CN118086256A - Medium-temperature alpha-amylase and gene and application thereof - Google Patents

Abstract

The present invention discloses a medium-temperature α-amylase AmyB and its gene and application. The present invention screened a new medium-temperature α-amylase gene AmyB from the Bacillus velezensis BL-LM strain isolated from Baijiu Daqu, and the medium-temperature α-amylase encoded by it has the following advantages: high catalytic activity under medium temperature conditions, good temperature stability and wide pH stability. The above advantages mean that the medium-temperature α-amylase AmyB of the present invention has great application potential in industries such as food, fermentation, feed, textile and medicine.

Description

A medium-temperature alpha-amylase and its gene and application

Technical Field

The invention relates to the field of biotechnology, and in particular to a medium-temperature alpha-amylase with high specific enzyme activity and a gene and application thereof.

Background technique

Starch is one of the most abundant polymers in nature, which is composed of glucose monomers connected by glycosidic bonds. In addition to being directly processed into various foods, starch hydrolyzates and related derivatives obtained by enzymatic or chemical methods using starch as raw materials are also widely used in various aspects of human production and life.

α-amylase (E.C.3.2.1.1) mostly belongs to the 13th family of glycoside hydrolases, which can randomly cut the α-1,4 glycosidic bonds inside the sugar chains of amylose (linear α-1,4 glycosidic bond-linked glucan) and amylopectin (linear α-1,4 glycosidic bond-linked main chain with α-1,6 glycosidic bond-linked branches) indiscriminately, and hydrolyze starch into dextrin, oligosaccharides, maltose and glucose. As an important industrial enzyme with a wide range of applications and large usage, α-amylase currently has important applications in the fields of food processing, fermentation, brewing, textile, papermaking, pharmaceutical, new bioenergy, etc. For example, α-amylase and pullulanase can be used to hydrolyze starch to generate resistant starch for use in the healthcare industry. In the dough fermentation process of the food processing industry, the addition of α-amylase can reduce the viscosity of the dough, increase the sugar content of the dough, increase the volume of the bread, and improve the color. In the liquor industry, the liquefaction power of Daqu is an important indicator to measure the quality of Daqu, and the level of Daqu liquefaction power is mainly related to the activity of α-amylase.

In industrial production, the use of amylases with high activity and good stability is extremely critical to improve the utilization rate of starch. Although amylases in nature are widely found in animals, plants and microorganisms, amylases from microorganisms are widely used due to their good stability and suitability for large-scale production. Bacillus bacteria, which are widely found in nature, are one of the main sources of microbial amylases. For example, Bacillus licheniformis is often used as a source of thermostable amylase. Therefore, it is of great significance to develop amylases from Bacillus bacteria in practical applications.

According to the different optimal reaction temperatures of the catalytic activity of α-amylase, α-amylase can be divided into high temperature, medium temperature and low temperature α-amylase. Medium temperature α-amylase is a kind of α-amylase with a wide range of applications, and its optimal reaction temperature is generally 50-70℃. At present, the demand for medium temperature α-amylase in industrial production is relatively large. A certain amount of medium temperature α-amylase needs to be added in the processes of starch liquefaction and processing, textile desizing, paper sizing and processing of starch-containing fermentation auxiliary materials.

Summary of the invention

The purpose of the present invention is to provide a medium-temperature alpha-amylase with high specific enzyme activity and a gene and application thereof.

In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

Provided is a medium-temperature alpha-amylase AmyB, whose amino acid sequence is shown in SEQ ID NO.1.

Provided is a mesophilic α-amylase gene AmyB, which encodes the mesophilic α-amylase AmyB according to claim 1.

Furthermore, the nucleotide sequence of the mesophilic α-amylase gene AmyB is shown in SEQ ID NO.2.

Provided is a recombinant plasmid containing a mesophilic alpha-amylase gene AmyB.

