CN101781641B - Optimization method of culture conditions of alkaline pectinase gene engineering bacteria - Google Patents
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Abstract
The invention relates to an optimization method of culture conditions of alkaline pectinase gene engineering bacteria, comprising the following steps: (1) adopting a single-factor experiment to optimize the fermentation medium formation of the alkaline pectinase gene engineering bacteria to obtain a preliminarily optimized fermentation medium; (2) optimizing a target strain medium with a response surface methodology to obtain the target strain medium; and (3) optimizing the fermentation culture conditions to the target strain by a post-optimizing medium to obtain the optimization culture conditions. The invention gropes a set of optimal fermentation culture conditions to ensure that enzymatic activity produced by engineering bacteria fermentation reaches 758.7825U/ml which is improved by 50 times compared with the activity of alkaline pectinase produced by original wide strains and is improved by 3 times compared with the enzymatic activity of alkaline pectinase produced by LB fermentation medium fermentation gene engineering bacteria, so that the invention has an important meaning for improving the market competitiveness of industrial enzyme.
Description
Technical field
The invention belongs to field of microorganism engineering, especially a kind of optimization method of culture conditions of alkaline pectinase gene engineering bacteria.
Background technology
Polygalacturonase (pectinase) is the general name of the enzyme of one type of decompose pectin matter, is the prozyme that contains various ingredients.Polygalacturonase is distributed widely in higher plant and the mikrobe, but extraction receives many restrictions of resource, state of the art and fails really to realize industrialization always from plant, and therefore microbe-derived polygalacturonase becomes the research focus.Research to acid pectase has both at home and abroad had certain basis, but less to the research report of alkaline pectase especially pectin lyase (pectate lyase).
Alkaline pectase is meant the polygalacturonase of the righttest action pH in alkaline range; Be the emerging content in the polygalacturonase research field, be mainly used in and be used as environment amenable biological catalyst in the textile industry, with the traditional alkali high temperature steaming technology of the biological concise replacement of soft enzyme; Realize green cleaner production; Both can reduce the alkali exhaust emission, can reduce heating energy consumption and rinsing water amount again, improve the processing and the use properties of yarn fabric simultaneously.Thereby along with the continuous fast development of biotechnology, the application potential of alkaline pectase in textile industry also more and more receives everybody concern.
From Horikoshi in 1972, K. delivered first piece of paper about genus bacillus production alkaline pectase and begins, and states such as Japan begin to be devoted to the research of alkaline pectase.India Mukesh Kapoor has reported production, purifying and the zymologic property of Bacillus sp.MG-cp-2 alkaline polygalacturonase in calendar year 2001; Confirm that this strain fermentation vigor is 47u/mL; Vigor promotes and is 98u/mL behind the medium optimization; Stable in pH7.0～12.0 scopes during room temperature, can effectively be applied to ramie and sunn comes unstuck; German scholar was measured the genome sequence of polygalacturonase bacterial classification Bacillus licheniformis DSM13 in 2004.The scholar of states such as the U.S., Holland, Britain, Spain also reports the contents such as microorganism resource, fermentation, purification process, zymologic property, secretion regulation and control and molecular biology of alkaline pectase respectively; Research object is except above-mentioned genus bacillus; Also comprise some phytopathogens, like chrysanthemum Erwinia, onion burkholderia etc.
In order to obtain high yield, fine alkaline pectin enzyme product; Since nineteen ninety; The foreign scholar produces alkaline pectase bacterial classification aspect to research emphasis from screening and progressively turns in the research of producing enzyme gene and genetic engineering bacterium; Especially (optimal ph is 7.5～9.5 along with Denmark Novo Nordisk (Novo Nordisk) alkaline pectase Bioprep in 1999 (be the zymin that a kind of genus bacillus of improvement of genes is processed through fermentation, its optimum pH is 7.5～9.5, and Applicable temperature is about 55 ℃) and alkaline pectate lyase ScourzymeL; Selecting for use then of temperature decided according to the pH value; At 8～8.5 o'clock, temperature was grasped at 55 ℃～60 ℃ like the pH value) product come out, people have turned to attention the research of enzyme gene and clone technology thereof more and more.Spain, France, Holland, Japanese scholar successfully express the alkaline pectase gene clone of Bacillus sp. (2000), Erwiniachrysanthemi3937, Thermotoga maritima (Thermotoga maritima, 2003), four kinds of bacterial strains of Bacillus subtilis (2002) in intestinal bacteria respectively.
