CN103555595A - Strain for producing cellulase and application of strain - Google Patents
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Abstract
The invention discloses a strain for producing cellulase. The strain has a collection number of CGMCC No.8339 in the China General Microbiological Culture Collection Center, and the classification name of the strain is Penillium piceum H16. The Penillium piceum is obtained through induced mutation treatment by taking a strain Penillium piceum 9-3 screened from straws as an original strain. The Penillium piceum 9-3 serves as the original strain for mutation breeding, the Penillium piceum H16 for producing high-activity cellulase is obtained through breeding, the enzyme activity of filter paper of cellulase crude enzyme liquid obtained by fermenting the Penillium piceum H16 reaches 7IU/mL, and the enzyme activity of beta-glucosidase reaches 50IU/mL. Different cellulase systems of the Penillium piceum H16 and Trichoderma reesei RUT-C30 are researched according to different proportions, the enzyme activity and the hydrolysis rate of microcrystalline celluloses are greatly improved at an optimal proportion, and the enzyme cost is reduced.
Description
Technical field
Microbiology of the present invention field, particularly strain cellulase producing strain and an application thereof.
Background technology
Since the mankind enter 21 century, at aspects such as the energy, resource, environment, be faced with more and more stern challenge.Mierocrystalline cellulose is recyclability organic resource maximum on the earth, utilize these cheapnesss, reproducible plant cellulose material produces thing base product and biomass energy next life, to the mankind's Sustainable development, is very favorable.Mierocrystalline cellulose is the main component of plant cell wall, to be that occurring in nature distributes the widest, content is maximum the more than 50% of a kind of polysaccharide ，Zhan vegitabilia carbon content.In China, there is every year the agricultural crop straw of several hundred million tons to be incinerated, not only polluted environment but also wasted resource.At present the method for saccharification of cellulose mainly contains acid hydrolyzation and enzymolysis process, but acid hydrolyzation have power consumption large, large to equipment loss, pollute the shortcomings such as large, so enzymolysis process is by the main trend that is cellulase saccharification.But because the production cost of cellulase is too high, cause ligocellulose degradation's high cost, make really to realize industrialization.Therefore, in order to improve enzymic hydrolysis efficiency, reduce the cost of industrial production cellulase, countries in the world scientist has launched research widely around cellulase, comprises the screening of bacterial strain, optimization of zymotechnique etc.
Cellulase is the general name that makes the glucogenic one group of enzyme of cellulose degradation, mainly comprises endoglucanase, exoglucanase and beta-glucosidase.Three components of cellulase, through synergy, are low-molecular-weight oligosaccharides, disaccharide or polysaccharide by macromolecular cellulose degradation.Cellulase is widely used in the aspects such as food-processing industry, Brewing industry, paper industry, fodder additives, textile industry, field of medicaments, Plant Cytomixis.
At present, the production method of cellulase is generally solid state fermentation and liquid state fermentation.Solid state fermentation cost is low, but easy pollution microbes is difficult for extraction, is difficult to carry out large-scale industrial production.And liquid state fermentation belongs to closed fermentation, in fermenting process, be difficult for polluting, and fermentation condition to be to control, later stage separation and Extraction enzyme liquid is easy, is therefore convenient to scale operation.Produce highly active cellulase except having good zymotechnique route, comprise substratum, fermentation time, leavening temperature etc., the factor of most critical is breeding high-yield bacterial strain.
The generation bacterium of cellulase comprises bacterium, fungi, yeast etc., but the bacterial classification that is mainly used at present cellulase production is mainly filamentous fungus.The cellulase systems that filamentous fungus produces is more complete, and the enzyme of its generation mostly is extracellular enzyme, is convenient to the separation and Extraction in later stage.At present the more bacterium of research comprise that wood is mould, aspergillus etc., for example Trichodermareesei, aspergillus niger, especially concerned with Trichoderma, and less to the report of Penicillium.The Penicillium bacterial strain that is a plant height cellulase-producing that the present invention explains.