Furthermore, the recombinant plasmid is obtained by cloning the α-amylase gene AmyB into the plasmid pET29a(+).

Provided is a recombinant strain containing a mesophilic alpha-amylase gene AmyB.

Furthermore, the recombinant strain uses Escherichia coli, Bacillus, yeast or Lactobacillus as a host bacteria.

Provided is a method for preparing a medium-temperature alpha-amylase, comprising the following steps:

1) transforming a host cell with a recombinant vector containing a medium-temperature α-amylase encoding gene to obtain a recombinant strain;

2) culturing the recombinant strain to induce the expression of mesophilic α-amylase;

3) Isolating and purifying the expressed medium-temperature α-amylase AmyB.

Provided is a medium-temperature alpha-amylase for use as an enzyme preparation for hydrolyzing starch in starch processing, fermentation, food industry or feed field.

Provided is a medium-temperature alpha-amylase gene for genetic engineering application in starch processing, fermentation, food industry or feed fields.

The beneficial effects of the present invention are:

The present invention separates a medium-temperature alpha-amylase AmyB from Bacillus velezensis BL-LM, which has high specific enzyme activity, good temperature stability and wide pH stability. The medium-temperature alpha-amylase of the present invention has hydrolysis activity on a variety of starch substrates, and provides a new choice for starch hydrolysis in industrial fields such as starch processing, food industry, brewing, fermentation, feed and medicine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an electrophoretic diagram of PCR amplification of the mesophilic α-amylase encoding gene AmyB;

1: DL5000 DNA Marker; 2: PCR amplification product of the mesophilic α-amylase gene AmyB;

FIG2 is an SDS-PAGE electrophoresis diagram of the purification of recombinant mesophilic α-amylase AmyB;

1: Standard protein marker; 2: Precipitate of induced E. coli fragmentation containing mesophilic α-amylase gene vector pET29a-AmyB; 3: Supernatant of induced E. coli fragmentation containing mesophilic α-amylase gene vector pET29a-AmyB; 4: Purified protein of recombinant mesophilic α-amylase AmyB;

FIG3 is a schematic diagram of the optimum pH for the mesophilic α-amylase AmyB;

FIG4 is a schematic diagram of pH stability of mesophilic α-amylase AmyB;

FIG5 is a schematic diagram of the optimum temperature of mesophilic α-amylase AmyB;

FIG6 is a schematic diagram of the temperature stability of the mesophilic α-amylase AmyB;

FIG7 is a schematic diagram showing the effect of metal ions on the activity of mesophilic α-amylase AmyB;

FIG8 is a schematic diagram showing the effect of different chemical reagents on the activity of mesophilic α-amylase AmyB;

FIG9 is a schematic diagram showing the effect of different substrate concentrations on the activity of mesophilic α-amylase AmyB;

Figure 10 is a thin layer chromatography TLC analysis of the degradation products of starch substrates from different sources by medium-temperature α-amylase AmyB; 1: soluble starch standard; 2: glucose standard; 3: maltose standard; 4-6: degradation reaction products of soluble starch, dextrin and corn starch plus medium-temperature α-amylase AmyB, respectively.

Detailed ways

The specific implementation modes of the present invention are described below to facilitate those skilled in the art to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

Example 1 Cloning of the medium-temperature α-amylase gene AmyB and construction of expression vector and recombinant strain

1.1 Cloning of α-amylase gene AmyB

According to the whole genome sequence information of BL-LM Bacillus velezensis isolated from the special-flavor liquor Daqu, an α-amylase gene AmyB was predicted. According to the predicted α-amylase gene sequence, the gene amplification primers were designed using DNAMAN software. The primer sequences are shown in the following table, where the underlined parts are the 15bp homology arms on both sides of the Nde I and Xho I restriction sites on the pET-29a (+) plasmid.