Domestic research to polygalacturonase starts from the sixties in 20th century; Begin at the beginning of nineteen ninety alkaline pectase is studied; Lot of documents reports the aspects such as industrial application of the screening of polygalacturonase bacterial classification, traditional selection by mutation, production technique and enzyme, but less about the research contents of fermentation, enzyme preparation and the molecular biology genetic breeding aspect of alkaline pectase.Bacterial classification mainly is a genus bacillus; Like bacillus gibsonii, gram Lloyd's genus bacillus; Subtilis No.46, XZ2, B13, LH-16, WSHB04-02 and bacillus pumilus WSH03-09, RK9 etc. have a liking for salt in addition in addition and have a liking for alkali bacterium Alkalibacterium sp.F26 etc.Chinese scholar in 2006 discriminate east wait dawn with the heat-resisting pectin lyase gene pel9A of Clostridium stercorarium (heat-resisting clostridium spp) be cloned into expression vector pET28 α then transformed into escherichia coli express; The same year, Zhu Gebin etc. have utilized temperature control vector construction alkaline pectin enzyme engineering bacteria.Increasing research unit has been applied to carry out under the industrial general trend effective trial of alkaline pectase molecular biology operation at genetic engineering bacterium; Mostly molecular biology operation or be main contents with the coli expression system; Or be research object with the subtilis expression system; But being chosen in when being purpose with production of big multichip carrier and host all shown certain deficiency, and for example the use meeting of temperature control carrier and shuttle plasmid is because of needing conditions such as temperature, inductor to increase production cost in the expression.
China's alkaline pectase research is totally started late; The industrial enzymes of zymin company 80% all is to be obtained by the genetic engineering bacterium fermentative prodn abroad; The present invention is after successfully making up a strain alkaline pectinase gene engineering bacteria; Make every effort to search out the higher fermentation culture method of yield of enzyme, with competition and the development space of widening the national industry zymin.
Summary of the invention
The object of the present invention is to provide the optimization method of the culture conditions of alkaline pectinase gene engineering bacteria that a kind of yield is high, method is simple, economical and practical, this method can effectively improve the market competitiveness of the enzymatic productivity and the industrial enzyme of alkaline pectinase gene engineering bacteria.
The technical scheme that the present invention takes is:
A kind of optimization method of culture conditions of alkaline pectinase gene engineering bacteria, step is following:
(1) adopts experiment of single factor that the fermention medium of alkaline pectinase gene engineering bacteria is formed and be optimized, obtain the fermention medium of initial optimization;
(2) through the response surface method fermention medium of optimizing is further optimized again, obtained the target bacterial classification and optimize substratum;
(3) adopt optimization back substratum, the target bacterial classification is carried out fermentation culture conditions optimization, culture condition is optimized.
And said experiment of single factor is formed the step that is optimized to the fermention medium of alkaline pectinase gene engineering bacteria and is:
(1) different carbon sources and concentration are confirmed optimum carbon source and optimum addition to producing the influence of enzyme;
(2) different nitrogen sources and concentration are confirmed optimum nitrogen source and optimum addition to producing the influence of enzyme;
(3) different metal ion and inorganic salt and concentration thereof are confirmed the kind and the optimum addition of best metal ion and inorganic salt to producing the influence of enzyme.
And said response surface method to the step that substratum is optimized is:
(1) carries out data analysis according to Plackett-Burman method contrived experiment step and utilization minitab software;
(2) confirm level of factor: according to the climbing experiment of Plackett-Burman experimental result design, filtering out has the factor of material impact to producing enzyme;
(3) adopting the center combination contrived experiment to optimize substratum forms: center combination principle of design and the experiment of climbing experiment gained consequence devised response surface analysis according to Box-Benhnken, important factor is optimized, and the final fermention medium of confirming is formed.
