CN112300953B - Bacillus subtilis and application thereof in fermentation production of adenylate deaminase - Google Patents

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

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CN112300953B
CN112300953B CN201910686571.8A CN201910686571A CN112300953B CN 112300953 B CN112300953 B CN 112300953B CN 201910686571 A CN201910686571 A CN 201910686571A CN 112300953 B CN112300953 B CN 112300953B
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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 adenylate deaminase. The invention provides a strain for producing adenylate deaminase, which is a bacillus subtilis strain (Bacillus subtilis ESP) and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2018827. The invention provides a method for producing adenylate deaminase, which uses the strain to carry out liquid fermentation to produce the adenylate deaminase. The fermentation enzyme activity of the obtained adenylate deaminase is far higher than that of the prior reported fermentation enzyme activity, the fermentation enzyme activity is 14000-18000U/mL, the liquid fermentation mode is easier for industrial production of products, the fermentation liquid fermentation enzyme activity is high, conventional and soluble raw materials are used for fermentation, the risk of bacterial contamination is low, and the products are easy to separate.

Description

Bacillus subtilis and application thereof in fermentation production of adenylate 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 adenylate deaminase.
Background
The yeast extract with high I+G (sodium 5 '-inosinate and sodium 5' -guanylate) is a yeast extract which strengthens the content of the flavor nucleotide substances in the product on the basis of the original yeast extract, can be used as a natural and nutritional high-end condiment to replace monosodium glutamate, has the functions of enhancing freshness, improving flavor, reducing salt and the like, and accords with the dietary consumption habit and the nutritional demand trend of modern people. The I+G content of the yeast extract directly affects the flavor quality and the production cost of the product. In domestic research, the content of I+G in yeast extract is about 10%, and foreign patents report on yeast extract with the content of I+G reaching 20%. The Angel yeast stock company improves the heat extraction time and pH of high-protein high-nucleic acid yeast, yeast RNA in an extract solution is improved to more than 25% from the original 13%, then more than 18% of I+G products can be directly produced through enzymolysis process improvement, and then ultrafiltration is carried out on the products, so that various properties of the products are obviously improved, various indexes of the products reach the quality level of European and American high-end products, and the method belongs to the first origins in China.
In the production of high I+G yeast extracts, single-stranded DNA and RNA are first hydrolyzed using 5 '-nucleotide phosphodiesterase to produce Adenylate (AMP) and Guanylate (GMP), and then reacted with 5' -adenylate deaminase to convert adenylate into inosinic acid (IMP). In the preparation process of the high I+G yeast extract, 5 '-adenylate deaminase plays a vital role, and the enzyme activity and the action performance of the 5' -adenylate deaminase directly influence the inosinic acid production and the quality of the high I+G yeast extract. Therefore, the obtained 5' -adenosine deaminase with high catalytic activity and high performance is one of key technologies for solving the production of the high I+G yeast extract, and has important research significance and market value.
The 5' -adenylate deaminase was originally prepared from murine skeletal muscle and human red blood cells, and enzymes used in modern industrial production were mostly prepared by fermentation with microorganisms such as yeast, penicillium, aspergillus and mucor, among which the most used species are aspergillus oryzae and aspergillus melleus, and in recent years, patent reports have been made by genetically engineered streptomyces. In the early days of deaminase production, solid state fermentation is generally used, and the method has the defects that pigments, proteins, sugar, inorganic salts and the like in solid raw materials are mixed into enzyme liquid to cause difficulty in extraction, purification and use of the enzyme, the enzyme activity of the solid enzyme is usually low, and then liquid fermentation is used for production, and the method can effectively overcome certain defects of solid state fermentation, but the activity and stability of the enzyme are still unresolved. At present, only Japanese wild enzyme Co., ltd. Internationally has mature technology and products, and the national academy of sciences of Guangxi also has 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' -adenosine deaminase are reported, and the research is still mainly focused on strain breeding and fermentation condition optimization. Liu Changjun and the like, and obtaining fresh yeast with the expression quantity of 1543.48u/g by using an Aspergillus oryzae solid state fermentation mode; screening a strain of adenylate deaminase honey aspergillus from a plurality of strains of penicillium and aspergillus for general people, and optimizing the ultraviolet rays, the composite mutagenesis treatment and the fermentation process to improve the deaminase expression level of the original strain by 16 times, so that the finally obtained deaminase expression level reaches 1300u/g; the expression level of Aspergillus oryzae deaminase is about 1560u/g obtained by solid state fermentation, and the enzyme is purified by salting out precipitation method and the enzymatic property is studied initially.
Chinese application number CN201510306409.0 discloses a liquid fermentation process of adenylate deaminase comprising the steps of: (1) strain activation: inoculating aspergillus melleus spores into a culture medium for culture; (2) seed preparation: inoculating the spore suspension into a seed shake flask culture medium for culture according to the inoculum size of 1-2.5%; (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 culturing according to the inoculation amount of 2-10%, wherein the rotation speed is 100-200rpm, the ventilation ratio is 1:0.3-1:1.2, and the enzyme activity of the obtained adenylate deaminase is 2000-3100U/mL.
The main source of the adenylate deaminase in the current market is Japanese wild enzyme preparation company, the domestic scale manufacturers are not more, and the fermentation enzyme activity is low, so that the production cost is high.
At present, the national production of the adenosine deaminase is mainly carried out by adopting an aspergillus solid state fermentation process, the activity of the fermentation enzyme is low, and the purity of the product is not enough; the yield in the separation and purification steps is low, and the total cost is too high. In addition, the single-time production yield is limited by the fermentation technology, and the production needs are far from being met by the site and environment.
Disclosure of Invention
For this reason, the technical problems solved by the present invention are: at present, the national production of the adenosine deaminase is mainly carried out by adopting an aspergillus solid state fermentation process, the activity of the fermentation enzyme is low, and the purity of the product is not enough; the yield in the separation and purification steps is low, and the total cost is too high.
