CN1181108A - Psychrophilic protease and psychrophilic bacteria - Google Patents
Psychrophilic protease and psychrophilic bacteria Download PDFInfo
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- CN1181108A CN1181108A CN96193145A CN96193145A CN1181108A CN 1181108 A CN1181108 A CN 1181108A CN 96193145 A CN96193145 A CN 96193145A CN 96193145 A CN96193145 A CN 96193145A CN 1181108 A CN1181108 A CN 1181108A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
A novel psychrophilic protease and a microorganism having the psychrophilic protease producing ability are disclosed. The protease acts on and decomposes casein and dimethylcasein but not on ribonuclease, has an optimal temperature of about 40 ~C. Under the condition of storage at pH 7 for 1 hour, it is inactivated scarcely at a temperature up to 30 ~C, but at 40 ~C it loses about 40 % of the activity. At 50 ~C, the protease is rapidly inactivated so that the activity is completely abolished in about 15 minutes. Flavobacterium balustinum having the protease producing ability is also disclosed.
Description
Background of invention
Technical field that the present invention belongs to
The present invention relates in temperature range, to have highly active proteolytic enzyme, its purposes and the psychrophilic bacteria that produces this proteolytic enzyme.
Background technology
People understand the existing segment length's of psychrophilic bacteria time, and the existence of these bacteriums can be confirmed in low temperature environment widely.Can be separated to psychrophilic bacteria from many low temperature environments (as soil, fishery products, milk preparation and artificial hypothermia's degree environment).From the needs of food microorganisms psychrophilic bacteria is studied, but the kind system that mainly is confined to related microorganism is studied.
Simultaneously, people's expectation is to have the cold enzyme of having a liking for of optimum temps low temperature range from the enzyme that psychrophilic bacteria obtains.Even the cold enzyme of having a liking for that effectively plays a role at low temperatures for example is believed to be incorporated in water at low temperature in the also operable washing agent.Also be considered to be used for the quality that has the chemical reaction of volatile organic solvent under the room temperature and be used under the incorrupt temperature of food, improving food.In addition, to the research of the enzyme that derives from psychrophilic bacteria to the physiological function that discloses psychrophilic bacteria and quite meaningful to cryogenic adaptation mechanism.
Summary of the invention
We have found to produce the new new bacterial isolates of having a liking for the cryoproteins enzyme now.
Therefore, the purpose of this invention is to provide a kind of new cryoproteins enzyme of having a liking for.
Another object of the present invention provides and produces the new microorganism of having a liking for the cryoproteins enzyme.
Also purpose of the present invention provides the method for having a liking for the cryoproteins enzyme with described new microbe preparation.
Have a liking for the cryoproteins enzyme and have following physico-chemical property according to of the present invention.
-activity specific and substrate specificity: it acts on casein and dimethyl casein and decomposes them but do not act on rnase;
-optimum temps: it has in about 40 ℃ the best use of temperature; With
-temperature stability: store at pH7 under 1 hour the condition, at temperature its inactivation hardly during up to 30 ℃, but in the time of 40 ℃ loss of activity about 40%.In the time of 50 ℃, its rapid inactivation so that activity completely lost in about 15 minutes.
In addition, according to the preferred embodiment of the invention, of the present inventionly have a liking for the cryoproteins enzyme and also have following physico-chemical property:
-best pH: it has the best use of when pH7.5; With
-stable p H scope: under 1 hour condition of 20 ℃ of storages, it is stable in the scope of pH6.0-10.0;
-molecular weight: about 38kDa (SDS-PAGE and gel filtration method);
-iso-electric point: about 4.5.
And, be to have the above-described flatfish Flavobacterium (Flavobacterium balustinum) that the cryoproteins enzyme produces ability of having a liking for according to new microbe of the present invention.
In addition, the method for having a liking for the cryoproteins enzyme of preparation invention comprises cultivates above-described flatfish Flavobacterium, has a liking for the cryoproteins enzyme from culture collection is said.
Accompanying drawing is briefly described
Fig. 1 is the result's of explanation embodiment 2 a chart, perhaps shows proteolytic enzyme and the temperature of proteolytic enzyme Subtilysin Carlsberg and the relation between the activity that derives from bacterial strain P104.
Fig. 2 is the result's of explanation embodiment 4 (2) a chart, shows that perhaps initial pH is to the activity of flatfish Flavobacterium P104 extracellular protease and the influence of generation.
Fig. 3 is the result's of explanation embodiment 4 (3) a chart, shows that perhaps culture temperature is to the activity of flatfish Flavobacterium P104 extracellular protease and the influence of generation.Fig. 3 (A), (B) and (C) result when being presented at 10 ℃, 20 ℃ and 30 ℃ respectively.
Fig. 4 is (B) middle gel-filtration wash-out result's a chart of explanation embodiment 5 (2).
Fig. 5 is (C) middle chromatography wash-out result's a chart of explanation embodiment 5 (2).
Fig. 6 illustrates the result of the SDS-PAGE of determining molecular weight among the embodiment 6.
Fig. 7 is the working curve of determining molecular weight among the embodiment 6.
Fig. 8 is the working curve of the gel-filtration of determining molecular weight among the embodiment 6.
Fig. 9 illustrates the result of isoelectric focusing among the embodiment 7.
Figure 10 is the working curve of isoelectric focusing among the embodiment 7.
Figure 11 is the result's of explanation embodiment 8 a chart, perhaps shows the influence of pH to the enzyme reaction of enzyme of the present invention.
Figure 12 is the result's of explanation embodiment 9 a chart, perhaps shows the stability of enzyme of the present invention to pH.
Figure 13 is the result's of explanation embodiment 10 a chart, and perhaps displays temperature is to the influence of the enzyme reaction of enzyme of the present invention.
Figure 14 is the result's of explanation embodiment 11 a chart, perhaps shows the stability of enzyme of the present invention to temperature.
Figure 15 is the result's of explanation embodiment 13 a chart, or explanation protein modification thing SDS is to the chart of the influence of enzyme of the present invention.
Figure 16 is the result's of explanation embodiment 13 a chart, or explanation protein modification thing urea is to the chart of the influence of enzyme of the present invention.
Figure 17 is the Lineweaver-Burk scheme of the enzyme of the present invention of check among the embodiment 16.Figure 17 (A) and (B) be presented in 0 to 2.0 scope respectively and the change of 1/v value in 0 to 0.2 scope.
Detailed Description Of The Invention
New protease-producing strain bacterium
According to new protease of the present invention by belonging to Flavobacterium (Flavobacterium) and having the microorganisms of the ability that produces the protease with the above character.
