CN101161811A - Method for sifting and cultivating bacillus thuringiensis of high-yield thermostable proteinase - Google Patents
Method for sifting and cultivating bacillus thuringiensis of high-yield thermostable proteinase Download PDFInfo
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- CN101161811A CN101161811A CNA2006100693392A CN200610069339A CN101161811A CN 101161811 A CN101161811 A CN 101161811A CN A2006100693392 A CNA2006100693392 A CN A2006100693392A CN 200610069339 A CN200610069339 A CN 200610069339A CN 101161811 A CN101161811 A CN 101161811A
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Abstract
A screening and cultivation method for Bacillus thuringiensis of high-yield high temperature resisting protease includes the following steps: high-yield high temperature resisting protease Bacillus thuringiensis Bt140 is obtained through adopting casein substrate agar to identify flat primary screen and fermenting enzyme activity measuring secondary screening; Plackett-Burman design and response surface analytical method are adopted to obtain optimal fermentation zymogenic formula and fermentation condition for Bt140 quickly; finally, shaking fermentation level reaches to 918.56U/mL. During adopting Bt140 to produce high temperature resisting protease, spore collection for producing pesticide after fermentation is not influenced; therefore, the Bacillus thuringiensis combines the production capacity of protease and pesticide. For example, when 1 ton Bacillus thuringiensis is used for fermentation, corresponding Bt pesticide is obtained along with 60 to 90 Kg/ megaunit enzyme powder.
Description
Affiliated technical field the present invention relates to the screening and the cultural method, particularly screening of high-yield thermostable proteinase Bacillus thuringiensis and cultural method of high proteinase yield bacterial strain.
Background technology
Proteolytic enzyme is a kind of important zymin, is widely used in food, medicine, washing composition, weaving and the leather industry.Advantages such as thermostable proteinase possesses heat-resisting, anti-denaturing agent, organic solvent-resistant, its research is particularly paid attention to.People are from Thermus aquatics, Bacillusthermoproteolyticus, B.stearothermophilus, Thermoactinomycesvulgaris, Sulfolobus solfataricus is separated to various types of high temperature proteolytic enzyme in the high temperature bacterium such as Pyrococus furiosus.The high temperature protein fungus yields poorly the production cost height at present.Bacillus thuringiensis (Bacillus thuringiensis is called for short Bt) is a kind of very important entomiasis indigenous bacteria, is the most successful up to now microbial pesticide.Its main application is not seen the report that utilizes Bt to produce high temperature resistant enzyme both at home and abroad as yet as biological pesticide.
The cycle of common fermentation optimization process need is longer, and workload is big.Adopt the method for statistical mathematics can comparatively fast reach optimization aim, can set up relevant mathematical model simultaneously, the prediction optimum value.Obtain optimal value and have more science, reliability and accuracy.The Plackett-Burman design of present two levels design is to be considered to the most effective statistical method in optimizing process with response surface analysis method (Response surface methodology is called for short RSM).
Summary of the invention
Purpose of the present invention: the Bt bacterial strain of screening high-yield thermostable proteinase, adopt the method for mathematical statistics to carry out this strain fermentation condition optimizing, obtain optimum substratum and culture condition.
1, bacterial strain screening method may further comprise the steps:
Primary dcreening operation: under 55 ℃ condition, utilize casein agar to identify that the dull and stereotyped bacterium colony agar block fermented liquid initial with it that primary dcreening operation Bt bacterial strain is provided that can buy market detects respectively, choosing caseinhydrolysate formation transparent circle is the primary dcreening operation bacterial strain than big and the tangible bacterial strain of hydrolysis circle;
Multiple sieve: to the checking of fermenting of described primary dcreening operation bacterial strain, be substrate with the casein, adopt the ultraviolet spectrometry assay method to carry out enzyme activity determination, the bacterial strain Bt140 that chooses high enzyme work is the bacterial strain of high-yield thermostable proteinase, and its initial fermentation level is 263.43U/mL.
2, fermentation condition optimization:
(1) Bt140 morphological specificity and physiological and biochemical property
Adopt microbiological analysis further to understand Bt140 cell morphological characteristic and common biochemical reactions feature.
