CN114591856B - Bacillus amyloliquefaciens for producing alkaline phosphatase and application thereof - Google Patents
Bacillus amyloliquefaciens for producing alkaline phosphatase and application thereof Download PDFInfo
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- 108020004774 Alkaline Phosphatase Proteins 0.000 title claims abstract description 88
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- 238000000034 method Methods 0.000 claims abstract description 24
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- 244000005700 microbiome Species 0.000 claims abstract description 10
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- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03001—Alkaline phosphatase (3.1.3.1)
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Abstract
The invention discloses bacillus amyloliquefaciens for producing alkaline phosphatase and application thereof, and belongs to the technical field of microorganisms. The purpose of the present invention is to obtain alkaline phosphatase with high yield and high enzyme activity. The invention provides bacillus amyloliquefaciens and a method for preparing alkaline phosphatase with high enzyme activity and high yield by using the bacillus amyloliquefaciens. The strain producing alkaline phosphatase is obtained by separating and purifying from soil, and the bacillus amyloliquefaciens with high purity can be obtained by carrying out plate streaking, seed culture, fermentation culture and centrifugal collection, so that the problems in research and production are solved, the step of separating and purifying enzyme is omitted, the enzyme has good stability and high product specificity, and the method has potential industrial application value.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to bacillus amyloliquefaciens for producing alkaline phosphatase and application thereof.
Background
Alkaline phosphatase (alkaline phosphatase, ALP) is a phosphomonoesterase with low substrate specificity and is a genetically common tool enzyme in molecular biology. The organisms producing alkaline phosphatase mainly include animals, plants and microorganisms, and commercial alkaline phosphatase mainly produces animal (bovine intestinal alkaline phosphatase) or plant (malt) with low yield, high price and poor heat stability, so that the use of alkaline phosphatase from animals and plants is limited.
Disclosure of Invention
The purpose of the present invention is to obtain alkaline phosphatase with high yield and high enzyme activity. The invention provides bacillus amyloliquefaciens (Bacillus amyloliquefaciens), which is deposited in the Guangdong province microorganism strain collection at 2022, 1 month and 17 days, and has the deposit number of GDMCC NO:62218, designated Bacillus amyloliquefaciens S-52.
The invention provides a microbial preparation containing the bacillus amyloliquefaciens.
The invention provides application of the bacillus amyloliquefaciens or the microbial preparation in preparing alkaline phosphatase.
The invention provides a method for preparing alkaline phosphatase, which comprises fermenting the bacillus amyloliquefaciens at 37 ℃ for at least 96 hours.
Further defined, characterized in that the carbon source of the medium used for fermentation is corn starch.
Further defined, the nitrogen source of the medium used for fermentation is a yeast extract.
Further defined, the medium for fermentation contains corn starch, yeast extract and Mg 2+ 。
Further defined as Mg 2+ Is magnesium sulfate.
Further defined, the medium used for fermentation contained 17.48g/L corn starch, 18.052g/L yeast extract and Mg2+0.744g/L.
Further defined, the inoculum size of the bacillus amyloliquefaciens fermentation is 5.59%.
The invention provides application of the method for preparing alkaline phosphatase in improving the enzyme activity of alkaline phosphatase.
The present invention provides an alkaline phosphatase obtained by the above-described method for producing an alkaline phosphatase.
The beneficial effects are that: the invention provides a high-yield alkaline phosphatase positive mutant strain S-52, and the activity of alkaline phosphatase is 5277.95U/L by using an alkaline phosphatase analysis method.
[ biological Material preservation ]
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) deposited at the microorganism strain collection, cantonese province, 1.17.2022 under the accession number GDMCC NO:62218, designated Bacillus amyloliquefaciens S-52, has the following deposit address: building 5, guangzhou city, first, middle road 100, university, 59, university of Guangdong, academy of sciences of China, and microbiological study.
