CN102676483A - Method for producing protease through one-bacterium multi-enzyme strain - Google Patents
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Abstract
The invention relates to a method for producing protease through a one-bacterium multi-enzyme strain. The method comprises the following steps of: collecting bacillus subtilis 1.1111 at China Center for Type Culture Collection with a collection number of CCTCC NO: M2011286; inoculating in a casein agar culture medium through stab inoculation; culturing in an incubator of 37 DEG C for 24 hours; and discovering that a hydrolytic circle is produced and the diameters of the hydrolytic circle and a colony can reach 4.80. According to the method, optimal enzyme activity conditions are determined through a method for determining enzyme activity through Folin phenol: the initial pH value of the culture medium is 7.0, the inoculum size is 3 percent, the inoculation age is 22 hours, the culture temperature is 37 DEG C, the fermentation time is 24 hours, and the enzyme activity of the produced protease reaches the maximum. Meanwhile, factors such as temperature, pH, thermal stability and metal ions affecting the enzyme activity of the protease are researched by the method for determining the enzyme activity through Folin phenol. Results show that the optimum operation temperature of the protease produced by the strain is 40 DEG C, the optimum operation pH is 5.7, Mn<2+> and Cu<2+> have certain promotion effect on the enzyme, and the Al<3+> has a strong inhibition effect on the enzyme. According to the method, a foundation is laid for making further exploration about related influencing factor of each enzyme, the optimum culture medium preparation, the optimum fermentation conditions and the optimum complex enzyme compatibility to expect the implementation of high-quality common fermentation production of the one-bacterium multi-enzyme strain.
Description
Technical field
The present invention relates to the method that an a kind of bacterium multienzyme bacterial strain produces proteolytic enzyme, be specifically related to the condition of bacterium multienzyme bacterial strain product proteolytic enzyme is optimized, and the proteolytic enzyme zymologic property is analyzed, belong to microbial technology field.
Background technology
Zymin is directly to extract as raw material with animal and plant at first, but because long, the influence of various factors such as raw material is limited, enzyme extraction complex process of animal and plant growth cycle are inappropriate for large-scale industrial production.And mikrobe has the incomparable meliority of other biological in production of enzyme preparation: mikrobe environment to external world has very strong flexibility and variation ability; People's various biology techniques capable of using are transformed mikrobe according to the wish of oneself, produce the purpose enzyme; Mikrobe is less demanding to nutritive substance, and major part is an agricultural product castoff, and is cheap, is easy to get, and production cost is low; Most of microbial reproduction is fast, and growth cycle is short, helps improving the output of zymin etc.
Summary of the invention
The object of the present invention is to provide an a kind of bacterium multienzyme bacterial strain to produce the method for proteolytic enzyme.
To achieve these goals; The method that method of the present invention has adopted an a kind of bacterium multienzyme bacterial strain to produce proteolytic enzyme may further comprise the steps: with subtilis (Bacillus subtilis) 1.1111, be preserved in Chinese typical culture collection center, its preserving number is CCTCC NO:M 2011286; Can produce 9 kinds of enzymes such as proteolytic enzyme; Wherein protease activity is high especially, and its thorn is planted on the casein agar substratum, in 37 ℃ of incubators, cultivates 24h; Discovery has the hydrolysis circle to produce, and hydrolysis circle and colony diameter reach 4.80.
In initial pH value of medium 7.0, inoculum size is 3%, plants 22h in age, 37 ℃ of culture temperature, and during fermentation time 24h, the proteolytic enzyme enzyme work that is produced reaches the highest.
With 1% Zulkovsky starch be optimum carbon source, 1% yeast to soak powder be optimum nitrogen source, make the fermention medium component reach optimum.
The proteolytic enzyme that is produced is better at temperature 25-40 ℃ of scope internal stability.
The proteolytic enzyme optimum temperature that is produced is 40 ℃.
The proteolytic enzyme that is produced is more stable under the pH5.5-6.5 condition.
The proteolytic enzyme that is produced is value 5.7 at ph optimum.
