CN107557314B - Protease-producing strain LS20-2-2 and method for producing low-temperature protease by using same - Google Patents
Protease-producing strain LS20-2-2 and method for producing low-temperature protease by using same Download PDFInfo
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Abstract
The invention relates to a protease-producing strainLS20-2-2 and the method for producing low temperature protease thereof can effectively solve the preparation problem of low temperature protease, and the technical scheme is as follows: a protease-producing strain LS20-2-2, which is classified and named Paenibacillus epidermidis (A)Paenibacillus peoriae) The strain is a strain separated and screened from a soil sample collected from the Qinghai-Tibet plateau, and the strain producing the protease at the low temperature is a method for producing the protease at the low temperature by using the strain LS20-2-2, which comprises strain activation, seed culture, fermentation culture and protease collection.
Description
Technical Field
The invention relates to the field of biology, in particular to a protease-producing strain LS20-2-2 and a method for producing low-temperature protease by using the same.
Background
Protease is an enzyme catalyzing protein hydrolysis, is one of the most widely used enzyme preparations in the world, and can be used for production of detergents, leathers, furs, protein hydrolysates, wine brewing, soy sauce, textiles, pharmaceuticals, cosmetics and the like and extraction of biological materials. Commercial proteases are classified by source into animal proteases, plant proteases and microbial proteases. Compared with the protease of animal and plant sources, the microbial protease has the advantages of relatively simple downstream technical treatment, low price, wide source, high yield and the like, and is easier to realize industrial production. The low-temperature protease is an enzyme which can hydrolyze peptide bonds of proteins at the low temperature of 0-40 ℃, and can still maintain high catalytic activity at the low temperature of 0-50 ℃, so that the low-temperature protease has great advantages in industrial application, such as the catalytic process at low temperature or room temperature, the strength of heating and cooling treatment is reduced, the production cost can be saved, and the production process is convenient to monitor. In addition, the low-temperature protease has thermal instability, and by utilizing the characteristic, the enzyme can be quickly inactivated by adopting mild heat treatment after the enzyme reaction is finished, so that the reaction is quickly and economically terminated, and the taste and the quality of food can be favorably kept in food processing. Therefore, the method has wide application prospect in industries such as food, washing and the like.
Paenibacillus genus (A), (B), (CPaenibacillus) 1993 by Ash et al on Bacillus (Bacillus) Analysis of the 16S rRNA gene sequence of the third group of species isolated from Bacillus. Belongs to the family of Bacillariophyceae, class Bacillariophyceae, order Bacillariophyceae. At present, 190 species are found and widely exist in various habitats such as soil, plant rhizosphere, air, water, food, animal bodies, south pole sediments and the like. It is reported that various species of Paenibacillus have plant disease prevention and plant growth promoting effects, and are safe to human and livestock without pollution. Many paenibacillus strain metabolites contain various available bioactive substances, and some strains can produce antibiotics, polysaccharides, peptides, proteins, pyrazines, phenols and other antibacterial substances with strong tolerance to heavy metals and antibiotics, and also produce enzymes such as alkaline pectinase, rennin, xylanase and the like. Some strains have also been demonstrated to function to produce proteases. Has potential application value in agriculture, food, environment, medicine and other aspects.
Disclosure of Invention
In view of the above situation, in order to solve the defects of the prior art, the invention aims to provide a protease-producing strain LS20-2-2 and a method for producing low-temperature protease thereof, which can effectively solve the preparation problem of the low-temperature protease.
The technical scheme of the invention is as follows: a protease-producing strain LS20-2-2, which is classified and named Paenibacillus epidermidis (A)Paenibacillus peoriae) The strain is preserved in China general microbiological culture Collection center in 2016, 9 and 28 months, the preservation number is CGMCC No.13053, and the preservation address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; is a strain separated and screened from a soil sample collected from the Qinghai-Tibet plateau.
