CN104711199B - The thermophilic salt aspergillus niger of one plant of cellulase-producing and its application - Google Patents

The thermophilic salt aspergillus niger of one plant of cellulase-producing and its application Download PDF

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CN104711199B
CN104711199B CN201510134172.2A CN201510134172A CN104711199B CN 104711199 B CN104711199 B CN 104711199B CN 201510134172 A CN201510134172 A CN 201510134172A CN 104711199 B CN104711199 B CN 104711199B
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aspergillus niger
cellulase
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salt
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CN104711199A (en
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刘开辉
张波
丁小维
陈文强
邓百万
杨方玉
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Shenzhen Zhongquan Technology Co., Ltd
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Abstract

The invention discloses the thermophilic salt aspergillus niger of one plant of cellulase-producing and its application, belong to microorganism field.The deposit number of disclosed Aspergillus niger strain is CGMCC9404, the Aspergillus niger strain is isolated from Shaanxi Province, China Yulin City in Northern Shaanxi Province Dingbian County dapple salt lake water pool deposit, the bacterial strain is aspergillus niger category by morphology and molecular biology identification, can be in the maize cob meal that carbon source is 2%, nitrogen source is 0.5% peptone, and starting pH is 5.5, and spore suspension inoculation volume fraction is 20%, cultivated 5 days for 28 DEG C in the culture medium that salinity is 2.5%, reach highest inulinase-producing activity.And the bacterial strain can produce cellulase under conditions of being 0%~15% in salinity.

Description

The thermophilic salt aspergillus niger of one plant of cellulase-producing and its application
Technical field
The invention belongs to biological technical field, and in particular to thermophilic the salt aspergillus niger and its cultural method of one plant of cellulase-producing And application.
Background technology
Cellulose substances are one of renewable resources most abundant on the earth, and it is by β-D-1,4 glucoside bond groups Into polysaccharide, molecular weight is up to hundreds thousand of or even millions of, it is impossible to directly utilizes for microbial cell.Cellulase is biological drop The general name of the glucogenic a kind of complex enzyme of cellulose of the solution containing β-Isosorbide-5-Nitrae glycosidic bond, is mainly made up of 3 kinds of hydrolases, point It is not beta glucan glycosides enzyme, cellobiohydrolase and beta-glucosidase.Beta glucan glycosides enzyme effect is in microfibre first Noncrystalline domain, it is exposed many ends and supply circumscribed-type enzyme effect, cellobiohydrolase decomposes successively from non reducing end, Cellobiose is produced, then the cellulose of Partial digestion is further made by beta glucan glycosides enzyme and cellobiohydrolase collaboration With decomposition generates the oligosaccharide such as cellobiose, trisaccharide, finally resolves into glucose by beta-glucosidase.
Cellulase is widely present in the organism of nature, bacterium, fungi, and cellulose can be produced in animal body Enzyme.Wherein microorganism is the main source of cellulase, as bacterium mainly has fusobacterium (Clostridium), cellulomonas cartae Belong to (Cellulomonas), high temperature zygosaccharomyces (Thermomonospora) etc.；Fungi mainly has trichoderma (Trichoderma), Penicillium (Penicillium), aspergillus (Aspergillus), white-rot fungi (P.chrysosporium), fragmentation Pseudomonas (Schizophyllum) etc..The cellulase for being generally used for production comes from fungi, At present, the microorganism for studying the more natural fiber that can degrade is mainly the fungies such as trichoderma, mould, aspergillus and whiterot fungi, example The complete Trichoderma viride of such as cellulase system (Trichoderma viride) and trichoderma reesei (T.reesei) bacterial strain, but wood A variety of mycotoxins in mold fermentation product be present, on the other hand its cellulase activity is relatively low, especially beta-glucosidase Vigor is very low, causes cellobiose to be accumulated in reaction system and influences enzymolysis efficiency, thus its application is restricted.So It is generally acknowledged safe microorganisms and aspergillus niger does not produce toxin.In the fungi of cellulase-producing, aspergillus niger produces cellulose It is higher with the vigor of cellobiohydrolase and beta-glucosidase in enzyme system, the particularly more general bacterium of beta-glucosidase enzyme activity Plant height.