Summary of the invention
The object of the invention is to provide a strain cellulase producing strain.The present invention utilizes mutafacient system to carry out mutagenesis screening to starting strain to go out the more cellulase strain of high yield, and the enzyme activity of described cellulase has obtained large increase.The present invention requires low, simple to operate to plant and instrument, reduced to a great extent and used enzyme cost.
For achieving the above object, the technical solution used in the present invention is:
The bacterial strain of one strain cellulase-producing, is characterized in that, described bacterial strain at the preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is: CGMCC No.8339, Classification And Nomenclature is: penicillium bacterial strain Penillium piceum H16.
The morphological feature of penicillium bacterial strain Penillium piceum H16 of the present invention is:
The morphological feature of Chinese juniper shape mould H16 of the present invention is: bacterial strain H16 30 ℃ of constant temperature culture 2d on PDA substratum can see white colony clearly, bacterium colony is carried out, velvet-like, during 4～5d, top layer the blackish green conidial powder of raw one deck, and the strain growth later stage back side is observed and is yellow.The color of spore is compared and is wanted dark compared with starting strain Penillium piceum 9-3, and starting strain is yellow-green colour, and the spore of H16 is blackish green.
Described penicillium bacterial strain H16 screens bacterial strain Penillium piceum 9-3 to obtain after mutafacient system is processed.
Described mutagenesis screening method comprises the following steps:
1) in the spore suspension of described bacterial strain Penillium piceum 9-3, add ethyl sulfate, described ethyl sulfate addition is that to make the mass percent concentration of ethyl sulfate in described spore suspension be 1%, afterwards at 10～30 ℃, 50～150rpm concussion reaction, 10～120min, obtain the mutagenic treatment liquid of described spore suspension, standby;
2) with the described reaction times, being as the criterion, is to take out the hypo solution that mutagenic treatment liquid is 25% with concentration at 1: 9 to mix at interval of 10min by volume, obtains mutagenesis stop buffer and numbers according to time sequence;
3) the described mutagenesis stop buffer of numbering is coated with respectively to PDA flat board in order, cultivates until grow single bacterium colony for 25～33 ℃;
4) described single bacterium colony is obtained to described penicillium bacterial strain H16 by contrast transparent circle on Congo red flat board with the size of the ratio of colony diameter and the method screening of fermentation culture mensuration crude enzyme liquid enzyme activity.
Described step 1) described reaction times is 60min.
Described penicillium bacterial strain H16 is applied to production of cellulose enzyme.
One strain application rights requires the method for cellulase producing strain cellulase-producing described in 1～5, it is characterized in that, for cultivating the component concentration of the substratum of described cellulase producing strain, be: Microcrystalline Cellulose 10～30g/L, glucose 1～10g/L, Dried Corn Steep Liquor Powder 10～20g/L, ammonium sulfate 1～10g/L, potassium primary phosphate 1～10g/L, sal epsom 1～10g/L and add water to 1L.
Preferably, described culture condition is: temperature is 26～32 ℃, and rotating speed is that 150～250rpm concussion is cultivated, and initial pH is 5.0～6.0, and inoculum size is 1～5%, and fermented incubation time is 96～144h.
Preferably, described culture condition is: initial pH is 5.5, and fermented incubation time is 120h.
The invention has the beneficial effects as follows the present invention by Penillium piceum 9-3 as starting strain selection by mutation, seed selection obtains the penicillium bacterial strain that high activity cellulase is produced in a strain, the cellulase crude enzyme liquid that described strain fermentation obtains, its filter paper enzyme activity reaches 7IU/mL, and beta-glucosidase enzyme activity reaches 50IU/mL.By the different cellulase system of described bacterial strain H16 and Trichodermareesei RUT-C30 are carried out to different proportion research, show that significantly high enzyme is lived and the percent hydrolysis to Microcrystalline Cellulose when optimal proportion, reduced and used enzyme cost.The present invention requires low, simple to operate to plant and instrument, reduced to a great extent and used enzyme cost.
