CN115786300B

CN115786300B - Bacillus amyloliquefaciens with low yield and application thereof

Info

Publication number: CN115786300B
Application number: CN202211461449.9A
Authority: CN
Inventors: 刘翔; 周其洋; 周斌; 廖艺楠; 高庭
Original assignee: Tianqu Biotechnology Co ltd; Foshan Haitian Flavoring and Food Co Ltd; Foshan Haitian Gaoming Flavoring and Food Co Ltd
Current assignee: Tianqu Biotechnology Co ltd; Foshan Haitian Flavoring and Food Co Ltd; Foshan Haitian Gaoming Flavoring and Food Co Ltd
Priority date: 2022-11-22
Filing date: 2022-11-22
Publication date: 2023-11-28
Anticipated expiration: 2042-11-22
Also published as: CN115786300A

Abstract

The invention relates to a low-yield bacillus amyloliquefaciens and application thereof. The invention can reduce the spore-producing ability in the liquid fermentation production of neutral protease, thereby greatly reducing the spore quantity in the fermentation liquor and improving the yield and activity of the neutral protease. The phosphoserine phosphatase amino acid sequence mutation and the locus mutation of the encoding gene rsbU of the bacillus amyloliquefaciens are key factors for making the bacillus amyloliquefaciens have low spore production characteristics. The fermentation liquor obtained by the low-spore-yield mutant strain is easy to separate and extract neutral protease in the follow-up process. After the fermentation liquor of the low-spore-yield mutant strain is subjected to the separation process, the recovery rate of the enzyme activity can reach 89%.

Description

Bacillus amyloliquefaciens with low yield and application thereof

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to low-yield bacillus amyloliquefaciens and application thereof.

Background

Bacillus is a common bacterium in nature that can withstand harsh external environments due to its ability to sporulate, such as: high temperature resistance, high salt resistance, radiation resistance, and the like. As such, bacillus is widely distributed and exists in the soil, water, air and animal intestinal tracts. Bacillus amyloliquefaciens is one of bacillus, and most of the research on Bacillus amyloliquefaciens is focused on the enzyme production capacity, especially the protease production capacity.

In the fermentation and enzyme production process of bacillus, especially after the thallus growth reaches a stable period, fermentation conditions change due to consumption of nutrients and accumulation of certain metabolites, and the thallus is easy to form spores, so that the bacillus responds to an environment unsuitable for growth in a form of spore dormancy. Once the spores are formed, the fermentation enzyme production is affected by a plurality of adverse effects, such as increased viscosity and foam of fermentation liquor, and material burst; the formed spores are in a dormant state, and the formation of a large number of spores can seriously influence the yield and the enzyme activity of protease and the acquisition of high-density nutrition thalli, so that the utilization rate of raw materials is reduced; the formation of spores is not only induced by the external growth environment, but also depends on the activation expression or inhibition of genes associated with sporulation in the cells. Because the process of sporulation of vegetative bacteria involves multiple steps, sporulation is a polygenic regulatory process.

It has been reported that phosphoserine phosphatase is a phosphatase capable of indirectly activating SigB (a stress-responsive RNA polymerase sigma factor), and that the activity of the coding gene rsbU can indirectly lead to the activity of SigB. SigB, a major responder to stress physiological signals (e.g., phosphate limitation, salt stress, acid shock, etc.) that may occur during growth or resting phase, can activate the expression of about 150 genes, and activation of SigB can result in a decrease in the spore-forming ability of the bacteria. Therefore, reducing the spore-forming ability of bacteria by activating the phosphoserine phosphatase-encoding gene rsbU becomes a novel means for reducing spore-forming ability of Bacillus.

Disclosure of Invention

The invention aims to obtain a low-yield mutant bacillus amyloliquefaciens (Bacillus amyloliquefaciens) by screening natural mutant strains of a phosphoserine phosphatase encoding gene rsbU, wherein the expression quantity of the phosphoserine phosphatase of the bacillus amyloliquefaciens is high. The strain 2022 is preserved in the microorganism strain collection center of Guangdong province for 7 months and 27 days, and addresses: building 5, building 59, national institute of first, middle road 100, guangzhou, guangdong, post code 510075, accession number GDMCC NO:62651, taxonomic name: bacillus amyloliquefaciens, it is named: bacillus amyloliquefaciens Bacillus amyloliquefaciens ZH806,806.

