CN112300953A - Bacillus subtilis and application thereof in fermentation production of adenosine deaminase - Google Patents

Bacillus subtilis and application thereof in fermentation production of adenosine deaminase Download PDF

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CN112300953A
CN112300953A CN201910686571.8A CN201910686571A CN112300953A CN 112300953 A CN112300953 A CN 112300953A CN 201910686571 A CN201910686571 A CN 201910686571A CN 112300953 A CN112300953 A CN 112300953A
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李汉文
俞学锋
李知洪
喻晨
胡悦
吴尧
姚鹃
余华顺
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Angel Enzyme Preparation Yichang Co ltd
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    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04017—Adenosine-phosphate deaminase (3.5.4.17)

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Abstract

The invention relates to the field of microorganism screening and fermentation, in particular to bacillus subtilis and application thereof in fermentation production of adenosine deaminase. The invention provides a bacterial strain for producing adenylate deaminase, which is a Bacillus subtilis ESP710 and is preserved in China center for type culture collection with the preservation number of CCTCC No. M2018827. The invention provides a method for producing adenylate deaminase, which uses the strain to carry out liquid fermentation production to obtain the adenylate deaminase. The obtained adenylate deaminase has the fermentation enzyme activity far higher than that of the fermentation enzyme activity reported at present, the fermentation enzyme activity is 14000-.

Description

Bacillus subtilis and application thereof in fermentation production of adenosine deaminase
Technical Field
The invention relates to the field of microorganism screening and fermentation, in particular to bacillus subtilis and application thereof in fermentation production of adenosine deaminase.
Background
The yeast extract with high I + G content (5 '-sodium inosinate and 5' -sodium ornithinate) is the yeast extract which strengthens the content of flavour development nucleotide substances in the product on the basis of the original yeast extract, can be used as a natural and nutritional high-end seasoning to replace monosodium glutamate, has the functions of increasing freshness, improving taste, reducing salt and the like, and accords with the dietary consumption habits and nutritional requirement trends of modern people. The content of I + G in yeast extract directly affects the flavor quality and production cost of the product. In domestic research, the content of I + G in yeast extract is about 10%, and foreign patents report that the content of I + G reaches 20%. By increasing the heat extraction time and pH improvement of high-protein and high-nucleic acid yeast, yeast RNA in an extract solution is increased to more than 25% from the original 13%, and then an I + G product with the concentration of more than 18% can be directly produced by the improvement of an enzymolysis process, and then the product is subjected to ultrafiltration, so that various performances of the product are obviously improved, various indexes of the product reach the quality level of European and American high-end products, and the method belongs to domestic initiative.
In the production of the high I + G yeast extract, first, single-stranded DNA and RNA are hydrolyzed with 5 '-nucleotide phosphodiesterase to produce Adenosine (AMP) and guanylic acid (GMP), and then adenosine is converted into inosinic acid (IMP) by the action of 5' -adenosine deaminase. In the preparation process of the high I + G yeast extract, 5 '-adenylate deaminase plays a crucial role, and the enzyme activity and the action performance of the 5' -adenylate deaminase directly influence the generation of inosinic acid and the quality of the high I + G yeast extract. Therefore, obtaining the 5' -adenylate deaminase with high catalytic activity and high performance is one of the key technologies for solving the production of the yeast extract with high I + G content, and has important research significance and market value.
5' -adenylic deaminase was first prepared from skeletal muscle of mouse and red blood cells of human, and most of enzymes used in modern industrial production were prepared by fermentation with microorganisms such as yeast, penicillium, aspergillus, and mucor, among which the most used species were aspergillus oryzae and aspergillus melleus, and recently, there have been patent reports on the production of streptomyces genetically engineered in japan. The solid fermentation production method is usually used in the early stage of deaminase production, the method has the defects that pigments, proteins, sugar, inorganic salts and the like in solid raw materials are mixed in enzyme liquid to cause difficulty in extraction, purification and use of the enzyme, the enzyme activity of the solid enzyme is often very low, and then the liquid fermentation production method is adopted, and the method can effectively overcome some defects of the solid fermentation, but the activity and the stability of the enzyme are still not solved. At present, only Japan wild enzyme company has mature technology and products internationally, and domestic Guangxi academy of sciences have products on trial sale, but the stability and application effect of the products are still to be further verified.
In recent years, few researches on 5' -adenylate deaminase are reported, and the research is mainly focused on the aspects of strain breeding and fermentation condition optimization. The Liuchangjun and the like are optimized by a strain screening and fermentation process, and fresh starter with the expression quantity of 5' -adenylate deaminase of 1543.48u/g is obtained by an aspergillus oryzae solid state fermentation mode; an adenylic deaminase aspergillus melleus producing strain is screened from multiple strains of penicillium and aspergillus by general citizens and the like, and the deaminase expression quantity of an original strain is improved by 16 times after ultraviolet ray and composite mutation treatment and fermentation process optimization, so that the deaminase expression quantity of the finally obtained deaminase reaches 1300 u/g; the expression level of the aspergillus oryzae deaminase obtained by the way of solid state fermentation such as segmental culture is about 1560u/g, and the enzyme is purified by using a salting-out precipitation method and the enzymology property is preliminarily researched.
Chinese application No. CN201510306409.0 discloses a liquid fermentation method of adenosine deaminase, which comprises the following steps: (1) activating strains: inoculating Aspergillus melleus spore into culture medium for culturing; (2) seed preparation: inoculating the spore suspension into a seed shake flask culture medium according to the inoculation amount of 1-2.5% for culture; (3) seed tank culture: inoculating the seed bacterial liquid into a seed tank for culture according to the inoculation amount of 0.1-0.5%, wherein the rotating speed is 50-150rpm, and the ventilation ratio is 1:0.3-1: 1.2; (4) fermentation tank production: inoculating the seed culture solution into a fermentation tank for culture according to the inoculation amount of 2-10%, wherein the rotation speed is 100-.
The adenosine deaminase on the market is mainly derived from Japanese Tianye enzyme preparations, so that domestic large-scale manufacturers are few, and the production cost is high due to low fermentation enzyme activity.
At present, the domestic production of adenylate deaminase is mainly carried out by using aspergillus to carry out a solid state fermentation process, the fermentation enzyme activity is low, and the product purity is insufficient; the yield in the separation and purification steps is low, and the total cost is overhigh. In addition, the yield of single production is limited by site and environment due to the limitation of fermentation process, and the production requirement is far from being achieved.
Disclosure of Invention
Therefore, the technical problem solved by the invention is as follows: at present, the domestic production of adenylate deaminase is mainly carried out by using aspergillus to carry out a solid state fermentation process, the fermentation enzyme activity is low, and the product purity is insufficient; the yield in the separation and purification steps is low, and the total cost is overhigh.
The invention provides a Bacillus subtilis ESP710 for producing adenylate deaminase, which is screened from soil, and mutagenized and domesticated by mutagenesis technologies such as ultraviolet, ARTP and the like to obtain a high-yield enzyme activity strain, wherein the strain is sent to the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CCTCC NO: M2018827.
The invention provides a method for producing adenylate deaminase by liquid fermentation, which uses the strain, wherein the activity level of the fermentation enzyme is 14000-18000U/mL; the fermentation enzyme has high activity, the product is easy to separate and extract, the production yield can be greatly improved, the production cost is reduced, and the product competitiveness is improved.