And; The step that said target bacterial classification optimization substratum carries out fermentation culture conditions optimization is: optimizing under the culture medium condition; Adopt experiment of single factor that the alkaline pectinase gene engineering bacteria fermentation culture conditions is optimized, confirm the best fermentation culture conditions of optimum target bacterial classification.
And said fermentation culture conditions comprises: bacterial classification kind age, initial pH, inoculum size, leavening temperature and shake a bottle rotating speed.
Advantage of the present invention and positively effect are:
1, the present invention is Object of Development with the engineering strain; Carry out the substratum fermentation test with experiment of single factor and response surface method, the utilization computer software is analyzed experimental data, and it is optimized comprehensively; And further fermentation culture conditions is optimized on this basis; Grope the optimum fermentation culture conditions of a cover, the enzyme work that engineering bacterium fermentation is produced reaches 758.7825U/ml, produces the alkaline pectin enzymic activity than original wild type strain and has improved 50 times; The vigor that produces alkaline pectase than LB substratum fermentation genetic engineering bacterium has improved 3 times, and the market competitiveness that improves industrial enzyme is had very important meaning.
2, the present invention is with alkaline pectinase gene engineering bacteria WB600/pWB-pelG
2521 are optimized the fermentation culture conditions of producing bacillus subtilis alkaline pectase for example; Set up the zymotechnique of alkaline pectinase gene engineering bacteria; Obtain best fermentation condition, this fermentation condition is applicable to industrialized production and application, has very big theory and using value.
Description of drawings
Fig. 1 is genetic engineering bacterium WB600/pWB-pelG of the present invention
2521 Electronic Speculum figure;
Fig. 2 and Fig. 3 are respectively X of the present invention
3=0 o'clock Y
1=f (X
1, X
2) stereoscopic analysis figure and isogram;
Fig. 4 and Fig. 5 are respectively X of the present invention
2=0 o'clock Y
1=f (X
1, X
3) stereoscopic analysis figure and isogram;
Fig. 6 and Fig. 7 are respectively X of the present invention
1=0 o'clock Y
1=f (X
2, X
3) stereoscopic analysis figure and isogram.
Embodiment
Below in conjunction with embodiment, the present invention is further specified, following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
One, description of test
1, experimental strain is engineering strain WB600/pWB-pelG
2521, this bacterial strain is subtilis (having six protease-deficients), and this bacterial classification and construction process are applied for a patent, and patent No. application is: 200910068617.6, and the applying date is: on April 24th, 2009.This produces bacterial strain 37 ℃ of constant temperature culture 24h on agar slant, and thalline is grown on the inclined-plane fully, is creamy white from seeing in appearance, is mostly rod-short in Electronic Speculum hypothallus shape, and 4 ℃ of preservations are for use.
2, experimental strain WB600/pWB-pelG
2521 slant mediums: peptone 1%, yeast extract 0.5%, NaCl1%, the pH value is 6.8～7.0, agar powder 1.5%.
3, experimental strain WB600/pWB-pelG
2521 seed culture mediums: peptone 1%, yeast extract 0.5%, NaCl1%, the pH value is 6.5～7.0, sterilising conditions is 121 ℃, 20min.
4, the pre-treatment of experimental raw
Wheat bran: get 50g wheat bran+about 500ml water, stir and boil 1h, be settled to 500ml, the centrifugal 5min of 4000r/min.Calculate by 100g/L when getting supernatant and getting 10% bran water extract and use.
Soybean cake powder: commercially available soybean cake powder is handled through 40 mesh sieves, gets 50g soybean cake powder+about 450ml water+0.02g neutral protease, 45 ℃ of water bath processing 45min; Be settled to 500mL; The centrifugal 5min of 4000r/min gets supernatant, calculates by 100g/L when obtaining 10% soybean cake powder water extract use.
Semen Maydis powder: commercially available Semen Maydis powder is handled through 40 mesh sieves, gets 50g Semen Maydis powder+about 450mL water+38 μ L α-middle temperature glycase, 65 ℃ of water bath processing 1h; Be settled to 500mL, the centrifugal 5min of 4000r/min gets supernatant; Obtain 10% Semen Maydis powder water extract, press 100g/L during use and calculate.