Accompanying drawing explanation
Fig. 1 is cellulase producing strain mutagenesis screening method schema of the present invention.
Fig. 2 is that the crude enzyme liquid of cellulase producing strain of the present invention fermentation gained crude enzyme liquid and Trichodermareesei RUT-C30 is according to the filter paper enzyme activity comparison diagram of different volumes ratio proportioning gained mixed enzyme solution.
Fig. 3 is the comparison diagram of the mixed enzyme extracting solution of cellulase producing strain of the present invention and Trichodermareesei RUT-C30 and the percent hydrolysis of Trichodermareesei enzyme RUT-C30 extracting solution.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, to make those skilled in the art can implement according to this with reference to specification sheets word.
Embodiment 1: the cultivation of starting strain
By Chinese juniper shape penicillium bacterial strain 9-3 activation twice continuously on PDA inclined-plane, in 30 ℃ of thermostat containers, cultivate 3-4 days, afterwards, 4 ℃ save backup.
Chinese juniper shape mould 9-3 (Penicillium piceum 9-3).Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preservation date: on October 9th, 2011, preserving number: CGMCC5314.
Embodiment 2: ethyl sulfate mutagenesis screening bacterial strain H16 (as shown in Figure 1)
1) ethyl sulfate mutagenesis
The spore of washing lower PDA slant culture with the phosphoric acid buffer that pH is 7, sterile glass beads is broken up, and two-layer aseptic lens wiping paper filters, and obtains spore suspension, blood counting chamber counting, adjusting spore concentration is 10
6individual/mL, get 4mL spore suspension, in the phosphoric acid buffer that the pH that joins 16mL is 7, add again 0.2mL ethyl sulfate, at 30 ℃, under 150r/min condition, reaction starts after 20min to extract reaction solution in the Sulfothiorine that concentration that 0.1mL joins 0.9mL is 25% and fully to mix every 5min, stops mutagenesis reaction, and then three gradients of mixed solution dilution being made to bacterial classification final concentration in mixed solution is 10
5individual/mL, 10
4individual/mL, 10
3individual/mL, the diluent 0.1mL coating PDA that gets respectively three kinds of concentration is dull and stereotyped, cultivates until grow single bacterium colony for 30 ℃, for detection of lethality rate and the positive mutation rate of mutagenesis.As shown in table 1, lethality rate during 55min (%) and positive mutation rate (%) almost reach 100%.
2) screening
1. congo red staining screening: ask while choosing different treatment on above-mentioned flat board, bacterium colony 100 strains of different shape, be inoculated into and contain peptone 10g/L, yeast powder 10g/L, sodium-chlor 5g/L, Xylo-Mucine 10g/L, potassium primary phosphate 1g/L, agar 18g/L, tween-80 2mL/L.Cultivate after 4-5 days the congo red staining 1h that is 1% by concentration for 30 ℃.On above-mentioned flat board, choose 9 of the bacterium colonies that transparent circle and colony diameter ratio are larger, be inoculated into PDA inclined-plane, cultivate after 4-5 days for 30 ℃ and preserve bacterial classification.
2. fermentation culture screening: the inoculation of above-mentioned preservation is cultivated to increment in seed culture medium, be then linked into fermention medium and induce and produce enzyme, and centrifugal acquisition crude enzyme liquid is carried out to enzyme activity determination, obtain 1 strain enzyme higher bacterial strain alive through contrast screening.
Described seed culture medium is: Microcrystalline Cellulose 20g/L, Dried Corn Steep Liquor Powder 17g/L, glucose 2g/L.
