CN101407800B - Neuter protease produced by using Paenibacillus polymyxa from sea and method thereof - Google Patents
Neuter protease produced by using Paenibacillus polymyxa from sea and method thereof Download PDFInfo
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- CN101407800B CN101407800B CN2008102344588A CN200810234458A CN101407800B CN 101407800 B CN101407800 B CN 101407800B CN 2008102344588 A CN2008102344588 A CN 2008102344588A CN 200810234458 A CN200810234458 A CN 200810234458A CN 101407800 B CN101407800 B CN 101407800B
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Abstract
The invention relates to a method for producing a neutral protease through Paenibacillus polymyxa L1-9 (Paenibacillus polymyxa isolate L1-9), which includes the steps as follows: the activation of a strain, the preparation of a seed liquid, the preparation of a medium for producing the protease and the preparation of a fermentation culturing and coarse protease liquid for producing the protease. The proper reaction temperature of the neutral protease produced by the method is 40 DEG C and the pH value is 7.0; the protease has protease activity when temperature preservation is carried out under the temperature of 50 DEG C; the protease activity is weakened when the temperature is above 60 DEG C; the protease activity is stable when the pH is between 5.0 and 7.0 and the protease activity isreduced when the pH value is below 4.0 or above 9.0; and the metal ions including Ag<+>, Zn<2+>, Cu<2+> and Fe<3+> can restrain the protease activity while Na<+> and K<+> can improve the protease activity.
Description
Technical field
The present invention relates to a kind of marine bacteria white enzyme method of laying eggs, particularly a kind of Paenibacillus polymyxa from the ocean produces the neutral protein enzyme method, the invention still further relates to neutral protease that this method is produced.
Background technology
Proteolytic enzyme is the general name of the enzyme of catalytic proteins hydrolysis.The proteolytic enzyme kind is a lot, and important have stomach en-, trypsinase, kethepsin, Sumizyme MP, papoid and a subtilisin etc.Proteolytic enzyme has strict selectivity to the reaction substrate that is acted on, and a kind of proteolytic enzyme only can act on peptide bond certain in the protein molecule, as the formed peptide bond of trypsinase catalytic hydrolysis basic aminoacids.Proteolytic enzyme distributes wide, mainly is present in the humans and animals digestive tube, and content is abundant in plant and microorganism.Because animal and plant resource is limited, industrial production protease preparation mainly utilizes microbial fermentation preparations such as Bacillus subtilus, aspergillus terricola.
Neutral protease can be widely used in leather depilation, proteolysis, proteinic removal in fruit wine, beer and the beverage.Bacteria protease has important effect in bacteria growth process simultaneously, it is not only relevant with the nutrition of bacterial growth, but also can destroy host protein, be bacterium opposing and the important substance of attacking other pathogens, body wall as insect and nematode contains protein, bacterium parasitizes back extracellular proteinase on insect or the nematode, protein in degraded insect or the nematode body wall and kill insects and pathogenic nematode, thereby proteolytic enzyme has vital role in the control of plant pathogeny line insect and plant epiphyte venereal disease evil.But the product proteolytic enzyme microorganism that kills insect and nematode effect that has of report mainly comes from the Lu Yuan microorganism at present.And the Lu Yuan microorganism is through for many years separation screening, and it is more and more lower to obtain the probability that produces novel substance or have new functional microorganism.
Summary of the invention
Technical problem to be solved by this invention is at the deficiencies in the prior art, and a kind of novel method that is used to produce from the Paenibacillus polymyxa of ocean neutral protease is provided, this method is simple to operate, the neutral protein enzymic activity of producing stronger.
The neutral protease that the present invention also provides the method as mentioned above of sowing to produce.
Bacterial strain Paenibacillus polymyxa L involved in the present invention
1-9 (Paenibacillus polymyxa isolateL
1-9) be public bacterial strain, this bacterial strain is open at Genebank on September 30th, 2008, and accession number is FJ178378, and the 16S rRNA sequence of this bacterial strain is open, and network address is:
Http:// www.ncbi.nlm.nih.gov/sites/entrez? db=nuccore﹠amp; Cmd=search﹠amp; Term=FJ1783 78The public if desired, Huaihai Institute of Technology oceanography institute laboratory can externally provide.And the contriver published the paper of a piece " separation screening of the marine bacteria of 3 strain anti-plant pathogenic fungis " by name in " Henan agricultural sciences " in 2007, also disclosed bacterial strain Paenibacillus polymyxa L involved in the present invention in this paper
1-9 (Paenibacillus polymyxa isolate L
1-9) (be referred to as L in the paper
1-9 bacterial strains) with and isolation cultivation method and antibacterial application.
The present invention is with above-mentioned Paenibacillus polymyxa L from the ocean
1-9 (Paenibacilluspolymyxa isolate L
1-9) bacterial strain is the basis, studies its method of producing neutral protease and the neutral protease that is produced thereof.
