CN107488615B

CN107488615B - Pseudomonas capable of producing lipase at high yield and fermentation enzyme production method thereof

Info

Publication number: CN107488615B
Application number: CN201710838176.8A
Authority: CN
Inventors: 王兴吉; 郭庆文; 刘文龙; 曹世源; 佟新伟
Original assignee: Shandong Lonct Enzymes Co ltd
Current assignee: Shandong Lonct Enzymes Co ltd
Priority date: 2017-09-18
Filing date: 2017-09-18
Publication date: 2020-01-14
Anticipated expiration: 2037-09-18
Also published as: CN107488615A

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to pseudomonas capable of producing lipase at high yield and a fermentation enzyme production method thereof. The Pseudomonas is specifically Pseudomonas (Pseudomonas sp.) LD-14, and the strain preservation number is CGMCC No. 14414. The enzyme activity of fermentation liquor for producing lipase by pseudomonas LD-14 fermentation reaches 25000U/mL to 26000U/mL; the produced lipase has the optimum pH range of 8.5-10.0, the optimum action temperature range of 30-40 ℃, and the residual enzyme activity of 80 percent after heat preservation for 1 day at 20 ℃, has obvious low temperature resistance and alkali resistance, and can be widely applied to industrial production.

Description

Pseudomonas capable of producing lipase at high yield and fermentation enzyme production method thereof

The technical field is as follows:

the invention belongs to the technical field of microorganisms, and particularly relates to pseudomonas capable of producing lipase at high yield and a fermentation enzyme production method thereof.

Background art:

lipases (EC 3.1.1.3) are a generic term for a class of enzymes capable of hydrolyzing triglycerides at the oil-water interface, which hydrolyze triacylglycerides to fatty acids, diglycerides, monoglycerides and glycerol, the natural substrates of which are generally water-insoluble acyl esters of long-chain fatty acids.

The lipase is mainly derived from plants, animals and microorganisms, wherein the microbial lipase is widely existed in bacteria, yeasts and molds, has the characteristics of multiple types, short period, quick propagation and easy genetic variation, has wider action temperature, action pH and substrate specificity than animal and plant lipases, can catalyze the hydrolysis, alcoholysis, acidolysis, ester exchange, synthesis and the like of ester compounds under the condition of no coenzyme, has the characteristics of mild catalysis condition, low energy consumption, less byproducts, high efficiency, high selectivity, environmental friendliness and the like, and changes the relatively harsh conditions of high temperature, strong acid, strong base and the like required by the traditional esterification or transesterification reaction. The microbial lipase is suitable for industrial production, can produce high-purity products, and has great value in basic theoretical research and practical application. At present, the production method of lipase mainly comprises an extraction method and a microbial fermentation method, wherein the microbial fermentation method is the preferred method for producing lipase with higher yield, lower cost and wide source of strain resources.

Because of the particularity of the enzymatic properties of lipase and modified preparations thereof, the lipase and modified preparations thereof are widely applied to many fields such as food and nutriment processing industry, grease chemical industry, daily chemical industry, medicine and agriculture, bioactive agent synthesis and the like. The wide application range and abundant resources of the method show more and more important positions in industrial production.

The optimum reaction temperature of the lipase in the prior art is generally 40 ℃ or higher, and the lipase is seriously inactivated below 30 ℃, so the lipase is restricted by enzyme activity and enzyme application conditions, and the lipase in the prior art still cannot meet the requirements of industrial production, so the pseudomonas strain with high lipase yield is bred, the fermentation product of the pseudomonas strain has good alkali resistance and low temperature resistance, the production cost is low, and the lipase has great significance for promoting mechanization of various industries on a large scale.

The invention content is as follows:

in order to solve the problems, the invention provides pseudomonas with high lipase yield, a method for producing lipase by fermentation of the pseudomonas and lipase, and the method can obviously improve the good alkali resistance and low temperature resistance of the lipase while ensuring the stability of the high lipase yield by fermentation of the pseudomonas.

The purpose of the invention is realized by the following technical scheme:

the Pseudomonas with high lipase yield is Pseudomonas (Pseudomonas sp.) LD-14, and the strain preservation number is CGMCC No. 14414.