Table 1 Bt 140 physio-biochemical characteristics
(2) the Plackett-Burman design screening material impact factor
Obtain producing relevant substratum and 8 factors of culture condition of enzyme by single factor shaker test with Bt140.Adopt Plackett-Burman design screening to produce the enzyme material impact factor, the design of selecting test number (TN) N=12 for use is to initial pH, loading amount, glucose (%), yeast powder (%), soybean cake powder (%), KH
2PO
4(%), MgSO
4(%) and CaCO
3(%) wait 8 factors to investigate, the results are shown in Table 2, table 3, find that confidence level is nutrient media components greater than 80% Several Factors.They to producing the enzyme influence are in proper order: soybean cake powder＞yeast powder＞glucose＞CaCO
3
The test design of the Plackett-Burman of table 2 N=12 and result
The main effect of each factor of table 3
(3) response surface analysis is optimized the substratum composition
To three important factors, be soybean cake powder, yeast powder, glucose, adopt the center combination design of Box-Behnken, designed 3 factors, 3 levels totally 15 test points, carry out response surface analysis optimization, set up quadratic response face regression model and and then sought the optimal response factor level.The estimated value of regression coefficient sees Table 4 in the regression equation, and it is as follows to obtain the match quadratic regression equation: Y=759.36+51.33*X1+7.21*X2-20.98*X3+12.45X1*X1-61.21X2*X2-74.52X3*X3-29.47X1*X2+1.50X2*X3-0.53X1*X3.Model credibility analysis in table 5.Quadratic response face regression model is remarkable (coefficient of determination R
2=0.9472), the model-fitting degree is fine, illustrate that these 3 factors and quadratic term thereof can explain that Y changes 94.72%, model returns P value=0.0104; The not enough P value of match=0.6561, it is not remarkable to illustrate that model loses plan, returns significantly, produces the prediction of enzymic fermentation Optimization Theory so this model can be used for Bt.The optimal medium prescription (weight percent) of being released high yield enzyme level by model is: soybean cake powder 1.8%, yeast powder 0.36%, glucose 0.14%, KH
2PO
40.25%, MgSO
47H
2O 0.035%, CaCO
30.05%, all the other are water.Initial pH value is 7.0.Model predication value is 826.66 U/mL, and the experimental verification value is 837.71U/mL, trial value and Model Calculation value differ+and 1.34%.
The estimated value of regression coefficient in table 4 regression equation
Table?6 Estimated?value?of?coefficient?in?the?regression?equation
Table 5 analysis of variance table
(4) culture condition optimization
Adopt single factor sieve method that the Bt140 condition of enzyme production is optimized, determine best fermentation condition: described condition of enzyme production comprises leavening temperature, liquid amount, inoculum size and fermentation time.Substratum loading amount 35mL/250mL triangular flask, 31 ℃ of culture temperature, shaking bottle rotating speed is 250r/min, inoculum size is a 0.75mL (bacteria suspension concentration 7 * 10
6Individual/mL), and initial pH7.0, fermentation time is 24h.
(5) Bt140 proteolytic enzyme enzymatic property
The most suitable operative temperature of Bt140 proteolytic enzyme is 55 ℃, the basic non-inactivation of 1h under 55 ℃ of conditions, and 1h still has the vigor more than 60% under 60 ℃ of conditions, is typical thermostable proteinase.
3, Bt140 fermentation condition optimization result
Adopt the soybean cake powder substratum, best product enzyme prescription is as follows by weight percentage: soybean cake powder 1.8%, yeast powder 0.36%, glucose 0.14%, KH
2PO
40.25%, MgSO
47H
2O 0.035%, CaCO
30.05%, all the other are water;
Best enzymatic production culture condition: substratum loading amount 35mL/250mL triangular flask, 31 ℃ of culture temperature, shaking bottle rotating speed is 250r/min, inoculum size is a 0.75mL (bacteria suspension concentration 7 * 10
6Individual/mL), and initial pH7.0, fermentation time is 24h.
Bt140 proteolytic enzyme enzymatic property studies show that it is a kind of typical thermostable proteinase.
The invention has the advantages that: 1. adopt the Bt bacterial strain to prepare thermostable proteinase first;
2. high temperature proteolytic enzyme has purposes widely because it possesses advantages such as heat-resisting, anti-denaturing agent, organic solvent-resistant in industry;
3.Bt140 when producing thermostable proteinase, do not influence fermentation ends and collect gemma production sterilant, make the throughput of this bacterium with proteolytic enzyme and two kinds of products of sterilant.With 1 ton of fermentation be example except obtaining corresponding Bt sterilant, also obtain the enzyme powder of 60～90Kg, 10,000 units.
4. adopt statistical method that Bt140 is produced enzyme process and be optimized, obtain optimum formula and fermentation condition, Bt140 produces enzyme level significant raising, brings up to 918.56U/mL by the 263.43U/mL of primary dcreening operation.
Specific embodiment
Embodiment one: a kind of bacillus thuringiensis screening method of high-yield thermostable proteinase may further comprise the steps:
Casein substrate agar is identified flat panel production: agar identifies that dull and stereotyped prescription (weight percent) is a casein 2%, agar 2%, and all the other are water, pH is 7.0-7.2, is prepared into the agar plate that thickness is 3mm.
