CN103468583A - Penicillium oxalicum WX-209 strain with high cellulase yield and enzyme producing method - Google Patents
Penicillium oxalicum WX-209 strain with high cellulase yield and enzyme producing method Download PDFInfo
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Abstract
The invention discloses a Penicillium oxalicum WX-209 strain with high cellulase yield and an enzyme producing method. The Penicillium oxalicum WX-209 is screened from rotten walnut skins in Wenxian County Gansu province, and the preservation number is CCTCC NO:M2013325. The method for preparation of a crude enzyme liquid by fermentation includes slant culture, ultrasonic pretreatment of a substrate (wheat bran), fermentation, enzyme liquid acquiring and other steps. The ultrasonic treatment of the substrate under ultrasonic power of 700-900W for 40-55min effectively improves the cellulase activity (in terms of CMCase enzyme) of the strain, and the improvement effect is significant compared with a non-pretreated control group. The strain provided in the invention has very good application prospects in cellulose degradation.
Description
Technical field
The present invention relates to plant height cellulase producing strain and an enzyme producing method, belong to strain fermentation and produce the zymotechnic field.
Background technology
Mierocrystalline cellulose is the renewable energy source material of quantity maximum on the earth, and annual global photosynthetic product is up to 1.5 * 10
11～2.0 * 10
11t, the crop residues (straw, stalk etc.) formed in agriculture production often is only by China just approximately 2,000,000,000 t.How to utilize these a considerable number of agriculture productions " tankage ", developing serial high value added product becomes world today's focal issue.Development along with biotechnology; domestic and international all multi-experts start to attempt utilizing microorganism to carry out fermentative degradation to Mierocrystalline cellulose; the cellulase of microorganisms can be low-molecular-weight hexose or pentose by cellulase hydrolysis; for the production of new feed, single cell protein etc.; grain, the energy and the ecocrisis of facing mankind will be alleviated; this behave is about to the Mierocrystalline cellulose that reserves are huge and becomes the resource that value is very considerable, and for present society, Economic development has great strategic importance.On the other hand, the cellulase produced in microorganism fermented cellulose process can be made zymin, important and purposes is widely also arranged in foodstuffs industry, can be used for the aspects such as garden stuff processing, oil crops processing, Tea Production, Alcohol Production, beer production, vinegar brewing, active substance extraction, the development of above-mentioned industry is had to huge pushing effect.
In order to obtain the enzyme higher wild cellulose-degrading bacteria of living, the scientific worker has started to do a large amount of work from 20 discipline the sixties both at home and abroad, has screened at present a lot of cellulose-degrading bacterias, mainly comprises bacterium, actinomycetes and filamentous fungus etc.Much the stronger bacterial strain to the Mierocrystalline cellulose effect of having reported is Trichoderma, Aspergillus, Penicillium.The applicant is by the congo red staining primary dcreening operation, and shake flask fermentation sieves again and obtains High Cellulase Production bacterial strain---penicillium oxalicum Penicillium oxalicum WX-209.And its enzymatic production is studied.
Mierocrystalline cellulose content in plant cell wall is higher, but vegetable cell cell walls complex structure, the cellulose molecular chain skin is wrapped in a large amount of hemicelluloses and a considerable number of xylogen, make cell walls have very strong rigidity and toughness, the mixture that simultaneously Multiple components forms also makes the single enzyme substrate of can't effectively degrading; And Mierocrystalline cellulose is with β-1 by D-Glucose, the macromolecular polysaccharide that 4 glycosidic links form, usually molecular weight is 50000～2500000, be equivalent to 300～15000 glucosyl groups, in its molecule and a large amount of hydrogen bonds of intermolecular formation, make structure more firm, cause microorganism to be difficult to utilize, biodegradation rate is low.Usually, by physics modes such as steam explosions, substrate is processed the macrostructure that can destroy cell walls, high but standard machinery is processed power consumption, efficiency is low, and effect is not remarkable.Ultrasonic wave can directly act on the microtexture of cell walls, its principle is owing to containing gas and minute impurities in solvent, while in solvent, launching ultrasonic wave, will between solvent and sample, form the sound wave cavatition, cause a large amount of micro-bubble formation, growth, explosion and compression in solution, sample is produced to destruction.The great force produced during ultrasonic cavitation can be destroyed cell wall structure, Mierocrystalline cellulose is exposed and fragment into to be easy to the small molecules utilized, and improves cellulosic degradation rate.The present invention, by ultrasonication wheat bran fibre element, makes its exposure and fragments into growth to be easy to the small molecules utilized, thereby improve growth and the cellulase-producing efficiency of bacterial classification.By existing report, preprocessing means is to prepare reducing sugar for improving biomass degradation mostly, and the report that improves the production by biological enzyme for degraded product but rarely has report.
