CN112375699B - Bacillus licheniformis for expressing lipase and fermentation enzyme production method thereof - Google Patents
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Abstract
A bacillus licheniformis for expressing lipase and a fermentation enzyme production method thereof are preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 20140. The enzyme activity of fermentation liquor for producing lipase by fermenting the bacillus licheniformis reaches 32000U/mL; the produced lipase has the optimum action temperature range of 30-40 ℃, the optimum pH range of 8.5-9.5, and the relative enzyme activity of 60 percent after heat preservation for 48 hours at 4 ℃, has wide optimum action range and obvious low temperature resistance and alkali resistance, can be applied to industrial production with different requirements, obviously expands the industrial application range of the lipase, and improves the application value of the lipase.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to Bacillus licheniformis (Bacillus licheniformis) for expressing lipase and a fermentation enzyme production method thereof.
Background
Lipases (1 ipase, e.c. 3.1.1.3), also known as lipolytic, glyceride hydrolase or triacylglycerol acylhydrolase, catalyze the hydrolysis of triglycerides to convert them to glycerol and fatty acids in the oil-water interface with the property and enantioselectivity of reversing the reaction in aqueous and non-aqueous media. Lipase has become one of the leading biocatalysts, and can be applied to a plurality of industrial fields such as food, medicine, detergent, textile, biodiesel, paper making, leather, cosmetics, environmental protection and the like.
Lipases are mainly derived from animals, plants and microorganisms. Due to the wide variety of microorganisms, rapid propagation, genetic variation, and broader pH, temperature range and substrate specificity of action than that of animals and plants, many microorganisms are regarded as potential producers of secreted extracellular lipases and are gaining continuous industrial interest, including bacteria, yeasts and fungi. Fungi are suitable for solid state fermentation, while bacteria and yeasts are suitable for submerged fermentation. The microbial lipase is an important source of industrial lipase, is suitable for industrial mass production and obtaining high-purity samples, and has important significance in the aspect of theoretical research.
The preparation method of lipase includes extraction method, chemical synthesis method and microbial fermentation method. The extraction method mainly aims at animals and plants, and has the defects of limited resources, complex process and low yield; the chemical synthesis cost is too high; the application prospect of the microbial fermentation method is far greater than that of the extraction method and the chemical synthesis method, the method is not influenced by the environment, the resources are rich, the enzyme production period is short, the product is simple, the cost is low, and the production is easy to manage. Commercial lipases are mainly derived from fermentation products of various microorganisms such as bacteria, yeasts and fungi, and some molds can be fermented by both solid state fermentation and liquid submerged fermentation.
The currently industrially synthesized lipase has limited types, low production efficiency, high production cost, small optimal reaction temperature and pH range, and is limited by enzyme activity and application conditions, so the requirements of industrial production cannot be met, a bacillus licheniformis strain with high lipase yield is absolutely necessary to be screened, the extracellular lipase has high activity, wide application range and low production cost, and the method has great significance for widening the application field of lipase in a large scale.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the bacillus licheniformis for expressing lipase, the fermentation enzyme production method thereof and the extracellular lipase, so that the yield and the activity of the lipase of the bacillus licheniformis in the fermentation process are improved, and the optimal reaction conditions of the lipase are remarkably widened.
The purpose of the invention is realized by the following technical scheme.
Bacillus licheniformis (Bacillus licheniformis) NCU CS-5 for expressing lipase is preserved in China general microbiological culture Collection center (address: Beijing Kogyo Xinyang district No. 1 Beichen Xilu No. 3, China academy of sciences microbiological research, postal code: 100101) in 28 days 6 and 2020, which is abbreviated as CGMCC and the preservation number is CGMCC No. 20140.
After the bacillus licheniformis strain NCU CS-5 is cultured on a nutrient broth agar culture medium for 24 hours, the bacterial colony is circular, flat, irregular in edge, rough and wrinkled in white surface, opaque, adhesive and 1.5-3.0 mm in diameter. Gram staining is positive, the cell shape and arrangement are observed under a microscope to be in a long rod shape, the diameter is about 0.6-0.9 mu m, and spores (generated in elliptic spores), flagella and no capsule are produced.
Another object of the present invention is to provide a method for producing an enzyme by fermentation of Bacillus licheniformis NCU CS-5.
