CN102206616A - Bacillus cereus fermentation method for producing phosphatidase C - Google Patents
Bacillus cereus fermentation method for producing phosphatidase C Download PDFInfo
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Abstract
本发明公开了一种蜡状芽孢杆菌发酵产磷脂酶C的方法。属于酶催化反应的技术领域。其用于解决在生产磷脂酶C所用菌株和工艺复杂的的问题。蜡状芽孢杆菌发酵产磷脂酶C的方法，是将蜡状芽孢杆菌接种到LB培养基中培养后得到种子液，将种子液接种到由牛肉膏、蛋白胨、NaCl、水、ZnSO4·7H2O或ZnCl2和卵磷脂或磷脂混合物(PC、PE、PI、PS等的混合)组成的发酵培养基中培养得到发酵液，对发酵液进行分离即得到磷脂酶C。采用这种蜡状芽孢杆菌发酵产磷脂酶C的方法，可广泛地应用于植物油脱胶的磷脂酶C生产。The invention discloses a method for producing phospholipase C by fermentation of bacillus cereus. It belongs to the technical field of enzyme-catalyzed reactions. It is used to solve the problem of complex strains and techniques used in the production of phospholipase C. The method for fermenting Bacillus cereus to produce phospholipase C is to inoculate Bacillus cereus into LB medium and cultivate it to obtain seed liquid, and inoculate the seed liquid into a mixture of beef extract, peptone, NaCl, water, ZnSO 4 7H 2 O or ZnCl 2 and lecithin or phospholipid mixture (mixture of PC, PE, PI, PS, etc.) in the fermentation medium to obtain fermentation broth, the fermentation broth is separated to obtain phospholipase C. The method for producing phospholipase C by fermenting bacillus cereus can be widely used in the production of phospholipase C for vegetable oil degumming.
Description
Technical field
The present invention relates to the method that Phospholipase C is produced in a kind of bacillus cereus fermentation.
Background technology
The Phospholipase C that the present invention relates to is the Vegetable oil lipoprotein that the is used for oil prodution industry production workshop section of coming unstuck.Phosphatide in the vegetables oil mainly contains phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositols (PI) and phosphatidylserine (PS).Phospholipase C is divided into PC-PLC (specificity hydrolysis PC and PE) and PI-PLC (specificity hydrolysis PI).
Phospholipase C extensively is present in animal, plant and the microorganism, but the complex process of extracting from animal, plant because accompaniment is many and the character more complicated, makes that the loss of activity of Phospholipase C is big in the process of extracting.Microbial fermentation has advantage with short production cycle, and tunning is easy to separate, and is the first-selection that obtains Phospholipase C.In the Phospholipase C that is obtained by bacillus cereus fermentation, PC-PLC (specificity hydrolysis PC and PE) and PI-PLC (specificity hydrolysis PI) have, and are suitable as very much the production bacterial classification of the Phospholipase C that is used for vegetable oil degumming.
Not can be used at present the Phospholipase C supply of vegetable oil degumming at home on the market, and 10 milligrams of the prices of Sigma SILVER REAGENT Phospholipase C are uneconomical economically about 2000 yuans, can not be as the usefulness of vegetable oil degumming.The practical use of considering Phospholipase C is in order to can be used in vegetable oil degumming, and required Phospholipase C purity does not need high especially, as long as can reach the effect of vegetable oil degumming.
The domestic researchist of having has screened the bacterial strain that can produce Phospholipase C, but its sieve bacterium process is loaded down with trivial details and the cycle is long, it is carried out strain identification after, numbering is not domestic or international uniform, can't buy this bacterial strain according to this numbering, does not have ubiquity in popularization.We screen from the existing bacterial classification in domestic microbial strains preservation center, and the numbering of bacterial strain uses therefor is clear and definite, shorten the sieve bacterium cycle greatly, and are easy to promote.
The researchist is also arranged by engineered method, utilize pichia spp to express, and then produce Phospholipase C, but, make that follow-up separation and Extraction process is very loaded down with trivial details because there are a large amount of inclusion bodys in pichia spp.Present method later separation is easy and simple to handle, is fit to very much amplify produce.