The present invention pays close attention to the bacterial strain of screening High Cellulase Production, and improves it by pre-treatment and produce enzyme efficiency.It is fermentation strain that the penicillium oxalicum Penicillium oxalicum WX-209 that screening obtains is take in the present invention, usings cheap wheat bran as fermentation substrate, and the simple exercisable ultrasonication of usining obtains the higher crude enzyme liquid of cellulase activity as preprocessing means.Therefore, the present invention has certain using value for efficient utilization and the product high added value Industrial products of waste fiber cellulosic biomass.
Summary of the invention
The purpose of this invention is to provide the penicillium oxalicum Penicillium oxalicum WX-209 of a plant height cellulase-producing, its deposit number is CCTCC NO:M2013325, and a kind of enzyme producing method that improves enzyme activity by the ultrasonic pretreatment substrate is provided.
Technical scheme of the present invention: rot within the border to separate the penicillium oxalicum Penicillium oxalicum WX-209 that obtains a plant height cellulase-producing Cortex walnut from Wenxian, Gansu, on July 9th, 2013, be preserved in the Chinese Typical Representative culture collection center of Wuhan, China Wuhan University, deposit number is CCTCC NO:M2013325, called after penicillium oxalicum WX-209 Penicillium oxalicum WX-209.The morphological specificity of penicillium oxalicum Penicillium oxalicum WX-209:
The dull and stereotyped upper 35 ℃ of cultivations of PDA are white hypha in 1 day, cultivate the bacterium colony that forms medium aquamarine Chan Bao district in outer white in 2 days, and cultivating colony diameter after 3 days is 17-19mm, and bacterium colony is blackish green, outer rim white (Fig. 1); Microscopic examination, mycelia interweaves, the tool tabula; Penicillus is the symmetric double wheel shape; The conidium ellipse is translucent.
Penicillium oxalicum WX-209 provided by the present invention can be used for preparing cellulase preparation, has higher product CMCase enzyme activity.Penicillium oxalicum Penicillium oxalicum WX-209 can the liquid nutrient medium that be sole carbon source at wheat bran on growth produce enzyme, the zymotechnique simple possible, the characteristics such as enzyme is rapid, and the utilization of waste wheat bran is abundant are produced in growth.
The method that adopts described penicillium oxalicum bacterial strain fermentation to produce high vigor enzyme comprises the following steps:
1) slant culture: penicillium oxalicum bacterial strain Penicillium oxalicum WX-209 spore inoculating, to the PDA slant medium, is cultivated 3～4 days, with spore under aseptic washing, made spore suspension standby for 35 ℃;
2) substrate pre-treatment: get Mandels salts solution 30ml and add the wheat bran that mass percent is 5～8%, carry out ultrasonication, the ultrasonic wave parameter is: 700～900W supersound process 40～55 minutes;
3) fermentation: the substrate autoclaving 20min after pre-treatment, cooling rear by 1 * 10
5～1 * 10
6the inoculum size of individual/mL access penicillium oxalicum spore suspension, 32 ℃～37 ℃, 150～180r/min are cultivated 72h.
Fermenting container is the 250mL shaking flask, and liquid amount is 30～80mL.
The inventive method is processed rear crude enzyme liquid and is obtained and enzyme activity determination: nutrient solution is carried out to suction filtration or the centrifugal 10min of 10000r/min, separate the supernatant fermented liquid obtained and be crude enzyme liquid, adopt the DNS method to measure the CMCase enzyme activity.
The CMCase enzyme activity determination: the enzyme liquid 0.5mL that gets the dilution of suitable damping fluid adds 1.5mL1.0%(W/V) CMC-Na solution (this solution is by pH=4.8, the configuration of concentration 0.05M citrate buffer solution) in 25mL tool plug test tube, add 2mL DNS termination reaction after 50 ℃ of reaction 30min, boiling water bath 5min, flowing water is cooling, with distilled water, keep the skin wet to scale, the 540nm place measures light absorption value.Blank, for before adding enzyme liquid, first adds the DNS termination reaction of 2mL, and other steps are identical.Enzyme is lived and defined: the 1mL crude enzyme liquid is enzyme unit (U) alive at 1min bottom exploded deposits yields 1 μ mol glucose.
