CN109161481A - A kind of mutation Aspergillus niger strain and application - Google Patents

A kind of mutation Aspergillus niger strain and application Download PDF

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Publication number
CN109161481A
CN109161481A CN201810940716.8A CN201810940716A CN109161481A CN 109161481 A CN109161481 A CN 109161481A CN 201810940716 A CN201810940716 A CN 201810940716A CN 109161481 A CN109161481 A CN 109161481A
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enzyme activity
strain
aspergillus niger
enzyme
producing
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陈�光
赵文萱
孙旸
王刚
陈欢
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Jilin Agricultural University
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Jilin Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/685—Aspergillus niger
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

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  • Virology (AREA)
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  • Enzymes And Modification Thereof (AREA)
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Abstract

The present invention provides a kind of mutation Aspergillus niger strain, deposit number is CCTCC NO:M2018150.By the enzyme activity determination for producing beta-glucosidase, reach producing enzyme peak within the mutant strain the 9th day, enzyme activity 104.305IU/mL, wild type reached producing enzyme peak in the 12nd day, enzyme activity 77IU/mL, enzyme activity is up to 140.023IU/mL after optimum culture condition, and mutant strain producing enzyme is accelerated and enzyme activity has no reduction.It is safer nontoxic compared with the bacterial strain is complete with current most cellulase systems and the higher trichoderma of enzyme activity such as Trichoderma viride and trichoderma reesei bacterial strain.Aspergillus niger is during Fermentative growth, it can produce a variety of organic acids such as a large amount of oxalic acid and citric acid and phytase, to enable organic phosphorus and Phos to be absorbed and utilized, the fertility that can increase soil when by the ingredient of Aspergillus Niger Mutant crop biological organic fertilizer of the invention improves crop yield.

