CN1670187A - Alkaline low-temperature protease and its preparation method - Google Patents
Alkaline low-temperature protease and its preparation method Download PDFInfo
- Publication number
- CN1670187A CN1670187A CN 200510062600 CN200510062600A CN1670187A CN 1670187 A CN1670187 A CN 1670187A CN 200510062600 CN200510062600 CN 200510062600 CN 200510062600 A CN200510062600 A CN 200510062600A CN 1670187 A CN1670187 A CN 1670187A
- Authority
- CN
- China
- Prior art keywords
- enzyme
- marinobacter
- protease
- liquid
- temperature
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
This invention provides to a basified cryoen-protease gaining from R2 strain as pure culture extracted from Antarctic abyssal ooze and its preparing process, with preservation number as CCTCCNO: M205002, comprising the following steps: a, activating strain R2 and linked to medium, culturing it until exponential growth phase, then transferring in medium, filling acillin and agedoite, culturing and fermenting and centrifuging; b, filling ammonia sulfate in enzyme liquid, then centrifuging them and picking up deposition to dialyze in cushioning liquid; c, centrifuging enzyme liquid , then freeze drying and eluting it, collecting enzyme active part to freeze dry; d, again resolving in cushioning liquid and dialyzing, eluting, merging active parts, and freeze drying; e, resolving sample in cushioning liquid and dialyzing to purify it, collecting active parts by freeze drying to acquire pure enzyme.
Description
Technical field
The proteolytic enzyme that the present invention relates to a kind of bacterium and obtain from bacterium especially relates to a kind of alkaline low-temperature protease that obtains from marinobacter (Marinobactersp.) R2 bacterial strain with from this bacterium and preparation method thereof.
Background technology
Existing Sumizyme MP can obtain from number of ways, wherein have some reports in the application of industries such as washing composition by microorganism acquisition Sumizyme MP and Sumizyme MP, for example Rohm in 1913 at first is used to wash soaking agent with pancreas islet proteolytic enzyme, 1945 Dr.Jaag (Switzerland) etc. found Studies on Microbial Alkaline Protease.And the Sumizyme MP commodity that are used for washing composition are also more and more, the Properase CT of U.S. GENENCOR international corporation for example, the Savinase 4.OT alkaline low-temperature protease of Denmark NOVO company etc.Notification number provides the microorganism of a kind of novel alkaline low-temperature protease, manufacture method, application and this proteolytic enzyme of generation for the patent of CN1109750C, relate to a kind of new Flavobacterium unknown species bacterial strain YS9412-130, novel alkaline low-temperature protease by this bacterial strain or the generation of its mutant, this proteolytic enzyme has low-activation energy and high reactivity ratio, effectively protein degradation class under cold condition; The enzyme genes encoding is made up of 792 Nucleotide, with serine protease in various degree homology is arranged, but is different from existing serine stretch protein enzyme family, belongs to a kind of newborn type serine stretch protein enzyme family.
Summary of the invention
The present invention aims to provide a kind of from acquisition of deep-sea, South Pole mud sample and bacterial strain marinobacter (Marinobacter sp.) R2 through being separated into pure growth.
Another object of the present invention is to provide a kind of alkaline low-temperature protease that obtains from marinobacter R2 bacterial strain and preparation method thereof.
The said bacterial strain marinobacter of the present invention (Marinobacter sp.) R2 is preserved in the Chinese typical culture collection center in Chinese Wuhan on January 5th, 2005, and deposit number is CCTCC NO:M205002.The source of sample is the mud sample of 900 meters depths, deep-sea, the South Pole.The screening and separating of bacterial strain and cultural method are after the mud sample is diluted with the extension rate of seawater by 10,100,1000 of sterilizing, coat screening culture medium, cultivate a week in 20 ℃, pick out single bacterium colony with transparent circle, further line separates, and getting single bacterium colony microscopy is purebred back preservation in-70 ℃ of Ultralow Temperature Freezers.Said substratum is as follows:
1, Gai Liang 2216E substratum: peptone 0.5%, yeast extract paste 0.1%, natural Chen Haishui, pH7～8.