(1) Preparing a first-level seed solution: a Bacillus licheniformis strain NCU CS-5 is selected and inoculated on a nutrient broth agar plate culture medium, and cultured at a constant temperature of 37 ℃ for 36 h.
(2) Preparing a secondary seed solution: taking a ring of first-level seeds from the agar plate, inoculating the first-level seeds into a seed culture medium, and culturing for 36h at 37 ℃ and 200r/min to obtain a second-level seed solution.
(3) High-density fermentation culture: inoculating thalli in the secondary seed liquid into a fermentation tank culture medium according to the proportion of 40% (w/v) of wet inoculation amount, rotating at 35-40 ℃, setting the rotation speed at 600r/min and the ventilation speed at 1.5L/min, adopting batch fermentation to maintain the pH of the fermentation liquid at 7.5 by using a feed medium in the whole fermentation process, wherein the fermentation period is 156h, and finally obtaining the finished product liquid lipase by a purification and refining method, wherein the lipase activity in the obtained fermentation liquid is as high as 32000U/mL.
The nutrient broth agar plate culture medium (g/L): 15.0 parts of agar, 10.0 parts of peptone, 3.0 parts of beef extract powder, 5.0 parts of sodium chloride, 7.2 +/-0.2 parts of pHs, and sterilizing at 121 ℃ for 20 min.
The seed culture medium (g/L): 40.0 parts of glucose, 5.0 parts of peptone, 5.0 parts of beef extract powder, 0.5 part of sodium chloride, 0.5 part of magnesium sulfate, 0.5 part of disodium hydrogen phosphate, 7.0 parts of pH, and sterilizing at 121 ℃ for 20 min.
The culture medium in the seeding tank (g/L): 80.0 parts of glucose, 20.0 parts of yeast powder, 5.0 parts of corn juice, 1.0 part of sodium chloride, 0.5 part of magnesium sulfate, 2.0 parts of monopotassium phosphate, 7.0 parts of pH and 30min of sterilization at 121 ℃.
The culture medium of the fermentation tank (g/L): 200.0 parts of glucose, 10.0 parts of peptone, 5.0 parts of yeast powder, 1.5 parts of sodium chloride, 0.5 part of magnesium sulfate, 20.0 parts of olive oil, 0.3 part of dipotassium hydrogen phosphate, 7.0 parts of pH, and sterilizing at 121 ℃ for 30 min.
The feed medium (g/L): glucose 500.0, sodium chloride 5.5, potassium dihydrogen phosphate 5.0, corn juice 7.0, olive oil 40.0, pH 7.0, and sterilizing at 121 deg.C for 30 min.
The invention also provides lipase produced by fermentation of the bacillus licheniformis strain NCU CS-5, wherein the optimal temperature range of the lipase is 30-40 ℃, the optimal pH range is 8.5-9.5, and the relative enzyme activity of the lipase is 60% after incubation for 48 hours at 4 ℃.
The purification and refining method of the lipase is as follows.
(1) And adding 5% (w/v) of diatomite filter aid into the finally obtained fermentation liquor, and performing filter pressing to obtain clarified filter-pressed enzyme liquor.
(2) And (3) carrying out ultrafiltration concentration on the clarified filter-pressed enzyme liquid by using an ultrafiltration membrane with molecular weight cutoff of 30000, and separating to obtain a lipase concentrated solution.
(3) Adding 20% (w/v) of glycerol and 0.5% (w/v) of preservative into the lipase concentrated solution: potassium sorbate: sodium benzoate =1:1 (w/w) pH8.5, and then filter-sterilized by a membrane sterile filtration system to obtain the final product, namely the liquid lipase.
The invention has the beneficial effects that: screening a bacillus licheniformis strain NCU CS-5 with high lipase yield, and optimizing the feeding conditions in the fermentation process to ensure that the enzyme activity of the produced lipase reaches about 32000U/mL. The components of the fermentation culture medium of the bacillus licheniformis strain NCU CS-5 are all derived from raw materials with lower cost, so that the fermentation cost is reduced, the fermentation capacity and the production efficiency are improved, and great help is brought to industrial production. The lipase obtained by fermenting the bacillus licheniformis strain NCU CS-5 has the optimum temperature range of 30-40 ℃, the optimum pH range of 8.5-9.5, and the relative enzyme activity of 60% after incubation for 48h at 4 ℃, 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.
