CN110656063A - Strain for producing high-temperature-resistant protease - Google Patents

Abstract

The invention belongs to the field of microorganisms, and particularly relates to a high temperature resistant protease producing strain, wherein the strain is Bacillus cereus, which is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 17 months, and the preservation number of the strain is as follows: CCTCC M2019836. The invention screens out a protease B.cereus strain HSU-2 by using a casein medium screening method, wherein the optimum reaction pH value and temperature of the protease are 7.0 and 60 ℃ respectively, and the protease is a high-temperature resistant neutral protease. The protease can resist the effects of 5.0% of hydrogen peroxide, 5.0% of sodium dodecyl sulfate and 5.0% of detergent Triton X-100, and the 5.0% of detergent Tween 80 can obviously improve the enzyme activity of the protease.

Description

Strain for producing high-temperature-resistant protease

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a strain for producing high-temperature resistant protease.

Background

The protease is a biocatalyst capable of hydrolyzing protein, and the reaction catalyzed by the protease has the advantages of high speed, mild condition, environmental protection and no pollution, and can be widely applied to industries such as leather making, food, medicine and health, cosmetics and the like. Protease is used in the leather-making process for removing residual meat, collagen interstitial protein and mucoid from raw leather, so that the treated leather is soft (huzhi, wangjun, 2008); in the food industry, it is mainly used to tenderize meat, hydrolyze protein to prepare seasoning liquid, and make cheese (Huzhi, Wangjun, 2008). In the field of medicine and health, neutral protease is used for hydrolyzing Tibetan sheep serum protein to prepare antioxidant peptide (sorghum dandan, etc. 2015), and can also be used for preparing anticancer drug intermediates (Teodora bavaro, Terreni,2016), and the like. In addition, neutral proteases can also be used in cosmetic development to remove aged keratin (Zhang Xiaoyan, Guo Li Dong, Liu Xiao Yan, 2018). Therefore, the development of new proteases has important application value.

Proteases are present in animal tissues, plant stems and leaves, fruits and various microorganisms. The protease preparation which is widely used for production in industry at present is mainly derived from microorganisms due to the restriction of animal and plant resources; the strains are mainly bacteria, mould, a small amount of yeast and actinomycetes. It has been shown that protease-producing strains of bacteria are mainly concentrated in the genera Bacillus subtilis, Brevibacillus (Brevibacillus) and Halomonas (Halomonas). The protease can be divided into three categories of acid (pH value is 2.0-6.0), neutral (pH value is 6.0-9.0) and alkaline protease (pH value is more than 9.0) according to the optimum reaction pH value, wherein the neutral protease is firstly and clearly researched; however, there are few reports in the literature that neutral proteases can tolerate high temperatures of 60 ℃ and above.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a strain for producing high-temperature resistant protease.

The technical scheme adopted by the invention for realizing the purpose is as follows:

the strain for producing the high-temperature resistant protease is Bacillus cereus (Bacillus cereus) which is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 17 months, and the preservation number of the strain is as follows: CCTCC M2019836.

Further, the 16S rDNA sequence of the strain is shown in SEQ ID NO. 1.

The invention also provides a preparation method of the protease, which comprises the steps of activating the strain for 12 hours by using a seed activation culture medium; inoculating the activated strain into a fermentation culture medium, and fermenting in a shaking table at 28 ℃ and 180rpm for 5d at constant temperature; the fermented broth was aspirated and centrifuged and the supernatant was aspirated.

The invention also provides protease, wherein the use condition of the protease is pH6.0-8.0, and the temperature is 50-70 ℃.

The invention also provides a fermentation product, which comprises the fermentation liquor or the filtrate of the fermentation liquor for producing the high-temperature resistant protease strain.

The invention also provides the application of the strain in producing detergents.

The invention also provides a detergent which comprises the fermentation product and 5.0% of detergent Tween 80.

Further, the detergent contains Ca²⁺。

The invention also provides a microbial inoculum for producing the high-temperature resistant protease, which comprises the strain.

