CN102120974A - Low-temperature alkaline proteinase marine bacteria strain, low-temperature alkaline proteinase and production method thereof - Google Patents
Low-temperature alkaline proteinase marine bacteria strain, low-temperature alkaline proteinase and production method thereof Download PDFInfo
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Abstract
The invention discloses a marine Pseudoalteromonas sp. B8453013 (strain collection number CGMCC No.3281) of low-temperature proteinase, a production method for producing low-temperature proteinase from the strain and a low-temperature proteinase. A system method for strain collection, rejuvenation and seed selection is built by alternating Pseudoalteromonas sp. B8453013; the method for producing the low-temperature proteinase is built by culture medium optimization screening and the optimized control of fermentation technology conditions; and the produced low-temperature proteinase has the advantages of good low-temperature activity and high enzyme activity. The low-temperature proteinase can be widely applied to industries, such as detergents, feed, fur, food processing and the like.
Description
Technical field
The present invention relates to microorganism and microbial fermentation field, be specifically related to a kind of bacteriogenic, have the proteolytic enzyme of low best enzyme reaction temperature, a method that produces the microbial strains of this enzyme and produce this enzyme.
Background technology
Proteolytic enzyme is to use maximum a kind of enzymes at present, accounts for 60～65% of global enzyme industrial market share.It all is middle temperature enzyme that the present proteolytic enzyme that uses is gone up substantially.Compare with middle temperature proteolytic enzyme, low-temperature protease (＜10 ℃) at low temperatures can keep active, K
CatValue and physiology efficient (K
Cat/ K
m) height, higher features such as catalytic activity are arranged, so warm proteolytic enzyme has more using value in the low-temperature protease ratio.It is industrial that low-temperature protease is applied to food, washing composition, makeup, aquatic feeds etc., the superiority that warm proteolytic enzyme can't replace in having.The low-temperature protease that derives from microorganism is used to wash industry more existing report, for example Savinase 4.oT alkaline low-temperature proteases of Denmark NOVO company.Domestic, publication number provides the microorganism of a kind of 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 strains YS9412-130, by the novel alkaline low-temperature protease of this bacterial strain or the generation of its mutant; Publication number relates to a kind of pseudomonas C15-3CGMCC No.1021, a kind of alkaline low-temperature protease and preparation method thereof, the proteolytic enzyme that relates to a kind of bacterium and obtain from bacterium for the patent of CN1611596A provides a kind of cold induced proteins and production method thereof.Publication number obtains and bacterial strain marinobacter R2 through being separated into pure growth, the alkaline low-temperature protease that obtains from the R2 bacterial strain and preparation method thereof from deep-sea, South Pole mud sample for the patent of CN1670187A provides a kind of.But generally, both at home and abroad at present the otherness between the bacterial classification is bigger, has the enzyme property difference of the bacterium that produces, comprises the suitableeest action condition, thermostability, to the hydrolysis properties of substrate etc.Under existing research level, can be very rare for the enzyme of bacterial classification of studying and generation thereof.
Summary of the invention
One of purpose of the present invention is to provide a kind of ocean bacterial strain of producing alkaline low-temperature protease.
Two of purpose of the present invention is to provide the alkaline low-temperature protease that has higher the enzyme activity under a kind of temperature of supporting.
The method that the ocean bacterial strain that three of purpose of the present invention is to provide a kind of utilization can produce alkaline low-temperature protease is produced alkaline low-temperature protease.
The present invention can produce the ocean bacterial strain of alkaline low-temperature protease, it is pseudoalteromonas (Pseudoalteromonassp.) B8453013, on September 14th, 2009 was stored in China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center, and deposit number is CGMCC No.:3281.Cultivation, separation and the screening of this bacterial strain by the seawater sample that derives from Canadian sea basin being carried out microbial strains, obtain a collection of (fitting) the cold bacterial strain of having a liking for, and therefrom screening obtains a strain low-temperature protease superior strain, label is B8453013, thereby provide a kind of low-temperature protease to produce bacterial strain, it has the good characteristic that produces low-temperature protease, identifies through 16SrRNA, belongs to Pseudoalteromonas and belongs to (Pseudoalteromonas).The B8453013 cell is rod-short (0.4 μ m * 2～3 μ m), single arrangement, single-ended living flagellum.Gramstaining is negative.This bacterial strain is in 2216 media surface, bacterium colony circle, low projection, glossy, oyster white, opaque.On the casein culture dish, can produce the obvious transparent circle.The low-temperature protease optimal reactive temperature that this bacterial classification produces is 25 ℃, and optimal pH is 9-10.
