuged (4 °C, 8000 rpm) to collect the cells and stored at -20 °C for later use. Example 3:

[0060] halophilic archaea α-amylase (α-Amy) Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap Nickel column purification (1) The collected bacterial cells were resuspended in 35 mL of cell lysis buffer (2 M NaCl, 20 mM Tris-HCl, pH 8.0) and sonicated in an ice bath at a power of 180 W for 3 s with 5 s intervals for a total of 30 min. After centrifugation at low temperature, the supernatant was collected for subsequent purification.

[0061] (2) After draining the preservation liquid (20% ethanol) from the nickel column, wash the column with 10 times the column bed volume of ddH2O and equilibrate the nickel column with 10 times the column bed volume of cell lysis buffer.

[0062] (3) Load the supernatant onto the column.

[0063] (4) Wash away the impurities with 25 mL of buffer I.

[0064] (5) Elute the target protein with 10 mL of buffer II, and store the enzyme solution at 4 °C.

[0065] The obtained enzyme solution was analyzed by SDS-PAGE and its enzyme activity was determined.

[0066] Figure 3 SDS-PAGE gel image after nickel column affinity chromatography (a: α-Amy) Har b: MAA Hap c:GA Hap In this ), lane M: Protein Marker; lane 1: supernatant after centrifugation; lane 2: sample flow-through solution; lane 3: contaminating proteins eluted by buffer I; and lane 4: target protein eluted by buffer II.

[0067] (6) Wash the column with 10 column bed volumes of buffer (1 M imidazole, 2 M NaCl, 20 mM Tris-HCl, pH 8.0) and 10 column bed volumes of ddH2O respectively; then soak the column in 20% ethanol and store at 4 °C. Example 4:

[0068] halophilic archaea α-amylase (α-Amy) Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap Enzyme activity assay α-Amy was determined using a 1% soluble starch solution as a substrate. Har and MAA Hap The catalytic activity of GA was determined using 1% dextrin solution as a substrate. HapEnzyme activity was measured. 30 μL of substrate solution was mixed with 2 μg of enzyme solution (enzyme concentration determined using a Bradford protein assay kit). The reaction mixture was then brought to 100 μL with enzyme activity assay buffer (2 M NaCl, 20 mM Tris-HCl, pH 8.0). The mixture was reacted at 37 °C for 30 min. Finally, an equal volume of DNS solution was added, and the mixture was boiled at 100 °C for 5 min. The mixture was then rapidly cooled on an ice-water mixture. The absorbance (OD) was measured using a microplate reader. 540 .

[0069] α-Amylase activity (U): Under optimal conditions, the amount of enzyme required to hydrolyze soluble starch to produce 1 mg of glucose per minute is defined as one enzyme activity unit.

[0070] Maltase activity (U): Under optimal conditions, the amount of enzyme required to hydrolyze soluble starch to produce 1 μg of maltose per minute is defined as one unit of enzyme activity.

[0071] Glucose enzyme activity (U): Under optimal conditions, the amount of enzyme that hydrolyzes dextrin to produce 1 μg of glucose per minute is defined as one unit of enzyme activity. Example 5:

[0072] halophilic archaea α-amylase (α-Amy) Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap Optimal reaction conditions analysis (1) Determination of the optimal reaction temperature like Figure 4 Different reaction temperatures were set as shown, with other conditions the same as in Example 4. The relative enzyme activity at other temperatures was calculated with the highest enzyme activity as 100%, and three parallel experiments were conducted for each group. Figure 4 Figure a shows the effect of temperature on enzyme catalytic activity (a: α-Amy). Har b: MAA Hap c:GA Hap The results showed that α-amylase (α-Amy) Har Maltose amylase (MAA) exhibits high enzyme activity within the temperature range of 35-60 ℃, with an optimal reaction temperature of 50 ℃. Hap ) exhibits high activity in the 30-50 ℃ range, with an optimal reaction temperature of 40 ℃; glucoamylase (GA) Hap It exhibits high activity in the range of 25-55 ℃, with the optimal reaction temperature being 45 ℃.

