CN103509721B - The method of one plant height cellulase producing strain and fermentation production neutral cellulase - Google Patents
The method of one plant height cellulase producing strain and fermentation production neutral cellulase Download PDFInfo
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- CN103509721B CN103509721B CN201210211311.3A CN201210211311A CN103509721B CN 103509721 B CN103509721 B CN 103509721B CN 201210211311 A CN201210211311 A CN 201210211311A CN 103509721 B CN103509721 B CN 103509721B
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Abstract
Belong to Richter scale wood enzyme bacterial strain Trichodema reesei QZ 5 the invention provides the wooden enzyme of a plant height cellulase-producing, be stored in China typical culture collection center, deposit number is：CCTCC NO：M2012204, preservation date on June 5th, 2012.It also invented simultaneously using the bacterium as the strain that sets out, through seed culture and with corn steep liquor, ammonium sulfate etc. for nitrogen source, it is the method that carbon source through fermentation produces neutral cellulase with glucose, wheat bran etc., zymotic fluid highest enzyme activity is up to more than 5000IU/L, produced neutral cellulase can be widely applied to the fields such as weaving, papermaking, feed, food, belong to biological technical field.
Description
Technical field
The present invention utilizes the bacterial strain (Trichodema reesei QZ-5) of a plant height cellulase-producing and utilizes the bacterial strain
It is carbon source through fermentation production neutral cellulase through seed culture and with corn steep liquor, ammonium sulfate etc. for nitrogen source, with glucose, wheat bran etc.
Method, prepared enzyme can be applied to the fields such as weaving, papermaking, feed, food, belong to biological technical field.
Background technology
Cellulase is the compound enzyme system of multicomponent that a class is capable of degraded cellulose into glucose, can be widely applied to energy
The fields such as source, weaving, papermaking, feed, food, in recent years, are protruded, cellulase as global resources energy shortage problem day is aobvious
Be using be distributed in nature most wide, content at most, cellulose the most cheap as regenerated resources main enzyme system so that raw
Production prepares the problem that the research of cellulase is more paid close attention into global researcher.
Cellulase is distributed widely in fungi, plant, animal, bacterium and insect.But the master for producing cellulase
If the wooden enzyme bacterium of Richter scale of wooden enzyme category.The cellulase that Richter scale wood enzyme is produced includes endoglucanase (EG), circumscribed glucan
Enzyme (CBH) and the glucuroides of β mono- (CB), according to application conditions, can be divided into acidic cellulase, neutral cellulase, alkalescence
Cellulase.
About 40,000 tons of China's neutral cellulase annual requirement (10000IU/g), emphasis be applied to weaving, food, feed,
The fields such as papermaking, current basic dependence on import.Although China turn into after the U.S., Japan, Denmark in the world the 4th can give birth to
The country of cellulase-producing, but China's neutral cellulase production technology is also relatively backward with respect to developed country at present, enzyme activity
Low, cost is high, small scale, and its root is not screen that producing enzyme lives high wild-type strain or genetic modification engineering bacteria technology is still located
In developing stage.
The content of the invention
The present invention is screened and there is provided the wild strain (Trichodema reesei QZ-5) of a plant height cellulase-producing
And be carbon source through fermentation production using the bacterial strain through seed culture and with corn steep liquor, ammonium sulfate etc. for nitrogen source, with glucose, wheat bran etc.
The method of neutral cellulase, prepared enzyme can be applied to the fields such as weaving, papermaking, feed, food, carry out fictitious hosts and hold high
Expensive import neutral cellulase.
Technical scheme：A kind of wooden enzyme of High Cellulase Production belongs to bacterial strain, and it is named as
Trichodemareesei QZ-5, have been stored in China typical culture collection center, and deposit number is CCTCC NO：
M2012204, preservation date on June 5th, 2012.
It is the strain that sets out with the bacterium, through seed culture and with corn steep liquor, ammonium sulfate etc. for nitrogen source, with glucose, wheat bran etc.
Neutral cellulase is produced for carbon source through fermentation；Technique is：
(1) seed culture
Seed culture medium：Glucose 15g/L, yeast extract 2g/L, peptone 3g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate 3g/
L, CaCl2.2H2O0.3g/L, MgSO4.7H2O0.3g/L, 115 DEG C of sterilizings are stand-by.