Drawings
FIG. 1 is a phylogenetic tree of S-1 strain and related species based on the 16S rRNA gene;
FIG. 2 is a graph showing the effect of carbon source on S-52 alkaline phosphatase activity;
FIG. 3 is a graph showing the effect of nitrogen source on the enzyme activity of S-52 alkaline phosphatase;
FIG. 4 is a graph showing the effect of metal ions on S-52 alkaline phosphatase activity;
FIG. 5 shows the results of alkaline phosphatase production for both predicted and actual values;
FIG. 6 is a graph of contour and response surface of the interaction of a and b in alkaline phosphatase production, wherein the abscissa is corn starch content, the ordinate is 17.483g/L, and the ordinate is yeast extract; the coordinates are 18.052g/L, the vertical axis is alkaline phosphatase content, and the coordinates are 12110.571U/L;
FIG. 7 is a graph of contour and response surface of the interaction of a and c in alkaline phosphatase production, wherein the abscissa represents corn starch content, the ordinate represents 17.483g/L, and the ordinate represents Mg2+; the coordinates are 0.744g/L, the vertical axis is alkaline phosphatase content, and the coordinates are 12110.571U/L;
FIG. 8 is a contour and response plot of the b and c interaction effects in alkaline phosphatase production, wherein the abscissa is yeast extract, the ordinate is 18.052g/L, and the ordinate is Mg2+; the coordinates are 0.744g/L, the vertical axis is alkaline phosphatase content, and the coordinates are 12110.571U/L;
FIG. 9 is a contour plot and response surface plot of b and d interactions in alkaline phosphatase production, wherein the abscissa is yeast extract, the ordinate is 18.052g/L, and the ordinate is temperature; the coordinate is 37.193 ℃, the vertical axis is alkaline phosphatase, and the coordinate is 12110.571U/L;
FIG. 10 is a graph of contour and response surface of b and e interactions in alkaline phosphatase production, wherein the abscissa is yeast extract, the ordinate is 18.052g/L, and the ordinate is inoculation ratio; the coordinates are 5.593%, the vertical axis is alkaline phosphatase, and the coordinates are 12110.571U/L;
FIG. 11 is a graph of contour and response surface of the c and e interactions in alkaline phosphatase production, wherein Mg2+ is on the abscissa and 0.744g/L is on the ordinate and the inoculation ratio is on the ordinate; the coordinates are 5.593%, the vertical axis is alkaline phosphatase, and the coordinates are 12110.571U/L.
Detailed Description
Alkaline phosphatase assay: the alkaline phosphatase activity was measured by the disodium phosphate microplate method. After 1mg/ml of Phenols (Phenol) was returned to room temperature, 0.1ml of the standard was dissolved in 1.9ml of ddH 2 O, the concentration was brought to 0.05mg/ml. 96. A set of standard curve wells 1-5 were made in the well plate, and 0, 10, 20, 30, 40, 50. Mu.L of standard working fluid and 55, 45, 35, 25, 15, 5. Mu.L of ddH2O were added, respectively. Three sample wells and one control well were provided for each sample to be tested. After adding 55 mu L of corresponding sample to be detected into three sample holes and adding 50 mu L ALP Assay buffer (containing aminoantipyrine) into a standard hole, a sample hole and a control hole, carrying out an incubation in a 37 ℃ incubator for 5min, immediately adding 50 mu L of ALP chromogenic solution (containing disodium phosphate solution) which is incubated in advance at 37 ℃ after taking out, carrying out an incubation in a 37 ℃ incubator for 15min, and adding 150 mu L of chromogenic base solution (containing potassium ferricyanide solution) after taking out to stop the reaction and obtaining red with different depths. And respectively adding 55 mu L of corresponding to-be-detected liquid into parallel holes arranged on each to-be-detected sample, and detecting absorbance at 510 nm.