The method for producing the low-temperature protease by the protease-producing strain LS20-2-2 comprises the following steps:
(1) strain activation: inoculating strain LS20-2-2 to LB plate culture medium containing skimmed milk powder with mass concentration of 0.8-1.2%, and culturing at 18-22 deg.C until transparent ring is generated;
(2) seed culture: inoculating the single colony generating the transparent ring in a seed culture medium, and culturing at 25-28 ℃ for 20-28h to obtain a seed solution; the seed culture medium is prepared from 0.8-1.2% of peptone, 0.2-0.8% of yeast powder, 0.8-1.2% of NaCl and the balance of distilled water by weight;
(3) fermentation culture: inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 2-5% of the volume for fermentation at the temperature of 25-28 ℃, the pH value of 7-9 and the rotation speed of 160-180r/min for 72-86h to obtain a fermentation liquid;
the fermentation medium is prepared from the following components in percentage by weight: carbon source 0.5-1.0%, nitrogen source 0.9-1.5%, K2HPO40 to 0.1 percent of enzyme production promoter, 0.02 to 0.05 percent of enzyme production promoter and the balance of distilled water; the carbon source is one of corn flour, starch, glucose and sucrose; the nitrogen source is one or two of peptone, casein, yeast powder, skimmed milk powder and soybean meal according to the volume ratio of 1.0-1.2: mixing at a ratio of 0.3-0.5; the enzyme production promoter is a metal ion and Tween series surfactant;
(4) collecting the protease: centrifuging the fermentation liquid at 3-5 deg.C and 8000-; slowly adding ammonium sulfate into the supernatant while stirring at 3-5 ℃, adding 472-662 g of ammonium sulfate into each liter of solution to ensure that the saturation degree of the ammonium sulfate in the supernatant reaches 70-90%, and salting out for 8-12h at 3-5 ℃ after full dissolution to obtain salting-out solution; centrifuging the salting-out solution at 3-5 deg.C and rotation speed of 5000-8000rpm for 20-30min, collecting precipitate, and dissolving the precipitate with phosphate buffer solution of pH7.2 and mass concentration of 20-50mmol/L to obtain crude protease solution; transferring the crude protease liquid into a 3500D dialysis bag with molecular weight cutoff, placing in 2.5-3.5L phosphoric acid buffer solution with mass concentration of 20-50mmol/L, dialyzing overnight for desalting, changing the buffer solution for 2-3 times, and freeze-drying the dialysate to obtain the low-temperature protease. The preparation process is simple and efficient, energy-saving and environment-friendly, the prepared protease has high enzyme activity, the protease can be effectively prepared at low temperature, the prepared protease has the enzyme activity of 80-95% at 30-50 ℃, the activity of more than 80% can be still maintained after the protease is kept for 60min at 0-40 ℃, and the low-temperature stability is high; the strain produces special fragrance in the fermentation process, and the produced protease is extracellular enzyme and is convenient to collect.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
The invention relates to a protease-producing strain LS20-2-2 which is classified and named as Paenibacillus epidermidis(Paenibacillus peoriae) The strain is preserved in China general microbiological culture Collection center (CGMCC) at 2016, 9 and 28, has a preservation number of CGMCC No.13053, and is a strain separated and screened from a soil sample collected from the Qinghai-Tibet plateau. The 16SrDNA gene sequence of the strain is shown in SEQ ID NO 1.
The strain screening method comprises the steps of separating and culturing microbial strains of soil samples from Qinghai-Tibet plateau, inoculating the separated strains to the surface of a primary screening culture medium, culturing for 3-5 days at 4 ℃, generating obvious transparent rings around the colonies, and selecting the strains with the largest diameter ratio of the transparent rings to the colonies as secondary screening strains. The secondary screening is a method for measuring the enzyme activity of fermentation liquor by inoculating the primary screened strain into a secondary screening culture medium for fermentation. The prescreening culture medium is preferably an LB culture medium containing 1.0% of skimmed milk powder, and the rescreening culture medium is preferably: casein protein1.0 percent, yeast powder 0.4 percent, glucose 1.0 percent and K2HPO40.1％，Mg2SO4·7H2O 0.02％。
A protease high-yield strain is obtained by screening and is named as LS 20-2-2. Thus, a protease-producing strain is provided which has excellent properties of producing protease under low temperature conditions. Identified by 16S rDNA sequence and belongs to Paenibacillus epidermidisPaenibacillus peoriae). LS20-2-2 was gram-positive, cells were short rods (0.5-1.0. mu. m.times.3.0-6.0. mu.m) and individually aligned. The bacterial strain forms a colony round, milky white and opaque colony on the surface of an LB culture medium; a clear transparent ring can be produced on the skimmed milk powder plate. The protease produced by the strain has the optimum reaction temperature of 40 ℃ and the optimum pH value of 7-9.
The protease producing strain can be an original strain screened naturally, a mutant strain mutated naturally or artificially, or an engineering strain constructed by obtaining a low-temperature protease gene from the original strain or an induced mutant strain through a molecular biology technology and transferring the gene into a recipient strain.
The protease-producing strain LS20-2-2 can be preserved by a conventional slant method, wherein the strain is inoculated on the surface of LB slant culture medium containing 1.0% of skimmed milk powder, and is preserved in a refrigerator at 4 ℃ after being cultured for 2-3 days at 20 ℃; or adding the strain into LB liquid culture medium to culture for 1-2 days at 20 ℃, centrifuging to obtain the strain, and preparing the strain suspension by using 20% of skimmed milk powder and 10% of glycerol.