Inoculating process：The ring of slant strains one point on PDA plate is taken to connect mycelia, 30 DEG C are cultivated 7 days, take a flat board 10ml
Aseptic water washing, and the triangular flask that uniform spore suspension takes 1ml spore suspensions to access 250ml specifications is broken into, liquid amount is
50ml。
Condition of culture：Rotating speed 200rpm, 30 degree of temperature, as incubated 2 days, the seed of ferment tank producing enzyme.
(2) producing enzyme culture
Culture medium：Corn steep liquor 30g/L, wheat bran 10g/L, glucose 10g/L, yeast extract 0.5g/L, peptone 3g/L,
Ammonium sulfate 2g/L, potassium dihydrogen phosphate 3g/L, CaCl2.2H2O0.3g/L, MgSO4.7H2O0.3g/L, FeSO4.7H2O0.005g/L,
MnSO4.H2O0.016g/L, ZnSO4.7H2O0.0014g/L, CoCl20.002g/L, Tween-80 0.2g/L, 115 DEG C of sterilizings are treated
With.
Fermentation tank：Specification is that capacity 7L. real attenuation liquid amounts are 5L.
Fermentation parameter：30 degree of rotating speed 200r/min. temperature throughput 1.4L/min/L. fermentation times 150 hours.
According to above-mentioned condition, fermented by 10% inoculum concentration in 7L fermentation tanks, the neutral cellulase of zymotic fluid is lived
Power is up to more than 5000U/L.
The present invention protects river levee domestic fungus cultivating base rotten soil sample, detritus straw, mushroom cultivation microbiological contamination material etc. from Jinshi City
The wild wooden enzyme that plant height production neutral cellulase is filtered out in the sample of collection belongs to the wooden enzyme bacterial strain of Richter scale, is named as
Trichodema reesei QZ-51, the screening of this bacterial strain：
1.1 flat board primary dcreening operations
The processing method of sample：By material with a small amount of 30 DEG C of immersion 24h of sterilized water, it is diluted to afterwards using gradient dilution method
10-8, take 1mL to be uniformly coated on 121 DEG C of sterilizing 20min Congo red screening flat board culture medium (MgSO40.50g/L, (NH4)2SO42.00g/L, KH2PO41.00g/L, NaCl0.50g/L, Congo red 0.20g/L, agar 20.00g/L, CMC2.00g/L, from
Right pH value) upper 28 DEG C it is incubated 3-4 days, picking have hydrolyze transparent circle and transparent circle compared with rounded edge bacterium colony clearly new
Congo red screening flat board culture medium on rule and be separately cultured 3-4 days, picking transparent circle rounded edge is clear and transparent loop diameter
Larger bacterium colony is as filtering out the higher inoculation of cellulase-producing vigor to PDA plate with colony diameter ratio (H/d)
Culture medium (potato 200g/L, glucose 20g/L, agar 20.00g/L) is used as secondary screening bacterial strain.
1.2 shaking flask secondary screenings
By the PDA plate for covering with mycelia 10mL aseptic water washings, 1mL access seed culture mediums (glucose 15g/ is drawn
L, yeast extract 2g/L, peptone 3g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate 3g/L, CaCl2.2H2O0.3g/L,
MgSO4.7H2O0.3g/L), seed culture medium liquid amount is dress 50mL culture mediums, cultivation temperature 30 DEG C, turn in 250mL triangular flasks
Incubated 48 hours of fast 200r/min shaking tables, take bacteria suspension (by inoculum concentration 10%) to access liquid state fermentation culture medium (beautiful
Rice ＆ peanut milk 30g/L, wheat bran 10g/L, glucose 10g/L, yeast extract 0.5g/L, peptone 3g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate
3g/L, CaCl2.2H2O0.3g/L, MgSO4.7H2O0.3g/L, FeSO4.7H2O0.005g/L, MnSO4.H2O0.016g/L,
ZnSO4.7H2O0.0014g/L, CoCl20.002g/L, Tween-80 0.2g/L), liquid state fermentation basal medium liquid amount is
50mL culture mediums, 30 DEG C of cultivation temperature, rotating speed 200r/min, incubated 8 days of shaking table are filled in 250mL triangular flasks.
