CN114134074B - Strain for producing low-temperature alkaline protease and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to a strain for producing low-temperature alkaline protease and a production method thereof. The strain is specifically Bacillus sp. UN-28, deposit No.: CGMCC No.23195. The strain is obtained by means of UV-NTG composite mutagenesis, the shake flask fermentation activity of the produced alkaline protease is 75336U/mL, and the enzyme activity of a fermentation tank is 150672U/mL. The alkaline protease has good activity at low temperature, the optimal action temperature range is 25-50 ℃, the optimal action pH range is 8.0-11.5, the alkaline protease belongs to alkaline protease, the alkaline protease has strong alkali resistance, the alkaline protease can maintain the enzyme activity of more than 90% after heat preservation for 1h at 35 ℃ under the condition that the pH is 6.5-11.5, and the alkaline protease has good pH compatibility with washing powder, detergents and the like, and has good application prospect and market potential in the detergent industry.
Description
Technical field:
the invention belongs to the technical field of microorganisms, and particularly relates to a strain for producing low-temperature alkaline protease and a method for producing the protease.
The background technology is as follows:
proteases are enzymes that act on proteins or polypeptides, catalyzing the hydrolysis of peptide bonds. Among them, proteases whose optimum pH of hydrolyzed protein is in the alkaline range are called alkaline proteases.
The alkaline protease is a popular washing additive in the market at present, can greatly improve the washing and decontamination capability, has unique washing effect on protein dirt such as sweat stain, milk stain, blood stain, oil stain and the like, does not contain phosphorus, has no pollution, does not need high water temperature, has high washing efficiency and saves energy, and therefore, the alkaline protease has been widely applied to the washing industry as an additive. It is reported that protease accounts for 60% -65% of the total amount of enzyme preparation sold worldwide, wherein alkaline protease is used as an additive of phosphate-free washing powder, and the sales thereof account for more than 1/3 of the whole protease market.
Because of the specificity of the environment in which detergents function with alkaline proteases, two conditions are required: firstly, high activity and stability can be maintained in a wide pH and temperature range; secondly, it must be combined with a washing powder containing various oxidants, chelating agents and surfactants. In addition, because the middle-temperature alkaline protease is mainly added in the enzyme-added detergent at present, the optimal temperature of the enzyme-added detergent is generally above 50 ℃, and unlike the condition that the washing is carried out by using hot water at 40-60 ℃ in the foreign habit, the washing is carried out by using cold water in the Chinese habit, so that the alkaline protease is required to have good activity under the low-temperature condition.
China is a large country of detergent production and demand, however, the main enzymatic properties of the current domestic alkaline protease can not meet the demands of domestic detergent industry development, and the large-scale industrial alkaline protease mainly depends on foreign import. Therefore, it is very necessary and important to find a high-yield strain capable of secreting alkaline protease, which has good enzymatic properties and is suitable for large-scale industrial production of detergents, and has good application prospect and market potential.
The invention comprises the following steps:
in order to solve the technical problems, the invention provides a strain for producing low-temperature alkaline protease and a method for producing the protease by adopting the strain.
The strain producing alkaline protease, in particular Bacillus sp.) UN-28, is a low temperature alkaline protease producing strain obtained by means of UV-NTG complex mutagenesis, and the strain UN-28 has been deposited in the general microbiological centre of the chinese microbiological bacterial deposit management committee at month 8 of 2021, address: beijing, chaoyang area, north Chen Xili No. 1, 3, department of microbiology, department of Chinese sciences, accession number: CGMCC No.23195.