It all is with through purifying repeatedly that the method for Phospholipase C is produced in fermentation at present, improve its enzyme and live and be purpose, and be not to carry out at the application-specific purpose.The Phospholipase C that present method is produced is at being used for vegetable oil degumming.Utilizing bacillus cereus to produce in the fermenting process of Phospholipase C, all be the LB substratum that adopts, and our rule has been added ZnSO targetedly
47H
2O or ZnCl
2, Yelkin TTS or mixture of phospholipids (mixing of PC, PE, PI, PS etc.) have improved the ability that bacillus cereus produces Phospholipase C greatly.
It is starting strain that this inventive method adopts the bacillus cereus at domestic microbial strains preservation center, screens by egg plate, finds out the bacterial strain that can produce Phospholipase C, carries out fermentation culture then, obtains can be used in the Phospholipase C of vegetable oil degumming.Listed bacterial strain CMCC63302 and GIM1.300 are with a kind of bacterial strain among the embodiment, and just numbering is different, and AS1.196 and GIM1.4 are with a kind of bacterial strain, and just numbering is different.
Summary of the invention
The present invention overcomes shortcoming of the prior art, provides a kind of strain number clear and definite, the method that Phospholipase C is produced in the bacillus cereus fermentation that throughput is strong.
In order to solve the problem of above-mentioned technology, the present invention is achieved by the following technical solutions: the method that Phospholipase C is produced in the bacillus cereus fermentation is bacillus cereus to be inoculated in the LB substratum obtain seed liquor after the cultivation; Seed liquor is inoculated in the fermention medium to cultivate obtains fermented liquid; Fermented liquid separated promptly obtain Phospholipase C.
The present invention also can be achieved through the following technical solutions: above-described fermention medium is by extractum carnis, peptone, NaCl, water, ZnSO
47H
2O and Yelkin TTS are formed.
The present invention also can be achieved through the following technical solutions: above-described fermention medium is by extractum carnis, peptone, NaCl, water, ZnCl
2Form with mixture of phospholipids.
The present invention also can be achieved through the following technical solutions: above-described bacillus cereus is the CMCC63302 bacterial strain.
The present invention also can be achieved through the following technical solutions: above-described bacillus cereus is the AS1.196 bacterial strain.
The present invention can also be achieved through the following technical solutions: above-described bacillus cereus is the GIMT1.104 bacterial strain.
Compared with prior art, the invention has the beneficial effects as follows: it is clear and definite that the method that adopts the bacillus cereus fermentation to produce Phospholipase C has strain number, and technology is easy, the advantage that throughput is strong.
Embodiment
Below the present invention is described in further detail: the method that adopts the bacillus cereus fermentation to produce Phospholipase C, be preparation: get one piece of fresh Countryside Egg by 50% yolk liquid, alcohol with 75% is cleaned appearance, knock eggshell gently open with the disinfectant tweezers, flow out egg white, use aseptic water washing yolk 1～2 time, draw 10 milliliters of yolk with the glass syringe after the sterilization, join in 100 milliliters the triangular flask adorning 10 ml sterile waters and shake up, sealing can make 50% yolk liquid.
The composition of egg plate: NaCl, 0.66%; Boric acid, 1.09%; Borax, 0.19%; Agar, 1.5%; Yolk liquid, 5%.Take by weighing NaCl, boric acid, borax earlier, soluble in water, the agar of adding 1.5%, heating is fully dissolved agar, and with a small amount of undissolved agar of gauze elimination, moisturizing is to accurately sterilizing behind the volume.Get 10 milliliter of 50% yolk liquid and be added in 90 milliliters of borax agar solutions of having sterilized and being cooled to about 55 degree, pour in the culture dish after shaking up while hot, promptly get egg plate after the cooled and solidified.
Configuration LB substratum: NaCl, 0.5%; Peptone, 0.5%; Extractum carnis, 0.3%.Adjusting pH to 7.0～7.2,121 degree sterilized 20 minutes.
In the Erlenmeyer flask that the LB substratum is housed, 37 degree, shaking table are cultivated and are obtained seed liquor with bacterial classification inoculation.The inoculation seed liquor is in the LB substratum, and 37 spend, and 180 rev/mins, shaking table was cultivated 2～30 hours, since the 2nd hour, every sampling in 1 hour once, and room temperature, 10000 rev/mins rotating speed centrifugal 20 minutes, is got supernatant liquor.Keep flat the Oxford cup on egg plate, draw 100 microlitre supernatant liquors and place cup, 37 degree were cultivated 24 hours in constant incubator, whether observe has the oyster white haloing to occur, if have, illustrate that then this bacterial strain can produce Phospholipase C after fermentation culture, can carry out the fermentation culture of next stage.
