CN102994399A - Aspergillus strain for producing beta-glucosaccharase as well as applications thereof - Google Patents

Aspergillus strain for producing beta-glucosaccharase as well as applications thereof Download PDF

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CN102994399A
CN102994399A CN2012105393718A CN201210539371A CN102994399A CN 102994399 A CN102994399 A CN 102994399A CN 2012105393718 A CN2012105393718 A CN 2012105393718A CN 201210539371 A CN201210539371 A CN 201210539371A CN 102994399 A CN102994399 A CN 102994399A
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aspergillus niger
beta
aspergillus
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glucosidase
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CN102994399B (en
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陈介南
张�林
刘进
陈石兰
王义强
石彩蕊
先天敏
石旺
马云中
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CHANGDE JOINT BIOENERGY INSTITUTE
CHANGSHA LUEN MEI BIOLOGICAL TECHNOLOGY Co Ltd
Central South University of Forestry and Technology
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CHANGDE JOINT BIOENERGY INSTITUTE
CHANGSHA LUEN MEI BIOLOGICAL TECHNOLOGY Co Ltd
Central South University of Forestry and Technology
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Abstract

The invention discloses an Aspergillus strain for producing beta-glucosaccharase as well as applications thereof, in particular to Aspergillus nigerC112 is preserved in China Center for Type Culture Collection in April, 23rd, 2012 with the preservation number of CCTCC No. M 2012129. After the Aspergillus nigerC112 provided by the invention is subjected to ultraviolet mutagenesis, the Aspergillus nigerC112 is subjected to primary screening and re-screening, and a mutation strain with genetic stability is obtained. Through prolonging the seed culturing time of the Aspergillus nigerC112, bran, yeast powder and the like are taken as raw materials, and through liquid state fermentation, the enzyme activity of beta-glucosaccharase can achieve 10IU/Ml above.