2, MM substratum: (NH
4)
2SO
40.1%, K
2HPO
40.7%, KH
2PO
40.3%, Trisodium Citrate 0.05%, MgSO
40.01%, glucose 0.5%, agar 1.5%～2.0%, distilled water preparation.
3, casein substratum: KH
2PO
40.04%, Na
2HPO
40.1%, NaCl 0.01%, ZnSO
40.002%, CaCl
20.0002%, casein 0.4%, casein hydrolysis amino acid 0.005%, agar 1.5%～2.0%, distilled water, pH7.0～7.2.
4, screening culture medium: skim-milk 1%, yeast extract paste 0.2%, agar powder 1.5%, natural Chen Haishui, pH7～8.
5, fermention medium: casein 1%, yeast extract paste 0.2%, KH
2PO
40.04%, Na
2HPO
40.1%, ZnSO
40.002%, CaCl
20.0002%, natural Chen Haishui, pH7～8.
Bacterial strain marinobacter R2 belongs to a novel species of marinobacter (Marinobacter sp.) after following method is identified.
1,16S rDNA The sequencing results shows that the R2 bacterial strain belongs to marinobacter (Marinobacter sp.).
2, DNA-DNA hybrid experiment result shows, the genomic dna of bacterial strain R2 and Marinobacterhydrocarbonoclasticus homology only are 70%.
3, morphological feature and resistant proof: oily mirror is observed down, and the R2 cell is shaft-like, single arrangement.Gramstaining is negative, and the thalline size is 10～15 μ m * 2～3 μ m.Bacterium colony is rounded, the surface irregularity tarnish, and marginal swell has tangible gauffer, and color is oyster white, and is opaque.On casein substratum plate, can produce the obvious transparent circle.The antibiotics resistance experiment shows that the R2 bacterial strain all has resistance to penbritin, kantlex and Streptomycin sulphate.
The said bacterial strain marinobacter of the present invention R2 alkaline low-temperature protease has following zymologic property:
1, the optimal reactive temperature of bacterial strain marinobacter R2 alkaline low-temperature protease is 20 ℃, at 15～25 ℃ of scope inner enzyme vigor height, shows tangible low-temperature protease characteristic.Bacterial strain marinobacter R2 alkaline low-temperature protease vigor in 0～20 ℃ is comparatively stable, enzyme work reduces to 8.3% after being incubated 24h under 30 ℃, behind 40 ℃ of insulation 1h enzyme live residue more than 7.6%, 50 ℃ 10min lose whole vigor substantially, meet low-temperature protease to heat sensitive characteristic.Ca
2+Improved the stability of enzyme to a certain extent.
2, the optimal pH of bacterial strain marinobacter R2 alkaline low-temperature protease is 9～10, belongs to Sumizyme MP.
3, bacterial strain marinobacter R2 alkaline low-temperature protease specific substrate N-Succ-(Ala) 2-Pro-Phe-pNA and N-Succ-(Ala) 2-Pro-Leu-pNA of Quimotrase that can degrade has the class chymotrypsin activity.
4, the single-minded inhibitor Phenylmethylsufonyl of sequestrant EDTA and serine protease fluoride (PMSF), 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) demonstrate stronger restraining effect to bacterial strain marinobacter R2 alkaline low-temperature protease, and single-minded inhibitor E-64 of L-Cysteine HCL Anhydrous and the single-minded inhibitor pepstatinA of aspartate protease are to the equal unrestraint effect of this enzyme.Show that bacterial strain marinobacter R2 alkaline low-temperature protease belongs to serine protease.
5, the denaturing agent of high density comprises sodium lauryl sulphate (SDS), urea, and Guanidinium hydrochloride, dithionthreitol (DTT), mercaptoethanol all has comparatively obvious suppression effect to bacterial strain marinobacter R2 alkaline low-temperature protease.
6, Ca
2+, Mn
2+, Cu
2+Bacterial strain marinobacter R2 alkaline low-temperature protease there is comparatively significantly activation, and Cd
2+, Co
2+Then can live by inhibitory enzyme.