Primary dcreening operation 1: plate isolation 34 strain Bt bacterial strains, after waiting to grow sparse small colonies, take out the long agar fritter that single bacterium colony is arranged with punch tool, each bacterial strain is got 3 agar blocks, 34 strain agar blocks are placed on the same evaluation flat board, identify dull and stereotypedly for totally three, place 37 ℃ to continue to cultivate 24h, big according to the proteolysis circle that forms around the agar block, and the high bacterial strain of transparency is a superior strain.
Primary dcreening operation 2: the hole that the size one of utilizing punch tool to get 34 1.5mm on casein substrate agar evaluation flat board is put, the fermenting enzyme liquid of adding 15 μ L34 strain bacterial strains in hole with identifying the dull and stereotyped incubator 24h that places 55 ℃, repeats 3 flat boards respectively.Big according to formed proteolysis circle around the hole, and the high bacterial strain of transparency is a superior strain.
Can accurately obtain thermostable proteinase high yield bacterium 4 strains among the 34 strain Bt in conjunction with primary dcreening operation 1 and primary dcreening operation 2.
Multiple sieve: the primary dcreening operation bacterial strain is obtained the checking of further fermenting of 4 strain Bt bacterial strains, and be substrate with the casein, adopt the ultraviolet spectrometry assay method to carry out enzyme activity determination, obtain the bacterial strain Bt140 of high enzyme work, its initial fermentation level is 263.43U/mL.
Embodiment two: Bt140 produces the preparation of enzymic fermentation liquid
The preparation of Bt140 fermented liquid comprises that bacterial strain selection, slant culture, first order seed, slant medium are beef-protein medium.The Bt140 slant culture places under 37 ℃, cultivate 2d after, be stored in 4 ℃ the refrigerator.Connect one from the inclined-plane and encircle to the 250mL triangular flask, the substratum loading amount is the 35mL/250mL triangular flask, and substratum is a beef extract-peptone, under 31 ℃ condition, places on the shaking table of 250r/min, cultivates 15h, obtains first order seed.By the counting to first order seed, the inoculum size of control fermentation is a 0.75mL (bacteria suspension concentration 7 * 10
6Individual/mL), fermentative medium formula (weight percent) is: soybean cake powder 1.8%, yeast powder 0.36%, glucose 0.14%, KH
2PO
40.25%, MgSO
47H
2O 0.035%, CaCO
30.05%, all the other are water.The enzymatic production culture condition is: substratum loading amount 35mL/250mL triangular flask, and 31 ℃ of culture temperature, shaking bottle rotating speed is 250r/min, bacteria suspension concentration is 7 * 10
6Individual/mL, every bottle of inoculum size is 0.75mL, initial pH7.0, and fermentation time is 24h, promptly obtains Bt140 and produces enzymic fermentation liquid.
Embodiment three: utilize Bt140 to produce enzymic fermentation liquid and prepare thermostable proteinase and Bt sterilant
1, preparation Bt sterilant: get as embodiment one described Bt140 and produce enzymic fermentation liquid 1000L, regulate pH to 5.0, under 50r/min stirred, the ratio in 3% added flocculating agent A l
2(SO
4)
3.18H
2O30kg behind the precipitation 2h, adopts the butterfly centrifugal machine, and centrifuge speed is 8000r/min, and centrifugally operated 15-20min carries out bacterium liquid and separates, the product enzymic fermentation liquid after obtaining wet thallus and removing thalline.Collect wet thallus vacuum-drying 8h, get the Bt sterilant after the pulverizing; Product enzymic fermentation liquid behind the removal thalline is used to prepare thermostable proteinase.
2, preparation food grade thermostable proteinase: remove except that the product enzymic fermentation liquid 1000L behind the thalline, adopt 100 purpose filter cloth filtering macrobeads, filtered liquid is carried out ultrafiltration and concentration, the aperture of ultra-filtration membrane is 10000 dalton, concentration ratio is 1: 4, obtains the concentrated solution of thermostable proteinase.Add 70kg solid additive W-Gum and carry out spraying drying.Adopt centrifugal spraying machine, temperature of inlet air is 80 ℃, and 40 ℃ of temperature outs finally prepare 10000U/g food grade thermostable proteinase enzyme powder.
