CN101070526A - Method for producing low-temperature alkaline protease by alternative pseudomonad - Google Patents

Abstract

A alternately false single afterbirth bacterium HH407( Pseudoalteromonas flavipulchra HH407) CCTCC N0: M207009. This bacterium can grow up under 0degree C, the growth temperature of this bacterium scope is 0-35 degree C, the most suitable growth temperature is 20degree C; Growth pH scope is 5.5-11, most suitable growth pH is 8.5; The NaCl density of growth is 0.5%-13%, the optimal growth of NaCl concentration of 4%, no growth without NaCl, it is a typically moderate marine Psychrophilic Halophilic microorganisms; bacteria can use pancreatic peptone, yeast extract, peptone, cellobiose, starch, gelatin, acetic acid, citric acid, methanol and ethanol. The present invention also opens an alternate use of low-temperature alkaline-producing Pseudomonas methods and by the method of low-temperature alkaline protease products. The low-temperature alkaline protease can be used as the main enzyme detergent additives can be directly room temperature washing, saves energy; and in food manufacturing, leather, silk, environmental protection, medicine and other fields also has broad application prospects.

Description

A kind of pseudoalteromonas produces the method and the product of low-temperature alkaline protease

Technical field

The present invention relates to a kind of separation from the pseudoalteromonas HH407 in marine site, Lianyun Harbour bacterial strain CCTCCNO:M 2070009 (be deposited in Chinese typical culture collection center C CTCC on January 29th, 2007, deposit number is CCTCC NO:M 207009); The invention still further relates to the method and the product of this bacterial strain product low-temperature alkaline protease.

Background technology

Cold-adapted enzyme has at low temperatures than the higher catalytic efficiency of middle temperature enzyme, can play the effect that cuts down the consumption of energy in the industrial processes, therefore in washing composition, textile industry, foodstuffs industry, brewage and aspects such as liquor industry, feed processing, bio-transformation and environmental organism improvement are with a wide range of applications.The low temperature enzyme that screening has using value from marine microorganism has become the important channel that Living marine resources are developed.

Sumizyme MP is the main additive of enzyme-containing detergent, and what mainly add in the washing powder at present is middle temperature Sumizyme MP.The generally the suitableeest enzyme of this fermentoid is lived usually more than 50 ℃, so will reach best washing effect, will add hot water.And the suitableeest enzyme of cold-adapted enzyme is lived generally below 40 ℃, and directly room temperature washing is saved the energy, and demonstrates application prospects in food mfg, process hides, silk, environmental protection, medicine and other fields.

More to the research report of low-temperature alkaline protease both at home and abroad, its bacterium producing multi enzyme preparation is mainly Rhodopseudomonas (Pseudomonas), Flavobacterium (Flavobacterium), Aeromonas (Aeromonas), Shiva Bordetella (Shewanella) and Vibrio (Vibrio).All there is not Pseudoalteromonas.flavipulchra to produce the bibliographical information of low-temperature alkaline protease at present both at home and abroad.

Summary of the invention

Technical problem to be solved by this invention is at the deficiencies in the prior art, belong to by a kind of new ocean psychrophilic bacteria pseudoalteromonas of seed selection, the flavipulchra bacterium produces low-temperature alkaline protease, other technical problem to be solved by this invention is the growth characteristics of this bacterial strain of research, and produces the method for low-temperature alkaline protease and the product of enzyme.

Technical problem to be solved by this invention is to realize by following technical scheme.

Feature of the present invention comprises pseudoalteromonas HH407 (Pseudoalteromonas flavipulchraHH407) bacterial strain itself, and the method for utilizing this bacterial strain to produce low-temperature alkaline protease, and the low-temperature alkaline protease product that utilizes this bacterial strain to produce.