Described fermention medium is: Microcrystalline Cellulose 20g/L, Dried Corn Steep Liquor Powder 17g/L, ammonium sulfate 5g/L, calcium carbonate 2.5g/L, potassium primary phosphate 6g/L, sal epsom 1g/L.At 30 ℃, 200 turn/min, initial pH5.5, inoculum size 3%, fermentation culture 120h.
| Time (min) | Lethality rate (%) | Positive mutation rate (%) |
| 20 | 5.12 | 0.14 |
| 25 | 16.53 | 4.83 |
| 30 | 25.25 | 7.99 |
| 35 | 46.77 | 12.12 |
| 40 | 59.53 | 21.24 |
| 45 | 72.18 | 31.88 |
| 50 | 90.12 | 45.26 |
| 55 | 99.98 | 100 |
Table 1
Embodiment 3: bacterial classification genetic stability
The bacterial strain H16 higher to the resulting activity of embodiment 2 carries out genetic stability investigation.Bacterial strain was transferred for 6 generations continuously, and in per generation, is all accessed fermentation culture in the fermention medium described in embodiment 2.As shown in table 2 below, bacterial strain H16 enzyme activity is higher and have good stability.
Table 2
Embodiment 4: cellulase activity is measured
The enzyme activity determination the present invention relates to comprises filter paper enzyme activity, beta-glucosidase enzyme activity.
The various enzyme activities that the present invention relates to are agreed to be defined as: at 50 ± 0.1 ℃, under the condition of pH4.8,1mL enzyme liquid hydrolysis substrate 1min produces the reducing sugar amount that is equivalent to 1 μ mol glucose, is 1 enzyme activity unit, with IU/mL, represents.
Described enzyme detection method alive is all carried out according to international method IUPAC.
The measuring method of filter paper enzyme activity: 1mL crude enzyme liquid, at 50 ± 0.1 ℃, under the condition of pH4.8, be hydrolyzed the amount of the reducing sugar that 50 ± 1mg filter paper 1h produces, by DNS method, measure the growing amount of reducing sugar.
The measuring method of beta-glucosidase enzyme activity: 1mL crude enzyme liquid, at 50 ± 0.1 ℃, under the condition of pH4.8, the cellobiose 30min of hydrolysis 1mL1%, the amount of the glucose producing, with the growing amount of biosensor assay glucose.
Embodiment 5: the enzyme activity of starting strain and mutagenic strain and protein secretion comparison
Chinese juniper shape mould starting strain 9-3 and mutagenic strain H16 are carried out to fermentation culture simultaneously, and as described in Example 2, culture condition is fermention medium: 30 ℃, and 200rpm, initial pH5.5, inoculum size 3%, fermentation culture 120h.Contrast two bacterial strain enzyme activities as shown in table 1:
Table 3
As shown in table 3, to compare with starting strain, product enzyme activity and the protein excretion ability of mutagenic strain H16 are all improved largely.
Embodiment 6: penicillium bacterial strain Penillium piceum H16 transforming glucose is the application of sophorose aspect
Penicillium bacterial strain Penillium piceum H16 is measured to the enzyme activity of beta-glucosidase enzyme according to above-mentioned culture condition and above-mentioned substratum fermentation gained crude enzyme liquid according to the method for said determination cellobiose enzyme activity, with the enzyme liquid of same protein content (3mg/mL), act on glucose substrate with type strain Trichodermareesei RUT C30, wherein the addition of mould enzyme liquid is 1mL, the addition of the mould enzyme liquid of wood is 0.8mL, the concentration of glucose is 400g/L, at 50 ℃ of reaction 30min, bio-sensing instrument is measured glucose residual content, and HPLC analyzes sophorose content in conversion fluid.Analysis shows, in reaction solution, sophorose concentration, up to 10.57g/L, is 6 times of Trichodermareesei RUT C30.Illustrate that this bacterial strain has advantage aspect the efficient inductor-sophorose of generation cellulase.