Description

Aspergillus niger strain and the application thereof of beta-glucosidase produced in one strain
Technical field
The invention belongs to technical field of enzyme engineering, be specifically related to the Aspergillus niger strain of plant height effect secretion beta-glucosidase, and relate to the application that Aspergillus niger strain produces beta-glucosidase.
Background technology
(cell-oligosaccharide of energy hydrolysis fiber disaccharides and short chain generates glucose to beta-glucosidase, and is very fast to the hydrolysis rate of cellobiose and procellose for EC3.2.1.21, β-glucosidase), also be known as cellobiase.In the cellulosic process of cellulase hydrolysis, beta-glucosidase hydrolyzable cellobiose, thus discharge glucose.At present, beta-glucosidase content nearly all very low (except aspergillus niger) in the bacterial strain of cellulase-producing, be less than 1% such as beta-glucosidase content in the cellulase of the mould secretion of wood, far do not reach the level of practical application, therefore, beta-glucosidase becomes the bottleneck in the cellulose degraded Mierocrystalline cellulose process.Except important role aspect degraded cellulose, beta-glucosidase is used also more and more extensive at other field, comprise the industries such as food, wine brewing, medicine, chemistry.In recent years, domestic and international many scientists are devoted to the research of beta-glucosidase, and expectation better improves the catalytic efficiency of cellulase, utilizes cellulose resource.
In improving the mode of producing enzyme efficient, selection by mutation is a kind of very effective means.Selection by mutation is to utilize induced mutations to bring out microbial gene sudden change, and output is high, the mutant of excellent property thereby filter out, have speed fast, bring notable results, the advantage such as method is simple.The chemomorphosis methods such as the new approaches of physical mutagenesis such as ultraviolet mutagenesis and ethyl sulfate (DES), ethylmethane sulfonate (EMS) are the focuses that scholars study always.
The trichoderma pseudokiningii that the people such as Song Xiaoyan preserve take the laboratory is as starting strain, process through ultraviolet mutagenesis, with the seed selection of glucose (gradient concentration) Mierocrystalline cellulose double-layer plate, obtain a strain glucose concn up to the 10% mutant strain UV III that still can produce the cellulose hydrolysis transparent circle containing glucose (4%) Mierocrystalline cellulose double-layer plate, cultivate 6d, the beta-glucoside enzyme activity of dry medium can reach 24U/g, 12.6 times of [journal of Shandong university (natural section version) of starting strain, 34 (4): 488-492,1999].Wang Depei [51]Deng the people by separation screening and ultraviolet mutagenesis method, apart from 30W ultraviolet lamp 15cm, irradiation time 0.5-3min, select a plant height cellulase-producing Aspergillus niger strain S13 and carried out the best solid-state research of sending out culture condition liquor-saturated, under top condition, its beta-glucoside enzyme activity reaches 801.18mg/ (hg) [Guangdong chemical industry, 2:102-104,1999].The people such as Su Xiangping carry out ultraviolet mutagenesis take Aspergillus niger strain S1 as original strain, under the 15W ultraviolet lamp, about irradiation distance 30cm, irradiation time is 9min, and screening obtains a plant height cellulase-producing Aspergillus niger strain S12, at pH6.0,28 ℃ of culture temperature, under the incubation time 96h condition, the beta-glucoside enzyme activity 1.13 times of [brewing science and technologies that the work of original strain enzyme has improved of comparing, 4:25-30,2009].Although, have manyly for Aspergillus Niger beta-glucosidase research report, it produces enzyme efficient and still also has great Improvement.
Ultraviolet ray is as a kind of physical mutagen commonly used, and its mutagenic frequency is high, and the recovery that is not easy to undergo mutation.The objective of the invention is to adopt the mode of ultraviolet mutagenesis that existing Aspergillus niger strain is carried out screen mutation, Aspergillus niger strain more stable to obtaining, high-yield beta-glucosidase.
Summary of the invention
The invention reside in provides a plant height to produce aspergillus niger dominant strain and the application thereof of beta-glucosidase.
The Aspergillus niger strain that beta-glucosidase is produced in one strain is aspergillus niger Aspergillus nigerC112, and its preserving number is CCTCCNO:M 2012129.
Described Aspergillus niger strain is used for producing beta-glucosidase.
Specifically with aspergillus niger Aspergillus nigerC112 through liquid fermenting, produce beta-glucosidase.
Described aspergillus niger Aspergillus nigerC112 produces enzyme process: the solid plate spore, through 25-30 ℃ of activation of PDA liquid nutrient medium 48-72 hour, press the 3-10% inoculum size, switching enters in the liquid culture medium, pH to 4.5-6.0 under 26-30 ℃, cultivated 96 hours to 144 hours.