Bacterial strain involved in the present invention is that a strain separates from the marine bacteria psychrophilic bacteria pseudoalteromonas HH407 in marine site, Lianyun Harbour (Pseudoalteromonas, flavipulchra HH407), and on January 29th, 2007 in China typical culture collection center C CTCC preservation, its deposit number is CCTCC NO:M 207009.

One, the feature of bacterial strain of the present invention and growth characteristics

1, morphologic observation: respectively bacterial strain is carried out opticmicroscope and Electronic Speculum (Agricultural University Of Nanjing) observation.

2, the growth characteristics of bacterial strain

2.1 the preparation of seed liquor: the inclined-plane inoculation that will be deposited in the 2216E substratum in fermention medium, rotating speed 180r/min, liquid amount 20% is cultivated 16h.

2.2 the influence of temperature to growing: seed liquor 2% is inoculated in fermention medium, the pH nature, and rotating speed 180r/min, liquid amount 20% is cultivated under differing temps respectively, measures cell concn.

2.3 the influence of pH: in fermention medium, add following different damping fluid (10mM) to growing, regulating the pH:pH 5.0-6.0 (MES) of fermention medium respectively, pH 7.0 (PIPES), pH 7.5-8.5 (HEPES), pH 9.0-11.0 (not with damping fluid), cultivated 24 hours at 20 ℃, other conditions are measured cell concn with 2.2.

2.4 the influence of NaCl to growing: (will use the trace quantity mineral salts solution instead with the seawater configuration replaces at fermention medium, every liter of substratum adds 10ml) in add NaCl, making it is 0% to 13% NaCl, cultivates 24 hours at 20 ℃, other conditions are measured cell concn with 2.2.

2.5 the utilization to carbon nitrogen source: carbon source experiment: the different final concentration of adding is 0.5% carbon source material (carbohydrate, organic acid, alcohols and amino acids) replacement glucose in minimum medium; The nitrogenous source experiment: the different final concentration of adding is 0.2% nitrogen source (yeast extract paste etc.) replacement peptone in minimum medium.Contrast is minimum medium, and 20 ℃, 180r/min shaking culture are measured cell concn every 12h, till measuring cell concn decline.

2.6 the mensuration of cell concn: adopt the petroff-Hausser bateria chamber to measure at the Olympus phase microscope.

3, about the substratum among the present invention.

2216E substratum (g/L): peptone 5, yeast powder 1, seawater configuration, pH8.5;

Casein substratum (g/L): casein food grade 10, seawater configuration, pH10.0;

Minimum medium (g/L): peptone 0.5, glucose 0.5, seawater configuration, pH8.5;

Fermention medium (g/L): peptone 5, yeast powder 3, glucose 5, seawater configuration, pH8.5

Trace quantity mineral salt (every liter): CuSO ₄5H ₂O, 0.01g; ZnSO ₄7H ₂O, 0.1g; CoCl ₂6H ₂O, 0.005g; MnCl ₂4H ₂O, 0.2g; Na ₂MoO ₄2H ₂O, 0.1g; KBr, 0.05g; KI, 0.05g; H ₃B0 ₃, 0.1g; NaF, 0.05g; LiCl, 0.05g; Al ₂(SO4) ₃, 0.05g; NiCl ₂6H ₂O, 0.01g; VoSO ₄2H ₂O, 0.005g; H ₂WO ₄2H ₂O, 0.002g; Na ₂SeO ₄, 0.005g; SrCl6H ₂O, 0.005g; BaCl ₂, 0.005g.

4, the morphological specificity of bacterial strain and growth characteristics

4.1 the morphological specificity of bacterial strain: bacterial strain is aerobic gram negative bacillus, no gemma, and no pod membrane, end is given birth to single flagellum, and size is 0.4-0.7 μ m * 1.0-1.8 μ m (Fig. 1).Bacterial strain casein dull and stereotyped 20 ℃ cultivate 4d after, colony diameter 3mm-4mm, oval golden yellow, moistening, the edge is smooth, intermediate recess, swell all around, translucent, easy picking, can produce the obvious transparent circle.Fig. 2 is the Electronic Speculum figure of bacterial strain.