Most of research for cellulase producing strain at present is all concentrated in conventional less salt microorganism, is seldom related to salt The research of lake microorganism cellulase-producing, at present it has been reported that only Vibrio sp.G21 (Gao Z et al.2010), Chromohalobacter sp.TPSV 101(Prakasha et al.2012)、Haloarcula sp.G10、 Haloarcula sp.LLSG7(Li X et al.2013)、Pestalotiopsis sp.NCi6(Arfi Y et Al.2013) etc..Because Salt Lake Environments are unique, contain the enzyme system of various extreme microorganism and its uniqueness.Halophilic microorganism enzyme one As still there is higher activity under high salt conditions, can be advantageous in efficient degradation cellulose under hypersaline environment, such a characteristic Solve the problems, such as that hypersaline environment inactivates enzyme in industrialized production, in addition, the NaCl of fermentation medium middle and high concentration can suppress big The growth and breeding of most microorganisms, prevent living contaminants in cellulase production processes.
Cellulase can be widely used for numerous necks such as food, weaving, feed, wine brewing, oil exploration, traditional Chinese medicine ingredients extraction Domain, particularly there is critical role in the commercial Applications such as weaving, washing, papermaking and bioenergy.The molecule of native cellulose Interior and intermolecular there is substantial amounts of hydrogen bond, while its aggregated structure is complicated, crystallinity is higher so that cellulose to enzyme can and Property it is low, dissolving is difficult, and reactivity worth is poor, and this directly affects the performance of cellulosics, and the pretreatment of cellulose is fine The important step that dimension element effectively utilizes in some industrial circles, during acid-base method pre-processes native cellulose, produce a large amount of Waste water, the high direct discharging of waste water of these salinity can cause organic matter to be enriched with, and halophilic cellulase can be under hypersaline environment Degraded cellulose, therefore improvement of the halophilic cellulase to waste water caused by pretreatment of fiber element is significant.It is in addition, fine It is to characterize the leading indicator of salt affected soil Carbon cycle to tie up plain enzyme enzyme activity, and the enzyme can be effectively increased the circulation speed of salt-soda soil carbon Degree, promote the fast quick-recovery of salt-soda soil fertility.
The content of the invention
It is an object of the invention to provide the thermophilic salt aspergillus niger of one plant of cellulase-producing and its cultural method and application.
The present invention is to be achieved through the following technical solutions：
A kind of thermophilic salt Aspergillus niger strain of cellulase-producing, the Aspergillus niger strain are preserved in Chinese microorganism strain preservation pipe Reason committee common micro-organisms center, preserving number are CGMCC No.9404.
The Aspergillus niger strain produces cellulase under conditions of being 0%~15% in salinity.
The carbon source that the Aspergillus niger strain produces the fermentation medium of cellulase is the corn of mass fraction 1%~3.5% Core powder, nitrogen source are the peptone of mass fraction 0.2%~1.0%；The Aspergillus niger strain produce cellulase cultivation temperature be 24~32 DEG C, incubation time is 1~7 day, and spore suspension inoculation volume fraction is 5%~25%, and medium pH is 4.5~8.5, Salinity is 0%~15%.
Preferably, the carbon source that the Aspergillus niger strain produces the fermentation medium of cellulase is the corn of mass fraction 2% Core powder, nitrogen source are the peptone of mass fraction 0.5%；The cultivation temperature that the Aspergillus niger strain produces cellulase is 28 DEG C, training It is 5 days to support the time, and spore suspension inoculation volume fraction is 20%, and culture medium starting pH is 5.5, salinity 2.5%.
Application of the thermophilic salt Aspergillus niger strain of cellulase-producing disclosed by the invention in cellulose waste water treatment is produced.