Potato dry powder: get 50g potato dry powder+about 450mL water+40 μ L α-middle temperature glycase, 65 ℃ of water bath processing 1h are settled to 500mL, and the centrifugal 5min of 4000r/min gets supernatant, calculate by 100g/L when obtaining 10% potato dry powder water extract use.
5, the mensuration of alkaline pectin enzyme activity
The enzyme activity unit definition: 1mL enzyme liquid is at 45 ℃, and pH is under 9.0 conditions, and PM makes the polygalacturonic acid cleavage produce the enzyme amount of the unsaturated polyester galacturonic acid of 1 μ mol.
Measuring method is following:
The preparation of enzyme liquid: the polygalacturonase bacterial classification is after fermentation culture finishes, and 8000 left the heart 5 minutes, got supernatant.Dilute different multiples and stoste replicate(determination) with damping fluid; The enzyme activity determination method: comprise thick enzyme diluent 20 μ L in the reaction system, (pH 9.0, contain the CaCl of 0.44mmol/L for the glycocoll-NaOH of 0.2% polygalacturonic acid (0.2mol/L) damping fluid
2) 2mL, reaction conditions is 45 ℃ of incubation 15min, with the phosphoric acid termination reaction of 3mL0.03mol/L, measures its absorbance at the 235nm place.Enzyme is blank: behind the substrate insulation 2min with the above-mentioned buffer system preparation of 2mL, add the phosphoric acid and the enzyme liquid of 20 μ L with the deactivation of the identical extension rate of experiment appearance, mixing of 3mL0.03mol/L successively.Other operations are identical with experiment appearance.
In the formula: 4600 (Lmol
-1Cm
-1)-unsaturated polyester galacturonic acid is at the molar absorptivity at 235nm place
T (min)-time of enzymatic reacting (in the linearity range of enzyme reaction)
B (cm)-cuvette thickness
Through simplifying: enzyme activity (U/mL)=3.6232 * extension rate * OD
235
Two, a kind of optimization method of culture conditions of alkaline pectinase gene engineering bacteria, the step of method is following:
1, experiment of single factor is to alkaline pectinase gene engineering bacteria (WB600/pWB-pelG
2521) fermention medium composition is optimized.
(1) different carbon sources and concentration are to producing the influence of enzyme
According to lot of documents of being consulted and breadboard condition; Glucose, sucrose, lactose, dextrin, Semen Maydis powder, sweet potato starch, W-Gum, potato dry powder, wheat bran and steeping water with identical carbon content is that carbon source is carried out fermenting experiment respectively; And compare with the LB substratum; Take all factors into consideration according to experimental result and raw materials cost, with wheat bran as carbon source, and addition when being 30g/L enzyme live the highest.
(2) different nitrogen sources and concentration are to producing the influence of enzyme
According to lot of documents of being consulted and breadboard condition, be carbon source with wheat bran (30g/L), respectively with peptone, soy peptone, yeast extract paste, steeping water, soybean cake powder, Semen Maydis powder, (NH
4)
2SO
4, KNO
3, yeast powder, urea and cottonseed meal be that nitrogenous source produces the enzyme experiment, take all factors into consideration according to experimental result and cost, with soybean cake powder as nitrogenous source, and addition during for 40g/L the product enzyme the highest.
(3) different metal ion and inorganic salt and concentration are to producing the influence of enzyme
Some metals ion can play certain influence to growth and the metabolism of bacterium, is carbon source with wheat bran (30g/L) therefore, and soybean cake powder (40g/L) is that nitrogenous source is respectively to Na
+, Fe
2+, Ca
2+, Mn
2+, Cu
2+, Zn
2+, K
+Deng investigating.
Through single factor optimization, confirmed that the enzymatic production substratum is: wheat bran 30g/L, soybean cake powder 40g/L, NaCl 5g/L, phosphoric acid salt 0.05mol/L, FeSO
40.005mol/L.
2, response surface method (RSM) is to optimizing substratum
(1) the Plackett-Burman design method is the part factorial method, and the design arrangement of experiment selected N=12 is to wheat bran (X
1), soybean cake powder (X
2), phosphoric acid salt (X
4), NaCl (X
5), FeSO
47H
2O (X
7) five factors investigate, each factor is got two levels respectively, and low-level is the result of experiment of single factor, and high level is got low-level 1.25 times, and two null term X are added in design
3, X
6, a central point, response value is that enzyme is lived (Y), experimental design and experimental result are seen table 1,2.