Specifically, the present invention proposes the following technical solutions.
The invention provides a bacterial strain for producing adenylate deaminase, which is a Bacillus subtilis ESP710 and is preserved in China center for type culture collection with the preservation number of CCTCC No. M2018827.
The invention provides the application of the strain in the field of fermentation, preferably the application in the fermentation production of adenosine deaminase.
The invention provides a method for producing adenylate deaminase, which uses the strain to carry out liquid fermentation production to obtain the adenylate deaminase.
Preferably, for the method described above, wherein the method comprises the steps of:
(1) activating strains: inoculating the strain of claim 1 in a culture medium to obtain an activated strain;
(2) seed preparation: inoculating the strain obtained in the step (1) into a seed shake flask culture medium for culture to obtain seed bacterial liquid;
(3) seed tank culture: inoculating the seed bacterial liquid obtained in the step (2) into a seed tank filled with a seed culture medium for culture to obtain a seed culture liquid;
(4) culturing in a fermentation tank: inoculating the seed culture solution into a fermentation tank for culturing, and obtaining fermentation liquor after the fermentation is finished.
Preferably, in the method as described above, wherein, in the step (1), the cultivation temperature is 30 to 40 ℃, and preferably, the cultivation time is 1 to 3 days.
Preferably, in the method described above, wherein, in the step (2), the culture temperature is 30 to 40 ℃, preferably 35 to 38 ℃; further preferably, the culture time is 16 to 24 hours, preferably 18 to 22 hours.
Preferably, for the above-mentioned method, wherein, in the step (3), the rotation speed is 50-150rpm, preferably 150-; preferably, the ventilation ratio is 1:0.5 to 1:1, preferably 1:0.8 to 1: 1; further preferably, the culture temperature is 30-40 ℃, preferably 35-37 ℃; further preferably, the culture time is 10-18 hours; it is further preferred that the inoculum size is between 0.1% and 0.5% by volume, preferably between 0.1 and 0.3% by volume.
Preferably, for the above-mentioned method, wherein, in the step (4), the rotation speed is 100-200rpm, preferably, the ventilation ratio is 1:0.5-1:1.2, preferably 1:0.8-1: 2; further preferably, the culture temperature is 30-40 ℃, preferably 35-37 ℃; further preferably, the culture time is 30-42 hours, preferably 34-36 hours; it is further preferred that the inoculum size is 2% to 10% by volume, preferably 6 to 10% by volume.
Preferably, the method described above, wherein, in step (4), the carbon source is supplemented after 3 to 6 hours of culture; preferably, the carbon source is a culture medium containing 30-60% glucose by mass/volume.
Preferably, in the method, the obtained fermentation broth is separated by plate-and-frame filtration to obtain a crude enzyme filtrate.
Preferably, in the method, the crude enzyme filtrate is concentrated, preferably, the concentration is 8 to 15 times to obtain a concentrated solution.
Preferably, for the method, the seed culture medium comprises 0.1-0.5% of yeast powder, 0.5-1.5% of peptone and 0.5-1.5% of sodium chloride, and the balance of water by mass-volume ratio.
Preferably, for the above method, wherein the fermentation medium comprises glucose 0.5-1.5%, peptone 1.5-4%, ammonium sulfate 0.15-0.65%, calcium chloride 0.01-0.15%, sodium citrate 0.01-0.2%, sodium dihydrogen phosphate 0.5-1.5%, disodium hydrogen phosphate 0.5-1.5% and defoaming oil 0.01-0.2%, and the balance of water, by mass to volume ratio.
The invention provides adenylate deaminase obtained by fermentation by using the method, wherein the enzyme activity is 14000-18000U/mL.
The beneficial effects obtained by the invention are as follows:
the invention provides bacillus subtilis for producing adenosine deaminase, and provides a method for producing the adenosine deaminase by adopting the strain to carry out liquid fermentation, wherein the fermentation enzyme activity in the method is far higher than that reported at present, the fermentation enzyme activity is 14000-18000U/mL, the liquid fermentation mode is easier for industrial production of products, the fermentation enzyme activity of the fermentation liquor is high, the fermentation uses conventional and soluble raw materials, the risk of producing contamination is small, and the products are easy to separate.
Information on the preservation of the strains
The Bacillus subtilis ESP710 used in the invention is preserved in China Center for Type Culture Collection (CCTCC) in 11 months and 26 days in 2018, the preservation number is CCTCC NO: M2018827, and the preservation address is as follows: china, wuhan university, zip code: 430072; telephone: (027) -68754052.
Detailed Description
As described above, the invention provides a Bacillus subtilis, the strain is separated from soil, and the separated strain is obtained by mutagenesis and domestication through mutagenesis technologies such as ultraviolet, ARTP and the like, the activity of the adenylate deaminase produced by fermentation of the obtained strain is high and is 14000-18000U/mL, the strain is sent to the China general microbiological preservation and management Committee, the preservation number is CCTCC No: m2018827.
The invention provides a liquid fermentation method for producing adenosine deaminase by using the strain, wherein the method comprises the following steps:
(1) activating strains: inoculating the strain of claim 1 in a culture medium to obtain an activated strain;
(2) seed preparation: inoculating the strain obtained in the step (1) into a seed shake flask culture medium for culture to obtain seed bacterial liquid;
(3) seed tank culture: inoculating the seed bacterial liquid obtained in the step (2) into a seed tank filled with a seed culture medium for culture to obtain a seed culture liquid;
(4) culturing in a fermentation tank: inoculating the seed culture solution into a fermentation tank for culturing, and obtaining fermentation liquor after the fermentation is finished.
In a preferred embodiment of the present invention, wherein, in the step (4), the fermentation pH is 6.5 to 7.5; preferably, Dissolved Oxygen (DO) is 40-60%; further preferably, the content of glucose in the fermentation process is 5-15 g/L.
In a preferred embodiment of the present invention, wherein, in step (4), the rotation speed is 100-200rpm, preferably, the ventilation ratio is 1:0.5-1:1.2, preferably 1:0.8-1: 2; further preferably, the culture temperature is 30-40 ℃, preferably 35-37 ℃; further preferably, the culture time is 30-42 hours, preferably 34-36 hours; it is further preferred that the inoculum size is 2% to 10% by volume, preferably 6 to 10% by volume.
Wherein the culture time is the sum of the time before feeding the carbon source and the fermentation time after feeding.
In a preferred embodiment of the present invention, the liquid fermentation process comprises the steps of:
(1) activating strains: one of the glycerol tube strains was inoculated into a fresh medium under aseptic conditions using an inoculating loop, and cultured at 37 ℃ for 1 day.
(2) Seed preparation: inoculating the activated and cultured seeds to a large ring single colony in a fresh seed shake flask culture medium, placing in a constant temperature shake flask at 37 ℃ for culturing for 16-24h,
(3) seed tank culture: inoculating a seed culture medium into a seed tank through a Karschner tank, wherein the inoculation amount is 0.1-0.5%, the culture temperature is 35-37 ℃, the rotating speed is 50-150rpm, and the ventilation ratio is 1:0.5-1:1, the culture period is about 10 to 18 hours,
(4) culturing in a fermentation tank: inoculating the seed culture solution into a fermentation tank according to the inoculation ratio of 2-8% for culture, wherein the culture temperature is 35-37 ℃, the rotation speed is 100-200rpm, the culture period is about 36 hours, and the ventilation ratio is 1:0.5-1:1.2, feeding carbon source after 5 hours of fermentation, and discharging the tank when the fermentation is finished, wherein the activity level of the tank is 14000u/ml to 18000 u/ml.