Application of the thermophilic salt Aspergillus niger strain of cellulase-producing disclosed by the invention in the reparation of salt-soda soil.
Compared with prior art, the present invention has technique effect beneficial below：
The invention provides a kind of thermophilic salt Aspergillus niger strain of new cellulase-producing, Aspergillus is named as Niger, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number CGMCC No.9404.The thermophilic salt Aspergillus niger strain is from being isolated from separating in the dapple salt lakes and marshes deposit of Shaanxi Province, China Yulin City Dingbian County Obtain.
The thermophilic salt Aspergillus niger strain of cellulase-producing disclosed by the invention can give birth in the culture medium of 0%~15% salinity Long to produce cellulase, halophilic cellulase can be located in advance in efficient degradation cellulose under hypersaline environment in papermaking and cellulose Managing in the improvement of waste water and the reparation in salt-soda soil has relatively broad application.Preservation explanation
The present invention has carried out following preservations to described thermophilic salt Aspergillus niger strain：
The preservation time：On July 1st, 2014, preservation place：China, Beijing.Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Number, Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)； Preserving number is CGMCC No.9404.
Brief description of the drawings
Fig. 1 is the micro-morphology photo of thermophilic salt Aspergillus niger strain；
Fig. 2 is the thermophilic salt Aspergillus niger strain of the present invention and has identified that the system of the ITS sequence structure of Aspergillus category is sent out Educate tree；
Fig. 3 is the block diagram that culture medium carbon source influences on CMCase；
Fig. 4 is the block diagram that culture media nitrogen source influences on CMCase；
Fig. 5 is the line chart that fermentation temperature influences on CMCase；
Fig. 6 is the line chart that fermentation time influences on CMCase；
Fig. 7 is the line chart that the volume fraction of spore suspension inoculation influences on CMCase；
Fig. 8 is the line chart that medium pH influences on CMCase；
Fig. 9 is the line chart that medium salts concentration influences on CMCase.
Embodiment
With reference to specific drawings and examples, the present invention is described in further detail, and described is to the present invention Explain rather than limit.
The strain isolation of the thermophilic salt cellulase-producing of the present invention sinks from Shaanxi Province, China Yulin City Dingbian County dapple salt lakes and marshes Product thing.The aspergillus niger category bacterial strain (Aspergillus niger), China General Microbiological bacterium is stored on July 01st, 2014 Kind preservation administrative center, depositary institution address：Institute of Microorganism, Academia Sinica of BeiJing, China, postcode 100101, preservation are compiled Number：CGMCC9404.
Screened by Congo red solid medium, the bacterial strain has an ability for producing cellulase, and to its morphology and ITS Phylogenetic Relationships are studied, and are defined as aspergillus niger (Aspergillus niger).Zymology Quality Research shows, is somebody's turn to do The most suitable carbon source that bacterial strain produces cellulase fermentations culture medium is the maize cob meal of mass fraction 2%, and most suitable nitrogen source is quality point The peptone of number 0.5%, most suitable cultivation temperature are 28 DEG C, and optimal incubation time is 5 days, and optimal spore suspension is inoculated with volume fraction For 20%, culture medium most optimal pH is 5.5, and most suitable salinity is 2.5%.
Present invention discover that the fungi of thermophilic salt cellulase-producing obtained through following specific steps：
1. the preliminary screening of cellulase
Using transparent circle method, fungi single bacterium colony is inoculated on Congo red culture medium, 28 DEG C of incubated 7d, treats that bacterium colony is grown Cheng Hou, transparent loop diameter (D) and colony diameter (d) are measured, inulinase-producing activity size is tentatively judged according to the big I of the two difference.
2. the identification of bacterial strain
1) Morphological Identification
Strain is inoculated into czapek's medium, 28 DEG C are inverted 7~14d of culture, colonial morphology are observed, by inoculation site Oblique cutting enters sterile cover slips, through cultivating after mycelia climbs full cover glass, takes out slide and is seen under Nikon high optics microscope Examine the morphological feature and conidial fructification of mycelia.