[0073] (2) Determination of optimal NaCl concentration for reaction like Figure 5The reaction systems shown were configured with different NaCl concentrations. The enzyme activity of each enzyme was measured at its optimal reaction temperature, with other conditions the same as in Example 4. The relative enzyme activities of other gradients were calculated with the highest enzyme activity as 100%, and each group was tested in triplicate. Figure 5 Figure a shows the effect of NaCl concentration on enzyme catalytic activity (a: α-Amy). Har b: MAA Hap c:GA Hap The results showed that α-amylase (α-Amy) Har Maltose amylase (MAA) can maintain more than 60% of its maximum enzyme activity in 1.0-4.5 M NaCl, with the optimal NaCl concentration for the reaction being 2.5 M. Hap Glucoamylase (GA) can maintain more than 60% of its maximum enzyme activity in 1.0-4.5 M NaCl, with the optimal NaCl concentration being 2.0 M; Hap The enzyme can maintain more than 50% of its maximum activity in 1.0-2.0 M NaCl, and the optimal NaCl concentration for the reaction is 1.0 M.

[0074] (3) Determination of the optimal reaction pH like Figure 6 Different pH settings were used, specifically: 0.1 M citrate buffer at pH 4.0, 4.5, 5.0, 5.5, and 6.0; 0.1 M phosphate buffer at pH 6.0, 6.5, 7.0, and 7.5; 0.1 M Tris-HCl buffer at pH 7.5, 8.0, 8.5, and 9.0; and 0.1 M CHES-NaOH buffer at pH 9.0, 9.5, 10.0, and 10.5. The effect of pH on enzyme activity was measured at the optimal reaction temperature and salinity, with other conditions the same as in Example 4. The relative enzyme activities of other gradients were calculated with the highest enzyme activity as 100%, and each group was tested in triplicate. Figure 6 Figure a shows the effect of pH on enzyme catalytic activity (a: α-Amy). Har b: MAA Hap c:GA Hap The results showed that α-amylase (α-Amy) Har It maintains over 60% of its maximum enzyme activity within a pH range of 5.0-8.0, with the optimal reaction pH being 6.0; maltose amylase (MAA) Hap It maintains over 60% of its maximum enzyme activity within a pH range of 6.0-7.0, with the optimal reaction pH being 6.0; glucoamylase (GA) Hap It maintains more than 60% of its maximum enzyme activity within a pH range of 5.0-6.0, with the optimal reaction pH being 5.5.

[0075] halophilic archaea α-amylase α-Amy Har The optimal reaction conditions are 50 °C, 2.5 M NaCl, and pH 6.0.

[0076] halophilic archaea maltose amylase (MAA) Hap The optimal reaction conditions are 40 °C, 2.0 M NaCl, and pH 6.0.

[0077] halophilic archaea glucoamylase GA Hap The optimal reaction conditions are 45 °C, 1.0 M NaCl, and pH 5.5. Example 6:

[0078] halophilic archaea α-amylase (α-Amy) Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap Stability analysis (1) Thermal stability analysis like Figure 7 As shown, the enzyme solution was incubated at different temperatures for 0, 30, 60, and 90 min, and the residual enzyme activity was measured under the optimal reaction conditions. Figure 7 The graph shows the results of the enzyme's thermostability analysis. Enzyme activity at 0 min incubation was considered 100%. The results indicate that α-amylase (α-Amy)... Har When incubated at 20, 40, and 50 °C for different times, the relative enzyme activity remained above 70%; maltose amylase (MAA) Hap When incubated at 20 and 40 °C for different times, the relative enzyme activity remained above 80%; glucoamylase (GA) Hap When incubated at 20, 40 and 45 °C for different times, the relative enzyme activity remained above 70%, indicating that the halophilic archaea amylase has good thermal stability.