At the proteolytic enzyme that is produced, Mn
2+And Cu
2+The enzyme that can significantly improve this proteolytic enzyme is lived Mg
2+, Ba
2+, Zn
2+This proteolytic enzyme also there are certain promoter action, Al
3+, Fe
2+This proteolytic enzyme is had restraining effect in various degree, and Al
3+Restraining effect obviously be better than Fe
2+
The present invention is optimized carbon source, the nitrogenous source in the fermentation condition that influences producing bacillus subtilis proteolytic enzyme such as pH value, inoculum size, liquid amount, kind age, culture temperature, the nutrient media components.The result shows that this bacterial strain produces the righttest fermentation time 24h of proteolytic enzyme, and optimum medium pH value 7.0, inoculum size are 3%, and liquid amount 15mL/100mL plants 22h in age, 37 ℃ of control culture temperature.Optimum carbon source is a Zulkovsky starch, and nitrogenous source is that yeast soaks powder.And the factors such as temperature, pH, thermostability and metals ion that influence the proteolytic enzyme enzyme and live are studied through Folin phenol method.The result shows that it is 40 ℃ that this bacterial strain produces the proteolytic enzyme optimum temperature, and the righttest action pH is 5.7, Mn
2+And Cu
2+Enzyme there are certain promoter action, Al
3+Enzyme there is had strong inhibitory effects.Related bacillus subtilis strain can produce 9 kinds of enzymes such as zytase, proteolytic enzyme, Sumizyme PHY, polygalacturonase, lypase, sweet dew LSD, saccharifying enzyme among the present invention; Wherein proteolytic enzyme, sweet dew LSD and saccharifying enzyme rate ratio are higher; And the constellation between each enzyme, inductive condition, the best compatibility of best condition of enzyme production and compound enzymic preparation all awaits further development research.The present invention is to the preparation of the righttest substratum of proteolytic enzyme, the righttest fermentation condition and influence the correlative factor that its enzyme lives and inquire into; In the hope of compound enzymic preparations such as the generation zytase that reaches high-quality, a large amount, proteolytic enzyme, Sumizyme PHY, polygalacturonase, lypase, sweet dew LSD, saccharifying enzyme, realize that bacterium multienzyme fermentation lays a good foundation.
The drafting of tyrosine typical curve
Get 6 test tubes,, get each 1.00mL of above-mentioned solution respectively according to the tyrosine standardized solution of table 1 preparation different concns; Respectively add 0.4mol/L yellow soda ash and dissolve and to tuck in 5.00mL, Folin phenol reagent diluent 1.00mL takes out after placing 40 ℃ ± 2 ℃ water-baths colour developing 20min; In wavelength 660nm, with 10mm cuvette colorimetric, is blank with No. 0 pipe that does not contain tyrosine with spectrophotometer; Measure its absorbancy respectively, record OD660 value.With the absorbancy is ordinate zou, and the concentration of tyrosine is X-coordinate, the drawing standard curve.According to mapping, calculate the amount of the tyrosine when absorbancy is 1, be extinction constant K value.
Table 1 tyrosine typical curve method for drafting
The calculation formula that enzyme is lived: proteinase activity (U/mL)=Δ OD660nm * N * K * 4/10
Δ OD660nm is the OD value of sample when being blank with the control sample;
K be optical density(OD) be 1.00 o'clock suitable tyrosine concentration;
N is the extension rate of enzyme liquid;
4 is the TV of reaction reagent;
10 is reaction times 10min.
Producing bacillus subtilis proteolytic enzyme Optimizing Conditions of Fermentation
1. the preparation of the activation of bacterial classification and seed liquor
Get freeze-drying Bacillus subtilis strain strok method and be inoculated on the plain agar substratum, cultivate 18-24h under 37 ℃ of conditions.And provoke single colony inoculation in liquid culture, and cultivate 18h under 37 ℃, 140rpm condition, preserve subsequent use.
2. fermentation culture conditions is to enzyme influence alive
(1) the different fermentations time is produced the influence of proteolytic enzyme to bacterial strain: seed liquor is inserted in the fermention medium by 2% inoculum size; 37 ℃ of 140rpm cultivate; Remove tunning respectively in 8h, 12h, 16h, 20h, 24h, 28h, its enzyme work is measured with improvement Folin phenol method.