Microbial strains of the present invention can produce alkaline low-temperature protease, and the nucleotide sequence of DNA of producing this proteolytic enzyme is as sequence＜400〉as described in 1, and with the homology of the 16S rDNA of B8453013 be 100% homology.This bacterial strain can be by the strain separating of preservation, or from ocean environment, cultivate, screening.In addition, this bacterial classification can be by the bacterial strain of nature and artificial induce variation, as long as the bacterial strain of the following property of protease of generation is all in this scope.
The microbial strains of generation low-temperature protease involved in the present invention, it belongs to Pseudoalteromonas and belongs to (Pseudoalteromonas) on significance for taxonomy, and bacterial classification of the present invention separates acquisition from Canadian sea basin seawater sample.
The low-temperature protease bacterial strain that the present invention relates to, its 16S rDNA sequence is as sequence＜400〉as described in 1.
The generation bacterial classification of low-temperature protease of the present invention can be the original strain of nature screening, or the dissociant that makes a variation by natural variation or artificial induction.
As the method for production of above-mentioned dissociant, comprise that physical mutagenesis is different, inject various rays such as processing, laser irradiation as treatment with uv radiation, co-60 radiation processing, particle and handle; Chemomorphosis is handled as chemomorphosises such as nitrosoguanidine, ethyl sulfates, filters out the good bacterial strain of production performance with conventional protease-producing bacterium separation screening substratum and method.
In addition, also can pass through Protocols in Molecular Biology, from original strain or induce variation bacterium, obtain the low-temperature protease gene, change gene over to recipient bacterium, make up genetically engineered and produce bacterial strain, also can produce as low-temperature protease of the present invention and produce bacterial classification.
Produce bacterial classification B8453013 as low-temperature protease of the present invention, can adopt following method to its preserve, rejuvenation and screening, and shake flask fermentation obtains alkaline low-temperature protease of the present invention.
Low-temperature protease of the present invention produces bacterial classification B8453013 can adopt the preservation of going down to posterity of conventional inclined-plane, this method is that the bacterial classification inoculation that will the present invention relates to is trained primary surface in the inclined-plane that is suitable for bacterial growth, cultivated 3-5 days for 10-15 ℃, place 4 ℃ of following preservations again, can deposit 3 months; Or utilizing vacuum freeze-drying method that bacterial classification of the present invention is made the dry powder bacterial classification, low temperature or preservation under room temperature can reach more than 1 year.
The bacterial strain activation of bacterial strain of the present invention comprises bacterial strain rejuvenation and bacterial strain screening, and is specific as follows:
The bacterial strain rejuvenation is in the rejuvenation media surface that is fit to its growth with alkaline low-temperature protease ocean inoculation, cultivated 3-5 days for 10-15 ℃, bacterial strain screening be with through the inoculation of rejuvenation to the screening and culturing primary surface, cultivated 3-5 days for 10-15 ℃, periphery of bacterial colonies produces the obvious transparent circle, and the picking transparent circle compares maximum bacterial strain as follow-up fermentation bacterial strain with colony diameter.Described rejuvenation substratum is preferably 2216 substratum, and described screening culture medium is preferably 2216 substratum that contain the 1wt% skim-milk; The composition of described 2216 substratum is by weight: peptone 0.5%, and yeast extract paste 0.1%, NaCl 11.738%, and KC1 0.332%, and KBr 0.048%, MgCl
26H
2O 5.305%, SrCl
26H
2O 0.020%, CaCl
22H
2O 0.7345%, Na
2SO
41.9585%, NaHCO
30.096%, H
3BO
30.013%, (NH
4)
2SO
40.5%, deionized water, pH9.0.