The protease produced has the characteristic of maintaining higher activity in low-temperature and alkaline environments, and the specific enzymology properties are as follows:
(1) The optimal pH range is 8.0-11.5, and the optimal pH is 11.0;
(2) The optimal action temperature is 25-50 ℃ and the optimal temperature is 35 ℃;
(3) Temperature stability: at pH11.0, the temperature was maintained for 30min at various bath temperatures: the enzyme can maintain more than 95% of enzyme activity within the range of 10-40 ℃, can maintain 65% of enzyme activity after being treated for 30min at 50 ℃, and has good thermal stability;
(4) pH stability: under different pH conditions, preserving the temperature at 35 ℃ for 1h: the enzyme can maintain the enzyme activity of more than 90% under the condition that the pH value is 6.5-11.5, and still maintains the enzyme activity of 60% after the temperature is kept for 1h at 35 ℃ under the condition that the pH value is 12.5.
The invention also provides an application of bacillus UN-28 in producing alkaline protease, in particular to an application in producing alkaline protease by fermentation, which comprises the following specific steps:
culturing primary seeds in a shaking bottle: inoculating the strain subjected to slant culture into 250ml seed shake flask culture medium, and culturing at 28-32deg.C for 18-20 hr with liquid loading amount of 50ml and rotary shaking table of 180-200 r/min;
shake flask culture of secondary seeds: inoculating the first-stage seeds into 500ml seed shaking flask culture medium according to the inoculation amount of 8-10%, and the liquid loading amount is 100ml, wherein the culture conditions are the same as those of the first-stage seeds;
seed pot culture: transferring the second-level seed liquid into a seed tank according to the inoculation amount of 8-10% of the volume ratio, continuously culturing for 18-24h at the culture temperature of 28-32 ℃, controlling the tank pressure to be 0.05-0.08MPa, and stirring at the rotation speed of 200-400r/min and the pH value to be 7.5-8.0;
fermentation culture: inoculating seed solution of a seed tank into a fermentation tank according to the inoculum size of 8-10% of the volume ratio, culturing at 28-32 ℃, wherein the tank pressure is 0.05-0.08Mpa, the stirring speed is 200-800r/min, the dissolved oxygen is controlled between 20-30%, and the pH of the fermentation liquid is controlled to be maintained within the range of 7.5-8.0 by adding a feed medium, ammonia water or dilute phosphoric acid in a coordinated manner in the fermentation process;
and (3) tank discharge: culturing for about 80-84h, slowly increasing or no longer increasing enzyme activity, and discharging when part of thallus is aged and autolyzed.
Extracting and refining: collecting fermentation liquor after tank discharge, adding 0.2% -0.3% of preservative, 3-5% of disodium hydrogen phosphate and 1-3% of calcium chloride for flocculation, then adding 2-4% of perlite filter aid, carrying out plate frame filter pressing to obtain clarified enzyme liquor, carrying out ultrafiltration on the clarified enzyme liquor by using 10000 molecular weight ultrafiltration membranes to obtain ultrafiltrate, regulating the pH value to 8.0, and then carrying out fine filtration and sterilization by using diatomite to obtain the alkaline protease liquid product.
The culture medium used in the above culture process was as follows:
seed shake flask medium (g/L): 4-6 parts of yeast powder, 10-12 parts of glucose and 3-5,K parts of tryptone 2 HPO 4 15-20, the rest is water, pH7.0-7.2, sterilizing at 121-124 deg.C for 20-30min.
Seeding tank medium (g/L): 5-10 parts of yeast powder, 1-5 parts of corn steep liquor, 80-120 parts of maltodextrin and 1-5,K parts of peptone 2 HPO 4 15-20，Mg 2 SO 4 0.8-1, the balance being water, pH7.5-8.0, 121-124 ℃, sterilizing for 30-40min.
Fermenter Medium (g/L): 8-12 parts of yeast powder, 15-25 parts of bean cake powder, 5-10 parts of corn steep liquor, 80-120 parts of corn meal, 2-5 parts of sodium citrate and CaCl 2 3-5、Mg 2 SO 4 0.8-1，K 2 HP0 4 15-20, the balance being water, pH7.5-8.0, 121-124 ℃, sterilizing for 30-40min.
Feed medium (g/L): 25-40 parts of bean cake powder, 150-200 parts of corn starch liquefied liquid, and the balance of water, wherein the pH is 7.5-8.0, the temperature is 121-124 ℃, and the sterilization is carried out for 30-40min.
