CN117305279B - Alpha-amylase mutant with high activity and high heat resistance as well as preparation method and application thereof - Google Patents
Alpha-amylase mutant with high activity and high heat resistance as well as preparation method and application thereof Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
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- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
The invention discloses an alpha-amylase mutant with high activity and high heat resistance, which is derived from pyrococcus hyperthermophiles Pyrococcus yayanosii, wherein the amino acid sequence of the alpha-amylase mutant is shown as SEQ ID NO.3, and the DNA sequence of the alpha-amylase mutant is shown as SEQ ID NO. 4. Has the advantages of high activity, good stability and the like, the activity of the mutant is 1.6 times of that of the wild type, and the stability at 110 ℃ is 2.4 times of that of the wild type. The specific activity of the alpha-amylase mutant is about 764U/mg and the yield is 1751U/ml after the alpha-amylase mutant is expressed in bacillus subtilis W800N. Is suitable for the application in the fields of food processing and the like.
Description
Technical Field
The invention relates to an alpha-amylase mutant with high activity and high heat resistance, and a preparation method and application thereof, and belongs to the technical field of protein expression.
Background
Alpha-amylase (alpha-amylase) is an enzyme capable of hydrolyzing alpha-1, 4-glycosidic bonds in starch molecules, which is widely distributed in organisms and is found in microorganisms, plants and animal tissues. The alpha-amylase has high application value in industry, is widely applied to hydrolysis reaction in the fields of food, feed, medicine and the like, and is one of research hotspots in the field of biological medicine. Alpha-amylases can be divided into four classes depending on the source: plant alpha-amylase, animal alpha-amylase, bacterial alpha-amylase and fungal alpha-amylase. The different sources of alpha-amylase have a certain difference in nature and structure, but all have the same action mechanism, namely, starch is decomposed by hydrolyzing alpha-1, 4-glycosidic bonds in starch molecules. Plant alpha-amylase is mainly present in seeds, tubers and fruits of plants, has higher affinity with substrate and wider substrate specificity. Animal alpha-amylase is mainly present in saliva, pancreas and digestive tract of animals, and has low affinity with substrate, but high catalytic efficiency. Bacterial alpha-amylases and fungal alpha-amylases have a variety of sources, with bacterial alpha-amylases having a broader substrate specificity and fungal alpha-amylases having a narrower substrate specificity.
The alpha-amylase has wide application prospect in the fields of food, feed, medicine and the like. In the food field, the alpha-amylase can be used for preparing sugar products such as syrup, glucose and the like, and also can be used for preparing novel sugar products such as oligosaccharide, functional sugar and the like. In the feed field, alpha-amylase can be used to increase feed utilization and animal growth rate. In the medical field, alpha-amylase can be used to treat diabetes and other sugar metabolic diseases. In particular, alpha-amylase can hydrolyze starch to form dextrin, oligosaccharide and the like in food processing, so that the mouthfeel and the nutritional value of the food are improved. In the feed industry, alpha-amylase can improve the digestibility of feed and promote animal growth. In the medical field, alpha-amylase can be used for preparing drug carriers and prodrugs, and can be used as a therapeutic agent and a diagnostic agent for treating and diagnosing diseases.
Although α -amylase has a broad application prospect in various fields, there are some problems that need to be further studied and solved. First, there are differences in the nature and function of α -amylase from different sources, so that further knowledge of their mechanism of action and structure-activity relationship is needed. Secondly, the industrial production of alpha-amylase is mainly dependent on a microbial fermentation method at present, but the problems of long enzyme production period, low enzyme production efficiency and the like exist in the fermentation process, so that new production methods and improvement of the enzyme production efficiency are required to be researched. In addition, care should be taken in adjusting parameters such as temperature, pH, etc. during the use of the alpha-amylase to avoid adverse effects on the substrate and the product. Although the alpha-amylase has wide application prospect, intensive researches are still needed in the aspects of knowing the action mechanism, improving the enzyme production efficiency, optimizing the use condition and the like. With the advancement of technology and the intensive research, it is believed that alpha-amylase will be more widely used and popularized in the future.