Alkaline low-temperature protease of the present invention, its zymetology is characterized as:
1) optimal reactive temperature is 25 ℃, and at 25 ℃ of-30 ℃ of scope inner enzyme vigor height, after temperature was higher than 60 ℃, enzyme did not possess vigor substantially, showed tangible low-temperature protease characteristic; Stable at 0 ℃ of-30 ℃ of inner enzyme vigor, 50 ℃, 60 ℃ and 70 ℃ handle 10min after, protease activities only be former active 70%, 42% and 7%, 25 ℃ and still keeps 100% activity after being incubated 30min, meets low-temperature protease to heat sensitive characteristic;
2) optimal pH is 9-10, belongs to Sumizyme MP;
3) this enzyme is suppressed by serpin PMSF, and inhibitors of metalloproteinase EDTA and cystatin E-64 do not have influence to protease activity, shows that this proteolytic enzyme belongs to serine protease;
4) metal ion Ni
2+, Zn
2+, Hg
2+, Cu
2+, Fe
3+, Ca
2+, and Mg
2+But the activity of part inhibitory enzyme, and reductive agent such as DTT and mercaptoethanol and tensio-active agents such as Triton X-100 and tween-80 do not make significant difference to enzymic activity.
The production method of alkaline low-temperature protease of the present invention is characterized in that may further comprise the steps:
A, bacterial strain activation;
B, fermentation; The bacterial strain direct inoculation is fermented in fermention medium, or bacterial strain is carried out the liquid multiplication culture earlier, be inoculated in the fermention medium and ferment; The condition of fermentation is: inoculum size 5-15%, leavening temperature are 5-30 ℃, and pH is 6-10, and rotating speed is 150-200 rev/min, and fermentation period is 72-120 hour;
C, collection alkaline low-temperature protease; Collecting alkaline low-temperature protease from fermentation gained nutrient solution gets product.
Described leavening temperature is preferably 10-15 ℃, and the pH in the described fermentation step is preferably 9-10.
As the carbon source of fermention medium, can be extensive use of the nutrition source that is generally used for microbial culture.For example, can use starch, sodium acetate, Semen Maydis powder, glucose, wort, sucrose, hydrolysis sugar, available polysaccharide etc.Its consumption is according to the difference of the selection difference of other nutrition source and culture condition and different.Carbon source in the fermention medium of the present invention is preferably Trisodium Citrate, and consumption is preferably the 0.5-1wt% of fermention medium total amount.
As the nitrogenous source of fermention medium, also can be extensive use of the nutrition source that is generally used for microbial culture, for example ammonium salt, nitrate, casein, corn steep liquor, yeast extract paste, dregs of beans, gelatin, peptone, urea, saltpetre etc.The selection of its nitrogenous source and consumption, can be according to the composition of other nutrition source and consumption different and different, produce the cultivation of bacterium and fermentation and produce and both can as long as be suitable for low-temperature protease of the present invention.Nitrogenous source in the fermention medium of the present invention is preferably casein, and consumption is preferably the 0.5-1wt% of fermention medium total amount.
As the product enzyme promotor of fermention medium, can add the tensio-active agent of tween series in right amount, addition is decided according to the difference of substratum, both can as long as be fit to promote culturing process to produce proteolytic enzyme of the present invention.Product enzyme promotor in the fermention medium of the present invention is preferably tween-80, and consumption is preferably the 0.05-0.1wt% of fermention medium total amount.
The composition of fermention medium of the present invention and weight composition are preferably: casein 0.5%, and Trisodium Citrate 0.5%, peptone 0.5%, yeast extract paste 0.1%, NaCl 11.738%, and KCl 0.332%, and KBr 0.048%, MgCl
26H
2O 5.305%, SrCl
26H2O 0.020%, CaCl
22H
2O 0.7345%, Na
2SO
41.9585%, NaHCO
30.096%, H
3BO
30.013%, (NH
4)
2SO
40.5%, Tween-80 0.05%, deionized water, pH9.0.
Through above-mentioned cultivation, mainly (in the nutrient solution) produces the target protein enzyme outside thalline.
From the nutrient solution of as above gained, collect low-temperature protease, can as ammonium sulfate precipitation, ultrafiltration and concentration, lyophilize, chromatographic separation etc., be undertaken according to the method that is generally used for gathering perienzyme by separation and purification.