Have the specific example that produces according to the microorganism of protease ability of the present invention and comprise flatfish Flavobacterium P104. This bacterial strain is the microorganism that separates from the salmon internal, is deposited in industrial science and the state-run life science of technical body and human body technical research institute on February 17th, 1995 with preserving number FERM 5006.
Below listed the bacteriology character according to flatfish Flavobacterium P104 of the present invention:
(1) morphological properties
This bacterial strain exists with the bacillus form of weak point with 0.4-0.5 * 1.7-1.9 μ m size.
(2) character on culture medium
This bacterial strain is in the agar medium growth and produce xanthein.
(3) optimum condition of growth
PH: this bacterial strain is grown under the pH of 5-9 scope, and optimum growh pH value is about neutrality.
Temperature: this bacterial strain is grown under the temperature of 10-30 ℃ of scope, and the optimum temperature of growth is about 20 ℃.
(4) difference between the aerobic and non-aerobic bacteria: aerobic bacteria.
(5) Gram’s staining: feminine gender.
(6) biochemical property
Flatfish Flavobacterium P104 has the main biochemical property shown in the following table 1: table 1 pilot project as a result galactosidase-arginine dihydrolase-lysine decarboxylase-ornithine decarboxylase-citric acid utilization-hydrogen sulfide generation-urase+tryptophan deaminase-indoles generation+gelatinase+glucose-D-mannital-inositol-D-glucitol-rhamnose-sucrose-D-melibiose-D-amygdalin-L-arbinol-oxidizing ferment-
Although can judge as if that from these character this bacterial strain is Flavobacteriumindolgenes, but the base sequence of the DNA by the coding 16S ribosome-RNA(rRNA) will be in following examples 3 described and the base sequence comparison of known microorganisms judge that it is more suitable for being categorized as the flatfish Flavobacterium.
The substratum of cultivating this bacterial strain can be liquid or solid medium, but adopts shaking culture or the aerated culture that carries out with liquid nutrient medium usually.
The substratum of cultivating described microorganism can be any substratum that is suitable for growing and can produces proteolytic enzyme.Specifically, the example of carbon source comprises glucose, trehalose, fructose, maltose, sucrose, starch and Fructus Hordei Germinatus widow-saccharides.The example in chlorine source comprises yeast extract paste, malt extract, beef extract, soyflour, cottonseed meal, corn steep liquor, each seed amino acid and their salt and nitrate.Also can utilize comprise suitable inorganic salt such as magnesium, calcium, sodium, potassium, iron and maganese phosphoric acid salt and other must nutraceutical synthetic medium or natural medium.
Culture condition such as pH and temperature can determine in producing the scope of proteolytic enzyme, and liquid oscilaltion cultivation or aeration-agitation are cultivated at pH under about 20 ℃ of the neutral approximately and temperature and carried out.
Proteolytic enzyme of the present invention and can be bacterial cell, thick enzyme (obtaining from bacterial cell or nutrient solution supernatant liquor) or that extract and enzyme form purifying in the cell walls of bacterial cell, within the cell, produce in the nutrient solution supernatant liquor.
The collection of enzyme
For collect from nutrient solution and purifying according to proteolytic enzyme of the present invention, can be used alone or in combination well-known method.
Mainly be secreted into the extracellular according to proteolytic enzyme of the present invention, promptly be secreted in the nutrient solution, therefore can obtain thick enzyme solution with for example filtration or the centrifugal bacterial cell of easily removing.Can be further purified thick enzyme solution with currently known methods.Described method preferably includes with saltouing that salt (as ammonium sulfate) carries out; The precipitation of carrying out with organic solvent (as methyl alcohol, ethanol or acetone); The raw starch absorption method; Ultrafiltration process; With various chromatography (as gel permeation chromatography or ion exchange chromatography).The specific examples of preferred method is described in following examples.
The character of proteolytic enzyme
Measured the character according to proteolytic enzyme of the present invention, the result is as follows:
(1) activity and substrate specificity
Enzyme of the present invention decomposes macro-molecular protein such as casein or dimethyl casein or denatured protein well.It also decompose gelatin (with casein comparison collagen ratio be about 50% denatured protein).Yet it has very little effect to other natural protein, and it does not act on rnase.
The proteolytic enzyme of industrial use such as subtilisin can have non-substrate specificity, almost act on all proteins.On the contrary, enzyme of the present invention only acts on macro-molecular protein or the relatively easy approaching denatured protein of enzyme.
A kind of similar proteolytic enzyme of the present invention, that derive from the psychrophilic bacteria Pseudomonas fluorescens has successfully decomposed macro-molecular protein such as casein or dimethyl casein or denatured protein.Yet this enzyme is different from enzyme of the present invention, and it also decomposes natural globular preteins such as oxyphorase and bovine serum albumin (comparing the ratio with about 40% with the dimethyl casein).Like this, enzyme of the present invention has the substrate specificity higher than known enzyme probably.
(2) optimum temps and equilibrium temperature
Enzyme of the present invention plays a role under about 40 ℃.
Store at pH7 under 1 hour the condition, at temperature its inactivation hardly during up to 30 ℃, but in the time of 40 ℃ loss of activity about 40%.In the time of 50 ℃, rapid inactivation of this proteolytic enzyme so that activity completely lost in about 15 minutes.
Therefore, enzyme of the present invention is called as has a liking for cold enzyme, and it demonstrates effective katalysis at low temperatures.
(3) best pH and stable pH range
Enzyme of the present invention has 7.5 best pH.
In addition, under 1 hour condition of 20 ℃ of storages, it is stable in the scope of pH6.0-10.0.
Thus, this enzyme is a neutral protease, its in extreme acidity or alkaline range with inoperative.And it can inactivation when storing under extreme acidity or alkaline range.
(4) molecular weight
With SDS-PAGE and gel filteration determining the time, enzyme of the present invention has the molecular weight of about 38 kDa.
(5) iso-electric point
When measuring with isoelectric focusing, enzyme of the present invention has about 4.5 iso-electric point.
(6) active inhibition
The protease activity of described enzyme is not suppressed by phenylmethylsulfonyl fluoride or iodo-acid amide, but be subjected to ethylenediamine tetraacetic acid (EDTA) significantly, 2,2-bipyridyl, citric acid or oxalic acid suppress.Find out that thus protease activity depends on metal ion, this shows that enzyme of the present invention is a metalloprotease.Be subjected to one of citric acid and oxalic acid to suppress to think that described activity depends on calcium from protease activity.
In addition, the activity of enzyme is subjected to metal ion such as Ag significantly
+, Cu
2+, Zn
2+, Co
2+And Fe
2+Suppress, particularly be subjected to Ag significantly
+Suppress.Yet it is not subjected to Mg
2+Perhaps Ca
2+Suppress.