In a more preferred embodiment of the present invention, the liquid fermentation method further comprises the steps of centrifugation, ultrafiltration concentration, lyophilization or powder spraying treatment.
Preferably, the centrifugation is performed by removing the cells by plate-and-frame separation.
Preferably, the ultrafiltration concentration refers to that the crude enzyme filtrate is concentrated by an ultrafiltration membrane concentration system, and the concentration multiple is controlled to be 8-15 times.
In a preferred embodiment of the present invention, wherein, the formulation of the seed culture medium is: 0.1-0.5% of yeast powder, 0.5-1.5% of peptone, 0.5-1.5% of sodium chloride and the balance of water;
the formula of the fermentation medium comprises the following components in percentage by mass: 0.5-1.5% of glucose, 1.5-4% of peptone, 0.15-0.65% of ammonium sulfate, 0.01-0.15% of calcium chloride, 0.01-0.2% of sodium citrate, 0.5-1.5% of sodium dihydrogen phosphate, 0.5-1.5% of disodium hydrogen phosphate, 0.01-0.2% of defoaming oil and the balance of water;
the carbon source comprises the following components in percentage by mass and volume: 30-60% of glucose and the balance of water.
The following examples are detailed descriptions of the technical solutions of the present invention.
Example 1 obtaining of the Strain
(1) Obtaining a wild strain: the strain is obtained from the periphery of a fermented bean curd or soy sauce factory or soil, and the specific steps are as follows:
1) collecting samples: collecting a sample from soil around a fermented bean curd or soy sauce factory or a semi-finished fermented bean curd or a semi-finished fermented soy sauce;
2) and (3) microorganism enrichment: spinning the collected sample in a constant temperature environment at 37 ℃ for more than 48 hours until the sample is dried; taking a proper amount of dried samples to inoculate into a fresh culture medium, and carrying out enrichment culture; the formula of the culture medium comprises 0.5 percent of glucose, 4 percent of peptone, 0.65 percent of ammonium sulfate, 0.15 percent of calcium chloride, 0.2 percent of sodium citrate, 0.5 percent of sodium dihydrogen phosphate, 0.5 percent of disodium hydrogen phosphate and the balance of water;
3) obtaining a single colony: and (4) carrying out plate culture on the enriched sample after gradient dilution to obtain a single colony.
(2) Mutagenesis
The starting strain is induced by adopting an ARTP (active technique for fragment) mutagenesis mode
Preparing equipment: normal operation according to the device operating instructions; (ARTP-IIS, Wuxi Yuan Qingtian Wood Biotech Co., Ltd.)
Preparing a bacterial suspension: centrifuging and collecting the bacteria liquid cultured to logarithmic phase, washing with normal saline for 2 times, and re-dissolving with normal saline until OD is about 0.5, wherein the bacteria amount is 10-6～10-8Left and right;
placing the metal slide on an alcohol lamp in an ultra-clean bench, burning for about 20s by outer flame, cooling the metal slide on a rear sterile flat plate, and uniformly coating 10 mu L of bacterial liquid on the slide;
moving the flat plate with the sample slide to an ARTP mutagenesis system operation bin, placing the slide at a corresponding hole position by using sterile forceps, adjusting a knob below a carrying platform to enable the slide to be positioned at a position 2mm away from an airflow port, and closing a bin gate;
the power is set to be 100W, the flow is 10SLM, and the time is 31 s;
after the sample is processed, placing the slide glass into a centrifuge tube filled with 1mL of sterile water by using sterile forceps; fully and uniformly shaking; forming a new bacterial suspension;
carrying out gradient dilution and coating on the new bacterial suspension to obtain a single bacterial colony; the detection lethality rate is 99.17%;
(3) physiological and biochemical identification and 16S rDNA identification
1) Physiological and biochemical experiments: liquefied gelatin, hydrolyzed starch, positive V.P test, reduced nitrate, negative lecithase test, gram-positive;
2) the morphology is specific: the colony is white, moist, irregular in shape, convex with emulsion dots, opaque, and round or irregular in edge; the cells are in a long rod shape or a chain shape;
3) identification of 16S rDNA: the primers are shown as SEQ ID NO.2 and SEQ ID NO.3, wherein the sequence of SEQ ID NO.2 is 5'-AGAGTTTGATCCTGGCTCAG-3', and the sequence of SEQ ID NO.3 is: 5'-ACGGTTACCTTGTTACGACTT-3', the primers are provided by Oko Dingsheng Biotechnology (Wuhan) Co., Ltd
The PCR reaction system is as follows:
Figure BDA0002146463640000081
PCR amplification conditions: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 deg.C for 1min, annealing at 50 deg.C for 1min, extension at 72 deg.C for 1min, and amplification for 30 cycles; 72 ℃ for 10min
The amplified sequence was sent to Oakware tripod Biotechnology (Wuhan) Inc. for sequencing, the 16S rDNA of which is shown as SEQ ID NO.1, and BLAST nucleotide alignment analysis was performed on the sequence using NCBI website, and the homology with the 16S rDNA sequence of Bacillus subtilis was 100% (Gene Bank: MK1383007.1 and NR 112116.2).
EXAMPLE 2-1 fermentative production of adenylate deaminase
(1) Strain activation: selecting a glycerol tube strain, activating the glycerol tube strain in a fresh plate culture medium at 37 ℃ for 1d to obtain a single colony, wherein the plate culture medium contains 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride in terms of 100L of water;
(2) seed preparation: selecting 1-2 activated single colonies, transferring the single colonies into a triangular flask (containing 100mL of liquid in a 500mL triangular flask) containing a fresh seed culture medium (the components and the content of the single colonies are the same as those in the step (1)), preparing a seed liquid, and culturing under the following conditions: culturing at 37 deg.C and 200rpm for 18 h;
(3) seed tank culture: inoculating the seed solution obtained in the step (2) into a 3L seed tank containing a seed culture medium at an inoculation amount of 0.2 w/v% for culturing to obtain mature seeds, wherein the culture temperature is 35 ℃, the rotation speed is 150rpm, and the ventilation ratio is 1:0.8, the culture period is 15 hours, and the seed culture medium comprises 0.1 percent of yeast powder, 1.5 percent of peptone and 1.5 percent of sodium chloride, and the balance of water according to the mass-volume ratio;
(4) culturing in a fermentation tank: inoculating mature seeds with an inoculation amount of 6 vol% into a 5L fermentation tank with a liquid loading amount of 3L, fermenting at 37 ℃, and feeding a carbon source in a flowing manner after fermenting for 5 hours, wherein the carbon source contains a culture medium containing 40% of glucose by mass-volume ratio, and the balance is water; controlling the content of glucose in fermentation liquor to be about 5g/L, controlling the pH to be 6.5, rotating speed to be 100rpm, ventilating ratio to be 1:0.8, controlling DO value of the fermentation liquor to be 40%, fermenting for 31h, then separating and removing thalli by adopting a plate frame to obtain crude enzyme filtrate, concentrating the crude enzyme filtrate by an ultrafiltration membrane concentration system by 8 times, and freeze-drying to obtain the solid adenylic acid deaminase, wherein a fermentation culture medium comprises 0.5% of glucose, 4% of peptone, 0.65% of ammonium sulfate, 0.15% of calcium chloride, 0.2% of sodium citrate, 0.5% of sodium dihydrogen phosphate, 0.5% of disodium hydrogen phosphate, 0.01% of defoaming oil and the balance of water by mass-volume ratio.