The invention may be embodied in the form of the following examples:
example 1
The method for producing the low-temperature protease by the protease-producing strain LS20-2-2 comprises the following steps:
(1) strain activation: inoculating the strain into LB plate culture medium containing 0.8% skimmed milk powder, and culturing at 18 deg.C until transparent ring is generated;
(2) seed culture: inoculating the single colony generating the transparent ring in a seed culture medium, and culturing at 25 ℃ for 20h to obtain a seed solution; the seed culture medium is prepared from 0.8% of peptone, 0.2% of yeast powder, 0.8% of NaCl and 98.2% of distilled water by weight;
(3) fermentation culture: inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 2% of the volume for fermentation, wherein the fermentation temperature is 25 ℃, the pH value is 7.0, the rotating speed is 160r/min, and the fermentation is carried out for 72 hours to obtain a fermentation liquid;
the fermentation medium is prepared from the following components in percentage by weight: corn flour 0.5%, peptone 0.9%, K2HPO40.01 percent, Tween-800.01 percent and distilled water 98.58 percent;
(4) collecting the protease: centrifuging the fermentation liquor at 3 deg.C and 12000rpm for 5min, and collecting supernatant; slowly adding ammonium sulfate into the supernatant while stirring at 3 ℃, adding 472 g of ammonium sulfate into each liter of solution to ensure that the saturation degree of the ammonium sulfate in the supernatant reaches 70%, and salting out for 8h at 3 ℃ after full dissolution to obtain salting-out solution; centrifuging the salting-out solution at 3 deg.C and 5000rpm for 30min, collecting precipitate, and dissolving the precipitate with 20mmol/L phosphate buffer solution with pH of 7.2 to obtain crude protease solution; transferring the crude protease liquid into a 3500D dialysis bag with molecular weight cutoff, placing the bag in 2.5L phosphoric acid buffer solution with mass concentration of 20mmol/L, dialyzing overnight to remove salt, changing the buffer solution for 2 times, and freeze-drying the dialysate to obtain the low-temperature protease.
Example 2
The method for producing the low-temperature protease by the protease-producing strain LS20-2-2 comprises the following steps:
(1) strain activation: inoculating the strain to LB plate culture medium containing 1.0% skimmed milk powder, and culturing at 20 deg.C until transparent ring is formed;
(2) seed culture: inoculating the single colony generating the transparent ring in a seed culture medium, and culturing at 26 ℃ for 24h to obtain a seed solution; the seed culture medium is prepared from 1.0% of peptone, 0.5% of yeast powder, 1.0% of NaCl and 97.5% of distilled water by weight;
(3) fermentation culture: inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 4% of the seed liquid by volume, fermenting for 80 hours at the fermentation temperature of 26 ℃, the pH value of 8 and the rotation speed of 170r/min to obtain fermentation liquid;
the fermentation medium is by weightThe following steps: 1.0 percent of glucose, 1.2 percent of casein, 0.3 percent of yeast powder and K2HPO40.1％，Mn2SO40.02%, Tween-800.01% and distilled water 97.37%;
(4) collecting the protease: centrifuging the fermentation liquid at 4 deg.C at 10000rpm for 8min, and collecting supernatant; slowly adding ammonium sulfate into the supernatant while stirring at 4 ℃, adding 561 g of ammonium sulfate into each liter of solution to ensure that the saturation degree of the ammonium sulfate in the supernatant reaches 80%, and salting out at 4 ℃ for 10h after full dissolution to obtain salting-out solution; centrifuging the salting-out solution at 4 deg.C at 7000rpm for 25min, collecting precipitate, and dissolving the precipitate with 30mmol/L phosphate buffer solution with pH of 7.2 to obtain crude protease solution; transferring the crude protease liquid into a 3500D dialysis bag with molecular weight cutoff, placing the bag in 3L phosphoric acid buffer solution with mass concentration of 30mmol/L, dialyzing overnight for desalting, changing the buffer solution for 2 times, and freeze-drying the dialysate to obtain the low-temperature protease.