Disclosure of Invention
The invention discloses an alpha-amylase PyaAMY with high activity and high heat resistance, which is derived from pyrococcus hyperthermophilus Pyrococcus yayanosii, wherein the amino acid sequence of the alpha-amylase is shown as SEQ ID NO. 1, and the DNA sequence is shown as SEQ ID NO. 2. The alpha-amylase PyaAMY is modified to obtain alpha-amylase mutant, the amino acid sequence is shown as SEQ ID NO.3, and the DNA sequence is shown as SEQ ID NO. 4. Mutation systems the pyamay sequences include, but are not limited to, G3R, N4G, K5V, T6D, I7P, A8S, T9R, F10K, A11K, A12K, A13K, A15K, A15K, A16K, A17K, A18K, A18K, A19K, A20K, A21K, A23K, A24K, A25K, A26K, A27K, A29K, A30K, A31K, A32K, A33K, A34K, A35K, A36K, A38K, A39K, A40K, A41K, A42K, A43K, A44K, A45K, A46K, A47K, A165K, A a.
The invention also discloses an expression vector of the alpha-amylase mutant.
The carrier is pHT01.
In the expression vector, the N-terminal of the alpha-amylase mutant is connected with a pelB signal peptide and a histidine tag.
The invention also discloses an expression host bacterium of the alpha-amylase mutant.
The host bacteria are bacillus subtilis W800N.
The invention also discloses a preparation method of the alpha-amylase mutant, which is characterized by comprising the following steps:
(1) Adding pelB signal peptide and histidine tag at the N end of the alpha-amylase mutant, constructing the encoding gene of the pelB signal peptide and histidine tag to a pHT01 vector in a seamless cloning mode, and obtaining an expression vector pHT 01-pelB-of the alpha-amylase mutant after transformation;
(2) Transforming an expression vector pHT 01-pelB-modified PyaAMY into bacillus subtilis W800N, and screening out positive monoclonal;
(3) Culturing, inducing and expressing the screened monoclonal, and purifying to obtain the alpha-amylase mutant.
The alpha-amylase mutant has higher alpha-amylase activity compared with wild type alpha-amylase PyaAMY and can be used for hydrolyzing starch molecules.
The invention provides an alpha-amylase mutant with high activity and high heat resistance, which is derived from pyrococcus hyperthermophiles Pyrococcus yayanosii, wherein the amino acid sequence of the alpha-amylase mutant is shown as SEQ ID NO.3, and the DNA sequence of the alpha-amylase mutant is shown as SEQ ID NO. 4. Has the advantages of high activity, good stability and the like, the activity of the mutant is 1.6 times of that of the wild type, and the stability at 110 ℃ is 2.4 times of that of the wild type. The specific activity of the alpha-amylase mutant is about 764U/mg and the yield is 1751U/ml after the alpha-amylase mutant is expressed in bacillus subtilis W800N. Is suitable for the application in the fields of food processing and the like.
Drawings
FIG. 1 results of structural clustering analysis of thermophilic bacteria-derived alpha-amylase.
FIG. 2 shows a schematic representation of pTH-pelB-PyaAMY expression vector.
FIG. 3SDS-PAGE shows the amount of PyaAMY expressed in the supernatant of the fermentation broth.
FIG. 4 determination of alpha-amylase PyaAMY activity in the supernatant of the fermentation broth.
FIG. 5PyaAMY purification results.
FIG. 6 enzyme activity assay of PyaAMY.
FIG. 7PyaAMY optimum reaction pH measurement.
FIG. 8 optimum temperature measurement of PyaAMY.
FIG. 9 half-life assay of PyaAMY at 110 ℃.
Figure 10 pyamay RMS stability score.
Figure 11 RMS stability score for pyamay after modification.
FIG. 12 schematic representation of pTH-pelB-engineered PyaAMY expression vector.
FIG. 13SDS-PAGE shows comparison of the expression level of PyaAMY after transformation in the supernatant of the fermentation broth.
FIG. 14 comparison of activity of alpha-amylase in supernatant of fermentation broth after modification with PyaAMY before modification.
FIG. 15 results of purification of PyaAMY after modification.
FIG. 16 comparison of PyaAMY enzyme activity assays after modification with those before modification.
FIG. 17 comparison of the optimal reaction pH after modification to PyaAMY before modification.
FIG. 18 comparison of optimum temperature after modification to PyaAMY before modification.
FIG. 19 comparison of half-life measurements in 110℃for PyaAMY after modification and before modification.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention.