The inventors have conducted intensive studies to achieve the above object, and have completed obtaining the present invention by repeating the study demonstration a plurality of times, specifically as follows:

in a first aspect, the present invention provides a phosphoserine phosphatase having an amino acid sequence of which at position 173 is changed from methionine to valine. The invention changes the 173 th amino acid sequence of the phosphoserine phosphatase of the bacillus amyloliquefaciens from methionine to valine by a mutagenesis technology. The expression level of the mutated phosphoserine phosphatase in the bacillus amyloliquefaciens is obviously improved, and the SigB factor is activated, so that the spore-forming ability of the bacillus amyloliquefaciens is weakened.

Further, the amino acid sequence of the phosphoserine phosphatase is shown in SEQ ID NO. 3. The amino acid sequence of the phosphoserine phosphatase after sequencing mutation is shown as SEQ ID NO. 3.

In a second aspect, the present invention provides a phosphoserine phosphatase encoding gene rsbU, the 517bp of the cDNA sequence of which is changed from A to G. The gene rsbU is found to have site mutation through gene identification, and mutation from A to G occurs in 517bp of the gene cDNA.

Further, the coding gene rsbU sequence of the phosphoserine phosphatase is shown in SEQ ID NO. 2. The cDNA sequence of the rsbU gene after sequencing mutation is shown as SEQ ID NO. 2.

In a third aspect, the invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) having the accession number GDMCC No. 62651. The strain is subjected to mutagenesis by a normal pressure room temperature plasma mutagenesis technology, so that the spore production capacity of the strain is greatly reduced, and the neutral protease production capacity is remarkably improved.

In some embodiments, the amino acid sequence 173 of the phosphoserine phosphatase of Bacillus amyloliquefaciens is changed from methionine to valine, and the amino acid sequence is shown as SEQ ID NO. 3.

In some embodiments, the gene identification shows that the gene rsbU of the bacillus amyloliquefaciens has site mutation, the 517bp of cDNA of the gene has mutation from A to G, and the mutated rsbU gene sequence is shown as SEQ ID NO. 2.

In a fourth aspect, the invention provides the use of the bacillus amyloliquefaciens in the preparation of neutral protease. Experiments prove that the neutral protease activity of the strain provided by the invention in fermentation liquor of a 10L fermentation tank after mutagenesis is improved by 196.5% compared with that of a wild strain.

In a fifth aspect, the invention provides the phosphoserine phosphatase, a gene encoding the same, and an application of the bacillus amyloliquefaciens strain containing the phosphoserine phosphatase and the gene in reducing spores of bacillus amyloliquefaciens fermentation broth. The invention carries out mutagenesis on bacillus amyloliquefaciens by mutagenesis technology to obtain a mutant strain, and the phosphoserine phosphatase of the strain obviously improves the expression quantity of the phosphoserine phosphatase due to mutation of the coding gene of the phosphoserine phosphatase, thereby indirectly improving the activity of SigB, reducing the spore production capacity of bacillus and greatly reducing the number of spores in fermentation liquor fermented by the strain. Experiments prove that the spore amount of the fermentation liquor of the strain in a 270L fermentation tank after mutagenesis is reduced by 99.89 percent compared with that of a wild strain.

In a sixth aspect, the invention provides a method for reducing the number of spores in a fermentation broth of bacillus amyloliquefaciens, which is a method for liquid fermentation using the bacillus amyloliquefaciens of the invention. Experiments prove that the bacillus amyloliquefaciens is used for producing neutral protease through fermentation, and the spore number in the fermentation liquid can be effectively reduced, so that the yield and the enzyme activity of the neutral protease are improved.

Compared with the prior art, the invention has the following beneficial effects:

1. the spore-producing ability of the bacillus amyloliquefaciens is reduced: the bacillus amyloliquefaciens is obtained by a normal pressure room temperature plasma mutagenesis technology and directional screening. The strain can reduce the spore production capacity in the liquid fermentation production of neutral protease, thereby greatly reducing the spore quantity in fermentation broth and achieving the effect of improving the yield and activity of the neutral protease. For example, fermentation in a 10L fermenter, the spore amount of the fermentation broth is reduced by 99.86% compared with the wild-type strain, and the neutral protease activity of the fermentation broth is improved by 196.5% compared with the wild-type strain; fermenting in 500L fermenter, the spore amount of the fermentation liquid is reduced by 99.89% compared with the wild strain, and the neutral protease activity of the fermentation liquid is improved by 190.2% compared with the wild strain.