Example 3
The method for producing the low-temperature protease by the protease-producing strain LS20-2-2 comprises the following steps:
(1) strain activation: inoculating the strain to LB plate culture medium containing 1.2% skimmed milk powder, and culturing at 22 deg.C until transparent ring is formed;
(2) seed culture: inoculating the single colony generating the transparent ring in a seed culture medium, and culturing at 28 ℃ for 28h to obtain a seed solution; the seed culture medium is prepared from peptone 1.2%, yeast powder 0.8%, NaCl1.2% and 96.8% distilled water by weight;
(3) fermentation culture: inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 5% of the volume for fermentation, wherein the fermentation temperature is 28 ℃, the pH value is 9, the rotating speed is 180r/min, and the fermentation time is 86 hours to obtain fermentation liquid;
the fermentation medium is prepared from the following components in percentage by weight: starch 1.0%, skimmed milk powder 1.5%, Mn2SO40.02%, Tween-800.03% and 97.45% distilled water;
(4) collecting the protease: centrifuging the fermentation liquor at 5 deg.C and 8000rpm for 10min, and collecting supernatant; slowly adding ammonium sulfate into the supernatant while stirring at 5 ℃, adding 662 g of ammonium sulfate into each liter of solution to ensure that the saturation degree of the ammonium sulfate in the supernatant reaches 90%, and salting out for 12h at 5 ℃ after full dissolution to obtain salting-out solution; centrifuging the salting-out solution at 5 deg.C and rotation speed of 8000rpm for 20min, collecting precipitate, and dissolving the precipitate with 50mmol/L phosphate buffer solution with pH of 7.2 to obtain protease crude enzyme solution; transferring the crude protease liquid into a 3500D dialysis bag with molecular weight cutoff, placing in 3.5L phosphoric acid buffer solution with mass concentration of 50mmol/L, dialyzing overnight to remove salt, changing the buffer solution for 3 times, and freeze-drying the dialysate to obtain low-temperature protease.
The invention relates to Paenibacillus epidermidis from Qinghai-Tibet plateau soilPaenibacillus peoriaeA method of producing protease. The method comprises the specific steps of screening strains, preparing seed liquid, preparing an enzyme production culture medium and preparing crude enzyme. The protease produced by the method has the enzymological characteristics that:
1. the optimal reaction temperature is 40 ℃, the enzyme activity is high within the range of 20-50 ℃, and after the temperature is higher than 50 ℃, the enzyme activity is rapidly reduced, and obvious low-temperature protease characteristics are shown; the enzyme activity is stable within 0-40 ℃, and 86.8 percent of activity is still kept after heat preservation for 1h at 40 ℃; after 1h of treatment at 60 ℃, 70 ℃ and 80 ℃, the activity of the protease is only 11.7 percent, 11.2 percent and 5.6 percent of the original activity, which is consistent with the characteristic that the low-temperature protease is sensitive to heat.
2. The optimum pH value is 7.0-9.0, the enzyme activity is stable when the pH value is 5.0-7.0, and the protease belongs to neutral protease;
3. metallic ion Fe2+、Zn2+Can partially inhibit the activity of the enzyme, and Mn2+And the surfactants Tween-20 and Tween-80 have obvious promotion effect on the enzyme activity.
The enzymology characteristics of the protease prepared by the invention are obtained by repeated experiments, and the related experimental data are as follows:
the enzyme assay method used in the present invention:
protease activity was determined using the Folin method: preheating substrate and enzyme solution at 40 deg.C for 2min, respectively, adding 0.5mL diluted enzyme solution, adding 0.5mL substrate, reacting at 40 deg.C for 10min, and adding 1mL 10% trichloroacetic acid to stop reaction. Keeping the temperature at 40 ℃ for 10min, and then rapidly cooling. Centrifuging at 12000rpm for 2min, adding 0.4mol/L sodium carbonate 5mL into 1mL of supernatant, mixing, adding lml forskolin reagent (Solarbio), mixing, and keeping the temperature at 40 deg.C for 20 min. And replacing the supernatant with a buffer solution to serve as a reagent blank to adjust the zero point of the instrument, measuring the OD value at 680nm by using a spectrophotometer, adding no substrate casein solution when reacting a control group, and adding trichloroacetic acid and then adding a substrate after reacting for 10 min. The standard curve was prepared with different concentrations of L-tyrosine.
Enzyme activity is defined as: the amount of enzyme that produces 1. mu.g of tyrosine per hydrolyzed casein per ml of enzyme solution under certain conditions of temperature and pH is one activity unit (U).
Experiment 1: screening of protease-producing strains at Low temperatures
And primarily screening and re-screening plateau bacteria to obtain the protease-producing strain at low temperature.
The method specifically comprises the following steps: separating and culturing the strain of soil sample collected from the farmland of Qinghai-Tibet plateau by a conventional gradient dilution method. Selecting a single colony point with a sterilized toothpick to be connected with a 1.0% skimmed milk powder plate (1 g of skimmed milk powder, 50mL of distilled water, steam sterilization at 115 ℃ for 15 min; 1.0g of peptone, 0.5g of yeast powder, 1.0g of NaCl, 1.5 g of agar, 50mL of distilled water, steam sterilization at 121 ℃ for 25min, uniformly mixing two bottles of solution and pouring the solution into the plate), culturing at 4 ℃ for 72h, and selecting a strain with a larger ratio as a primary screened protease-producing strain according to the ratio of the radius of a generated hydrolysis ring to the radius of a bacterial colony.