The Pseudomonas (Pseudomonas sp.) LD-14 is obtained by performing normal-temperature normal-pressure plasma mutagenesis on an original Pseudomonas strain, wherein the Pseudomonas LD-14 is preserved in China general microbiological culture Collection center (CGMCC for short) in 7-13.2017, with the preservation number of CGMCC No.14414 and the preservation address of: western road No.1, north chen, west road, 3, china academy of sciences, zip code: 100101.

the colony morphology characteristics of the pseudomonas LD-14 are as follows: round, milky white, regular periphery, lenticular, smooth surface, colored, opaque, odorous.

Another objective of the invention is to provide a method for producing enzyme by fermentation of Pseudomonas LD-14, which comprises the following steps:

culturing in a fermentation tank: inoculating the seed solution into a fermentation tank culture medium according to the proportion of 6% (v/v) of the inoculation amount, keeping the temperature at 35 ℃, setting the rotation speed at 200r/min, and setting the ventilation: 0 to 5 hours of 0.25vvm, 5 to 20 hours of 0.5vvm, 20 hours of 1.0vvm after the fermentation is finished, controlling the pH value of the fermentation liquor to be 6.8 by using a supplemented medium in the whole fermentation process, finishing the fermentation until the enzyme activity is slowly increased and the thallus autolysis is serious, wherein the fermentation period is 150 hours, and obtaining the final fermentation liquor;

the enzyme activity of the fermentation liquor of the lipase can reach: 25000U/mL to 26000U/mL;

and (4) extracting and refining the final fermentation liquor to obtain the finished product liquid enzyme preparation of the lipase.

Further, the preparation method of the seed liquid comprises the following steps: inoculating the secondary seed liquid into a seed tank culture medium according to the proportion of 6% (v/v) of the inoculum size, and culturing for 12-15h at the constant temperature of 35 ℃ and the rotating speed of 200 r/min;

further, the preparation method of the secondary seed liquid comprises the following steps: taking a ring of the first-stage seeds, inoculating the ring of the first-stage seeds into a seed culture medium, and culturing for 48 hours at the constant temperature of 35 ℃ and the rotating speed of a shaking table of 200r/min to obtain a second-stage seed solution;

further, the preparation method of the first-class seeds comprises the following steps: selecting a ring of pseudomonas LD-14, inoculating to a solid slant culture medium, and culturing at constant temperature of 35 ℃ for 36h to obtain first-stage seeds;

further, the extraction and refining method of the lipase comprises the following steps:

(1) adding 1-5% of perlite filter aid into the final fermentation liquor, and performing filter pressing to obtain clarified filter-pressed enzyme liquor;

(2) carrying out ultrafiltration concentration on the clarified filter-pressed enzyme liquid by using a 20000 molecular weight ultrafiltration membrane to obtain a concentrated solution;

(3) adding 20% (m/v) stabilizer and 0.45% (m/v) preservative into the above concentrated solution, adjusting pH to 8.5, and filtering for sterilization to obtain lipase finished product liquid enzyme preparation.

Preferably, the stabilizer is glycerin, and the preservative is a mixture of 1: 2 potassium sorbate and sodium benzoate.

The solid slant culture medium (g/L): peptone 12, yeast extract 6, agar 15, sodium chloride 1 and the balance of water, the pH value is 7.0, and the sterilization is carried out for 20min at the temperature of 121 ℃;

the seed culture medium (g/L): malt syrup 40, peptone 5, yeast extract 5, sodium chloride 0.8, magnesium sulfate 0.5, calcium chloride 0.1, disodium hydrogen phosphate 0.5, and water in balance, pH 6.8, sterilizing at 121 deg.C for 20 min;

the seeding tank medium (g/L): 80 parts of glucose, 15 parts of yeast extract, 5 parts of corn steep liquor, 1 part of ammonium sulfate, 0.5 part of magnesium sulfate, 1.5 parts of monopotassium phosphate, 0.1 part of calcium chloride, 6.8 parts of pH, and sterilization at 123 ℃ for 30 min;

the fermenter medium (g/L): maltose 180, peptone 10, yeast extract 5, ammonium sulfate 1.5, magnesium sulfate 0.5, corn oil 20, calcium chloride 0.1, dipotassium hydrogen phosphate 0.3, pH 6.8, sterilizing at the temperature of 121-;

the feed medium (g/L): malt syrup 450, KH₂PO₄5, 7 parts of corn steep liquor, 40 parts of corn oil and 5 parts of calcium chloride, wherein the pH value is 4.0, and the sterilization is carried out at the temperature of 121-.