2. Mutation of specific sites of the spore-forming regulatory gene: through the analysis and identification of whole genome sequencing, the phosphoserine phosphatase encoding gene rsbU of the bacillus amyloliquefaciens strain disclosed by the invention is subjected to site mutation, the mutation from A to G is generated in 517bp of cDNA of the gene, so that the amino acid sequence of the phosphoserine phosphatase affecting the spore production level is also subjected to mutation, and the 173 th methionine is changed into valine. The phosphoserine phosphatase amino acid sequence mutation and the locus mutation of the encoding gene rsbU of the bacillus amyloliquefaciens are key factors for making the bacillus amyloliquefaciens have low spore production characteristics. It should be noted that any mutation of the amino acid sequence or the coding gene sequence of the phosphoserine phosphatase does not achieve the purpose of reducing Bacillus amyloliquefaciens. The reason is that SigB is the main responder of stress physiological signals occurring in the growth or resting period, and the bacterial spore-producing ability is reduced due to the activation of SigB, and the phosphoserine phosphatase can indirectly activate SigB, so that the bacterial spore-producing ability is indirectly regulated. Thus, the ability of the mutated phosphoserine phosphatase to be recognized by SigB is a prerequisite for the modulation of spore production, if the mutant phosphoserine phosphatase has altered its SigB recognition site or structure, sigB cannot be activated. In addition, the higher the phosphoserine phosphatase expression level, the stronger the signal activating SigB, the more active the SigB signal factor, and the stronger the ability to inhibit bacterial spore production. The mutant phosphoserine phosphatase gene rsbU of the invention not only reserves the SigB recognition site, but also can improve the expression quantity of the enzyme, thereby achieving the effect of enhancing and activating the SigB signal, and further achieving the purpose of reducing the spore production capability of bacteria once the SigB is activated. Therefore, the mutated sequence (amino acid sequence or coding gene sequence) not only has the site recognized by SigB, but also has the effect of improving the expression quantity of phosphoserine phosphatase.

3. Easy separation and extraction of products: the fermentation liquor obtained by the low-spore-yield mutant strain is easy to separate and extract neutral protease in the follow-up process. The feeding pressure is low in the plate-and-frame filter pressing process, the whole feeding pressure is maintained at 0.5MPa, a good separation effect can be realized by only using 5% of perlite filter aid, the turbidity of filter pressing clear liquid is less than 5NTU, and the ultrafiltration concentration flux is slow to attenuate; the filter cloth is easy to be blocked when the wild strain fermentation liquor is subjected to plate-frame filter pressing separation, and the feeding pressure can be increased to 1.0MPa in a short time, so that the separation is difficult to carry out; in order to reduce the feeding pressure, the dosage of the perlite filter aid needs to be increased to 10 percent, the turbidity of the obtained filter pressing clear liquid is more than 20NTU, and the turbid clear liquid is easier to block the membrane during ultrafiltration, so that the production efficiency is influenced.

4. The recovery rate of enzyme activity is high: after the fermentation liquor of the low-spore-yield mutant strain is subjected to the separation process, the recovery rate of enzyme activity can reach 89%; after the fermentation broth of the wild strain is subjected to the separation process, the recovery rate of the enzyme activity is only 78%.

Drawings

FIG. 1 is a morphology of streaked isolated plate colonies of strains of the invention;

FIG. 2 shows a microscopic cell morphology of the strain of the invention.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

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. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

EXAMPLE 1 screening and identification of strains of the order

1. Isolation and purification of strains

Solid medium: 1% peptone, 0.5% yeast extract, 1% sodium chloride, 2.5% agar powder, and water, and sterilizing at 121deg.C for 15min.

5g of the traditional soy sauce soybean starter sample is taken and added into 50g of sterile water, and the mixture is shaken in a shaking table at 180rpm for 1 hour and fully mixed. Sucking 2mL of supernatant onto folded filter paper, and filtering to obtain bacterial liquid. The bacterial liquid is diluted to proper concentration in a gradient way and then is placed in a water bath kettle with the temperature of 80 ℃ for incubation for 30 minutes. And sucking 50ul of bacterial liquid, coating the bacterial liquid on a solid culture medium plate, and culturing for 15 hours at 30 ℃, wherein single bacterial colony on the obtained plate is the pure bacillus strain.