Preparation method's step of the said bacterial strain marinobacter of the present invention R2 alkaline low-temperature protease is:
1) crude enzyme liquid prepares: transfer in liquid casein substratum after bacterial strain R2 is activated with minimal medium, shaking culture is to exponential phase of growth under 5～30 ℃ condition, the bacterium liquid that will be in exponential phase of growth is transferred in liquid fermentation medium, in liquid fermentation medium, add penbritin, shaking culture under 5～25 ℃ of temperature, add l-asparagine after 1～4 day, continue that shaking culture to the solid casein particle in the substratum disappears and nutrient solution is light yellow and is and produces the enzyme peak period, fermented liquid is centrifugal, get supernatant, get crude enzyme liquid;
2) crude enzyme liquid adds ammonium sulfate to 30%～40% saturation ratio, and the centrifuging and taking supernatant continues to add ammonium sulfate to 70%～80% saturation ratio, centrifuging and taking precipitation, dialysed overnight in the Tris-HCl damping fluid;
3) the enzyme liquid that will dialyse later is centrifugal, and supernatant liquor concentrates with lyophilize, is splined on the DEAE-52 post of having crossed with Tris-HCl damping fluid pre-equilibration again, wash-out, the lyophilize of collection enzymic activity part;
4) be dissolved in the enzyme grain weight after the lyophilize in the Tris-HCl damping fluid and dialysed overnight, be splined on then, and, merge active part, lyophilize with identical buffer solution elution with same damping fluid equilibrated Sephadex G-75 post;
5) sample after the freeze-drying is dissolved in the Tris-HCl damping fluid and dialysed overnight, separation and purification again, collecting has the lyophilize of vigor part, is pure enzyme.
In step 1), the minimal medium during activated spawn is selected from the MM substratum: (NH
4)
2SO
40.1%, K
2HPO
40.7%, KH
2PO
40.3%, Trisodium Citrate 0.05%, MgSO
40.01%, glucose 0.5%, agar 1.5%～2.0%, distilled water preparation.Adding the penbritin final concentration during fermentation is 0.05%, and the final concentration of l-asparagine is 0.005%.
In step 3), said wash-out adopts the Tris-HCl buffer solution elution that contains 0～2mol/LNaCl linear gradient.
In step 5), used damping fluid adopts 0.01mol/L Tris-HCl damping fluid, pH8.0～9.0 during said separation and purification.The electrophoresis purifying is adopted in said separation and purification, and the condition of electrophoresis purifying is: resolving gel concentration 14%, the high 5cm of post, voltage 300V; Concentrated gum concentration is 4%, the high 1cm of post, voltage 240V.
Proteinase activity is measured and is adopted Folin phenol reagent development process to carry out.
The characteristic and the advantage of bacterial strain marinobacter alkaline low-temperature protease that R2 produces are:
1, the proteolytic enzyme that produced of R2 bacterial strain belongs to alkaline low-temperature protease, and its optimal reactive temperature is 20 ℃, is minimum in the existing low-temperature protease, has excellent application value.
2, most proteolytic enzyme can be by Cu
2+, Ag
+, Pb
2+Suppress, and the proteolytic enzyme that bacterial strain R2 is produced is not only to Ag
+, Pb
2+Insensitive, on the contrary can be by Cu
2+Activate, show that this enzyme has special mechanism.
3, the R2 bacterial strain has resistance to multiple microbiotic, and this point especially has advantage in application, because add the pollution that microbiotic can suppress other bacterium in fermentation, thereby simplifies step, saves cost.
4, the direct fermentation at normal temperatures of R2 bacterial strain produces low-temperature protease, but the vigor of occurs at low temperatures ferment is higher.Cost inputs such as this characteristic in actual applications also can save energy, equipment.
