CN103045484B - Penicillium strain producing cellulase and application in cellulose enzymatic hydrolysis thereof - Google Patents
Penicillium strain producing cellulase and application in cellulose enzymatic hydrolysis thereof Download PDFInfo
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Abstract
The invention belongs to the field of microorganism, and particularly relates to a microorganism penicillium strain producing high-activity cellulase. The penicillium strain has a classification name of Penicillium decumbens PD-G3-08, and is preserved in Wuhan University China Center for Type Culture Collection with a preservation number of CCTCC M2011195. The invention also provides an application of the strain in cellulose enzymatic hydrolysis. Through mutation breeding, the penicillium strain producing high-activity cellulase is bred; cellulase crude enzyme liquid is obtained through fermentation of the strain, which has filter paper enzyme activity of up to 10 IU/ml, endoglucanase activity of up to 30 IU/ml, exoglucanase activity of up to 1.5 IU/ml and beta-glucosidase activity of up to 8 IU/ml, respectively being 3 times, 4 times, 4.5 times, and 4 times higher than the activities of an original strain; when the strain is applied to enzymatic hydrolysis of cellulose raw materials, the cellulose conversion rate in 3 days is up to more than 80%, and the cellulase has extremely high activity.
Description
Technical field
The invention belongs to microorganism field, be specifically related to a kind of microorganism penicillium bacterial strain and the cellulosic application of enzymolysis thereof of producing high activity cellulase.
Background technology
Along with the exhaustion day by day of Global Oil deposit, liquid fuel shortage will seriously restrict the development of the mankind, and the alternate resources finding recyclability has become the urgent task maintaining human social.The renewable resources that Devoting Major Efforts To Developing is new is also that China keeps the precondition of Sustainable development in 21 century.With renewable resources the abundantest on the earth--plant cellulose has been subject to extensive concern both domestic and external as raw material production bioenergy and bio-based chemical, and is considered to one of optimal path of 21 century reply fossil resource exhaustion.Mierocrystalline cellulose is by β-1, the linear polymeric polymkeric substance that 4-glycosidic link is formed by connecting, being degraded into monose is and then the key of the bio-based chemical such as preparing ethanol by fermentation, this step needs to have been come by cellulase, and therefore producing high efficiency cellulase becomes a committed step in bio-ethanol path.
Cellulase (Cellulase) is the general name that degraded cellulose is converted into the various enzymes of glucose, is divided into fungal cellulase and bacteria cellulose enzyme.Cellulase is by endoglucanase (EG), 1,4-BETA-D-glucancellobio-hydrolase (CBH), and beta-glucosidase (BG) forms.The industries such as it is widely used in weaving, feed, brewage, food-processing, environmental protection, papermaking, effectively can improve quality product, improve product production; Cellulase is utilized to carry out bio-finishing and enzyme liberating arrangement to cellulose base fiber fabric; Mierocrystalline cellulose Knitted fabrics appearance performance can be improved, the pliability of fortifying fibre cellulose fiber, make fabric sheen and colour brightness be improved significantly, application is more and more wider, and market requirement is also increasing.
The production method of cellulase generally adopts solid fermentation and liquid fermenting two kinds of processing methodes: although solid fermentation process fixed investment is few, production stability is poor, is difficult to carry out large-scale industrial production; And liquid fermentation process production stability is high, culture condition production efficiency easy to control is high, is convenient to scale operation, but its technical bottleneck is that the enzyme of fermented liquid is lived generally on the low side.
Producing high active cellulase key is: one is breeding high-yield bacterial classification, as production bacterial classification; Two is zymotechnique routes, and substratum is selected, fermentation time, temperature etc., the filtration of fermentation secondary fermentation liquid, and the concentrated extraction process etc. waited all exists considerable influence to the activity of cellulase.
Microorganism at present for the production of cellulase belongs to fungi mostly, and the cellulase produced because of it mostly is extracellular enzyme, and the technique of separation and Extraction is also relatively simple.Study the more bacterial strain having Trichoderma (Trichodema sp.), Aspergillus (Aspergillus sp.), Penicillium (Penicillium sp.) and the mould genus of branch top spore (Acremonium sp.).Wherein occupy high with the output of Trichoderma, and relatively less to the report of Penicillium notatum (Penicillium sp.), and general vigor is all lower than the enzyme that Trichoderma (Trichodema sp.) is produced.