2. Shaking flask fermentation screening of starting strain

Liquid medium: 1% peptone, 0.5% yeast extract, 1% sodium chloride, the other water, and sterilizing at 121deg.C for 15min.

Single colony is selected from a strain separation and purification plate and inoculated into a triangular flask containing 50mL of liquid culture medium, the culture is carried out for 48 hours at 180rpm in a shaking table at 30 ℃, and the supernatant is taken after the fermentation broth is centrifuged, thus obtaining crude enzyme liquid.

Measurement of neutral protease enzyme activity: and properly diluting the crude enzyme solution obtained after centrifugation to ensure that the absorbance is between 0.2 and 0.8 when the enzyme activity is finally measured. The neutral protease activity was determined by the Furling method according to GB/T23527-2009 protease preparation.

And selecting a strain with the highest enzyme activity as an initial strain according to the determined enzyme activity result, and carrying out subsequent mutation breeding.

3. Mutant strains

And (3) carrying out mutagenesis on the strain obtained in the step (2) by adopting an atmospheric pressure room temperature plasma (ARTP) mutagenesis technology.

Preparing equipment: ARTP-IIIS, qingshan biological technology Co., ltd. Without tin source, operates normally according to the instruction of equipment use.

Experimental operation: the bacterial liquid cultured to the logarithmic phase is collected by centrifugation, washed 3 times with physiological saline, and resuspended to an OD600 of about 0.5 with physiological saline. 10. Mu.L of the bacterial suspension was spread evenly on ARTP metal slides in an ultra clean bench, and the slides were then transferred to an ARTP tray. Setting power 120W, gas quantity 10SLM, mutagenesis time 30s on the control panel, clicking "start" to process the sample. After the sample is processed, transferring the metal slide into a centrifuge tube containing 1mL of sterile physiological saline, and fully oscillating and uniformly mixing to form a new bacterial suspension. After the bacterial suspension was diluted appropriately, it was spread on a solid medium.

4. Shaking flask fermentation screening of mutant strains

Single bacterial colonies are respectively picked from a mutant strain separation plate and inoculated into a triangular flask containing 50mL of liquid culture medium, the culture is carried out for 48 hours at 180rpm in a shaking table at 30 ℃, uniform bacterial liquid is taken for spore quantity detection, and supernatant fluid is taken for enzyme activity detection.

The detection of the enzyme activity of the neutral protease is the same as that of the step 2.

The method for detecting the number of spores comprises the following steps: taking 10mL of uniform bacterial liquid, placing in a water bath at 80 ℃ for 30 minutes, then taking out, carrying out 10-time serial dilution by using sterile physiological saline, selecting 2-3 sample homogenates with proper dilutions as inoculation samples according to the estimation of the number of spores, and sucking 100ul of sample homogenates and coating the sample homogenates on a solid culture medium. Culturing for 12-15 h in an incubator at 37 ℃ in an inversion way, selecting a proper flat plate for colony counting, and calculating the number of spores in the original bacterial liquid.

According to the measurement result, the strains with high enzyme activity and small spores are comprehensively selected as target strains.

5. Identification of species

The bacillus strain genome DNA obtained by the screening is extracted according to the specification of a Tiangen biochemical technology (Beijing) limited company bacterial genome DNA extraction kit (DP 302) and is sent to a biological engineering (Shanghai) limited company for 16S rDNA identification. The identification result shows that the homology of the strain and the 16S rDNA sequence of the bacillus amyloliquefaciens is 100 percent (Gene Bank: CP 054415.1), and the measured 16S rDNA sequence is shown as SEQ ID NO. 1.

The strain 2022 is preserved in the microorganism strain collection center of Guangdong province for 7 months and 27 days, and addresses: building 5, building 59, national institute of first, middle road 100, guangzhou, guangdong, post code 510075, accession number GDMCC NO:62651, taxonomic name: bacillus amyloliquefaciens, it is named: bacillus amyloliquefaciens Bacillus amyloliquefaciens ZH806,806. The colony morphology of the strain plate is shown in figure 1, and the microscopic cell morphology is shown in figure 2.

Example 2 mesh Strain characterization

1. Research on spore production condition of shake flask fermentation

(1) Activating target strains: dipping target strain bacterial liquid in glycerol pipe, streaking and inoculating on a solid culture medium plate, and culturing at 37 ℃ for 15h to obtain single colony.