That is, can obtain low-temperature protease of the present invention: from nutrient solution, obtain supernatant liquor or filtrate by filtration method or centrifugal separation, these parting liquids are carried out or do not concentrate, add the solubility salt, make the sedimentary salting-out process of enzyme by following method; Add hydrophilic organic solvent, make the sedimentary organic solvent precipitation method of enzyme or inclusion; Make the absorb-elute method of spent ion exchange resin etc.; Gel filtration method; Add and stablize auxiliary or do not add the spray-drying process of stablizing auxiliary; Independent or a plurality of separation or process for purification such as freeze-drying of being used in combination.
By implementing the concrete technical scheme of the present invention, realize content of the present invention, can reach following beneficial effect.
False unit cell (Pseudoalteromonas sp.) bacterial strain B8453013 and bacterial strain or its variant equal with it thereof of replacing of the present invention can be effectively applied to produce low-temperature protease of the present invention.Secondly, this low-temperature protease (25 ℃-30 ℃) at low temperatures has the high enzyme activity, and has higher low-temperature stability (still keeping 100% activity behind 25 ℃ of insulation 30min); In addition, the invention has the advantages that the method for using described bacterial strain manufacturing to have the low-temperature protease of above-mentioned character can produce described low-temperature protease effectively, its enzyme work can reach 80-110U/mL, and, be convenient to collect refining because proteolytic enzyme that this method is produced is stored in the nutrient solution.
The preservation explanation
1, pseudoalteromonas (Pseudoalteromonas sp.) B8453013, on September 14th, 2009 was stored in China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center, and deposit number is CGMCC No.:3281.
Description of drawings
Fig. 1 is the low-temperature protease optimal reaction pH value chart that replaces false unit cell (Pseudoalteromonas sp.) bacterial strain B8453013 generation of the present invention.
Fig. 2 is the low-temperature protease optimal reactive temperature chart that replaces false unit cell (Pseudoalteromonas sp.) bacterial strain B8453013 generation of the present invention.
Fig. 3 is the cold induced proteins enzyme heat stability chart that replaces false unit cell (Pseudoalteromonas sp.) bacterial strain B8453013 generation of the present invention.
Embodiment
Below in conjunction with the drawings, the invention will be further described for sequence table and embodiment.
Embodiment 1
Low-temperature protease produces the activation culture of bacterium (B8453013)
Low-temperature protease is produced bacterium B8453013 be inoculated in 2216 media surface, cultivated 3 days, and carried out rejuvenation for 15 ℃.It is inoculated in 2216 substratum that contain the 0.1wt% skim-milk and screens, cultivate for 15 ℃ and can produce the obvious transparent circle in periphery of bacterial colonies in 3 days, the picking transparent circle compares maximum bacterial strain as follow-up fermentation bacterial strain with colony diameter.The composition of 2216 substratum is by weight: peptone 0.5%, and yeast extract paste 0.1%, NaCl 11.738%, and KCl 0.332%, and KBr 0.048%, MgCl
26H
2O 5.305%, SrCl
26H2O 0.020%, CaCl
22H
2O 0.7345%, Na
2SO
41.9585%, NaHCO
30.096%, H
3BO
30.013%, (NH
4)
2SO
40.5%, deionized water, pH9.0,0.1Mpa steam sterilizing 15 minutes.
Embodiment 2: low-temperature protease produces fermentation culture-I of bacterium (B8453013)
Present embodiment is that the fermentation of screening gained is cultivated in fermention medium with the bacterial strain direct inoculation.Be specially:
To ferment is inoculated in the fermention medium with the bacterial strain inclined-plane, and every 250mL shakes bottled fermention medium 100mL, and culture condition is: 15 ℃, and 150 rev/mins, fermentation culture 72 hours, enzyme activity reaches 97U/mL.The composition of fermention medium is by weight: casein 0.5%, and Trisodium Citrate 0.5%, peptone 0.5%, yeast extract paste 0.1%, NaCl11.738%, KCl 0.332%, and KBr 0.048%, MgCl
26H
2O 5.305%, SrCl
26H2O 0.020%, CaCl
22H
2O 0.7345%, Na
2SO
41.9585%, NaHCO
30.096%, H
3BO
30.013%, (NH
4)
2SO
40.5%, Tween-800.05%, deionized water, pH9.0,0.1Mpa steam sterilizing 15 minutes.