[0079] (2) Stability analysis of NaCl like Figure 8 As shown, the enzyme was placed in different optimal pH buffers of NaCl and incubated at room temperature for 0, 30, 60 and 90 min, and the residual enzyme activity was measured under the optimal reaction conditions. Figure 8 The graph shows the results of NaCl stability analysis of the enzyme. Enzyme activity at 0 min incubation is considered 100%. The results indicate that α-amylase (α-Amy)... Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap The three types of amylases maintained more than 80% of their activity after incubation in 1.0-4.5 M NaCl concentrations for 90 min, indicating that they have excellent NaCl stability.

[0080] (3) pH stability analysis like Figure 9 As shown, the enzyme was incubated in optimal NaCl buffer at different pH values ​​for 0, 30, 60, and 90 min at room temperature, and the residual enzyme activity was measured under optimal reaction conditions. Figure 9 The graph shows the results of the enzyme's pH stability analysis. Enzyme activity at 0 min incubation was considered 100%. The results indicate that α-amylase (α-Amy)... Har At pH 5.0-pH 9.5, maltose amylase (MAA) Hap After incubation at pH 5.5–8.0 for 90 min, the relative enzyme activity remained above 80%; glucoamylase (GA) Hap After incubation at pH 5.5-pH 7.0 for 90 min, the relative enzyme activity remained above 70%, indicating that all three enzymes have good pH stability. Example 7:

[0081] halophilic archaea α-amylase (α-Amy) Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap Tolerance analysis (1) Tolerance analysis to different metal ions Ni with a final concentration of 5 mM was added to the reaction system. 2+ Cu 2+ Ca 2+ 、Sr 2+ Mg 2+ Mn 2+ Zn 2+ Fe 3+ Ba 2+ K + and Co 2+ Enzyme activity was determined under optimal reaction conditions, with ddH₂O used instead of metal ions in the positive control system. The positive control result was defined as 100% enzyme activity. The relative enzyme activity of the reaction systems with different metal ions was calculated, and each group was tested in triplicate. Figure 10 Figure a shows the effect of different metal ions on enzyme catalytic activity (a: α-Amy). Har b: MAA Hap c:GA Hap The results showed that α-amylase (α-Amylase) was affected by different metal ions. Har The relative enzyme activity of all enzymes remained above 95%, with Ni 2+ Cu 2+ Ca 2+ 、Sr 2+ Mn 2+ Fe 3+ Ba 2+ K + and Co 2+ It promotes the activity of this enzyme; Ca 2+ 、Sr 2+ Mg 2+ Mn 2+ Ba 2+ and K + maltose amylase (MAA) Hap Ni has a promoting effect on activity. 2+ Cu 2+ and Zn 2+ MAA Hap It has a significant inhibitory effect; glucosamine (GA) Hap In solutions containing all of the above metal ions, the relative enzyme activity can be maintained at over 75%, with Ni being the most abundant. 2+ Cu 2+ Mn 2+ Ba 2+ and Co 2+ Zn has a significant promoting effect on this enzyme. 2+ and Fe 3+ It can inhibit enzyme activity by up to 20%. Mn 2+ For α-amylase (α-Amy) Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap The activity-promoting effect is strongest. The above data indicate that halophilic archaea amylases have superior tolerance to most metal ions.