Fermentative routes is as follows:
(1) configuration fermention medium: extractum carnis, 3～15 grams; Peptone, 5～10 grams; NaCl, 5～8 grams; Water, 1000 grams; ZnSO
47H
2O or ZnCl
2, 0～100 milligram; Yelkin TTS or mixture of phospholipids (mixing of PC, PE, PI, PS etc.), 0～10 gram; PH, 4～10.121 degree sterilizations 20 minutes.
(2) microbial fermentation: in the Erlenmeyer flask that the LB substratum is housed, 37 degree, shaking table are cultivated and are obtained seed liquor with bacterial classification inoculation.Seed liquor with volume ratio 0.01～15% is inoculated in the Erlenmeyer flask that fermention medium is housed then, and 20～50 degree were cultivated on 70～260 rev/mins the shaking table 1～24 hour, obtained fermented liquid.Fermented liquid can separate by following several modes:
1. fermented liquid is spent 4～35, and 3000～10000 rev/mins, centrifugal 5～45 minutes, get supernatant, be the enzyme liquid of Phospholipase C, survey enzyme and live.The relatively environmental protection of this method, environmental pollution is little and loss enzyme is little.
2. adjust pH to 6～8, add solid (NH
4)
2SO
4To 55～80%, stirring at room 0.5～2 hour, 2～15 spend the precipitation at night, 2～15 degree, 3000～10000 rev/mins, centrifugal 5～45 minutes, particle is suspended from 10 mmoles of fermentating liquid volume 0.1～10%/rise (pH6～8) in the Tris-HCl damping fluid, with 10 mmoles of 30～1000 times of volumes/rising Tris-HCl damping fluid (pH6～8) carries out 2～10 dialysis, obtains the enzyme liquid of Phospholipase C, survey enzyme and live.This method is use in the purifying of enzyme maximum, classic methods very, but because to use (NH in a large number
4)
2SO
4, be disadvantageous to environment.
3. fermented liquid is at 4～35 degree, 3000～10000 rev/mins, centrifugal 5～45 minutes, get supernatant, change in molecular weight cut-off 8000～40000 daltonian dialysis tubings, under vacuum tightness 0.1～20000 handkerchief condition, carry out the suction filtration dialysis, migrate out the liquid in the dialysis tubing, be the enzyme liquid of Phospholipase C, survey enzyme and live.The relatively environmental protection of this method, environmental pollution is little and loss enzyme is little.
Three kinds of production processes of numbering bacterial strains are adopted in narration respectively below:
(1) strain number: CMCC63302
Shift a ring bacterial strain in 50 milliliters LB substratum from plate culture medium, 37 degree, 150 rev/mins, shaking table was cultivated 4 hours, as seed liquor.Inoculate 1.5 milliliters of seed liquor in 100 milliliters of LB substratum, carry out 37 degree, 180 rev/mins, shaking table was cultivated 4 hours, room temperature, and 10000 rev/mins rotating speed centrifugal 20 minutes, is got supernatant liquor.Keep flat the Oxford cup on egg plate, draw 100 microlitre supernatant liquors and place cup, 37 degree were cultivated 24 hours in constant incubator, had observed the oyster white haloing and had occurred.
Shift a ring bacterial strain in 50 milliliters LB substratum from plate culture medium, 37 degree, 150 rev/mins, shaking table was cultivated 4 hours, as seed liquor.Inoculate 1.5 milliliters of seed liquor in 100 milliliters of fermention mediums, carry out 37 degree, 150 rev/mins, shaking table was cultivated 4 hours, 4 degree, and 9000 rev/mins, the supernatant that obtains is collected in centrifugation 20 minutes, adjusts pH to 6.5, adds solid (NH
4)
2SO
4To 55%, stirring at room 1 hour, 5 spend the precipitation at night, 4 degree, 8000 rev/mins, centrifugal 25 minutes, particle is suspended from 10 mmoles of fermentating liquid volume 10%/rise (pH6.5) in the Tris-HCl damping fluid, with 10 mmoles of 300 times of volumes/rising Tris-HCl damping fluid (pH6.5) carries out 3 dialysis, obtains the enzyme liquid of Phospholipase C, surveying enzyme work is 8.1 units per ml.