The genomic DNA of pure culture of B. velezensis BL-LM strain was extracted using the OMEGA Bacterial Genome Extraction Kit (D3350). The specific operation was referred to the kit instructions and verified by agarose gel electrophoresis. The extracted genomic DNA was used as a template, and primers 29a-AmyB-F and 29a-AmyB-R were used to perform PCR amplification with Phanta Super-Fidelity DNA polymerase. The amplification system was as follows:

The PCR amplification program was set as follows, and the number of reaction cycles was 35 cycles.

The PCR amplification product was verified by 1% agarose gel electrophoresis, and the results are shown in Figure 1. The full length of the amplified AmyB gene is about 2000 bp, which is consistent with the sequencing result of 1980 bp. The target gene was recovered using the DNA gel recovery kit (TSP601) of Qingke Company. The specific operation refers to the kit manual, and the recovered product was verified by 1% agarose gel electrophoresis.

1.2 Enzyme-linked transformation

The pET-29a(+) plasmid was extracted using a plasmid miniprep kit (D1100) from Solebo Company, and the pET-29a(+) plasmid was double-digested with Nde I and Xho I restriction endonucleases. The digestion system is as follows:

The enzyme digestion system was reacted at 37°C overnight, and the digestion products were verified by agarose gel electrophoresis. The linearized pET-29a(+) vector was recovered using the DNA gel recovery kit (TSP601) of Qingke Company. The recovered target gene and the recovered linearized vector pET-29a(+) after double enzyme digestion were linked by enzyme using the ClonExpressII one-step cloning kit (C112) of Novozyme Company. The enzyme linkage system is as follows:

1.3 Transformation of enzyme-linked products into E. coli DH5α competent cells

The enzyme-linked product was transformed into Escherichia coli DH5α competent cells by heat shock. After heat shock, it was coated with LB solid medium plates containing 50μg/ml kanamycin and cultured at 37℃ overnight. The next day, a single colony was picked and cultured in LB liquid medium at 37℃ and 180rpm for 12h. The extracted plasmid was verified by gel electrophoresis, and the plasmid with the correct size was sent to Qingke Biotechnology Company for DNA sequencing.

The sequencing results showed that the nucleotide sequence of the α-amylase AmyB gene cloned into the expression vector was shown in SEQ ID NO.2.

The full length of the AmyB gene sequence is 1980bp, encoding 659 amino acids and a stop codon. The predicted protein isoelectric point is 6.14 and the molecular weight is 72.43kDa. It is predicted that the AmyB protein contains a signal peptide sequence of 33 amino acids. The properties of α-amylase genes with a similarity greater than 50% to AmyB in the GenBank database have not been studied.

Example 2 High-efficiency expression of α-amylase AmyB gene in Escherichia coli BL21 (DE3)

2.1 Construction of α-amylase AmyB expression strain

The expression vector plasmid with correct sequencing was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, and then coated on LB solid plate containing 50 μg/ml kanamycin, cultured at 37°C overnight, and single colonies were picked up the next day and cultured in LB liquid medium at 37°C and 180rpm for 12h. The plasmid was extracted and verified by DNA sequencing to verify that the correct expression strain can be used for the inducible expression of α-amylase AmyB. Thus, the expression strain E.coli BL21 (pET29a-AmyB) of recombinant α-amylase AmyB was successfully constructed.