Embodiment
Embodiment 1
The preparation crude enzyme liquid is transferred in liquid casein substratum after the bacterial strain R2 of preservation activated with minimal medium, and substratum is selected from the MM substratum: (NH
4)
2SO
40.1%, K
2HPO
40.7%, KH
2PO
40.3%, Trisodium Citrate 0.05%, MgSO
40.01%, glucose 0.5%, agar 2.0%, distilled water preparation.Shaking culture is to exponential phase of growth under 20 ℃ condition.The bacterium liquid that will be in exponential phase of growth is transferred in liquid fermentation medium, and adding penbritin, final concentration is 0.05%, shaking culture under 15 ℃ of temperature, add l-asparagine after 2 days, the final concentration of l-asparagine is 0.005%, continues shaking culture, observe the solid casein particle degraded situation in the substratum, particle disappearance and nutrient solution are light yellow and are product enzyme peak period.Fermented liquid is centrifugal, get supernatant, get crude enzyme liquid.Add ammonium sulfate to 32% saturation ratio in crude enzyme liquid, the centrifuging and taking supernatant continues to add ammonium sulfate to 75% saturation ratio, centrifuging and taking precipitation, dialysed overnight in the Tris-HCl damping fluid.Dialysis enzyme liquid later is centrifugal, supernatant liquor concentrates with lyophilize, be splined on the DEAE-52 post of having crossed with Tris-HCl damping fluid pre-equilibration then,, collect the lyophilize of enzymic activity part with the Tris-HCl buffer solution elution that contains 0～2mol/LNaCl linear gradient.Be dissolved in the enzyme grain weight after the lyophilize in the Tris-HCl damping fluid and dialysed overnight, be splined on then, and, merge active part, lyophilize with identical buffer solution elution with same damping fluid equilibrated Sephadex G-75 post.Be dissolved in the Tris-HCl damping fluid sample after the freeze-drying and dialysed overnight, used damping fluid adopts 0.01mol/L Tris-HCl damping fluid during separation and purification, and pH 8.0.Utilize SDS-PAGE to prepare electrophoresis apparatus and carry out separation and purification.The condition of electrophoresis purifying is: resolving gel concentration 14%, the high 5cm of post, voltage 300V; Concentrated gum concentration is 4%, the high 1cm of post, voltage 240V.Elutriant is the Tris-HCl damping fluid.Collection has the lyophilize of vigor part, is pure enzyme.The mensuration of proteinase activity adopts Folin phenol reagent development process to carry out.
Embodiment 2
Similar to Example 1, when its difference was to prepare crude enzyme liquid, substratum was selected from the MM substratum: (NH
4)
2SO
40.1%, K
2HPO
40.7%, KH
2PO
40.3%, Trisodium Citrate 0.05%, MgSO
40.01%, glucose 0.5%, agar 1.5%, distilled water preparation.Shaking culture is to exponential phase of growth under 15 ℃ condition.The bacterium liquid that will be in exponential phase of growth is transferred in liquid fermentation medium, and adding penbritin, shaking culture under 25 ℃ of temperature, add l-asparagine after 3 days, add ammonium sulfate to 35% saturation ratio in crude enzyme liquid, the centrifuging and taking supernatant continues to add ammonium sulfate to 72% saturation ratio, the centrifuging and taking precipitation, dialysed overnight in the Tris-HCl damping fluid.Be dissolved in the Tris-HCl damping fluid sample after the freeze-drying and dialysed overnight, used damping fluid adopts 0.01mol/L Tris-HCl damping fluid during separation and purification, and pH 8.5.
Embodiment 3
Similar to Example 1, when its difference was to prepare crude enzyme liquid, substratum was selected from the MM substratum: (NH
4)
2SO
40.1%, K
2HPO
40.7%, KH
2PO
40.3%, Trisodium Citrate 0.05%, MgSO
40.01%, glucose 0.5%, agar 1.8%, distilled water preparation.Shaking culture is to exponential phase of growth under 5 ℃ condition.The bacterium liquid that will be in exponential phase of growth is transferred in liquid fermentation medium, and adding penbritin, shaking culture under 20 ℃ of temperature, add l-asparagine after 1 day, add ammonium sulfate to 30% saturation ratio in crude enzyme liquid, the centrifuging and taking supernatant continues to add ammonium sulfate to 80% saturation ratio, the centrifuging and taking precipitation, dialysed overnight in the Tris-HCl damping fluid.Be dissolved in the Tris-HCl damping fluid sample after the freeze-drying and dialysed overnight, used damping fluid adopts 0.01mol/L Tris-HCl damping fluid, pH9 during separation and purification.