And the secondary screening is to inoculate the strains obtained by the primary screening into a secondary screening culture medium for culture. The method specifically comprises the following steps: inoculating single colony of strain capable of producing protease at low temperature with sterilized toothpick to double-sieving culture medium (casein 1.0%, yeast powder 0.4%, glucose 1.0%, K)2HPO40.1％，Mg2SO4·7H2O0.02%, natural pH, steam sterilization at 115 ℃ for 20 min), shaking per 250mL of shaking bottle to fill 100mL of re-screening culture medium, fermenting for 72h, centrifuging, taking supernatant, measuring protease activity in fermentation liquor, and selecting the strain LS20-2-2 with the highest activity as a fermentation strain. The strain LS20-2-2 is isolated from rhizosphere soil of highland barley plants in the suburb of the Lassa.
Experiment 2: identification of protease-producing bacterium (LS20-2-2)
And identifying the screened strains to determine the taxonomic status of the strains.
The LS20-2-2 strain was subjected to physiological and biochemical identification tests according to Bergey's Manual of identification of bacteria and Manual of identification of bacteria systems, and the results are shown in Table 1, and the bacteria were identified as gram-positive bacteria: LS20-2-2 strain can hydrolyze casein, gelatin and starch, and reduce nitrate.
TABLE 1 physiological and biochemical identification of LS20-2-2 Strain
| Feature(s) | Results | Feature(s) | Results |
| Oxidase enzyme | - | Casein hydrolysis | + |
| Contact enzyme | + | Hydrolysis of gelatin | + |
| V.P. | - | Starch hydrolysis | + |
| MR | - | Nitrate reduction | + |
| Hydrogen sulfide test | - | Formation of indoles | - |
Note: "+" indicates a positive reaction; "-" indicates that the reaction was negative.
16S rDNA sequence analysis, which comprises the following steps:
(1) extracting genome DNA: the procedure was carried out according to the instruction manual of the bacterial total DNA extraction kit (Promega).
(2) And (4) PCR amplification. The amplification system was 50. mu.L, including 19. mu.L of sterile double distilled water, 2. mu.L each of the forward and reverse primers, 2. mu.L of template DNA, 25. mu.L of 2 XEs Taq MasterMix (Kangshi).
Reaction conditions are as follows: each reaction is carried out for 30 cycles, each cycle comprises denaturation at 94 ℃ for 30S, annealing at 56 ℃ for 30S and extension at 72 ℃ for 1.5 min; finally, extension is carried out for 10min at 72 ℃, and pre-denaturation is carried out for 2min at 95 ℃ in the first cycle.
PCR amplification primers: 27F (5'-AGAGTTTGATCCTGGCTCA-3'); 1492R (5'-GGTTACCTTGTTACGACTT-3').
(3) Agarose gel cutting purification
And (3) recovering the target fragment of about 1500bp by using a DNA gel recovery kit, and detecting the purity and the content of the purified DNA by electrophoresis.
(4) TA cloning and screening of fragments of interest
With pGEM®T is a vector for TA ligation, the linker is 10. mu.L, and comprises 2 × Rapid ligation buffer 5. mu.L of pGEM®-T Vector 1μL，PCR product 3μL，T4DNA Ligase 1. mu.L was transformed into E.coli DH5 α strain at 42 ℃ and finally plated on LB plate containing ampicillin positive clones were screened by blue-white spots.
(5) Plasmid DNA extraction and PCR identification
Plasmid extraction was performed according to the procedure of the plasmid extraction kit of OMEGA. The PCR primers used were SP6 (5'-ATTTAGGTGACACTATAG-3') and T7 (5'-TAATACGACTCACTATAGGG-3').
(6) DNA sequencing
And detecting the PCR amplification product by agarose gel electrophoresis, and sequencing the PCR amplification product by using the Huada gene. Similarity analysis was done by GenBank data.
Experiment 3: fermentation culture of protease-producing bacterium (LS20-2-2)
Inoculating the screened strain for fermentation into a seed culture medium for amplification, and then inoculating the strain for fermentation into a fermentation culture medium for fermentation. The method specifically comprises the following steps: inoculating LS20-2-2 strain slant in seed culture medium, culturing at 25 deg.C and 180rpm for 24 hr, adding seed solution at 5% inoculum size into fermentation culture medium (casein 1.2%, yeast powder 0.3%, glucose 1.0%, K)2HPO40.1％，Mn2SO40.02%, Tween-800.01%, distilled water, pH7.0, steam sterilization at 115 deg.C, 20 min). The fermentation culture is 100mL of fermentation medium in each 250mL shake flask, and the culture conditions are as follows: fermenting and culturing at 25 ℃ and 180rpm for 72 hours until the enzyme activity reaches 202U/mL.