2.2 Induced expression and purification of α-amylase AmyB

The expression strain E.coli BL21 (pET29a-AmyB) was inoculated into 5 ml LB liquid medium containing 50 μg/ml kanamycin at 37°C, 200 rpm and cultured overnight, and then inoculated into 180 ml LB liquid medium containing 50 μg/ml kanamycin at a ratio of 1% (v/v) at 37°C, 200 rpm and cultured until OD ₆₀₀ = 0.6, and IPTG with a final concentration of 0.2 mmol/l was added, and induced culture was carried out at 16°C, 200 rpm for 24 hours. The culture solution of the expression strain after induction culture was centrifuged at 6000 r/min for 10 minutes, the bacteria were collected and ultrasonically broken for 30 minutes, and then centrifuged at 12000 r/min for 10 minutes, and the supernatant was collected. The supernatant crude enzyme solution after centrifugation was purified by Ni ²⁺ -NAT affinity chromatography column, and stained with Coomassie Brilliant Blue R-250 after SDS-PAGE electrophoresis, and observed after decolorization with a decolorizing solution, and the results are shown in Figure 2. From the results of SDS-PAGE electrophoresis, it can be seen that the expression strain E. coli BL21 (pET29a-AmyB) expressed the recombinant α-amylase AmyB protein. The size of the purified protein band was about 72 kD, which was consistent with the predicted size of α-amylase AmyB.

Example 3 Determination of Enzymatic Properties of α-Amylase AmyB

3.1 Determination of α-amylase AmyB activity using 3,5-dinitrosalicylic acid (DNS) method

Take 5 μl of the purified and 10-fold diluted enzyme solution and add it to 1.5 ml of PBS buffer (pH 7.0) dissolved with corn starch at a final concentration of 0.5%, react at 50°C for 10 minutes, use the inactivated enzyme as a control, add 1 ml of DNS after the reaction, boil in boiling water for 5 minutes, cool to room temperature and dilute 2 times with deionized water, measure the absorbance at 540 nm and calculate the enzyme activity. One unit of enzyme activity (U) is defined as the amount of enzyme required to hydrolyze starch to produce 1 μmol of reducing sugar per minute under given conditions.

3.2 Determination of the optimal pH and pH stability of starch hydrolysis by α-amylase AmyB

Determination of the optimum pH: Take 2ul of purified and 10-fold diluted recombinant α-amylase AmyB enzyme solution and add it to the reaction system with different pH values to determine the enzyme activity of AmyB. The reaction buffer is citric acid buffer (pH 3.0-6.0), Tris-HCl buffer (pH 7.0-9.0) and PBS buffer (pH 6.5-7.5), and the concentration of the buffer is 20mM. The enzyme activity is determined after reacting for 10min at 50℃, and each condition is repeated three times. The maximum enzyme activity measured is used as the control (100%), and the relative enzyme activity under different pH conditions is calculated. As shown in Figure 3, the recombinant α-amylase AmyB has the maximum enzyme activity when the pH of the PBS buffer is 6.5, and it also has a high enzyme activity at about pH 7.0. The relative enzyme activity at pH 3.0 is 20%, and the relative enzyme activity at pH 9.0 is 21%. The results show that the optimum pH of α-amylase AmyB is 6.5.

Determination of pH stability of enzyme: The purified recombinant α-amylase AmyB enzyme solution was mixed with buffer solutions of different pH and concentration of 20mM at a ratio of 1:9 and treated at 4°C for 24h. Then 2ul of the diluted enzyme solution was added to the enzyme activity assay system, and the enzyme activity was measured after reacting for 10min under pH 6.5 and 70°C, and each condition was repeated three times. The results are shown in Figure 4. α-amylase AmyB can maintain more than 75% of its enzyme activity in the pH 4.0-9.0 range, and the enzyme activity is almost zero when pH <3.0, indicating that the enzyme has good pH stability in the pH 4.0-pH 9.0 range.

3.3 Determination of the optimal temperature and temperature stability of starch hydrolysis by α-amylase AmyB

Determination of the optimum temperature: Take 2 μl of purified and 10-fold diluted recombinant α-amylase AmyB enzyme solution and add it to 1.5 ml of PBS buffer with a pH of 6.5. Place the reaction system at 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C and 100°C respectively. After 10 minutes of reaction, measure the enzyme activity of α-amylase AmyB in hydrolyzing starch. Repeat each condition three times. Take the maximum enzyme activity measured as the control (100%) to calculate the relative enzyme activity under different temperature conditions. As shown in Figure 5, the recombinant α-amylase AmyB has the maximum enzyme activity at 70°C. When the temperature is higher than 70°C, the enzyme activity decreases rapidly. When the temperature is 60°C, the relative enzyme activity is above 50%, and when the temperature is 50°C or below, the relative enzyme activity is less than 40%. The results show that the optimum temperature of α-amylase AmyB is 70°C, and the enzyme is a medium-temperature α-amylase.