Chinese patent CN101717728A discloses a penicillium and the purposes for catalytic hydrolysis lignocellulose thereof, the cellulase utilizing this bacterial strain to obtain can play biocatalysis efficacy, decompose the biomass of rich cellulose and generate monose, the hydrolyzate of gained can be used as carbon source for the synthesis of bioenergy and bio-based chemical.The cellulase of gained is 48% at the cellulolytic transformation efficiency of shake flask scale, is 64% at the cellulolytic transformation efficiency of fermentor tank scale.Although the cellulase utilizing this bacterial strain to obtain has good hydrocellulose ability, but there is the Mierocrystalline cellulose close to half be not hydrolyzed and waste after all, be therefore badly in need of developing the cellulase with better hydrolysis ability and can the bacterial strain of this cellulase of high yield.
Summary of the invention
For this reason, technical problem to be solved by this invention is the problem of Penicillium notatum hydrocellulose limited ability in prior art, and then provides a kind of penicillium bacterial strain that can produce effectively hydrolyzing cellulase.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
The present inventor is through selection by mutation, select a strain of penicillium bacterial strain, its Classification And Nomenclature is Penicillium decumbens PD-G3-08, has been preserved in Wuhan University's China typical culture collection center (being called for short CCTCC), depositary institution address: Wuhan University's preservation center.The numbering of registering on the books is CCTCC M 2011195, and preservation date is on June 13rd, 2011.With this bacterial strain for the cellulosic bacterial strain of enzymolysis.
This bacterial strain has following character: the bacterium colony of described penicillium bacterial strain is in rose pink on wort flat board, and single bacterium colony is rounded, and there is a small amount of fold on surface, and basis of microscopic observation mycelia is sturdy, interweaves intensive and seldom ruptures.
The invention also discloses a kind of cellulase of being encoded by above-mentioned penicillium bacterial strain.
The invention also discloses the cultural method of described penicillium bacterial strain Penicillium decumbens PD-G3-08, comprise the steps:
(1) actication of culture: described inoculation is cultivated activation in the solid medium being suitable for microorganism growth, in 25-30 DEG C, aerobic cultivation 5-7 days;
Described solid medium is the solid medium being applicable to mould growth common to those skilled in the art preferably, further the preferred substratum containing, for example lower component and consumption: wheat bran 80-100g/L, glucose 10-20g/L, agar powder 15-20g/L, all the other are water;
(2) bacterial classification propagation: the bacterial classification activated in step (1) is dug together with solid medium the liquid seed culture medium that block access is suitable for microorganism growth, 25-30 DEG C, aerobic cultivation 30-60 hour, obtains seed liquor;
Described liquid seed culture medium is the liquid seed culture medium being applicable to mould growth common to those skilled in the art preferably, further the preferred liquid seed culture medium containing, for example lower component and consumption: xylose residue 10-30g/L, wheat bran 20-50g/L, peptone 1-4g/L, ammonium sulfate 2-4g/L, all the other are water.
Component in described seed culture medium and consumption be more preferably: xylose residue 20g/L, wheat bran 40g/L, peptone 3g/L, ammonium sulfate 3g/L, all the other are water.
The invention also discloses the method being suitable for above-mentioned penicillium bacterial strain Penicillium decumbens PD-G3-08 cellulase-producing: under the culture condition being suitable for cellulase expression, cultivate described penicillium bacterial strain Penicillium decumbens PD-G3-08.
Described culturing step carries out in the substratum containing, for example lower content component: carbon source fibrous material 30-50g/L, wheat bran 20-50g/L, produce enzyme inducer 4-8g/L, inorganic nitrogen-sourced 2-5g/L, inorganic phosphate 2-4g/L, inorganic magnesium salt 0.4-0.6g/L, all the other are water.
Described carbon source fibrous material is agricultural or the industrial residue of cellulose, described product enzyme inducer is Microcrystalline Cellulose and/or carboxymethyl cellulose, described inorganic nitrogen-sourced be one or more in ammonium sulfate, urea or ammonium chloride, described inorganic phosphate is potassium primary phosphate, dipotassium hydrogen phosphate or Sodium phosphate dibasic, and described inorganic magnesium salt is magnesium sulfate, magnesium chloride.