(2) different initial pH value of medium are produced the influence of proteolytic enzyme to bacterial strain: it is respectively in 5.4,6.2,7.0,7.8,8.6 fermention mediums that seed liquor is inserted the pH value by 2% inoculum size; 37 ℃ of 140rpm shaking table shaking culture; With the above-mentioned time be optimum incubation time, sampling is measured its enzyme work with improveing Folin phenol method respectively.
(3) the different vaccination amount is produced the influence of proteolytic enzyme to bacterial strain: seed liquor is inoculated in the fermention medium by 1%, 2%, 3%, 4%, 5% respectively; 37 ℃ of 140rpm cultivate with above-mentioned optimal time, optimal ph, and sampling is measured its enzyme work with improvement Folin phenol method respectively.
(4) different cell ages are produced the influence of proteolytic enzyme to bacterial strain: get the seed liquor cell age respectively and be 10h, 13h, 16h, 19h, 22h, 25h and be inoculated in the fermention medium; 37 ℃ of 140rpm cultivate sample thief respectively with optimal time, pH value, inoculum size, with improvement Folin phenol method its enzyme work are measured.
(5) differing temps is produced the influence of proteolytic enzyme to bacterial strain: the seed liquor of best cell age is inoculated in the appropriate media by optimum inoculation amount; Respectively under 27 ℃, 32 ℃, 37 ℃, 42 ℃, 47 ℃; 140rpm cultivates, and sampling is respectively measured its enzyme work with improvement Folin phenol method.
3. fermention medium is formed the influence that enzyme is lived
(1) different carbon sources are produced the influence of proteolytic enzyme to bacterial strain: process substratum with the sucrose in 1% glucose, Zulkovsky starch, lactose and the SANMALT-S equivalent substitution basis fermented liquid respectively.Under optimal culture condition, carry out fermentation culture, sample thief is measured its enzyme work with improvement Folin phenol method respectively.
(2) different nitrogen sources is produced the influence of proteolytic enzyme to bacterial strain: adopt optimum carbon source; Nitrogenous source is used 1% urea, ammonium chloride, peptone, Carnis Bovis seu Bubali cream, yeast instead and is soaked powder and substitute 1% peptone in the basic fermentation culture; Under optimal culture condition, carry out fermentation culture; Sample thief is measured its enzyme work with improvement Folin phenol method respectively.
The research of Validase TSP Concentrate II influence factor
1.pH influence to protease activity
1 times of dilution of damping fluid enzyme liquid with different pH values; Make pH and be respectively 5.0,5.5,6.0,6.5,7.0 enzyme liquid; Be incubated 30min down at 40 ℃, and prepare casein solution as substrate, its enzyme work is measured with improvement Folin phenol method with the damping fluid of corresponding pH value.
2. temperature is to the influence of protease activity
After the 1 times of dilution of phosphoric acid buffer of enzyme liquid with pH 7.5, under 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, be substrate respectively with casein solution, measure enzyme activity with improvement Folin phenol method.
3. common metal ion is to the influence of protease activity
Respectively with CuSO
4, FeSO
4, MgSO
4, MnSO
4, ZnSO
4, BaCl
2, AlCl
3Be made into the solution of 0.01mol/L, respectively get in the 1mL enzyme liquid that 1mL joins purifying respectively and (be about to 2 times of enzyme liquid dilutions), under optimum temperuture, handle 10min after, and compare with the enzyme liquid that does not add metals ion, measure the residual protein enzyme activity with improvement Folin phenol method.
Subtilis of the present invention (Bacillus subtilis) 1.1111 is preserved in the Chinese typical culture collection center that is positioned at Wuhan University, and its preserving number is CCTCC NO:M 2011286, and preservation date is on August 11st, 2011.