Precipitating protein in the fermentation liquor by using 80% ammonium sulfate, centrifuging, taking the precipitate, desalting by a conventional method to obtain enzyme liquid, and freeze-drying the enzyme liquid to obtain protease raw powder.
Experiment 4: optimum temperature and thermal stability of protease
Performing enzymatic reaction at 0 deg.C, 10 deg.C, 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, and 80 deg.C for 10min, respectively, and determining protease activity. The optimal reaction temperature of the protease prepared by the invention is 40 ℃, the enzyme activity is high within the range of 20-50 ℃, and the enzyme activity is rapidly reduced after the temperature is higher than 50 ℃.
The diluted enzyme solution is respectively subjected to water bath heat preservation at the temperature of 0 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for 1h, and then is put into an ice-water mixture, and compared with the enzyme activity which is not subjected to heat preservation treatment, the residual activity of the protease is measured. The protease prepared by the invention has stable enzyme activity at 0-40 ℃, and the activity of the protease is only 11.7%, 11.2% and 5.6% of the original activity after 1h of treatment at 60 ℃, 70 ℃ and 80 ℃.
Experiment 5: optimum pH and pH stability of low temperature protease
Buffer solutions with pH of 3.0 (citric acid-sodium citrate), pH of 5.0 (phosphoric acid), pH of 7.0 (phosphoric acid), pH of 9.0 (glycine-sodium hydroxide) and pH of 11.0 (carbonate) are prepared, and the buffer solutions are prepared into 1% casein solutions with different pH values to be used as substrates respectively, so that the protease activity is measured. Diluting the enzyme solution with buffer solutions with different pH values, storing at 4 ℃ for 20h, and measuring the residual enzyme activity.
The protease secreted by the strain has activity at pH5.0-11.0, and shows high activity at pH7.0-9.0. The enzyme solution is kept stable under the condition of pH5.0-7.0, and the enzyme activity of more than 98% is still kept after treatment, belonging to neutral protease.
Experiment 6: effect of various inhibitors on enzyme Activity
Under the conditions of optimal temperature and optimal pH of the enzyme reaction, adding different inhibitors into the reaction system to ensure that the ion concentration in the reaction system reaches 2 mu mol/mL, taking the enzyme activity without the inhibitors as the contrast of 100 percent, and adding Mn2+Tween-80 and Tween-20 have obvious activation effect on enzyme activity, and the relative enzyme activity reaches 134.6%, 127.1% and 115.4%; there is a difference in the effect of various metal ions on the enzyme activity, among which Zn is present2+Has the strongest inhibiting effect, the relative enzyme activity is reduced to 78 percent, and Fe is used for the second time2+EDTA has weak inhibition effect on enzyme activity.
The preparation process is simple and efficient, energy-saving and environment-friendly, the prepared protease has high enzyme activity which can reach 202U/mL at most, and the protease can be effectively prepared at low temperature, the prepared protease has high enzyme activity at 30-50 ℃, the activity of more than 80% can be still maintained after the temperature is kept for 60min at 0-40 ℃, and the low-temperature stability is high; the strain produces special fragrance in the fermentation process, and the produced protease is extracellular enzyme and is convenient to collect.