And drawing a standard curve by taking the Kirschner units (10, 20, 30, 40 and 50U/L) corresponding to the standard numbers 1-5 as the abscissa and the corresponding standard products as the ordinate. And (3) taking the difference value of the measured mean value minus the control as an actual absorbance value, and comparing the difference value with a standard curve to obtain the enzyme activity unit.
Definition of gold activity units 100ml of the sample to be tested was subjected to a reaction for 15min at 37℃without chromogenic substrate (substance contained in ALP chromogenic solution) to yield 1mg of phenol as one gold unit (22), where 1 gold unit = 7.14U/L.
The formula of the LB medium: 10g of tryptone (tryptone); 5g of yeast extract (yesat extract); 10g of NaCl; constant volume to 1L and natural pH.
Example 1 obtaining alkaline phosphatase-producing Bacillus amyloliquefaciens
1. Soil containing nutrients (5 cm-15cm depth) was taken from the university of northeast forestry and the composition of the medium (LB medium) used to culture the bacteria: 10.0g/L tryptone, 5.0g/L yeast extract and 10.0g/L sodium chloride, adding sterile water into the soil to prepare soil leaching solution, sealing the leaching solution, placing the soil leaching solution in a water bath at 100 ℃ for 5min, culturing in a 37 ℃ incubator for 24h, coating the soil leaching solution on an agar plate culture medium by a dilution coating method, culturing for 24h, and streaking single bacterial colony on an LB culture medium by a plate streaking method repeatedly for a plurality of times to ensure purity.
2. Strain identification and phylogenetic analysis
Through subsequent research, a strain is screened. The 16S rRNA gene is synthesized by using universal primers 27F (5-AGAGTTTGATCCTGGCTCAG-3, the sequence of which is shown as SEQ ID NO: 1) and 1492R (5-GGTTACCTTGTTACGACTT-3, the sequence of which is shown as SEQ ID NO: 2) and using a Polymerase Chain Reaction (PCR) method. Sequencing PCR products in Rui Boxing family limited technology company, and the sequence of the strain 16S rRNA is shown as SEQ ID NO. 3;
(AGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGA GTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAAT ACCGGATGCTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACG ATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGA CTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACA AGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCG TAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTG TAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGT CTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGC AGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGG A) the gene sequences were sequenced in the BLAST program of the national center for Biotechnology information. Then, a phylogenetic tree is constructed by using a MEGA 7.0 (21) program neighborhood join method.
Results: the S-1 16S rRNA gene sequences were aligned with NCBI public gene library (http:// www.ncbi.nlm.nih.gov). Phylogenetic tree was constructed using MEGA 7.0 software. As shown in FIG. 1, the 16S rRNA gene sequence was very similar to that of Bacillus amyloliquefaciens. From this result, the strain was considered to be Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), which was named Bacillus amyloliquefaciens strain (Bacillus amyloliquefaciens). Designated as S-1, the alkaline phosphatase activity was 2594.73U/L.
ARTP mutagenesis parameters
The Bacillus amyloliquefaciens strain was inoculated into LB liquid medium (50 mL/250 mL), and cultured at 37℃and 150rpm to logarithmic phase. Absorbing a certain amount of bacterial liquid, centrifuging at 4000rpm for 5min, washing the bacterial cells with sterile water for 2 times, finally re-suspending in a certain amount of sterile water and glycerol, and regulating the bacterial concentration to 106-108/mL and the final glycerol concentration to 5%. Sucking 10 mu L of bacterial suspension to a slide, uniformly coating, setting mutagenesis parameters according to mutagenesis conditions, and starting mutagenesis. Fixed parameters: the power is 120w, the air quantity is 10slm, the treatment distance is 2mm, and the set temperature of the cooling water circulation machine is 20 ℃; the treatment time is as follows: 0s, 10s, 20s, 30s, 40s, 50s, 60s, 80s, 100s, 120s. Placing the mutagenized slide glass into an EP tube (containing 1mL of sterile water), vibrating the EP tube filled with the slide glass for 1min, diluting and coating a flat plate, culturing at 37 ℃, calculating the lethality after bacterial colonies grow out, and drawing a lethality curve, wherein the lethality calculation formula is as follows:
mortality (%) =100
A-the number of colonies grown on plates without mutagenesis treatment;
b-number of colonies grown on the mutagenized plates.