Technical problem to be solved by this invention is to realize by following technical scheme.The present invention is a kind of Paenibacillus polymyxa L from the ocean
1-9 (Paenibacillus polymyxa isolate L
1-9) produce the neutral protein enzyme method, be characterized in that its step is as follows:
(1) bacterial strain activation: under the super clean bench aseptic technique, get Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolate L
1-9) bacterial strain, streak inoculation 25 ℃ of following constant temperature culture 24h to the PDA slant medium; Consisting of of described PDA slant medium: potato 200g, glucose 20g, agar 15-20g, seawater 1000ml;
(2) preparation of seed liquor: under aseptic condition, 5ml PD liquid nutrient medium joined the good Paenibacillus polymyxa L of the described activation of step (1)
1-9 (Paenibacillus polymyxa isolate L
1-9) on the PDA inclined-plane, the following lawn of flushing is also poured in the 250ml triangular flask that the 45mlPD liquid nutrient medium is housed, and 25 ℃, the condition of 190r/min, shaking culture 24h gets seed liquor; Consisting of of described PD liquid nutrient medium: potato 200g, glucose 20g, seawater 1000ml;
(3) preparation of product enzyme substratum: press soybean cake powder 2%, wheat bran 3%, Semen Maydis powder 4%, Na with seawater
2HPO
412H
2O 0.4%, KH
2PO
40.03%, CaCl
20.1%, NaCO
310H
2The weight percent of O0.1% is mixed with and produces the enzyme liquid nutrient medium, and regulating the pH value is 6.0, is divided in the 500mL triangular flask, and every bottled liquid measure is 70mL, 121.5 ℃ of sterilization 20min;
(4) produce that enzyme is cultivated and the preparation of crude enzyme liquid: the inoculum size by 5% under aseptic condition is inoculated into seed liquor in the product enzyme liquid nutrient medium, is that 25 ℃, rotating speed are under the 190r/min condition in temperature, shaking culture 42h; Under 4 ℃, 10000r/min condition, centrifugal 20min collects supernatant liquor and promptly gets crude enzyme liquid with the bacterium liquid after cultivating.
Technical problem to be solved by this invention can also further realize by following technical scheme.The invention also discloses a kind of as the described Paenibacillus polymyxa L of above technical scheme from the ocean
1-9 (Paenibacillus polymyxa isolate L
1-9) produce neutral protein neutral protease that enzyme method produces, be characterized in, it is 40 ℃ that this enzyme is fit to temperature of reaction, and the pH value is 7.0; This enzyme has enzymic activity at the 20min of insulation below 50 ℃, weakens in enzymic activity more than 60 ℃; Stable in pH value 5.0～7.0 o'clock, in pH value 4.0 below or 9.0 work of enzyme when above declines; Metal ion Ag
+, Zn
2+, Cu
2+, Fe
3+The activity of energy arrestin enzyme, Na
+, K
+Can improve protease activity.
Below be the Paenibacillus polymyxa L that the contriver did
1-9 (Paenibacilluspolymyxa isolateL
1-9) related experiment and result thereof.
1, about the neutral protease fermentation condition.
1.1, different substratum is to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolateL1-9) produce the influence of neutral protease.
Choose 5 kinds of substratum conducts to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxaisolate L
1-9) be optimized the substratum that sets out that produces enzyme, the medium pH value is made as 7.0.Cultivate basigamy 100mL for every kind and divide in two triangular flasks of packing into bottling amount 50ml.By the inoculation of 5% inoculum size, in the shaking table shaking culture, rotating speed is 180r/min, 28 ℃ of temperature, and the time is 44h.Cultivate end back centrifuging and taking supernatant liquor and carry out enzyme activity determination, thereby filter out optimal medium.
Five kinds of prescriptions:
(1) extractum carnis 5g/L, peptone 10g/L, yeast extract paste 5g/L, NaCl 5g/L.
(2) soybean cake powder 2%, wheat bran 3%, Semen Maydis powder 4%, Na
2HPO
412H
2O 0.4%, KH
2PO
40.03%, CaCl
20.1%, NaCO
310H
2O 0.1%.
(3) Semen Maydis powder 5%, soybean cake powder 3%, Na
2HPO
40.4%, KH
2PO
40.03%, MgSO
47H
2O 0.02%, CaCl
20.3%.
(4) sucrose 0.1%, soybean cake powder 0.4%, yeast extract paste 0.1%, casein 1%, MgSO
40.05%, CaCO
30.4%, KH
2PO
40.03%.
(5) skim-milk 1%.
1.2, different incubation time is to Paenibacillus polymyxa L
1-9 (Paenibacilluspolymyxa isolateL
1-9) influence of product neutral protease.