4.2 temperature, pH and the NaCl influence to growing: this bacterium can be 0 ℃ of slowly growth down, and 15 ℃～20 ℃ quick down growths, the growth temperature range of this bacterium is 0-35 ℃, and optimum growth temperature is 20 ℃; Growth pH scope is 5.5-11, and the suitableeest growth pH is 8.5; The NaCl concentration of growth is 0.5%-13%, and the NaCl concentration of suitable growth is 4%, and no NaCl does not grow.Belong to typical ocean and have a liking for cold moderate halophilic microorganism (seeing Fig. 3, Fig. 4 and Fig. 5).

4.3 bacterial strain is to the utilization of carbon nitrogen source.

HH407 the results are shown in Table 1 to the utilization of carbon nitrogen source, HH407 can well utilize complex proteins such as Tryptones, yeast extract paste, peptone, next is cellobiose, starch, gelatin, can utilize organic acid (acetate, citric acid) and alcohols (methyl alcohol, ethanol), but relatively poor to sugar and the amino acid whose ability of utilization.

The various carbon nitrogen source materials of table 1 are to the growth effect of bacterium HH407

Substrate	Growth(10 7)	Substrate	Growth(10 7)
				Control yeast extract gelatin sucrose starch cellobiose glycogen galactose arabinose rhamnose cellulose citrate methanol glycerol sorbitol L-Glycine L-Alanine L-Arginine	1.20 120 53.5 15.0 72.0 75.0 11.25 3.20 6.75 1.20 5.20 11.20 24.0 1.40 1.35 8.50 7.80 5.20	tryptone peptone casein glucose maltose glycogen melibiose lactose mannose Mannitol xylose acetic acid lactic acid ethanol inositol sodium pyruvate L-Lysine L-tryptophan	140 99.5 5.00 12.8 1.20 10.0 1.30 1.20 4.15 1.10 1.30 22.0 9.50 6.80 1.25 6.75 1.10 1.15

Two, the evaluation of bacterial strain

2.1 bacterial strain HH407 biochemical reactions

The biochemical reactions of bacterial strain HH407 the results are shown in Table 2: oxydase, catalase are all positive, unfermentable glucose and utilize Poly-salt, methyl red, VP, ornithine decarboxylase, lysine decarboxylase, phenylalanine decarboxylase are negative, edwardsiella hoshinae.HH407 can utilize sucrose, semi-lactosi, Citrate trianion, tyrosine, pyruvic acid, and can not utilize maltose, melibiose.Pass through Bergey ' s Manualof Systematic Bacteriology (second edition) and analyze relatively, this bacterial strain of preliminary judgement is that Pseudoalteromonas belongs to.The comparative result that other bacterium colony produces between the xanthein kind in belonging to sees Table 3, and the result shows the feature basically identical of HH407 with P.flavipulchra.

Table 2 physio-biochemical characteristics

Characteristic	Result	Characteristic	Result
				Oxidase Catalase VP Indole Methyl red Gram staining	+ + - - - -	accumulate poly-β-hydroxybutyrate require Na + forgrowth Lysine decarboxylase Tryptophan deaminase Ornithine decarboxylase fermentation of D-glucose	- + - - - -

+：positive；-：negative.

Table 3 bacterial strain compares with the characteristic that pseudoalteromonas belongs to other kind of bacterium colony product xanthein

Characteristic	HH407	P.flavipulchra	P.piscicida	p.citrea	p.aurantia	p.peptidolytica
							Requirement for organic growth factors Pigmentation Growth at： 4℃ 37℃ 12％NaCl 15％NaCl Production of： Chitinase Caseinase Alginase Utilization of： Maltose D-Galactose Sucrose Melibiose Citrate Pyruvate L-Arginine L-Tyrosine	- + + + + - + + ND - + + - + + - +	- + - + + - + + ND + + + - + + - +	ND + - + - - - ND ND - + - - + + ND ND	+ + - - - - - + ND - - - - - - - +	+ + + - - - - - - - - - - - ND + -	- + - + - - + - + + - - ND - ND ND ND

+：positive；-：negative；ND：Not determined.