The beneficial effects are that:
the invention obtains the low-temperature alkaline protease production strain UN-28 by carrying out UV-NTG composite mutagenesis screening on the original strain, and the produced alkaline protease can still show higher enzyme activity level under low-temperature alkaline conditions.
The ability of producing alkaline protease by fermenting the mutagenesis strain UN-28 is obviously improved, the enzyme activity reaches 150672U/mL, and the enzyme activity is improved by 30% compared with the original strain.
The enzyme has good activity at low temperature, the optimal action temperature range is 25-50 ℃, the enzyme activity of more than 80% can be maintained, the enzyme activity of 68% is maintained at 20 ℃, when the enzyme is added into a detergent for washing clothes, the washing effect can be exerted without soaking the clothes in hot water at 40-60 ℃, the energy is saved, the washing mode of household tap water in Asia, particularly in developing China is met, and the global development of the washing industry towards low temperature and water saving is promoted.
The pH of the enzyme is 8.0-11.5, the enzyme has strong alkali resistance, and the enzyme activity of more than 90% can be maintained after heat preservation for 1h at 35 ℃ under the condition that the pH is 6.5-11.5, and the enzyme has good pH compatibility with washing powder, liquid laundry detergent and the like.
The enzyme solution with the concentration of 4g/L and the standard washing powder solution with the concentration of 4g/L are respectively prepared, are uniformly mixed in a ratio of 1:1, and have good compatibility with detergents after heat preservation for 1h at 35 ℃ and have good application prospects in the detergent industry. The standard washing powder comprises the following formula: sodium alkyl benzene sulfonate: 15 parts of sodium tripolyphosphate: 17 parts of sodium silicate: 10 parts of sodium carbonate: 3 parts of sodium carboxymethylcellulose (CMC): 1 part of sodium sulfate: 58 parts.
Description of the drawings:
FIG. 1 shows the relative enzyme activity change curves at different reaction pH values;
FIG. 2 shows the relative enzyme activity change curves at different reaction temperatures;
FIG. 3 is a graph of thermostability versus enzyme activity;
FIG. 4 pH stability versus enzyme activity curve of enzyme.
The specific embodiment is as follows:
the invention will now be described in more detail by way of specific examples, which are given by way of illustration only and are not intended to limit the scope of the invention. Modifications which would occur to those skilled in the art based on the principles of this invention are also considered to be within the scope of this invention.
EXAMPLE 1 mutagenesis screening of strains
(1) UV-NTG complex mutagenesis
Inoculating the original strain AP-01 into a seed culture medium, and culturing for 18 hours at a culture temperature of 30 ℃ by a rotary shaking table of 180r/min to obtain a bacterial suspension to be mutagenized. The supernatant was removed by centrifugation, the cells were washed with physiological saline for 2 times, then resuspended in 0.05mol/L phosphate buffer pH7.4, and NTG was added to a final concentration of 0.4g/L, followed by treatment at 30℃for 20min under 100r/min of shaking back and forth. Centrifuging, removing supernatant, centrifuging with phosphate buffer solution for 3 times to remove NTG, re-suspending with buffer solution, pouring 10mL of bacterial solution into sterile culture dish, respectively irradiating with ultraviolet lamp (15 w, distance 30 cm) in dark for 30s, 60s, 90s, 120s, gradient diluting with 200 μL, coating 100 μL on screening culture medium plate, and culturing at 30deg.C in dark for 36 hr.
The composition of the screening culture medium is (g/L): yeast powder 2, peptone 5, beef extract 7, casein 2,K 2 HP0 4 10 parts of sodium chloride 1, skim milk powder 20, agar powder 18 and the balance of water. The skimmed milk powder and other nutrient components are sterilized respectively, and then mixed, and sterilized at pH7.5 and 121 ℃ for 20min.