The materials or instruments used in the following examples, if not specifically described, were available from conventional commercial sources.
Example 1 search for novel alpha-amylases with high activity and high thermostability.
In this example, we have searched and analyzed highly active and thermostable α -amylase using bioinformatics technology. The specific flow is as follows:
by means of structure clustering, proteins containing amylase domains were searched in an extreme microbial database, extremeDB (http:// extrem. Igib. Res. In), to find 72 thermostable species of alpha-amylase (see FIG. 1). Wherein, the alpha-amylase PyaAMY (the amino acid sequence of which is shown in SEQ ID NO:1 and the DNA sequence of which is shown in SEQ ID NO: 2) derived from the enterococcus hyperthermophilus Pyrococcus yayanosii has not been reported before, and is an ideal target for screening novel alpha-amylase.
Example 2 expression of pyamay.
In order to detect the performance of PyaAMY, bacillus subtilis is used for expressing PyaAMY, and in order to ensure that alpha-amylase PyaAMY can be secreted outside cells, pelB signal peptide and histidine tag are added to the N end of protein of PyaAMY (the amino acid sequence of pelB signal peptide is shown as SEQ ID NO:5, the DNA sequence is shown as SEQ ID NO:6, the amino acid sequence of histidine tag is shown as SEQ ID NO:7, and the DNA sequence is shown as SEQ ID NO: 8). The fused genes were synthesized in Guangzhou Ai Ji organism and cloned seamlessly from Guangzhou Ai Ji organism into the common expression vector pHT01 of Bacillus subtilis (purchased from biological wind). The plasmid map is shown in FIG. 2.
We transformed the synthesized plasmid into a competent Bacillus subtilis Bacillus subtilis WB N (purchased from Horgene), spread on TB solid medium containing 50mg/L chloramphenicol, and incubated overnight at 37 ℃. The monoclonal is selected and cultured in TB solid culture medium containing 50mg/L chloramphenicol at 37 ℃ under shaking at 200rpm until OD value reaches 0.8-1.0. IPTG was added at a final concentration of 1mM, and the culture was performed at 37℃for 12 hours with shaking at 200 rpm. The supernatant was checked for protein expression using SDS-PAGE and the enzyme activity was determined using an alpha-amylase activity detection kit (available from Soy pal) at 90℃above the optimal growth temperature for Pyrococcus thermophilus.
SDS-PAGE results are shown in FIG. 3, in which pyamay containing pelB was significantly expressed in the culture supernatant, whereas pyamay containing no pelB was not significantly expressed in the culture supernatant.
The results of the alpha-amylase activity measurement are shown in FIG. 4, and the concentration of alpha-amylase in the culture supernatant of pyamay containing pelB was 1206U/ml, and the concentration of alpha-amylase in the culture supernatant of pyamay containing no pelB was 176U/ml. The concentration of alpha-amylase in the culture supernatant was 6.8 times higher for pyamay containing pelB than for pyamay without pelB.
Example 3 purification of pyamay.
To examine the performance and stability of pyamay, we performed expression purification of pyamay expressed in bacillus subtilis supernatant. 100ml of the culture supernatant was ultrafiltered to a volume of 10ml by a 10kD filter, 10ml PBS (pH 7.5) was added, ultrafiltered to a volume of 10ml, and 10ml PBS (pH 7.5) was added. Until no apparent medium color was present.
The treated culture supernatant was loaded into a Ni-NTA 6FF His tag protein purification kit (purchased from a biological organism) and subjected to protein purification and desalting according to the instructions thereof. Purified proteins were subjected to SDS-PAGE. See fig. 5.
Example 4 enzymatic property determination of pyamay.
To examine the performance and stability of pyamay, we performed enzymatic property examination of purified pyamay in bacillus subtilis supernatant.
Enzyme specific activity assay: we used BCA protein concentration assay kit (from Bio Inc.) to determine the protein concentration of purified PyaAMY and then used alpha-amylase activity assay kit (from Soy Bao) to determine the activity of purified PyaAMY at different concentrations at 90 ℃.
The results are shown in FIG. 6, and the enzyme specific activity of PyaAMY is 487U/mg.
Determination of optimum pH: at various reaction pH's, we measured the activity of the purified enzyme (10 ug) at 90℃using an alpha-amylase activity assay kit (available from Soy).