Determination of enzyme temperature stability: A certain amount of purified and 10-fold diluted α-amylase AmyB enzyme solution was placed at 50°C, 60°C, 70°C, and 80°C, and samples were taken at regular intervals and added to 1.5 ml of PBS buffer with a pH of 6.5. After reacting for 10 minutes, the residual enzyme activity was determined. The enzyme activity of the enzyme solution without heat treatment was used as the control (100%). Each condition was repeated three times, and the relative enzyme activity after different temperature treatments for different times was calculated. As shown in Figure 6, the enzyme activity of the recombinant α-amylase AmyB was almost reduced to zero after being treated at 70°C and 80°C for 20 minutes, and there was still more than 60% residual enzyme activity after being treated at 60°C for 40 minutes, and the enzyme activity became zero after being treated at 60°C for 6 hours; the enzyme still had more than 80% residual enzyme activity after being treated at 50°C for 6 hours. This shows that the medium-temperature α-amylase AmyB is relatively stable at 50°C.

3.4 Effects of metal ions on AmyB amylase activity

Different common metal ions were added to the activity test system to make the final concentration of 1 mM. The system without any metal ions was used as a control. After reacting at pH 6.5 and 70°C for 10 minutes, the enzyme activity under different metal ion treatments was measured.

In the activity test system containing different metal ions at a final concentration of 1 mM, the system without any metal ions was used as the control (100%), and the results are shown in Figure 7. Among them, Co ²⁺ , K ⁺ , Li ⁺ , Mg ²⁺ , Ni ⁺ , and Ca ²⁺ have a certain activation effect on the activity of the enzyme, and Li ⁺ and Mg ²⁺ have the most obvious enhancement effect. The remaining ions have an inhibitory effect on the enzyme activity, and Cu ²⁺ has the greatest impact on the activity of the enzyme.

3.5 Effect of organic solvents on AmyB amylase activity

10% methanol, ethanol, isopropanol, acetonitrile, glycerol, 20 mg/ml tween-80, 10 mM EDTA, and 2 mg/ml SDS were added to the activity test system. The system without any organic reagents was used as the control. 2 μl of 10-fold diluted enzyme solution was added and reacted at 70°C for 30 minutes. The activity under the influence of different organic reagents was measured and the relative enzyme activity was calculated.

Different organic reagents were added to the activity test system to determine the effects of different organic reagents on the enzyme activity. The activity test system without any organic reagents was used as the control (100%). The results are shown in Figure 8. Methanol, ethanol, isopropanol, acetonitrile, glycerol, Tween-80, SDS, and EDTA all had inhibitory effects on the activity of the enzyme, among which acetonitrile had the most obvious inhibitory effect on the enzyme. SDS denaturant could not completely denature the enzyme. During SDS-PAGE electrophoresis, the enzyme solution should be heated in a boiling water bath for 5 minutes before loading onto the gel.

3.6 Determination of specific activity of AmyB recombinant enzyme and kinetic parameters of enzymatic reaction

Use purified protein to determine the kinetic parameters of amylase. Prepare substrates of different concentrations, and measure enzyme activity under optimal conditions while keeping the enzyme reaction system unchanged. Measure the protein concentration to calculate the specific activity of the enzyme. Specific activity is defined as the units of enzyme activity per milligram of enzyme protein. Make a double reciprocal graph with 1/[S] as the horizontal axis and 1/[V] as the vertical axis.