(2) Flat plate primary screen
And (5) performing primary screening by using the size of the transparent ring. Observing the morphology of the grown colonies and the generation of casein hydrolysis rings around the colonies, picking the colonies with changed morphology and larger casein hydrolysis rings, separating and purifying by a plate streaking method, repeating for 3 times, picking single colonies, inoculating the single colonies into a slant culture medium, culturing at 30 ℃ for 48 hours, and preserving in a refrigerator at 4 ℃.
(3) Shaking bottle enzyme production compound sieve
Inoculating the bacterial colony obtained by primary screening into shake flask secondary screening culture medium, culturing at 30deg.C for 72 hr, centrifuging the fermentation broth at 4deg.C and 8000r/min for 10min to obtain crude enzyme solution, and measuring the activity and enzymatic characteristic curve of alkaline protease.
The shaking bottle rescreening culture medium comprises the following components (g/L): peptone 5, yeast extract 5, grape farm chemical 8,K 2 HPO 4 10, the rest is water, pH7.5, 121 ℃ and sterilization for 30min.
The invention adopts Folin reagent color development method to determine the enzyme activity of alkaline protease, namely, the enzyme quantity required by hydrolyzing casein to generate 1 mu g tyrosine per minute with 1mL enzyme solution is defined as 1 enzyme activity unit U/mL at the pH of 11.0 and 35 ℃.
The mutant strain is obtained through shaking bottle re-screening, the enzyme activity of alkaline protease in crude enzyme liquid is 75336U/mL, which is 25% higher than that of the original strain (60269U/mL), and the strain can maintain higher activity in low temperature and alkaline environment, and is named as bacillus (UN-28).
Example 2 stability passage test of strains
And carrying out a passage experiment on the mutant strain obtained by re-screening, and calculating the relative enzyme activity by taking the shake flask fermentation enzyme activity of the first-generation strain as 100%. As can be seen from Table 1, the strain has little change in enzyme productivity after 6 continuous passages and good passage stability.
TABLE 1 genetic stability test results of mutagenized Strain
Example 3 Strain fermentation enzyme production
Culturing primary seeds in a shaking bottle: inoculating the strain subjected to slant culture into a seed culture medium, shaking 250mL, filling the culture medium with a liquid volume of 50mL, and rotating a shaking table for 200r/min at a culture temperature of 30 ℃ for 18h;
shake flask culture of secondary seeds: the first-level seeds are inoculated into 500mL seed shaking flask culture medium according to the inoculation amount of 8 percent, the liquid loading amount is 100mL, and the culture conditions are the same as those of the first-level seeds;
seed pot culture: transferring the secondary seed liquid into a seed tank according to the inoculation amount of 8% by volume ratio, continuously culturing for 18h at a culture temperature of 30 ℃, controlling the tank pressure to be 0.05-0.08MPa, stirring at a rotation speed of 200-400r/min and a pH value of 7.5-8.0;
fermentation culture: inoculating seed solution of a seed tank into a fermentation tank according to an inoculum size of 8% by volume, culturing at 30 ℃, and stirring at a rotation speed of 200-800r/min under a tank pressure of 0.05-0.08 Mpa;
dissolved oxygen control: adjusting the stirring speed to control the dissolved oxygen to be between 20 and 30 percent;
pH control: the pH of the fermentation liquid is controlled by the coordination and the supplement of a feed culture medium, ammonia water or dilute phosphoric acid, so that the pH of the fermentation liquid is maintained within the range of 7.5-8.0;
and (3) tank discharge: culturing for about 84 hours, slowly growing enzyme activity, and placing the cells in a tank when partial cells age and autolyze.
The culture medium used in the above culture process was as follows:
seed medium (g/L): yeast powder 6, glucose 10, tryptone 4, K 2 HPO 4 18, the balance being water, pH7.0-7.2, sterilizing at 121-124 ℃ for 20min.
Seeding tank medium (g/L): yeast powder 6, corn steep liquor 5, maltodextrin 100, peptone 4, K 2 HPO 4 18，Mg 2 SO 4 0.8, the balance being water, pH7.5-8.0, 121-124 ℃, sterilizing for 35min.