The invention provides a bacillus subtilis strain (Bacillus subtilis ESP) for producing adenylate deaminase, which is screened from soil, and subjected to mutagenesis and domestication 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 China general microbiological culture collection center (CCTCC) with the preservation number of M2018827.
The invention provides a method for producing adenylate deaminase by liquid fermentation, which uses the strain, and the fermentation enzyme activity level is 14000-18000U/mL; the fermentation enzyme activity is high, 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 invention provides the following technical scheme.
The invention provides a strain for producing adenylate deaminase, which is a bacillus subtilis strain (Bacillus subtilis ESP) and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2018827.
The invention provides application of the strain in the field of fermentation, preferably in the fermentation production of adenylate deaminase.
The invention provides a method for producing adenylate deaminase, which uses the strain to carry out liquid fermentation to produce the adenylate deaminase.
Preferably, for the method described above, wherein the method comprises the steps of:
(1) Activating strains: inoculating the strain into 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 culturing to obtain seed bacterial liquid;
(3) Seed pot 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 fermentation.
Preferably, for the above method, wherein, in step (1), the culture temperature is 30 to 40 ℃, preferably, the culture time is 1 to 3 days.
Preferably, for the above method, wherein, in step (2), the culture temperature is 30 to 40 ℃, preferably 35 to 38 ℃; further preferably, the incubation time is 16 to 24 hours, preferably 18 to 22 hours.
Preferably, for the above method, wherein in step (3), the rotation speed is 50-150rpm, preferably 150-200rpm; preferably, the ventilation ratio is 1:0.5-1:1, preferably 1:0.8-1:1; further preferably, the cultivation temperature is 30-40deg.C, preferably 35-37deg.C; further preferably, the incubation time is 10 to 18 hours; it is further preferred that the inoculum size is 0.1% to 0.5% by volume, preferably 0.1% to 0.3% by volume.
Preferably, for the above method, 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 cultivation temperature is 30-40deg.C, preferably 35-37deg.C; further preferably, the incubation time is 30 to 42 hours, preferably 34 to 36 hours; it is further preferred that the inoculum size is 2% to 10% by volume, preferably 6% to 10% by volume.
Preferably, for the method described above, wherein, in step (4), the carbon source is supplemented after 3 to 6 hours of cultivation; preferably, the carbon source is a medium containing 30-60% glucose by mass volume.
Preferably, in the above method, the obtained fermentation broth is separated by plate-and-frame filtration to obtain a crude enzyme filtrate.
Preferably, in the above method, the crude enzyme filtrate is concentrated, preferably by 8-15 times, to obtain a concentrated solution.
Preferably, for the above method, the seed culture medium comprises, by mass volume, 0.1-0.5% yeast powder, 0.5-1.5% peptone, 0.5-1.5% sodium chloride, and the balance water.
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% by mass volume, and the balance being water.
The invention provides the adenylate deaminase obtained by fermentation by the method of any one of the above, 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 adenylate deaminase, and provides a method for producing adenylate deaminase by adopting the strain for liquid fermentation, wherein the fermentation enzyme activity is far higher than that of the prior reported fermentation enzyme activity, the fermentation enzyme activity is 14000-18000U/mL, the liquid fermentation mode is easier for industrial production of products, the fermentation liquid fermentation enzyme activity is high, conventional and soluble raw materials are used for fermentation, the risk of producing bacteria contamination is low, and the products are easy to separate.
Strain preservation information
The strain bacillus subtilis (Bacillus subtilis ESP, 710) used in the invention is preserved in China Center for Type Culture Collection (CCTCC) in the period of 11 and 26 of 2018, and the preservation number is CCTCC NO: M2018827, and the preservation address is: chinese, wuhan, university of Wuhan, postal code: 430072; telephone: (027) -68754052.
Detailed Description
As described above, the invention provides a bacillus subtilis, the strain is separated from soil, the strain obtained by separating is subjected to mutagenesis and domestication by mutagenesis technology such as ultraviolet and ARTP, the activity of the adenylate deaminase produced by fermenting the obtained strain is higher and is 14000-18000U/mL, the strain is sent to China general microbiological culture collection center (CCTCC) No: m2018827.
The invention provides a liquid fermentation method for producing adenylate deaminase by using the strain, wherein the method comprises the following steps:
(1) Activating strains: inoculating the strain into 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 culturing to obtain seed bacterial liquid;
(3) Seed pot 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 fermentation.
In a preferred embodiment of the invention, wherein in step (4), the fermentation pH is from 6.5 to 7.5; preferably, the Dissolved Oxygen (DO) is 40-60%; further preferably, the glucose content during fermentation is 5-15g/L.
In a preferred embodiment of the invention, wherein in step (4) the rotational speed is 100-200rpm, preferably the ventilation ratio is 1:0.5-1:1.2, preferably 1:0.8-1:2; further preferably, the cultivation temperature is 30-40deg.C, preferably 35-37deg.C; further preferably, the incubation time is 30 to 42 hours, preferably 34 to 36 hours; it is further preferred that the inoculum size is 2% to 10% by volume, preferably 6% to 10% by volume.
Wherein, for the cultivation time, it 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 strain of glycerol tube was inoculated into fresh medium with an inoculating loop under aseptic conditions and cultured at 37℃for 1 day.
(2) Seed preparation: inoculating a large ring of single colony to fresh seed shake flask culture medium, culturing in shake flask at 37deg.C for 16-24 hr,
(3) Seed pot culture: inoculating seed culture medium into seed tank via Karsch tank with inoculation amount of 0.1-0.5%, culturing temperature of 35-37 deg.c, rotation speed of 50-150 rpm and ventilation ratio of 1:0.5 to 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 for culture according to 2-8%, wherein the culture temperature is 35-37 ℃, the rotation speed is 100-200 rpm, the culture period is about 36 hours, and the ventilation ratio is 1:0.5 to 1:1.2, feeding carbon source after fermenting for 5 hours, and placing the tank to keep the enzyme activity level at 14000 u/ml-18000 u/ml after fermenting.