Described PDA liquid nutrient medium is: potato 200g/L, and glucose 20g/L, all the other are water, natural pH value.
Described liquid culture medium is: wheat bran 10g/L, yeast powder 5g/L, ammonium sulfate 2.8g/L, KH 2PO 44g/L, MgSO 47H 2O 0.9g/L, CaCl 20.9g/L, tween 2mL/L, all the other are water.
Aspergillus niger Aspergillus niger C112 of the present invention, on April 23rd, 2012 was preserved in " Chinese Typical Representative preserved material center ", and the address is: China. Wuhan. Wuhan University, its preserving number is CCTCC NO:M2012129.
Described Aspergillus niger strain Aspergillus niger C112 is characterized as: bacterial strain is on the FDA seed culture medium; 25-30 ℃ of lower the cultivation 96-144 hour; mycelium becomes velvet shape or cotton-shaped; the globe-roof capsule is formed on the top; cover one deck metulae and one deck stigma on it, long on the stigma have bunchiness memnonious spherical comprehensively.Conidial head is spherical, and a large amount of conidiums are arranged, and conidium can educate comprehensively, just be white, after become aureus until black heavy fleece shape, chocolate, without transudate, the colourless or central slightly tawny in the bacterium colony back side is faint yellow.
Description of drawings
Fig. 1 is aspergillus niger Aspergillus nigerC112 solid plate full face of the present invention;
Fig. 2 is aspergillus niger Aspergillus nigerC112 solid plate of the present invention back side photo;
Fig. 3 is aspergillus niger Aspergillus nigerC112 microscopically mycelium photo of the present invention (400x);
Fig. 4 is aspergillus niger Aspergillus nigerC112 microscopically spore photo of the present invention (400x).
Embodiment
Be intended to further specify the present invention below in conjunction with embodiment, and unrestricted the present invention.
The present invention carries out ultraviolet mutagenesis with Aspergillus niger strain Aspergillus nigerACCC No 30786,
Aspergillus niger strain Aspergillus nigerACCC No 30786 is 28 ℃ of cultivation 5-7d on the PDA plate culture medium.The PDA substratum is potato 200g, glucose 20g, agar 20g, water 1000mL.The plate bacterial classification of making even adds stroke-physiological saline solution and washes spore, and four layers of filtered through gauze are to the aseptic triangular flask that fills granulated glass sphere, and 28 ℃ of vibration 2h disperse spore, and adjusting its concentration is 10 6/ mL.
Above-mentioned bacteria suspension is got lmL be diluted to 10 -4, 10 -5, 10 -6, l0 -7Spread plate doubly, and then to get l0mL be in the culture dish of 9cm in diameter, apart from 20W ultraviolet lamp 35cm, processes 50min under the magnetic agitation.Postradiation bacterium liquid dilution spread on separating plate, behind the cultivation 2d, is begun to form single bacterium colony in separating plate.Isolation medium (w/v): potato 20%, glucose 2%, agar 1.5%, glycerine 10%, sodium deoxycholate 0.1%, natural pH.Cultivate 1.5-2d for 28 ℃.
Isolated single bacterium colony on the plate is chosen in the screening and culturing ware with toothpick, behind 28 ℃ of training 5d, sprayed 1mol/LNa 2CO 3Colour developing, the darker bacterial strain of picking yellow directly carries out multiple sieve.Screening culture medium: potato 20%, glucose 2%, agar 1.5%, natural pH, p-NPG0.1% (sterilization is cooled to about 60 ℃ and adds).
The enzyme that primary dcreening operation obtains is lived relatively high bacterial strain by shake flask fermentation, and each bacterial strain connects 1 bottle, and 28 ℃ of cultivations are surveyed its beta-glucosidase enzyme and lived behind the 5d.Culture medium: wheat bran 1%, corn cob 2.15%, yeast powder 0.5%, ammonium sulfate 0.68%, KH 2(PO 4) 0.4%, MgSO 47H 2O 0.09%, CaCl 20.09%, 2 droplets/bottle of tween-80s, regulating pH is 5.2.
The higher bacterial strain of product enzyme that multiple sieve is obtained sieves again again, 3 bottles of every strains, and 28 ℃ of cultivations are surveyed its beta-glucosidase enzyme and are lived behind the 5d.
The highly active mutant strain that screening is obtained carries out succeeding transfer culture, and subculture is 8 times continuously, surveys its beta-glucosidase enzyme and lives, and observes its stability, has obtained the enzyme aspergillus niger Aspergillus nigerC112 higher, inheritance stability that lives.Behind the continuous subculture 8 times, its beta-glucosidase enzyme work reaches 6.510U/mL, has improved 49.31% with respect to starting strain.
Utilizing the pNPG method to measure the beta-glucosidase enzyme lives: get the crude enzyme liquid that 0.1mL has diluted suitable multiple, add the 0.05mol/L citric acid solution of 1mLpH4.8, in 50 ℃ of water-bath preheating 10min; Add the 0.9mL5mmol/LpNPG solution of preheating 10min, timing adds 1mL 1mol/LNa immediately behind the 10min 2CO 3The solution termination reaction: add the distilled water constant volume to 25mL, room temperature is placed 5min, surveys absorbance A in the 410nm place; Under these conditions, 1mL enzyme liquid lmin hydrolysis produces the enzyme activity of the p-NP of 1umol, is defined as enzyme unit alive.