Fermenter Medium (g/L): yeast powder 10, bean cake powder 20, corn steep liquor 7, corn flour 100g, sodium citrate 4 and CaCl 2 4g、Mg 2 SO 4 0.8，K 2 HP0 4 18g, the balance of water, pH7.5-8.0, 121-124 ℃, and sterilizing for 30min.
Feed medium (g/L): bean cake powder 30, corn starch liquefied liquid 180, the balance of water, pH7.5-8.0, 121-124 ℃, and sterilizing for 35min.
The enzyme activity of alkaline protease was determined by Folin reagent color development, i.e., the amount of enzyme required to hydrolyze casein at pH11.0 at 35℃in 1mL of enzyme solution per minute to produce 1. Mu.g of tyrosine was defined as 1 enzyme activity unit (U/mL).
In the embodiment, the enzyme activity of alkaline protease in the fermentation broth of the UN-28 tank is 150672U/mL, which is improved by 30% compared with that of the strain AP-01 (115912U/mL).
Extracting and refining: collecting fermentation liquor after tank placement, adding 0.2% of preservative, 3% of disodium hydrogen phosphate and 1% of calcium chloride for flocculation, adding 2% of perlite filter aid, and carrying out plate-frame filter pressing to obtain clarified enzyme liquid. Ultrafiltering the clarified enzyme solution by using an ultrafiltration membrane with 10000 molecular weight to obtain ultrafiltrate. And regulating the pH value to 8.0, and then finely filtering and sterilizing by using diatomite to obtain the liquid product of the alkaline protease.
Example 4 pH range for optimum action of alkaline protease
With NaH of 0.5mol/L respectively 2 PO 4 -Na 2 HPO 4 (pH6.0-8.5)、Na 2 B 4 O 7 Buffer system composed of NaOH (pH9.0-12.5), buffer solutions with different pH values are prepared at intervals of 0.5, substrate solutions with different pH values are prepared by using the corresponding buffer solutions, finished alkaline protease products prepared in example 3 are taken, and enzyme activities under different pH conditions are respectively measured under the water bath condition of 35 ℃ to determine the optimal reaction pH. And drawing a relative enzyme activity change curve under different pH conditions by taking the highest enzyme activity as 100%. As shown in FIG. 1, the pH of the optimum reaction is 11.0, and the enzyme activity of more than 80% can be maintained in the pH range of 8.0-11.5, so that the reaction is suitable for washing alkaline environment.
Example 5 optimal temperature range for alkaline protease
Taking the alkaline protease finished product prepared in the example 3, respectively measuring the enzyme activity at the temperature of different water baths at intervals of 5 ℃ in the range of 10-65 ℃ under the condition of pH11.0 so as to determine the optimal reaction temperature. And drawing a relative enzyme activity change curve under different temperature conditions by taking the highest enzyme activity as 100%. As shown in figure 2, the optimal reaction temperature is 35 ℃, and the enzyme activity is maintained at a higher level between 25 and 45 ℃, and the enzyme activity is still more than 45% at 15 ℃, belonging to low-temperature enzymes.
Example 6 thermostability of alkaline protease
Taking the finished alkaline protease product prepared in the example 3, respectively preserving heat for 30min at 10-65 ℃ at intervals of 5 ℃ under the condition of pH11.0, and measuring the enzyme activity at the condition of pH11.0 at 35 ℃ after cooling. The thermostability curve was drawn with the initial enzyme activity (150672U/mL) as 100%. As shown in FIG. 3, the enzyme was able to maintain 65% of the enzyme activity after 30min of treatment at 50 ℃.