The invention also provides a lipase produced by fermentation of the pseudomonas LD-14, wherein the lipase has the following characteristics:

(1) temperature: the optimal action temperature range is 30-40 ℃;

(2) pH: the optimum pH range is 8.5-10.0;

(3) low temperature resistance: the residual enzyme activity after the heat preservation for 1 day at 20 ℃ is 80 percent.

Has the advantages that:

the invention breeds a mutant strain LD-14 with high lipase yield by carrying out plasma mutagenesis on an original strain LA-32 of pseudomonas at normal temperature and normal pressure, optimizes the feeding conditions in the fermentation process of the pseudomonas LD-14 to ensure that the enzyme activity of the mutant strain LD-14 reaches 25000U/mL to 26000U/mL, and the highest enzyme activity of the original strain of the pseudomonas is 19500U/mL after liquid fermentation. Thus, the enzyme activity of the pseudomonas LD-14 is obviously higher than that of the original pseudomonas strain.

The components of the culture medium in the fermentation method of the strain are all derived from raw materials with lower cost, and the fermentation cost is greatly reduced while the fermentation productivity is improved and the production efficiency is improved, thereby being greatly helpful for production.

The lipase obtained by fermenting pseudomonas LD-14 has the optimum pH range of 8.5-10.0, the optimum action temperature range of 30-40 ℃, and the residual enzyme activity of 80 percent after heat preservation for 1 day at 20 ℃, has obvious low temperature resistance and alkali resistance, can be widely applied to industrial production, obviously expands the industrial application range of the lipase, and improves the application value of the lipase.

Description of the drawings:

FIG. 1: relative enzyme activity at different pH;

FIG. 2: relative enzyme activity at different temperatures;

FIG. 3: relative enzyme activity after heat preservation for 1 day at 20 ℃.

The specific implementation mode is as follows:

the invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

EXAMPLE 1 mutagenic Breeding of Pseudomonas LD-14

Taking two original strains LA-32 fresh slant, eluting thallus with sterile water, shaking in test tube with glass beads to disperse thallus, centrifuging to collect thallus, resuspending thallus with 5% glycerol, and counting with blood counting plate until the concentration is 10⁷-10⁸And (4) one/mL, and taking the obtained product as a starting bacterial suspension.

Starting the normal temperature and pressure plasma system, wiping the inside and outside of the operating room with alcohol cotton, and starting the ultraviolet lamp for sterilization for 30 min. After the sterilization in the operating room of the system is finished, 10 μ L of the bacterial suspension is spotted on the rough surface of the slide, and the slide is transferred to the table top of the operating room by tweezers under the aseptic condition. And opening a helium valve, and setting the air flow and the mutagenesis time for mutagenesis. The mutagenesis time was set to 90s, 120s, 150s, 180s, and 210s, respectively. After each mutagenesis, the slide glass is placed in an EP tube containing 990 mu L of sterile physiological saline and vortexed for 1 min. After dilution and coating, the mixture is placed in an incubator at 30 ℃ for culture.

Enrichment medium (g/L): 25 parts of glucose, 10 parts of tryptone, 5 parts of yeast extract powder, 10 parts of sodium chloride, 30 parts of olive oil and the balance of water, wherein the pH value is 6.8;

plate rescreening medium (g/L): maltose 20, peptone 5, beef extract 2, rhodamine 0.5, agar 20 and the balance of water, wherein the pH value is 6.8;

enzyme production medium (g/L): maltose 20, peptone 6, beef extract 3, olive oil 30, ammonium sulfate 1, magnesium sulfate 0.8, potassium dihydrogen phosphate 0.3 and the balance of water, wherein the pH value is 6.8;

primary screening of strains:

preparing a flat plate re-screening culture medium, selecting a single bacterial colony for streaking, culturing for 36h at 35 ℃, and selecting a bacterial colony with orange-yellow fluorescence around under an ultraviolet lamp with a wavelength of 365 nm.

Strain metabolic enzyme production test (preliminary screening): primarily screening by using a tributyrin flat transparent ring method, firstly, inoculating the screened strain into a 500mL triangular flask containing 100mL enzyme-producing culture medium, carrying out shake cultivation at 35 ℃ for 72h at 200r/min, centrifuging at 10000r/min for 20min, and collecting supernatant for later use; secondly, preparing 100g/L tributyrin emulsion by using 3% polyethylene glycol (PVA) solution by volume, adding 20mL of tributyrin emulsion into 180mL of Tris-HCl buffer solution (pH 8.0), then adding 1.5% agar, sterilizing by high pressure steam, pouring plates, punching a small hole with the diameter of 0.8cm in each plate, taking 50 mu L of the supernatant into the small hole, reacting for 24 hours at 30 ℃, measuring the size of a transparent ring, and taking the clear transparent ring to measure the enzyme activity.