In a preferred embodiment of the present invention, the liquid fermentation method further comprises a step of centrifugation, ultrafiltration concentration, lyophilization or powder injection.
Preferably, the centrifugation is performed by removing cells by plate-and-frame separation.
Preferably, the ultrafiltration concentration refers to concentration of the crude enzyme filtrate 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, the formula of the seed culture medium is as follows, based on mass to volume ratio: yeast powder 0.1-0.5%, peptone 0.5-1.5%, sodium chloride 0.5-1.5%, and water in balance;
the formula of the fermentation medium is as follows: 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%, defoaming oil 0.01-0.2%, and water in balance;
the formula of the carbon source is as follows: 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 strains
(1) Obtaining a wild-type strain: the strain is obtained from the periphery or soil of fermented bean curd or soy sauce factories, and comprises the following specific steps:
1) Sample collection: collecting samples from soil around a fermented bean curd or soy sauce factory or from the fermented bean curd semi-finished product or soy sauce fermentation semi-finished product;
2) Enrichment of microorganisms: placing the collected sample in a constant temperature environment at 37 ℃ for more than 48 hours until the collected sample is dried; inoculating a proper amount of dried sample into a fresh culture medium, and carrying out enrichment culture; the formula of the 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 and the balance of water;
3) Single colonies were obtained: the enriched sample is subjected to plate culture after gradient dilution, and single colony is obtained.
(2) Mutagenesis
Mutagenesis of the starting strain by means of ARTP mutagenesis
Preparing equipment: normal operation according to the equipment instruction; (ARTP-IIS, qingshan Ski biotechnology Co., ltd., without tin source)
Preparing a bacterial suspension: centrifugally collecting bacterial liquid cultured to logarithmic phase, washing with physiological saline for 2 times, re-dissolving with physiological saline until OD is about 0.5, and the bacterial load is 10 6 ～10 8 Left and right;
placing a metal slide on an ultra-clean bench, burning the metal slide for about 20 seconds by using an alcohol lamp outer flame, cooling, and uniformly coating 10 mu L of bacterial liquid on the slide on a sterile flat plate;
moving a flat plate with a sample slide to an operation bin of an ARTP mutagenesis system, placing the slide into a corresponding hole site by using sterile forceps, adjusting a knob below a carrier to enable the slide to be positioned at a position of 2mm of an airflow port, and closing a bin door;
setting the power to 100W, the flow rate to 10SLM and the time to 31s;
after the sample treatment, placing the slide glass into a centrifuge tube filled with 1mL of sterile water by using sterile forceps; fully and uniformly oscillating; forming a new bacterial suspension;
carrying out gradient dilution coating on the new bacterial suspension to obtain single bacterial colony; the detection mortality rate is 99.17%;
(3) Physiological and biochemical identification and 16S rDNA identification
1) Physiological and biochemical experiments: liquefying gelatin, hydrolyzing starch, V.P test positive, reducing nitrate, lecithin enzyme test negative, gram positive;
2) Morphology-specific: the colony is white, moist, irregular in shape, provided with punctate protrusions, opaque, round or irregular in edge; the cells are in a long rod shape or a chain shape;
3) Identification of 16S rDNA: the primer is shown as SEQ ID NO.2 and SEQ ID NO.3, wherein the sequence of SEQ ID NO.2 is 5'-AGAGTTTGATCCTGGCTCAG-3' SEQ ID NO. 3: 5'-ACGGTTACCTTGTTACGACTT-3' the primers are provided by October Dingsheng Biotechnology (Wuhan Co., ltd.)
The PCR reaction system is as follows:
Figure GDA0003831986840000081
PCR amplification conditions: pre-denaturation at 94℃for 4min; denaturation at 94℃for 1min, annealing at 50℃for 1min, extension at 72℃for 1min, amplification for 30 cycles; 72 ℃ for 10min
The amplified sequence was sent to October biosciences (Wuhan) limited for sequencing, whose 16SrDNA was shown in SEQ ID NO.1, and the sequence was subjected to BLAST nucleotide alignment analysis using NCBI website, with 100% homology to the Bacillus subtilis 16S rDNA sequence (Gene Bank: MK-183007.1 and NR-112116.2).
Example 2-1 fermentative production of adenylate deaminase
(1) Strain activation: selecting glycerol tube strains, and activating for 1d in a fresh plate culture medium at 37 ℃ to obtain single colonies, wherein the plate culture medium contains 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride by 100L of water;
(2) Seed preparation: 1-2 single colonies after activation were selected and transferred to a flask (100 mL of liquid in 500mL flask) containing fresh seed medium (the composition and content of which are the same as those in step (1)), and seed liquid was prepared under culture conditions: culturing at 37 ℃ and 200rpm for 18 hours;
(3) Seed pot culture: inoculating the seed solution obtained in the step (2) into a 3L seed tank containing a seed culture medium in an inoculum size of 0.2% by volume for culturing to obtain mature seeds, wherein the culture temperature is 35 ℃, the rotation speed is 150rpm, and the aeration ratio is 1:0.8, a culture period of 15 hours, wherein the seed culture medium comprises 0.1% of yeast powder, 1.5% of peptone, 1.5% of sodium chloride and the balance of water according to mass-volume ratio;
(4) Culturing in a fermentation tank: inoculating mature seeds to a fermentation tank with 5L of liquid loading capacity of 3L according to the inoculation amount of 6% by volume, fermenting at 37 ℃ for 5 hours, and then adding a carbon source in a flowing manner, wherein the carbon source is a culture medium containing 40% glucose according to the mass-volume ratio, and the balance is water; controlling the content of glucose in the fermentation liquor to be about 5g/L, wherein the pH value is 6.5, the rotating speed is 100rpm, the ventilation ratio is 1:0.8, the DO value of the fermentation liquor is 40%, the re-fermentation time is 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, concentrating by a factor of 8, and freeze-drying to obtain the solid adenylate deaminase, wherein the fermentation culture medium comprises 0.5% of glucose, 0.65% of peptone, 0.15% 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 and 0.01% of defoaming oil, and the balance of water according to the mass-volume ratio.