2.2 16S rRNA pcr amplification and sequential analysis: adopt the Takara test kit to propose the genomic dna of high purity bacterium.Forward primer P1:5 '-GAGAGTTTGATCCTGGCTCAG-3 ', reverse primer P2:5 '-CGGCTACCTTGTTACGAC-3 '.Reaction system 50 μ l, Taq enzyme 2U.Reaction conditions is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 1min, and 50 ℃ of annealing 30s, 72 ℃ are extended 60s, circulate 35 times, and 72 ℃ are extended 7min.The purifying of PCR product, clone, order-checking are finished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, and the row that check order submit to GenBank to carry out homology relatively.

2.3 the phylogenetic tree of bacterial strain HH407 makes up: choose the correlated series that homology is 95-99%, adopt the adjacent method of MEGA3.1 software to construct evolutionary tree, and carry out degree of confidence with the bootstrapping analytical method and detect, bootstrapping data set 1000 times.

2.3 the 16S rDNA sequence (number of registration of GenBank is EF136442) of HH407 bacterial strain is carried out homology relatively with the sequence in the ncbi database.Blast analyzes and shows that this Pseudomonas belongs in pseudoalteromonas, homology is more than 95%, evolutionary tree analysis and Pseudoalteromonas.ftavipulchra have 99.9% homology and are in the same branch, preliminary judgement and Pseudoalteromonas flavipulchra are of the same race, this bacterial strain called after Pseudoalteromonas flavipulchra HH407.Pseudoalteromonas belongs to (1995) as the marine bacteria of a new classification, and domestic research is just at the early-stage, and Pseudoalteromonas flavipulchra does not all have the related experiment report both at home and abroad as the kind of the new classification of report in 2002

Three, bacterial strain produces the method for low-temperature alkaline protease

Connect bacterial strain HH407 inclined-plane 1 ring bacterium and put into the 250ml triangular flask of 50ml fermention medium, 180r/min, cultivates 16h by 20 ℃.Insert in the fermention medium by 2% inoculum size, 180r/min, cultivates 36h by 20 ℃.With cultured fermented liquid 10000r/min, centrifugal 10min, supernatant liquor adds 60% ammonium sulfate, leave standstill 3h after, the centrifugal 30min of 10000r/min, dissolve with Tris damping fluid (pH8.0), then 4 ℃ the dialysis 24h, in the middle of change dialyzate (Tris pH8.0,20mmol) 4 times,-40 ℃ of cold storage preservations are standby, obtain low-temperature alkaline protease through DEAE-sepharose Flat Flow and Sephacryl S-200 method again.

Four, the character of low-temperature alkaline protease

4.1 the enzyme operative temperature is to the influence of enzymic activity: select to measure in 0～70 ℃ of scope the enzyme activity under the differing temps, the results are shown in Figure 6, the suitableeest enzyme operative temperature is 35 ℃, and enzyme still keeps certain enzyme activity when low temperature, enzyme is lived and is descended slowly, still has 22% catalytic efficiency at 0 ℃.

4.2 the thermostability of enzyme: with enzyme liquid water bath heat preservation different time under 25 ℃～65 ℃ temperature condition respectively, compare with the enzyme activity of not insulation processing, measure the residual vigor of proteolytic enzyme, the results are shown in Figure 7, enzyme is relatively poor in thermostability more than 45 ℃, the enzyme of 55 ℃ of insulation 30min loss 55% is lived, 65 ℃ of insulation 10min enzymes total losses alive.Enzyme is highly stable 25 ℃, 35 ℃, 45 ℃, insulation 60min, and enzyme is lived all more than 80%.