3, preparation technical grade thermostable proteinase enzyme powder: remove except that the product enzymic fermentation liquid 1000L behind the thalline, regulate below the pH to 4.0 with hydrochloric acid, adding ammonium sulfate makes its concentration be controlled at 55-60%, behind the precipitation 24h, the supernatant liquor that inclines will precipitate by link-suspended basket centrifuge, and centrifugal 20min removes mother liquor under the condition of 4000r/min, precipitation is placed vacuum-drying under 40 ℃ of conditions, wear into powder after the oven dry and prepare 5000U/g technical grade thermostable proteinase enzyme powder.
Claims (2)
1. the bacillus thuringiensis screening method of a high-yield thermostable proteinase may further comprise the steps:
(1) primary dcreening operation: under 55 ℃ condition, utilize casein agar to identify that flat board detects respectively the bacterium colony agar block fermented liquid initial with it for primary dcreening operation Bt bacterial strain, choosing caseinhydrolysate formation transparent circle is the primary dcreening operation bacterial strain than big and the tangible bacterial strain of hydrolysis circle;
(2) multiple sieve: to the checking of fermenting of described primary dcreening operation bacterial strain, be substrate with the casein, adopt the ultraviolet spectrometry assay method to carry out enzyme activity determination, the bacterial strain Bt140 that chooses high enzyme work is the bacterial strain of high-yield thermostable proteinase.
2. one kind is utilized the bacillus thuringiensis of high-yield thermostable proteinase to prepare the method for producing enzymic fermentation liquid, it is characterized in that
(1) adopt the Bt140 bacterial strain of high-yield thermostable proteinase as claimed in claim 1 for producing bacterial strain;
(2) adopt the soybean cake powder substratum, it is as follows to fill a prescription by weight percentage: soybean cake powder 1.8%, yeast powder 0.36%, glucose 0.14%, KH
2PO
40.25%, MgSO
47H
2O 0.035%, CaCO
30.05%, all the other are water;
(3) the enzymatic production culture condition is: substratum loading amount 35mL/250mL triangular flask, and 31 ℃ of culture temperature, shaking bottle rotating speed is 250r/min, bacteria suspension concentration is 7 * 10
6Individual/mL, every bottle of inoculum size is 0.75mL, initial pH7.0, and fermentation time is 24h.
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| CN101864390A (en) * | 2010-06-13 | 2010-10-20 | 福建农林大学 | Mosquito killing bacillus thuringiensis liquid culture medium |
| CN101869181A (en) * | 2010-04-09 | 2010-10-27 | 武汉科诺生物科技股份有限公司 | Preparation method of bacillus polymyxa raw powder with 100 billion live spores per gram |
| CN102559537A (en) * | 2011-11-15 | 2012-07-11 | 中国科学院微生物研究所 | Bacillus and application of bacillus in protease production |
| CN104140941A (en) * | 2014-07-22 | 2014-11-12 | 华南理工大学 | Bacillus thuringiensis and application thereof |
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| CN109609399A (en) * | 2018-11-27 | 2019-04-12 | 四川师范大学 | A kind of bacillus thuringiensis Dav IV and its application |
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| CN101869181A (en) * | 2010-04-09 | 2010-10-27 | 武汉科诺生物科技股份有限公司 | Preparation method of bacillus polymyxa raw powder with 100 billion live spores per gram |
| CN101869181B (en) * | 2010-04-09 | 2013-06-05 | 武汉科诺生物科技股份有限公司 | Preparation method of bacillus polymyxa raw powder with 100 billion live spores per gram |
| CN101864390A (en) * | 2010-06-13 | 2010-10-20 | 福建农林大学 | Mosquito killing bacillus thuringiensis liquid culture medium |
| CN101864390B (en) * | 2010-06-13 | 2012-04-18 | 福建农林大学 | Mosquito killing bacillus thuringiensis liquid culture medium |
| CN102559537A (en) * | 2011-11-15 | 2012-07-11 | 中国科学院微生物研究所 | Bacillus and application of bacillus in protease production |
| CN104140941A (en) * | 2014-07-22 | 2014-11-12 | 华南理工大学 | Bacillus thuringiensis and application thereof |
| CN106489997A (en) * | 2016-01-13 | 2017-03-15 | 中弘新缘生物科技（天津）有限公司 | A kind of process for preparing microbial insecticide |
| CN105603031A (en) * | 2016-03-09 | 2016-05-25 | 广东海洋大学 | Aquatic protein active peptide preparing method without adopting high-temperature treatment |
| CN109609399A (en) * | 2018-11-27 | 2019-04-12 | 四川师范大学 | A kind of bacillus thuringiensis Dav IV and its application |
| CN110484598A (en) * | 2019-08-30 | 2019-11-22 | 贵州大学 | Application of the casein plate method in the quantitative analysis of proteinase activity |
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