Description

A kind of mutation Aspergillus niger strain and application
Technical field
The present invention relates to a kind of microorganism fields, and in particular to a kind of mutation Aspergillus niger strain and application.
Background technique
China is a large agricultural country, and all kinds of stalk resources are very rich, and nearly 800,000,000 tons of annual output, stalk is that one kind has The very big resource using space.But most of stalk resource does not develop and use reasonably, and the overwhelming majority is thrown aside field Or be incinerated, cause the waste of resource and the serious pollution of environment.
With the swift and violent continuous improvement increased with living standards of the people of population, energy crisis is short of food, environmental pollution Etc. social concerns get worse.Find and develop the new energy, save grain, reduce environmental pollution it is more and more important, therefore add The comprehensive utilization of strong stalk is essential link.
Straw-returning is considered as a kind of effective farmland measures for building, and straw resource utilization economy and sustainable Mode.The main component of corn stover is cellulose, hemicellulose and lignin, a small amount of protein and ash content.
Main polysaccharide class product of the cellulose as photosynthesis of plant is renewable resource the most abundant on the earth. From the angle of sustainable development, the effective way that cellulose is thoroughly degraded without can cause environmental pollution is Using the hydrolysis of cellulase, it can make a large amount of cellulose resources and city cellulose wastes be transformed into the object of necessary for human Matter has positive far reaching significance.Up to the present, cellulase is widely used in the fields such as work, agriculture, poultry, doctor, and is taken Obtained first-stage success.Although China has the history of decades to the research of cellulase, since the enzyme system of cellulase forms Considerably complicated, activity is not high, and production cost is excessively high and its application is caused still to be restricted.Therefore the cellulase of breeding high activity It is a vital task of China's enzyme preparation research.
Research about cellulase production bacterial strain at present, the overwhelming majority concentrate on that cellulase system is complete and enzyme activity compared with It is toxic on high trichoderma such as Trichoderma viride and trichoderma reesei bacterial strain, but there are a variety of mycotoxins in Trichoderma tunning Property suspicion；Another aspect beta-glucosidase enzyme activity is very low, and cellobiose is caused to accumulate in the reaction system, and cellobiose is fine The inhibitor of other enzymes in plain enzyme system is tieed up, so that the hydrolytic process of entire cellulose is suppressed, therefore β-grape glucuroide water Solution cellobiose is to release the step that glucose becomes crucial.And it is in cellulose that beta-glucosidase expression quantity is low at present Bottleneck in enzyme degraded cellulose saccharifying, therefore raising beta-glucosidase expression quantity and producing enzyme rate are to promote stalk drop The effective way of solution.
Summary of the invention
The purpose of the present invention is to provide a kind of mutation Aspergillus niger strains, and provide the fermentation condition and fermented and cultured of optimization Based component makes mutant strain generate more beta-glucosidases.
The purpose of the present invention is what is be achieved through the following technical solutions:
A kind of mutation Aspergillus niger strain, it is characterised in that:
The mutation Aspergillus niger strain (Aspergillusniger) of production beta-glucosidase of the invention is on March 23rd, 2018 In China typical culture collection center preservation, depositary institution address is the Hubei China province Wuchang District Wu Shi Luo Jia Shan street 16 force Chinese university, deposit number are CCTCC NO:M 2018150.
Strain inoculated of the invention on P-NPG culture medium, P-NPG be sole carbon source screening and culturing medium on can hydrolyze P- NPG generates P-NP, and in Na2CO3Yellow light ring can be formed under effect, 28 DEG C of culture 72h spray 1mol/L Na2CO3It is shown Color, mutant strain of the invention have obvious yellow light ring.
It is that the present invention obtains the utility model has the advantages that
The present invention provides a kind of mutation Aspergillus niger strain for producing beta-glucosidase, 1, by enzyme activity determination, the mutant strain Reach within 9th day producing enzyme peak, enzyme activity 104.305IU/mL, wild type reached producing enzyme peak, enzyme activity 77IU/ in the 12nd day ML, to enzyme activity after Aspergillus niger strain optimum culture condition of the present invention up to 140.023IU/mL.2, provided by the present invention black Aspergillus strain with most cellulase systems are complete used in cellulase production bacterial strain at present and enzyme activity is higher Trichoderma such as Trichoderma viride is compared safer nontoxic with bacterial strains such as trichoderma reeseis.3, aspergillus niger can produce during Fermentative growth A variety of organic acids and the phytase such as raw a large amount of oxalic acid and citric acid incite somebody to action this so that organic phosphorus and Phos be enable to be absorbed and utilized The fertility that soil can be increased when the ingredient of the Aspergillus Niger Mutant crop biological organic fertilizer of invention improves crop yield.
Detailed description of the invention
Fig. 1: wild-type strain and mutant strain growth curve and beta-glucosidase producing enzyme course.
Specific embodiment
1 bacterial strain screening of embodiment
Using stochastical sampling method, the surface layer of soil is rooted out with sterilizing, takes soil 100g in aseptic plastic bag, 1g soil sample is claimed to add Enter into 10mL sterile water, shake up, draws 1mL in liquid PDA culture medium (yeast powder 10g/L, peptone 20g/L, agar In 20g/L), 28 DEG C of 180r/min shaking table cultures are for 24 hours.After the enriched culture of bacterial strain, dilution 104-107Times, respectively take 50 μ l dilutions It is coated in PDA culture medium, in 28 DEG C of culture 3d, picking single colonie is used for strain primary dcreening operation.
Primary dcreening operation: beta-glucosidase bacterial strain is produced with P-NPG(p-nitrophenyl-β-D- glucopyranoside 20g/L, ferment Female powder 10g/L, peptone 20g/L, agar 20g/L) for P-NPG generation P-NP can be hydrolyzed on the screening and culturing medium of sole carbon source, And in Na2CO3Yellow light ring can be formed under effect, aperture size shade can tentatively reflect enzymatic activity height.Therefore, will divide From to strain inoculated in PDA culture medium 28 DEG C of activation be seeded on screening and culturing medium afterwards for 24 hours, 28 DEG C of culture 72h, spray 1mol/L Na2CO3It develops the color, the apparent bacterial strain of yellow light ring is aimed strain.Beta-glucosidase strain isolation will be produced, It is inoculated in liquid fermentation medium, for screening high-yield beta-glucosidase bacterial strain.
Secondary screening: the aimed strain that primary dcreening operation is obtained is inoculated into preliminary fermentation culture medium, with 180r/min, 28 DEG C of shaking tables Culture.Per fermentation liquid 10mL is taken for 24 hours, in 4 DEG C, 8000r/min is centrifuged 10min, collects supernatant, measures the enzyme activity in supernatant Power therefrom screens the higher bacterial strain of enzyme activity.
Enzyme activity determination method: the drafting of standard curve weighs p-nitrophenol 139.0mg, and distilled water is settled to 1000mL, Respectively draw 0.1,0.2,0.3,0.4,0.5,0.6mL in 25mL volumetric flask, be added 5mL 1mol/L Na2CO3Solution, with It is mixed after distilled water constant volume, its light absorption value is surveyed at 400nm.Using p-nitrophenol concentration as abscissa, light absorption value is ordinate, Draw standard curve.
β-glucosidase activity is measured using P-NPG method, 0.1mL is taken to dilute the crude enzyme liquid of suitable multiple, is added The 0.05mmol/L citric acid solution of 1mLPH4.8 preheats 10min in 50 DEG C of water-baths, and the 0.9mL of warmed-up 10min is added 5mL 1mol/LNa is added after timing 10min in 5mmol/L P-NPG solution immediately2CO3Solution terminates reaction, and it is fixed that distilled water is added Hold 25mL, be placed at room temperature for 5min, absorbance is surveyed at 400nm.Under the above conditions, 1mL enzyme solution 1min hydrolysis generates 1umol P-nitrophenol enzyme activity, be defined as an enzyme-activity unit.
2 ARTP mutagenesis screening mutant strain of embodiment
Aspergillus niger strain is inoculated in PDA culture medium, 30 DEG C of culture 96h.The physiological saline that 10mL sterilizing is added elutes spore, Spore suspension is drawn in EP pipe, spore concentration is adjusted to (106~107) cfu/mL is as mutagenesis stoste.
Mutagenesis is carried out to aspergillus niger using normal temperature and pressure (ARTP) plasma breeding machine.Working gas when mutagenesis is helium Gas.According to processing power 100W, helium gas flow 12SLM, sample and plasma generator export distance 2mm, and processing sample is 10ul, 150s the mutagenic exposure time handle steamed stuffed bun suspension.
To treated, spore suspension dilutes 10-1-10-6Times, it takes 100 μ L dilutions to be coated in PDA plate, is placed in 30 It is very fast that the mutant strain cultivated in DEG C incubator is relatively large in diameter the speed of growth, carries out shaking flask culture and simultaneously ferments to stalk.
3 mutant strain enzyme activity of embodiment is measured
The mutant strain that this experiment obtains carries out shake flask fermentation, and is measured to enzyme activity, and measuring method is the same as embodiment 2, gained A plant mutant strain growth, producing enzyme quickening and the enzyme activity arrived is stablized.After passage is determined as stablizing positive mutating strain, to itself and open country Raw type bacterial strain compares, and measures its growth curve and beta-glucosidase enzyme activity.Mutant strain reached in the 9th day as shown in Figure 1: The mycelial growth limit, strain growth logarithmic phase are 4-7 days, and wild type reached the mycelial growth limit in the 12nd day, and strain is raw Long logarithmic phase is 7-10 days, and mutant strain reaches logarithmic phase 3 days better than wild type in advance.Mutant strain reached producing enzyme top in the 9th day Peak, enzyme activity 104.305IU/mL；Wild type reached producing enzyme peak, enzyme activity 77IU/mL in the 12nd day, and mutant strain producing enzyme is accelerated And enzyme activity increases.
4 mutant strain fermented maize stalk of embodiment produces beta-glucosidase fermented and cultured
Aspergillus niger Shake flask medium fermented maize stalk condition are as follows: initial pH value 4.98, glucose additive amount 14.94g/L, albumen Peptone additive amount 14.82g/L, Mn2+Additive amount 1g/L, 28 DEG C of cultivation temperature, revolving speed 150r, inoculum concentration 2%, the addition of straw powder Amount is 40g/L.
It is matched according to this and carries out shaking flask culture, carry out experimental verification, as the result is shown measured β-glucose under this condition Glycosides enzyme activity is 140.023IU/mL, obtains optimal condition of culture.