The method for measuring the enzyme activity of the adenylate deaminase comprises the following steps:
accurately weighing 13.9mg of 5' -adenylate dissolved in 1mL of 0.08 mol.L -1 NaHCO of (C) 3 In solution as original substrate, then using 0.1 mol.L -1 Succinic acid-NaOH (pH 6.0) was diluted 400-fold to give a concentration of 0.1X10 -3 mol·L -1 Is a substrate for a reaction. Adding 3mL of reaction substrate into a test tube, placing the test tube in a water bath at 37 ℃ for heat preservation for 5min, adding 100 mu L of pre-diluted adenylate deaminase solution for immediate timing, and uniformly mixing the reaction substrate and the enzyme solutionAfter 15min, the reaction was stopped by adding 3mL of 10% perchloric acid solution. The control experiment method is the same as that of the control experiment method, the reaction substrate is precooled by ice water after being in water bath at 37 ℃, 3mL of 10% perchloric acid is added, 100 mu L of enzyme solution is added for uniform mixing, an ultraviolet spectrophotometer is used for measuring the absorbance value at 265nm by taking deionized water as a reference, and a quartz cuvette is used for measuring the absorbance value.
Enzyme activity was defined as the change of absorbance at 265nm wavelength per minute of 0.001 as one activity unit of enzyme under the above conditions.
The calculation formula is as follows: enzyme activity (U.mL) -1 )＝(△A265×K×10)/(0.001×T)。
Delta A265 is the difference between the absorbance of the reaction solution and the absorbance of the control group; k is the dilution multiple of the enzyme solution; t is the reaction time, min
The enzyme activity of the resulting adenylate deaminase was 15800U/mL as measured by the method described above.
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 single colonies, wherein the components and the contents of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) Seed preparation: 1-2 single colonies after activation were selected and transferred to a triangular flask (100 mL of 500mL triangular flask liquid) containing fresh seed medium (the components and content of the seed medium are the same as those in step (2) of example 2-1), and seed liquid was prepared under the culture conditions: culturing at 37 ℃ and 200rpm for 20 hours;
(3) Seed pot culture: inoculating the seed solution obtained in the step (2) into a 3L seed tank containing a seed culture medium at an inoculum size of 0.3w/v% and culturing to obtain mature seeds, wherein the culture temperature is 36 ℃, the rotation speed is 200rpm, and the aeration ratio is 1:0.9, a culture period of 18 hours, wherein the seed culture medium comprises 0.5% of yeast powder, 0.5% of peptone, 1.5% of sodium chloride and the balance of water according to mass-volume ratio;
(4) Culturing in a fermentation tank: inoculating mature seeds to a fermentation tank with 5L of liquid loading capacity of 3L according to the inoculation amount of 6% by volume, fermenting at 37 ℃ for 5 hours, and then adding a carbon source in a flowing manner, wherein the carbon source is a culture medium containing 30% glucose according to the mass-volume ratio, and the balance is water; controlling the glucose content 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, and the DO of the fermentation liquor to be 40%; fermenting for 31 hours, 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, wherein the concentration multiple is 10 times, and performing freeze-drying treatment to obtain the solid adenosine deaminase, wherein the fermented culture medium 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 and 0.01% of defoaming oil, and the balance of water according to the mass-volume ratio.
The resulting adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the resulting adenylate deaminase had a fermentation enzyme activity of 16800U/mL.
EXAMPLES 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 single colonies, wherein the components and the contents of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) Seed preparation: 1-2 single colonies after activation were selected and transferred to a triangular flask (100 mL of 500mL triangular flask liquid) containing fresh seed medium (the components and content of the seed medium are the same as those in step (2) of example 2-1), and seed liquid was prepared under the culture conditions: culturing at 37 ℃ and 200rpm for 20 hours;
(3) Seed pot culture: inoculating the seed liquid obtained in the step (2) into a 5L seed tank with the liquid loading capacity of 1L according to the inoculation amount of 0.1% by volume to culture to obtain mature seeds, wherein the culture temperature is 37 ℃, the rotation speed is 200rpm, the ventilation ratio is 1:1, the culture period is 12 hours, and the seed culture medium comprises 0.4% of yeast powder, 1% of peptone and 1% of sodium chloride and the balance of water according to the mass volume ratio;
(4) Culturing in a fermentation tank: inoculating mature seeds into a 50L fermentation tank with a liquid loading amount of 25L according to an inoculation amount of 6.8% by volume, starting feeding a carbon source after fermenting for 5 hours, wherein the carbon source is a culture medium containing 50% glucose according to a mass-volume ratio, and the balance is water; controlling the glucose content 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 fermentation liquor DO to be 40%; and (3) fermenting for 36 hours, 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, wherein the concentration multiple is 15 times, and performing powder spraying treatment to obtain the solid adenylate deaminase, wherein the fermented culture 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 resulting adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the resulting adenylate deaminase had a fermentation enzyme activity of 14500U/mL.
EXAMPLES 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 single colonies, wherein the components and the contents of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) Seed preparation: 1-2 single colonies after activation are selected and transferred into a triangular flask (100 mL of 500mL triangular flask liquid) filled with fresh seed culture medium, seed liquid is prepared, and culture conditions are as follows: culturing at 37 ℃ and 200rpm for 20 hours;
(3) Seed pot culture: inoculating the seed liquid obtained in the step (2) into a 5L seed tank with the liquid loading capacity of 1L according to the inoculation amount of 0.1% by volume to culture 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 14 hours, and the seed culture medium comprises 5% of yeast powder, 0.5% of peptone and 0.5% of sodium chloride and the balance of water according to the mass volume ratio;
(4) Culturing in a fermentation tank: inoculating mature seeds into a 50L fermentation tank with a liquid loading amount of 30L according to an inoculation amount of 8% by volume, starting feeding a carbon source after fermenting for 4.5 hours, wherein the carbon source is a culture medium containing 60% glucose according to a mass-volume ratio, and the balance is water; controlling the glucose content in the fermentation liquor to be about 5g/L, the pH value to be 6.5, the rotating speed to be 120rpm, the ventilation ratio to be 1:0.5, and the fermentation liquor DO to be 40%; and (3) fermenting for 36 hours, 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, wherein the concentration multiple is 12 times, and performing powder spraying treatment to obtain the solid adenylate deaminase, wherein the fermented culture medium comprises, by mass and volume ratio, 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.