SEQUENCE LISTING
<110> institute of biological sciences, Inc. of Henan province
<120> protease-producing strain LS20-2-2 and method for producing low temperature protease by using same
<130>2016
<160>1
<170>PatentIn version 3.5
<210>1
<211>1548
<212>DNA
<213> Paenibacillus pickettii (Paenibacillus peiorae)
<400>1
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agcctgccca caagacaggg ataactaccg gaaacggtag ctaatacccg atacatcctt 180
ttcctgcatg ggagagggag gaaagacgga gcaatctgtc acttgtggat gggcctgcgg 240
cgcattagct agttggtggg gtaaaggcct accaaggcga cgatgcgtag ccgacctgag 300
agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta 360
gggaatcttc cgcaatgggc gaaagcctga cggagcaacg ccgcgtgagt gatgaaggtt 420
ttcggatcgt aaagctctgt tgccagggaa gaacgtcttg tagagtaact gctacaagag 480
tgacggtacc tgagaagaaa gccccggcta actacgtgcc agcagccgcg gtaatacgta 540
gggggcaagc gttgtccgga attattgggc gtaaagcgcg cgcaggcggc tctttaagtc 600
tggtgtttaa tcccgaggct caacttcggg tcgcactgga aactggggag cttgagtgca 660
gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc 720
agtggcgaag gcgactctct gggctgtaac tgacgctgag gcgcgaaagc gtggggagca 780
aacaggatta gataccctgg tagtccacgc cgtaaacgat gaatgctagg tgttaggggt 840
ttcgataccc ttggtgccga agttaacaca ttaagcattc cgcctgggga gtacggtcgc 900
aagactgaaa ctcaaaggaa ttgacgggga cccgcacaag cagtggagta tgtggtttaa 960
ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc ctctgaccgg tctagagata 1020
gacctttcct tcgggacaga ggagacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg 1080
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cgtaacaagg taaccaatca ctagtgcggc cgcctgcagg tcgaccat 1548
Claims (4)
1. A method for producing low-temperature protease by protease-producing strain LS20-2-2 is characterized in that the protease-producing strain LS20-2-2 is classified and named as Paenibacillus epidermidis (Paenibacillus peyae), is preserved in the China general microbiological culture Collection center in 28 months in 2016 (9.9.9.28.s), has the preservation number of CGMCC No.13053, is a strain separated and screened from soil samples collected from Qinghai-Tibet plateau, and comprises the following steps:
(1) strain activation: inoculating strain LS20-2-2 to LB plate culture medium containing skimmed milk powder with mass concentration of 0.8-1.2%, and culturing at 18-22 deg.C until transparent ring is generated;
(2) seed culture: inoculating the single colony generating the transparent ring in a seed culture medium, and culturing at 25-28 ℃ for 20-28h to obtain a seed solution; the seed culture medium is prepared from 0.8-1.2% of peptone, 0.2-0.8% of yeast powder, 0.8-1.2% of NaCl and the balance of distilled water by weight;
(3) fermentation culture: inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 2-5% of the volume for fermentation at the temperature of 25-28 ℃, the pH value of 7-9 and the rotation speed of 160-180r/min for 72-86h to obtain a fermentation liquid;
the fermentation medium is prepared from the following components in percentage by weight: carbon source 0.5-1.0%, nitrogen source 0.9-1.5%, K2HPO40 to 0.1 percent of enzyme production promoter, 0.02 to 0.05 percent of enzyme production promoter and the balance of distilled water; the carbon source is one of corn flour, starch, glucose and sucrose; the nitrogen source is one or two of peptone, casein, yeast powder, skimmed milk powder and soybean meal according to the volume ratio of 1.0-1.2: mixing at a ratio of 0.3-0.5; the enzyme production promoter is a metal ion and Tween series surfactant;
(4) collecting the protease: centrifuging the fermentation liquid at 3-5 deg.C and 8000-; slowly adding ammonium sulfate into the supernatant while stirring at 3-5 ℃, adding 472-662 g of ammonium sulfate into each liter of solution to ensure that the saturation degree of the ammonium sulfate in the supernatant reaches 70-90%, and salting out for 8-12h at 3-5 ℃ after full dissolution to obtain salting-out solution; centrifuging the salting-out solution at 3-5 deg.C and rotation speed of 5000-8000rpm for 20-30min, collecting precipitate, and dissolving the precipitate with phosphate buffer solution of pH7.2 and mass concentration of 20-50mmol/L to obtain crude protease solution; transferring the crude protease liquid into a 3500D dialysis bag with molecular weight cutoff, placing in 2.5-3.5L phosphoric acid buffer solution with mass concentration of 20-50mmol/L, dialyzing overnight for desalting, changing the buffer solution for 2-3 times, and freeze-drying the dialysate to obtain the low-temperature protease.
2. The method for producing low temperature protease according to claim 1 by protease producing strain LS20-2-2, comprising the steps of:
(1) strain activation: inoculating the strain into LB plate culture medium containing 0.8% skimmed milk powder, and culturing at 18 deg.C until transparent ring is generated;
(2) seed culture: inoculating the single colony generating the transparent ring in a seed culture medium, and culturing at 25 ℃ for 20h to obtain a seed solution; the seed culture medium is prepared from 0.8% of peptone, 0.2% of yeast powder, 0.8% of NaCl and 98.2% of distilled water by weight;
(3) fermentation culture: inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 2% of the volume for fermentation, wherein the fermentation temperature is 25 ℃, the pH value is 7.0, the rotating speed is 160r/min, and the fermentation is carried out for 72 hours to obtain a fermentation liquid;
the fermentation medium is prepared from the following components in percentage by weight: corn flour 0.5%, peptone 0.9%, K2HPO40.01 percent, Tween-800.01 percent and distilled water 98.58 percent;
(4) collecting the protease: centrifuging the fermentation liquor at 3 deg.C and 12000rpm for 5min, and collecting supernatant; slowly adding ammonium sulfate into the supernatant while stirring at 3 ℃, adding 472 g of ammonium sulfate into each liter of solution to ensure that the saturation degree of the ammonium sulfate in the supernatant reaches 70%, and salting out for 8h at 3 ℃ after full dissolution to obtain salting-out solution; centrifuging the salting-out solution at 3 deg.C and 5000rpm for 30min, collecting precipitate, and dissolving the precipitate with 20mmol/L phosphate buffer solution with pH of 7.2 to obtain crude protease solution; transferring the crude protease liquid into a 3500D dialysis bag with molecular weight cutoff, placing the bag in 2.5L phosphoric acid buffer solution with mass concentration of 20mmol/L, dialyzing overnight to remove salt, changing the buffer solution for 2 times, and freeze-drying the dialysate to obtain the low-temperature protease.