The enzyme activity determination method of the adenosine deaminase comprises the following steps:
accurately weighing 13.9mg of 5' -adenylic acid dissolved in 1mL of 0.08 mol.L-1NaHCO of3In solution as the original substrate, then 0.1 mol.L-1Succinic acid-NaOH (pH 6.0) diluted 400-fold to give a concentration of 0.1X 10-3mol·L-1A reaction substrate of (1). Adding 3mL of reaction substrate into a test tube, placing the test tube in a water bath kettle at 37 ℃ for heat preservation for 5min, adding 100 mu L of pre-diluted adenosine deaminase solution, immediately timing, uniformly mixing the reaction substrate and the enzyme solution, and adding 3mL of 10% perchloric acid solution after 15min to terminate the reaction. The control experiment method is as above, the reaction substrate is pre-cooled by ice water after being water-bathed at 37 ℃, then 100 mu L of enzyme solution is added after 3mL and 10% perchloric acid are added, the mixture is mixed evenly, and the light absorption value is measured by a quartz cuvette at 265nm by an ultraviolet spectrophotometer with deionized water as a reference.
The enzyme activity is defined as that the change of 0.001 of the absorbance per minute at the wavelength of 265nm under the above conditions is one activity unit of the enzyme.
The calculation formula is as follows: enzyme activity (U.mL)-1)＝(△A265×K×10)/(0.001×T)。
Delta A265 is the difference of the absorbance of the reaction solution and the control group; k is the dilution multiple of the enzyme solution; t is the reaction time, min
The enzyme activity of the obtained adenylate deaminase was 15800U/mL as determined by the above method.
EXAMPLE 2-2 fermentative production of adenylate deaminase
(1) Activating strains: selecting a glycerol tube strain, and activating the glycerol tube strain in a fresh plate culture medium at 37 ℃ for 1d to obtain a single colony, wherein the components and the content of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) seed preparation: 1-2 activated single colonies were picked and transferred to a flask (500mL flask containing 100mL) containing fresh seed medium (the components and contents of the seed medium are the same as those in step (2) of example 2-1), and seed solution was prepared under the following culture conditions: culturing at 37 deg.C and 200rpm for 20 h;
(3) seed tank culture: inoculating the seed solution obtained in the step (2) into a 3L seed tank containing a seed culture medium in an inoculation amount of 0.3 w/v% for culturing to obtain mature seeds, wherein the culture temperature is 36 ℃, the rotation speed is 200rpm, and the ventilation ratio is 1:0.9, the culture period is 18 hours, and the seed culture medium comprises 0.5 percent of yeast powder, 0.5 percent of peptone, 1.5 percent of sodium chloride and the balance of water according to the mass-volume ratio;
(4) culturing in a fermentation tank: inoculating mature seeds with an inoculation amount of 6 vol% into a 5L fermentation tank with a liquid loading amount of 3L, fermenting at 37 ℃, and feeding a carbon source in a flowing manner after fermenting for 5 hours, wherein the carbon source contains a culture medium with glucose of 30% by mass and volume ratio, and the balance of water; controlling the content of glucose in the fermentation liquor to be about 5g/L, the pH to be 7, the rotating speed to be 150rpm, the ventilation ratio to be 1:1, and the DO to be 40 percent; fermenting for 31 hours, separating and removing thalli by adopting a plate frame to obtain a crude enzyme filtrate, concentrating the crude enzyme filtrate by an ultrafiltration membrane concentration system, wherein the concentration multiple is 10 times, and performing freeze-drying treatment to obtain the solid adenosine deaminase, wherein a culture medium for fermentation comprises 1.5% of glucose, 1.5% of peptone, 0.5% of ammonium sulfate, 0.15% of calcium chloride, 0.01% of sodium citrate, 0.5% of sodium dihydrogen phosphate, 1.5% of disodium hydrogen phosphate, 0.01% of defoaming oil and the balance of water according to the mass-volume ratio.
The obtained adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the fermentation enzyme activity of the obtained adenylate deaminase was 16800U/mL.
EXAMPLE 2-3 fermentative production of adenylate deaminase
(1) Activating strains: selecting a glycerol tube strain, and activating the glycerol tube strain in a fresh plate culture medium at 37 ℃ for 1d to obtain a single colony, wherein the components and the content of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) seed preparation: 1-2 activated single colonies were picked and transferred to a flask (500mL flask containing 100mL) containing fresh seed medium (the components and contents of the seed medium are the same as those in step (2) of example 2-1), and seed solution was prepared under the following culture conditions: culturing at 37 deg.C and 200rpm for 20 h;
(3) seed tank culture: inoculating the seed solution obtained in the step (2) into a 5L seed tank with the liquid containing volume of 1L with the inoculation amount of 0.1 volume percent for culturing to obtain mature seeds, wherein the culture temperature is 37 ℃, the rotating speed is 200rpm, the ventilation ratio is 1:1, the culture period is 12h, and the seed culture medium comprises 0.4 percent of yeast powder, 1 percent of peptone and 1 percent of sodium chloride, and the balance of water according to the mass volume ratio;
(4) culturing in a fermentation tank: inoculating mature seeds with an inoculation amount of 6.8 volume percent into a 50L fermentation tank with a liquid loading amount of 25L, starting to feed a carbon source after fermenting for 5 hours, wherein the carbon source contains a culture medium with the glucose of 50 percent by mass volume ratio, and the balance is water; controlling the content of glucose in the fermentation liquor to be about 5g/L, the pH value to be 7.5, the rotating speed to be 200rpm, the ventilation ratio to be 1:0.5 and the DO value of the fermentation liquor to be 40 percent; the fermentation period is 36 hours, then the thalli are removed by adopting plate-and-frame separation to obtain a crude enzyme filtrate, the crude enzyme filtrate is concentrated by an ultrafiltration membrane concentration system, the concentration multiple is 15 times, and then powder spraying treatment is carried out to obtain the solid adenosine deaminase, wherein the fermentation medium comprises 0.5% of glucose, 1.5% of peptone, 0.65% of ammonium sulfate, 0.15% of calcium chloride, 0.2% of sodium citrate, 1.5% of sodium dihydrogen phosphate, 1.5% of disodium hydrogen phosphate, 0.2% of defoaming oil and the balance of water according to the mass-volume ratio.
The adenylate deaminase obtained was subjected to an enzyme activity test as described in example 2-1, and the fermentation enzyme activity of the adenylate deaminase obtained was 14500U/mL.