4.3 the enzyme action pH is to the influence of enzymic activity: enzyme liquid is detected enzyme activity under the condition of each pH value, damping fluid (concentration is 50mM) is Sodium phosphate dibasic-citric acid (pH5.0～8.0), Tris-HCl (pH7.5～8.5), Glycine (pH8.5～10.5), and NaOH-Sodium phosphate dibasic (pH11.0～12.0), mensuration vigor behind 4 ℃ of preservation 1h～2h.Result such as Fig. 8, the suitableeest action pH of enzyme is 10.0, and is higher at the pH9.0-11 enzyme activity, and enzyme activity is more than 80% relatively, and the action pH scope is very wide, and pH5-12 all has enzyme to live.

4.4 enzyme pH stability: damping fluid is measured enzyme activity with 4.3,30 ℃ of insulations behind the 30min, is contrast with uninsulated enzyme liquid, result such as Fig. 9, enzyme stability is best when pH9.0.

4.5 metal ion is to the influence of enzymic activity: enzyme liquid is mixed with metal ion, and making the concentration of metal ions in the reaction solution is 5mM, measures the enzyme work down of various ionic systems behind 35 ℃ of insulation 30min, is contrast with what do not add metal ion, calculates relative vigor.The results are shown in Table 4, Sr ²⁺, Ba ²⁺, Cu ²⁺, Mn ²⁺, K ⁺Enzyme there is activation, wherein with Mn ²⁺, Cu ²⁺The most obvious; Mg ²⁺, Na, Ca ²⁺, Ag ⁺, Pb ²⁺Act on not remarkable; Ni ²⁺, Co ²⁺, Fe ³⁺, Li ²⁺Restraining effect is arranged, Hg ²⁺, Al ³⁺, Zn ²⁺The strongly inhibited enzyme activity.

Table 4 metal ion influences proteinase activity

Metal ions(5mM)	Relative activity(％)	Metal ions(5mM)	Relative activity (％)
				None Cu 2+ Ba 2+ Pb 2+ Ca 2+ Na + Co 2+ Fe 3+ Al 3+	100.0 145.3 113.0 102.5 99.6 99.2 73.9 33.6 1.34	Mn 2+ Sr 2+ K + Ag + Mg 2+ Li 2+ Ni 2+ Zn 2+ Hg 2+	182.3 115.2 105.4 101.3 99.6 80.37 59.4 26.8 0.5

4.6 inhibitor, organic solution, denaturing agent are to the influence of enzymic activity: with enzyme liquid and various inhibitor mixed, measure enzyme behind 35 ℃ of insulation 30min and live, calculate relative vigor.The results are shown in Table 5.The work of PMSF strongly inhibited enzyme, EDTA still has 106.6% enzyme work, shows that it is a kind of serine protease.H ₂O ₂(1%), β-Mercaptoethanol (2mM), Urea (2M) and DTT (10mM) can improve enzyme (improving 4.8%, 8.2%, 20.1%, 83.3% respectively) alive, SDS there is very strong tolerance, 0.1%SDS lives to enzyme does not have influence, and work has promoter action a little to tensio-active agent (Tween20, Tween80, Triton X-100) to enzyme.

Table 5 inhibitor, organic solution, denaturing agent influence proteinase activity

Additives	Concentration	Relative activity(％)
			None EDTA PMSF DTT Iodoacetamide β-Mercaptoethanol	- 5mM 10mM 10mM 10mM 2mM	100.0 106.6 5.1 183.3 110.9 108.2

SDS Urea H 2O 2 Triton X-100 Tween-20 Tween-80

0.1％ 2M 1％ 1％ 1％ 1％

101.3 120.1 104.8 107.8 107.5 101.9

5, proteolytic enzyme enzyme activity determination.

5.1 proteolytic enzyme enzyme activity determination method: protease activity adopts the Folin method to measure.