3. The method for producing low temperature protease according to claim 1 by protease producing strain LS20-2-2, comprising the steps of:
(1) strain activation: inoculating the strain to LB plate culture medium containing 1.0% skimmed milk powder, and culturing at 20 deg.C until transparent ring is formed;
(2) seed culture: inoculating the single colony generating the transparent ring in a seed culture medium, and culturing at 26 ℃ for 24h to obtain a seed solution; the seed culture medium is prepared from 1.0% of peptone, 0.5% of yeast powder, 1.0% of NaCl and 97.5% of distilled water by weight;
(3) fermentation culture: inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 4% of the seed liquid by volume, fermenting for 80 hours at the fermentation temperature of 26 ℃, the pH value of 8 and the rotation speed of 170r/min to obtain fermentation liquid;
the fermentation medium is prepared from the following components in percentage by weight: 1.0 percent of glucose, 1.2 percent of casein, 0.3 percent of yeast powder and K2HPO40.1％，Mn2SO40.02%, Tween-800.01% and distilled water 97.37%;
(4) collecting the protease: centrifuging the fermentation liquid at 4 deg.C at 10000rpm for 8min, and collecting supernatant; slowly adding ammonium sulfate into the supernatant while stirring at 4 ℃, adding 561 g of ammonium sulfate into each liter of solution to ensure that the saturation degree of the ammonium sulfate in the supernatant reaches 80%, and salting out at 4 ℃ for 10h after full dissolution to obtain salting-out solution; centrifuging the salting-out solution at 4 deg.C at 7000rpm for 25min, collecting precipitate, and dissolving the precipitate with 30mmol/L phosphate buffer solution with pH of 7.2 to obtain crude protease solution; transferring the crude protease liquid into a 3500D dialysis bag with molecular weight cutoff, placing the bag in 3L phosphoric acid buffer solution with mass concentration of 30mmol/L, dialyzing overnight for desalting, changing the buffer solution for 2 times, and freeze-drying the dialysate to obtain the low-temperature protease.
4. The method for producing low temperature protease according to claim 1 by protease producing strain LS20-2-2, comprising the steps of:
(1) strain activation: inoculating the strain to LB plate culture medium containing 1.2% skimmed milk powder, and culturing at 22 deg.C until transparent ring is formed;
(2) seed culture: inoculating the single colony generating the transparent ring in a seed culture medium, and culturing at 28 ℃ for 28h to obtain a seed solution; the seed culture medium is prepared from peptone 1.2%, yeast powder 0.8%, NaCl1.2% and 96.8% distilled water by weight;
(3) fermentation culture: inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 5% of the volume for fermentation, wherein the fermentation temperature is 28 ℃, the pH value is 9, the rotating speed is 180r/min, and the fermentation time is 86 hours to obtain fermentation liquid;
the fermentation medium is prepared from the following components in percentage by weight: starch 1.0%, skimmed milk powder 1.5%, Mn2SO40.02%, Tween-800.03% and 97.45% distilled water;
(4) collecting the protease: centrifuging the fermentation liquor at 5 deg.C and 8000rpm for 10min, and collecting supernatant; slowly adding ammonium sulfate into the supernatant while stirring at 5 ℃, adding 662 g of ammonium sulfate into each liter of solution to ensure that the saturation degree of the ammonium sulfate in the supernatant reaches 90%, and salting out for 12h at 5 ℃ after full dissolution to obtain salting-out solution; centrifuging the salting-out solution at 5 deg.C and rotation speed of 8000rpm for 20min, collecting precipitate, and dissolving the precipitate with 50mmol/L phosphate buffer solution with pH of 7.2 to obtain protease crude enzyme solution; transferring the crude protease liquid into a 3500D dialysis bag with molecular weight cutoff, placing in 3.5L phosphoric acid buffer solution with mass concentration of 50mmol/L, dialyzing overnight to remove salt, changing the buffer solution for 3 times, and freeze-drying the dialysate to obtain low-temperature protease.
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