Strain metabolic enzyme production test (rescreening): and re-screening the strains obtained in the preliminary screening, inoculating each strain into 5 bottles, inoculating the strains into 500mL triangular bottles containing 100mL of enzyme production culture medium, performing shake culture at 35 ℃ for 72h at 200r/min, and taking fermentation liquor to determine the enzyme activity.

The list of 5 mutant strains with higher enzyme activity screened by the shake flask is as follows:

TABLE 1 comparison of enzyme Activity of original strains with mutants having higher enzyme Activity

Bacterial strains	Original bacteria	LD-05	LD-13	LD-14	LD-85	LD-231
							Enzyme activity (U/mL)	2432	2571	2192	3068	2605	2832

And finally, carrying out shake flask fermentation again to select the LD-14 strain with the highest stable enzyme activity.

EXAMPLE 2 Pseudomonas LD-14 fermentation for enzyme production

The fermentation enzyme production method of pseudomonas LD-14 mainly comprises the following steps:

slant culture: selecting a ring of pseudomonas LD-14, inoculating to a solid slant culture medium, and culturing at constant temperature of 35 ℃ for 36h to obtain first-stage seeds;

and (3) shake flask culture: taking a ring of the first-stage seeds, inoculating the ring of the first-stage seeds into a seed culture medium, and culturing for 48 hours at the constant temperature of 35 ℃ and the rotating speed of a shaking table of 200r/min to obtain a second-stage seed solution;

seed tank culture: inoculating the secondary seed liquid into a seed tank culture medium according to the proportion of 6% (v/v) of the inoculum size, and culturing for 12h at the constant temperature of 35 ℃ and the rotating speed of 200 r/min;

culturing in a fermentation tank: inoculating the seed liquid in the seed tank into a fermentation tank culture medium according to the proportion of 6% (v/v) of the inoculation amount, keeping the temperature at 35 ℃, rotating at the speed of 200r/min, and setting the ventilation: 0 to 5 hours of 0.25vvm, 5 to 20 hours of 0.5vvm, 20 hours of 1.0vvm after the fermentation is finished, controlling the pH value of the fermentation liquor to be 6.8 by using a supplemented medium in the whole fermentation process, finishing the fermentation until the enzyme activity is slowly increased and the thallus autolysis is serious, wherein the fermentation period is 150 hours, and obtaining the final fermentation liquor;

Solid slant medium (g/L): peptone 12, yeast extract 6, agar 15, sodium chloride 1 and the balance of water, the pH value is 7.0, and the sterilization is carried out for 20min at the temperature of 121 ℃;

seed medium (g/L): malt syrup 40, peptone 5, yeast extract 5, sodium chloride 0.8, magnesium sulfate 0.5, calcium chloride 0.1, disodium hydrogen phosphate 0.5, and water in balance, pH 6.8, sterilizing at 121 deg.C for 20 min;

seeding tank medium (g/L): 80 parts of glucose, 15 parts of yeast extract, 5 parts of corn steep liquor, 1 part of ammonium sulfate, 0.5 part of magnesium sulfate, 1.5 parts of monopotassium phosphate, 0.1 part of calcium chloride, 6.8 parts of pH, and sterilization at 123 ℃ for 30 min;

fermenter Medium (g/L): maltose 180, peptone 10, yeast extract 5, ammonium sulfate 1.5, magnesium sulfate 0.5, corn oil 20, calcium chloride 0.1, dipotassium hydrogen phosphate 0.3, pH 6.8, sterilizing at the temperature of 121-;

feed medium (g/L): malt syrup 450, KH₂PO₄5, 7 parts of corn steep liquor, 40 parts of corn oil and 5 parts of calcium chloride, wherein the pH value is 4.0, and the sterilization is carried out for 30min at the temperature of 121-.