Described culture condition is: control temperature 25-30 DEG C, pH 4.0-6.0, air flow 0.5-0.6vvm, mixing speed 100-150rpm, and fermentation time is 80-136 hour.
The step be separated by the cellulase obtained also optionally is comprised: penicillium bacterial strain Penicillium decumbens PD-G3-08 is adopted the such as centrifugal or ultrafiltration of this area conventional means through the fermented liquid that suitable culture medium culturing obtains in enzyme producing method of the present invention, namely obtain the crude enzyme liquid containing required cellulase after removing thalline, cellulosic enzymolysis can be directly used in and transform.
The invention also discloses the cellulase that the method for applying above-mentioned penicillium bacterial strain Penicillium decumbens PD-G3-08 production of cellulose enzyme obtains.
The invention also discloses the application of above-mentioned cellulase in enzymolysis Mierocrystalline cellulose, contact with cellulose substrate by described cellulase.
Described cellulase to be contacted with cellulose substrate enzymolysis according to the cellulosic addition of 10-15FPU/g, preferred enzymolysis conversion condition is: cellulose concentration of substrate is 50-200g/L, control temperature 45-55 DEG C, control pH4-5, mixing speed 50-100rpm, enzymolysis transforms 3-7 days, to cellulosic transformation efficiency up to more than 80%.
The invention also discloses a kind of cellulosic method of enzymolysis, it comprises the step contacted with Mierocrystalline cellulose by penicillium bacterial strain Penicillium decumbens PD-G3-08.
Cellulase of the present invention is a kind of cellulase complex (i.e. cellulase system), under the synergy of various enzyme, make cellulose degradation, so be referred to as cellulase.In general, the polycomponent enzyme system in cellulase comprises circumscribed β-Isosorbide-5-Nitrae-dextranase, inscribe β-Isosorbide-5-Nitrae-dextranase and cellobiase.
The enzyme activity determination related to of the present invention comprises filter paper enzyme activity (FPU), endo-glucanase enzyme activity, cellobiose enzyme activity, exoglucanase vigor.
The various enzyme activity unified definitions that the present invention mentions are: 1ml enzyme liquid, 50 ± 0.1 DEG C, under pH4.8 condition, per minute hydrolysis substrate, produces the reducing sugar amount being equivalent to 1 μm of ol glucose, is 1 enzyme activity unit, represents with IU/ml.
The detection method used all is carried out according to GB formulating method.
The mensuration of filter paper enzyme activity: 1ml liquid enzymes, under (50 ± 0.1) DEG C, pH4.8 condition, 1h is hydrolyzed 50 ± 1mg filter paper, then measures the growing amount of reducing sugar by DNS method.
The mensuration of endo-glucanase enzyme activity: 1ml liquid enzymes, under (50 ± 0.1) DEG C, pH4.8 condition, 1h is hydrolyzed 2ml 1%CMC-Na, then measures the growing amount of reducing sugar by DNS method.
The mensuration of cellobiose enzyme activity: 1ml liquid enzymes, under (50 ± 0.1) DEG C, pH4.8 condition, 30min is hydrolyzed 1ml 1% saligenin, then measures the growing amount of reducing sugar by DNS method.
The mensuration of exoglucanase vigor: 1ml liquid enzymes, under (50 ± 0.1) DEG C, pH4.8 condition, 24h is hydrolyzed 50 ± 1mg absorbent cotton, then measures the growing amount of reducing sugar by DNS method.
Technique scheme of the present invention has the following advantages compared to existing technology, 1, contriver passes through selection by mutation, seed selection obtains the penicillium bacterial strain that high active cellulase is produced in a strain, this strain fermentation is utilized to obtain cellulase crude enzyme liquid, its filter paper enzyme activity reaches 10IU/ml, and endo-glucanase enzyme activity reaches 30IU/ml, and exoglucanase vigor reaches 1.5IU/ml, beta-glucoside enzyme activity reaches 8IU/ml, improves 3 times, 4 times, 4.5 times and 4 times respectively according to starting strain vigor; 2, utilize this bacterial strain to carry out enzymolysis to cellulosic material, within 3 days, cellulose conversion rate can reach more than 80%, has high activity; 3, select agricultural or industrial residue such as xylose residue as the main nutrient composition of cultivating this bacterial strain, partly can play induction and produce enzyme and promote active effect, make use of the waste material in other production technique, economic environmental protection simultaneously.