Choosing the best product of above-mentioned experiment proteolytic enzyme substratum is fermention medium, ferments liquid amount 5ml, inoculum size 5% with test tube, 28 ℃ of temperature, rotating speed 180r/min, cultivation total time is 48h, wherein take out 2 test tubes every 2h, the centrifuging and taking supernatant is surveyed enzyme and is lived, and draws best producing the enzyme time.
1.3, different culture temperature is to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolateL1-9) produce the influence of neutral protease.
The culture temperature of fermented liquid is adjusted to 15 ℃ respectively, and 20 ℃, 25 ℃, 28 ℃, 30 ℃, 35 ℃, 37 ℃, the medium pH value is 7.0, bottling amount 50ml, and inoculum size 5%, rotating speed is 180r/min, incubation time is a Best Times.
The fermented liquid of getting differing temps shaking table cultivation acquisition carries out enzyme activity determination, by relatively obtaining optimum temps.
1.4, different pH value is to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolateL
1-9) influence of product neutral protease.
The obtaining liq optimal medium, do not transfer pH value to be respectively with PHS-3C type PH score: 4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10.0,10.5,11.0 each pH value do respectively 2 parallel.Bottling amount 50ml, inoculum size is 5%, and shaking speed is 180r/min, and culture temperature is 28 ℃, and incubation time is a Best Times.
The fermented liquid of getting different pH shaking tables cultivation acquisitions carries out enzyme activity determination, by relatively obtaining best pH.
1.5, the different vaccination amount is to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolateL1-9) produce the influence of neutral protease.
The obtaining liq optimal medium is adjusted to 1%, 3%, 5%, 7%, 9%, 11%, 13%, 15% respectively with the inoculum size of fermented liquid.All the other culture condition are constant.
The fermented liquid of getting different vaccination amount shaking table cultivation acquisition carries out enzyme activity, by relatively obtaining inoculum size.
1.6, different liquid amount is to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolateL
1-9) influence of product neutral protease.
The obtaining liq optimal medium ferments with the triangular flask of 500ml, and the liquid amount of fermented liquid is adjusted to 50,60,70,80,90,100,110 respectively, the 120ml/ bottle, and all the other culture condition are constant.
The fermented liquid of getting different bottling amounts cultivation acquisitions carries out enzyme activity determination, by relatively obtaining best liquid amount.
1.7, different shaking speed is to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolateL
1-9) influence of product neutral protease.
Obtaining liq optimal medium, shaking speed are adjusted to 160,170,180,190,200,210 respectively, 220r/min, and all the other culture condition are constant.
The fermented liquid of getting different rotating speeds shaking table cultivation acquisition carries out enzyme activity, by relatively obtaining optimum revolution.
2, about the character of neutral protease.
2.1, temperature is to the live influence of reaction of enzyme.
Ready enzyme reaction system respectively under 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ of temperature reacting phase seasonable between the back measure its activity.Relatively the activity of enzyme under the differing temps is determined best enzyme reaction temperature.
2.2, the mensuration of enzyme heat stability.
The reaction system that is ready to enzyme is respectively at 0 ℃, and 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ are incubated 20min down, and the back is the corresponding time of reaction under the corresponding standard temperature, measures enzymic activity.Obtain the temperature range of enzyme remain stable.
2.3, pH is to the live influence of reaction of enzyme.
Getting the pH value is 3.0,4.0,5.0,6.0, and 7.0,8.0,9.0,10.0 different damping fluids replace standard buffer solution to carry out enzyme reaction, measure protease activity.Obtain the optimum pH of enzyme reaction.
2.4, ph stability.
Enzyme liquid is respectively 3.0,4.0 in the pH value, 5.0,6.0,7.0,8.0, handle 2h for 40 ℃ in 9.0,10.0 the damping fluid, again enzyme liquid pH value is transferred to neutrality after, carry out enzyme reaction by standard conditions, measure its enzymic activity.Obtain the pH value stabilization scope of enzyme.
2.5, metal ion is to the influence of enzymic activity.
Choose the compound of ten kinds of different ions: NaCl, CaCcl, Kcl, FeCl
3.6H
2O, FeSO
4.7H
2O, Co (NO
3) .6H
2O, ZnSO
4.H
2O, AgNO
3, MnCl
2.4H
2O, CuSO
4.5H
2O, compound is made into 0.1M concentration with the standard buffer solution of reaction, and then is diluted to 10mM, replaces standard buffer solution to carry out enzyme reaction with this solution, and making the metal ion final concentration is 5mM.With the damping fluid that does not add metal ion is contrast, measures the enzymic activity of different treatment.
3, colorimetric method for determining Paenibacillus polymyxa L
1-9 (Paenibacilluspolymyxa isolate L
1-9) produce the neutral protein enzymic activity.
3.1 experiment reagent.