Has the advantages that:

the enzyme preparation industry in China has been developed for nearly 60 years, and has been in the line of the great countries for producing enzyme preparations, and the enzyme preparations produced in large scale have more than 30 kinds, but have certain gap with the large scale production of more than 60 kinds in the world. At present, enzyme preparations in China mainly comprise 9 types of saccharifying enzymes, amylases, proteases, cellulases and the like; while the largest consumer of proteases is the detergent industry. The proportion of the enzyme-added washing powder used in Europe is up to 85 percent, and Europe people are used to soak the fabric in hot water at the temperature of 40-60 ℃ before washing. The protease produced by the strain HSU-2 in the research can tolerate the high temperature of 60 ℃, and the protease can tolerate the action of 5.0% SDS, so that the protease is suitable for developing enzymatic laundry powder used in European regions to improve the industrial value of the laundry powder.

The invention screens out a protease B.cereus strain HSU-2 by using a casein medium screening method, wherein the optimum reaction pH value and temperature of the protease are 7.0 and 60 ℃ respectively, and the protease is a high-temperature resistant neutral protease. The protease can resist the effects of 5.0% of hydrogen peroxide, 5.0% of sodium dodecyl sulfate and 5.0% of detergent Triton X-100, and the 5.0% of detergent Tween 80 can obviously improve the enzyme activity of the protease. Meanwhile, the result of the influence of the metal ions on the protease activity shows that Mg²⁺、Zn²⁺、K⁺、Ni²⁺And Cu ²⁺5 metal ions can obviously reduce the activity of the protease, wherein Zn²⁺The inhibition effect is strongest; and Ca²⁺The ion can improve the activity of the protease. The invention lays a theoretical foundation for the industrial application and large-scale production of the protease-producing strain HSU-2.

Drawings

FIG. 1 is a colony of strain HSU-2;

FIG. 2 is a molecular evolutionary tree for constructing strain HSU-2 based on the results of 16S rDNA sequence alignment;

FIG. 3 is a casein standard curve;

FIG. 4 is the effect of pH on enzyme activity;

FIG. 5 is a graph showing the effect of temperature on enzyme activity;

FIG. 6 is a graph showing the effect of hydrogen peroxide on enzyme activity;

FIG. 7 shows the effect of detergents (A) sodium dodecylsulfate, (B) octyl polyethylene glycol phenyl ether X-100 and (C) Tween 80 on enzyme activity;

FIG. 8 is a graph showing the effect of metal ions on enzyme activity.

Detailed Description

The embodiments of the present invention will be described in further detail with reference to the drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Soil sample: soil for the experiment is collected under a forest in a campus of Huangshan college and is 5cm away from surface soil; after the surface soil is dug by a sampling shovel, the rhizosphere soil is taken out and put into a sterilized conical flask and is taken back to the laboratory for standby.

An experimental instrument: super clean bench (ZHJH-C2109B, Shanghai Zhicheng), constant temperature incubator (MQD-B2R, Shanghai Min spring), autoclave (SQ510C, Chongqing Yamatuo), incubator (ZGP-2050, Shanghai Zhicheng), three-hole constant temperature electric hot water tank (DK-8D, Shanghai Kubei industry), ultraviolet visible spectrophotometer (UV-765, Shanghai precision scientific instruments), centrifuge (Avanti J-E, Beckkurt company, USA).

Reagent: trypticase, casein, peptone, glucose, yeast extract, starch, methyl red, ammonium oxalate crystal violet, safranine and iodine solution, hydrogen peroxide (H)₂O₂) Sodium Dodecyl Sulfate (SDS), polyethylene glycol octyl phenyl ether (Triton X-100), Tween 80(Tween 80) and the like are purchased from Shanghai Biotech company; NaCl, ZnCl₂、Na₂HPO₄·7H₂O、MgSO₄·7H₂O、CaCl₂、FeSO₄、KH₂PO₄Glycerol and 95% ethanol, etc. are available from Shanghai pharmaceutical group.

The casein culture medium is prepared by the following method, and the specific concentration is as follows: KH2PO4 (3.6%), MgSO4 & 7H2O (5%), ZnCl2 (0.14%), Na2HPO4 & 7H2O (10.7%), NaCl (1.6%), CaCl2 (0.02%), FeSO4 (0.02%), casein (4%) (4 g of casein is weighed and placed in a 50mL beaker, 8mL of 1 mol/L NaOH is accurately transferred and taken by a liquid transfer gun and added, the mixture is heated in a water bath for 20min, and a volumetric flask is placed to 100mL after all solids are completely dissolved, so that the required target solution and Trypticase (0.5%) are obtained.