Embodiment
Embodiment 1: the cultivation of starting strain
To have the penicillium bacterial strain Secondary Culture of cellulase generation ability, after activating 3 times continuously, in access liquid nutrient medium, under 30 DEG C of conditions, shaking culture is spent the night.
Component in described substratum and consumption are: wheat bran 100g/L, glucose 10g/L, and all the other are water.
Embodiment 2: the pre-treatment of bacterial classification
The bacterium liquid centrifugal segregation medium liquid that embodiment 1 is obtained, by the resuspended configuration bacteria suspension of thalline physiological saline obtained, being connected to the tiling that sterilising treatment crosses has in the triangular flask of granulated glass sphere, oscillation treatment 5min under room temperature, makes somatic cells disperse, and uses the filtered through gauze after sterilizing, gradient dilution, counting is 106-107/ml to cell concn, for subsequent use.
Embodiment 3: ultraviolet mutagenesis
Within 20 minutes, open ultraviolet lamp tube with stable light wave in advance.
The bacteria suspension 5-6ml that Example 2 obtains joins in 9cm culture dish, ensures bacterium liquid thickness 2-3cm, culture dish is put on magnetic stirring apparatus, in culture dish, has put into aseptic stirring rod.
Open culture dish lid, distance ultraviolet lamp tube 20-30cm, irradiate 30 while stirring respectively, 60,90,120,150,180s.
Choose the bacteria suspension 0.1ml crossed through different time uv irradiating respectively to join in 0.9ml stroke-physiological saline solution and dilute, put into ice bath 1-2h.
The bacteria suspension got after 0.1ml ice bath is coated and is carried out primary dcreening operation containing 100g/L wheat bran juice with the flat board of 1-5g/L lithium chloride and be separated, and cultivates 3 days for 30 DEG C.
Above-mentioned flat board is chosen the bacterium colony about 200 of different shape, is inoculated into and carries out multiple sieve containing 100g/L wheat bran juice, 20g/L glucose and the cellulosic flat board of 4-8g/L, cultivate 3-5 days for 30 DEG C.
Above-mentioned flat board is chosen transparent circle and the larger bacterium colony about 20 of colony diameter ratio, is inoculated into the slat chain conveyor containing 100g/L wheat bran juice and 10g/L glucose, cultivate after 3-5 days for 30 DEG C and preserve.
The bacterial strain plating of above-mentioned preservation is cultivated propagation in seed culture medium, then accesses fermention medium and carry out induction product enzyme, and Enzyme activity assay is carried out to the crude enzyme liquid obtained, obtain 1 strain enzyme higher bacterial strain alive through contrast screening.
Component in described seed culture medium and consumption are: xylose residue 20g/L, wheat bran 40g/L, peptone 3g/L, ammonium sulfate 3g/L, all the other are water.
In described fermention medium, each amounts of components is: xylose residue 40g/L, wheat bran 30g/L, Microcrystalline Cellulose 6g/L, ammonium sulfate 3g/L, potassium primary phosphate 3g/L, magnesium sulfate 0.5g/L, all the other are water.
Embodiment 4: chemomorphosis
What Example 3 screening obtained has high enzyme bacterial strain alive, carries out the pre-treatment of spawn culture and bacterial classification according to the method described in embodiment 1 and embodiment 2, obtained required bacteria suspension.
In stink cupboard, take 20mgNTG in brown bottle, add 1ml acetone and make it dissolve, then add 0.2M phosphoric acid buffer 9ml, be mixed with 2mg/ml NTG mother liquor, and by this mother liquor dilution respectively to 0.5,1.0,1.5mg/ml.
Above-mentioned pretreated bacteria suspension 0.9ml is mixed with chemical mutagen NTG solution with the ratio of volume ratio 9: 1, the sample of each NTG solution gradient processes 20 respectively, 40,60, termination reaction after 80min.
Get the reacted bacteria suspension of 0.1ml to coat and carry out primary dcreening operation containing 100g/L wheat bran juice with the flat board of 3g/L lithium chloride and be separated, cultivate 3 days for 30 DEG C.