1.3 finally screen one plant of of a relatively high bacterial strain of neutral cellulase vigor through the screening of above two method, put down
Plate bacterium colony (Fig. 1), Electronic Speculum (Fig. 2), 18S rDNA Sequence Identifications (see sequence table) simultaneously identify the Richter scale being defined as in wooden enzyme Pseudomonas
Wooden enzyme, is named as Trichodema reesei QZ-51.
2nd, the assay method of enzyme activity
2.1 enzyme activity are defined
At 50 DEG C, pH is under the conditions of 6.0, per minute to be released from concentration to be degraded in 5mg/mL carboxymethylcellulose sodium solution
Enzyme amount required for putting 1 μm of ol reduced sugar is a unit of activity (IU), and reduced sugar is with glucose equivalent.
2.2 principle
Cellulose hydrolyzation cellulose produces monose and oligosaccharides under the conditions of certain temperature and pH.With reducing end under neutral
Oligosaccharides and have reduction group monose under the conditions of boiling water bath with DNS reagents occur chromogenic reaction, the intensity of reaction solution color with
The amount that enzymolysis produces reduced sugar is directly proportional, and reduced sugar growing amount is directly proportional to reaction solution cellulase vigor, passes through splitting ratio
Color determines reaction solution absorbance, can calculate cellulase activity.
2.3 instrument and equipment：
2.3.1 assay balance：Sensibility reciprocal 0.0001g
2.3.2 accurate pH meter：It is accurate to 0.01
2.3.3 magnetic force heating stirrer
2.3.4 spectrophotometer：GB9721 pertinent regulations, 5mm cuvettes should be met
2.3.5 electric-heated thermostatic water bath：30~100 DEG C, ± 0.5 DEG C
2.3.6 timer：Error is no more than five seconds per hour
2.3.7 pipettor：100~1000 μ L
2.3.8 microsyringe：0~50 μ L
2.4 reagents and solution
2.4.1 reagent：
2.4.2 prepare：
A.0.5M sodium dihydrogen phosphate：
Weigh 39.0g sodium dihydrogen phosphates to be completely dissolved in 400ml distilled water, be settled to 500ml.Labeled as A test solutions.
B.0.5M disodium phosphate soln：
Weigh 89.5g disodium hydrogen phosphates to be completely dissolved in 400ml distilled water, be settled to 500ml.Labeled as B test solutions.
C.0.5M phosphate buffer：
342.5ml A test solutions and 157.5ml B test solutions is taken to mix.Labeled as C test solutions
D.0.05M neutral buffered liquid：
C test solutions 500ml is taken to be added in 4000ml distilled water.Its pH value is determined with Accurate pH meter, if necessary with 3M's
Hydrochloric acid solution or 3M sodium hydroxide solutions adjust pH value to being settled to 5000ml again after 6.0.Mark is neutral buffered after shaking up
Liquid, while marking pH=6.0, preparing the date.Preserved under the conditions of 4 DEG C.
E.200g/L sodium hydroxide solution：Weigh sodium hydroxide 200.0g to be dissolved in water, be settled to 1000ml.
2.4.310mg/ml CMC solution：
Precision weighs CMC (sigma c-5678) 1.0000g and is slowly added into 80mL0.05M neutral buffered liquid (d),
Magnetic agitation is to being completely dissolved (at room temperature 2 hours or so).PH value is adjusted to 6.0 with 3M hydrochloric acid solutions or 3M sodium hydroxide solutions,
Again 100mL is settled to 0.05M neutral buffered liquid (d).Labeled as 10mg/ml CMC solution, while marking pH and preparing day
Phase.Preserved under the conditions of 4 DEG C, the term of validity 7 days.
2.4.410mg/ml Standard glucose solution：
Precision weighs DEXTROSE ANHYDROUS 1.0000g and puts stirring and dissolving in 80ml distilled water, is settled to until completely dissolved
100ml.Labeled as 10mg/ml Standard glucose solution, preservation under the conditions of date, 4 DEG C is prepared while marking.