The resulting adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the resulting adenylate deaminase had a fermentative enzyme activity of 15500U/mL.
EXAMPLES 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 single colonies, wherein the components and the contents of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) Seed preparation: 1-2 single colonies after activation were selected and transferred to a triangular flask (400 mL of 2000mL triangular flask liquid) containing fresh seed medium (the composition and content of the seed medium are the same as those in step (2) of example 2-1), and seed liquid was prepared under culture conditions: culturing at 37 ℃ and 200rpm for 20 hours;
(3) Seed pot culture: inoculating the seed solution obtained in the step (2) to a liquid loading amount of 1.5m at an inoculation amount of 0.2% by volume 3 5m of (5) 3 The mature seeds are obtained by culturing in a seed tank, wherein the culture temperature is 37 ℃, the rotation speed is 150rpm, and the ventilation ratio is 1:0.8, a culture period of 12 hours, wherein the seed culture medium comprises 0.4% of yeast powder, 1% 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 to 15m of liquid loading amount with 10% of volume ratio of inoculation amount 3 25m of (2) 3 In a fermentation tank, after fermenting for 5 hours, starting to feed a carbon source, wherein the carbon source is a culture medium containing 30% of glucose according to mass-volume ratio, and the balance is water; the content of glucose in the fermentation liquor is controlled to be about 5g/L, the pH value is 7.5, the rotating speed is 100rpm,ventilation ratio 1:0.5, fermentation liquor DO is 40%; and (3) fermenting for 34 hours, 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, wherein the concentration multiple is 8 times, and performing powder spraying treatment to obtain the solid adenylate deaminase, wherein the fermented culture 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 and 0.01% of defoaming oil, and the balance of water according to the mass-volume ratio.
The resulting adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the resulting enzyme activity of the fermentation of the adenylate deaminase was 17700U/mL.
EXAMPLES 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 single colonies, wherein the components and the contents of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) Seed preparation: 1-2 single colonies after activation were selected and transferred to a triangular flask (400 mL of 2000mL triangular flask liquid) containing fresh seed medium (the composition and content of the seed medium are the same as those in step (2) of example 2-1), and seed liquid was prepared under culture conditions: culturing at 37 ℃ and 200rpm for 18 hours;
(3) Seed pot culture: inoculating the seed solution obtained in the step (2) to a liquid loading amount of 1.5m at an inoculation amount of 0.3% by volume 3 5m of (5) 3 The mature seeds are obtained by culturing in a seed tank, wherein the culture temperature is 37 ℃, the rotation speed is 150rpm, and the ventilation ratio is 1:0.8, a culture period of 11 hours, wherein the seed culture medium comprises 0.5% of yeast powder, 1.5% 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 to 15m of liquid loading amount with 10% of volume ratio of inoculation amount 3 25m of (2) 3 In a fermentation tank, starting to feed a carbon source after fermenting for 4.5 hours, wherein the carbon source is a culture medium containing 45% of glucose according to mass-volume ratio, and the balance is water; the content of glucose in the fermentation liquor is controlled to be about 5g/L,pH 7, rotation speed 150rpm, ventilation ratio 1:1, fermentation liquor DO is 40%; and (3) fermenting for 34 hours, 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, wherein the concentration multiple is 15 times, and performing freeze-drying treatment to obtain the solid adenosine deaminase, wherein the fermented culture medium comprises, by mass and volume ratio, 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.
The resulting adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the resulting adenylate deaminase had a fermentation enzyme activity of 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 single colonies, wherein the components and the contents of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) Seed preparation: 1-2 single colonies after activation were selected and transferred to a triangular flask (400 mL of 2000mL triangular flask liquid) containing fresh seed medium (the composition and content of the seed medium are the same as those in step (2) of example 2-1), and seed liquid was prepared under culture conditions: culturing at 37 ℃ and 200rpm for 22 hours;
(3) Seed pot culture: inoculating the seed solution obtained in the step (2) to a liquid loading amount of 1.5m at an inoculation amount of 0.1% by volume 3 5m of (5) 3 The mature seeds are obtained by culturing in a seed tank, wherein the culture temperature is 37 ℃, the rotation speed is 150rpm, and the ventilation ratio is 1:0.8, a culture period of 16 hours, wherein 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 to 15m of liquid loading amount with 10% of volume ratio of inoculation amount 3 25m of (2) 3 In a fermentation tank, after fermenting for 5 hours, starting to feed a carbon source, wherein the carbon source is a culture medium containing 40% of glucose according to mass-volume ratio, and the balance is water; controlling the glucose content in the fermentation brothAbout 5g/L, pH 7, rotation speed 200rpm, ventilation ratio 1:1.2, fermenting the fermentation liquor DO of 40 percent for 36 hours, 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, wherein the concentration multiple is 12 times, and then performing freeze-drying treatment to obtain the solid adenylate deaminase, wherein the fermentation culture medium comprises 0.5 percent of glucose, 3 percent of peptone, 0.65 percent of ammonium sulfate, 0.15 percent of calcium chloride, 0.2 percent of sodium citrate, 1.5 percent of sodium dihydrogen phosphate, 0.5 percent of disodium hydrogen phosphate, 0.01 percent of defoaming oil and the balance of water according to the mass-volume ratio.
The resulting adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the resulting enzyme activity of the fermentation of the adenylate deaminase was 17900U/mL.