[0082] (2) Analysis of resistance to organic solvents and surfactants Add 15% (v / v) of organic solvent (DMSO, glycerol, methanol, ethanol, acetone, DMF, and isopropanol) or surfactant (Tween 20, Triton X-100, SDS, Tween 80, and PEG600) to the reaction system. Determine enzyme activity under optimal reaction conditions. In the positive control system, ddH2O replaces the organic solvent or surfactant. Calculate the relative enzyme activity containing the organic solvent or surfactant, using the positive control result as 100% enzyme activity. Perform three parallel experiments for each group. Figure 11 Figure a shows the effect of different organic solvents and surfactants on enzyme catalytic activity (a: α-Amy). Har b: MAA Hap c:GA Hap The results showed that DMSO, methanol, ethanol, acetone, isopropanol, and Tween 80 had an effect on α-Amy.Har Acetone has varying degrees of promoting effect on α-Amy. Har The promoting effect was most prominent, increasing enzyme activity by approximately 20%. This indicates that α-amylase (α-Amylase) has a significant promoting effect. Har It exhibits good resistance to organic solvents and surfactants; except for methanol, maltose amylase (MAA) Hap The catalytic activity of all enzymes was inhibited by other organic solvents, but they could maintain more than 80% of their activity in surfactants, and Triton X-100 and Tween 80 could promote enzyme activity; all organic solvents could significantly inhibit glucosamine (GA) Hap The catalytic activity of Tween 20, Triton X-100, Tween 80, and PEG600 can promote GA. Hap The enzyme activity indicates that maltose amylase (MAA) has increased. Hap ) and glucoamylase (GA) Hap It has poor tolerance to organic solvents but strong tolerance to surfactants. Example 8:

[0083] halophilic archaea α-amylase (α-Amy) Har ), maltose amylase (MAA) Hap ) and glucoamylase (GA) Hap Enzyme kinetic parameter analysis Determination of α-Amy using soluble starch solution as substrate Har and MAA Hap Enzyme kinetic curves were obtained, and GA was determined using dextrin solution. Hap Enzyme kinetic curves were obtained. The specific procedure involved adding soluble starch or dextrin solutions of different final concentrations to a 100 μL reaction system and measuring enzyme activity under optimal reaction conditions. The kinetic constants were calculated using the Michael-Mentene equation. K m and maximum reaction rate V max . Figure 12 The enzyme kinetics curve (a: α-Amy) Har b: MAA Hap c:GA Hap ), per milligram of α-amylase (α-Amy Har The maximum reaction rate when soluble starch is used as the substrate V max The kinetic constant is 12.48 mg / min. K m It was 16.48 mg / mL; per milligram of maltose amylase (MAA) HapThe maximum reaction rate when soluble starch is used as the substrate V max The kinetic constant is 27.80 μg / min. K m It was 5.42 mg / mL; per milligram of glucosamine (GA) Hap Maximum reaction rate when dextrin is used as substrate V max The kinetic constant is 9.39 μg / min. K m It was 3.39 mg / mL. Example 9:

[0084] The effect of combined use of three amylases on soy sauce Soy sauce starter culture was mixed with a saline solution (18% NaCl) at a ratio of 1:4 (w / v). The experimental group was supplemented with 0.02% α-amylase (α-Amylase). Har 0.01% maltose amylase (MAA) Hap ) and 0.01% glucoamylase (GA) Hap The control group did not receive any exogenous amylase. Fermentation was carried out at 40 ℃ for 30 days, with samples taken every 5 days to monitor changes in reducing sugar content, yellow index, and red index in the soy sauce supernatant. After fermentation, the types and contents of free amino acids were determined.

[0085] Figure 13 The graph shows the changes in reducing sugar content during fermentation. The results indicate that the reducing sugar content in soy sauce first increases and then decreases. In the early stages of fermentation, the activity of salt-tolerant amylase significantly increases the reducing sugar content; by day 10, the enzyme-added group had approximately 32% higher reducing sugar content than the control group. As fermentation time increases, enzyme activity gradually decreases, and the reducing sugars are utilized by microorganisms and undergo Maillard reactions with amino acids to form soy sauce pigments, leading to a gradual decrease in the reducing sugar content. Figure 14 The graph shows the changes in the yellow and red indices of soy sauce. During fermentation, both the yellow and red indices of the enzyme-added group were higher than those of the control group. After fermentation, the yellow index of the enzyme-added group (8.36) was nearly 4.0% higher than that of the control group (8.04), and the red index of the enzyme-added group (5.19) was nearly 3.4% higher than that of the control group (5.02). This indicates that the reducing sugar, a hydrolysis product of high-salt-tolerant amylase, can participate in the Maillard reaction with amino acids, which has a certain effect on improving the yellow and red indices of soy sauce, making the soy sauce brighter in color. Figure 15This is a heatmap showing the content of various free amino acids in soy sauce after fermentation. The total amount of free amino acids in the enzyme-added group (42.96 mg / mL) was 6.42% higher than that in the control group (40.37 mg / mL). It is speculated that this may be because amylase hydrolyzes starch into reducing sugars such as glucose, which makes the growth of microorganisms (such as Aspergillus oryzae, yeast, and lactic acid bacteria) more active, thereby enhancing the synthesis and secretion of proteases.