The composition of used culture medium：
Water is added to be settled to 1L, pH is natural；121 DEG C of sterilizing 30min.
2) molecular biology identification
A. the genomic DNA of the bacterial strain is extracted using CTAB methods, is dissolved in 40 μ L TE buffer solutions, it is standby in -20 DEG C of preservations With.
B. fungi ITS universal primer sequences are used：
ITS1(5′-TCCGTAGGTGAACCTGCGG-3′)
ITS2 (5 '-TCCTCCGCTTATTGATATGC-3 '), enter performing PCR amplification by template of the strain gene group DNA.
C. obtained fragment is expanded, after electrophoresis is verified, does glue purification.
D. send and be sequenced by Shanghai life work after purification, Phylogenetic Analysis is carried out to sequence.Referring to Fig. 1, according to form And ITS Phylogenetic Relationships, it is aspergillus niger (Aspergillus niger) to determine the fungal strain.
Bacterial strain 6MA1 ITS sequence has been filed on GeneBank, accession number KP987453.Referring to Fig. 2, sequence alignment point Analysing result is：The coverage of sequence is submitted more than 100%.With Aspergillus category aspergillus nigers (Aspergillus niger) Homologous similarity more than 99%, wherein homologous similarity is up to 100%.Bacterial strain and A.niger (accession number： JF436883), A.niger (accession number：KF496080), A.niger (KC341970) etc. is in same branch, homologous to compare knot Fruit shows that its homologous degree reaches 100%, determines a kind of new strains that the bacterial strain is aspergillus niger.
The composition of used culture medium：
Water is added to be settled to 1L, pH is natural；121 DEG C of sterilizing 30min.
6th, morphological feature
Bacterial strain 6MA1 cultivates 4d in czapek's medium and produces the radial white hypha (shown in Figure 1A) to stretch out, Mycelia is combined more firm with culture medium, and whole bacterium colony front is in villiform, then produces black conidial head, bacterium colony reverse side is in light Yellowish-brown, there are wrinkle.Micro- Microscopic observation (Figure 1B), mycelia width are 9.5-12.5um, and conidiophore is upright by one Mycelia generates, and top capsule is in homogeneous spheroidal, and size is 2.5 × 2.5um, and wall is bordering on smooth or slightly coarse.It is special according to its form Sign, can be primarily determined that as aspergillus niger (Aspergillus niger).
7th, the cellulose zymetology nature examination of the thermophilic salt aspergillus niger of the present invention
The screening of the optimum carbon source of the cellulase producer Aspergillus niger 21 fermentation medium of embodiment 1
1. seed liquor is inoculated in carbon source as maize cob meal, wheat bran, cellulose, wood chip, rice respectively in 10% inoculum concentration In the fermentation medium of chaff, 28 DEG C, 160rmin-1Under the conditions of cultivate 72h.
2. by zymotic fluid 4000rmin-110min is centrifuged, supernatant crude enzyme liquid is obtained, takes 1mL crude enzyme liquids, add 9mL pH 4.8,0.05mol/L citrate buffer solutions, it is configured to dilute enzyme liquid.2 20mL test tubes are taken, it is dilute to add 0.5mL in a test tube Enzyme liquid and 1.5mL 0.5%CMC solution are released, 2.0mL 0.5%CMC solution is added in another control tube, dilutes enzyme liquid test tube Often pipe does 3 repetitions.
3. 50 DEG C of water-bath 30min, add 3mL DNS reagents.
4. boiling water bath 5min, it is immediately placed on after taking-up in frozen water and is cooled to room temperature, be settled to 20mL, shaken up.
5. determine light absorption value under the conditions of wavelength 540nm.Block diagram is drawn to different carbon source with enzyme activity value, referring to Fig. 3, enzyme The property learned Quality Research shows that the most suitable carbon source that the bacterial strain produces cellulase fermentations culture medium is the corncob of mass fraction 2% Powder.