(2) Seed liquid culture: the activated single colonies were picked from the plates and transferred to 50mL Erlenmeyer flasks containing seed medium and incubated in a shaker at 37℃and 200rpm for 14h.

Seed culture medium: 1% peptone, 0.5% yeast extract, 1% sodium chloride, the other water, and sterilizing at 121deg.C for 15min.

(3) Shake flask culture: inoculating the seed solution obtained in the step (2) into a 500mL triangular flask containing 100mL of fermentation medium according to the inoculum size of 1% by volume, and culturing for 48h at 30 ℃ and 200rpm in a shaking table.

Fermentation medium: 1% glucose, 1% yeast extract, 1% corn steep liquor, 0.1% sodium carbonate, the other is water, and sterilized at 121℃for 15min.

(4) Spore detection and enzyme activity determination

The spore amount detection and the neutral protease activity measurement are carried out according to the method of the example 1, and the spore amount of the obtained fermentation broth is 120CFU/mL, which is reduced by 99.78% compared with a wild strain; the neutral protease activity of the fermentation broth is 189U/mL, which is improved by 39.4% compared with the wild strain.

2. Whole genome sequencing analysis

Through whole genome sequencing analysis, the phosphoserine phosphatase encoding gene rsbU of the low-yield bacillus amyloliquefaciens strain disclosed by the invention is subjected to site mutation, the mutation from A to G is generated in 517bp of cDNA of the gene, and the mutated rsbU gene sequence is shown as SEQ ID NO. 2.

The 173 th position of the amino acid sequence of the phosphoserine phosphatase is changed from methionine to valine due to mutation of the gene locus, and the mutated amino acid sequence is shown as SEQ ID NO. 3. The mutation improves the expression quantity of the phosphoserine phosphatase in the low-yield bacillus amyloliquefaciens, and the SigB factor is activated, so that the spore-producing capacity of the strain is greatly weakened, the yield of neutral protease produced by fermenting the strain is greatly improved, and the production cost is reduced.

EXAMPLE 3 purpose Strain 10L fermentor neutral protease production

1. Activating target strains: dipping the bacterial liquid in the glycerol pipe, streaking and inoculating on a solid culture medium plate, and culturing for 15h at 37 ℃ to obtain single bacterial colony.

Solid medium: 1% peptone, 0.5% yeast extract, 1% sodium chloride, 2.5% agar powder, and sterilizing at 121deg.C for 15min.

2. Culturing primary seed liquid: the activated single colonies were picked from the plates and transferred to 50mL Erlenmeyer flasks containing 10mL of seed medium and incubated in a shaker at 37℃and 200rpm for 14h.

Seed culture medium: 1% peptone, 0.5% yeast extract, 1% sodium chloride, and sterilized at 121℃for 15min.

3. Culturing a secondary seed solution: inoculating the primary seed solution obtained in the step 2 into a 250mL triangular flask containing 50mL of seed culture medium according to the volume ratio of 2%, and culturing for 8h at 37 ℃ and 200rpm in a shaking table. The seed culture medium is step 2.

4. Culturing in a fermentation tank: inoculating the secondary seed liquid obtained in the step 3 into a 10L fermentation tank containing 7L fermentation medium according to the volume ratio of 0.5%, wherein the fermentation temperature is 30 ℃, the stirring rotation speed is 180rpm, the aeration rate is 3V/V/min, and the fermentation is performed for 40h.

Fermentation medium: 5% molasses, 2% yeast extract, 1% corn steep liquor dry powder, 0.5% potassium dihydrogen phosphate, 0.5% dipotassium hydrogen phosphate, 0.05% magnesium sulfate and the balance water.

5. Spore detection and enzyme activity determination

The number of spores was measured and the activity of neutral protease was measured in the same manner as in example 1, and the number of spores in the obtained fermentation broth was 2.63X10 ³ CFU/mL, 99.86% less than wild-type strain; the neutral protease activity of the fermentation broth is 1467U/mL, and is improved by 196.5% compared with a wild strain.

EXAMPLE 4 production of neutral protease by 500L Strain of fermentor

1. Activating strains: dipping the bacterial liquid in the glycerol pipe, streaking and inoculating on a solid culture medium plate, and culturing for 15h at 37 ℃ to obtain single bacterial colony.