The extraction and refining method of lipase is as follows:

firstly, adding 1% perlite filter aid into the final fermentation liquor, and performing filter pressing to obtain clarified filter-pressed enzyme liquor;

carrying out ultrafiltration concentration on the clarified filter-pressed enzyme liquid by using a 20000 molecular weight ultrafiltration membrane to obtain a concentrated solution;

adding 20 percent (m/v) of glycerol, 0.15 percent (m/v) of potassium sorbate and 0.3 percent (m/v) of sodium benzoate into the concentrated solution by mass percentage, adjusting the pH to 8.5, and then carrying out filtration sterilization to obtain the finished product liquid enzyme preparation of the lipase.

Example 3 Pseudomonas LD-14 fermentation Performance validation

A50L fermentation tank verification experiment is carried out according to the fermentation enzyme production method of pseudomonas LD-14 in example 2, the fermentation period is 150h, the average enzyme production level of fermentation enzyme production of 6 batches is 25558U/mL, and Table 2 shows that the strain not only produces high-yield lipase, but also has remarkable stability in fermentation performance and enzyme activity of the produced lipase.

TABLE 26 fermentation enzyme production of high lipase producing strains of batches

Batches of	Fermentation period (h)	Fermentation vigor (U/mL)
			1	150	25825
2	150	25560
			3	150	25025
4	150	25590
			5	150	25400
6	150	25950

Example 4 method for measuring lipase Activity

(1) Preparation of enzyme solution: and (4) centrifuging the fermentation liquor at 6000r/min for 15min, and obtaining supernatant fluid which is the crude enzyme liquid.

(2) Definition of enzyme activity: 1g of solid enzyme powder or 1mL of liquid enzyme is defined, and under the conditions of certain temperature and pH value, 1min hydrolyzes a substrate to generate 1 mu mol of titratable fatty acid, namely an enzyme activity unit.

(3) And (3) enzyme activity determination:

sucking 1-2mL of enzyme sample, diluting with pH 7.5 phosphate buffer solution, controlling enzyme solution concentration during measurement, controlling the difference between the alkali consumption of the sample and the alkali consumption of a control within 1-2mL, and taking out the enzyme solution after shaking up the enzyme solution during sucking the sample.

Potentiometric titration method: the instrument calibration was performed according to the pH meter using instructions;

taking two 100mL beakers, adding 40mL of substrate diluent and 5mL of phosphate buffer solution into a blank cup and a sample cup respectively, adding 15mL of 95% ethanol into the blank cup, preheating in 40 plus-minus 0.2-degree water bath for 5 minutes, then adding 1mL of enzyme solution to be detected into the blank cup and the sample cup respectively, mixing uniformly immediately, timing, reacting accurately for 15 minutes, adding 15mL of ethanol into the sample cup immediately, stopping reaction, and taking out;

a rotor was added to the beaker, and the beaker was placed on an electromagnetic stirrer and titrated with 0.05mol/L sodium hydroxide standard solution while stirring until pH 10.3, and the end point of the titration was recorded as the volume of the sodium hydroxide standard solution consumed.

X1, enzyme activity U/mL of the sample;

v1 volume of sodium hydroxide standard solution consumed in milliliters (mL) upon titration of the sample;

v2: the volume of sodium hydroxide standard solution consumed in titrating the blank, in milliliters (mL);

c: sodium hydroxide standard solution concentration, unit mole per liter (mol/L);

50: 1.00mL of 0.05mol/L sodium hydroxide solution is equivalent to 50 μmol of fatty acid;

n 1: dilution factor of the sample;

0.05: the conversion coefficient of the concentration of the sodium hydroxide standard solution;

the reaction time is 15min, and is counted as 1 min;

example 5 optimum pH Range for Lipase

The lipase with the enzyme activity of 25500U/mL of the fermentation broth produced by the invention is taken as a sample, the enzyme activity is measured under the conditions of different pH values (7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5 and 11.0) at 35 ℃, the measured relative enzyme activity change curve is shown in figure 1, and the optimum action pH range of the enzyme is 8.5-10.0.

Example 6 optimum temperature Range for Lipase

The lipase with the enzyme activity of 25500U/mL of the fermentation broth produced by the invention is taken as a sample, the enzyme activity is measured under the condition that the pH value is 9.7 and different temperatures (20, 25, 30, 35, 40, 45 and 50) respectively, the measured relative enzyme activity change curve is shown in figure 2, and the optimal action temperature range of the enzyme is 30-40 ℃.