Embodiment 4
Similar to Example 1, when its difference was to prepare crude enzyme liquid, substratum was selected from the MM substratum: (NH
4)
2SO
40.1%, K
2HPO
40.7%, KH
2PO
40.3%, Trisodium Citrate 0.05%, MgSO
40.01%, glucose 0.5%, agar 1.5%, distilled water preparation.Shaking culture is to exponential phase of growth under 25 ℃ condition.The bacterium liquid that will be in exponential phase of growth is transferred in liquid fermentation medium, and adding penbritin, shaking culture under 30 ℃ of temperature, add l-asparagine after 4 days, add ammonium sulfate to 36% saturation ratio in crude enzyme liquid, the centrifuging and taking supernatant continues to add ammonium sulfate to 77% saturation ratio, the centrifuging and taking precipitation, dialysed overnight in the Tris-HCl damping fluid.Be dissolved in the Tris-HCl damping fluid sample after the freeze-drying and dialysed overnight, used damping fluid adopts 0.01mol/L Tris-HCl damping fluid during separation and purification, and pH 8.
Embodiment 5
Similar to Example 1, when its difference was to prepare crude enzyme liquid, substratum was selected from the MM substratum: (NH
4)
2SO
40.1%, K
2HPO
40.7%, KH
2PO
40.3%, Trisodium Citrate 0.05%, MgSO
40.01%, glucose 0.5%, agar 1.7%, distilled water preparation.Shaking culture is to exponential phase of growth under 30 ℃ condition.The bacterium liquid that will be in exponential phase of growth is transferred in liquid fermentation medium, and adding penbritin, shaking culture under 5 ℃ of temperature, add l-asparagine after 3 days, add ammonium sulfate to 40% saturation ratio in crude enzyme liquid, the centrifuging and taking supernatant continues to add ammonium sulfate to 70% saturation ratio, the centrifuging and taking precipitation, dialysed overnight in the Tris-HCl damping fluid.Be dissolved in the Tris-HCl damping fluid sample after the freeze-drying and dialysed overnight, used damping fluid adopts 0.01mol/L Tris-HCl damping fluid during separation and purification, and pH 8.5.
Claims (8)
1, a kind of bacterial strain marinobacter (Marinobacter sp.) R2, on January 5th, 2005 was preserved in the Chinese typical culture collection center in Chinese Wuhan, and deposit number is CCTCC NO:M205002.
2, a kind of alkaline low-temperature protease that obtains from marinobacter R2.
3, a kind of alkaline low-temperature protease that obtains from marinobacter R2 as claimed in claim 2, its zymologic property is:
1) optimal reactive temperature is 20 ℃, at 15～25 ℃ of scope inner enzyme vigor height, show tangible low-temperature protease characteristic, vigor is stable in 0～20 ℃, enzyme work reduces to 8.3% after being incubated 24h under 30 ℃, behind 40 ℃ of insulation 1h enzyme live residue more than 7.6%, 50 ℃ 10min lose whole vigor substantially, meet low-temperature protease to heat sensitive characteristic;
2) optimal pH is 9～10, belongs to Sumizyme MP;
3) can degrade specific substrate N-Succ-(Ala) 2-Pro-Phe-pNA and N-Succ-(Ala) 2-Pro-Leu-pNA of Quimotrase have the class chymotrypsin activity;
4) the single-minded inhibitor Phenylmethylsufonyl of sequestrant EDTA and serine protease fluoride, 4-(2-Aminoethyl) benzenesulfonyl fluoride demonstrate stronger restraining effect to bacterial strain marinobacter R2 alkaline low-temperature protease, and single-minded inhibitor E-64 of L-Cysteine HCL Anhydrous and the single-minded inhibitor pepstatir of aspartate protease A show that to the equal unrestraint effect of this enzyme bacterial strain marinobacter R2 alkaline low-temperature protease belongs to serine protease;
5) denaturing agent of high density comprises sodium lauryl sulphate, urea, and Guanidinium hydrochloride, dithionthreitol, thioglycol all has comparatively obvious suppression effect to bacterial strain marinobacter R2 alkaline low-temperature protease;
6) Ca
2+, Mn
2+, Cu
2+Bacterial strain marinobacter R2 alkaline low-temperature protease there is tangible activation, and Cd
2+, Co
2+Then can live by inhibitory enzyme.