2. Culturing primary seed liquid: the activated single colonies were picked from the plates and transferred to 500mL Erlenmeyer flasks containing 100mL of seed medium and incubated in a shaker at 37℃and 200rpm for 14h.

3. Culturing a secondary seed solution: inoculating the first-stage seed liquid obtained in the step 2 into a 10L fermentation tank containing 6L seed culture medium according to the inoculation amount of 1.5% by volume, and culturing for 7h at 37 ℃ with stirring rotation speed of 200 rpm.

Seed culture medium: 2% peptone, 0.5% yeast extract, 1% sodium chloride, the other water, and sterilizing at 121deg.C for 30min.

4. Culturing in a fermentation tank: inoculating the secondary seed liquid obtained in the step 3 into a 500L fermentation tank containing 270L fermentation medium according to the volume ratio of 2% of inoculum size. Fermenting for 0-8h, controlling the temperature at 37 ℃, stirring at 250rpm, and controlling the ventilation rate at 30 square/h. After 8 hours of fermentation, the temperature is controlled at 30 ℃, the stirring speed is 200rpm, the aeration rate is 25 square/h, and the fermentation is performed for 32 hours.

Fermentation medium: 10% glucose, 4% yeast extract, 2% corn steep liquor dry powder, 0.5% potassium dihydrogen phosphate, 0.5% dipotassium hydrogen phosphate, 0.1% magnesium sulfate and the balance water.

5. Spore detection and enzyme activity determination

The number of spores was measured and the activity of neutral protease was measured in the same manner as in example 1, and the number of spores in the obtained fermentation broth was 7.14X10 ³ The CFU/mL is reduced by 99.89% compared with the wild strain; the activity of the neutral protease is 2652U/mL, and is improved by 190.2 percent compared with the wild strain.

Example 5 isolation and extraction of neutral protease from fermentation broth

1. The separation and extraction method comprises the following steps: and separating and extracting neutral protease in the fermentation liquor through two steps of plate-and-frame filter pressing and ultrafiltration concentration.

2. Separation and extraction effects:

the fermentation liquor obtained by utilizing the low spore-producing mutant strain is easy to separate, the feeding pressure in the plate-and-frame filter pressing process is low, the whole feeding pressure is maintained at 0.5MPa, a good separation effect can be realized by only using 5% perlite filter aid, the turbidity of filter pressing clear liquid is less than 5NTU, and the ultrafiltration concentration flux is slow to attenuate. The filter cloth is easy to be blocked when the wild strain fermentation liquor is subjected to plate-frame filter pressing separation, and the feeding pressure can be increased to 1.0MPa in a short time, so that the separation is difficult to carry out; in order to reduce the feeding pressure, the dosage of the perlite filter aid needs to be increased to 10 percent, the turbidity of the obtained filter pressing clear liquid is more than 20NTU, and the turbid clear liquid is easier to block the membrane during ultrafiltration, so that the production efficiency is influenced.

3. Enzyme activity recovery effect:

after the fermentation liquor of the low-spore-yield mutant strain is subjected to the separation process, the recovery rate of enzyme activity can reach 89%; after the fermentation broth of the wild strain is subjected to the separation process, the recovery rate of the enzyme activity is only 78%.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. The phosphoserine phosphatase is characterized in that the amino acid sequence of the phosphoserine phosphatase is shown in SEQ ID NO. 3.

2. The coding gene rsbU of the phosphoserine phosphatase is characterized in that the cDNA sequence of the gene rsbU is shown as SEQ ID NO. 2.

3. The coding gene rsbU of claim 2, wherein said gene rsbU edits the amino acid sequence of claim 1.

4. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) characterized in that the Bacillus amyloliquefaciens has a deposit number of GDMCC No. 62651.

5. The use of the bacillus amyloliquefaciens of claim 4 for the preparation of neutral protease.

6. Use of the phosphoserine phosphatase according to claim 1 or the gene rsbU encoding the phosphoserine phosphatase according to claim 2 or the bacillus amyloliquefaciens according to claim 4 for reducing spores in a bacillus amyloliquefaciens fermentation broth.

7. A method for reducing the number of spores in a fermentation broth of Bacillus amyloliquefaciens, comprising the step of fermenting the fermentation broth with the Bacillus amyloliquefaciens of claim 4.