EXAMPLES 2-8 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 30 ℃ for 3d to obtain single colonies, wherein the components and the contents of the plate culture medium are the same as those in the step (1) of the example 2-1;
(2) Seed preparation: 1-2 single colonies after activation were selected and transferred to a triangular flask (400 mL of 2000mL triangular flask liquid) containing fresh seed medium (the composition and content of the seed medium are the same as those in step (2) of example 2-1), and seed liquid was prepared under culture conditions: culturing at 30 ℃ and 200rpm for 24 hours;
(3) Seed pot culture: inoculating the seed solution obtained in the step (2) to a liquid loading amount of 1.5m at an inoculation amount of 0.5% by volume 3 5m of (5) 3 The mature seeds are obtained by culturing in a seed tank, wherein the culture temperature is 30 ℃, the rotation speed is 50rpm, and the ventilation ratio is 1:1, a culture period of 18 hours, 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 mass volume ratio;
(4) Culturing in a fermentation tank: inoculating mature seeds to 15m of liquid loading amount with 10% of volume ratio of inoculation amount 3 25m of (2) 3 In a fermentation tank, after fermentation for 6 hours, starting to feed a carbon source, wherein the carbon source is a culture medium containing 60% of glucose according to mass-volume ratio, and the balance is water; controlling fermentation brothThe glucose content in the mixture is about 5g/L, the pH value is 6.5, the rotating speed is 150rpm, and the ventilation ratio is 1:1, fermenting fermentation liquor DO of 60 percent for 36 hours, 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, wherein the concentration multiple is 10 times, and performing freeze-drying treatment to obtain solid adenylate deaminase, wherein the fermentation culture medium comprises 1.5 percent of glucose, 2 percent of peptone, 0.15 percent of ammonium sulfate, 0.15 percent of calcium chloride, 0.01 percent of sodium citrate, 0.5 percent of sodium dihydrogen phosphate, 1.5 percent of disodium hydrogen phosphate and 0.2 percent of defoaming oil and the balance of water according to the mass-volume ratio.
The resulting adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the resulting adenylate deaminase had a fermentative enzyme activity of 14800U/mL.
EXAMPLES 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 single colonies after activation were selected and transferred to a triangular flask (400 mL of 2000mL triangular flask liquid) containing fresh seed medium (the composition and content of the seed medium are the same as those in step (2) of example 2-1), and seed liquid was prepared under culture conditions: culturing at 40 ℃ and 200rpm for 16 hours;
(3) Seed pot culture: inoculating the seed solution obtained in the step (2) to a liquid loading amount of 1.5m at an inoculation amount of 0.1% by volume 3 5m of (5) 3 The mature seeds are obtained by culturing in a seed tank, wherein the culture temperature is 40 ℃, the rotation speed is 100rpm, and the ventilation ratio is 1:0.5, a culture period of 10 hours, wherein the seed culture medium comprises 0.5% of yeast powder, 1.2% 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 to 15m of liquid loading amount with 2% of inoculation amount by volume 3 25m of (2) 3 In a fermentation tank, after fermentation for 3 hours, starting to feed a carbon source, wherein the carbon source is a culture medium containing 50% of glucose according to mass-volume ratio, and the balance is waterThe method comprises the steps of carrying out a first treatment on the surface of the Controlling the content of glucose in the fermentation liquor to be about 5g/L, pH to be 7.5, rotating speed to be 100rpm, and ventilation ratio to be 1:1.2, fermentation liquor DO is 50%; fermenting for 27h, 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, wherein the concentration multiple is 15 times, and performing powder spraying treatment to obtain the solid adenylate deaminase, wherein the fermented culture medium 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 and 0.2% of defoaming oil, and the balance of water according to the mass-volume ratio.
The resulting adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the resulting adenylate deaminase had a fermentation enzyme activity of 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 2d to obtain single colonies, 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 single colonies after activation were selected and transferred to a triangular flask (400 mL of 2000mL triangular flask liquid) containing fresh seed medium (the composition and content of the seed medium are the same as those in step (2) of example 2-1), and seed liquid was prepared under culture conditions: culturing at 35 ℃ and 200rpm for 20 hours;
(3) Seed pot culture: inoculating the seed solution obtained in the step (2) to a liquid loading amount of 1.5m at an inoculation amount of 0.4% by volume 3 5m of (5) 3 The mature seeds are obtained by culturing in a seed tank, wherein the culture temperature is 33 ℃, the rotation speed is 155rpm, and the ventilation ratio is 1:0.9, a culture period of 12 hours, wherein the seed culture medium comprises 0.1% of yeast powder, 0.5% of peptone, 0.5% of sodium chloride and the balance of water according to mass volume ratio;
(4) Culturing in a fermentation tank: inoculating mature seeds to 15m of liquid loading amount with 5% of inoculation amount by volume 3 25m of (2) 3 In a fermentation tank, after fermentation for 4 hours, starting feeding a carbon source, wherein the carbon source is a culture containing 30% of glucose according to mass-volume ratioNourishing medium, and water in balance; the glucose content in the fermentation broth is controlled to be about 5g/L, the pH value is 7, the rotating speed is 200rpm, and the ventilation ratio is 1:0.5, fermentation liquor DO is 40%; fermenting for 34 hours, 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, wherein the concentration multiple is 8 times, and performing freeze-drying treatment to obtain the solid adenosine deaminase, wherein the fermented culture medium 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-volume ratio.