Results: the ARTP mutagenesis result is shown in figure 2, the mortality of the strain is in an ascending trend along with the increase of the mutagenesis time, the experimental result meets the expectations, and when the mutagenesis time is 40S, the mutagenesis time with 85% -95% of the mortality is selected as the optimal mutagenesis time of the strain, so that the bacillus amyloliquefaciens mutant S-52 is obtained.
The 16S rRNA gene sequence is very similar to that of Bacillus amyloliquefaciens. From this result, the strain was considered to be Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), which was named Bacillus amyloliquefaciens strain (Bacillus amyloliquefaciens).
EXAMPLE 2 cultivation of Bacillus amyloliquefaciens mutant strain
The bacillus amyloliquefaciens mutant strain is subjected to continuous 20-generation culture, and is cultured by taking an LB (LB) culture medium as a basic culture medium to obtain a high-yield alkaline phosphatase positive mutant strain S-52, wherein the high-yield alkaline phosphatase positive mutant strain S-52 is preserved, and the activity of alkaline phosphatase is 5277.95U/L by an alkaline phosphatase analysis method.
EXAMPLE 3 alkaline phosphatase production Using Bacillus amyloliquefaciens S-52
1. Culturing Bacillus amyloliquefaciens S-52
After determining the optimal selection of the optimal carbon source, nitrogen source and metal ion, the composition of the alkaline phosphatase-producing medium was optimized by using Design Expert software version 12.0 and the Response Surface Method (RSM), taking into consideration the appropriate conditions (T1) for commercial alkaline phosphatase production. Five major factors that affect the fermentative production of alkaline phosphatase were optimized, respectively, (a) corn starch, (b) yeast extract, (c) magnesium ion, (d) fermentation temperature, and (e) inoculation ratio. The upper limit (+1) and the lower limit (-1) of each factor are shown in Table 1. A 2n factorial Center Combination Design (CCD) was developed using design Expert software (version 12.0) as shown in table 1 for a total of 50 trials of different combinations of selected variables. All experiments were performed in 500 ml Erlenmeyer flasks at 160rpm and 37℃and the initial pH was maintained at 7 for 4 days. The optimal concentration of each variable was obtained by regression analysis and graphical analysis.
TABLE 1 optimization of concentration ranges of parameters for screening using response surface method
| Factors of | Name of the name | Unit (B) | (-1) | (+1) | |
| a | Corn starch | g/ |
10 | 20 | |
| b | Yeast extract | g/ |
10 | 20 | |
| c | Mg 2+ | g/L | 0.5 | 1 | |
| d | Temperature (temperature) | ℃ | 36 | 38 | |
| e | | ％ | 3 | 7 |
S-52 selection of optimal carbon source, nitrogen source and metal ion
As shown in FIG. 2, corn starch, yeast extract, M g2+ The most suitable carbon source, nitrogen source and metal ion of the strain S-52 are respectively. In the selection of the carbon source, corn starch and soluble starchIs a good carbon source for producing alkaline phosphatase, but the corn starch has better effect. In the selection of nitrogen source, small molecular organic substances such as ammonium nitrate and urea have an inhibition effect on enzyme production. In the selection of metal ions, mg 2+ And Ca 2+ Is the activator of alkaline phosphatase produced by the strain S-52, however, K + ，Mn 2+ ，Fe 2+ Inhibiting alkaline phosphatase production by strain S-52.