2.4.5DNS the preparation of reagent
Weigh 3,5- dinitrosalicylic acids 31.50g to be slowly added in 5000ml distilled water, be stirred continuously, water-bath to 45 DEG C,
1000ml sodium hydroxide solutions (e) are gradually added, is stirred continuously to solution is as clear as crystal and (is adding sodium hydroxide solution process
In, solution temperature does not exceed 48 DEG C).Rochelle salt 910.0g, phenol 25.0g, anhydrous sulfurous acid are gradually added again
Sodium 25.0g, continues 45 DEG C of heating water baths, while moisturizing 3000ml, is stirred continuously, after being completely dissolved to above-mentioned substance, is cooled to
Room temperature, and constant volume is to 10000ml.With filter-cloth filtering, filtrate is stored in brown bottle, mark title and preparation date, lucifuge,
Room temperature preservation can be used after 7 days, the term of validity 6 months.
Beneficial effects of the present invention：
Provide firstly a plant height production neutral cellulase bacterial strain and with the bacterium be starting strain use liquid state fermentation system
The method of standby neutral cellulase.The strain and enzymatic production method of the present invention has following features：
1st, fermenting property is stable, and thalline is easy to culture.
2nd, the producing enzyme cycle is short, can stop fermentation at the 6th day.
3rd, producing enzyme vigor is high, reaches as high as more than 5000IU/L.
4th, neutral cellulase content turns into leading product extracellular in crude enzyme liquid, is easy to isolate and purify.
5th, thick enzymatic property is stable, and optimum temperature is 55 degree, more heat-resisting.
6th, substrate specificity and condition milder are extensive, with broader practice prospect.
7th, filled up that China's neutral cellulase fermentation level is low to need the blank of dependence on import.
Therefore the neutral cellulase that the method for the bacterial strain of the present invention and fermentation production neutral cellulase is produced can
It is widely used in the fields such as weaving, papermaking, feed, food, and alternative expensive import neutral cellulase, therefore this
The strain and enzymatic production method of invention have extensive industrial application value and significant economic benefit prospect.
Accompanying drawing table explanation
Fig. 1 and Fig. 2 are respectively Trichodema reesei QZ-5 bacterial strain flat boards bacterium colonies and ESEM form.
Sequence table is the 18S rDNA Sequence Identifications of Trichodema reesei QZ-5 bacterial strains.
Biological material specimens preservation
Trichodema reesei QZ-5, which have been stored in, has been stored in China typical culture collection center, deposit number
For：CCTCC NO：M 2012204, preservation date on June 5th, 2012.
Embodiment
Embodiment 1
The ring of slant strains one point on PDA plate is taken to connect mycelia, 30 DEG C are cultivated 7 days, take a flat board 10ml sterilized waters to rush
Wash, and be broken into uniform spore suspension, take the three of 250ml specification of the 1ml spore suspensions access equipped with 50ml seed culture mediums
In the bottle of angle, prepare several pieces.Condition of culture：Rotating speed 200rpm, 30 degree of temperature, as incubated 3 days, fermentation seed liquid.
Load 5L culture mediums in 7L fermentation tanks, add 2.5ml defoamers, 121 DEG C sterilize 30 points, after cooling,
By 10% inoculum concentration, the above-mentioned fermentation seed liquids of 500ml are added, condition of culture is：Rotating speed 200r/min, 30 DEG C of temperature, throughput
1.2L/min/L, fermented incubation time extracts crude enzyme liquid, and survey enzyme activity, fermentation broth enzyme vigor by enzyme activity method is surveyed after 150 hours
Up to 5080IU/L.
Culture medium is corn steep liquor 30g/L, wheat bran 10g/L, glucose 10g/L, yeast extract 0.5g/L, peptone 3g/
L, ammonium sulfate 2g/L, potassium dihydrogen phosphate 3g/L, CaCl2.2H2O0.3g/L, MgSO4.7H2O0.3g/L, FeSO4.7H2O0.005g/
L, MnSO4.H2O0.016g/L, ZnSO4.7H2O0.0014g/LCoCl20.002g/L, Tween-80 0.2g/L.