(7) enzymatic reaction kinetics
To substrate (as casein) concentration, enzyme of the present invention demonstrates Michaelis-Menten type speed of reaction.Along with the increase of temperature, the Km value reduces, and the Vmax value increases.In addition, in 10 to 40 ℃ scope, the Kcat value of enzyme is very high.In the presence of excessive substrate, enzyme is suppressed usually.Yet, under the situation of enzyme of the present invention, when system during near the best use of temperature Kcat value increase.Therefore, noticeable with regard to the restraining effect of degradation production generation with regard to not observing, described enzyme is favourable.Can believe also that from Lineweaver-Burk figure the restraining effect that is caused by temperature is a kind of complex form, that is, Temperature Influence is non-competitive inhibition, and the influence of degradation production is a competitive inhibition.
The purposes of enzyme
Have a liking for the cryoproteins enzyme and in low temperature range, have optimum temps according to of the present invention.Like this, have a liking for for the cryoproteins enzyme for of the present invention, decomposing protein is possible at low temperatures.For example, even by proteolytic enzyme of the present invention being mixed into washing agent preparation of compositions also operable washing agent in water at low temperature.Of the present inventionly have a liking for the cryoproteins enzyme except mixing, this detergent compositions can prepare according to ordinary method.In brief, can be by proteolytic enzyme of the present invention and common washing agent component, make up as tensio-active agent, SYNTHETIC OPTICAL WHITNER or the washing assistant of washing agent and to prepare.
In addition, can carry out at low temperatures according to the enzyme reaction of having a liking for the cryoproteins enzyme of the present invention.Like this, even reactive system comprises volatile organic solvent under the room temperature, reaction also can be carried out under the not volatile low temperature of described organic solvent.And when being intended to when improving food quality according to proteolytic enzyme of the present invention, it is favourable using proteolytic enzyme of the present invention, decomposes because carry out preventing effectively food at low temperatures.
In addition, owing to provide, be hopeful in the research of the physiological mechanism of psychrophilic bacteria and their application system, to make progress according to proteolytic enzyme of the present invention.
Following with reference to the present invention of specific embodiment detailed description, but the present invention is not subjected to the restriction of these embodiment.
In this part, unless other explanation is arranged, with protein staining method and Bio-Rad protein determination quantitative assay protein, by the protein component of eluate in the absorption measurement chromatography process in the ultraviolet range when the 280nm.
In addition, protease activities is measured as described below:
(a) proteolytic activity that obtains with azo-casein
A 0.05ml sample enzyme solution is added in the 0.067M phosphate buffered saline buffer (pH7.0) that 0.3ml comprises 1% (W/V) azo-casein (azocasein), and mixture kept 30 minutes down at 30 ℃.Then with 6% trichoroacetic acid(TCA) solution termination reaction.After 15 minutes, with reaction mixture in room temperature 14, under the 000rpm centrifugal 5 minutes.Measure the specific absorption of supernatant liquor at 340nm.Enzymic activity is definite for the basis with ACU (azo-casein digestion unit refers to that the per minute light absorption ratio increases by 0.001 under 340nm).
(b) proteolytic activity that obtains with improved Anson method
A 0.05ml sample enzyme solution is added in the 0.067M phosphate buffered saline buffer (pH7.0) that 0.3ml comprises 1% (W/V) azo-casein (azocasein), and mixture kept 30 minutes down at 30 ℃.Then with 7.5% trichoroacetic acid(TCA) solution termination reaction.After 30 minutes, with reaction mixture in room temperature and 14, under the 000rpm centrifugal 5 minutes.Measure the specific absorption of supernatant liquor at 280nm.Enzymic activity is definite for the basis with AU (improved Anson unit refers to that per minute produces the tyrosine of 1 μ mol amount).
(1) the new bacterial isolates of screening
On Agar Plating, carry out the separation of new bacteria bacterial strain.The sample separation of organ in the salmon is suspended in the physiological saline, supernatant liquor is used as stock solution.Make 102 extent of dilution from stock solution.With stock solution and 102 diluents each 0.2ml spray cloth (3 grams per liter polyprotein peptones (polypeptone), 10 grams per liter yeast extract pastes, 10 grams per liter sodium caseinate food grades, 0.2 grams per liter MgSO on the screening Agar Plating
47H
2O, 2.0 grams per liter agar are on the 9cm culture dish), cultivated 3 days in 10 ℃.Select well-grown bacterium colony in the bacterium colony of having grown on agar plate, succeeding transfer culture also is inoculated on the agar plate for storing.
On Agar Plating, measure the extracellular enzyme activity.With the isolated bacterial bacterial strain through streak inoculation to above-described screening Agar Plating, cultivated 3 days in 10 ℃.Then 10% trichoroacetic acid(TCA) solution is sprayed cloth to Agar Plating, bacterium grows on this substratum, determines the protease-producing bacterium by the existence of clear plaque.
(2) bacterial isolates of culture of isolated and generation enzyme
To be inoculated into the pre-culture medium of 25ml (extract (endoextract), 0.2 grams per liter MgSO in 10 grams per liter polyprotein peptones, 10 grams per liters from the storage medium separation of bacterial
47H
2O, pH7.0 are in the 100ml Erlenmeyer flask) on, for the growth activity of stable bacterial, rotational oscillation was cultivated 48 hours under 10 ℃ and 150rpm in TAITECNR-80.Under conventional culture condition, the pre-culture solution of 0.25ml is inoculated into 25ml produces on the substratum (5 grams per liter polyprotein peptones, 2.5 grams per liter yeast extract pastes, 5 grams per liter sodium caseinate food grades, 0.2 grams per liter MgSO47H2O, pH7.0 is in the 100ml Erlenmeyer flask) of enzyme.Under 10 ℃ and 150rpm on rotary shaker rotation-shaking culture 72 hours.Substratum is in advance at 1.2kgf/cm
2Steam sterilizing is 15 minutes under the scale (gauge) (121 ℃).
The isolated bacterial bacterial strain is stored in the Agar Plating so that 10 ℃ of storages.Succeeding transfer culture after 2 weeks to 1 month.
(3) measure protease activity
Clarify the nutrient solution that above step (2) obtains with centrifugal (17,000 * g, 4 ℃, 15 minutes).Supernatant liquor is used as thick enzyme solution.Resolution measurement protease activity by azo-casein.The thick enzyme solution of a 0.05ml is added in the 0.067M phosphate solution that 0.3ml comprises 1% (W/V) azo-casein (pH7.0), and mixture kept 30 minutes down at 20 ℃.With 6% trichoroacetic acid(TCA) solution termination reaction, after 15 minutes, in room temperature and 14, centrifugal reaction mixture is 5 minutes under the 000rpm then.Measure the specific absorption of supernatant liquor with spectrophotometer (Beekman DU640) at 340 nm.Enzymic activity is definite for the basis with ACU (azo-casein digestion unit refers to that the per minute specific absorption increases by 0.001 under 340nm).