EXAMPLE 2-4 fermentative production of adenylate deaminase
(1) Activating strains: selecting a glycerol tube strain, and activating the glycerol tube strain in a fresh plate culture medium at 37 ℃ for 1d to obtain a single colony, wherein the components and the content of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) seed preparation: selecting 1-2 activated single colonies, transferring the single colonies into a triangular flask (500mL triangular flask liquid 100mL) filled with a fresh seed culture medium, preparing a seed liquid, and culturing under the following conditions: culturing at 37 deg.C and 200rpm for 20 h;
(3) seed tank culture: inoculating the seed solution obtained in the step (2) into a 5L seed tank with the liquid containing volume of 1L with the inoculation amount of 0.1 volume percent for culturing to obtain mature seeds, wherein the culture temperature is 37 ℃, the rotation speed is 200rpm, the ventilation ratio is 1:0.9, the culture period is 14h, and the seed culture medium comprises 5 percent of yeast powder, 0.5 percent of peptone and 0.5 percent of sodium chloride by mass volume ratio, and the balance is water;
(4) culturing in a fermentation tank: inoculating mature seeds with an inoculation amount of 8 volume percent into a 50L fermentation tank with a liquid loading amount of 30L, starting to feed a carbon source after fermenting for 4.5 hours, wherein the carbon source contains a culture medium with glucose of 60 percent by mass volume ratio, and the balance is water; controlling the content of glucose in the fermentation liquor to be about 5g/L, the pH to be 6.5, the rotating speed to be 120rpm, the ventilation ratio to be 1:0.5 and the DO to be 40 percent; and (2) carrying out a fermentation period of 36h, then separating and removing thalli by adopting a plate-and-frame separation to obtain a crude enzyme filtrate, concentrating the crude enzyme filtrate by using an ultrafiltration membrane concentration system, wherein the concentration multiple is 12 times, and then carrying out powder spraying treatment to obtain the solid adenosine deaminase, wherein the fermentation medium comprises 1% of glucose, 1.5-3% of peptone, 0.5% of ammonium sulfate, 0.1% of calcium chloride, 0.1% of sodium citrate, 1% of sodium dihydrogen phosphate, 1% of disodium hydrogen phosphate, 0.1% of defoaming oil and the balance of water by mass volume ratio.
The obtained adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the fermentation enzyme activity of the obtained adenylate deaminase was 15500U/mL.
EXAMPLE 2-5 fermentative production of adenylate deaminase
(1) Activating strains: selecting a glycerol tube strain, and activating the glycerol tube strain in a fresh plate culture medium at 37 ℃ for 1d to obtain a single colony, wherein the components and the content of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) seed preparation: 1-2 activated single colonies were picked and transferred to a flask (2000mL of flask) containing fresh seed medium (the components and contents of the seed medium are the same as those in step (2) of example 2-1) to prepare a seed solution, and the culture conditions were as follows: culturing at 37 deg.C and 200rpm for 20 h;
(3) seed tank culture: inoculating the seed liquid obtained in the step (2) to a liquid containing amount of 1.5m in an inoculation amount of 0.2 vol%35m of3Culturing in a seed tank to obtain mature seeds, wherein the culture temperature is 37 ℃, the rotation speed is 150rpm, and the ventilation ratio is 1:0.8, the culture period is 12 hours, and the seed culture medium comprises 0.4 percent of yeast powder, 1 percent of peptone and 1 percent of sodium chloride, and the balance of water according to the mass-volume ratio;
(4) culturing in a fermentation tank: inoculating mature seeds with an inoculum size of 10 vol% to a liquid loading of 15m325m of3In a fermentation tank, starting to flow and add a carbon source after fermenting for 5 hours, wherein the carbon source contains a culture medium with 30% of glucose by mass volume ratio, and the balance of water; controlling the content of glucose in the fermentation liquor to be about 5g/L, the pH value to be 7.5, the rotating speed to be 100rpm, the ventilation ratio to be 1:0.5, fermentation liquor DO is 40%; the fermentation period is 34 hours, then the thalli are removed by adopting plate-and-frame separation to obtain a crude enzyme filtrate, the crude enzyme filtrate is concentrated by an ultrafiltration membrane concentration system, the concentration multiple is 8 times, and then powder spraying treatment is carried out to obtain the solid adenosine deaminase, wherein the fermentation medium comprises 1.5% of glucose, 3% of peptone, 0.65% of ammonium sulfate, 0.15% of calcium chloride, 0.01% of sodium citrate, 1.5% of sodium dihydrogen phosphate, 1.5% of disodium hydrogen phosphate, 0.01% of defoaming oil and the balance of water by mass volume ratio.
The adenylate deaminase thus obtained was subjected to an enzyme activity test as described in example 2-1, and the fermentation enzyme activity of the adenylate deaminase thus obtained was 17700U/mL.
EXAMPLE 2-6 fermentative production of adenylate deaminase
(1) Activating strains: selecting a glycerol tube strain, and activating the glycerol tube strain in a fresh plate culture medium at 37 ℃ for 1d to obtain a single colony, wherein the components and the content of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) seed preparation: 1-2 activated single colonies were picked and transferred to a flask (2000mL of flask) containing fresh seed medium (the components and contents of the seed medium are the same as those in step (2) of example 2-1) to prepare a seed solution, and the culture conditions were as follows: culturing at 37 deg.C and 200rpm for 18 h;
(3) seed tank culture: inoculating the seed liquid obtained in the step (2) to a liquid containing amount of 1.5m in an inoculation amount of 0.3 volume percent35m of3Culturing in a seed tank to obtain mature seeds, wherein the culture temperature is 37 ℃, the rotation speed is 150rpm, and the ventilation ratio is 1:0.8, the culture period is 11h, and the seed culture medium comprises 0.5% of yeast powder, 1.5% of peptone and 1% of sodium chloride, and the balance of water according to mass-to-volume ratio;
(4) culturing in a fermentation tank: inoculating mature seeds with an inoculum size of 10 vol% to a liquid loading of 15m325m of3In a fermentation tank, starting to flow and add a carbon source after fermenting for 4.5 hours, wherein the carbon source contains a culture medium with 45% of glucose by mass-volume ratio, and the balance of water; controlling the content of glucose in the fermentation liquor to be about 5g/L, the pH value to be 7, the rotating speed to be 150rpm, the ventilation ratio to be 1:1, fermentation liquor DO is 40%; the fermentation period is 34h, then the thalli are removed by adopting plate-and-frame separation to obtain a crude enzyme filtrate, the crude enzyme filtrate is concentrated by an ultrafiltration membrane concentration system, the concentration multiple is 15 times, and freeze-drying treatment is carried out to obtain the solid adenosine deaminase, wherein the fermentation medium comprises 1% of glucose, 4% of peptone, 0.5% of ammonium sulfate, 0.01% of calcium chloride, 0.01% of sodium citrate, 0.5% of sodium dihydrogen phosphate, 1.5% of disodium hydrogen phosphate, 0.2% of defoaming oil and the balance of water according to the mass-volume ratio.
The adenylate deaminase obtained was subjected to an enzyme activity test as described in example 2-1, and the fermentation enzyme activity of the adenylate deaminase obtained was 16700U/mL.