[0086] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

[0087] α-amy Har Nucleotide sequence: SEQ ID No. 1 α-Amy Har Amino acid sequence: SEQ ID No. 2 VGSSAVYQYYHTDWSEVESDLSTIAAQGYDAIQVPPAQYSRLDRSHQQGVTDPPLGYQPIDFKNFDSVFGTEAEYQSMVNEAHNQGLDVIADAVVNHMAANDDFRGAPGITFADLPYFSERDFHPESSIDYSDPESVENGWLVGLKDLKQESSYVRGQLQDYVQKYADLGVDGIRWDAAKHVPEWFFNDYANQWADNLGLYTVGEVLQGGVDYCQGYADTGMSVTDYPLYYTMKEDVFHQSGDMTALDGAGLVNQSPYQSLTFVSNHDSGPPQYEKLAYAYILTYEGYPRVYSNRIAVDDGDIQNLLWIKNNLAGGTAYQRHADQNLYIFERNNNLLVGLNRSGSWRSKWVYTTWTNQSLHDYTGSSGDVTTNGDGWVQISIPPQGWVCYAP maa Hap Nucleotide sequence: SEQ ID No. 3 MAA Hap Amino acid sequence: SEQ ID No. 4 MHTADPPRFTMVGTAVELAPHDPDPDGEYEWHIETRPDGSSAVVDDTTVIHFTPDVPGVYRLRLSAPDGDHERTVRAFPDERRPARFELPLSDLPIPHEELESVCIAGPFNEHLVGRDRPTYENGTYVYETELPPGEHPYGFLLNDDLTQQVQGTLTVPGPGRPRIHLDATLEADSIVVTATTESAPDSEFEDGELEVEFYEDGSRAVGHGPRTVRVSRNDIGDGVRIHAVAVGERHSVADCVEIGGRSELGLSERSGSKQSGSERSEVSWSERVGIHRPNDPPEWAESATVYEIFVRSFAGETVDTTFRELERRLPYLEWLGVDCVWLTPILGSPTDHGYHTTNYFETAEDLGTRAEFESFVSACHDAGIRVVFDLVINHSSRDHPAFQMSAAGVPEYRDWYIWEEDGETGEPKAQRYFNWDRIPNYNFDSLAVRRFLLDVVDEWAGLVDGFRCDVAWGVPHEFWKEVAALVPDGFLLLDETIPRDPAYHEGEFTMHYDTTLYGTLREIGTGEKPATTVFDALADAERAGFPESAVHLRYVENHDESRYLDDCDRDALKAAAAATFTLPGAPMIYYGQERGMTEYRGTMRWDDGDDELTAFHRSLIEARNEHPVLKGGAVERIGWESESSSVVAFARDDDDSRVVVVLNFADGSETVRIDERVEVVNLVTGERVPTAAGSDSKTELSVSDVVVVRTTEESK ga Hap Nucleotide sequence: SEQ ID No. 5 GA Hap Amino acid sequence: SEQ ID No. 6 MRICEGLFHQVFYYSDSWCYLMTLRDALSDFKRHRGNATHFPGERRTTAGRFSGFDARLVHVSPHGSLRDYSYPLSGRYGLDRSRFGVRRDGNADIVWFDAVDSTQRYHEGSALVVTEHGTTDWSITQYDLTFGDFHFTHFAVDDANENDAALDELVAYFGFAPDGRDDRIGQLRHGDVVEVYHTEEHDYLGMSAEPTEIDGLVSADFDALVSERSNAVPDISDTGRYEESRLSGELVVSLPLDGSTTFVSLLADHRELPREDALRRVRELALEYDSTARIRSLANDETSRNPLDATLDAPMTDAITDDLRVLSFLSAETGARIAGPDFDPFYQYSGGYGYTWFRDDAEISRFLLTADDHFDLSLADWHEKSARLYCETQRPDGTWPHRVWPRDGTIAPGWANARVEAGDGADYQADQTGSVIAFLATYLRTSDLDTELEAEIEGTLRAALDGLDDTLEADGLPIHCQNAWENMTGRFAHTAATYLEAYAELARAPVSDDVRDRALEGTKAVLAGLDRLWTGEFYALRLTEDDRDERRDSSALALAAAHRAAASVVDLDSEQVDRLVSHTEAVLDGLWRDPEESEVRGLARFDGDNWRVREQTTPKIWTVSTAWGANAANALADLLSERDDRRADEFARRSRDLLSLLLPGGSLCAKTGYLPEQYFDVGNADSATPLGWPHAIRLATVASLDERGALESKDVKPSLD