1. Folin reagent: in 1L volumetrical ground Backflow bottle, add 10g sodium wolframate (Na
2WO
42H
2O), 2.5g Sodium orthomolybdate (Na
2MoO
42H
2O), 70mL distilled water, 5mL85% phosphoric acid and 10mL concentrated hydrochloric acid, fully little fiery boiling reflux 10h behind the mixing.Take off reflux cooler, in stink cupboard, add 5g Lithium Sulphate, 2.5mL distilled water and number drop of liquid bromine (99%), opening continues boiling 15min, so that remove unnecessary bromine (boiling if still have green to add bromine water again after the cooling), adds the deionized water constant volume after the cooling to 200mL.Mixing filters, and the reagent that makes should be golden yellow liquid, places the brown bottle dark place to preserve.
Use solution: 1 part of Folin reagent mixes with two parts of water, shakes up.
2. 0.4mol/L sodium carbonate solution (Na2CO3)
Take by weighing anhydrous sodium carbonate 4.244g, with deionized water dissolving and be settled to 100mL.
3. 0.4mol/L trichoroacetic acid(TCA) (CCl3.COOH)
Take by weighing trichoroacetic acid(TCA) 6.54g with water dissolution and be settled to 100mL.
4. 1% casein solution: take by weighing casein 1.000g and be accurate to 0.001g, with a small amount of 0.5mol/L sodium hydroxide solution moistening after, the phosphate buffer soln 80mL that slowly adds pH7.2, in boiling water bath, heat while stirring, until dissolving fully, after the cooling, change in the 100mL volumetric flask, be settled to scale with distilled water, this solution is at freezer storage, and validity period is 3d.
3.2 preparation tyrosine typical curve.
1. get 10 test tubes, numbering, the tyrosine solution of according to the form below preparation different content.
2. draw different concns tyrosine solution 1mL respectively and put into 10 test tubes in addition, add 0.4mol/L sodium carbonate solution 5mL, Folin reagent uses liquid 1mL, fully shakes up, and is incubated 20min in 40 ℃ of water-baths.
3. use UV-754 type spectrophotometer, compare, measure light absorption value at the 680nm place with No. 0 pipe.
4. be ordinate zou with the optical density(OD), tyrosine content (micrograms) is X-coordinate, the drawing standard curve.
The preparation of table 1 different concns tyrosine standardized solution
3.3 the active mensuration of neutral protease.
Adopt Folin-phenol reagent process to measure the neutral protein enzymic activity.In test tube, add crude enzyme liquid 1.00mL, 40 ℃ of water-bath preheating 2min, adding 1% casein solution 1.00mL again shakes up, in 40 ℃ of water-baths, accurately be incubated 10min, add 0.4mol/L trichoroacetic acid(TCA) solution 2.00mL and also shake up, take out and leave standstill 10min, the centrifugal 5min of 3500r/min then, get supernatant 1.00mL and add the 5.00mL0.4mol/L sodium carbonate solution, add Folin reagent and use liquid 1.00mL, 40 ℃ of water-bath colour developing 20min.Control group adds trichoroacetic acid(TCA) solution earlier and adds casein solution again, and other steps are identical.Use UV-754 type spectrophotometer to measure its absorption photometric under the 680nm wavelength more at last, producing the required enzyme amount of 1 μ g tyrosine with every min is enzyme unit alive.
4, result and analysis.
4.1 tyrosine typical curve.
Concentration with tyrosine is X-coordinate, and the OD value can draw the typical curve of tyrosine for ordinate zou, and its regression equation is y=0.0149x-0.0301, R
2=0.9982.With reference to Fig. 1.
4.2 different substratum are to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolateL
1-9) influence of product neutral protease.
Be numbered X-coordinate with substratum, the enzyme activity unit size of being measured with every milliliter of fermented liquid is an ordinate zou, makes Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolate L
1-9) the column type figure of product enzyme situation in different substratum.With reference to Fig. 2.
The result shows that different substratum are to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxaisolate L
1-9) produce the neutral protease ability and considerable influence is arranged, the enzymatic productivity difference of bacterial strain in different substratum.Mensuration by enzyme activity comparison shows that, Paenibacillus polymyxa L
1-9 (Paenibacilluspolymyxa isolate L
1-9) producing enzyme at No. 2 substratum and be up to 70.4U, secondly is that No. 3 substratum reach 44.4U.In No. 1 substratum, do not produce enzyme substantially, the end of enzymic activity.Show No. 2 substratum for producing the optimal medium of proteolytic enzyme, and No. 2 substratum are soybean cake powder 2%, wheat bran 3%, Semen Maydis powder 4%, Na
2HPO
412H
2O 0.4%, KH
2PO
40.03%, CaCl
20.1%, NaCO
310H
2O 0.1%.Be natural medium, raw material is easily sought, the cost cheapness, be convenient to making in laboratory, can be as the product enzyme culture medium prescription of this experiment.So selected No. 2 substratum are Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolate L
1-9) optimal medium.
4.3 different incubation times are to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolateL
1-9) influence of product neutral protease.