(2) strain number: AS1.196
Shift a ring bacterial strain in 50 milliliters LB substratum from plate culture medium, 37 degree, 150 rev/mins, shaking table was cultivated 4 hours, as seed liquor.Inoculate 1.5 milliliters of seed liquor in 100 milliliters of LB substratum, carry out 37 degree, 180 rev/mins, shaking table was cultivated 4 hours, room temperature, and 10000 rev/mins rotating speed centrifugal 20 minutes, is got supernatant liquor.Keep flat the Oxford cup on egg plate, draw 100 microlitre supernatant liquors and place cup, 37 degree were cultivated 24 hours in constant incubator, had observed the oyster white haloing and had occurred.
Shift a ring bacterial strain in 50 milliliters LB substratum from plate culture medium, 37 degree, 150 rev/mins, shaking table was cultivated 4 hours, as seed liquor.Inoculate 1 milliliter of seed liquor in 100 milliliters of fermention mediums, carry out 37 degree, 150 rev/mins of shaking tables were cultivated 12 hours, 25 degree, and 10000 rev/mins, the supernatant that obtains is collected in centrifugation 20 minutes, and surveying enzyme work is 21.2 units per ml.
(3) strain number: GIMT 1.104
Shift a ring bacterial strain in 50 milliliters LB substratum from plate culture medium, 37 degree, 150 rev/mins, shaking table was cultivated 4 hours, as seed liquor.Inoculate 1.5 milliliters of seed liquor in 100 milliliters of LB substratum, carry out 37 degree, 180 rev/mins, shaking table was cultivated 4 hours, room temperature, and 10000 rev/mins rotating speed centrifugal 20 minutes, is got supernatant liquor.Keep flat the Oxford cup on egg plate, draw 100 microlitre supernatant liquors and place cup, 37 degree were cultivated 24 hours in constant incubator, had observed the oyster white haloing and had occurred.
Shift a ring bacterial strain in 50 milliliters LB substratum from plate culture medium, 37 degree, 150 rev/mins, shaking table was cultivated 4 hours, as seed liquor.Inoculate 2 milliliters of seed liquor in 100 milliliters of fermention mediums, carry out 37 degree, 150 rev/mins of shaking tables were cultivated 8 hours, 25 degree, 10000 rev/mins, centrifugation 20 minutes, the supernatant that collection obtains changes in the molecular weight cut-off 8000 daltonian dialysis tubings, carries out the suction filtration dialysis under vacuum tightness 15000 handkerchief conditions, migrate out the liquid in the dialysis tubing, surveying enzyme work is 13.2 units per ml.
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Cited By (10)
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| CN104419651A (en) * | 2013-08-19 | 2015-03-18 | 丰益(上海)生物技术研发中心有限公司 | Phospholipase C and bacterial strain generating same |
| CN104560764A (en) * | 2013-10-17 | 2015-04-29 | 丰益(上海)生物技术研发中心有限公司 | Bacillus pumilus for producing phospholipase C and phospholipase C produced by bacillus pumilus |
| CN104560763A (en) * | 2013-10-15 | 2015-04-29 | 丰益(上海)生物技术研发中心有限公司 | Bacillus licheniformis for producing phospholipase C and phospholipase C produced by bacillus licheniformis |
| CN104560910A (en) * | 2013-10-16 | 2015-04-29 | 丰益(上海)生物技术研发中心有限公司 | Growth medium and method for increasing yield of PLC (phospholipase C) through fermentation of Pichia pastoris |
| WO2015067161A1 (en) * | 2013-11-07 | 2015-05-14 | 丰益(上海)生物技术研发中心有限公司 | Phospholipase c mutant and use thereof |
| CN104630176A (en) * | 2013-11-13 | 2015-05-20 | 丰益(上海)生物技术研发中心有限公司 | Method for improving yield of phospholipase C produced by fermentation method |