Claims

1. An amylase derived from halophilic archaea, characterized in that, The amylase specifically includes α-amylase, maltose amylase, and glucoamylase. The nucleotide sequence of the α-amylase is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2; the nucleotide sequence of the maltose amylase is shown in SEQ ID No. 3, and the amino acid sequence is shown in SEQ ID No. 4; the nucleotide sequence of the glucoamylase is shown in SEQ ID No. 5, and the amino acid sequence is shown in SEQ ID No.

6.

2. The method for preparing amylase from halophilic archaea according to claim 1, characterized in that, The steps are as follows: (1) Construction of recombinant plasmid pTA04- α-amy Har pTA04- maa Hap and pTA04- ga Hap ; Based on halophilic archaea Haloarcula By comparing the whole genome sequence of sp. ZJK10-12, a halophilic archaea α-amylase gene was obtained. α-amy Har ; Based on halophilic archaea Haladaptatus By comparing the whole genome sequence of sp. ZJK29-3, a maltose amylase gene was obtained. maa Hap and a glucosidase gene ga Hap Amplifying the target gene fragment using PCR technology α-amy Har , maa Hap and ga Hap Then, the target gene is ligated to the pTA04 vector using molecular cloning technology to construct the recombinant plasmid pTA04- α-amy Har pTA04- maa Hap and pTA04- ga Hap ; (2) Recombinant plasmid pTA04- α-amy Har pTA04- maa Hap and pTA04- ga Hap Transformation of prokaryotic hosts and expression; The recombinant plasmid constructed in step (1) was transformed into a prokaryotic host bacterium using the PEG-mediated transformation method. Haloferax volcanii H1424 was used to obtain recombinant cells, which were then inoculated into Hv-YPC liquid medium and cultured until the growth stabilized. The cells were then collected by centrifugation and stored for later use. (3) Purification of halophilic archaea α-amylase, maltose amylase and glucoamylase; In step (2), the bacterial cells were resuspended in cell lysate, the cells were disrupted by an ultrasonic disruptor, the supernatant was collected after centrifugation, and the protein was purified by nickel affinity chromatography to obtain high purity halophilic archaea α-amylase, maltose amylase and glucoamylase.

3. The method for preparing amylase derived from halophilic archaea according to claim 2, characterized in that, The halophilic archaea described in step (1) Haloarcula sp. ZJK10-12 was obtained from the China General Microbiological Culture Collection Center, accession number CGMCC 1.62770; the halophilic archaea described Haladaptatus sp. ZJK29-3 was obtained from the China General Microbiological Culture Collection Center, with accession number CGMCC 1.62772.