Drawings
FIG. 1 shows relative enzyme activities at different temperatures.
FIG. 2 shows relative enzyme activities at different pH values.
FIG. 3 shows the relative enzyme activity after incubation for 48h at 4 ℃.
Detailed Description
The present invention will be further described with reference to the following specific examples.
Example 1. A screening method of Bacillus licheniformis NCU CS-5.
(I) preparation of experimental materials.
1. The isolated source is derived from seeds of Cinnamomum camphora which have been collected from a laboratory and partially deteriorated during storage after shelling.
2. And (4) a culture medium.
Nutrient broth medium (g/L): 10.0 parts of peptone, 3.0 parts of beef extract powder, 5.0 parts of sodium chloride, and finally, 7.2 +/-0.2 parts of pH, sterilizing at 121 ℃ for 15min under high pressure, and cooling to normal temperature for later use.
Agar broth culture medium: agar with the final mass concentration of 15.0-20.0g/L is added into the nutrient broth culture medium.
Preparation of olive oil emulsion: mixing oleum Olivarum and 0.1g/mL Arabic Gum at a ratio of 1:4 (v/v), and emulsifying with high speed homogenizer for 10min to obtain oleum Olivarum emulsion.
Enrichment medium (g/L): glucose 25.0, soybean peptone 5.0, tryptone 15.0, sodium chloride 5.0, and olive oil emulsion 2% (v/v), adjusting pH to 7.2, and sterilizing at 121 deg.C for 20 min.
Primary screening culture medium (g/L) of lipase: 5.0 parts of soybean peptone, 15.0 parts of tryptone, 5.0 parts of sodium chloride, 3.0 parts of yeast extract, 20.0 parts of agar powder and 10.0 parts of neutral tributyrin, adjusting the pH value to 7.5 +/-0.2, sterilizing at 121 ℃ for 15min, cooling to about 60 ℃, shaking, mixing uniformly, and pouring into a flat dish while the mixture is hot.
Lipase rescreening medium (g/L): soybean peptone 5.0, tryptone 15.0, sodium chloride 5.0, olive oil emulsion 5% (v/v), adjusting pH to 7.2, adding agar powder 20.0, sterilizing at 121 deg.C for 20min, cooling to about 60 deg.C, adding filtered and sterilized rhodamine B solution with final concentration of 0.5g/L, shaking, mixing, and pouring into a flat dish while it is hot.
Enzyme production medium (g/L): 5.0 parts of yeast powder, 0.5 part of magnesium sulfate, 3.0 parts of potassium dihydrogen phosphate, 5.5 parts of sodium chloride and 1 percent (v/v) of olive oil, adjusting the pH value to 7.2, and sterilizing at 121 ℃ for 20 min.
3. And (4) experimental equipment.
A constant temperature oscillator, a biochemical incubator, an ultra-clean workbench, an autoclave, a pH meter, a high-speed homogenizer and a 721 type ultraviolet spectrophotometer.
And (II) screening strains.
(1) Enrichment of high-yield lipase strains: weighing about 1g/mL of separation source sample, and preparing different dilutions (10 times dilution) by using enrichment medium and 10 times dilution method -1 ～10 -9 ) The dilution of (4) was pipetted 100. mu.L of each dilution and spread evenly onto agar broth plates. 37Culturing at constant temperature for 24h, and selecting a plate with about 10 colonies for later use.
(2) Primary screening of high-yield lipase strains: adopting a dibbling method, using an aseptic toothpick to live and dibble the bacterial colony obtained in the last step onto a lipase primary screening culture medium plate, culturing for 24H at a constant temperature of 37 ℃, carrying out primary screening on the lipase-producing strain according to the diameter of a transparent ring of the bacterial colony in the culture medium and the diameter ratio (H/C) of the bacterial colony, and selecting the strain with larger H/C for later use.
(3) Re-screening the high-yield alkaline lipase strains.