As the result of step (1)-(3), be separated to bacterial isolates P104 with protease activity.This bacterial isolates has the protease activity shown in the following table.
In table, by relatively obtaining the growth velocity of described bacterial strain with yellow Cytophaga bacterial strain (Cytophaga xantha) growth phase of IFO14972 of separating.
Table 2
Bacterial isolates with protease activity
| Bacterial strain | Growth velocity | Protease activity (* 10 3ACU/ml) |
| ????P104 | ????+++ | ????0.982 |
Embodiment 2
The protease activity of P104
In 0-60 ℃ of temperature range, measure the influence of temperature to enzymic activity with protease-producing bacterial strain P104.Also use Subtilisin Carlsberg (sigma) (the commercially available proteolytic enzyme that derives from Bacillus licheniformis) to measure the temperature dependency of enzymic activity in an identical manner.
The result shows that in Fig. 1 in Fig. 1, the specific enzyme activity of bacterial strain P104 represents that with the specific enzyme activity of Subtilisin Carlsberg is represented with Δ.
The optimum temps of bacterial strain P104 extracellular protease (exoprotease) and Subtilisin Carlsberg is respectively 40 ℃ and 60 ℃ or higher.When about 20 ℃ of temperature, when the extracellular protease of bacterial strain P104 keeps optimum temps active 40% or higher protease activity, about 10% of the protease activity when SubtilisinCarlsberg only keeps optimum temps.
In addition, at 10 to 40 ℃ of scopes activation energy of these extracellular protein enzyme reactions of letting it pass of falling into a trap.The results are shown in the following table: table 3
| Bacterial cell | Activation energy (KJ/mol) |
| ????P104 | ????39.8 |
| Subtilis | ????58.3 |
Base sequence strain identification P104 by the DNA of coding 16S ribosome-RNA(rRNA)
The nutrient solution that embodiment 2 is obtained is sampled in the 1.5ml Eppendorf tube, through centrifugal collecting cell.The extracting genomic dna is so that through the base sequence of the DNA of PCR (polymerase chain reaction) amplification coding 16S ribosome-RNA(rRNA).Relatively determine base sequence with Sanger method and GenBank database for discriminating.The primer that uses is listed in following, and 1F-Link and 5R-Link are used for PCR.
Primer: 1F-Link:5 '-TGTAAAACGACGGCCAGTAGTITGATCATGGCTCAG-3 ' 3R-Link:5 '-CAGGAAACAGCTATGACCCGTCAATTCATTTGAGTT-3 ' 3F-Link:5 '-TGTTAAACGACGGCCAGTGTAGCGGTGAAATGCGTA-3 ' 5R-Link:5 '-TGTAAAACGACGGCCAGTAAGTCCCGCAACGAGCGCAA-3 '.
Showing in the tabulation down with GenBank database result relatively.In this table, the inquiry representative derives from the 16S ribosomal RNA gene of bacterial strain P104, and object is represented flatfish Flavobacterium S16 ribosome-RNA(rRNA) (FVBRR16SH).Bacterial isolates is differentiated and is the flatfish Flavobacterium as a result.This bacterial strain is called as flatfish Flavobacterium P104 thus.(a) use primer 1F-Link identity property=185/204 (90%), positive=185/204 (90%)
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Object: 945 G A T A C G C G A G G A A C C T T A C C
Inquiry: 261 A N G N T T N N T T N G G G T C N T C C
:?????:????????:?????????????????:??:??:???????????:
Object: 965 A A G G C T T A A A T G G G A A T T G A
Inquiry: 281 C A G C C T T C G T C T N T A C T T G T
:??:??:????????:?:??????:??????????????????????????:?????:
Object: 985 C A G G T T T A G A A A T A G A C T T T
Inquiry: 301 G N T T C G G A C A
:??:??:??:??:??:??:??:
Object: 1005 T C T T C G G A C A (c) use primer 3R-Link identity property=128/162 (79%), positive=128/162 (79%)
Inquiry: 162 T T N T T G G G N A T A A N A G G G N C
:??:????????:?????:??:????????:??:??:?????:??:??:??:?????:
Object: 552 T T T A T T G G G T T T A A A G G G T C
Inquiry: 142 C N T C N G C G G A C C T G T A A A T C
:?????:????????:??:??:??:??:?????:??:??:??:??:??:??:??:??:
Object: 572 C G T A G G C G G A T C T G T A A G T C
Inquiry: 122 A T T G G T G A T A T C T C A G A G C C
:?????:??:??:??:??:??:?????:??:??:??:??:??:?????:??:??:
Object: 592 A G T G G T G A A A T C T C A T A G C T
Inquiry: 102 T G T C T A T G G G A C T A C C A T T G
:????????:??:??:??:??:????????:??:??:?????:??:??:??:??:??:
Object: 612 T A A C T A T G A A A C T G C C A T T G
Inquiry: 82 A T G C T C C A G G T C A T G A G T C T
:??:?????:??:?????:??:??:??:??:??:?????:??:??:??:??:
Object: 632 A T A C T G C A G G T C T T G A G T A A
Inquiry: 62 A G C A G G A G T G G C T G G A A T A A
:??:?????:??:?????:??:??:??:??:??:??:??:??:??:??:??:??:??:
Object: 652 A G T A G A A G T G G C T G G A A T A A
Inquiry: 42 G T A C T G T A A C G G T G T A A T G C
:??:??:?????:??:??:??:?????:??:??:??:??:?????:??:??:??:??:
Object: 672 G T A G T G T A G C G G T G A A A T G C
Inquiry: 22 A T A G A T A T T A C T C A G A A C C C
:??:??:??:??:??:??:??:??:??:??:??:?????:??:??:??:??:?????:
Object: 692 A T A G A T A T T A C T T A G A A C A C
Inquiry: 2 C A
:??:
Object: 712 C A (d) use primer 5R-Link identity property=237/273 (86%), positive=237/273 (86%)
Inquiry: 273 C C G G T A C N C C T T G G G C C A C A
:??:????????:??:??:?????:??:??:??:??:??:??:??:??:??:??:??:
Object: 1193 C C C T T A C G C C T T G G G C C A C A
Inquiry: 253 C A C G T A A T A C A A T G N C A A G N
:??:??:??:??:??:??:??:??:??:??:??:??:??:?????:?????:??:
Object: 1213 C A C G T A A T A C A A T G G C C A G T
Inquiry: 233 A C A G A G N G T A G C N A C C A G N C
:??:??:??:??:??:?????:??:??:??:??:?????:??:??:??:??:?????:
Object: 1233 A C A G A G G G C A G C T A C C A G G C
Inquiry: 213 G N C T G G A T G C G A A T C T C G A A
:?????:??:??:??:??:??:??:??:??:??:??:??:??:??:??:??:??:??:
Object: 1253 G A C T G G A T G C G A A T C T C G A A
Inquiry: 193 A N C T G C N C T C A G A N C G G A N T
:?????:??:?????:?????:??:??:??:??:??:?????:??:??:??:?????:
Object: 1273 A G C T N G N C T C A G T T C G G A T T
Inquiry: 173 G G A G T C T G C A A C T C G A C T C T
:??:??:??:??:??:??:??:??:??:??:??:??:??:??:??:??:??:??:??:
Object: 1293 G G A G T C T G C A A C T C G A C T C T
Inquiry: 153 A T G A A A C T G G A T N C G C T A G T
:??:??:??:??:?????:??:??:??:??:????????:??:??:??:??:??:??:
Object: 1313 A T G A A G C T G G A A T C G C T A G T inquiry: 133 A A T C G C A T A T C A G N C A T G A T
::::::::::::::::::: object: 1333 A A T C G C A T A T C A G C C A T G A T inquiry: 113 N C G G T G A A N A C G T T G C C N G G
:::::::::::::::: object: 1353 G C G G T G A A T A C G T T C C C N G G inquiry: 63 C C T T G N A A A C A C C G C C C G T C
::::::::::::::::: object: 1373 C C T T G T A C A C A C C G C N C G T C inquiry: 73 A A C C C A T G G A A G T T T G G G G T
::::::::::::::::::: object: 1393 A A G C C A T G G A A G T T T G G G G T inquiry: 53 A C C T G N A G T C G G T G A C C G T A
::::::::::::::::::: object: 1413 A C C T G A A G T C G G T G A C C G T A inquiry: 33 A C N G G A C C T N C C T A G G G T A N
:::::::::::::::: object: 1433 A C A G G A G C T G C C T A G G G T A A inquiry: 13 N A C A A G T A A C T A G
:::::::::::: object: 1453 A A C A A G T A A C T A G embodiment 4
Cultivate flatfish Flavobacterium P104
(1) density of mensuration bacterial cell
With the nutrient solution of physiological saline dilution embodiment 2 acquisitions, so that in the cell counting pond, comprise 0-5 cell.With optics microscopic counting cell.The concentration of cultivating diluent at 660nm through spectrographic determination is to obtain the relation between cell and the concentration.Following equation is represented the relation between turbidity and the flatfish Flavobacterium P104 bacterial cell concentration:
(cell/ml)=1.13 * 10
9* Abs.660nm
Wherein 1.13 * 10
9It is the factor that obtains from working curve.
(2) influence of pH
Culturing bacterium bacterial strain under the various pH of the protease-producing substratum in 5 to 9 scopes is to determine the influence of initial pH to extracellular protein enzymic activity and its generation.
The result shows in Fig. 2.
In alkaline pH, propagation obviously reduces, and protease activity also reduces.Yet, in the acid pH scope, observe the propagation or the obvious difference of protease activity.Bacterial isolates propagation is good in the neutral pH scope, and protease activity remains on the highest level.
(3) influence of culture temperature
Cultivate flatfish Flavobacterium P104 under all temps in 10-30 ℃ of scope, to measure along with the increase and decrease of carrying out extracellular protein enzymic activity and propagation of cultivating.
The result shows in Fig. 3.
Though bacterial isolates well-grown under the temperature of 10 ℃ and 20 ℃, 30 ℃ multiplication rates be the pacts of 10 ℃ or 20 ℃ stationary phases half.In the time of 20 ℃, strains expressed goes out the highest multiplication rate, so the optimum temps of cultivating is considered to about 20 ℃.
Purifying derives from the proteolytic enzyme of flatfish Flavobacterium P104
(1) culturing bacterium bacterial strain
The isolated bacterial inoculation that will obtain from following storage cell culture medium is in the following pre-culture medium of 25ml (the 100ml Erlenmeyer flask), and rotational oscillation was cultivated 48 hours under the 150rpm in 10 ℃ of TAITEC NR-80.By inoculation 0.25ml pre-nutrient solution in the following conventional substratum in the 10ml Erlenmeyer flask, in 10 ℃ of rotational oscillation culture solution 72 hours under 150rpm.
Storage medium:
Polyprotein peptone 3g/l,
Enzyme extract 2.5g/l,
Sodium caseinate food grade 20g/l,
MgSO
4·7H
2O?0.2g/l，
Agar 20g/l,
pH7.0
Pre-culture medium:
Polyprotein peptone 3g/l,
Enzyme extract 2.5g/l,
Sodium caseinate food grade 1g/l,
MgSO
4·7H
2O?0.2g/l，
pH7.0
Conventional substratum
Polyprotein peptone 3g/l,
Enzyme extract 2.5g/l,
Sodium caseinate food grade 5g/l,
KH
2PO
4·7H
2O3g/l
MgSO
4·7H
2O?0.2g/l，
pH7.0
At 1.2kgf/cm
2Under the scale (120 ℃), use high pressure steam autoclaving material (for example substratum) 15 minutes.
P104 is inoculated on the agar plate with the flatfish Flavobacterium, in 10 ℃ of storages, and succeeding transfer culture 2 thoughtful 1 month.
(2) purifying protein enzyme
(a) use ammonium sulfate precipitation
Clarify through the nutrient solution that centrifugal (17,000 * g 15 minutes) obtains above step (1).Supernatant liquor is used as enzyme solution.Ammonium sulfate is added in the thick enzyme solution, so that solution contains the ammonium sulfate of saturation concentration 50%.Slowly stir after 1 hour, centrifugal (17,000 * g 15 minutes) sedimentation solution provides the saturated component of 0-50%.During 25 ℃ of saturation concentrations, the addition of ammonium sulfate is as the amount of the ammonium sulfate that adds.
(b) gel-filtration
Then on HiLoad 16/60 Superdex 200 preparation classification (prep grade) posts, carry out gel-filtration.Operation in all column chromatographies is all carried out as chromatographic system with HiLoad system 50 (FarmaciaBiotec Co.) down in 4 ℃.