In the liquid of protease (being fermentation culture) of gained, add sulfate of ammoniac to 70% saturation ratio, the centrifuging and taking precipitation, after the method desalination with routine, cooling drying gets the former powder of low-temperature protease again.
Embodiment 3: low-temperature protease produces fermentation culture-II of bacterium (B8453013)
Present embodiment is that the fermentation of screening gained is carried out the liquid multiplication culture earlier with bacterial strain, is inoculated in the fermention medium and ferments.Be specially:
Low-temperature protease is produced strain inclined plane be inoculated in 2216 substratum and breed, 15 ℃, 150rpm cultivated after 24 hours, culture is joined in the fermention medium with 15% inoculum size ferment.Identical among 2216 medium components and the embodiment 1.Fermentation culture based component and sterilising method are with fermentation culture based component and sterilising method among the embodiment 2.Fermentation culture is that every 250mL shakes bottled fermention medium 50mL, and culture condition is: 15 ℃, and 200 rev/mins, fermentation culture 72 hours, enzyme activity reaches 110U/mL.
In the liquid of protease (being fermentation culture) of gained, add sulfate of ammoniac to 70% saturation ratio, the centrifuging and taking precipitation, after the method desalination with routine, cooling drying gets the former powder of low-temperature protease again.
Embodiment 4: the measuring method of enzymic activity
Get 1mL dilution enzyme liquid (50mmol/L TrisHCl (pH7.5) dilutes), be incubated 1min down at 40 ℃, the substrate (2% casein of using the preparation of 50mmol/L TrisHCl (pH8.0) damping fluid is as substrate) that adds the same temperature of 1mL, behind the reaction 10min, add 2mol/L 10% trichoroacetic acid(TCA) termination reaction under 40 ℃.Behind 40 ℃ of insulation 15min, the centrifugal 15min of 12000rpm gets the 1mL supernatant liquor and adds 5mL 0.4mol/L yellow soda ash and 1mL Folin reagent mixing, behind 40 ℃ of insulation 20min, with the spectrophotometric determination 660nm OD of place value.Enzyme activity is defined as under 40 ℃, and the enzyme amount that every mL enzyme liquid catalysis casein hydrolysis forms 1 μ g tyrosine is 1 unit (U/mL).
Embodiment 5: the low-temperature protease optimal pH
The dilution enzyme liquid of above-mentioned preparation is joined sodium-acetate (pH5) respectively, citric acid phosphoric acid salt (pH5～7), phosphoric acid (pH6～8), TrisCl (pH8, pH9), glycine sodium hydroxide (pH9, pH10) and carbonate (pH10, pH11) in 6 solution, 4 ℃ preserve 2h after, press the vigor of the protease activity determination method mensuration enzyme among the embodiment 4.The proteolytic enzyme enzyme optimum pH of the present invention preparation as shown in Figure 1, strains A rcB82306 excretory proteolytic enzyme of the present invention all has activity at pH5-11, and shows higher vigor about pH9, belongs to Sumizyme MP.
Embodiment 6: the low-temperature protease optimum temperuture
0 ℃, 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 40 ℃, 45 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃ 15 thermogrades are set, press the proteinase activity under the protease activity determination method mensuration differing temps among the embodiment 4, to determine its optimum temperuture by above-mentioned steps preparation of the present invention.Proteolytic enzyme enzyme optimum temperuture as shown in Figure 2, the proteolytic enzyme optimal reactive temperature of the present invention preparation is 25 ℃, at 25～30 ℃ of scope inner enzyme vigor height, after temperature was higher than 60 ℃, enzyme did not possess vigor substantially.
Embodiment 7: the thermostability of low-temperature protease
With the dilution enzyme liquid of above-mentioned preparation water bath heat preservation 10min, 20min, 30min, 40min and 50min successively under 15 ℃, 25 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃ of 6 temperature condition respectively, put into mixture of ice and water then, compare with the enzyme activity of not insulation processing, the protease activity determination method of pressing among the embodiment 4 is measured proteolytic enzyme residue vigor.The thermostability of low-temperature protease as shown in Figure 3, the proteolytic enzyme of the present invention preparation is stable at 0～30 ℃ of inner enzyme vigor, 50 ℃, 60 ℃ and 70 ℃ handle 10min after, protease activities only is former active 70%, 42% and 7%.