The resulting adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the resulting adenylate deaminase had a fermentative enzyme activity of 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 single colonies after activation were selected and transferred to a triangular flask (400 mL of 2000mL triangular flask liquid) containing fresh seed medium (the composition and content of the seed medium are the same as those in step (2) of example 2-1), and seed liquid was prepared under culture conditions: culturing at 38 ℃ and 200rpm for 22 hours;
(3) Seed pot culture: inoculating the seed solution obtained in the step (2) to a liquid loading amount of 1.5m at an inoculation amount of 0.3% by volume 3 5m of (5) 3 The mature seeds are obtained by culturing in a seed tank, wherein the culture temperature is 37 ℃, the rotation speed is 200rpm, and the ventilation ratio is 1:1, a culture period of 14 hours, wherein the seed culture medium comprises 0.4% of yeast powder, 1.5% 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 to 15m of liquid loading amount with 10% of volume ratio of inoculation amount 3 25m of (2) 3 In a fermentation tank, after fermentation for 4.5 hours, starting to add a carbon source, wherein the carbon source contains 45 percent ofGlucose culture medium, and water in balance; the glucose content in the fermentation broth is controlled to be about 5g/L, the pH value is 7, the rotating speed is 140rpm, and the ventilation ratio is 1:1.2, fermenting liquor DO is 55%, the fermentation period is 31.5 hours, then removing thalli by adopting plate and frame separation to obtain crude enzyme filtrate, concentrating the crude enzyme filtrate by an ultrafiltration membrane concentration system, wherein the concentration multiple is 15 times, and then performing freeze-drying treatment to obtain the solid adenylate deaminase, wherein the fermentation culture 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, and the balance is water according to the mass-volume ratio.
The resulting adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the resulting adenylate deaminase had a fermentative enzyme activity of 16527U/mL.
Comparative example 1
(1) Activating strains: selecting a glycerol tube strain (wild type strain, i.e., the strain prior to mutation in example 1) and activating for 1d at 37℃in fresh plate medium having the same composition and content as in step (1) of example 2-1 to obtain single colonies;
(2) Seed preparation: 1-2 single colonies after activation were selected and transferred to a triangular flask (100 mL of 500mL triangular flask liquid) containing fresh seed medium (the components and content of the seed medium are the same as those in step (2) of example 2-1), and seed liquid was prepared under the culture conditions: culturing at 37 ℃ and 200rpm for 20 hours;
(3) Seed pot culture: inoculating the seed liquid obtained in the step (2) into a 5L seed tank with the liquid loading capacity of 1L according to the inoculation amount of 0.1% by volume to culture to obtain mature seeds, wherein the culture temperature is 37 ℃, the rotation speed is 200rpm, the ventilation ratio is 1:1, the culture period is 12 hours, and the seed culture medium comprises 0.4% of yeast powder, 1% of peptone and 1% of sodium chloride and the balance of water according to the mass volume ratio;
(4) Culturing in a fermentation tank: inoculating mature seeds into a 50L fermentation tank with a liquid loading amount of 25L according to an inoculation amount of 6.8% by volume, starting feeding a carbon source after fermenting for 5 hours, wherein the carbon source is a culture medium containing 50% glucose according to a mass-volume ratio, and the balance is water; controlling the glucose content 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 fermentation liquor DO to be 40%; and (3) fermenting for 36 hours, 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, wherein the concentration multiple is 15 times, and performing powder spraying treatment to obtain the solid adenylate deaminase, wherein the fermented culture 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 resulting adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the resulting adenylate deaminase had a fermentation enzyme activity of 3233U/mL.
Comparative example 2
(1) Activating strains: one strain of the glycerol tube was selected, one loop was picked with a 10. Mu.L inoculating loop, 200g PDA medium was inoculated in a sterile environment, placed in a 28℃environment for 3 days, and eluted with 500mL sterile water to give a single colony.
(2) Seed preparation: and (3) inoculating the single colony into 2.7L of seed shake flask culture medium according to the inoculation amount of 2%, and culturing at 28 ℃ for 22 hours to obtain 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 pot culture: according to the inoculation amount of 0.3%, inoculating seed bacterial liquid into a seed tank (storage amount is 1 ton) filled with 900L of seed culture medium, and culturing to obtain seed culture liquid, wherein the culture temperature is 30 ℃, the rotation speed is 100rpm, the ventilation ratio is 1:0.7, and the culture period is 26 hours. Wherein the seed culture medium is prepared from the following components in percentage by weight: 10% of soybean juice, 2% of sucrose and K 2 HP0 4 0.5 percent of yeast powder 0.2 percent, and the balance of water, and the pH value is adjusted to 6.0 before sterilization.
(4) Fermentation production: according to the inoculation amount of 6%, inoculating the seed culture solution into a fermentation tank (storage amount is 25 tons) filled with 15 tons of fermentation medium for culturing, wherein the culture temperature is 29 ℃, the rotation speed is 150rpm, the ventilation ratio is 1:0.7, and the culture period is 115 hours When the fermentation is finished, the enzyme activity level is measured by placing the fermentation tank. Wherein the fermentation medium is prepared from the following components in percentage by weight: glucose 3%, peptone 2%, disodium citrate trihydrate 0.3%, mgSO 4 ·7H 2 0.05% of O, 5% of trace element mother liquor, 0.12% of Tween-80, and the balance of water, and adjusting pH to 6.0 before sterilization. The trace element mother liquor is prepared from the following components in percentage by weight: zinc sulfate 2%, calcium chloride 2% and water in balance.
(5) And (3) plate frame treatment: removing thalli by plate frame filtration to obtain crude enzyme filtrate.
(6) Ultrafiltration concentration: concentrating the crude enzyme filtrate by ultrafiltration concentration equipment, wherein the concentration multiple is controlled to be 10 times, and obtaining concentrated solution.
(7) And (3) freeze-drying: and (3) carrying out freeze-drying treatment on the concentrated solution to obtain the solid deaminase.
The resulting adenylate deaminase was subjected to an enzyme activity test as described in example 2-1, and the resulting enzyme activity of the fermentation of the adenylate deaminase was 3466U/mL.
The difference between comparative example 1 and examples 2 to 3 is that the strain used is different, the strain used in comparative example 1 is the strain before mutagenesis, i.e., the wild-type strain, and the strain used in examples 2 to 3 is the strain after mutagenesis, and the enzyme activity of the resulting adenylate deaminase is significantly different, the enzyme activity of the resulting adenylate deaminase in examples 2 to 3 is 14500U/mL, and the enzyme activity of the resulting adenylate deaminase in comparative example 1 is 3233U/mL, indicating that the enzyme activity of the resulting adenylate deaminase is higher using the strain after mutagenesis.