By under about 60cm/ hour linear velocity, crossing 20mM Bis-Tris damping fluid (pH6.0) balance HiLoad 16/60 Superdex 200 preparation fractionation columns to the proportional flow of gel volume with at least 3 (400ml).5ml component with the sample enzyme solution of ammonium sulfate precipitation is loaded on the post with Superloop.With 20mM Bis-Tris damping fluid (pH6.0) is elutriant, with about 60cm/ hour linear velocity wash-out post, collects the 5ml component.
Elution curve shows in Fig. 4.
As exclusion limit, wash-out is a kind of to be included in and to have high-molecular weight glassy yellow protein in the nutrient solution.As follow-up component with 280nm uv-absorbing, a kind of water white transparency component of wash-out.As if be removed attached to the fragment on the proteolytic enzyme, because in this component, have protease activity.
(c) column chromatography
Then carry out ion exchange chromatography with Q sepharose HP post, φ 0.7 * 12.5cm post of being made up of HiTrapQ (1ml) post of 5 series connections is used as chromatography column.By under about 35cm/ hour linear velocity, crossing 20mMBis-Tris damping fluid (pH6.0) balance columns to the proportional flow of gel volume with at least 5 (25ml).
The component that will advance the gel-filtration wash-out is loaded on the post with Superloop with about 17.5cm/ hour linear speed with the sample enzyme solution with protease activity.The 20mM Bis-Tris damping fluid that has the 1M NaCl that adds to wherein with 80ml is collected the 2ml component by the linear speed wash-out post of linear ion intensity increase gradient (0-150mM) with about 35cm/ hour.
Elution curve shows in Fig. 5.
In addition, purification process described above is summarised in the following table.
Table 4
Protease purification is summed up
| Amount (ml) | Protein (mg) | Enzymic activity (* 10 3ACU) | Specific activity (* 10 3ACU·mg -1) | Resorption rate (%) | Purifying rank (*) | |
| Cultivate material | ??270 | ??13.0 | ????365 | ????28.1 | ??100 | ????1 |
| Use ammonium sulfate precipitation | ??5 | ??3.38 | ????273 | ????80.8 | ??74.8 | ????2.9 |
| Gel-filtration | ??25 | ??0.752 | ????77.7 | ????103 | ??21.3 | ????3.7 |
| Ion exchange chromatography | ??18 | ??0.154 | ????29.3 | ????190 | ??8.03 | ????6.8 |
Proteolytic enzyme purity and molecular weight determination
(1) electrophoresis
(a) SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Use has 10% polyacrylamide gel of 1mm thickness.The 20mA electric current is administered to carries out electrophoresis on the gel, (BPB) reaches bottom up to tetrabromophenol sulfonphthalein.With the aqueous mixture dyeing gel slab with 30% methyl alcohol that is dissolved in 0.02% coomassie brilliant blue R250 wherein and 10% acetate 1 hour, then, decolour with discoloring agent (30% methyl alcohol and 10% acetate) and to spend the night.
Measure described protease molecule amount with having the Starch phosphorylase, albumin, Protalbinic acid, carbonic anhydrase, trypsin inhibitor and the alpha-lactalbumin that serve as a mark through SDS-PAGE.
The result of SDS-PAGE and working curve demonstrate in Fig. 6 and 7.Described enzyme demonstrates single band, can think single protein thus, rather than subunit structure.
(b) gel-filtration
With Hiprep 16/60 sephacryl S-100 HR through the gel filteration determining molecular weight.By under about 30cm/ hour linear velocity, crossing 50mM phosphate buffered saline buffer (pH7.0) balance columns to the proportional flow of gel volume with the 0.15M NaCl that adds to wherein with at least 3 (400ml).The sample enzyme solution of 1ml is loaded on the post, with as above identical damping fluid linear velocity wash-out with about 3030cm/ hour, collection 2ml component.With albumin (67kDa), Protalbinic acid (43kDa), chymotrypsinogen A (25kDa), ribonuclease A (13.7kDa) measure the withdrawal volume of blue dextran (Blue Dextran) 2000 as standard protein.
The results are shown in the table 8.In addition, put down in writing ultraviolet absorption value and the enzymic activity of each protein at 280nm.Proteinic ultraviolet absorption curve also demonstrates the maximum absorption (no visible absorbance) at 278nm.Can think that from these observationss described protein has the desirable purity of its character of mensuration.
Isoelectric focusing
Carry out isoelectric focusing with Phast system (Farmacia-Biotec company).Use the IEF3-9 gel, sample is loaded on the point of anode 3cm.2,000V carries out electrophoresis under the condition of 2.5mA when 15 ℃ and 410Vh.In 20 ℃ through with dull and stereotyped 5 minutes of the fixing dyeing of 20%TCA solution gel, washed 2 minutes with flushing and de-inking solution (30% methyl alcohol and 10% acetate).The most finally 50 ℃ with having the solution flushing that is dissolved in 0.02% coomassie brilliant blue R250 wherein and decolouring 10 minutes.Broad pI Calibration test kit (Farmacia-Biotec company) is with marking.
The result of isoelectric focusing and working curve are presented among Fig. 9 and Figure 10.
Described enzyme is colored the single band for the iso-electric point with pH4.5 basically.
PH is to the influence of enzyme reaction
Under various pH, decompose azo-casein (azocasein) with enzyme.
Damping fluid in the reaction mixture has the concentration of 67mM, and comprises acetate buffer (pH4-4.5), KH respectively
2PO
4-NaH
2PO
4(pH6.0-8.0), NaB
4O
7-HCl (pH8.0-9.0), NaB
4O
7-NaOH (pH9.5-10) and Na
2HPO
4-NaOH (pH10.5-12.0).
The results are shown among Figure 11.
Be maintained in the pH scope of relative reactivity 6.0 to 10.0 of the enzyme under the pH7.5 of best pH on about 80% the level.Find that thus described enzyme works in the quite wide scope that with the neutral pH is the center.Yet, pH5.5 or lower or 10.5 or higher scope described in the effect of enzyme unsatisfactory, it is inactivated when pH12, the forfeiture protease activity.
Embodiment 9
The pH stability of proteolytic enzyme
Has mensuration enzyme under the various pH that contain Econo-Pac (Biorad Co.).The buffered soln that uses has the concentration of 67mM, and comprises acetate buffer (pH4-5), KH respectively
2PO
4-NaH
2PO
4(pH6-8), glycine-NaOH (pH9-10) and Na
2HPO
4-NaOH (pH11-12).
The results are shown among Figure 12.
Discovery is under 20 ℃ of conditions of 1 hour, and described enzyme is stable in 6.0 to 10.0 pH scope.
Stable influence to enzyme reaction
Under 0 to 70 ℃ temperature of reaction and various pH, decompose azo-casein with enzyme.