1. Test method

1.1 isolation and purification of protease-producing strains

Weighing 1.0g of collected soil, adding into a conical flask containing an activation culture medium, shaking uniformly, and placing in a water bath kettle at 80 ℃ for heat preservation for 10 min; then placing the mixture in a shaking incubator at 28 ℃ and 180r/min for constant temperature culture for 24 h. And (5) sealing and storing the residual soil sample for later use. Placing the culture solution after 24h culture in 80 deg.C water bath again, keeping the temperature for 10min, and sequentially diluting the culture solution after proliferation to 10 deg.C by ten-fold dilution method^-3、10^-4And 10^-5100. mu.L of each dilution was transferred and spread on a casein solid medium, and incubated at 28 ℃ for 24 hours in an incubator. Observing a transparent hydrolysis ring around a bacterial colony on a casein solid culture medium, measuring the diameter (H) of the transparent hydrolysis ring and the diameter (C) of the bacterial colony, and selecting the bacterial colony with a large ratio (H/C) for purification; and (4) carrying out purification on the bacterial colony for multiple times by using a scribing method until a single bacterial colony is purified and the characteristic of the bacterial colony is stable. The purified colonies with large H/C ratio are numbered and stored for later use.

1.2 identification of protease-producing strains

And (3) carrying out colony morphology feature observation on the purified strain HSU-2 with the maximum H/C ratio, extracting the genome DNA of the strain HSU-2, and amplifying and determining the 16S rDNA sequence of the strain by using primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'. The 16SrDNA sequence obtained by sequencing is put into an NCBI database for comparison, a strain sequence with the sequence consistency of more than 99.43 percent is selected, a molecular evolutionary tree is constructed by utilizing sequence comparison software Clustal X2.1 and evolution analysis software MEGA 6.06(Tamura, et al,2013), and the type of the strain HSU-2 is identified.

1.3 Casein Standard Curve

Casein standard solution was prepared, and the absorbance of the solution at 280nm was measured and repeated 3 times. And drawing a casein standard curve according to the experimental result.

1.4 fermentation of Strain HSU-2 and protease extraction

Taking out the preserved strain HSU-2 from a refrigerator at-80 deg.C, and activating with seed activation culture medium for 12 hr; then inoculating the activated strain into a fermentation culture medium, and placing the fermentation culture medium in a shaking table at 28 ℃ and 180rpm for constant-temperature fermentation for 5 d. Sucking the culture solution after fermentation, and centrifuging for 5min at 8000rpm in a centrifuge; carefully sucking the supernatant to obtain the crude enzyme solution.

1.5 optimal reaction pH of protease

The optimal reaction pH for the protease was determined as follows: respectively preparing reaction buffer solutions with pH values of 4, 5, 6, 7, 8 and 9, respectively taking 3.5mL of the 6 pH buffer solutions, adding 0.5mL of 10mg/mL casein solution, then adding 1mL of the crude enzyme solution obtained in the step 1.2.4, uniformly mixing, and placing the reaction solution in a water bath kettle at 37 ℃ for constant-temperature reaction for 20 min. The reaction mixture was taken out, and 1mL of 20% (w/v) trichloroacetic acid (TCA) was added thereto and mixed well to terminate the reaction. The crude enzyme solution was inactivated with TCA before casein addition as a control. The reaction solution after TCA termination reaction was equilibrated, centrifuged at 12000rpm for 5min, the supernatant carefully aspirated and the absorbance measured at 280nm, each experiment was repeated three times, and the average value was taken for analysis.

1.6 optimal reaction temperature for protease

The optimal reaction temperature of the protease is determined at the optimal reaction pH determined in step 1.2.5, with reference to the method of step 1.2.5, which is briefly as follows: preparing a buffer solution with the pH value of 7.0, adding 52.5mL of the buffer solution into a 250mL beaker, then sequentially adding 7.5mL of 10mg/mL casein solution and 15mL of the crude enzyme solution obtained in the step 1.2.4 into the buffer solution, uniformly mixing, and subpackaging into 5mL test tubes. Placing the test tubes in constant-temperature water baths at 30, 40, 50, 60 and 70 ℃ respectively for reaction for 20min, and then sequentially adding 1mL of 20% TCA to terminate the reaction; after balancing, centrifuging at 12000rpm for 5min, sucking supernatant, measuring light absorption value at 280nm, repeating for three times, and taking average value for analysis.