In ultrasonic pretreatment substrate step, the JY92-II that the instrument used is manufactured for the new sesame bio tech ltd in Ningbo, 40～55 minutes best results of power 700～900W supersound process of rising to the Western Paradise.
The present invention has following advantage:
1) cost is low: whole fermenting process mild condition, and not high and consume energy lowly to equipment requirements, use Testa Tritici as fermentation substrate simultaneously, take full advantage of the scrap stock in agriculture production, both compress the production cost of cellulase, turned waste into wealth again, taken full advantage of resource;
2) enzyme activity is higher: bacterial strain involved in the present invention has stronger cellulose degradation ability, by the ultrasonic pretreatment substrate, can more obviously improve cellulase activity.
3) application prospect is good: on the earth, the Mierocrystalline cellulose reserves are huge, there is huge potential value, can utilize cellulose resource to there is great strategic significance to socio-economic development, the present invention is based on the problems referred to above, cellulosic utilization is attempted, obtain desirable result, there is good prospect.
The accompanying drawing explanation
Fig. 1 is the bacterium colony figure that bacterial strain PDA of the present invention cultivates 3 days.
Embodiment
Following examples are intended to illustrate the present invention rather than limitation of the invention further, and the present invention can implement by the described either type of summary of the invention.
Example 1: screening and the authentication method of penicillium oxalicum Penicillium oxalicum WX-209
A, primary dcreening operation: 18 collected specimens such as branch of rotting from the mouldy walnut of Wenxian, Gansu, Yue Lu mountain soil sample, tangerine garden mould sample, kiwi fruit tree garden soil sample, Yue Lu mountain.Sample thief 5g adds 100mL2%(W/V) CMC-Na liquid enrichment medium, under 35 ℃, 170r/min shaking table condition, enrichment culture, after 3 days, is got the 1mL nutrient solution and is diluted to 10
6-10
8get the 0.2mL diluent and be uniformly coated on the screening plate culture medium that CMC-Na is carbon source, after cultivating 3 days, by the method for congo red staining, filter out totally 20 of the fungal strains that transparent circle is larger, further draw dull and stereotyped purifying bacterial strain, finally be forwarded on the PDA slant medium and cultivate and preserve.
B, multiple sieve: preparation Mandels nutritive medium, 250mL triangular flask liquid amount 50mL, add the CMC-Na of mass percent 2%, 121 ℃ of autoclaving 15min, inoculation primary dcreening operation spore suspension to 1 * 10
6spore/mL, cultivate 72h in constant-temperature table, 35 ℃ of temperature, natural pH, rotating speed 170r/min.After fermented liquid is centrifugal for detection of the CMCase enzyme activity.Obtain by screening the bacterial strain Penicillium oxalicum WX-209 that the CMCase enzyme activity is higher.
C, identification of strains: by ITS sequence amplification identify, show that it is 99% that this sequence and Penicillium oxalicum belong to similarity, prove the bacterial strain of penicillium oxalicum, final called after Penicillium oxalicum WX-209.
Embodiment 2:
By the penicillium oxalicum spore inoculating, to slant medium, 35 ℃ of growths, after 4 days, with spore under aseptic washing, are made 4 ℃ of refrigerations of spore suspension standby.
Take wheat bran 2.4g and put in 30ml Mandels nutritive medium, fully infiltrate.Carry out ultrasonic pretreatment after infiltration, under ultrasonic power 800W condition, supersound process is 50 minutes.After processing, autoclaving 20min, sterilizing is cooled to room temperature after finishing, and under aseptic condition, access penicillium oxalicum spore, make inoculating spores concentration reach 1 * 10
6individual/mL.Postvaccinal shaking flask is placed in to 35 ℃ of constant-temperature tables and cultivates 72h, it is 170r/min that rotating speed is set.By nutrient solution suction filtration or centrifugal, obtain crude enzyme liquid, detect enzyme liquid CMCase enzyme activity by the DNS method, record the CMCase enzyme and live as 7.15U, and do not carry out ultrasonic pretreatment group (6.0U) and contrast vigor and improve 15.2%.
Embodiment 3:
By the penicillium oxalicum spore inoculating, to slant medium, 35 ℃ of growths, after 3 days, with spore under aseptic washing, are made 4 ℃ of refrigerations of spore suspension standby.