2. Medium condition optimization
Corn starch, yeast extract, magnesium sulfate, fermentation temperature and inoculation ratio are believed to affect the alkaline phosphatase production by bacillus amyloliquefaciens among various parameters. The influence effect of the influence parameters is optimized by a response surface method. Experiments were designed using a center-combined design (CCD), run 50 times, and the results were included in the response parameter positions according to the design, as shown in Table 2. Experimental observations were made regarding the maximum yield of alkaline phosphatase. To determine the interaction relationship between the variables, a no-transformation model, a quadratic model, and a polynomial model are selected. Model F18.95, p (0.0003) less than 0.05, indicated that the model term was significant. Analysis of variance (ANOVA) was used to arrive at a final regression equation representing the empirical relationship between alkaline phosphatase production and the other five variables.
TABLE 2 design of response surface method and corresponding experimental and predictive results
The relation of the coding factors is shown as the following formula:
Y＝+11856.02+309.67*A+449.78*B++100.53*C+121.69*D++198.04*E+80.83*AB-71.32*AC+34.05*AD- 28.23*AE-72.59*BC+72.63*BD+35.21*BE-3.99*CD-125.63*CE+32.26*DE-376.67*A 2 -421.86*B 2 -405.52*C 2 - 503.15*D 2 -362.48*E 2 wherein Y is the corresponding alkaline phosphatase activity yield (U/L),A. b, C, D, E the components are corn starch, yeast extract and Mg 2+ The fermentation temperature and the inoculation ratio (the ratio of the volume of the seed solution to the culture solution after inoculation, unit ml/ml).
The predicted and actual alkaline phosphatase yields are shown in FIG. 5. The results show that the points are relatively close to linear distribution, and the experimental design of CCD can effectively optimize alkaline phosphatase producing conditions.
Fitting model analysis of alkaline phosphatase activity as shown in table 3, model terms with "p-value" less than 0.05 were significant. The model f value is 307.76, and the p value is less than 0.0001, which shows that the model has portability and can be well fit with the experimental process. The chance of interference producing such a large "f-mode value" is less than 0.01%. The f-number of lack-of-fit was 2.52, the corresponding p-number was 10.57%, and was not significant, indicating the goodness of fit of the model to simulate the relationship between alkaline phosphatase production and conditions. The well-fitted model regressed significantly, with Lack-of-Fit not significantly, reflecting that the majority of the variation was caused by the regression model and the remainder of the variation was mainly caused by the residual.
TABLE 3 analysis of variance of fitting model
As shown in table 3, model term A, B, C, D, E was significant for alkaline phosphatase production in this study. As shown in FIGS. 6-11, corn starch, yeast extract and inoculum size have a significant effect on alkaline phosphatase yield, mg 2+ And temperature, but not corn starch, yeast extract and inoculum size. A is that 2 、B 2 、C 2 、D 2 And E is 2 All are significant, illustrating that the quadratic term for 5 variables has a significant effect on the response. AB. AC, BC, BD, BE and CE interact significantly, indicating that their interaction has a significant impact on the reaction. AD. AE (AE),CD. DE interaction was insignificant, indicating corn starch and temperature, corn starch and inoculation ratio, mg 2+ The interaction with temperature, temperature and inoculation ratio has no significant effect on the reaction. As shown in the equation, the coefficient absolute value of the significant term is larger than that of the non-significant term, which also coincides with the data of the table.
Determining coefficients (Coefficient of determination, R) 2 ) The ratio of the interpreted variance to the total variance is defined as a measure of fitness. R is R 2 value is 99.53%, indicating that the model well defines the true behavior of the system. And R is 2 (adj) (adjustment determination coefficient) is very high (99.21%), R 2 (pre) (predict R 2 The difference between =98.03%) was less than 0.2, demonstrating the significance of the model.
3. Verification of optimal conditions determined by a response surface method: the optimal process conditions determined by the response surface method are verified through experiments. The formula of the starter culture medium is as follows: the corn starch concentration is 17.38g/L, the yeast extract is 18.05g/L, and the Mg 2+ 0.745 g/L. The fermentation temperature was 37.2℃and the fermentation time was 96 hours, with an inoculum size of 5.6%.