Table 1 Plackett-Burman experimental design and result
Regression analysis is carried out in the experimental design of utilization minitab software his-and-hers watches 1, and carries out the variance analysis check, and the Several Factors of confidence level＞95% is: soybean cake powder, phosphoric acid salt and FeSO
4, and the confidence level of other Several Factors is all＜95%.Analytical results is following:
Table 2 PB experiment analysis results
Effect?Estimates?for?Y1
| Term | Estimate | Std?Err | t | Pr＞|t| |
| -3.446667 | 6.530464 | -0.52778 | 0.634196 | |
| X1 | 8.3223333 | 3.265232 | 2.548773 | 0.084033 |
| X2 | -30.21867 | 3.265232 | -9.25468 | 0.002669 |
| X3 | -5.742333 | 3.265232 | -1.75863 | 0.17688 |
| X4 | -16.11733 | 3.265232 | -4.93605 | 0.015945 |
| X5 | -19.70833 | 3.265232 | -6.03581 | 0.009119 |
| X6 | -7.505333 | 3.265232 | -2.29856 | 0.105127 |
| X7 | 7.611 | 3.265232 | 2.330922 | 0.102062 |
Fit?Statistics?for?Y1
| Mean | 671.7695 |
| R-square | 98.24％ |
| Adj.R-square | 93.53％ |
| RMSE | 5.655547 |
| CV | 0.841888 |
(2) confirm level of factor
Can know that by the Plackett-Burman experiment in the fermenting process of alkaline pectase, these three factors of soybean cake powder, phosphoric acid salt and NaCl have than significant effects producing enzyme; And be negative effect; Should reduce, according to the climbing experiment of Plackett-Burman experimental result design, result such as table 3.
Table 3 climbing experimental design and result
(3) adopting the center combination contrived experiment to optimize substratum forms
Center combination principle of design according to Box-Benhnken is respectively got 3 levels with climbing experiment gained result 3 factors, has designed 3 factors, 3 levels response surface analysis experiment and the result and the analysis (like table 4,5) of totally 15 test points.
Choosing of table 4 center combination design factor level
Design of table 5 center combination and result
Through analyses such as recurrence and variances, the result is following: Effect Estimates for Y1
| Term | Estimate | Std?Err | t | Pr＞|t| |
| X1 | -14.10075 | 4.487011 | -3.14257 | 0.025592 |
| X2 | 12.016625 | 4.487011 | 2.678091 | 0.04392 |
| X3 | -15.86413 | 4.487011 | -3.53557 | 0.016641 |
| X1*X1 | -27.02225 | 6.604702 | -4.09137 | 0.009434 |
| X1*X2 | -2.28025 | 6.345592 | -0.35934 | 0.734015 |
| X1*X3 | -13.28325 | 6.345592 | -2.0933 | 0.090519 |
| X2*X2 | -50.204 | 6.604702 | -7.60125 | 0.000626 |
| X2*X3 | 4.9805 | 6.345592 | 0.784876 | 0.468066 |
| X3*X3 | -16.3265 | 6.604702 | -2.47195 | 0.056395 |
ANOVA?for?Y1
| ?Source | DF | SS | MS | F | Pr＞F |
| ?Model | 9 | 17319.9 | 1924.433 | 11.94809 | 0.006926 |
| ?(Linear) | 3 | 4759.207 | 1586.402 | 9.849384 | 0.015369 |
| ?(Quadratic) | 3 | 11734.89 | 3911.632 | 24.28587 | 0.00208 |
| ?(Cross?Product) | 3 | 825.7986 | 275.2662 | 1.709026 | 0.279855 |
| ?Error | 5 | 805.3308 | 161.0662 | ||
| ?(Lack?of?fit) | 3 | 740.6978 | 246.8993 | 7.640037 | 0.117936 |
| ?(Pure?Error) | 2 | 64.633 | 32.3165 |
Fit?Statistics?for?Y1
| Mean | 703.9012 |
| R-square | 95.56％ |
| Adj.R-square | 87.56％ |
| RMSE | 12.69118 |
| CV | 1.802978 |
Explain through analytical resultss such as recurrence and variances: once item is to Y
1Remarkably influenced is arranged, and the influence of P=0.277 explanation cross-product term is not remarkable.R
2=95.56%, the expression confidence level is very high, and it is also very little to lose the F value of intending item, and it is fine to explain that this equation fits experiment, and experimental error is little.Obtain regression equation by experimental result: Y
1=-7519.67+153.0928*X
1+ 178277.3*X
2+ 705.4752*X
3-1.688891*X
1* X
1-114.0125*X
1* X
2-6.641625*X
1* X
3-2008160*X
2* X
2+ 1992.2*X
2* X
3-65.306*X
3* X
3