4, the preparation method of a kind of alkaline low-temperature protease that obtains from marinobacter R2 as claimed in claim 2 the steps include:
1) crude enzyme liquid prepares: transfer in liquid casein substratum after bacterial strain R2 is activated with minimal medium, shaking culture is to exponential phase of growth under 5～30 ℃ condition, the bacterium liquid that will be in exponential phase of growth is transferred in liquid fermentation medium, and adding penbritin, shaking culture under 5～25 ℃ of temperature, add l-asparagine after 1～4 day, continue that shaking culture to the solid casein particle in the substratum disappears and nutrient solution is light yellow and is and produces the enzyme peak period, fermented liquid is centrifugal, get supernatant, get crude enzyme liquid;
2) crude enzyme liquid adds ammonium sulfate to 30%～40% saturation ratio, and the centrifuging and taking supernatant continues to add ammonium sulfate to 70%～80% saturation ratio, centrifuging and taking precipitation, dialysed overnight in the Tris-HCl damping fluid;
3) the enzyme liquid that will dialyse later is centrifugal, and supernatant liquor concentrates with lyophilize, is splined on the DEAE-52 post of having crossed with Tris-HCl damping fluid pre-equilibration again, wash-out, the lyophilize of collection enzymic activity part;
4) be dissolved in the enzyme grain weight after the lyophilize in the Tris-HCl damping fluid and dialysed overnight, be splined on then, and, merge active part, lyophilize with identical buffer solution elution with same damping fluid equilibrated Sephadex G-75 post;
5) sample after the freeze-drying is dissolved in the Tris-HCl damping fluid and dialysed overnight, separation and purification again, collecting has the lyophilize of vigor part, is pure enzyme.
5, the preparation method of a kind of alkaline low-temperature protease that obtains from marinobacter R2 as claimed in claim 4, it is characterized in that in step 1), minimal medium during activated spawn is selected from the MM substratum: (NH4) 2,SO4 0.1%, K2HPO40.7%, KH2PO4 0.3%, Trisodium Citrate 0.05%, MgSO4 0.01%, glucose 0.5%, agar 1.5%～2.0%, distilled water preparation.The penbritin that adds during fermentation, final concentration are 0.05%, and the final concentration of l-asparagine is 0.005%.
6, the preparation method of a kind of alkaline low-temperature protease that obtains from marinobacter R2 as claimed in claim 4 is characterized in that in step 3), and said wash-out adopts the Tris-HCl buffer solution elution that contains 0～2mol/L NaCl linear gradient.
7, the preparation method of a kind of alkaline low-temperature protease that obtains from bacterial strain marinobacter R2 as claimed in claim 4, it is characterized in that in step 5), used damping fluid adopts 0.01mol/L Tris-HCl damping fluid, pH8.0～9.0 during said separation and purification.
8, the preparation method of a kind of alkaline low-temperature protease that obtains from marinobacter R2 as claimed in claim 4, it is characterized in that in step 5) the electrophoresis purifying is adopted in said separation and purification, the condition of electrophoresis purifying is: resolving gel concentration 14%, the high 5cm of post, voltage 300V; Concentrated gum concentration is 4%, the high 1cm of post, voltage 240V.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100626001A CN1296475C (en) | 2005-03-28 | 2005-03-28 | Alkaline low-temperature protease and its preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100626001A CN1296475C (en) | 2005-03-28 | 2005-03-28 | Alkaline low-temperature protease and its preparation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1670187A true CN1670187A (en) | 2005-09-21 |
| CN1296475C CN1296475C (en) | 2007-01-24 |
Family
ID=35041627
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100626001A Expired - Fee Related CN1296475C (en) | 2005-03-28 | 2005-03-28 | Alkaline low-temperature protease and its preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1296475C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946573A (en) * | 2015-07-16 | 2015-09-30 | 青岛农业大学 | Bacterial strain for producing low temperature alkaline protease |