Similarly react with commercially available enzyme, described enzyme is Subtilisin Carlsberg, V8 proteolytic enzyme (it is the proteolytic enzyme that derives from Staphylococcus aurcub V8), Sabinase (Novonordisc) and Alkalase (Novonordisc) for example.
The result's (there is excessive substrate in supposition, thereby enzyme all exists with the form of enzyme-substrate complex) who is represented by Kcat provides in Figure 13.In Figure 13, represents described enzyme, ◆ represent V8 proteolytic enzyme, zero represents Subtilisin Carlsberg, and △ represents Sabinase, and represents Alkalase.
Find that described enzyme has the optimum temps at 40 ℃, surpassing rapid inactivation under the enzyme reaction temperature of optimum temps.And find that all commercially available proteolytic enzyme has 50 ℃ or higher optimum temps, in 10 to 40 ℃ scope, the Kcat value of enzyme of the present invention is higher than any relatively proteolytic enzyme.
Embodiment 11
The temperature stability of proteolytic enzyme
In 20 to 50 ℃ temperature range, keep enzyme of the present invention.Activity is shown among Figure 14 over time, and wherein represents 20 ℃ variation, ◆ represent 30 ℃ variation, zero represents 40 ℃ variation, and △ represents 50 ℃ variation.
At 20 ℃ or 30 ℃ of enzymes inactivation hardly, but 40 ℃ of inactivation to 20 ℃ or 30 ℃ of enzymic activitys about 60% gradually after 1 hour, at 50 ℃ of about 15 minutes rapid complete deactivations.
Enzyme of the present invention is considered to thermally labile, because above temperature is lower than the optimum temps of the comparison proteolytic enzyme that uses in the former experiment.
Embodiment 12
The influence of inhibitor
The phenylmethylsulfonyl fluoride (PMSF) that serine protease is worked, the iodo-acid amide (IAA) that L-Cysteine HCL Anhydrous is worked, ethylenediamine tetraacetic acid (EDTA) (EDTA), O-phenanthroline, 2 that metalloprotease is worked, the 2-bipyridyl and act on the Citrate trianion of calcium specifically and oxalate as inhibitor.After the inhibitor of various concentration adds in the enzymatic reaction system, keep 1 hour to detect the protease activity of surviving at 20 ℃.
The result shows in following table.
Table 5
The influence of inhibitor
| Inhibitor | The activity (%) that concentration is remaining |
| No PMSF iodoacetamide EDTA o-phenanthroline 2,2 '-bipyridyl citrate oxalates | ????10mM????100 ????10mM????99 ????10mM????94 ????10mM????8 ????10mM????91 ????10mM????39 ????10mM????41 ????100mM???0 ????10mM????45 ????100mM???0 |
The protease activity of described enzyme is not suppressed by PMSF or LA-A, but significantly by EDTA, 2,2-bipyridyl, Citrate trianion or oxalate suppress.Find that from these observationss described protease activity is that metal ion is dependent.Thus, enzyme of the present invention is a metalloprotease.From being suppressed to think that also described protease activity depends on calcium by Citrate trianion or oxalate.
Embodiment 13
The influence of protein denaturant
SDS and urea are as protein denaturant.Behind the protein denaturant that adds various concentration, enzymatic reaction system is kept 1 hour to detect remaining protease activity at 20 ℃.
About the result of SDS and urea is presented at respectively in Figure 15 and 16.
Even under quite low concentration, described proteolytic enzyme is suppressed by SDS also, when 0.25%SDS by complete inactivation.Up to 2M concentration the time, described proteolytic enzyme is not suppressed by urea yet, but is suppressed to about 40%, by 4M urea complete inactivation by 3M urea.
Embodiment 14
The influence of metal ion
AgNO
3, CuSO
4, ZnSO
4, CoSO
4, FeSO
4, MnSO
4, CaCl
2And MgSO
4As metal source.Adding metal-salt with after guaranteeing that ultimate density is 1mM, enzymatic reaction system is being kept 1 hour to detect remaining protease activity at 20 ℃.
The result is showing in the tabulation down.
Table 6
The influence of metal ion
| Metal ion | Remaining activity (%) |
| Do not have | ????100 |
| ????Ag 2+ | ????11 |
| ????Cu 2+ | ????44 |
| ????Zn 2+ | ????61 |
| ????Co 2+ | ????63 |
| ????Fe 2+ | ????76 |
| ????Mn 2+ | ????95 |
| ????Ca 2+ | ????100 |
| ????Mg 2+ | ????100 |
Enzyme of the present invention is by Ag
2+, Cu
2+, Zn
2+, Co
2+And Fe
2+The degree of depth suppresses.When by Ag
2+During inhibition, more than only 10% protease activity be retained.Enzyme of the present invention is not at all by Mg
2+Or Ca
2+Suppress.
Embodiment 15
Substrate specificity
Casein (Hammarsten), dimethyl casein, gelatin, oxyphorase, bovine serum albumin and rnase are as the substrate protein white matter, so that measure proteolytic activity through improved Anson method.Azo-casein and azoalbumin are as the azoprotein modifying protein.The result shows in following table.
Table 7
Substrate specificity
| Substrate (1%) | Percent hydrolysis (%) |
| Casein (Hammarsten) dimethyl casein gelatin oxyphorase bovine serum albumin rnase | ????100 ????93 ????53 ????18 ????17 ????0 |
| The azo-casein azoalbumin | ????100 ????20 |
Enzyme of the present invention is decomposing macromolecular protein and denatured protein such as casein and dimethyl casein well, also decompose as the proteinic gelatin of collagenous degeneration (based on casein about 50%).Enzyme acts on other natural protein hardly, and particularly it does not act on the rnase.
Embodiment 16
With caseic enzymatic reaction kinetics
The Lineweaver-Burk that obtains under 5 to 40 ℃ all temps with the solution that comprises 0.05-1% casein (as substrate) schemes.This is illustrated among Figure 17, and wherein last figure (A) illustrates the change of 1/v value in 0 to 2.0 scope, and figure below (B) illustrates the change of 1/v value in 0 to 0.2 scope.In the figure, represents 5 ℃ curve, and ◇ represents 10 ℃ curve, and zero represents 20 ℃ curve, and △ represents 30 ℃ curve, and represents 40 ℃ curve.The enzyme reaction power constant obtains from Lineweaver-Burk figure, and is as shown in the table.