5.2 enzyme activity unit definition: under 35 ℃, the condition of pH10.0, the catalysis of every milliliter of enzyme liquid per minute

The enzyme amount that casein hydrolysis generates 1 μ g tyrosine is 1 unit (U/mL).

Five, the application of low-temperature alkaline protease product.

Low-temperature alkaline protease of the present invention can be used as the main additive of enzyme-containing detergent, and directly room temperature washing is saved the energy.Low-temperature alkaline protease of the present invention also is with a wide range of applications in food mfg, process hides, silk, environmental protection, medicine and other fields.

Description of drawings

Fig. 1 is bacterial strain opticmicroscope figure (10 * 100).

Fig. 2 is the Electronic Speculum figure of bacterial strain.

Fig. 3 is the influence figure of temperature to strain growth.

Fig. 4 is the influence figure of pH to strain growth.

Fig. 5 is the influence figure of NaCl to strain growth.

Fig. 6 is the influence figure of enzyme operative temperature to enzymic activity.

Fig. 7 is the thermostability figure of enzyme.

Fig. 8 is the influence figure of enzyme action pH to enzymic activity.

Fig. 9 is an enzyme pH stability diagram.

Embodiment

Embodiment 1.A kind of pseudoalteromonas HH407 (Pseudoalteromonas flavipulchra HH407) CCTCC NO:M 207009.This bacterial strain has following feature: this bacterium can be 0 ℃ of growth down, and the growth temperature range of this bacterium is 0-35 ℃, and optimum growth temperature is 20 ℃; Growth pH scope is 5.5-11, and the suitableeest growth pH is 8.5; The NaCl concentration of growth is 0.5%-13%, and the NaCl concentration of suitable growth is 4%, and no NaCl does not grow, and belongs to typical ocean to have a liking for cold moderate halophilic microorganism; This bacterium can utilize Tryptones, yeast extract paste, peptone, cellobiose, starch, gelatin, acetate, citric acid, methyl alcohol and ethanol.

Embodiment 2.A kind of method of utilizing embodiment 1 described a kind of pseudoalteromonas HH407 CCTCCNO:M 207009 to produce low-temperature alkaline protease, its step is as follows,

(1) connect the 250ml triangular flask that bacterial strain inclined-plane 1 ring bacterium is put into the 50ml fermention medium, 180r/min, cultivates 16h by 20 ℃; Insert in the fermention medium by 2% inoculum size, 180r/min, cultivates 36h by 20 ℃;

(2) will cultivate good fermented liquid 10000r/min, centrifugal 10min, supernatant liquor add 60% ammonium sulfate, leave standstill 3h after, the centrifugal 30min of 10000r/min is 8.0 Tris damping fluid dissolving with an amount of pH, then 4 ℃ of 24h that dialyse; Obtain low-temperature alkaline protease through DEAE-sepharose Flat Flow and SephacrylS-200 method again.

Embodiment 3.A kind of embodiment 2 low-temperature alkaline proteases that described method is produced, this low-temperature alkaline protease has following feature:

(1) the enzyme optimum temperature is 35 ℃, still has 22% catalytic efficiency at 0 ℃; Enzyme is stablized at 25 ℃, 35 ℃, 45 ℃, and insulation 60min, enzyme live all more than 80%, and the enzymes of 55 ℃ of insulation 30min loss 55% are alive; Be a kind of cryogenic proteolytic enzyme;

(2) the suitableeest action pH of enzyme is 10.0, and more than 80%, pH5-12 all has enzyme to live at the relative enzyme activity of pH9.0-11; The most stable pH of enzyme is 9.0; Be a kind of proteolytic enzyme of alkalescence;

(3) Sr ²⁺, Ba ²⁺, Cu ²⁺, Mn ²⁺, K ⁺Enzyme there is activation, Hg ²⁺, Al ³⁺, Zn ²⁺, Ni ²⁺, Co ²⁺, Fe ³⁺, Li ²⁺Restraining effect is arranged;

(4) PMSF inhibitory enzyme work, EDTA still has 106.6% enzyme work, shows that it is a kind of serine protease; H ₂O ₂, β-Mercaptoethanol, Urea and DTT can improve enzyme and live, improve 4.8%, 8.2%, 20.1%, 83.3% respectively, enzyme has tolerance to SDS, and 0.1%SDS lives to enzyme does not have influence, and work has promoter action to enzyme for tensio-active agent Tween20, Tween80, Triton X-100.