Adopt improved method of CTAB to extract the genomic dna of bacterial strain.Carry out PCR by the design primer, utilize PCR product direct Sequencing to obtain to express the gene order of beta-glucosidase, sequence analysis is obtained the CDS sequence of encoding beta-glucosidase.
Embodiment 1
Preparation PDA liquid nutrient medium: potato 200g, glucose 20g, water 1000mL.Be in the 250mL triangular flask of 50mLPDA liquid nutrient medium at liquid amount, inoculation one ring aspergillus niger spore is cultivated the seed liquor that 60h obtains for 28 ℃, and the inoculum size with 5% is inoculated in the aspergillus niger liquid culture medium and produces enzyme; One ring spore and a fritter (nail cover size) are set respectively simultaneously contain spore PDA solid medium inoculated aspergillus niger culture medium as producing the enzyme contrast, at 28 ℃, cultivate 5d under the same terms, measure enzyme and live.
The liquid culture medium is: wheat bran 10g/L, yeast powder 5g/L, ammonium sulfate 2.8g/L, KH 2PO 44g/L, MgSO 4-7H 2O 0.9g/L, CaCl 20.9g/L, tween 2mL/L, all the other are water.
After measured, the aspergillus niger Aspergillus nigerC112 behind the activation 60h, its beta-glucosidase enzyme work reaches 7.96U/mL, has improved 82.56% with respect to starting strain.
Embodiment 2
Preparation PDA liquid nutrient medium: potato 200g, glucose 20g, water 1000mL.Be in the 250mL triangular flask of 50mLPDA liquid nutrient medium at liquid amount, inoculation one ring aspergillus niger spore is cultivated the seed liquor that 60h obtains for 28 ℃, inoculum size with 5% is inoculated in the aspergillus niger liquid culture medium, respectively at 26 ℃, 28 ℃, 30 ℃, pH to 5.0 cultivates 5d, measures enzyme and lives.
The liquid culture medium is: wheat bran 10g/L, yeast powder 5g/L, ammonium sulfate 2.8g/L, KH 2PO 44g/L, MgSO 4-7H 2O 0.9g/L, CaCl 20.9g/L, tween 2mL/L, all the other are water.
After measured, after cultivating 5d, respectively at the aspergillus niger Aspergillus of 26 ℃, 28 ℃, 30 ℃ growths nigerC112, its beta-glucosidase enzyme work is respectively 10.248U/mL, 11.57U/mL, 9.52U/mL, has improved respectively 135.04%, 165.36%, 118.34% with respect to starting strain.
Embodiment 3
Preparation PDA liquid nutrient medium: potato 200g, glucose 20g, water 1000mL.Be in the 250mL triangular flask of 50mLPDA liquid nutrient medium at liquid amount, inoculation one ring aspergillus niger spore is cultivated the seed liquor that 60h obtains for 28 ℃, inoculum size with 5% is inoculated in the aspergillus niger liquid culture medium, at 28 ℃, pH to 5.0 cultivates respectively 96h, 120h, 144h, measures enzyme and lives.
The liquid culture medium is: wheat bran 10g/L, yeast powder 5g/L, ammonium sulfate 2.8g/L, KH 2PO 44g/L, MgSO4-7H2O 0.9g/L, CaCl 20.9g/L, tween 2mL/L, all the other are water.
After measured, after cultivating respectively 96h, 120h, 144h, the beta-glucosidase enzyme work of aspergillus niger Aspergillus nigerC112 is respectively 9.33U/mL, 11.57U/mL, 11.44U/mL, has improved respectively 113.99%, 165.36%, 162.38% with respect to starting strain.
Embodiment 4
Modified CTAB method is extracted DNA: 1. use the liquid nitrogen grinding aspergillus niger, the centrifuge tube of packing into.2. add 700 μ L and contain the DNA extraction liquid of 2%CTAB and the beta-mercaptoethanol of 20 μ l, in 65 ℃ of water-bath 1h.3. add 700 μ L phenol: chloroform: primary isoamyl alcohol, the centrifugal 5min of 12000r/min gets supernatant liquor behind the mixing, adds 600 μ L chloroforms: primary isoamyl alcohol, the centrifugal 5min of 12000r/min behind the mixing.Supernatant liquor is added 500 μ L Virahols, and-20 ℃ leave standstill 1h.4. the centrifugal 5min of 12000r/min abandons supernatant.Ethanol washing and precipitating with 70% adds 300 μ L H afterwards 2The O dissolution precipitation adds the 3mol/L of 1/5 volume and the dehydrated alcohol of 2 times of volumes, leaves standstill 30min.5. the centrifugal 5min of 12000r/min is with air-dry after 70% the ethanol washing and precipitating.Precipitation is dissolved in the 30 μ L TE damping fluids.Adopt survey OD value and electrophoresis to detect DNA purification quality.
The listed primer of use table 1 carries out PCR, obtain the PCR product, the PCR product is directly checked order, each PCR system is recorded sequence splices, obtain the gene order of aspergillus niger Aspergillus niger C112 beta-glucosidase, total 2934bp base, wherein exon 2 583bp, comparison is new gene order by analysis, can prove that this bacterial strain is new bacterial strain.
Table 1.PCR the primer
The primer title Sequence
1F ttcgtttctg?tcctcccta
1R gttcgcactt?ccactctc
2F tggaactatg?tgttggtcag
2R cttgtagccg?tattcatcct?c
3F gctgtgatgt?gctcctac
3R cagagtgaat?agcaacgatt
4F tattcaccgc?caccgata
4R cttcatcacc?agcaacctt
5F agttcggcta?tggtctga
5R gaggaagaag?gctgaagag
Figure IDA00002577767500011
Figure IDA00002577767500021
Figure IDA00002577767500031