Above-mentioned flat board is chosen transparent circle and the larger bacterium colony about 200 of colony diameter ratio, is inoculated into and carries out multiple sieve containing 100g/L wheat bran juice, 20g/L glucose and the cellulosic flat board of 6g/L, cultivate 3-5 days for 30 DEG C.
Above-mentioned flat board is chosen transparent circle and the larger bacterium colony about 20 of colony diameter ratio, is inoculated into the slat chain conveyor containing 100g/L wheat bran juice and 10g/L glucose, cultivate after 3-5 days for 30 DEG C and preserve.
The bacterial strain plating of above-mentioned preservation is cultivated propagation in seed culture medium, then accesses fermention medium and carry out induction product enzyme, and Enzyme activity assay is carried out to the crude enzyme liquid obtained, obtain 1 strain enzyme higher bacterial strain alive through contrast screening.
Component in described seed culture medium and consumption are: xylose residue 30g/L, wheat bran 30g/L, peptone 2g/L, ammonium sulfate 4g/L, all the other are water.
In described fermention medium, each amounts of components is: xylose residue 50g/L, wheat bran 40g/L, Microcrystalline Cellulose 4g/L, ammonium sulfate 4g/L, potassium primary phosphate 2g/L, magnesium sulfate 0.6g/L, all the other are water.
Embodiment 5: the chemically composited mutagenesis of ultraviolet
The bacterial strain through chemomorphosis embodiment 4 obtained repeats 4 complex mutations according to the method for the spawn culture described in embodiment 1 to embodiment 4, bacterial classification pre-treatment, ultraviolet mutagenesis and chemomorphosis respectively, finally obtain the strain of enzyme activity the highest cellulase producing strain 10, and serial number, and be stored in respectively in the solid medium containing 100g/L wheat bran, 10g/L glucose and 20g/L agar powder.
Embodiment 6: bacterial classification genetic stability is investigated
The bacterial strain that 10 strain vigor embodiment 6 obtained are higher carries out genetic stability investigation.Each bacterial strain was transferred for 10 generations continuously, often carry out cultivation and fermentation for all accessing described fermention medium, fermentation culture conditions is: the initial pH of substratum is 5.0-6.0, leavening temperature 25-30 DEG C, air flow 0.5-0.6vvm, mixing speed 100-150rpm, fermentation 80-136 hour, bacterial strain shows stable leavening property.The bacterial strain that wherein enzyme activity is higher, stability is best is PD-G3-08, and by this culture presevation in Wuhan University's China typical culture collection center (being called for short CCTCC), deposit number is CCTCC M2011195.
Embodiment 7: before and after strain improvement, leavening property compares
Mutagenic strain PD-G3-08, starting strain and commercially available penicillium bacterial strain CICC 40361 are carried out laboratory 250ml shake-flask culture respectively, and the crude enzyme liquid obtained respectively carries out Enzyme activity assay.Component in described seed culture medium and consumption are: xylose residue 30g/L, wheat bran 50g/L, peptone 4g/L, ammonium sulfate 4g/L, all the other are water.The vigor result of crude enzyme liquid before and after contrast mutagenesis, sees the following form:
As can be seen from upper watch test, the bacterial strain enzyme activity after mutagenesis not only strengthens greatly with reference to starting strain, and compared with the penicillium bacterial strain of commercially available cellulase-producing, its enzymic activity also strengthens greatly.
Embodiment 8: bacterial classification multiplication culture
Mutagenic obtained blast resistance PD-G3-08 is seeded to containing xylose residue 30g/L, wheat bran 20g/L, peptone 4g/L, ammonium sulfate 2g/L, all the other are in the seed culture medium of water, 30 DEG C, 150rpm cultivates 30-60 hour, for subsequent use.
Above-mentioned seed liquor is linked into the inoculum size of 5% (v/v) and activates through containing in the fermentor tank of above-mentioned seed culture medium of 121 DEG C of sterilizing 30min, keep tank pressure 0.02-0.05MPa, air flow 0.5vvm, mixing speed 100-150rpm, 30 DEG C of cultivation 30-60 hour, obtain the seed liquor after activating.