EXAMPLES 2-7 fermentative production of adenylate deaminase
(1) Activating strains: selecting a glycerol tube strain, and activating the glycerol tube strain in a fresh plate culture medium at 37 ℃ for 1d to obtain a single colony, wherein the components and the content of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) seed preparation: 1-2 activated single colonies were picked and transferred to a flask (2000mL of flask) containing fresh seed medium (the components and contents of the seed medium are the same as those in step (2) of example 2-1) to prepare a seed solution, and the culture conditions were as follows: culturing at 37 deg.C and 200rpm for 22 h;
(3) seed tank culture: inoculating the seed liquid obtained in the step (2) to a liquid containing amount of 1.5m in an inoculation amount of 0.1 volume percent35m of3Culturing in a seed tank to obtain mature seeds, wherein the culture temperature is 37 ℃, the rotation speed is 150rpm, and the ventilation ratio is 1:0.8, the culture period is 16h, and the seed culture medium comprises 0.3% of yeast powder, 0.8% of peptone, 1% of sodium chloride and the balance of water according to mass-volume ratio;
(4) culturing in a fermentation tank: inoculating mature seeds with an inoculum size of 10 vol% to a liquid loading of 15m325m of3In a fermentation tank, starting to flow and add a carbon source after fermenting for 5 hours, wherein the carbon source contains a culture medium with the glucose content of 40% and the balance of water according to the mass-volume ratio; controlling the content of glucose in the fermentation liquor to be about 5g/L, the pH value to be 7, the rotating speed to be 200rpm, the ventilation ratio to be 1:1.2, fermentation liquor DO is 40%, fermentation period is 36h, then a plate frame is adopted to separate and remove thalli to obtain crude enzyme filtrate, the crude enzyme filtrate is concentrated by an ultrafiltration membrane concentration system, the concentration multiple is 12 times, and freeze-drying treatment is carried out to obtain solid adenosine deaminase, wherein a fermentation medium comprises 0.5% of glucose, 3% of peptone, 0.65% of ammonium sulfate, 0.15% of calcium chloride, 0.2% of sodium citrate, 1.5% of sodium dihydrogen phosphate, 0.5% of disodium hydrogen phosphate and 0.01% of defoaming oil in mass-volume ratio, and the balance is water.
The adenylate deaminase thus obtained was subjected to an enzyme activity test as described in example 2-1, and the fermentation enzyme activity of the adenylate deaminase thus obtained was 17900U/mL.
EXAMPLES 2-8 fermentative production of adenylate deaminase
(1) Activating strains: selecting a glycerol tube strain, activating the glycerol tube strain in a fresh plate culture medium at 30 ℃ for 3 days to obtain a single colony, wherein the components and the content of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) seed preparation: 1-2 activated single colonies were picked and transferred to a flask (2000mL of flask) containing fresh seed medium (the components and contents of the seed medium are the same as those in step (2) of example 2-1) to prepare a seed solution, and the culture conditions were as follows: culturing at 30 deg.C and 200rpm for 24 hr;
(3) seed tank culture: inoculating the seed liquid obtained in the step (2) to a liquid containing amount of 1.5m in an inoculation amount of 0.5 volume percent35m of3Culturing in a seed tank to obtain mature seeds, wherein the culture temperature is 30 ℃, the rotating speed is 50rpm, and the ventilation ratio is 1:1, culturing for 18h, wherein the seed culture medium comprises 0.2% of yeast powder, 1.2% of peptone and 1.2% of sodium chloride, and the balance of water according to the mass-volume ratio;
(4) culturing in a fermentation tank: inoculating mature seeds with an inoculum size of 10 vol% to a liquid loading of 15m325m of3In a fermentation tank, starting to flow and add a carbon source after fermenting for 6 hours, wherein the carbon source contains a culture medium with 60% of glucose by mass volume ratio, and the balance of water; controlling the content of glucose in the fermentation liquor to be about 5g/L, the pH value to be 6.5, the rotating speed to be 150rpm, the ventilation ratio to be 1:1, fermenting for 36 hours again with 60% of fermentation liquor DO, then separating and removing thalli by adopting a plate frame to obtain crude enzyme filtrate, concentrating the crude enzyme filtrate by an ultrafiltration membrane concentration system with the concentration multiple of 10 times, and performing freeze-drying treatment to obtain solid adenosine deaminase, wherein a fermentation medium comprises 1.5% of glucose, 2% of peptone, 0.15% of ammonium sulfate, 0.15% of calcium chloride, 0.01% of sodium citrate, 0.5% of sodium dihydrogen phosphate, 1.5% of disodium hydrogen phosphate, 0.2% of defoaming oil and the balance of water according to the mass-volume ratio.
The adenylate deaminase obtained was subjected to an enzyme activity test as described in example 2-1, and the fermentation enzyme activity of the adenylate deaminase obtained was 14800U/mL.
EXAMPLE 2-9 fermentative production of adenylate deaminase
(1) Activating strains: selecting a glycerol tube strain, and activating the glycerol tube strain in a fresh plate culture medium at 40 ℃ for 1d to obtain a single colony, wherein the components and the content of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) seed preparation: 1-2 activated single colonies were picked and transferred to a flask (2000mL of flask) containing fresh seed medium (the components and contents of the seed medium are the same as those in step (2) of example 2-1) to prepare a seed solution, and the culture conditions were as follows: culturing at 40 deg.C and 200rpm for 16 h;
(3) seed tank culture: inoculating the seed liquid obtained in the step (2) to a liquid containing amount of 1.5m in an inoculation amount of 0.1 vol%35m of3Culturing in a seed tank to obtain mature seeds, wherein the culture temperature is 40 ℃, the rotating speed is 100rpm, and the ventilation ratio is 1:0.5, the culture period is 10 hours, and the seed culture medium comprises 0.5 percent of yeast powder, 1.2 percent of peptone and 1 percent of sodium chloride, and the balance of water according to the mass-volume ratio;
(4) culturing in a fermentation tank: inoculating mature seeds with an inoculum size of 2 vol% to a liquid loading of 15m325m of3In a fermentation tank, starting to flow and add a carbon source after fermenting for 3 hours, wherein the carbon source contains a culture medium with the glucose content of 50% and the balance of water according to the mass-volume ratio; controlling the content of glucose in the fermentation liquor to be about 5g/L, the pH value to be 7.5, the rotating speed to be 100rpm, the ventilation ratio to be 1:1.2, the DO of the fermentation liquor is 50 percent; and fermenting for 27 hours, separating and removing thalli by adopting a plate frame to obtain a crude enzyme filtrate, concentrating the crude enzyme filtrate by using an ultrafiltration membrane concentration system, wherein the concentration multiple is 15 times, and performing powder spraying treatment to obtain the solid adenosine deaminase, wherein a culture medium for fermentation comprises 0.5% of glucose, 4% of peptone, 0.3% of ammonium sulfate, 0.15% of calcium chloride, 0.2% of sodium citrate, 1.5% of sodium dihydrogen phosphate, 1.5% of disodium hydrogen phosphate, 0.2% of defoaming oil and the balance of water according to the mass-volume ratio.
The adenylate deaminase obtained was subjected to an enzyme activity test as described in example 2-1, and the fermentation enzyme activity of the adenylate deaminase obtained was 15700U/mL.