Claims (4)

1, a kind of pseudoalteromonas HH407 (Pseudoalteromonas flavipulchra HH407) CCTCC NO:M 207009.

2, a kind of pseudoalteromonas HH407 CCTCC NO:M207009 according to claim 1, this bacterial strain has following feature: this bacterium can be 0 ℃ of growth down, and the growth temperature range of this bacterium is 0-35 ℃, and optimum growth temperature is 20 ℃; Growth pH scope is 5.5-11, and the suitableeest growth pH is 8.5; The NaCl concentration of growth is 0.5%-13%, and the NaCl concentration of suitable growth is 4%, and no NaCl does not grow, and belongs to typical ocean to have a liking for cold moderate halophilic microorganism;

This bacterium can utilize Tryptones, yeast extract paste, peptone, cellobiose, starch, gelatin, acetate, citric acid, methyl alcohol and ethanol.

3, a kind of method of utilizing claim 1 or 2 described a kind of pseudoalteromonas HH407 CCTCCNO:M 207009 product low-temperature alkaline proteases is characterized in that its step is as follows,

(1) connect the 250ml triangular flask that bacterial strain inclined-plane 1 ring bacterium is put into the 50ml fermention medium, 180r/min, cultivates 16h by 20 ℃; Insert in the fermention medium by 2% inoculum size, 180r/min, cultivates 36h by 20 ℃;

(2) will cultivate good fermented liquid 10000r/min, centrifugal 10min, supernatant liquor add 60% ammonium sulfate, leave standstill 3h after, the centrifugal 30min of 10000r/min is 8.0 Tris damping fluid dissolving with an amount of pH, then 4 ℃ of 24h that dialyse; Obtain low-temperature alkaline protease through DEAE-sepharose Flat Flow and Sephacryl S-200 method again.

4, a kind ofly it is characterized in that by claim 3 low-temperature alkaline protease that described method is produced this low-temperature alkaline protease has following feature:

(1) the enzyme optimum temperature is 35 ℃, still has 22% catalytic efficiency at 0 ℃; Enzyme is stablized at 25 ℃, 35 ℃, 45 ℃, and insulation 60min, enzyme live all more than 80%, and the enzymes of 55 ℃ of insulation 30min loss 55% are alive; Be a kind of cryogenic proteolytic enzyme;

(2) the suitableeest action pH of enzyme is 10.0, and more than 80%, pH5-12 all has enzyme to live at the relative enzyme activity of pH9.0-11; The most stable pH of enzyme is 9.0; Be a kind of proteolytic enzyme of alkalescence;

(3) Sr ²⁺, Ba ²⁺, Cu ²⁺, Mn ²⁺, K ⁺Enzyme there is activation, Hg ²⁺, Al ³⁺, Zn ²⁺, Ni ²⁺, Co ²⁺, Fe ³⁺, Li ²⁺Restraining effect is arranged;

(4) PMSF inhibitory enzyme work, EDTA still has 106.6% enzyme work, shows that it is a kind of serine protease; H ₂O ₂, β-Mercaptoethanol, Urea and DTT can improve enzyme and live, improve 4.8%, 8.2%, 20.1%, 83.3% respectively, enzyme has tolerance to SDS, and 0.1%SDS lives to enzyme does not have influence, and work has promoter action to enzyme for tensio-active agent Tween20, Tween80, Triton X-100.

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