Embodiment 9: ferment tank produces enzyme
What seed liquor embodiment 8 obtained accessed sterilizing with the inoculum size of 10% (v/v) is equipped with in the 5L fermentor tank of 3L fermention medium, add defoamer in fermenting process and control foaming, keep tank pressure 0.02-0.05MPa, air flow 0.5-0.6vvm, mixing speed 100-150rpm, 30 DEG C of cultivation 80-136 hour, obtain producing enzymic fermentation liquid.
In described fermention medium, each amounts of components is respectively: xylose residue 50g/L, wheat bran 20g/L, Microcrystalline Cellulose 4g/L, ammonium sulfate 5g/L, potassium primary phosphate 2g/L, magnesium sulfate 0.4g/L, all the other are water, and the initial pH of substratum is 5.0-6.0.
Embodiment 10: crude enzyme liquid transforms cellulosic application
Fermented liquid 8000rpm centrifugal segregation thalline embodiment 9 obtained obtains supernatant liquor, obtain the crude enzyme liquid containing cellulase, and then use the crude enzyme liquid of gained cellulase to contain multiple cellulosic substrate according to 10-15FPU/g cellulosic addition enzymolysis.
The condition of described enzymolysis step is: the crude enzyme liquid of cellulase is added into respectively containing 50,100, in the reaction solution of 200g/L cellulosic substrate, in 45-55 DEG C, control pH5.0, mixing speed 50-100rpm, enzymolysis transforms 3-7 days, and whole enzymolysis process is without the need to pressurize.In the conversion fluid obtained glucose content be equivalent to Mierocrystalline cellulose be respectively 44.4,86.2,167.2g/L, average conversion is all higher than 80%, and the bacterial classification that visible mutagenesis of the present invention obtains has high cellulase hydrolysis vigor.
Embodiment 11: penicillium bacterial strain PD-G3-08 transforms the application of Mierocrystalline cellulose aspect
Mutagenic obtained blast resistance PD-G3-08 is seeded to containing xylose residue 10g/L, wheat bran 50g/L, glucose 1g/L, ammonium chloride 4g/L, all the other are in the seed culture medium of water, 30 DEG C, 150rpm cultivates 30-60 hour, for subsequent use.
Above-mentioned seed liquor is linked into the inoculum size of 5% (v/v) and activates through containing in the fermentor tank of above-mentioned seed culture medium of 121 DEG C of sterilizing 30min, keep tank pressure 0.04-0.05MPa, air flow 0.5vvm, mixing speed 100-150rpm, 30 DEG C of cultivation 30-60 hour, obtain the seed liquor after activating.
What above-mentioned seed liquor was accessed sterilizing with the inoculum size of 10% (v/v) is equipped with in the 5L fermentor tank of 3L fermention medium, add defoamer in fermenting process and control foaming, keep tank pressure 0.04-0.05MPa, air flow 0.5-0.6vvm, mixing speed 100-150rpm, 30 DEG C of cultivation 80-136 hour, obtain producing enzymic fermentation liquid.
In described fermention medium, each amounts of components is respectively: furfural dregs 30g/L, wheat bran 50g/L, carboxymethyl cellulose 8g/L, ammonium nitrate 2g/L, dipotassium hydrogen phosphate 4g/L, magnesium chloride 0.4g/L, all the other are water, and the initial pH of substratum is 5.0-6.0.
Fermented liquid obtained above is obtained supernatant liquor with 8000rpm centrifugal segregation thalline, obtains the crude enzyme liquid containing cellulase, and then use the crude enzyme liquid of gained cellulase to contain multiple cellulosic substrate according to 12FPU/g cellulosic addition enzymolysis.
The condition of described enzymolysis step is: be added into by the crude enzyme liquid of cellulase obtained above in the reaction solution containing 100g/L cellulosic substrate, and in 50 DEG C, control pH5.0, mixing speed 100rpm, enzymolysis transforms 5 days, and whole enzymolysis process is without the need to pressurize.In the conversion fluid obtained, glucose content is equivalent to Mierocrystalline cellulose and is respectively 84.2g/L, and transformation efficiency is all up to 84.2%, and the bacterial classification that visible mutagenesis of the present invention obtains has high cellulase hydrolysis vigor.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (8)
1. a penicillium bacterial strain, its Classification And Nomenclature is
penicillium decumbenspD-G3-08, has been preserved in Wuhan University's China typical culture collection center, and its deposit number is CCTCC M 2011195.