The optimum nitrogen source screening of the cellulase producer Aspergillus niger 21 fermentation medium of embodiment 2
1. determining optimal carbon source, it is respectively nitrogen source with (NH4) 2SO4, analysis for soybean powder, beef extract, yeast extract, peptone, will plants Sub- liquid is inoculated in fermentation medium in 10% inoculum concentration, 28 DEG C, 160rmin-1Under the conditions of cultivate 72h.
2. -5. step is the same as embodiment 1.Block diagram is drawn to different nitrogen sources with enzyme activity value, referring to Fig. 4, zymologic property is ground Study carefully and show, the optimum nitrogen source that the bacterial strain produces cellulase fermentations culture medium is the peptone of mass fraction 0.5%.
The optimum temperature screening of the cellulase producer Aspergillus niger 21 fermented and cultured of embodiment 3
1. determining optimum carbon source, the culture medium of nitrogen source, seed liquor is inoculated in fermentation medium in 10% inoculum concentration, Respectively at 24 DEG C, 26 DEG C, 28 DEG C, 30 DEG C, 32 DEG C, 160rmin-1Under the conditions of cultivate 72h.
2. -5. step is the same as embodiment 1.Line chart is drawn to different temperatures with enzyme activity value, referring to Fig. 5, zymologic property is ground Study carefully and show, the optimum temperature that the bacterial strain produces cellulase fermentations culture medium is 28 DEG C.
The Best Times screening of the cellulase producer Aspergillus niger 21 fermented and cultured of embodiment 4
1. determining optimum carbon source, nitrogen source and cultivation temperature, seed liquor is inoculated in fermentation medium in 10% inoculum concentration In, since fermentation the 2nd day, sampled every 24h and carry out enzyme activity determination, until terminating for the 7th day.
2. -5. step is the same as embodiment 1.Line chart is drawn to incubation time with enzyme activity value, referring to Fig. 6, zymologic property is ground Study carefully and show, the Best Times that the bacterial strain produces cellulase fermentations culture medium are 5 days.
The volume fraction of the optimal spore suspension inoculation of the cellulase producer Aspergillus niger 21 fermented and cultured of embodiment 5
1. determining optimum carbon source, nitrogen source, cultivation temperature and time, seed liquor is pressed 5%, 10%, 15%, 20%, 25% Inoculum concentration be inoculated in fermentation medium, optimum temperature and cultivated under the conditions of the time.
2. -5. step is the same as embodiment 1.The volume fraction being inoculated with enzyme activity value to spore suspension draws line chart.Referring to figure 7, zymology Quality Research shows, the bacterial strain produces the volume fraction of the optimal spore suspension inoculation of cellulase fermentations culture medium For 20%.
The most suitable starting pH of the cellulase producer Aspergillus niger 21 fermented and cultured of embodiment 6
1. determining optimum carbon source, nitrogen source, cultivation temperature, time and the volume fraction of spore suspension inoculation, 0.05mol/L is used Citrate buffer prepares the nutrient solution that pH is 4.5,5.5,6.5,7.5,8.5 and cultivates pH as starting.
2. -5. step is the same as embodiment 1.Line chart is drawn to starting pH with enzyme activity value, referring to Fig. 8, zymology Quality Research Show, the most suitable starting pH that the bacterial strain produces cellulase fermentations culture medium is 5.5.
The most suitable salinity of the cellulase producer Aspergillus niger 21 fermented and cultured of embodiment 7
1. determining optimum carbon source, nitrogen source, cultivation temperature, time, inoculum concentration and starting pH, matter is added respectively in the medium Measure the NaCl that fraction is 0,2.5%, 5%, 7.5%, 10%, 12.5%, 15%.
2. -5. step is the same as embodiment 1.Line chart, referring to Fig. 9, zymologic property are drawn to different salinity with enzyme activity value Research shows that the most suitable salinity that the bacterial strain produces cellulase fermentations culture medium is 2.5%.