Description of drawings
Fig. 1 is the tyrosine canonical plotting;
Fig. 2 is fermentation time produces proteolytic enzyme to bacterial strain influence;
Fig. 3 is that different initial pH value of medium are to producing the influence of enzyme;
Fig. 4 is that the different vaccination amount is to producing the influence of enzyme;
Fig. 5 is that age not of the same race is to producing the influence of enzyme;
Fig. 6 is that differing temps is to producing the influence of enzyme;
Fig. 7 is that different carbon sources are to producing the influence of enzyme;
Fig. 8 is that different nitrogen sources is to producing the influence of enzyme;
The influence that the different pH of Fig. 9 live to enzyme;
Figure 10 is the influence that temperature is lived to enzyme;
The influence that Figure 11 metals ion is lived to enzyme.
Embodiment
(1) the tyrosine typical curve is drawn embodiment
According to experimental result, be ordinate zou with the absorbancy, the concentration of tyrosine is X-coordinate, draws the tyrosine typical curve.As shown in Figure 1, according to slope K=92.98 that figure calculates equation of linear regression, promptly the extinction constant is 92.98.
(2) producing bacillus subtilis proteolytic enzyme Optimizing Conditions of Fermentation embodiment
1. fermentation culture conditions is to the influence of producing bacillus subtilis proteolytic enzyme
(1) fermentation time produces the influence of proteolytic enzyme to bacterial strain
Seed liquor is inserted in the fermention medium by 2% inoculum size, and 37 ℃ of 140rpm cultivate, and remove tunning respectively in 8h, 12h, 16h, 20h, 24h, 28h, and it is as shown in Figure 2 to measure enzyme slip-knot fruit with improvement Folin phenol method.
Can be known that by Fig. 2 cultivation initial stage subtilis begins to produce proteolytic enzyme, along with the prolongation of incubation time, enzymic activity raises gradually.Cultivate the 24h enzymic activity and reach the highest, be 23.39U/mL, activity ratio 24h's is low behind the 28h.Therefore the best incubation time of this bacterial strain generation proteolytic enzyme is 20-24h, and enzyme activity begins to descend subsequently, produces proteolytic enzyme and thalli growth and carries out synchronously, and this proteolytic enzyme biosynthesizing type belongs to synchronous synthesis type.
(2) initial pH value of medium is produced the influence of proteolytic enzyme to bacterial strain
It is respectively in 5.4,6.2,7.0,7.8,8.6 fermention mediums that seed liquor is inserted the pH value by 2% inoculum size, and 37 ℃ of 140rpm shaking table shaking culture are cultivated 24h, and sampling is measured its enzyme work with improvement Folin phenol method respectively, and the result is as shown in Figure 3.
Can know that by Fig. 3 the too high or too low generation that all can influence proteolytic enzyme of the initial pH of substratum is especially when the pH value reaches 8.6; Generation to proteolytic enzyme has very big influence, and pH is 6.2 o'clock, and enzyme is lived and is 17.39U/mL; PH is 7.0 o'clock, and product enzyme effect is best, and enzyme work reaches 19.67U/mL.
(3) inoculum size is produced the influence of proteolytic enzyme to bacterial strain
Seed liquor is inoculated in the pH7.0 fermention medium by 1%, 2%, 3%, 4%, 5% respectively, 37 ℃ of 140rpm24h, and sampling is measured its enzyme work with improvement Folin phenol method respectively, and its result sees Fig. 4.
Can know that by Fig. 4 during inoculum size 3%, its enzyme is lived the highest.Inoculum size is that 1%, 2% o'clock yield of enzyme is lower; Inoculum size is 4%, 5% o'clock, and proteolytic enzyme output is also lower.
(4) different cell ages are produced the influence of proteolytic enzyme to bacterial strain
Get the seed liquor cell age respectively and be 10h, 13h, 16h, 19h, 22h, 25h and be inoculated in the pH7.0 fermention medium by 3%, 37 ℃ of 140rpm24h, sample thief is measured its enzyme work with improveing Folin phenol method respectively, and its result sees shown in Figure 5.
Can know that by Fig. 5 the seed liquor yield of enzyme of inoculation 13h cell age is minimum, also low than 10h kind age; This possibly cause by experiment condition, and like the fermentation flask dissolved oxygen amount etc., inoculation 19h is to the seed liquor of 22h cell age; The strain enzyme-producing amount increases greatly gradually, and yield of enzyme reduces greatly gradually behind the 22h.