The activity of the enzyme under different substrate concentrations was measured, and the substrate concentration and the reciprocal of the reaction rate were plotted, as shown in Figure 9. The specific enzyme activity of the enzyme was 27003.37 U/mg, and the Km value was 10.998 mg/ml. The Kcat was 1.73×10 ⁶ s ^-1, and the catalytic efficiency was (Kcat/Km) 1.57×10 ⁵ ml(s.mg).

Example 4 Degradation of substrate by recombinase AmyB

Product verification of the effect of recombinant enzyme AmyB on starch degradation

Take an appropriate amount of recombinant enzyme AmyB and add it to the antisense system of 20mM PBS (pH7.0) containing 1% corn starch, soluble starch and dextrin to fully react at 50°C. After the reaction, maltose and glucose were used as controls, and the control solution and the reaction solution were spotted on the TCL silica gel plate. The TCL silica gel plate was separated using a developing agent (n-butanol: methanol: water = 8:4:3), and after the layer was developed, a color developer (concentrated sulfuric acid: methanol = 1:3) was used at 70°C.

α-Amylase has a cleavage effect on the α-1,4-glycosidic bond of starch, and the degradation products may be dextrin, oligosaccharides and monosaccharides. In order to study the hydrolysis effect of recombinant enzyme AmyB on various substrates, the recombinant enzyme was fully reacted with each substrate, and then TLC thin layer chromatography analysis was performed. The results are shown in Figure 10. It can be seen from the figure that the recombinant enzyme AmyB has a hydrolysis effect on soluble starch, dextrin and corn starch (sequence numbers 4, 5, and 6). According to the Rf value of the standard product and the Rf value of the recombinant enzyme hydrolyzing soluble starch, dextrin and corn starch products, it can be seen that the hydrolysis products are maltose and glucose.

The present invention screens a new mesophilic α-amylase gene AmyB from the Bacillus velezensis BL-LM strain isolated from Baijiu Daqu, and the mesophilic α-amylase encoded by the gene has the following advantages: high catalytic activity under mesophilic conditions, good temperature stability and wide pH stability. The above advantages mean that the mesophilic α-amylase AmyB of the present invention has great application potential in industries such as food, fermentation, feed, textile and medicine.

BL-LM, classified as Bacillus velezensis, was deposited in the General Microbiology Center of China Culture Collection Administration on March 25, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a postal code of 100101 and a deposit number of CGMCC No. 30130.

It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims (10)

1. A medium-temperature alpha-amylase AmyB is characterized in that the amino acid sequence is shown in SEQ ID NO. 1.

2. A mesophilic alpha-amylase gene AmyB, characterized in that it encodes a mesophilic alpha-amylase AmyB according to claim 1.

3. The medium temperature alpha-amylase gene AmyB according to claim 2, wherein the nucleotide sequence is shown in SEQ ID No. 2.

4. A recombinant plasmid comprising the mesophilic alpha-amylase gene AmyB according to claim 2.

5. The recombinant plasmid according to claim 4, wherein the recombinant plasmid is obtained by cloning the alpha-amylase gene AmyB according to claim 3 into plasmid pET29a (+).

6. A recombinant strain comprising the mesophilic alpha-amylase gene AmyB according to claim 2.

7. The recombinant strain according to claim 6, wherein E.coli, bacillus, yeast or Lactobacillus is used as host.

8. A method for preparing the medium temperature alpha-amylase according to claim 1, comprising the steps of:

1) Transforming host cells with a recombinant vector containing a medium-temperature alpha-amylase encoding gene to obtain recombinant strains;

2) Culturing the recombinant strain, and inducing the expression of the medium-temperature alpha-amylase;

3) And separating and purifying the expressed medium-temperature alpha-amylase AmyB.

9. Use of the medium-temperature alpha-amylase according to claim 1 as an enzyme preparation for the production of starch by hydrolysis in starch processing, fermentation, food industry or feed.

10. The use of the medium temperature alpha-amylase gene of claim 2 in the genetic engineering of starch processing, fermentation, food industry or feed.