In summary, the Aspergillus niger strain disclosed by the invention that cellulase can be produced under hypersaline environment, the present invention Obtained by following method：Preserving number CGMCC9404 thermophilic salt aspergillus niger is isolated from Shaanxi Province, China Yulin City in Northern Shaanxi Province Dingbian County Dapple salt lake marshy land deposit, there is the ability for producing cellulase by Congo red solid medium Preliminary Identification bacterial strain, And morphology and ITS molecular biology identifications are carried out to it, it is defined as aspergillus niger (Aspergillus niger).Through zymology Quality Research, the most suitable carbon source that the bacterial strain produces the fermentation medium of cellulase is the maize cob meal of mass fraction 2%, most suitable Carbon source is the peptone of mass fraction 0.5%, and most suitable cultivation temperature is 28 DEG C, and optimal incubation time is 5 days, optimal spore suspension It is 20% to be inoculated with volume fraction, and culture medium starting pH is 5.5, and most suitable salinity is 2.5%.The bacterial strain is caused by under salt environment Cellulase can keep higher activity, will have in papermaking and the improvement of cellulose pretreated waste water and the reparation in salt-soda soil Wide application prospect.

Claims (5)

  1. A kind of 1. thermophilic salt aspergillus niger (Aspergillus niger) bacterial strain of cellulase-producing, it is characterised in that the black-koji mould Strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC No.9404；This is thermophilic Salt Aspergillus niger strain is separated from the dapple salt lakes and marshes deposit of Shaanxi Province, China Yulin City Dingbian County and obtained.
  2. 2. utilize the method for the thermophilic salt Aspergillus niger strain cellulase-producing described in claim 1, it is characterised in that the black-koji mould Strain produces cellulase under conditions of being 0%~15% in salinity mass volume ratio；
    The carbon source for producing the fermentation medium of cellulase is the maize cob meal of mass fraction 1%~3.5%, and nitrogen source is quality point The peptone of number 0.2%~1.0%；The cultivation temperature that the Aspergillus niger strain produces cellulase is 24~32 DEG C, incubation time For 1~7 day, spore suspension inoculation volume fraction was 5%~25%, and medium pH is 4.5~8.5, salinity mass volume ratio For 0%~15%.
  3. 3. the method for cellulase-producing as claimed in claim 2, it is characterised in that the Aspergillus niger strain produces cellulase The carbon source of fermentation medium is the maize cob meal of mass fraction 2%, and nitrogen source is the peptone of mass fraction 0.5%；The aspergillus niger The cultivation temperature that bacterial strain produces cellulase is 28 DEG C, and incubation time is 5 days, and spore suspension inoculation volume fraction is 20%, training It is 5.5 to support base starting pH, and salinity mass volume ratio is 2.5%.
  4. 4. the thermophilic salt Aspergillus niger strain of the cellulase-producing described in claim 1 answering in the waste water for administering production cellulose With.
  5. 5. application of the thermophilic salt Aspergillus niger strain of the cellulase-producing described in claim 1 in the reparation of salt-soda soil.
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Publication number Priority date Publication date Assignee Title
CN102010855A (en) * 2010-07-30 2011-04-13 浙江大学 Liquid fermentation method for producing salt-tolerant cellulase from sea aspergillus niger
CN102010856A (en) * 2010-07-30 2011-04-13 浙江大学 Solid fermentation method for producing salt-tolerant cellulase by using oceanic Aspergillus niger

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102010855A (en) * 2010-07-30 2011-04-13 浙江大学 Liquid fermentation method for producing salt-tolerant cellulase from sea aspergillus niger
CN102010856A (en) * 2010-07-30 2011-04-13 浙江大学 Solid fermentation method for producing salt-tolerant cellulase by using oceanic Aspergillus niger

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
一株耐盐纤维素酶海洋曲霉的筛选及产酶条件研究;刘杰凤等;《食品与生物技术学报》;20120715;第31卷(第7期);摘要，第714页图3(b)，第715页图4(b)、图5、图7，第716页图8，第717页图11，第717页右栏第1段 *

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