| CN104694394A (en) * | 2013-12-05 | 2015-06-10 | 丰益(上海)生物技术研发中心有限公司 | Cladosporium-expressed phosphatidase C and produced bacterial strain thereof |
| CN104726356A (en) * | 2013-12-20 | 2015-06-24 | 丰益(上海)生物技术研发中心有限公司 | Bacillus subtilis mutant strain capable of producing phospholipase |
| CN104726357A (en) * | 2013-12-20 | 2015-06-24 | 丰益(上海)生物技术研发中心有限公司 | Bacillus amyloliquefaciens mutant strain capable of generating phosphatidase and phosphatidase C |
| CN104962499A (en) * | 2015-07-17 | 2015-10-07 | 陈明锴 | Bacillus cereus mutant strain for high production of phospholipase C as well as fermentation method and application of bacillus cereus mutant strain |
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Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104419651A (en) * | 2013-08-19 | 2015-03-18 | 丰益(上海)生物技术研发中心有限公司 | Phospholipase C and bacterial strain generating same |
| CN104419651B (en) * | 2013-08-19 | 2018-12-25 | 丰益(上海)生物技术研发中心有限公司 | Phospholipase C and its producing bacterial strain |
| CN104560763A (en) * | 2013-10-15 | 2015-04-29 | 丰益(上海)生物技术研发中心有限公司 | Bacillus licheniformis for producing phospholipase C and phospholipase C produced by bacillus licheniformis |
| CN104560763B (en) * | 2013-10-15 | 2018-12-25 | 丰益(上海)生物技术研发中心有限公司 | Produce the bacillus licheniformis of phospholipase C and its phospholipase C of generation |
| CN104560910A (en) * | 2013-10-16 | 2015-04-29 | 丰益(上海)生物技术研发中心有限公司 | Growth medium and method for increasing yield of PLC (phospholipase C) through fermentation of Pichia pastoris |
| CN104560764B (en) * | 2013-10-17 | 2018-12-25 | 丰益(上海)生物技术研发中心有限公司 | Produce the bacillus pumilus of phospholipase C and its phospholipase C of generation |
| CN104560764A (en) * | 2013-10-17 | 2015-04-29 | 丰益(上海)生物技术研发中心有限公司 | Bacillus pumilus for producing phospholipase C and phospholipase C produced by bacillus pumilus |
| WO2015067161A1 (en) * | 2013-11-07 | 2015-05-14 | 丰益(上海)生物技术研发中心有限公司 | Phospholipase c mutant and use thereof |
| US10144919B2 (en) | 2013-11-07 | 2018-12-04 | Wilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd | Phospholipase C mutant and use thereof |
| CN104630176A (en) * | 2013-11-13 | 2015-05-20 | 丰益(上海)生物技术研发中心有限公司 | Method for improving yield of phospholipase C produced by fermentation method |
| CN104630176B (en) * | 2013-11-13 | 2020-05-01 | 丰益(上海)生物技术研发中心有限公司 | Method for increasing yield of phospholipase C produced by fermentation method |
| CN104694394A (en) * | 2013-12-05 | 2015-06-10 | 丰益(上海)生物技术研发中心有限公司 | Cladosporium-expressed phosphatidase C and produced bacterial strain thereof |
| CN104694394B (en) * | 2013-12-05 | 2019-07-12 | 丰益(上海)生物技术研发中心有限公司 | The phospholipase C and its producing bacterial strain of a kind of mould expression of spore |
| CN104726357A (en) * | 2013-12-20 | 2015-06-24 | 丰益(上海)生物技术研发中心有限公司 | Bacillus amyloliquefaciens mutant strain capable of generating phosphatidase and phosphatidase C |
| CN104726356A (en) * | 2013-12-20 | 2015-06-24 | 丰益(上海)生物技术研发中心有限公司 | Bacillus subtilis mutant strain capable of producing phospholipase |
| CN104726357B (en) * | 2013-12-20 | 2019-07-09 | 丰益(上海)生物技术研发中心有限公司 | The bacillus amyloliquefaciens mutagenic strain and phospholipase C of one plant of generation phosphatidase |
| CN104962499A (en) * | 2015-07-17 | 2015-10-07 | 陈明锴 | Bacillus cereus mutant strain for high production of phospholipase C as well as fermentation method and application of bacillus cereus mutant strain |
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Application publication date: 20111005 |