Embodiment 7: the crude enzyme liquid enzyme of penicillium bacterial strain Penillium piceum H16 and Trichodermareesei RUT-C30 is the filter paper enzyme activity after proportioning
By penicillium bacterial strain Penillium piceum H16 according to the crude enzyme liquid of above-mentioned culture condition and above-mentioned substratum fermentation gained crude enzyme liquid and Trichodermareesei RUT-C30 according to different ratios proportioning, gained mixed enzyme solution is surveyed to filter paper enzyme activity according to above-mentioned filter paper enzyme activity measuring method, result as shown in Figure 2, when Trichodermareesei and penicillium bacterial strain H16 are 5: 1 proportionings by volume, filter paper enzyme activity improves 37% compared with Trichodermareesei self enzyme liquid.
Embodiment 8: the hydrolysis effect after the crude enzyme liquid proportioning of penicillium bacterial strain Penillium piceum H16 and Trichodermareesei RUT-C30
By penicillium bacterial strain Penillium piceum H16 according to the crude enzyme liquid of culture condition described in embodiment 2 and substratum fermentation gained crude enzyme liquid and Trichodermareesei RUT-C30 according to best filter paper enzyme activity ratio proportioning, gained mixed enzyme solution is reacted according to 10IU/g Microcrystalline Cellulose, with Trichodermareesei self enzyme liquid in contrast, at 50 ℃ of hydrolysis 12h, 24h, 36h, 48h, 60h, the hydrolysis gained residual sugar amount of take is standard, as shown in Figure 3, the mixed enzyme of Trichodermareesei and penicillium bacterial strain H16 system improves 22% compared with Trichodermareesei self percent hydrolysis to results of hydrolysis.
Although embodiment of the present invention are open as above, but it is not restricted to listed utilization in specification sheets and embodiment, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other modification, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and shown here embodiment.
Claims (9)
1. the bacterial strain of a strain cellulase-producing, is characterized in that, described bacterial strain at the preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center is: CGMCC No.8339, Classification And Nomenclature is: penicillium bacterial strain Penillium piceum H16.
2. the bacterial strain of cellulase-producing as claimed in claim 1, is characterized in that, described penicillium bacterial strain H16 screens bacterial strain Penillium piceum 9-3 to obtain after mutafacient system is processed.
3. the mutagenesis screening method of strain cellulase producing strain as claimed in claim 2, is characterized in that, to adding ethyl sulfate in the spore suspension of described bacterial strain Penillium piceum 9-3, carries out 10～120min mutagenesis reaction.
4. the mutagenesis screening method of cellulase-producing as claimed in claim 3, is characterized in that, comprises the following steps:
1) in the described spore suspension of described bacterial strain Penillium piceum 9-3, add ethyl sulfate, described ethyl sulfate addition is that to make the mass percent concentration of ethyl sulfate in described spore suspension be 0.8～1.2%, afterwards at 10～30 ℃, 50～150rpm concussion reaction, 10～120min, obtain the mutagenic treatment liquid of described spore suspension, standby;
2) with the described reaction times, be as the criterion, at interval of 8～12min, take out 0.1～0.3mL mutagenic treatment liquid and mixes ethyl sulfate in the described mutagenic treatment liquid of neutralization with hypo solution with termination mutagenesis reaction, acquisition mutagenesis stop buffer is also numbered according to time sequence;
3) the described mutagenesis stop buffer of numbering is coated with respectively to PDA flat board in order, cultivates until grow single bacterium colony for 25～33 ℃;
4) described single bacterium colony is obtained to described penicillium bacterial strain H16 by contrast transparent circle on Congo red flat board with the size of the ratio of colony diameter and the method screening of fermentation culture mensuration crude enzyme liquid enzyme activity.
5. the mutagenesis screening method of the bacterial strain of cellulase-producing as claimed in claim 3, is characterized in that described step 1) mass percent concentration of described ethyl sulfate is 1%, the reaction times is 60min.