Select No. 2 substratum for supplying the examination substratum.Measure Paenibacillus polymyxa L respectively
1-9 (Paenibacillus polymyxa isolate L
1-9) the neutral protein enzymic activity of cultivation different time.With the incubation time is X-coordinate, and the enzyme activity unit size of being measured with fermented liquid is an ordinate zou, makes Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolate L
1-9) broken line graph of the product neutral protease situation of cultivating at different time.With reference to Fig. 3.
The result shows that beginning just has small amounts of protease to produce, and later enzymic activity raises gradually, and the enzymic activity of cultivating 42h reaches the highest, is 38.39U, and enzymic activity begins to reduce backward again.
4.4. different culture temperature are to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxaisolate L
1-9) influence of product enzyme.
Temperature is to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolate L
1-9) produce neutral protease and have a significant impact, measure Paenibacillus polymyxa L under the different culture temperature
1-9 (Paenibacillus polymyxa isolate L
1-9) size of the enzyme activity of fermented liquid.With the culture temperature is X-coordinate, and the enzyme activity unit size of being measured with every milliliter of fermented liquid is an ordinate zou, makes the broken line graph of bacterial strain in the product neutral protease situation of differing temps cultivation, sees Fig. 4.
The result shows that when culture temperature was 25 ℃, the enzyme activity unit of fermented liquid reached 35.25U, reached the maximum value of each temperature section.Yield of enzyme descends when temperature raises or reduce, and therefore can determine Paenibacillus polymyxa L
1The best product of-9 bacterial strains enzyme temperature is 25 ℃.
4.5 different pH values are to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolateL
1-9) influence of product enzyme.
To cultivate the pH value is X-coordinate, and the enzyme activity unit size of being measured with every milliliter of fermented liquid is an ordinate zou, makes Paenibacillus polymyxa L
1-9 (Paenibacilluspolymyxa isolate L
1-9) bacterial strain produces the broken line graph of enzyme situation under different pH value culture condition, sees Fig. 5.
Measurement result shows, occurs two and produce the enzyme peak when the pH value of substratum is 6.0 and 9.0, and 6.0 enzymic activitys reach 43.98U, and the pH9.0 enzymic activity reaches 41.94U.Initial pH is 4.0 o'clock enzyme minimum 18.30U that are alive, shows that this bacterium is unfavorable for producing enzyme in the environment of acid.Enzyme was lived and is changed gently when initial pH4.5 was above, showed that pH is little to the product enzyme influence of this bacterium.Two product enzyme peaks occur and show that this bacterium can secrete the multiple protein enzyme, under different initial pH, express different proteolytic enzyme.
4.6 the different vaccination amount is to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolateL
1-9) influence of product enzyme.
By measuring Paenibacillus polymyxa L under the different vaccination amount
1-9 (Paenibacillus polymyxaisolate L
1-9) enzyme of fermented liquid is lived, by comparison shows that Paenibacillus polymyxa L
1-9 (Paenibacilluspolymyxa isolate L
1-9) enzymatic productivity difference to some extent under the different vaccination amount.
With the inoculum size is X-coordinate, and the enzyme activity unit size of being measured with every milliliter of fermented liquid is an ordinate zou, makes Paenibacillus polymyxa L
1-9 (Paenibacilluspolymyxa isolate L
1-9) bacterial strain produces the broken line graph of proteolytic enzyme situation under different vaccination amount culture condition, sees Fig. 6.
The result shows that when inoculum size was 5%, the enzyme activity unit of fermented liquid reached 44.35U, reached the maximum value of each inoculum size.
4.7 different liquid amounts are to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolateL
1-9) influence of strain enzyme-producing.
With the liquid amount is X-coordinate, and the enzyme activity unit size of being measured with every milliliter of fermented liquid is an ordinate zou, makes Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolate L
1-9) bacterial strain produces the broken line graph of neutral protease situation under different liquid amount culture condition.See Fig. 7.
Measurement result shows, when liquid amount is 70ml (triangular flask is the 500ml capacity), the enzyme of the fermented liquid 40.16U that lives reaches the maximum value of each liquid amount.Therefore determine Paenibacillus polymyxa L
1-9 (Paenibacilluspolymyxa isolate L
1-9) the suitableeest liquid amount of strain enzyme-producing is 70ml.
4.8 shaking speed is to Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolateL
1-9) influence of strain enzyme-producing.
Can reach the purpose that changes oxygen-supply quantity by changing shaking speed.Measure the enzyme of different shaking speed bottom fermentation liquid and live, and, draw corresponding best shaking speed according to enzyme big or small comparison alive.
With the shaking speed is X-coordinate, and the enzyme activity unit size of being measured with every milliliter of fermented liquid is an ordinate zou, makes Paenibacillus polymyxa L
1-9 (Paenibacilluspolymyxa isolate L
1-9) broken line graph of the bacterial strain product enzyme situation of cultivating under different shaking speed is seen Fig. 8.