(5) differing temps is produced the influence of proteolytic enzyme to bacterial strain
The seed liquor of 22h cell age is inoculated in the pH7.0 fermention medium by 3%, places 27 ℃, 32 ℃, 37 ℃, 42 ℃, 47 ℃ respectively, 140rpm shaking table vibration 24h, sampling is measured its enzyme work with improvement Folin phenol method respectively, and its result sees shown in Figure 6.
Can be known that by Fig. 6 when culture temperature was lower than 27 ℃, enzymic activity was lower, along with the rising of temperature, enzymic activity improves gradually; When culture temperature reached 37 ℃, the enzymic activity of fermented liquid was the highest, was 23.39U/mL; Along with temperature raises, enzymic activity begins to descend
2. fermention medium is formed the influence that enzyme is lived
(1) different carbon sources are produced the influence of proteolytic enzyme to bacterial strain
Process substratum with the sucrose in 1% glucose, Zulkovsky starch, lactose and the SANMALT-S equivalent substitution basis fermented liquid respectively, place 37 ℃ of 140rpm to cultivate 24h down, sample thief is measured its enzyme work with improvement Folin phenol method respectively, and its result sees Fig. 7.
Can be known that by Fig. 7 enzyme is lived the highlyest when being carbon source with the Zulkovsky starch, sucrose takes second place, and SANMALT-S during as carbon source yield of enzyme minimum.
(2) different nitrogen sources is produced the influence of proteolytic enzyme to bacterial strain
The employing Zulkovsky starch is a carbon source; Nitrogenous source is used 1% urea, ammonium chloride, peptone, Carnis Bovis seu Bubali cream, yeast instead and is soaked powder and substitute 1% peptone in the basic fermentation culture, places 37 ℃ of 140rpm to cultivate 24h, sample thief respectively down; With improvement Folin phenol method its enzyme work is measured, the result sees Fig. 8.
Can be known that by Fig. 8 inorganic nitrogens such as the relative urea of organonitrogens such as peptone, Carnis Bovis seu Bubali cream, ammonium chloride produce the proteolytic enzyme enzyme and live higherly, enzyme is lived the highest when especially soaking powder and be nitrogenous source with yeast.
(3) the research embodiment of Validase TSP Concentrate II influence factor
1.pH influence to protease activity
Prepare casein solution as substrate with 1 times of dilution of damping fluid enzyme liquid of different pH values and with the damping fluid of corresponding pH value, with improvement Folin phenol method its enzyme work is measured, its result sees Fig. 9.
As shown in Figure 9, pH is when 5.0-5.7, and enzymic activity raises along with the rising of pH value; During pH5.7, it is the highest that enzymic activity reaches; PH＞5.7 o'clock, enzymic activity is on a declining curve.Therefore, this enzyme is more stable under weak acid environment, and the ph optimum of enzyme is 5.7.
2. temperature is to the influence of protease activity
After the 1 times of dilution of phosphoric acid buffer of enzyme liquid with pH 7.5, under 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, be substrate respectively with casein solution, measure enzyme activity with improvement Folin phenol method, its result sees Figure 10.
Show that like Figure 10 between 25-40 ℃, enzymic activity rises along with the rising of temperature; In the time of 40 ℃, it is the highest that enzymic activity reaches; After surpassing 40 ℃, enzymic activity begins to descend.Therefore, the optimal reactive temperature of proteolytic enzyme is 40 ℃.
3. common metal ion is to the influence of protease activity
Respectively with CuSO
4, FeSO
4, MgSO
4, MnSO
4, ZnSO
4, BaCl
2, AlCl
3Be made into the solution of 0.01mol/L; Respectively get in the 1mL enzyme liquid that 1mL joins purifying respectively and (be about to 2 times of enzyme liquid dilutions), 40 ℃ handle 10min after, and compare with the enzyme liquid that does not add metals ion; Measure the residual protein enzyme activity with improvement Folin phenol method, its result sees Figure 11.