1.7 Effect of Hydrogen peroxide on protease Activity

The addition of different concentrations of H was tested at the optimum reaction pH and temperature determined in step 1.2.5 and step 1.2.6₂O₂And at different placesThe influence of processing time on the activity of protease enzyme is as follows: h was added to the buffer at pH 7.0 at concentrations of 1%, 5% and 10% (v/v), respectively₂O₂And fixing the volume to 3.5mL, adding 1mL of the crude enzyme solution extracted in the step 1.2.4, uniformly mixing, and placing the solution on ice for respectively treating for 20min, 40min and 60 min; then respectively adding 0.5mL of 10mg/mL casein solution, uniformly mixing, placing the reaction solution in a water bath at 60 ℃ for reacting for 20min, and adding 1mL of 20% TCA to terminate the reaction; after the trimming, the mixture was centrifuged at 12000rpm for 5min, and the supernatant was aspirated and the absorbance at 280nm was measured, which was repeated three times. At the same time, with no addition of H₂O₂The treated crude enzyme solution was used as a control, the enzyme activity was measured and set to 100% according to the above procedure, and comparison H was carried out₂O₂The effect of treatment on the protease activity.

1.8 Effect of detergents on protease Activity

The effect of different detergents, different concentrations and different treatment times on protease enzyme activity was tested according to the procedure of step 1.2.7, briefly described as follows: adding SDS, Triton X-100 or Tween 80 with the concentration of 0.5%, 1.0%, 2.5% and 5.0% (w/v) into a buffer solution with the pH of 7.0, fixing the volume to 3.5mL, adding 1mL of the crude enzyme solution extracted in the step 1.2.4, uniformly mixing, and placing the solution on ice for respectively treating for 20min, 40min and 60 min; the effect of detergent on the protease activity was compared according to step 1.2.7.

1.9 Effect of Metal ions on protease Activity

Addition of 1mM of Mg as a metal ion, determined by the method of step 1.2.7²⁺、Zn²⁺、K⁺、Ni²⁺And (3) enzyme activity after 5min treatment is carried out, and the influence of metal ions on the enzyme activity of the protease is compared.

2. Results and analysis

2.1 molecular characterization of Strain HSU-2

A strain HSU-2 (figure 1) producing high temperature resistant protease is screened from the soil in campus by a transparent circle method. As can be seen from FIG. 1, the strain forms a clear hydrolysis loop on the casein screening solid medium, and the ratio of the diameter (H) of the clear loop to the diameter (C) of the colony is about 4.1. The bacterial colony formed by the strain on a casein solid culture medium is round and dry, and is whitish in the middle and yellowish at the periphery. Partial physiological and biochemical index detection shows that the starch hydrolysis experiment, the catalase experiment and the methylred experiment of the strain are all positive, NaCl is not needed or can only grow on a NaCl culture medium with the concentration of 1% (table 1) in the growth process of the strain, and the NaCl with the concentration of 5% or 10% can inhibit the growth of the strain.

TABLE 1 results of physiological and biochemical experiments of the strain HSU-2

The 16S rDNA sequence of the strain HSU-2 was amplified using the universal primers 27F and 1492R for sequencing analysis, and the result showed that the 16S rDNA sequence of the strain HSU-2 was about 1407 bp. And (3) putting the sequence obtained by sequencing into an NCBI database, and performing homology analysis by using a BLAST tool, wherein the alignment result shows that the strain with higher sequence homology with the strain HSU-2 is derived from Bacillus (Bacillus sp.). From the alignment results, a strain sequence having a sequence identity of 99.43% or more to the HSU-2 strain was selected, and the sequence was subjected to multiple sequence alignment using the software ClustalX 2.1, and a molecular evolutionary tree was constructed by the method reported in Cummit et al (2018) (FIG. 2). From the constructed evolutionary tree results, the strain HSU-2 and 5 strains of Bacillus cereus strain JCM2152 and the like are located on the same evolutionary tree branch. The bacterial colony, partial physiological and biochemical characteristics and 16S rDNA evolution analysis result of the bacterial strain are combined, and the bacterial strain HSU-2 is identified as Bacillus cereus HSU-2(Bacillus cereusstrain HSU-2).