6. the bacterial strain of cellulase-producing as claimed in claim 2, is characterized in that, described penicillium bacterial strain H16 is applied to production of cellulose enzyme.
7. a strain application rights requires the method for the cellulase producing strain cellulase-producing described in any one in 3～5, it is characterized in that, for cultivating the component concentration of the substratum of described cellulase producing strain, be: Microcrystalline Cellulose 10～30g/L, glucose 1～10g/L, Dried Corn Steep Liquor Powder 10～20g/L, ammonium sulfate 1～10g/L, potassium primary phosphate 1～10g/L, sal epsom 1～10g/L and add water to 1L.
8. the method for cellulase producing strain cellulase-producing as claimed in claim 7, is characterized in that, described culture condition is: temperature is 26～32 ℃, rotating speed is that 150～250rpm concussion is cultivated, initial pH is 5.0～6.0, and inoculum size is 1～5%, and fermented incubation time is 96～144h.
9. the method for cellulase producing strain cellulase-producing as claimed in claim 8, is characterized in that, described culture condition is: initial pH is 5.5, and fermented incubation time is 120h.
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Cited By (5)
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| CN103710326A (en) * | 2013-12-11 | 2014-04-09 | 中国科学院天津工业生物技术研究所 | Beta-glucosaccharase and application thereof |
| CN104263658A (en) * | 2014-09-26 | 2015-01-07 | 中国科学院天津工业生物技术研究所 | Trichoderma reesei strain and application thereof |
| CN104560917A (en) * | 2014-10-30 | 2015-04-29 | 中国科学院天津工业生物技术研究所 | Beta-glucosaccharase, beta-glucosaccharase mutant and application |
| CN105602854A (en) * | 2014-11-24 | 2016-05-25 | 勐海茶业有限责任公司 | Bacterial strain of Paecilomyces lilacinus and application thereof to production of Pu'er tea |
| CN106434447A (en) * | 2016-09-26 | 2017-02-22 | 浙江大学舟山海洋研究中心 | Strain capable of producing salt-tolerant neutral cellulase, and screening method and application thereof |
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| CN113151006B (en) * | 2021-03-29 | 2022-04-26 | 中国科学院天津工业生物技术研究所 | Trichoderma reesei strain capable of producing cellulase with improved activity and application thereof |
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| CN103710326A (en) * | 2013-12-11 | 2014-04-09 | 中国科学院天津工业生物技术研究所 | Beta-glucosaccharase and application thereof |
| CN103710326B (en) * | 2013-12-11 | 2016-01-20 | 中国科学院天津工业生物技术研究所 | A kind of beta-glucosidase and application thereof |
| CN104263658A (en) * | 2014-09-26 | 2015-01-07 | 中国科学院天津工业生物技术研究所 | Trichoderma reesei strain and application thereof |
| CN104560917A (en) * | 2014-10-30 | 2015-04-29 | 中国科学院天津工业生物技术研究所 | Beta-glucosaccharase, beta-glucosaccharase mutant and application |
| CN104560917B (en) * | 2014-10-30 | 2018-12-18 | 中国科学院天津工业生物技术研究所 | A kind of beta-glucosidase and β-glucosidase mutant and application |
| CN105602854A (en) * | 2014-11-24 | 2016-05-25 | 勐海茶业有限责任公司 | Bacterial strain of Paecilomyces lilacinus and application thereof to production of Pu'er tea |
| CN106434447A (en) * | 2016-09-26 | 2017-02-22 | 浙江大学舟山海洋研究中心 | Strain capable of producing salt-tolerant neutral cellulase, and screening method and application thereof |
| CN106434447B (en) * | 2016-09-26 | 2019-08-13 | 浙江大学舟山海洋研究中心 | The bacterial strain and screening technique of production salt tolerant neutral cellulase and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104312928A (en) | 2015-01-28 |
| CN104312928B (en) | 2017-10-31 |
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