Can know by Figure 11, through Al
3+, Fe
2+After the processing, enzymic activity is lower than contrast, shows that these two kinds of pair ion protease activities have restraining effect in various degree, and Al
3+Restraining effect obviously be better than Fe
2+Through Cu
2+, Mg
2+, Mn
2+, Ba
2+, Zn
2+After the processing, enzymic activity is higher than contrast, shows that these several metal ion species have promoter action to protease activity, wherein Mn
2+And Cu
2+Promoter action especially strong, enzyme is lived and have been increased by 42.4% and 38.9% respectively, Mg
2+, Zn
2+, Ba
2+Promoter action close, enzyme is lived and have been increased by 9.4%, 8.2% and 7.6% respectively.
Claims (8)
1. a bacterium multienzyme bacterial strain produces the method for proteolytic enzyme; It is characterized in that: may further comprise the steps: with subtilis (Bacillus subtilis) 1.1111; Be preserved in Chinese typical culture collection center, its preserving number is that CCTCCNO:M 2011286 thorns are planted on the casein agar substratum, in 37 ℃ of incubators, cultivates 24h; Discovery has the hydrolysis circle to produce, and hydrolysis circle and colony diameter reach 4.80.
2. produce the method for proteolytic enzyme according to a bacterium multienzyme bacterial strain described in claims 1, it is characterized in that: in initial pH value of medium 7.0, inoculum size is 3%, plants 22h in age, 37 ℃ of culture temperature, and during fermentation time 24h, the proteolytic enzyme enzyme work that is produced reaches the highest.
3. the method for producing proteolytic enzyme according to the said bacterium multienzyme bacterial strain of claims 1 is characterized in that: with 1% Zulkovsky starch be optimum carbon source, 1% yeast to soak powder be optimum nitrogen source, make the fermention medium component reach optimum.
4. the method for producing proteolytic enzyme according to claims 1 said bacterium multienzyme bacterial strain, it is characterized in that: the proteolytic enzyme that is produced is better at temperature 25-40 ℃ of scope internal stability.
5. the method for producing proteolytic enzyme according to claims 4 said bacterium multienzyme bacterial strains, it is characterized in that: the proteolytic enzyme optimum temperature that is produced is 40 ℃.
6. the method for producing proteolytic enzyme according to claims 1 said bacterium multienzyme bacterial strain, it is characterized in that: the proteolytic enzyme that is produced is more stable under the pH5.5-6.5 condition.
7. the method for producing proteolytic enzyme according to claims 6 said bacterium multienzyme bacterial strains is characterized in that: the proteolytic enzyme that is produced is value 5.7 at ph optimum.
8. the method for producing proteolytic enzyme according to claims 1 said bacterium multienzyme bacterial strain is characterized in that: at the proteolytic enzyme that is produced, and Mn
2+And Cu
2+The enzyme that can significantly improve this proteolytic enzyme is lived Mg
2+, Ba
2+, Zn
2+This proteolytic enzyme also there are certain promoter action, Al
3+, Fe
2+This proteolytic enzyme is had restraining effect in various degree, and Al
3+Restraining effect obviously be better than Fe
2+
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Cited By (3)
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| CN104328097A (en) * | 2014-07-17 | 2015-02-04 | 河南科技大学 | Method for producing cellulase, mannase and glucoamylase from bacillus subtilis |
| CN104762229A (en) * | 2015-03-24 | 2015-07-08 | 福建省农业科学院农业工程技术研究所 | A bacillus subtilis strain and applications thereof |
| CN110257305A (en) * | 2019-07-24 | 2019-09-20 | 浙江海洋大学 | The breeding method of the Shewanella of high yield alkali protein |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104328097A (en) * | 2014-07-17 | 2015-02-04 | 河南科技大学 | Method for producing cellulase, mannase and glucoamylase from bacillus subtilis |
| CN104762229A (en) * | 2015-03-24 | 2015-07-08 | 福建省农业科学院农业工程技术研究所 | A bacillus subtilis strain and applications thereof |
| CN104762229B (en) * | 2015-03-24 | 2018-01-09 | 福建省农业科学院农业工程技术研究所 | A kind of bacillus subtilis strain and its application |
| CN110257305A (en) * | 2019-07-24 | 2019-09-20 | 浙江海洋大学 | The breeding method of the Shewanella of high yield alkali protein |
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Application publication date: 20120919 |