2.2 drawing of Casein Standard Curve

The absorbance was measured at different casein concentrations and a standard curve was drawn (FIG. 3). From the measurement results, the absorbance of the casein solution was positively correlated with the concentration thereof, and tended to increase. The regression equation of the concentration (X) of the casein solution and the light absorption value (Y) thereof is as follows: Y1.1647X +0.0061 (R)²0.9998), the linear correlation of the standard curve is very good, and the standard curve can be used for measuring the enzyme activity.

2.3 Effect of pH on protease Activity

The effect of pH on protease activity in strain HSU-2 was tested using a series of buffers with pH values of 4, 5, 6, 7, 8 and 9 as reaction solutions (FIG. 4). According to the experimental results, the protease produced by the strain HSU-2 has lower activity under the acidic condition (pH is less than 5); when the pH value of the reaction solution is increased from 5 to 7, the enzyme activity is obviously increased and reaches the maximum value at the pH value of 7; when the pH value is continuously increased to be slightly alkaline (pH value is 8-9), the protease activity is influenced to a certain extent; the protease can be maintained at a higher level under more alkaline conditions than under more acidic conditions. The results show that the optimal reaction pH value of protease produced by the strain HSU-2 is 7, and the strain has alkali resistance in a certain range.

2.4 Effect of temperature on protease Activity

The effect of different temperatures (30, 40, 50, 60 and 70 ℃ respectively) on the protease activity was determined under the condition that the optimum reaction pH of the protease produced by the strain HSU-2 was 7 as determined in the above experiment (FIG. 5). The experimental result shows that the enzyme activity of the protease is increased along with the increase of the temperature before the temperature is 60 ℃, and the enzyme activity is maximum when the temperature is 60 ℃; when the temperature continues to rise, the enzyme activity begins to decrease. Therefore, the optimal reaction temperature for protease production by the strain HSU-2 is 60 ℃.

2.5 Effect of other factors on protease Activity

2.5.1 Effect of Hydrogen peroxide on protease Activity

Detecting hydrogen peroxide (H) with different concentrations by using protease activity of 100% at pH 7.0 and reaction temperature of 60 deg.C₂O₂) The results of the effect on the protease activity produced by the strain HSU-2 are shown in FIG. 6. The experimental results show that the concentration of H is 1%₂O₂Can improve the enzyme activity of protease produced by the strain HSU-2, and the concentration of H is 5 percent or 10 percent₂O₂Can reduce the enzyme activity of protease produced by the strain HSU-2. At the same time, with H₂O₂Extended treatment time, 1%, 5% and 10% H₂O₂All reduce the enzyme activity of protease produced by the strain HSU-2 to different degrees.

2.5.2 Effect of three detergents on protease Activity

The effect of 3 different kinds and concentrations of detergent on the protease activity of the strain HSU-2 was examined with the protease activity at pH 7.0 and a reaction temperature of 60 ℃ being 100%, and the results are shown in FIG. 7. From the results of FIG. 7A, it can be seen that when the concentration of Sodium Dodecyl Sulfate (SDS) as a detergent is 0.5%, 1.0% and 2.5%, respectively, the protease is not significantly affected by the treatment for 20 min; and the 3 concentrations of SDS still have no significant influence on the enzyme activity of the protease as the treatment time is prolonged to 40 and 60 min. When the concentration of SDS is increased to 5.0%, the enzyme activity of the protease is slightly reduced after different time of treatment, but the enzyme activity is still maintained to be more than 95%.

FIG. 7B shows that the enzyme activity of protease produced by the strain HSU-2 can be slightly improved by the detergent Triton X-100 with concentrations of 0.5, 1.0 and 5.0%, respectively; and the enzyme activity of the protease can be maintained at more than 100% after the treatment time is prolonged to 40min and 60 min.

From the results of FIG. 7C, it can be seen that the enzyme activity of the protease can be increased to 115.8% -122.7% when the protease is treated with the detergent Tween 80 at concentrations of 0.5, 1.0 and 5.0% for 20 min; after the treatment for 40min, the enzyme activity of the protease is improved to 123.3-133.6%; after 60min of treatment, the enzyme activity of the protease can still be maintained at 119.6-126.6%.