Take wheat bran 2.4g and put in 30ml Mandels nutritive medium, fully infiltrate.Carry out ultrasonic pretreatment after infiltration, under ultrasonic power 800W condition, supersound process is 55 minutes.After processing, autoclaving 20min, sterilizing is cooled to room temperature after finishing, and under aseptic condition, access penicillium oxalicum spore, make inoculating spores concentration reach 1 * 10
5individual/mL.Postvaccinal shaking flask is placed in to 35 ℃ of constant-temperature tables and cultivates 72h, it is 180r/min that rotating speed is set.By nutrient solution suction filtration or centrifugal, obtain crude enzyme liquid, detect enzyme liquid CMCase enzyme activity by the DNS method, record the CMCase enzyme and live as 6.70U, and do not carry out ultrasonic pretreatment group (5.94U) and contrast vigor and improve 12.8%.
Claims (4)
1. the penicillium oxalicum bacterial strain Penicillium oxalicum WX-209 of a plant height cellulase-producing, deposit number is CCTCC NO:M2013325.
2. a method that adopts penicillium oxalicum bacterial strain fermentation claimed in claim 1 to produce high activity cellulase, is characterized in that, comprises the following steps:
(1) slant culture: penicillium oxalicum bacterial strain Penicillium oxalicum WX-209 spore inoculating, to the PDA slant medium, is cultivated 3～4 days, with spore under aseptic washing, made spore suspension standby for 35 ℃;
(2) substrate pre-treatment: get the wheat bran that Mandels salts solution 30ml adds mass percent 5～8%, carry out ultrasonication, the ultrasonic wave parameter is: 700～900W supersound process 40～55 minutes;
(3) fermentation: the substrate autoclaving 20min after pre-treatment, cooling rear by 1 * 10
5～1 * 10
6the inoculum size of individual/mL access penicillium oxalicum spore suspension, 32 ℃～37 ℃, 150～180r/min are cultivated 72h.
3. method according to claim 2, it is characterized in that: fermenting container is the 250mL shaking flask, liquid amount is 30～80mL.
4. method according to claim 2 is characterized in that: supplementing peptone on Mandels nutritive medium basis is quick-acting nitrogenous sources, and concentration is 1g/L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103911296A (en) * | 2014-04-21 | 2014-07-09 | 山东大学 | Penicillium oxalicum strain for increasing enzyme activities of cellulase and hemicellulase |
| CN104911110A (en) * | 2015-05-31 | 2015-09-16 | 中国烟草总公司郑州烟草研究院 | Functional bacterial strain capable of degrading cellulose and application of functional bacterial strain in tobacco |
| CN107502553A (en) * | 2017-07-11 | 2017-12-22 | 南京中医药大学 | A kind of cellulase producing bacteria for being resistant to liquorice dregs and the method applied to liquorice dregs cellulase-producing |
| CN113512501A (en) * | 2021-04-19 | 2021-10-19 | 中国科学院广州能源研究所 | Penicillium oxalicum XZH-2 and application thereof |
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- 2013-08-13 CN CN201310350621.8A patent/CN103468583B/en active Active
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| 刘春辉等: "纤维素酶高产菌的选育及其酶制取的研究进展", 《农产品加工 创新版》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103911296A (en) * | 2014-04-21 | 2014-07-09 | 山东大学 | Penicillium oxalicum strain for increasing enzyme activities of cellulase and hemicellulase |
| CN103911296B (en) * | 2014-04-21 | 2016-02-24 | 山东大学 | A kind of penicillium oxalicum bacterial strain improving cellulase and hemicellulase enzymic activity |
| CN104911110A (en) * | 2015-05-31 | 2015-09-16 | 中国烟草总公司郑州烟草研究院 | Functional bacterial strain capable of degrading cellulose and application of functional bacterial strain in tobacco |
| CN104911110B (en) * | 2015-05-31 | 2018-07-24 | 中国烟草总公司郑州烟草研究院 | It is a kind of can degraded cellulose function stem and its application in tobacco |
| CN107502553A (en) * | 2017-07-11 | 2017-12-22 | 南京中医药大学 | A kind of cellulase producing bacteria for being resistant to liquorice dregs and the method applied to liquorice dregs cellulase-producing |
| CN113512501A (en) * | 2021-04-19 | 2021-10-19 | 中国科学院广州能源研究所 | Penicillium oxalicum XZH-2 and application thereof |
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