Table 8
Substrate specificity
| Substrate (1%) | Hydrolysis rate (%) |
| Casein (Hammarsten) | ????100 |
| The dimethyl casein | ????93 |
| Gelatin | ????53 |
| Oxyphorase | ????18 |
| Bovine serum albumin | ????17 |
| Rnase | ????0 |
| Azo-casein | ????100 |
| Azoalbumin | ????20 |
Can find out obviously that from last table described enzyme shows Michaelis-Mentne type speed of reaction to casein concentration.Along with the increase of temperature, the Km value reduces, and the Vmax value increases.When 5 ℃ and 10 ℃, in the high density casein, enzymic activity is suppressed.In general, enzyme tends to be suppressed in excessive concentration of substrate.Yet under the temperature of reaction that increases, in 1% casein, described enzyme is not suppressed.This is considered to because the Kcat value increases near optimum temps and the little restraining effect of casein degradation production.Can think also that from Lineveaver-Burk figure the suppressor mode of temperature is a mixed type.That is, think that Temperature Influence is noncompetitive or noncompetitive suppresses, the influence of casein degradation production is a competitive inhibition.
Claims (10)
1. have a liking for the cryoproteins enzyme for one kind, this proteolytic enzyme has following physico-chemical property:
-activity specific and substrate specificity: it acts on casein and dimethyl casein and decomposes them but do not act on rnase;
-optimum temps: it has in about 40 ℃ the best use of temperature; With
-temperature stability: store at pH7 under 1 hour the condition, its inactivation hardly when temperature is up to 30 ℃, but in the time of 40 ℃ loss of activity about 40%.In the time of 50 ℃, its rapid inactivation so that activity completely lost in about 15 minutes.
2. have a liking for the cryoproteins enzyme as claim 1 is desired, this proteolytic enzyme also has following physico-chemical property:
-best pH: it has the best use of when pH7.5; With
-stable p H scope: under 1 hour condition of 20 ℃ of storages, it is stable in the scope of pH6.0-10.0.
3. have a liking for the cryoproteins enzyme as claim 1 is desired, wherein said proteolytic enzyme has the molecular weight of about 38kDa with SDS-PAGE and gel filteration determining the time.
4. have a liking for the cryoproteins enzyme as claim 1 is desired, wherein said proteolytic enzyme has about 4.5 iso-electric point when measuring with isoelectric focusing.
5. isolating microorganism that belongs to flatfish Flavobacterium (Flavobacterium balustinum), this microorganism can produce the arbitrary desired proteolytic enzyme as claim 1 to 4.
6. as the desired isolating microorganism that belongs to the flatfish Flavobacterium of claim 5, wherein said microorganism preferably grows in 10 to 20 ℃ scope.
7. as the desired isolating microorganism that belongs to the flatfish Flavobacterium of claim 5, this microorganism is flatfish Flavobacterium P104 (FERM BP-5006).
8. one kind prepares the method for having a liking for the cryoproteins enzyme, and this method may further comprise the steps:
Cultivate as the desired flatfish Flavobacterium of claim 5 and
From culture, collect as claim 1 is desired and have a liking for the cryoproteins enzyme.
9. one kind prepares the method for having a liking for the cryoproteins enzyme, and this method may further comprise the steps:
Cultivate as the desired flatfish Flavobacterium of claim 6 and
From culture, collect as claim 1 is desired and have a liking for the cryoproteins enzyme.
10. one kind prepares the method for having a liking for the cryoproteins enzyme, and this method may further comprise the steps:
Cultivate as the desired flatfish Flavobacterium of claim 7 and
From culture, collect as claim 1 is desired and have a liking for the cryoproteins enzyme.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP29904/95 | 1995-02-17 | ||
| JP7029904A JPH08214878A (en) | 1995-02-17 | 1995-02-17 | Low temperature protease and low temperature bacteria |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1181108A true CN1181108A (en) | 1998-05-06 |
Family
ID=12288976
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96193145A Pending CN1181108A (en) | 1995-02-17 | 1996-02-16 | Psychrophilic protease and psychrophilic bacteria |
Country Status (10)
| Country | Link |
|---|---|
| JP (1) | JPH08214878A (en) |
| CN (1) | CN1181108A (en) |
| BR (1) | BR9607452A (en) |
| CA (1) | CA2211938A1 (en) |
| CZ (1) | CZ258097A3 (en) |
| HU (1) | HUP9802061A3 (en) |
| IL (1) | IL117164A0 (en) |
| MA (1) | MA23810A1 (en) |
| TR (1) | TR199700776T1 (en) |
| ZA (1) | ZA961237B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109749978A (en) * | 2019-03-07 | 2019-05-14 | 自然资源部第一海洋研究所 | Promote the method for bacterial growth using low-temperature protease |
| CN110208411A (en) * | 2019-06-10 | 2019-09-06 | 杭州必益泰得医学科技有限公司 | Carboxylesterase inhibitor preparation for drug metabolism detection |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2019080493A (en) * | 2017-10-27 | 2019-05-30 | プリマハム株式会社 | Protease, detergent composition, washing method, and microorganism |
-
1995
- 1995-02-17 JP JP7029904A patent/JPH08214878A/en not_active Withdrawn
-
1996
- 1996-02-16 TR TR97/00776T patent/TR199700776T1/en unknown
- 1996-02-16 CA CA002211938A patent/CA2211938A1/en not_active Abandoned
- 1996-02-16 MA MA24167A patent/MA23810A1/en unknown
- 1996-02-16 ZA ZA961237A patent/ZA961237B/en unknown
- 1996-02-16 CZ CZ972580A patent/CZ258097A3/en unknown
- 1996-02-16 HU HU9802061A patent/HUP9802061A3/en unknown
- 1996-02-16 CN CN96193145A patent/CN1181108A/en active Pending
- 1996-02-16 BR BR9607452A patent/BR9607452A/en not_active Application Discontinuation
- 1996-02-18 IL IL11716496A patent/IL117164A0/en unknown
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109749978A (en) * | 2019-03-07 | 2019-05-14 | 自然资源部第一海洋研究所 | Promote the method for bacterial growth using low-temperature protease |
| CN110208411A (en) * | 2019-06-10 | 2019-09-06 | 杭州必益泰得医学科技有限公司 | Carboxylesterase inhibitor preparation for drug metabolism detection |
| CN110208411B (en) * | 2019-06-10 | 2021-12-24 | 浙江龙传生物医药科技有限公司 | Carboxylesterase inhibitor formulations for drug metabolism detection |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2211938A1 (en) | 1996-08-22 |
| MA23810A1 (en) | 1996-10-01 |
| ZA961237B (en) | 1996-08-27 |
| HUP9802061A2 (en) | 1998-12-28 |
| HUP9802061A3 (en) | 1999-07-28 |
| TR199700776T1 (en) | 1998-02-21 |
| JPH08214878A (en) | 1996-08-27 |
| IL117164A0 (en) | 1996-06-18 |
| CZ258097A3 (en) | 1998-01-14 |
| BR9607452A (en) | 1998-06-30 |
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