As a result, as shown in FIG. 7, pyaAMY had an optimum pH of 6.5 and a high enzyme activity in the pH range of 4.5 to 8.5.
Determination of optimum temperature: at various temperatures, we measured the activity of the purified enzyme (10 ug) using the alpha-amylase activity assay kit (from soribao).
As a result, as shown in FIG. 8, pyaAMY has an optimum reaction temperature of 80℃and a high enzyme activity in the temperature range of 70-100 ℃.
Half-life determination: we measured the enzyme activity (10 ug) after treatment using an alpha-amylase activity assay kit (available from solebao) at 110 ℃ for various times.
The results are shown in FIG. 9, where PyaAMY has a half-life of about 20 minutes at 110 ℃.
Example 5 pyamay stability analysis and modification.
To further improve the stability of pyamay, we performed a Root Mean Square (RMS) analysis of the amino acid sequence of pyamay at 110 ℃. As a result, see FIG. 10, we found that the N-terminal RMS score of the PyaAMY protein was higher, affecting the stability of PyaAMY. Then we use foldx to predict and modify the stability of N-terminal and middle region of PyaAMY, the amino acid sequence of modified PyaAMY is shown as SEQ ID NO 3, and the DNA sequence is shown as SEQ ID NO 4. We performed RMS analysis of the amino acid sequence of the modified pyamay at 110 ℃. The results are shown in FIG. 11, where the modified pyamay has significantly lower RMS score than the wild type pyamay, with better stability, both at the N-terminus and on the whole protein sequence.
Example 6 expression of pyamay after engineering.
In order to detect the performance of the modified PyaAMY, bacillus subtilis is used for expressing the modified PyaAMY, and in order to ensure that the alpha-amylase PyaAMY can be secreted out of cells, the modified PyaAMY protein N end is added with
The pelB signal peptide and the histidine tag (the amino acid sequence of the pelB signal peptide is shown in SEQ ID NO:5, the DNA sequence is shown in SEQ ID NO:6, the amino acid sequence of the histidine tag is shown in SEQ ID NO:7, and the DNA sequence is shown in SEQ ID NO: 8). The fused genes were synthesized in Guangzhou Ai Ji organism and cloned seamlessly from Guangzhou Ai Ji organism into the common expression vector pHT01 of Bacillus subtilis (purchased from biological wind). The plasmid map is shown in FIG. 12.
We transformed the synthesized plasmid into a competent Bacillus subtilis Bacillus subtilis WB N (purchased from Horgene), spread on TB solid medium containing 50mg/L chloramphenicol, and incubated overnight at 37 ℃. The monoclonal is selected and cultured in TB solid culture medium containing 50mg/L chloramphenicol at 37 ℃ under shaking at 200rpm until OD value reaches 0.8-1.0. IPTG was added at a final concentration of 1mM, and the culture was performed at 37℃for 12 hours with shaking at 200 rpm. The supernatant was checked for protein expression using SDS-PAGE and the enzyme activity was determined using an alpha-amylase activity detection kit (available from Soy pal) at 90℃above the optimal growth temperature for Pyrococcus thermophilus.
SDS-PAGE results are shown in FIG. 13, and the expression level of PyaAMY in the culture medium supernatant after transformation is basically equivalent to that before transformation.
The results of the alpha-amylase activity assay are shown in FIG. 14, and the alpha-amylase concentration in the culture supernatant before the modification PyaAMY was 1206U/ml. The concentration of alpha-amylase in the culture medium supernatant after transformation was 1751U/ml, which was 1.46 times that before transformation.
Example 7 purification of modified pyamay.
To examine the performance and stability of the modified pyamay, we performed expression purification of the modified pyamay expressed in bacillus subtilis supernatant. 100ml of the culture supernatant was ultrafiltered to a volume of 10ml by a 10kD filter, 10ml PBS (pH 7.5) was added, ultrafiltered to a volume of 10ml, and 10ml PBS (pH 7.5) was added. Until no apparent medium color was present.
The treated culture supernatant was loaded into a Ni-NTA 6FF His tag protein purification kit (purchased from a biological organism) and subjected to protein purification and desalting according to the instructions thereof. Purified proteins were subjected to SDS-PAGE. See fig. 15.