Example 7 Low temperature resistance of Lipase

The lipase with the enzyme activity of 25500U/mL of the fermentation broth produced by the invention is taken as a reference, the residual enzyme activity is measured after heat preservation at 20 ℃ under the condition that the pH value is 9.7, as shown in figure 3, the residual enzyme activity is 80% after heat preservation at 20 ℃ for 1d, the lipase has good low-temperature-resistant preservation activity, can be widely applied to low-temperature industrial production, obviously expands the industrial application range of the lipase, and improves the application value of the lipase.

Claims

1. The Pseudomonas with high lipase yield is Pseudomonas (Pseudomonas sp.) LD-14, and the strain preservation number is CGMCC No. 14414.

2. Use of the pseudomonads of claim 1 for producing lipase.

3. The use of Pseudomonas bacteria in the production of lipase as claimed in claim 2, wherein the fermentation method of Pseudomonas bacteria LD-14 for lipase preparation is as follows: inoculating the seed solution into a fermentation tank culture medium according to the proportion of 6% of the inoculation amount, keeping the temperature at 35 ℃, setting the rotation speed at 200r/min, and setting the ventilation: 0 to 5 hours of the fermentation broth are 0.25vvm, 5 to 20 hours of the fermentation broth are 0.5vvm, 20 hours of the fermentation broth are 1.0vvm after the fermentation is finished, the pH value of the fermentation broth is controlled to be 6.8 by using a supplemented medium in the whole fermentation process, the fermentation is finished when the enzyme activity is slowly increased and the bacterial autolysis is serious, and the fermentation period is 150 hours;

the culture medium of the fermentation tank is as follows: 180g/L of maltose, 10g/L of peptone, 5g/L of yeast extract, 1.5g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 20g/L of corn oil, 0.1g/L of calcium chloride, 0.3g/L of dipotassium hydrogen phosphate, pH 6.8, and sterilization at the temperature of 123 ℃ for 30 min;

the feed culture medium comprises: malt syrup 450g/L, KH₂PO₄5g/L, 7g/L of corn steep liquor, 40g/L of corn oil, 5g/L of calcium chloride, pH4.0, and sterilization at the temperature of 121-.

4. The use of Pseudomonas bacteria in the production of lipase as claimed in claim 3, wherein the seed liquid is prepared by the following method: inoculating the secondary seed liquid into a seed tank culture medium according to the proportion of 6 percent of the inoculation amount, and culturing for 12-15h at the constant temperature of 35 ℃ and the rotating speed of 200 r/min;

the preparation method of the secondary seed liquid comprises the following steps: taking a ring of the first-stage seeds, inoculating the ring of the first-stage seeds into a seed culture medium, and culturing for 48 hours at the constant temperature of 35 ℃ and the rotating speed of a shaking table of 200r/min to obtain a second-stage seed solution;

the preparation method of the first-class seeds comprises the following steps: selecting Pseudomonas circulans LD-14, inoculating to solid slant culture medium, and culturing at constant temperature of 35 deg.C for 36 hr to obtain first-stage seed.

5. The use of Pseudomonas bacteria in the production of lipase according to claim 4, wherein the solid slant medium is: peptone 12g/L, yeast extract 6g/L, agar 15g/L, sodium chloride 1g/L, and water in balance, pH7.0, sterilizing at 121 deg.C for 20 min;

the seed culture medium is as follows: 40g/L of malt syrup, 5g/L of peptone, 5g/L of yeast extract, 0.8g/L of sodium chloride, 0.5g/L of magnesium sulfate, 0.1g/L of calcium chloride, 0.5g/L of disodium hydrogen phosphate and the balance of water, wherein the pH value is 6.8, and the malt syrup is sterilized at 121 ℃ for 20 min;

the culture medium of the seeding tank is as follows: 80g/L glucose, 15g/L yeast extract, 5g/L corn steep liquor, 1g/L ammonium sulfate, 0.5g/L magnesium sulfate, 1.5g/L potassium dihydrogen phosphate, 0.1g/L calcium chloride, pH 6.8, and sterilization at 123 ℃ for 30 min.

6. The use of Pseudomonas as claimed in claim 3, wherein the extraction and purification method of lipase is as follows:

(3) adding 20% of stabilizer and 0.45% of preservative into the concentrated solution, adjusting the pH to 8.5, and then carrying out filtration sterilization to obtain a finished liquid lipase preparation;

the stabilizer is glycerin, and the preservative is prepared from the following components in a mass ratio of 1: 2 potassium sorbate and sodium benzoate.