Claims (2)
1. one plant of cellulase-producing Li's Trichoderma strains (Trichoderma reesei) QZ-5, it is preserved in Chinese Typical Representative culture
Thing collection, deposit number is CCTCC NO：M 2012204, preservation date 2012 year 06 month 05 day.
2. the method for neutral cellulase is produced with the strain fermentation described in claim 1, it is characterised in that with the bacterial strain to set out
Strain, is that carbon source through fermentation production is neutral fine with glucose, wheat bran etc. through seed culture and with corn steep liquor, ammonium sulfate etc. for nitrogen source
Plain enzyme is tieed up, technique is：
(1) seed culture
Seed culture medium：Glucose 15g/L, yeast extract 2g/L, peptone 3g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate 3g/L,
CaCl2·2H2O 0.3g/L、MgSO4·7H2O 0.3g/L；
Inoculating process：The ring of slant strains one point on PDA plate is taken to connect mycelia, 30 DEG C are cultivated 7 days, take a flat board 10ml sterile
Water is rinsed, and is broken into uniform spore suspension, takes 1ml spore suspensions to access the triangular flask of 250ml specifications, and liquid amount is
50ml；
Condition of culture：Rotating speed 200rpm, 30 DEG C of temperature, as incubated 2 days, the seed of enzymatic production；
(2) producing enzyme culture
Culture medium：Corn steep liquor 30g/L, wheat bran 10g/L, glucose 10g/L, yeast extract 0.5g/L, peptone 3g/L, sulfuric acid
Ammonium 2g/L, potassium dihydrogen phosphate 3g/L, CaCl2·2H2O 0.3g/L、MgSO4·7H2O0.3g/L、FeSO4·7H2O 0.005g/
L、MnSO4·H2O 0.016g/L、ZnSO4·7H2O 0.0014g/L、CoCl20.002g/L, Tween-80 0.2g/L；
pH：It is natural；
Fermentation tank：Specification is 7L capacity, and real attenuation liquid amount is 5L；
Fermentation parameter：Inoculum concentration 10%, rotating speed 200r/min, 30 DEG C of temperature, throughput 1.2L/min/L, incubation time 150 is small
When.
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| CN111876335A (en) * | 2020-07-31 | 2020-11-03 | 深圳市顺盛农业科技发展有限公司 | Fungus capable of degrading cellulose and separation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107603884A (en) * | 2016-07-12 | 2018-01-19 | 青岛蔚蓝生物集团有限公司 | One plant height produces the trichoderma reesei mutant strain of neutral cellulase |
| CN107475223B (en) * | 2017-09-22 | 2020-10-02 | 宜昌东阳光生化制药有限公司 | Production method of acidic cellulase |
| CN109019874B (en) * | 2018-09-08 | 2022-12-30 | 佛山市森昂生物科技有限公司 | Biological growth promoter for papermaking wastewater and preparation method thereof |
| CN110150468A (en) * | 2019-04-30 | 2019-08-23 | 海南久纳生物科技开发有限公司 | A kind of dedicated production technology of herbage biological fermentation feed |
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| CN101280330A (en) * | 2008-05-08 | 2008-10-08 | 南昌航空大学 | Method for preparing chitosan oligosaccharide with trichoderma reesei cellulase |
| CN101735993A (en) * | 2010-01-19 | 2010-06-16 | 浙江大学 | Method for efficiently producing cellulase |
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|---|---|---|---|---|
| CN101280330A (en) * | 2008-05-08 | 2008-10-08 | 南昌航空大学 | Method for preparing chitosan oligosaccharide with trichoderma reesei cellulase |
| CN101418289B (en) * | 2008-11-28 | 2011-05-04 | 天津科技大学 | Trichoderma reesei liquid submerged fermentation cellulase and enzymatic beating process thereof |
| CN101735993A (en) * | 2010-01-19 | 2010-06-16 | 浙江大学 | Method for efficiently producing cellulase |
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| CN111876335A (en) * | 2020-07-31 | 2020-11-03 | 深圳市顺盛农业科技发展有限公司 | Fungus capable of degrading cellulose and separation method and application thereof |
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