2.5.3 Effect of Metal ions on protease Activity

The final concentration of metal ion Mg is 1mM according to the protease activity of 100 percent at the pH of 7.0 and the reaction temperature of 60 DEG C²⁺、Zn²⁺、K⁺、Ni²⁺Etc. on the protease activity (FIG. 8). The experimental result shows that Mg²⁺、Zn²⁺、K⁺、Ni²⁺And Cu ²⁺5 metal ions can obviously reduce the enzyme activity of the protease, wherein Zn²⁺The inhibition effect is strongest, and the enzyme activity is reduced to only 34.0 percent; ni²⁺And Cu²⁺The enzyme activity is respectively reduced to 50.5 percent and 45.2 percent after the secondary inhibition; mg (magnesium)²⁺And K⁺The inhibition effect is weak, and the enzyme activity is respectively reduced to 85.8 percent and 81.0 percent. In contrast, Ca²⁺The ion can improve the enzyme activity of the protease to 116.5%.

Sequence listing

<110> Huangshan college

<120> a strain for producing high temperature resistant protease

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1407

<212> DNA

<213> Bacillus cereus (Bacillus cereus)

<400> 1

tgcagtcgag cgaatggatt aagagcttgc tcttatgaag ttagcggcgg acgggtgagt 60

aacacgtggg taacctgccc ataagactgg gataactccg ggaaaccggg gctaataccg 120

gataacattt tgaaccgcat ggttcgaaat tgaaaggcgg cttcggctgt cacttatgga 180

tggacccgcg tcgcattagc tagttggtga ggtaacggct caccaaggca acgatgcgta 240

gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg 300

aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag 360

tgatgaaggc tttcgggtcg taaaactctg ttgttaggga agaacaagtg ctagttgaat 420

aagctggcac cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg 480

cggtaatacg taggtggcaa gcgttatccg gaattattgg gcgtaaagcg cgcgcaggtg 540

gtttcttaag tctgatgtga aagcccacgg ctcaaccgtg gagggtcatt ggaaactggg 600

agacttgagt gcagaagagg aaagtggaat tccatgtgta gcggtgaaat gcgtagagat 660

atggaggaac accagtggcg aaggcgactt tctggtctgt aactgacact gaggcgcgaa 720

agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct 780

aagtgttaga gggtttccgc cctttagtgc tgaagttaac gcattaagca ctccgcctgg 840

ggagtacggc cgcaaggctg aaactcaaag gaattgacgg gggcccgcac aagcggtgga 900

gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca tcctctgaaa 960

accctagaga tagggcttct ccttcgggag cagagtgaca ggtggtgcat ggttgtcgtc 1020

agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt 1080

gccatcatta agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg 1140

atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggacggt 1200

acaaagagct gcaagaccgc gaggtggagc taatctcata aaaccgttct cagttcggat 1260

tgtaggctgc aactcgccta catgaagctg gaatcgctag taatcgcgga tcagcatgcc 1320

gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgag agtttgtaac 1380

acccgaagtc ggtggggtaa ccttttt 1407

Claims (9)

1. The strain for producing the high-temperature resistant protease is characterized by being Bacillus cereus (Bacillus cereus) which is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 10 and 17 months, wherein the preservation number of the strain is as follows: CCTCC M2019836.

2. The hyperthermostable protease producing strain according to claim 1, wherein the 16S rDNA sequence of the strain is represented by SEQ ID No. 1.

3. A process for producing protease, characterized by activating the strain of claim 1 with a seed activation medium for 12 hours; inoculating the activated strain into a fermentation culture medium, and fermenting in a shaking table at 28 ℃ and 180rpm for 5d at constant temperature; the fermented broth was aspirated and centrifuged and the supernatant was aspirated.

4. The protease according to claim 3, wherein the protease is used at a pH of 6.0 to 8.0 and a temperature of 50 to 70 ℃.

5. A fermented product comprising the fermentation broth or filtrate of the fermentation broth of the hyperthermostable protease producing strain of claim 1.

6. Use of the strain according to claim 1 for the production of detergents.

7. A detergent comprising the fermented product of claim 5 and 5.0% detergent Tween 80.

8. The detergent according to claim 7, wherein the detergent contains Ca²⁺。

9. A bacterial agent producing a high-temperature resistant protease, comprising the strain according to claim 1.

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