4. And (3) enzyme liquid collection: isolation and purification of alkaline phosphatase
(1) Ammonium sulfate fractional precipitation: the extracted crude enzyme solution is subjected to fractional precipitation by adopting an ammonium sulfate method, standing is carried out at 4 ℃ for 2h, centrifugation is carried out at 10000r/min for 20min, precipitation under 60% saturation is collected, active precipitation is dissolved by using a Tris-HCl (pH 7.5) buffer solution with the concentration of 50mmol/L as few as possible, the obtained solution is placed in the same buffer solution at 4 ℃ for dialysis overnight, no white precipitation is detected in the dialyzate by Ba2+, and the obtained solution is frozen and dried and stored at-20 ℃ for standby.
(2) DEAE-52 ion exchange chromatography: dissolving 0.2g of lyophilized enzyme powder in 5mL of Tris-HCl buffer solution, filtering with 0.45 μm filter membrane, loading onto DEAE-52 ion exchange chromatography column (2.6X10 cm) equilibrated with buffer solution, continuously gradient eluting with Tris-HCl buffer solution containing 0.1mol/L NaCl at flow rate of 0.5mL/min, recovering 5mL of eluate, measuring enzyme activity and protein content of each tube, combining tube number with high enzyme activity, dialyzing at 4deg.C for desalting, freeze drying, and storing at-20deg.C for use.
(3) Sephadex G-200 gel filtration chromatography: according to the method in 2.3.2, the sample was applied to a Sephadex G-200 gel filtration chromatography column (1.6X10 cm), the column was eluted with Tris-HCl buffer containing 0.1mol/L NaCl at a flow rate of 0.2mL/min, 3mL per tube was collected, the number of tubes with high enzyme activity was combined, and after dialysis for desalting and freeze-drying, it was stored at-20℃for use.
(4) Enzyme purity identification and relative molecular weight determination: purity identification is carried out on the purified enzyme by SDS-PAGE electrophoresis, a standard curve is obtained by plotting the mobility of standard proteins with known molecular weights against the logarithm of the molecular weights, and the molecular weights of the purified products are obtained on the standard curve according to the relative mobility of the purified products by electrophoresis under the same conditions.
5. Enzyme activity detection:
(1) And (3) collecting the extracted alkaline phosphatase crude enzyme liquid for enzyme activity detection, wherein the enzyme activity of the alkaline phosphatase is 12110.6U/L, and the yield of the alkaline phosphatase obtained by experiments is 12076.4 +/-105.3U/L.
(2) The fermentation broth of the bacillus amyloliquefaciens is used for detecting the enzyme activity, the enzyme activity of alkaline phosphatase is 12110.6U/L, and the yield of the alkaline phosphatase obtained by experiments is 12076.4 +/-105.3U/L.
Example 4 methods for increasing alkaline phosphatase production Using Bacillus amyloliquefaciens S-52
1. Preparing a fermentation medium: the corn starch concentration is 17.38g/L, the yeast extract is 18.05g/L, and the Mg 2+ 0.745 g/L.
2. And (3) fermenting in the fermentation medium obtained in the step (1) by using bacillus amyloliquefaciens S-52 at the temperature of 37.2 ℃ for 96 hours, wherein the inoculation amount is 5.6%.
3. (1) And (3) collecting the extracted crude enzyme liquid for enzyme activity detection, wherein the enzyme activity of alkaline phosphatase is 12110.6U/L, and the yield of the alkaline phosphatase obtained by experiments is 12076.4 +/-105.3U/L.
(2) The fermentation broth of the bacillus amyloliquefaciens is used for detecting the enzyme activity, the enzyme activity of alkaline phosphatase is 12110.6U/L, and the yield of the alkaline phosphatase obtained by experiments is 12076.4 +/-105.3U/L.