| CN114787195A (en) * | 2019-12-27 | 2022-07-22 | 深圳市天道医药有限公司 | Preparation method of heparinase III |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1109750C (en) * | 2000-08-15 | 2003-05-28 | 中国水产科学研究院黄海水产研究所 | New type low temperature akaline protease and its producing method and use and microbe for producing said protease |
-
2005
- 2005-03-28 CN CNB2005100626001A patent/CN1296475C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946573A (en) * | 2015-07-16 | 2015-09-30 | 青岛农业大学 | Bacterial strain for producing low temperature alkaline protease |
| CN104946573B (en) * | 2015-07-16 | 2017-11-10 | 青岛农业大学 | A kind of bacterial strain for producing low-temperature alkaline protease |
| CN114787195A (en) * | 2019-12-27 | 2022-07-22 | 深圳市天道医药有限公司 | Preparation method of heparinase III |
| CN114787195B (en) * | 2019-12-27 | 2024-01-12 | 深圳市天道医药有限公司 | Preparation method of heparinase III |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1296475C (en) | 2007-01-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Nam et al. | Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe | |
| Li et al. | Flocculation mechanism of the actinomycete Streptomyces sp. hsn06 on Chlorella vulgaris | |
| Ravindran et al. | Solid-state fermentation for the production of alkaline protease by Bacillus cereus 1173900 using proteinaceous tannery solid waste | |
| Feby et al. | Sponge-associated bacteria of Lakshadweep coral reefs, India: resource for extracellular hydrolytic enzymes | |
| Wang et al. | Screening and characterization of the alkaline protease isolated from PLI-1, a strain of Brevibacillus sp. collected from Indonesia’s hot springs | |
| Li et al. | Experimental evidence for long‐term coexistence of copiotrophic and oligotrophic bacteria in pelagic surface seawater | |
| Mehta et al. | Isolation of a novel feather-degrading bacterium and optimization of its cultural conditions for enzyme production | |
| Ire et al. | Effects of some cultural conditions on keratinase production by Bacillus licheniformis strain NBRC 14206 | |
| WO2022257391A1 (en) | Strain capable of degrading collagen and use thereof | |
| CN101240254B (en) | Organic solvent resisting proteinase high-yield bacterium, gene and application of the organic solvent resisting proteinase | |
| CN118185841A (en) | Vibrio cellulosae strain NN19 and application thereof | |
| CN1296475C (en) | Alkaline low-temperature protease and its preparation method | |
| Hashim et al. | Characterization of marine bioflocculant-producing bacteria isolated from Biofloc of Pacific Whiteleg shrimp, Litopenaeus vannamei culture ponds | |
| Alrumman et al. | Isolation of thermoalkalophilic-α-amylase producing bacteria and optimization of potato waste water medium for enhancement of α-amylase production | |
| Boltyanskaya et al. | Halonatronomonas betaini gen. nov., sp. nov., a haloalkaliphilic isolate from soda lake capable of betaine degradation and proposal of Halarsenatibacteraceae fam. nov. and Halothermotrichaceae fam. nov. within the order Halanaerobiales | |
| Jain et al. | Production and partial characterization of collagenase of Streptomyces exfoliatus CFS 1068 using poultry feather | |
| JP2013027374A (en) | Basidiomycetous yeast from the south pole having milk fat decomposition ability and use of the same | |
| CN1280186A (en) | New type low temperature akaline protease and its producing method and use and microbe for producing said protease | |
| Boyadzhieva et al. | Diversity of heterotrophic halophilic bacteria isolated from coastal solar salterns, Bulgaria and their ability to synthesize bioactive molecules with biotechnological impact | |
| CN114540252B (en) | Microbacterium P6 for converting livestock and poultry breeding waste and application | |
| KR101509326B1 (en) | Microbial agent having degradation of organic compounds and endosulfan and method for preparation thereof | |
| CN108018276B (en) | Deep sea bacterium keratinase, coding gene thereof, enzyme protein production engineering bacterium and application | |
| CN102206657B (en) | Gene and its application in treatment of quinoline contaminant | |
| Urzo et al. | Screening and identification of polyester polyurethane-degrading fungi | |
| Yang et al. | Family-level diversity of extracellular proteases of sedimentary bacteria from the South China Sea |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070124 Termination date: 20170328 |