Measurement result shows, Paenibacillus polymyxa L
1-9 (Paenibacilluspolymyxa isolate L
1-9) bacterial strain is an aerobic bacteria, and the rotating speed of shaking table is more remarkable to the influence of producing enzyme, and when shaking speed was set at 190r/min, the enzyme of its fermented liquid was lived and is 56.85U, reaches the maximum value of each rotating speed, illustrates that product enzyme situation is ideal.Along with the quickening of rotating speed or slow down, descending all appears in the value of mensuration, shows that the strain enzyme-producing ability reduces.Therefore its best shaking speed is 190r/min.
5. the research of neutral protease properties.
5.1 the influence that temperature is lived and reacted enzyme.
The enzyme reaction system is reacted in different temperature environments respectively, measure protease activities.With the temperature is X-coordinate, is ordinate zou with enzyme work, makes the broken line graph of temperature to enzyme reaction influence alive, with reference to Fig. 9.Fig. 9 shows that temperature is more remarkable to the influence of enzyme reaction alive.Along with the rising of temperature, protease activity rises gradually, and protease activity reaches the highest in the time of 40 ℃, is 56.36U.Enzymic activity began to descend when temperature raise again.This experiment shows that the optimal reaction temperature that separates the neutral protease that obtains is 40 ℃.
5.2 the mensuration of enzyme heat stability.
To be ready to crude enzyme liquid and under different temperature, be incubated 20min respectively, under the corresponding standard temperature, react, measure enzymic activity.The result shows that the Temperature Treatment below 40 ℃ is little to the activity influence of enzyme, and the activity of enzyme begins to descend during 50 ℃ of processing, and 80 ℃ of enzymic activitys are very low.Show this enzyme more stable below 40 ℃, performance is unstable more than 50 ℃.With reference to Figure 10.
5.3 the influence that pH lives and reacts enzyme.
Neutral protease liquid is reacted the different enzymic activity differences of pH in the damping fluid reaction system of different pH.The beginning enzymic activity raises along with the rising of pH, and enzymic activity is up to 56.95U when pH8.0, and enzyme work begins to descend during pH9.0, reduces with the rising of pH.This result shows that the best pH of the reaction of enzyme is 8.0.With reference to Figure 11.
5.4 ph stability.
With crude enzyme liquid in the damping fluid of different pH values 40 ℃ handle 2h, again enzyme liquid pH value is transferred to neutrality after, carry out enzyme reaction by standard conditions, measure its enzymic activity.The result shows that protease activity is the highest during pH7.0, reaches 47.55U.This enzymic activity raises with the rising of pH during initial pH3, and pH sharply descended at 8.0 o'clock, illustrated that this enzyme is more stable in neutral environment.With reference to Figure 12.
5.5 metal ion is to the influence of enzymic activity.
Choose the compound of ten kinds of different ions, measure the protease activity in the reaction system that contains 5mmol different ions damping fluid.The result shows that the activity that does not add the enzyme of metal ion is 33.78U, contains Na
+, K
+, Co
2+Be higher than contrast during ion processing, be respectively 39.32U, 45.26U, 45.15U illustrates that this three metal ion species has promoter action to protease activity.Mn
2+Little to the enzymic activity influence.Metal cations Fe
3+, Ag
+Influence to proteolytic enzyme is bigger, and restraining effect is arranged.Ca
2+, Fe
2+, Cu
2+, Zn
2+Ion takes second place.With reference to Figure 13.
5.6 Paenibacillus polymyxa L of the present invention
1-9 (Paenibacilluspolymyxa isolate L
1-9) the neutral protease that produces the same with other neutral protease, can be widely used in leather depilation, proteolysis, proteinic removal in fruit wine, beer and the beverage.Simultaneously can also destroy host protein, be bacterium opposing and the important substance of attacking other pathogens, body wall as insect and nematode contains protein, and bacterium parasitizes on insect or the nematode back extracellular proteinase, the protein in degraded insect or the nematode body wall and kill insects and pathogenic nematode.
Below be the Paenibacillus polymyxa L of the present invention that the contriver did
1-9 (Paenibacilluspolymyxa isolate L
1-9) the neutral protease that produces to the experiment of the lethal effect of plant pathogeny line insect.
In the recessed glass ware that 1.5ml different concns enzyme liquid is housed, add 40 of sweet potato stem nematode, Meloidogyne incognitas respectively, recessed glass ware is put into big culture dish preserves moisture, in 25 ℃ of constant incubators, cultivate, the death condition of observation nematode behind 48h, the life or death of nematode is identified and is adopted NaCl detection method, calculation correction mortality ratio.If the blank enzyme substratum that produces is contrast, every processing repeats 3 times.