Claims (6)

1. the Aspergillus niger strain of beta-glucosidase is produced in a strain, it is characterized in that, described Aspergillus niger strain is aspergillus niger AspergillusnigerC112, and its preserving number is CCTCC NO:M 2012129.
2. the application method of Aspergillus niger strain claimed in claim 1 is characterized in that, is used for producing beta-glucosidase.
3. the application method of Aspergillus niger strain according to claim 2 is characterized in that, described aspergillus niger AspergillusnigerC112 produces beta-glucosidase through liquid fermenting.
4. the application method of Aspergillus niger strain according to claim 3, it is characterized in that, described aspergillus niger AspergillusnigerC112 produces enzyme process: the solid plate spore, through 25-30 ℃ of activation of PDA liquid nutrient medium 48-72 hour, 3-10% inoculum size by volume, switching enter in the liquid culture medium, pH to 4.5-6.0, under 26-30 ℃, cultivated 96 hours to 144 hours.
5. the application method of Aspergillus niger strain according to claim 4 is characterized in that, described PDA liquid nutrient medium is: potato 200g/L, and glucose 20g/L, all the other are water, natural pH value.
6. the application method of Aspergillus niger strain according to claim 5 is characterized in that, described liquid culture medium is: wheat bran 10g/L, yeast powder 5g/L, ammonium sulfate 2.8g/L, KH 2PO 44g/L, MgSO 47H 2O 0.9g/L, CaCl 20.9g/L, tween 2mL/L, all the other are water.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104293861A (en) * 2014-09-10 2015-01-21 中南林业科技大学 Method for improving enzymatic saccharification of wood fibre by combination of dilute phosphoric acid and steam explosion pretreatment
CN106755029A (en) * 2017-01-22 2017-05-31 湖北工业大学 It is a kind of to change the method that aspergillus niger genome partial structurtes improve cellulase expression amount
CN110699263A (en) * 2019-10-29 2020-01-17 浙江工业大学 Aspergillus niger YH-6 and application thereof in improving content of icaritin in epimedium

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104293861A (en) * 2014-09-10 2015-01-21 中南林业科技大学 Method for improving enzymatic saccharification of wood fibre by combination of dilute phosphoric acid and steam explosion pretreatment
CN106755029A (en) * 2017-01-22 2017-05-31 湖北工业大学 It is a kind of to change the method that aspergillus niger genome partial structurtes improve cellulase expression amount
CN106755029B (en) * 2017-01-22 2019-11-08 湖北工业大学 A method of changing aspergillus niger genome partial structurtes and improves cellulase expression amount
CN110699263A (en) * 2019-10-29 2020-01-17 浙江工业大学 Aspergillus niger YH-6 and application thereof in improving content of icaritin in epimedium

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