2. a method for production of cellulose enzyme, it cultivates the step of penicillium bacterial strain according to claim 1 under being included in the substratum and culture condition being suitable for cellulase expression;
Described substratum is containing, for example lower content component: carbon source fibrous material 30-50g/L, wheat bran 20-50g/L, produce enzyme inducer 4-8g/L, inorganic nitrogen-sourced 2-5g/L, inorganic phosphate 2-4g/L, inorganic magnesium salt 0.4-0.6g/L, all the other are water;
Described culture condition is: control temperature 25-30 DEG C, pH 4.0-6.0, air flow 0.5-0.6vvm, mixing speed 100-150rpm, fermentation time 80-136 hour.
3. the method for production of cellulose enzyme according to claim 2, it is characterized in that: described carbon source fibrous material is agricultural or the industrial residue of cellulose, described product enzyme inducer is Microcrystalline Cellulose and/or carboxymethyl cellulose, described inorganic nitrogen-sourced be one or more in ammonium sulfate, urea or ammonium chloride, described inorganic phosphate is potassium primary phosphate, dipotassium hydrogen phosphate or Sodium phosphate dibasic, and described inorganic magnesium salt is magnesium sulfate, magnesium chloride.
4. the cellulase that the method for the production of cellulose enzyme according to Claims 2 or 3 obtains.
5. the application of cellulase according to claim 4 in enzymolysis Mierocrystalline cellulose.
6. the application of cellulase according to claim 5 in enzymolysis Mierocrystalline cellulose, is characterized in that: contacted with cellulose substrate by described cellulase.
7. the application of cellulase according to claim 6 in enzymolysis Mierocrystalline cellulose, is characterized in that: to be contacted with cellulose substrate enzymolysis according to the cellulosic addition of 10-15FPU/g by described cellulase.
8. the cellulosic method of enzymolysis, it comprises the step contacted with Mierocrystalline cellulose by penicillium bacterial strain according to claim 1.
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| CN103725660B (en) * | 2013-12-04 | 2015-07-29 | 青岛蔚蓝生物集团有限公司 | A kind of endoglucanase and application thereof |
| EP3628751A1 (en) | 2018-09-27 | 2020-04-01 | INDIAN OIL CORPORATION Ltd. | Method for preparation and screening of fungal mutant with high hydrolytic activity and catabolite derepressed character |
| US11149261B2 (en) | 2018-11-28 | 2021-10-19 | Indian Oil Corporation Limited | Provident method of cellulases enzymes production by Penicillium funiculosum MRJ-16 using low cost media components |
| BR102019027128A2 (en) | 2018-12-19 | 2020-10-06 | Indian Oil Corporation Limited | METHOD FOR PREPARING THE FUNGAL MUTANT WITH HIGH HYDROLYTIC ACTIVITY |
| CN112725194B (en) * | 2021-01-21 | 2021-10-22 | 南京林业大学 | Fungus Flavodon sp.x10 for high yield of cellulase and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1157853A (en) * | 1996-11-13 | 1997-08-27 | 山东大学 | Preparing method of high active cellulase |
| CN102108347A (en) * | 2009-12-25 | 2011-06-29 | 中国科学院生态环境研究中心 | Method for increasing enzyme activity of cellulase produced from fungal |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1157853A (en) * | 1996-11-13 | 1997-08-27 | 山东大学 | Preparing method of high active cellulase |
| CN102108347A (en) * | 2009-12-25 | 2011-06-29 | 中国科学院生态环境研究中心 | Method for increasing enzyme activity of cellulase produced from fungal |
Non-Patent Citations (3)
| Title |
|---|
| Cellulase Production from Spent Sulfite Liquor and Paper-Mill Waste Fiber;QU YINBO et al.;《Applled Biochemistry and Biotechnology》;19911231;第28-29卷;第363-368页 * |
| 一株产纤维素酶真菌的筛选、鉴定及降解效果;李保深等;《东北林业大学学报》;20110731;第39卷(第7期);第134-137页 * |
| 抗黑液毒性的纤维素酶产生菌选育的探讨;赵昕等;《纤维素科学与技术》;19931231;第1卷(第2期);第28-32页 * |
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| CN103045484A (en) | 2013-04-17 |
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