And (3) adopting a dibbling method, dibbling the strain obtained by primary screening in the last step onto a lipase rescreened culture medium plate, culturing at a constant temperature of 37 ℃ for 24 hours, and observing whether a yellow transparent hydrolysis ring is generated around a bacterial colony. And (4) re-screening the lipase-producing strains according to the diameter ratio (H/C) of the yellow transparent circle of the bacterial colony to the bacterial colony in the culture medium, and selecting the strains with larger H/C for later use. Inoculating the strains obtained by re-screening into nutrient broth culture medium, and shake-culturing at 37 deg.C and 220r/min for 24h to obtain seed solution. Adding the seed solution into a fermentation culture medium with an inoculum size of 4%, shaking-culturing at 37 deg.C and 220r/min for 51h, taking the fermentation liquid, centrifuging at 8000r/min for 10min, and collecting the supernatant as the crude enzyme solution. The lipase activity of the crude enzyme liquid is determined by an acid-base titration method specified in GB/T23535-2009 lipase preparation enzyme activity determination method, and a strain with high lipase production activity is selected.
And (III) bacterial colony morphological characteristics and physiological and biochemical characteristics of the bacterial strain.
After bacillus licheniformis NCU CS-5 is cultured on the nutrient broth agar culture medium for 24 hours, the bacterial colony is circular and flat, irregular in edge, rough and wrinkled in white surface, opaque, adhesive and 1.5-3.0 mm in diameter. Gram staining is positive, the cell shape and arrangement are observed under a microscope to be in a long rod shape, the diameter is about 0.6-0.9 mu m, and spores (generated in elliptic spores), flagella and no capsule are produced.
The 16SrDNA gene sequence of the strain NCU CS-5 is shown in an instruction nucleotide and amino acid sequence table. The 16SrDNA gene sequence is compared with a GenBank database by adopting a BLAST method, and the homology of the strain NCU CS-5 and the bacillus licheniformis is up to 99 percent. The results of physiological, biochemical and molecular identification of the strain are integrated, and the strain NCU CS-5 is named as Bacillus licheniformis NCU CS-5. The screening and application of high-yield lipase bacillus licheniformis are not reported by referring to relevant literature data, and the bacillus licheniformis is delivered to China general microbiological culture collection center (CGMCC) for preservation in 6-28 months in 2020 with the preservation number of 20140.
Example 2. The bacillus licheniformis NCU CS-5 produces enzyme by fermentation.
A fermentation enzyme production method of Bacillus licheniformis NCU CS-5 mainly comprises the following steps.
Plate culture: selecting a ring of Bacillus licheniformis NCU CS-5, inoculating to nutrient broth agar plate culture medium, and culturing at 37 deg.C for 36h to obtain first-stage seed.
And (3) shake flask culture: and (3) taking a ring of the first-level seeds to be inoculated into a seed culture medium, and culturing for 36h at 37 ℃ and 200r/min to obtain a second-level seed solution.
Seed tank culture: the thalli in the secondary seed liquid is inoculated into a seed tank culture medium according to the proportion of 40 percent (w/v) of the wet weight inoculum concentration, and cultured for 12 hours 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, setting the rotation speed at 600r/min and the ventilation rate at 1.5L/min, adopting batch fermentation to maintain the pH of the fermentation liquid at 7.5 by using a fed-batch culture medium in the whole fermentation process, wherein the fermentation period is 156h, and the lipase activity in the finally obtained fermentation liquid is as high as 32000U/mL.
And (4) purifying and refining the final fermentation liquor to obtain the finished product liquid lipase.
Nutrient broth agar plate medium (g/L): agar 15, peptone 10.0, beef extract 3.0, sodium chloride 5.0, pH7.2 + -0.2, sterilizing at 121 deg.C for 20 min.
Seed medium (g/L): 40.0 parts of glucose, 5.0 parts of peptone, 5.0 parts of beef extract powder, 0.5 part of sodium chloride, 0.5 part of magnesium sulfate, 0.5 part of disodium hydrogen phosphate, 7.0 parts of pH, and sterilizing at 121 ℃ for 20 min.
Seeding tank medium (g/L): 80.0 parts of glucose, 20.0 parts of yeast powder, 5.0 parts of corn juice, 1.0 part of sodium chloride, 0.5 part of magnesium sulfate, 2.0 parts of monopotassium phosphate, 7.0 parts of potassium dihydrogen phosphate and 30min of sterilization at 121 ℃.