Example 8 comparison of enzymatic Properties of PyaAMY before and after modification.
To compare the performance and stability of pyamay before and after modification, we performed enzymatic property tests on purified pyamay in bacillus subtilis supernatant.
Enzyme specific activity assay: we used BCA protein concentration assay kits (from Bio) to determine protein concentration of PyaAMY before and after modification, and used alpha-amylase activity assay kits (from Soy) to determine activity of purified PyaAMY before and after modification at 90 ℃.
The results are shown in FIG. 16, and the enzyme specific activity of PyaAMY before transformation is 487U/mg. The enzyme specific activity of the modified PyaAMY is 764U/mg, which is 1.58 times that of the modified PyaAMY.
Determination of optimum pH: at various reaction pH's, we measured the activity of the purified enzyme (10 ug) at 90℃using an alpha-amylase activity assay kit (available from Soy).
The results are shown in FIG. 17, where the response to pH was relatively similar before and after modification of PyaAMY.
Determination of optimum temperature: at various temperatures, we measured the activity of the purified enzyme (10 ug) using the alpha-amylase activity assay kit (from soribao).
As a result, as shown in FIG. 18, the optimum reaction temperature of PyaAMY after modification was 100℃and slightly higher than that before modification, and the activity was reduced to a smaller extent at 110 ℃.
Half-life determination: we measured the enzyme activity (10 ug) after treatment using an alpha-amylase activity assay kit (available from solebao) at 110 ℃ for various times.
The results are shown in FIG. 19, where the half-life of PyaAMY after modification is about 49min at 110deg.C, which is more than 2 times that of PyaAMY before modification.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. An alpha-amylase mutant with high activity and high heat resistance is characterized in that the amino acid sequence of the alpha-amylase mutant is shown as SEQ ID No. 3.
2. The α -amylase mutant encoding gene of claim 1.
3. The coding gene according to claim 2, wherein the nucleotide sequence is shown in SEQ ID No. 4.
4. An expression vector for the alpha-amylase mutant of claim 1.
5. The expression vector of claim 4, wherein the vector is pHT01.
6. The expression vector of claim 5, wherein the α -amylase mutant has a pelB signal peptide and a histidine tag attached to the N-terminus.
7. The expression host bacterium of the alpha-amylase mutant of claim 1.
8. The expression host bacterium according to claim 7, wherein the host bacterium is Bacillus subtilis W800N.
9. The method for producing an alpha-amylase mutant according to claim 1, comprising the steps of:
(1) Adding pelB signal peptide and histidine tag at the N end of the alpha-amylase mutant as claimed in claim 1, constructing the encoding gene of the pelB signal peptide and histidine tag into a pHT01 vector in a seamless cloning mode, and obtaining an expression vector pHT 01-pelB-modified pyaary of the alpha-amylase mutant;
(2) Transforming an expression vector pHT 01-pelB-modified PyaAMY into bacillus subtilis W800N, and screening out positive monoclonal;
(3) Culturing, inducing and expressing the screened monoclonal, and purifying to obtain the alpha-amylase mutant.
10. Use of the alpha-amylase mutant according to claim 1 for hydrolyzing starch molecules.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IE902369L (en) * | 1989-06-29 | 1990-12-29 | Gist Brocades Nv | Mutant microbial alpha-amylases |
| CN113583996A (en) * | 2021-09-29 | 2021-11-02 | 翌圣生物科技(上海)股份有限公司 | Bst DNA polymerase recombinant mutant, coding DNA thereof and ultra-fast magnetic bead LAMP detection method |
| WO2022060775A1 (en) * | 2020-09-15 | 2022-03-24 | Board Of Regents, The University Of Texas System | Recombinant proteins with increased solubility and stability |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IE902369L (en) * | 1989-06-29 | 1990-12-29 | Gist Brocades Nv | Mutant microbial alpha-amylases |
| WO2022060775A1 (en) * | 2020-09-15 | 2022-03-24 | Board Of Regents, The University Of Texas System | Recombinant proteins with increased solubility and stability |
| CN113583996A (en) * | 2021-09-29 | 2021-11-02 | 翌圣生物科技(上海)股份有限公司 | Bst DNA polymerase recombinant mutant, coding DNA thereof and ultra-fast magnetic bead LAMP detection method |
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