Embodiment 8: the influence that various inhibitor are lived to enzyme
Under the optimal reaction temperature and optimum response pH value condition of enzyme, in the enzyme reaction system, add different inhibitor, the protease activity determination method among the embodiment 4 of pressing is measured enzyme and is lived, and is 100% with the enzyme activity of inhibiting not, calculates the remaining vigor that adds behind the Chemical Composition.The influence that various inhibitor are lived to the enzyme of the proteolytic enzyme of the present invention's preparation the results are shown in Table 1.As can be seen from the table, serpin (phenylmethylsulfonyl fluoride PMSF) effect is the strongest; The inhibitors of metalloproteinase edta edta, and cystatin E-64 does not have influence to the protease activity that the present invention prepares.Each metal ion species also there are differences the influence of protease activity, wherein with Cu
2+Restraining effect the strongest, secondly be Fe
3+, and Mg
2+And Zn
2+Restraining effect the most weak.Reductive agent [as dithiothreitol (DTT) DTT and beta-mercaptoethanol (β-mercaptoethanol)] and tensio-active agent [as triton x-100 (Triton X-100) and tween-80 (Tween-80)] to enzyme while still alive property do not make significant difference, and denaturing agent (as sodium lauryl sulphate SDS) is stronger to the restraining effect of proteolytic enzyme.
The influence that the various inhibitor of table 1 are lived to enzyme
Claims (10)
1. alkaline low-temperature protease ocean bacterial strain, it is pseudoalteromonas (Pseudoalteromonas sp.) B8453013, on September 14th, 2009 was stored in China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center, and deposit number is CGMCC No.:3281.
2. alkaline low-temperature protease, the nucleotide sequence of DNA of producing this proteolytic enzyme is as sequence＜400〉as described in 1.
3. alkaline low-temperature protease according to claim 2, its zymetology is characterized as:
1) optimal reactive temperature is 25 ℃, and at 25 ℃ of-30 ℃ of scope inner enzyme vigor height, after temperature was higher than 60 ℃, enzyme did not possess vigor substantially, showed tangible low-temperature protease characteristic; Stable at 0 ℃ of-30 ℃ of inner enzyme vigor, 50 ℃, 60 ℃ and 70 ℃ handle 10min after, protease activities only be former active 70%, 42% and 7%, 25 ℃ and still keeps 100% activity after being incubated 30min, meets low-temperature protease to heat sensitive characteristic;
2) optimal pH is 9-10, belongs to Sumizyme MP;
3) this enzyme is suppressed by serpin PMSF, and inhibitors of metalloproteinase EDTA and cystatin E-64 do not have influence to protease activity, shows that this proteolytic enzyme belongs to serine protease;
4) metal ion Ni
2+, Zn
2+, Hg
2+, Cu
2+, Fe
3+, Ca
2+, and Mg
2+But the activity of part inhibitory enzyme, and reductive agent such as DTT and mercaptoethanol and tensio-active agents such as Triton X-100 and tween-80 do not make significant difference to enzymic activity.
4. production method as claim 2 or 3 described alkaline low-temperature proteases is characterized in that may further comprise the steps:
A, bacterial strain activation; The bacterial strain activation comprises bacterial strain rejuvenation and bacterial strain screening, the bacterial strain rejuvenation is in the rejuvenation media surface that is fit to its growth with alkaline low-temperature protease ocean inoculation, cultivated 3-5 days for 10-15 ℃, bacterial strain screening be with through the inoculation of rejuvenation to the screening and culturing primary surface, cultivated 3-5 days for 10-15 ℃, periphery of bacterial colonies produces the obvious transparent circle, and the picking transparent circle compares maximum bacterial strain as follow-up fermentation bacterial strain with colony diameter;
B, fermentation; The bacterial strain direct inoculation is fermented in fermention medium, or bacterial strain is carried out the liquid multiplication culture earlier, be inoculated in the fermention medium and ferment; The condition of fermentation is: inoculum size 5-15%, leavening temperature are 5-30 ℃, and pH is 6-10, and rotating speed is 150-200 rev/min, and fermentation period is 72-120 hour;
C, collection alkaline low-temperature protease; Collecting alkaline low-temperature protease from fermentation gained nutrient solution gets product.