4. The method for preparing amylase derived from halophilic archaea according to claim 2, characterized in that, The gene described in step (1) α-amy Har The F-terminal primer is 5'-CGCGAATTCGTCGGGTCCAGTGCGGTG-3', where GAATTC is... Eco The restriction enzyme site for RI is 5'-ATAGGATCCCGGCGCGTAGCAGACCCA-3', and GGATCC is... Bam HI restriction enzyme site; maa Hap The F-terminal primer is 5'-ATAGGTACCCACACTGCTGATCCCCCG-3', and the GGTACC is... Kpn The restriction enzyme site for I, the R-terminal primer is 5'-ATAGCATGCTTTACTTTCCTCGGTGGT-3', and GCATGC is... Sph I restriction enzyme site; ga Hap The F-terminal primer is 5'-CGCAGATCTAGAATCTGTGAAGGATTA-3', where AGATCT is... Bgl The restriction enzyme site for II has an R-terminal primer of 5'-ATAGGATCCGTCCAACGACGGTTTCAC-3', and GGATCC is... Bam HI restriction enzyme site.

5. The method for preparing amylase from halophilic archaea according to claim 2, characterized in that, The prokaryotic host expression described in step (2) is based on strain. Haloferax volcanii The constructed uracil-thymidine auxotrophic mutant strain is denoted as Haloferax volcanii H1424; Haloferax volcanii Purchased from the Japan Microbial Culture Collection, culture number JCM 8879; The Hv-YPC culture medium consists of the following components per liter: yeast extract 5.0 g, soybean peptone 1.0 g, acid-hydrolyzed casein 1.0 g, KCl 4.2 g, MgSO4·7H2O 33.0 g, MgCl2·6H2O 30.0 g, NaCl 144.0 g, 1 M Tris-HCl (pH 7.5) 12.0 mL, and CaCl2 0.33 g. The above components are diluted to 1 L with distilled water, the pH is adjusted to 7.2-7.5, and the medium is sterilized at 115 °C for 30 min. The culture conditions were 37 ℃, 200 rpm, for 3-4 days; the centrifugation conditions were 4 ℃, 8000 rpm, for 10-15 min.

6. The method for preparing amylase from halophilic archaea according to claim 2, characterized in that, The cell lysis buffer in step (3) consists of 2 M NaCl, 20 mM Tris-HCl, and pH 8.0; the conditions for ultrasonic cell disruption are: ultrasonic time 3 s, interval 5 s, power 180 W, and total time 30 min.

7. The method for preparing amylase derived from halophilic archaea according to claim 2, characterized in that, The steps of nickel column affinity chromatography described in step (3) are as follows: drain the 20% ethanol used to preserve the nickel column, wash the column with 10 column volumes of ddH2O, and then equilibrate the column with 10 column volumes of cell lysis buffer; load the supernatant collected after centrifugation onto the column; wash away contaminating proteins with 10 column volumes of buffer I; and elute the target protein with 5 column volumes of buffer II. The components of buffer I are 40 mM imidazole, 2 M NaCl, 20 mM Tris-HCl, pH 8.0; The buffer II consisted of 100 mM imidazole, 2 M NaCl, 20 mM Tris-HCl, and pH 8.

0.

8. The application of the halophilic archaea-derived amylase according to claim 1 or the halophilic archaea-derived amylase prepared by any one of claims 2-7 in the preparation of soy sauce, characterized in that, The applications include increasing the reducing sugar content, color, and total free amino acid content of soy sauce.

9. The application according to claim 8, characterized in that, The steps are as follows: After mixing soy sauce starter with a brine solution, α-amylase, maltose amylase, and glucoamylase are added. After fermentation, the reducing sugar content, color, and total free amino acid content of the soy sauce are improved.

10. The application according to claim 9, characterized in that, The ratio of soy sauce starter to brine is 1g:4ml, and the brine concentration is 18%. The amounts of α-amylase, maltose amylase, and glucoamylase added are 0.02%, 0.01%, and 0.01% of the total fermentation volume, respectively. The fermentation temperature is 30-40℃, and the fermentation time is 25-30 days.

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