The result shows: the nemic death rate after enzyme is also handled is apparently higher than contrast.The proteolytic enzyme of different concns is to the lethal effect difference of nematode, the mortality ratio of the high more nematode of enzyme concn is high more, and the corrected mortality of sweet potato stem nematode, Meloidogyne incognita has reached the mortality ratio of 68.5% and 73.3% dilution after 5 times respectively and also reached 40.2% and 50.1% respectively behind the enzyme liquid stoste 48h.Illustrate that this proteolytic enzyme has stronger lethal effect to two kind of plant pathogenic nematodes.
The corrected mortality (%) of two kinds of nematodes behind the table 1 different concns enzyme liquid processing 48h
| Enzyme liquid concentration | Sweet potato stem nematode | Meloidogyne incognita |
| Stoste | 68.5 | 73.3 |
| 2 times of diluents | 52.3 | 66.4 |
| 5 times of diluents | 40.2 | 50.1 |
Description of drawings
Fig. 1 is the tyrosine canonical plotting.
Fig. 2 is that different substratum are to Paenibacillus polymyxa L
1-9 strain enzyme-producing Effect on Performance are figure as a result.
Fig. 3 is that different incubation times are to Paenibacillus polymyxa L
1-9 bacterial strains produce the figure as a result that influences of proteolytic enzyme.
Fig. 4 is that different culture temperature are to Paenibacillus polymyxa L
1-9 strain enzyme-producings influence figure.
Fig. 5 is that different pH values are to Paenibacillus polymyxa L
1-9 strain enzyme-producings influence figure as a result.
Fig. 6 is that the different vaccination amount is to Paenibacillus polymyxa L
1-9 strain enzyme-producings influence figure as a result.
Fig. 7 is that different liquid amounts are to Paenibacillus polymyxa L
1-9 strain enzyme-producings influence figure as a result.
Fig. 8 is that shaking speed is to Paenibacillus polymyxa L
1-9 strain enzyme-producings influence figure as a result.
Fig. 9 is the figure as a result that influences that temperature is lived and reacted enzyme.
Figure 10 is the measurement result figure of enzyme heat stability.
Figure 11 is the figure as a result that influences that pH lives and reacts enzyme.
Figure 12 is the measurement result figure of enzyme ph stability.
Figure 13 is influence the as a result figure of metal ion to enzymic activity.
Embodiment
Below further describe concrete technical scheme of the present invention,, and do not constitute restriction its right so that those skilled in the art understands the present invention further.
Embodiment 1.A kind of Paenibacillus polymyxa L from the ocean
1-9 (Paenibacillus polymyxaisolate L
1-9) produce the neutral protein enzyme method, its step is as follows:
(1) bacterial strain activation: under the super clean bench aseptic technique, get Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolate L
1-9) bacterial strain, streak inoculation 25 ℃ of following constant temperature culture 24h to the PDA slant medium; Consisting of of described PDA slant medium: potato 200g, glucose 20g, agar 15-20g, seawater 1000ml;
(2) preparation of seed liquor: under aseptic condition, 5ml PD liquid nutrient medium is joined in the good PDA slant medium of the described activation of step (1), the following lawn of flushing is also poured in the 250ml triangular flask that the 45mlPD liquid nutrient medium is housed, 25 ℃, the condition of 190r/min, shaking culture 24h gets seed liquor; Consisting of of described PD liquid nutrient medium: potato 200g, glucose 20g, seawater 1000ml;
(3) preparation of product enzyme substratum: press soybean cake powder 2%, wheat bran 3%, Semen Maydis powder 4%, Na with seawater
2HPO
412H
2O 0.4%, KH
2PO
40.03%, CaCl
20.1%, NaCO
310H
2The weight percent of O0.1% is mixed with and produces the enzyme liquid nutrient medium, and regulating the pH value is 6.0, is divided in the 500mL triangular flask, and every bottled liquid measure is 70mL, 121.5 ℃ of sterilization 20min;
(4) produce that enzymic fermentation is cultivated and the preparation of crude enzyme liquid: the inoculum size by 5% under aseptic condition is inoculated into seed liquor in the product enzyme liquid nutrient medium, is that 25 ℃, rotating speed are under the 190r/min condition in temperature, shaking culture 42h; Under 4 ℃, 10000r/min condition, centrifugal 20min collects supernatant liquor and promptly gets crude enzyme liquid with the bacterium liquid after the fermentation culture.
Embodiment 2.A kind of as embodiment 1 described Paenibacillus polymyxa L from the ocean
1-9 (Paenibacillus polymyxa isolate L
1-9) produce neutral protein neutral protease that enzyme method produces, it is 40 ℃ that this enzyme is fit to temperature of reaction, and the pH value is 7.0; This enzyme has enzymic activity at the 20min of insulation below 50 ℃, weakens in enzymic activity more than 60 ℃; Stable in pH value 5.0～7.0 o'clock, in pH value 4.0 below or 9.0 work of enzyme when above declines; Metal ion Ag
+, Zn
2+, Cu
2+, Fe
3+The activity of energy arrestin enzyme, and Na
+, K
+Can improve protease activity.