Claims (2)

1. a kind of mutation Aspergillus niger strain, deposit number is CCTCC NO:M2018150.
2. application of the Aspergillus niger strain as described in claim 1 in the microbial inoculum for preparing degraded cellulose.
CN201810940716.8A 2018-08-17 2018-08-17 A kind of mutation Aspergillus niger strain and application Pending CN109161481A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113403207A (en) * 2021-08-23 2021-09-17 中国科学院天津工业生物技术研究所 Aspergillus niger strain for high yield of beta-glucosidase and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102399803A (en) * 2011-09-30 2012-04-04 南京林业大学 Improved beta-glucosidase gene and preparation of recombinase thereof
CN107488601A (en) * 2017-09-18 2017-12-19 山东隆科特酶制剂有限公司 One plant height produces bacterial strain and its application of the resistance to acidproof β glucuroides of sugar

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102399803A (en) * 2011-09-30 2012-04-04 南京林业大学 Improved beta-glucosidase gene and preparation of recombinase thereof
CN107488601A (en) * 2017-09-18 2017-12-19 山东隆科特酶制剂有限公司 One plant height produces bacterial strain and its application of the resistance to acidproof β glucuroides of sugar

Non-Patent Citations (2)

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LI-NIAN CAI 等: "Directed expression of halophilic and acidophilic β-glucosidases by introducing homologous constitutive expression cassettes in marine Aspergillus niger", 《JOURNAL OF BIOTECHNOLOGY》 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113403207A (en) * 2021-08-23 2021-09-17 中国科学院天津工业生物技术研究所 Aspergillus niger strain for high yield of beta-glucosidase and application thereof

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