EXAMPLE 7 pH stability of alkaline protease
The alkaline protease finished product prepared in example 3 was taken and kept at 35℃in a buffer solution at pH6.0-12.5 for 1 hour, respectively, and then the enzyme activity was measured at 35℃and pH 11.0. The pH stability curve was drawn with the initial enzyme activity (150672U/mL) taken as 100%. As shown in FIG. 4, the enzyme can maintain the enzyme activity of more than 90% under the condition that the pH value is 6.5-11.5, and still maintains the enzyme activity of 60% after the temperature is kept for 1h at 35 ℃ under the condition that the pH value is 12.5, so that the alkali resistance is high.
EXAMPLE 8 compatibility of alkaline protease with Standard washing powder
Taking the finished alkaline protease product prepared in the example 3, preparing a solution with the concentration of 4g/L, uniformly mixing the solution with a standard washing powder solution with the concentration of 4g/L in a ratio of 1:1, preserving the temperature at 35 ℃ for 1h, measuring the activity of the alkaline protease under the conditions of 35 ℃ and pH11.0, and calculating the activity of the residual enzyme by taking the initial activity as 100%, wherein the activity of the residual enzyme is 95%.
The standard washing powder comprises the following formula: sodium alkyl benzene sulfonate: 15 parts of sodium tripolyphosphate: 17 parts of sodium silicate: 10 parts of sodium carbonate: 3 parts of sodium carboxymethylcellulose (CMC): 1 part of sodium sulfate: 58 parts.
The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that, for a person skilled in the art, the above embodiments may also make several variations, combinations and improvements, without departing from the scope of the present patent. Therefore, the protection scope of the patent is subject to the claims.
Claims (4)
1. The Bacillus for producing the low-temperature alkaline protease is characterized by specifically comprising Bacillus UN-28 and the preservation number of CGMCC No.23195.
2. Use of Bacillus sp. UN-28 according to claim 1 for the production of alkaline protease.
3. Use according to claim 2, wherein the alkaline protease is produced by fermentation in the following way:
inoculating 8% -10% of seed liquid into a fermentation culture medium, culturing at 28-32 ℃, carrying out tank pressure of 0.05-0.08Mpa, stirring at a speed of 200-800r/min, controlling dissolved oxygen between 20% -30%, and controlling pH of the fermentation liquid to be maintained within 7.5-8.0 by adding the feed culture medium, ammonia water or dilute phosphoric acid in a coordinated manner in the fermentation process; culturing for 80-84h, slowly increasing or no longer increasing enzyme activity, and discharging when partial thallus is aged and autolyzed.
4. Use according to claim 3, characterized in that the fermenter medium consists of, in g/L: 8-12 parts of yeast powder, 15-25 parts of bean cake powder, 5-10 parts of corn steep liquor, 80-120 parts of corn meal, 2-5 parts of sodium citrate and CaCl 2 3-5、MgSO 4 0.8-1，K 2 HPO 4 15-20, the balance being water, pH7.5-8.0, 121-124 ℃, sterilizing for 30-40min;
the feed medium consists of the following components in g/L: 25-40 parts of bean cake powder, 150-200 parts of corn starch liquefied liquid, and the balance of water, wherein the pH is 7.5-8.0, the temperature is 121-124 ℃, and the sterilization is carried out for 30-40min.
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| CN102925388A (en) * | 2012-10-26 | 2013-02-13 | 天津科技大学 | Alkaline proteinase high-producing strain and alkaline proteinase being produced from same |
| CN103409347A (en) * | 2013-07-25 | 2013-11-27 | 山东隆科特酶制剂有限公司 | Bacterial strain capable of producing alkali protease and industrialized liquid fermentation method of bacterial strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102925388A (en) * | 2012-10-26 | 2013-02-13 | 天津科技大学 | Alkaline proteinase high-producing strain and alkaline proteinase being produced from same |
| CN103409347A (en) * | 2013-07-25 | 2013-11-27 | 山东隆科特酶制剂有限公司 | Bacterial strain capable of producing alkali protease and industrialized liquid fermentation method of bacterial strain |
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| Title |
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| 重离子诱变技术选育碱性蛋白酶高产菌株;薛林贵等;微生物学通报;第37卷(第6期);第845-851页 * |
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