Claims (2)
1. Paenibacillus polymyxa L from the ocean
1-9 (Paenibacillus polymyxa isolateL
1-9) produce the neutral protein enzyme method, it is characterized in that its step is as follows:
(1) bacterial strain activation: under the super clean bench aseptic technique, get Paenibacillus polymyxa L
1-9 (Paenibacillus polymyxa isolate L
1-9) bacterial strain, streak inoculation 25 ℃ of following constant temperature culture 24h to the PDA slant medium; Consisting of of described PDA slant medium: potato 200g, glucose 20g, agar 15-20g, seawater 1000ml;
(2) preparation of seed liquor: under aseptic condition, 5ml PD liquid nutrient medium joined the good Paenibacillus polymyxa L of the described activation of step (1)
1-9 (Paenibacillus polymyxa isolate L
1-9) on the PDA inclined-plane, the following lawn of flushing is also poured in the 250ml triangular flask that the 45mlPD liquid nutrient medium is housed, and 25 ℃, the condition of 190r/min, shaking culture 24h gets seed liquor; Consisting of of described PD liquid nutrient medium: potato 200g, glucose 20g, seawater 1000ml;
(3) preparation of product enzyme substratum: press soybean cake powder 2%, wheat bran 3%, Semen Maydis powder 4%, Na with seawater
2HPO
412H
2O 0.4%, KH
2PO
40.03%, CaCl
20.1%, Na
2CO
310H
2The weight percent of O0.1% is mixed with and produces the enzyme liquid nutrient medium, and regulating the pH value is 6.0, is divided in the 500mL triangular flask, and every bottled liquid measure is 70mL, 121.5 ℃ of sterilization 20min;
(4) produce that enzyme is cultivated and the preparation of crude enzyme liquid: the inoculum size by 5% under aseptic condition is inoculated into seed liquor in the product enzyme liquid nutrient medium, is that 25 ℃, rotating speed are under the 190r/min condition in temperature, shaking culture 42h; Under 4 ℃, 10000r/min condition, centrifugal 20min collects supernatant liquor and promptly gets crude enzyme liquid with the bacterium liquid after cultivating.
2. Paenibacillus polymyxa L from the ocean as claimed in claim 1
1-9 (Paenibacillus polymyxa isolate L
1-9) produce neutral protein neutral protease that enzyme method produces, it is characterized in that, it is 40 ℃ that this enzyme is fit to temperature of reaction, and the pH value is 7.0; This enzyme has enzymic activity at the 20min of insulation below 50 ℃, weakens in enzymic activity more than 60 ℃; Stable in pH value 5.0～7.0 o'clock, in pH value 4.0 below or 9.0 work of enzyme when above declines; Metal ion Ag
+, Zn
2+, Cu
2+, Fe
3+The activity of energy arrestin enzyme, Na
+, K
+Can improve protease activity.
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| CN103014078A (en) * | 2012-11-16 | 2013-04-03 | 淮海工学院 | Method for extracting DEHP (di(2-ethylhexyl)phthalate) from fermentation product of ocean paenibacillus polymyxa L1-9 and application |
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| CN102559537B (en) * | 2011-11-15 | 2013-06-12 | 中国科学院微生物研究所 | Bacillus and application of bacillus in protease production |
| CN103250741B (en) * | 2013-03-05 | 2015-02-18 | 吉林农业大学 | Preparation method of ginseng endophytic Paenibacillus polymyxa biological control microbial inoculum |
| CN103740605B (en) * | 2013-10-31 | 2017-01-18 | 华南理工大学 | Bacillus aquimaris and application thereof |
| CN106591203B (en) * | 2017-01-23 | 2019-02-26 | 华中农业大学 | A kind of Paenibacillus polymyxa KM2501-1 and its application |
| CN107247143A (en) * | 2017-03-27 | 2017-10-13 | 天津国际生物医药联合研究院 | Influence of the bivalent metal ion to zika virus non-structural protein enzymatic activity |
| CN106929465A (en) * | 2017-04-10 | 2017-07-07 | 吉林农业大学 | A kind of Cu2+Ion promotes Paenibacillus polymyxa propagation and its application process |
| CN109825463A (en) * | 2019-04-11 | 2019-05-31 | 淮海工学院 | The preparation method and purposes of Paenibacillus polymyxa BMF02 and its wettable powder |
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Cited By (2)
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| CN103014078A (en) * | 2012-11-16 | 2013-04-03 | 淮海工学院 | Method for extracting DEHP (di(2-ethylhexyl)phthalate) from fermentation product of ocean paenibacillus polymyxa L1-9 and application |
| CN103014078B (en) * | 2012-11-16 | 2014-09-10 | 淮海工学院 | Method for extracting DEHP (di(2-ethylhexyl)phthalate) from fermentation product of ocean paenibacillus polymyxa L1-9 and application |
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