Fermenter Medium (g/L): 200.0 parts of glucose, 10.0 parts of peptone, 5.0 parts of yeast powder, 1.5 parts of sodium chloride, 0.5 part of magnesium sulfate, 20.0 parts of olive oil, 0.3 part of dipotassium hydrogen phosphate, 7.0 parts of pH, and sterilizing at 121 ℃ for 30 min.
Feed medium (g/L): glucose 500.0, sodium chloride 5.5, potassium dihydrogen phosphate 5.0, corn juice 7.0, olive oil 40.0, pH 7.0, and sterilizing at 121 deg.C for 30 min.
The purification and purification method of lipase is as follows.
And adding 5% (w/v) of diatomite filter aid into the final fermentation liquor, and performing filter pressing to obtain clarified filter-pressed enzyme liquor.
And (3) carrying out ultrafiltration concentration on the clarified filter-pressed enzyme liquid by using an ultrafiltration membrane with molecular weight cutoff of 30000, and separating to obtain a lipase concentrated solution.
Adding 20% (w/v) of glycerol and 0.5% (w/v) of preservative into the concentrated solution: potassium sorbate: sodium benzoate =1:1 (w/w) pH8.5, and then subjected to filtration sterilization by a membrane type sterile filtration system to obtain a final product, namely liquid lipase.
Example 3. The optimum temperature range for lipase.
The lipase with the enzyme activity of 32000U/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 (20-50), the variation curve of the measured relative enzyme activity is shown in figure 1, and the optimal action temperature range of the enzyme is 30-40 ℃.
Example 4. The optimum pH range for lipase action.
The lipase with the enzyme activity of 32000U/mL of the fermentation broth produced by the invention is taken as a sample, the enzyme activity is measured under the condition of 35 ℃ and different pH values (3-12.0), the measured relative enzyme activity change curve is shown in figure 2, and the optimum action pH range of the enzyme is 8.5-9.5.
Example 5. Low temperature resistance of lipase.
The lipase with the enzyme activity of 32000U/mL of the fermentation liquor produced by the invention is taken as a sample, the residual enzyme activity is determined after heat preservation at 4 ℃ under the condition that the pH value is 9.0, as shown in figure 3, the residual enzyme activity is 60% after heat preservation at 4 ℃ for 48h, 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.
Sequence listing
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Claims (3)
1. Bacillus licheniformis for expressing lipase (b)Bacillus licheniformis) The preservation number is CGMCCNo.20140.
2. The method for producing an enzyme by fermenting Bacillus licheniformis expressing a lipase as claimed in claim 1, characterized in that the method comprises the following steps:
(1) preparing a first-level seed solution: selecting a ring of bacillus licheniformis strain, inoculating the bacillus licheniformis strain to a nutrient broth agar plate culture medium, and culturing at the constant temperature of 37 ℃ for 36 hours;
(2) preparing a secondary seed solution: taking a ring of first-stage seeds from an agar plate, inoculating the first-stage seeds into a seed culture medium, and culturing for 36 hours at 37 ℃ and 200r/min to obtain a second-stage seed solution;
(3) high-density fermentation culture: and inoculating the thalli in the secondary seed liquid into a fermentation tank culture medium according to the weight-volume ratio of the wet inoculation amount of 40%, rotating at 35-40 ℃ at 600r/min and setting the ventilation rate to be 1.5L/min, adopting batch fermentation to maintain the pH of the fermentation liquid to be 7.5 by using a fed-batch culture medium in the whole fermentation process, wherein the fermentation period is 156h, and obtaining the finished product liquid lipase through purification and refining.
3. The method of claim 2, wherein the purification and refining step comprises the steps of:
(1) adding a diatomite filter aid with the weight volume ratio of 5% into the finally obtained fermentation liquor, and performing filter pressing to obtain clarified filter-pressed enzyme liquid;
(2) carrying out ultrafiltration concentration on the clarified filter-pressed enzyme liquid by using an ultrafiltration membrane with molecular weight cutoff of 30000, and separating to obtain a lipase concentrated solution;
(3) adding 20% by weight and volume of glycerol and 0.5% of preservative into the lipase concentrated solution: potassium sorbate: the weight ratio of sodium benzoate is 1:1, the pH value is 8.5, and then a membrane type sterile filtration system is carried out for filtration sterilization, so as to obtain the final product, namely the liquid lipase.
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