Example 5 use of Bacillus amyloliquefaciens S-52 to produce alkaline phosphatase
The application of bacillus amyloliquefaciens S-52 to producing alkaline phosphatase,
alkaline phosphatase (alkaline phosphatase, ALP) is a phosphomonoesterase with low substrate specificity and is a genetically common tool enzyme in molecular biology. The organisms producing alkaline phosphatase mainly include animals, plants and microorganisms, and commercial alkaline phosphatase mainly produces animal (bovine intestinal alkaline phosphatase) or plant (malt) with low yield, high price and poor heat stability, so that the use of alkaline phosphatase from animals and plants is limited. Microbial cells are receiving increasing attention from microorganisms for commercial production of alkaline phosphatase by isolating and screening enzyme-producing strains from the microorganisms due to their fast growth, short cycle and diversity of populations.
The research adopts the normal pressure room temperature plasma mutagenesis technology and the center combination design to maximize the alkaline phosphatase yield on the basis of screening out an alkaline phosphatase-producing strain. The bacillus amyloliquefaciens S-52 is obtained by screening and mutagenizing from soil, so that the strain can not generate toxic byproducts in the future agricultural production, and meanwhile, the bacillus amyloliquefaciens has certain effects of improving the plant immunity and secreting antibacterial peptides. Alkaline phosphatase is an enzyme that removes phosphate groups from substrates and is effective in removing phosphate groups from various molecules including nucleotides, proteins and alkaloids, converting phosphorus into soluble compounds for uptake by plants. In soils with low phosphate content, alkaline phosphatase can effectively hydrolyze the phosphate groups of microorganisms to make them available phosphate ions.
SEQUENCE LISTING
<110> northeast agricultural university
<120> Bacillus amyloliquefaciens for producing alkaline phosphatase and application thereof
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Synthesis
<400> 1
<210> 2
<211> 19
<212> DNA
<213> Synthesis
<400> 2
ggttaccttg ttacgactt 19
<210> 3
<211> 867
<212> DNA
<213> Bacillus amyloliquefaciens
<400> 3
agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg tgagtaacac 60
gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa taccggatgc 120
ttgtttgaac cgcatggttc agacataaaa ggtggcttcg gctaccactt acagatggac 180
ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcgacgat gcgtagccga 240
cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 300
gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 360
aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtgccgtt caaatagggc 420
ggcaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta 480
atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc 540
ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa ctggggaact 600
tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta gagatgtgga 660
ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga gcgaaagcgt 720
ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg 780
ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg cctggggagt 840
acggtcgcaa gactgaaact caaagga 867
Claims (8)
1. Bacillus amyloliquefaciens @Bacillus amyloliquefaciens) The bacillus amyloliquefaciens is characterized in that the bacillus amyloliquefaciens is deposited in the microorganism strain collection of Guangdong province at 2022, 1 month and 17 days, and the deposit number is GDMCCNO:62218, designated Bacillus amyloliquefaciens S-52.
2. A microbial preparation comprising the Bacillus amyloliquefaciens of claim 1.
3. Use of the bacillus amyloliquefaciens of claim 1 or the microbial preparation of claim 2 for the preparation of alkaline phosphatase.
4. A process for producing alkaline phosphatase, characterized in that Bacillus amyloliquefaciens according to claim 1 is fermented at 37℃for at least 96 hours.
5. The method according to claim 4, wherein the carbon source of the medium for fermentation is corn starch and the nitrogen source of the medium for fermentation is yeast extract.
6. The method according to claim 4, wherein the medium for fermentation comprises corn starch, yeast extract and Mg 2+ 。
7. The method according to claim 6, wherein the medium for fermentation contains 17.48g/L of corn starch, 18.052g/L of yeast extract and Mg 2+ 0.744 g/L。
8. The method of claim 4, wherein the bacillus amyloliquefaciens is fermented at an inoculum size of 5.59% by volume.
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