Stereoscopic analysis figure and isogram that software drew have shown the influence of the addition of soybean cake powder, phosphoric acid salt and NaCl to the product enzyme very intuitively, and have shown Y
1Optimum value is arranged, and ridge is analyzed through the mountain range, and soybean cake powder adds 35.3345g/L, and phosphoric acid salt adds 0.04552mol/L, and when NaCl added 4.2988g/L, enzyme work had peak, and peak is 758.7825U/mL.
Final definite fermention medium consists of: wheat bran 30g/L, soybean cake powder 35.3345g/L, phosphoric acid salt 0.04552mol/L, NaCl 4.2988g/L, FeSO
40.005mol/L.
3, fermentation culture conditions optimization method
Adopt experiment of single factor to alkaline pectinase gene engineering bacteria (WB600/pWB-pelG
2521) the kind age in the fermenting experiment, initial pH, inoculum size, leavening temperature and shake fermentation condition such as bottle rotating speed and optimize.The result shows that be 12h kind of age, and initial pH is 7.0, and inoculum size is 4%, and leavening temperature is 37 ℃, and when shaking bottle rotating speed and being 200r/min, enzyme work can reach 780U/mL.
Claims (2)
1. the optimization method of a culture conditions of alkaline pectinase gene engineering bacteria, it is characterized in that: step is following:
(1) adopts experiment of single factor that the fermention medium of alkaline pectinase gene engineering bacteria is formed and be optimized, obtain the fermention medium of initial optimization;
(2) through the response surface method fermention medium of optimizing is further optimized again, obtained the target bacterial classification and optimize substratum;
(3) the target bacterial classification is optimized substratum and carry out fermentation culture conditions optimization, the culture condition that is optimized,
Said experiment of single factor is formed the step that is optimized to the fermention medium of alkaline pectinase gene engineering bacteria:
(1) different carbon sources and concentration are confirmed optimum carbon source and optimum addition to producing the influence of enzyme;
(2) different nitrogen sources and concentration are confirmed optimum nitrogen source and optimum addition to producing the influence of enzyme;
(3) different metal ion and inorganic salt and concentration thereof are confirmed the kind and the optimum addition of best metal ion and inorganic salt to producing the influence of enzyme,
Said response surface method to the step that substratum is optimized is:
(1) carries out data analysis according to Plackett-Burman method contrived experiment step and utilization minitab software;
(2) confirm level of factor: according to the climbing experiment of Plackett-Burman experimental result design, filtering out has the factor of material impact to producing enzyme;
(3) adopting the center combination contrived experiment to optimize substratum forms: center combination principle of design and the experiment of climbing experiment gained consequence devised response surface analysis according to Box-Benhnken, important factor is optimized, and the final fermention medium of confirming is formed,
The step that said target bacterial classification optimization substratum carries out fermentation culture conditions optimization is: optimizing under the culture medium condition; Adopt experiment of single factor that the alkaline pectinase gene engineering bacteria fermentation culture conditions is optimized, confirm the best fermentation culture conditions of optimum target bacterial classification.
2. the optimization method of a kind of culture conditions of alkaline pectinase gene engineering bacteria according to claim 1, it is characterized in that: said fermentation culture conditions comprises: bacterial classification kind age, initial pH, inoculum size, leavening temperature and shake a bottle rotating speed.
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