Examples 2-10 fermentative production of adenylate deaminase
(1) Activating strains: selecting a glycerol tube strain, and activating the glycerol tube strain in a fresh plate culture medium at 35 ℃ for 2 days to obtain a single colony, wherein the components and the content of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) seed preparation: 1-2 activated single colonies were picked and transferred to a flask (2000mL of flask) containing fresh seed medium (the components and contents of the seed medium are the same as those in step (2) of example 2-1) to prepare a seed solution, and the culture conditions were as follows: culturing at 35 deg.C and 200rpm for 20 h;
(3) seed tank culture: inoculating the seed liquid obtained in the step (2) to a liquid containing amount of 1.5m in an inoculation amount of 0.4 volume percent35m of3Culturing in a seed tank to obtain mature seeds, wherein the culture temperature is 33 ℃, the rotation speed is 155rpm, and the ventilation ratio is 1:0.9, the culture period is 12 hours, and the seed culture medium comprises 0.1 percent of yeast powder, 0.5 percent of peptone and 0.5 percent of sodium chloride, and the balance of water according to the mass-volume ratio;
(4) culturing in a fermentation tank: inoculating mature seeds with an inoculum size of 5 vol% to a liquid loading of 15m325m of3In a fermentation tank, starting to flow and add a carbon source after fermenting for 4 hours, wherein the carbon source contains a culture medium with 30% of glucose by mass volume ratio, and the balance of water; controlling the content of glucose in the fermentation liquor to be about 5g/L, the pH value to be 7, the rotating speed to be 200rpm, the ventilation ratio to be 1:0.5, fermentation liquor DO is 40%; fermenting for 34 hours, separating and removing thalli by adopting a plate frame to obtain a crude enzyme filtrate, concentrating the crude enzyme filtrate by an ultrafiltration membrane concentration system, wherein the concentration multiple is 8 times, and performing freeze-drying treatment to obtain the solid adenosine deaminase, wherein a culture medium for fermentation comprises 1.5% of glucose, 4% of peptone, 0.65% of ammonium sulfate, 0.08% of calcium chloride, 0.2% of sodium citrate, 0.5% of sodium dihydrogen phosphate, 1% of disodium hydrogen phosphate, 0.2% of defoaming oil and the balance of water according to the mass-to-volume ratio.
The resulting adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the fermentation enzyme activity of the resulting adenylate deaminase was 17770U/mL.
Examples 2-11 fermentative production of adenylate deaminase
(1) Activating strains: selecting a glycerol tube strain, and activating the glycerol tube strain in a fresh plate culture medium at 38 ℃ for 1d to obtain a single colony, wherein the components and the content of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) seed preparation: 1-2 activated single colonies were picked and transferred to a flask (2000mL of flask) containing fresh seed medium (the components and contents of the seed medium are the same as those in step (2) of example 2-1) to prepare a seed solution, and the culture conditions were as follows: culturing at 38 deg.C and 200rpm for 22 h;
(3) seed tank culture: inoculating the seed liquid obtained in the step (2) to a liquid containing amount of 1.5m in an inoculation amount of 0.3 volume percent35m of3Culturing in a seed tank to obtain mature seeds, wherein the culture temperature is 37 ℃, the rotation speed is 200rpm, and the ventilation ratio is 1:1, a culture period of 14h, wherein the seed culture medium comprises 0.4% of yeast powder, 1.5% of peptone and 1% of sodium chloride, and the balance of water according to mass-volume ratio;
(4) culturing in a fermentation tank: inoculating mature seeds with an inoculum size of 10 vol% to a liquid loading of 15m325m of3In a fermentation tank, starting to flow and add a carbon source after fermenting for 4.5 hours, wherein the carbon source contains a culture medium with 45% of glucose by mass-volume ratio, and the balance of water; controlling the content of glucose in the fermentation liquor to be about 5g/L, the pH value to be 7, the rotating speed to be 140rpm, the ventilation ratio to be 1:1.2, fermentation liquor DO is 55%, fermentation period is 31.5h, then a plate frame is adopted to separate and remove thalli to obtain crude enzyme filtrate, the crude enzyme filtrate is concentrated by an ultrafiltration membrane concentration system, the concentration multiple is 15 times, and freeze-drying treatment is carried out to obtain solid adenosine deaminase, wherein a fermentation medium comprises 1% of glucose, 1.5% of peptone, 0.5% of ammonium sulfate, 0.1% of calcium chloride, 0.2% of sodium citrate, 1.5% of sodium dihydrogen phosphate, 1.5% of disodium hydrogen phosphate and 0.01% of defoaming oil in mass-volume ratio, and the balance is water.
The adenylate deaminase thus obtained was subjected to an enzyme activity test in the same manner as in example 2-1, and the fermentation enzyme activity of the adenylate deaminase thus obtained was 16527U/mL.
Comparative example 1
(1) Activating strains: selecting a glycerol tube strain (a wild strain, namely the strain before mutation in the example 1) and activating the glycerol tube strain for 1d in a fresh plate culture medium at 37 ℃ to obtain a single colony, wherein the components and the content of the plate culture medium are the same as those in the step (1) in the example 2-1;
(2) seed preparation: 1-2 activated single colonies were picked and transferred to a flask (500mL flask containing 100mL) containing fresh seed medium (the components and contents of the seed medium are the same as those in step (2) of example 2-1), and seed solution was prepared under the following culture conditions: culturing at 37 deg.C and 200rpm for 20 h;
(3) seed tank culture: inoculating the seed solution obtained in the step (2) into a 5L seed tank with the liquid containing volume of 1L with the inoculation amount of 0.1 volume percent for culturing to obtain mature seeds, wherein the culture temperature is 37 ℃, the rotating speed is 200rpm, the ventilation ratio is 1:1, the culture period is 12h, and the seed culture medium comprises 0.4 percent of yeast powder, 1 percent of peptone and 1 percent of sodium chloride, and the balance of water according to the mass volume ratio;
(4) culturing in a fermentation tank: inoculating mature seeds with an inoculation amount of 6.8 volume percent into a 50L fermentation tank with a liquid loading amount of 25L, starting to feed a carbon source after fermenting for 5 hours, wherein the carbon source contains a culture medium with the glucose of 50 percent by mass volume ratio, and the balance is water; controlling the content of glucose in the fermentation liquor to be about 5g/L, the pH value to be 7.5, the rotating speed to be 200rpm, the ventilation ratio to be 1:0.5 and the DO value of the fermentation liquor to be 40 percent; and (2) performing fermentation for 36h, separating and removing thalli by using a plate frame to obtain a crude enzyme filtrate, concentrating the crude enzyme filtrate by using a super concentrator, wherein the concentration multiple is 15 times, and performing powder spraying treatment to obtain the solid adenosine deaminase, wherein the fermentation medium comprises 0.5% of glucose, 1.5% of peptone, 0.65% of ammonium sulfate, 0.15% of calcium chloride, 0.2% of sodium citrate, 1.5% of sodium dihydrogen phosphate, 1.5% of disodium hydrogen phosphate, 0.2% of defoaming oil and the balance of water according to the mass-volume ratio.
The adenylate deaminase thus obtained was subjected to an enzyme activity test as described in example 2-1, and the fermentation enzyme activity of the adenylate deaminase thus obtained was 3233U/mL.
Comparative example 2
(1) Activating strains: selecting one strain of the glycerinum tube, selecting one loop by using a 10 mu L inoculating loop, inoculating 200g of PDA culture medium in a sterile environment, culturing for 3 days at 28 ℃, and eluting by using 500mL of sterile water to obtain a single colony.