Comparison of comparative example 2 and examples 2-3 is different in that the liquid fermentation method is different in the enzyme activity of the obtained adenylate deaminase, the enzyme activity of the adenylate deaminase obtained in comparative example 2 is 3466U/mL, and the enzyme activity of the adenylate deaminase obtained in examples 2-3 is 14500U/mL, which is 4 times or more than that of comparative example 2, indicating that the strain of the present invention has high enzyme activity of the adenylate deaminase obtained under the process conditions of the fermentation method of the present invention.
The above description is not intended to limit the invention in any way, but is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Figure IDA0002146463720000011
Figure IDA0002146463720000021

Claims (46)

1. A strain for producing adenylate deaminase is characterized in that the strain is a bacillus subtilis strainBacillus subtilis) ESP710, which is preserved in China center for type culture Collection, has a preservation number of CCTCC NO: M2018827.
2. Use of the strain of claim 1 for the fermentative production of adenylate deaminase.
3. A method for producing an adenylate deaminase, wherein the adenylate deaminase is produced by liquid fermentation using the strain of claim 1.
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 culturing to obtain seed bacterial liquid;
(3) Seed pot 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 fermentation.
5. The method according to claim 4, wherein in the step (1), the culture temperature is 30 to 40 ℃.
6. The method according to claim 4, wherein, in the step (1), the culturing time is 1 to 3 days.
7. The method according to claim 4, wherein in the step (2), the culture temperature is 30 to 40 ℃.
8. The method according to claim 5, wherein in the step (2), the culture temperature is 30 to 40 ℃.
9. The method according to claim 4, wherein in the step (2), the culture temperature is 35 to 38 ℃.
10. The method according to claim 4, wherein in the step (2), the culturing time is 16 to 24 hours.
11. The method according to claim 5, wherein, in the step (2), the culturing time is 16 to 24 hours.
12. The method according to claim 7, wherein, in the step (2), the culturing time is 16 to 24 hours.
13. The method according to claim 4, wherein in the step (2), the culturing time is 18 to 22 hours.
14. The method according to claim 4, wherein in the step (3), the rotation speed is 50 to 150rpm.
15. The method according to claim 5, wherein in the step (3), the rotation speed is 50-150rpm.
16. The method according to claim 7, wherein in the step (3), the rotation speed is 50-150rpm.
17. The method according to claim 10, wherein in step (3), the rotation speed is 50-150rpm.
18. The method according to claim 4, wherein in the step (3), the rotation speed is 150-200rpm.
19. The method of claim 4, wherein in step (3), the ventilation ratio is 1:0.5-1:1.
20. The method of claim 5, wherein in step (3), the ventilation ratio is 1:0.5-1:1.
21. The method of claim 7, wherein in step (3), the ventilation ratio is 1:0.5-1:1.
22. The method of claim 10, wherein in step (3), the ventilation ratio is 1:0.5-1:1.
23. The method of claim 14, wherein in step (3), the ventilation ratio is 1:0.5-1:1.
24. The method of claim 4, wherein in step (3), the ventilation ratio is 1:0.8-1:1.
25. The method according to claim 4, wherein in the step (3), the culture temperature is 30 to 40 ℃.
26. The method according to claim 4, wherein in the step (3), the culture temperature is 35 to 37 ℃.
27. The method according to claim 4, wherein in the step (3), the culturing time is 10 to 18 hours.
28. The method of claim 4, wherein in step (3), the inoculum size is 0.1% -0.5% by volume.
29. The method according to claim 4, wherein in the step (3), the inoculation amount is 0.1 to 0.3% by volume.
30. The method according to any one of claims 4 to 29, wherein in step (4), the rotational speed is 100 to 200rpm.
31. The method of any one of claims 4-29, wherein in step (4), the ventilation ratio is 1:0.5-1:1.2.
32. The method according to any one of claims 4 to 29, wherein in step (4), the ventilation ratio is 1:0.8-1:2.
33. the method according to any one of claims 4 to 29, wherein in step (4), the culture temperature is 30 to 40 ℃.
34. The method according to any one of claims 4 to 29, wherein in step (4), the culture temperature is 35 to 37 ℃.
35. The method according to any one of claims 4 to 29, wherein in step (4), the incubation time is 30 to 42 hours.
36. The method according to any one of claims 4 to 29, wherein in step (4), the incubation time is 34 to 36 hours.
37. The method according to any one of claims 4 to 29, wherein in step (4) the inoculum size is 2% to 10% by volume.
38. The method according to any one of claims 4 to 29, wherein in step (4) the inoculum size is 6 to 10% by volume.
39. The method according to any one of claims 4 to 29, wherein in step (4), the carbon source is supplemented after 3 to 6 hours of cultivation.
40. The method of claim 39, wherein the carbon source is a medium containing 30-60% glucose by mass volume.
41. The method of any one of claims 4-29, wherein the resulting fermentation broth is separated by plate and frame filtration to provide a crude enzyme filtrate.
42. The method of claim 41, wherein the crude enzyme filtrate is concentrated.
43. The method of claim 42, wherein the concentration factor is 8-15 to obtain a concentrate.
44. The method according to any one of claims 4 to 29, wherein the seed medium comprises, by mass volume, yeast powder 0.1 to 0.5%, peptone 0.5 to 1.5% and sodium chloride 0.5 to 1.5%, the balance being water.
45. The method according to any one of claims 4 to 29, wherein the fermentation medium comprises glucose 0.5 to 1.5%, peptone 1.5 to 4%, ammonium sulfate 0.15 to 0.65%, calcium chloride 0.01 to 0.15%, sodium citrate 0.01 to 0.2%, sodium dihydrogen phosphate 0.5 to 1.5%, disodium hydrogen phosphate 0.5 to 1.5% and defoaming oil 0.01 to 0.2% by mass volume, and the balance being water.
46. The adenylate deaminase fermented by the process of any of claims 4-45 wherein the enzyme activity is 14000-18000U/mL.
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