5. production method according to claim 4 is characterized in that described leavening temperature is 10-15 ℃, and the pH in the described fermentation step is 9-10.
6. according to claim 4 or 5 described production methods, it is characterized in that described rejuvenation substratum is 2216 substratum, described screening culture medium is 2216 substratum that contain the 1wt% skim-milk; The composition of described 2216 substratum is by weight: peptone 0.5%, and yeast extract paste 0.1%, NaCl 11.738%, and KCl 0.332%, and KBr 0.048%, MgCl
26H
2O 5.305%, SrCl
26H
2O 0.020%, CaCl
22H
2O 0.7345%, Na
2SO
41.9585%, NaHCO
30.096%, H
3BO
30.013%, (NH
4)
2SO
40.5%, deionized water, pH9.0.
7. according to claim 4 or 5 described production methods, it is characterized in that the carbon source in the described fermention medium is a Trisodium Citrate, consumption is the 0.5-1wt% of fermention medium total amount.
8. according to claim 4 or 5 described production methods, it is characterized in that the nitrogenous source in the described fermention medium is a casein, consumption is the 0.5-1wt% of fermention medium total amount.
9. according to claim 4 or 5 described production methods, it is characterized in that the product enzyme promotor in the described fermention medium is tween-80, consumption is the 0.05-0.1wt% of fermention medium total amount.
10. according to claim 4 or 5 described production methods, it is characterized in that the weight in the described fermention medium consists of: casein 0.5%, Trisodium Citrate 0.5%, peptone 0.5%, yeast extract paste 0.1%, NaCl 11.738%, KCl0.332%, KBr 0.048%, MgCl
26H
2O 5.305%, SrCl
26H2O 0.020%, CaCl
22H
2O 0.7345%, Na
2SO
41.9585%, NaHCO
30.096%, H
3BO
30.013%, (NH
4)
2SO
40.5%, Tween-80 0.05%, deionized water, pH9.0.
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| CN104004737A (en) * | 2014-05-23 | 2014-08-27 | 北京市农林科学院 | Low-temperature proteinase derived from Collimonas pratensis, and correlated biological material and application thereof |
| CN104894024A (en) * | 2015-06-11 | 2015-09-09 | 中南大学 | Pseudoalteromonas mutant strain and application thereof |
| 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 |
| CN110684692A (en) * | 2019-10-24 | 2020-01-14 | 湖北大学 | Stenotrophomonas maltophilia780 and application thereof |
| CN112608915A (en) * | 2020-12-21 | 2021-04-06 | 自然资源部第一海洋研究所 | Low-temperature alkaline protease NJXD01, gene and application |
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| CN101037661A (en) * | 2007-02-12 | 2007-09-19 | 浙江大学 | Pseudoalteromonas and its usage |
| CN101070526A (en) * | 2007-02-12 | 2007-11-14 | 淮海工学院 | Method for producing low-temperature alkaline protease by alternative pseudomonad |
| CN101531974A (en) * | 2009-03-21 | 2009-09-16 | 国家海洋局第一海洋研究所 | Arctic bacteria strain for highly efficiently degrading crude oil and application thereof |
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Cited By (7)
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| CN104004737A (en) * | 2014-05-23 | 2014-08-27 | 北京市农林科学院 | Low-temperature proteinase derived from Collimonas pratensis, and correlated biological material and application thereof |
| CN104894024A (en) * | 2015-06-11 | 2015-09-09 | 中南大学 | Pseudoalteromonas mutant strain and application thereof |
| CN104894024B (en) * | 2015-06-11 | 2018-01-12 | 中南大学 | One plant of Pseudoalteromonas mutant strain and its application |
| 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 |
| CN110684692A (en) * | 2019-10-24 | 2020-01-14 | 湖北大学 | Stenotrophomonas maltophilia780 and application thereof |
| CN110684692B (en) * | 2019-10-24 | 2021-08-17 | 湖北大学 | Stenotrophomonas maltophilia780 and application thereof |
| CN112608915A (en) * | 2020-12-21 | 2021-04-06 | 自然资源部第一海洋研究所 | Low-temperature alkaline protease NJXD01, gene and application |
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| CN102120974B (en) | 2013-09-11 |
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