(2) Seed preparation: and (3) inoculating the single colony into a seed shake flask culture medium accounting for 2.7L in total according to the inoculation amount of 2%, and culturing at 28 ℃ for 22 hours to obtain a seed bacterial liquid, wherein the formula of the seed shake flask culture medium is the same as that of the seed culture medium in the step (3).
(3) Seed tank culture: inoculating the seed bacterial liquid into a seeding tank (the storage capacity is 1 ton) filled with 900L of seed culture medium according to the inoculation amount of 0.3 percent, and culturing to obtain a seed culture solution, wherein the culture temperature is 30 ℃, the rotating speed is 100rpm, the ventilation ratio is 1:0.7, and the culture period is 26 hours. Wherein the seed culture medium comprises the following components in percentage by weight: 10% of soybean juice, 2% of cane sugar and K2HP040.5 percent, 0.2 percent of yeast powder and the balance of water, and the pH value is adjusted to 6.0 before sterilization.
(4) Fermentation production: inoculating the seed culture solution into a fermentation tank (the storage capacity is 25 tons) filled with 15 tons of fermentation medium according to the inoculation amount of 6 percent, culturing at the temperature of 29 ℃, rotating speed of 150rpm, ventilation ratio of 1:0.7, culturing for 115 hours, and placing the tank after fermentation to measure the enzyme activity level. Wherein the fermentation medium is prepared from the following components in percentage by weight: 3% of glucose, 2% of peptone, 0.3% of disodium citrate trihydrate and MgSO4·7H20.05% of O, 5% of a microelement mother solution, tween-800.12% and the balance of water, and adjusting the pH value to 6.0 before sterilization. The microelement mother liquor is prepared from the following components in percentage by weight: 2% of zinc sulfate, 2% of calcium chloride and the balance of water.
(5) Plate frame treatment: and filtering with a plate-and-frame filter to remove thallus to obtain a crude enzyme filtrate.
(6) And (3) ultrafiltration concentration: concentrating the crude enzyme filtrate by ultrafiltration concentration equipment, wherein the concentration multiple is controlled at 10 times to obtain a concentrated solution.
(7) And (3) freeze-drying treatment: and (4) carrying out freeze-drying treatment on the concentrated solution to obtain the solid deaminase.
The adenylate deaminase obtained was subjected to an enzyme activity test as described in example 2-1, and the fermentation enzyme activity of the adenylate deaminase obtained was 3466U/mL.
Comparing comparative example 1 with examples 2 to 3, which are different in the strain used, comparative example 1 is a strain before mutagenesis, i.e., a wild-type strain, while examples 2 to 3 are strains after mutagenesis, and liquid fermentation is performed, the enzyme activity of the resulting adenylate deaminase is significantly different,
the enzyme activity of the adenylate deaminase obtained in example 2-3 was 14500U/mL, and the enzyme activity of the adenylate deaminase obtained in comparative example 1 was 3233U/mL, indicating that the enzyme activity of the adenylate deaminase obtained using the strain after mutagenesis was higher.
Comparing the comparative example 2 with the examples 2-3, the difference is that the liquid fermentation method is different, the obtained adenylate deaminase has different enzyme activity, the enzyme activity of the adenylate deaminase obtained in the comparative example 2 is 3466U/mL, while the enzyme activity of the adenylate deaminase obtained in the examples 2-3 is 14500U/mL, which is more than 4 times that of the comparative example 2, thus the enzyme activity of the adenylate deaminase obtained by the strain of the invention under the fermentation method process condition of the invention is high.
The foregoing is considered as illustrative and not restrictive in character, and that various modifications, equivalents, and improvements made within the spirit and principles of the invention are intended to be included within the scope of the invention.
Figure IDA0002146463720000011
Figure IDA0002146463720000021

Claims (14)

1. The bacterial strain for producing the adenylate deaminase is characterized by being a Bacillus subtilis ESP710 which is preserved in China center for type culture collection with the preservation number of CCTCC No. M2018827.
2. Use of a strain according to claim 1 in the field of fermentation, preferably in the fermentative production of adenylate deaminase.
3. A method for producing adenylate deaminase, wherein the strain of claim 1 is used for liquid fermentation to produce adenylate deaminase.
4. A method according to claim 3, wherein the method comprises the steps of:
(1) activating strains: inoculating the strain of claim 1 in a culture medium to obtain an activated strain;
(2) seed preparation: inoculating the strain obtained in the step (1) into a seed shake flask culture medium for culture to obtain seed bacterial liquid;
(3) seed tank culture: inoculating the seed bacterial liquid obtained in the step (2) into a seed tank filled with a seed culture medium for culture to obtain a seed culture liquid;
(4) culturing in a fermentation tank: inoculating the seed culture solution into a fermentation tank for culturing, and obtaining fermentation liquor after the fermentation is finished.
5. The method according to claim 4, wherein, in the step (1), the culture temperature is 30-40 ℃, and preferably, the culture time is 1-3 days.
6. The method according to claim 4 or 5, wherein, in step (2), the culture temperature is 30-40 ℃, preferably 35-38 ℃; further preferably, the culture time is 16 to 24 hours, preferably 18 to 22 hours.
7. The process according to any one of claims 4 to 6, wherein in step (3), the rotation speed is 50 to 150rpm, preferably 150 and 200 rpm; preferably, the ventilation ratio is 1:0.5 to 1:1, preferably 1:0.8 to 1: 1; further preferably, the culture temperature is 30-40 ℃, preferably 35-37 ℃; further preferably, the culture time is 10-18 hours; it is further preferred that the inoculum size is between 0.1% and 0.5% by volume, preferably between 0.1 and 0.3% by volume.
8. Process according to any one of claims 4 to 7, wherein in step (4) the rotation speed is 100-200rpm, preferably the aeration ratio is 1:0.5-1:1.2, preferably 1:0.8-1: 2; further preferably, the culture temperature is 30-40 ℃, preferably 35-37 ℃; further preferably, the culture time is 30-42 hours, preferably 34-36 hours; it is further preferred that the inoculum size is 2% to 10% by volume, preferably 6 to 10% by volume.
9. The method according to any one of claims 4 to 8, wherein, in step (4), the carbon source is supplemented after 3 to 6 hours of culture; preferably, the carbon source is a culture medium containing 30-60% glucose by mass/volume.
10. The process according to any one of claims 4 to 9, wherein the obtained fermentation broth is separated by plate and frame filtration to obtain a crude enzyme filtrate.
11. The process according to claim 10, wherein the crude enzyme filtrate is concentrated, preferably by a factor of 8-15, to obtain a concentrate.
12. The method according to any one of claims 4 to 11, wherein the seed culture medium comprises 0.1 to 0.5% by mass of yeast powder, 0.5 to 1.5% by mass of peptone, and 0.5 to 1.5% by mass of sodium chloride, and the balance of water.
13. The method according to any one of claims 4 to 12, wherein the fermentation medium comprises glucose 0.5-1.5%, peptone 1.5-4%, ammonium sulfate 0.15-0.65%, calcium chloride 0.01-0.15%, sodium citrate 0.01-0.2%, sodium dihydrogen phosphate 0.5-1.5%, disodium hydrogen phosphate 0.5-1.5%, and antifoam oil 0.01-0.2%, and the balance water, by mass to volume ratio.
14. An adenylate deaminase obtainable by fermentation according to any of claims 3 to 13, wherein the enzyme activity is 14000-18000U/mL.
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