CN109679866A - A kind of efficient endo-xylanase produces screening technique and its application of bacterium - Google Patents

A kind of efficient endo-xylanase produces screening technique and its application of bacterium Download PDF

Info

Publication number
CN109679866A
CN109679866A CN201811600816.2A CN201811600816A CN109679866A CN 109679866 A CN109679866 A CN 109679866A CN 201811600816 A CN201811600816 A CN 201811600816A CN 109679866 A CN109679866 A CN 109679866A
Authority
CN
China
Prior art keywords
screening
culture
bacterial strain
fermentation
bacterium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201811600816.2A
Other languages
Chinese (zh)
Other versions
CN109679866B (en
Inventor
姚健
沈爱喜
周希平
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Agricultural Application Microbe Institute Of Jiangxi Academy Of Agricultural Sciences (jiangxi Rural Energy Research Center)
Original Assignee
Agricultural Application Microbe Institute Of Jiangxi Academy Of Agricultural Sciences (jiangxi Rural Energy Research Center)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Agricultural Application Microbe Institute Of Jiangxi Academy Of Agricultural Sciences (jiangxi Rural Energy Research Center) filed Critical Agricultural Application Microbe Institute Of Jiangxi Academy Of Agricultural Sciences (jiangxi Rural Energy Research Center)
Priority to CN201811600816.2A priority Critical patent/CN109679866B/en
Publication of CN109679866A publication Critical patent/CN109679866A/en
Application granted granted Critical
Publication of CN109679866B publication Critical patent/CN109679866B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N1/02—Separating microorganisms from their culture media
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention provides screening technique and its application of a kind of efficient endo-xylanase production bacterium, comprising: (1) sample source；(2) bacterial strain screening；(3) preparation of zytase fermentation liquid, the present invention carries out being separately cultured for bacterial strain in growth medium first, double-layer agar technique is recycled to carry out the screening of bacterial strain, Azo-xylan (Birchwood) is added in the culture medium of upper layer as screening substrate, this method, which can solve, first cultivates microorganism, it adds Congo red solution and carries out the problem of mixing during color reaction between bacterium colony, it can also solve to be added when preparing plate Congo red, then carry out the false positive issue occurred in microbial cultivation process.

Description

A kind of efficient endo-xylanase produces screening technique and its application of bacterium
Technical field
The invention belongs to field of biotechnology, and in particular to it is a kind of effect endo-xylanase production bacterium screening technique and its Using.
Background technique
Hemicellulose is to be present in resource very rich in nature, accounts for about the 35% of plant dry weight, is widely present in In agricultural and sideline product such as corncob, wheat bran, rice bran, stalk and bagasse, content is only second to cellulose in nature.Xylan is The important component of hemicellulose is to constitute main chain, main chain through β-Isosorbide-5-Nitrae-glucosides key connection by β-D- pyranoid form xylose units It is upper different with a variety of different substituent groups, source or branch degrees such as acetyl group, arabinose residues and glucose residues The xylan molecule of main side chain and the complexity that different substituent groups collectively forms and multiplicity.The degradable needs of xylan are a variety of The common participation of enzyme, including β-Isosorbide-5-Nitrae-endo-xylanase (EC3.2.1.8), xylobiase, (EC3.3.1.37) and asafoetide Acid esters enzyme (EC3.1.1.73) etc., wherein β-Isosorbide-5-Nitrae-endo-xylanase acts on xylan backbone and generates different chain length Oligosaccharides is the enzyme of most critical in xylanolytic enzyme system, at the same be also study at present it is more, using wide xylan degrading Enzyme.There is highly selective cultivation effect to Bifidobacterium etc. in animal intestinal tract using the xylo-oligosaccharide of Production by Enzymes, works as in reduction Cholesterol maintains intestinal health and promotion calcium uptake etc. to have important role.In addition, zytase pulping and paper-making, Food, feed addictive and bioconversion etc. all play an important role.
The screening of xylan degrading bacterial strain is mostly used congo red staining method at present, and mainly by two ways, a kind of mode is Microorganism is first cultivated, Congo red solution is added and carries out color reaction, then decolourize to form wood not of uniform size by sodium chloride The advantages of Polyose degradation transparent circle, this mode is the effect that the color reaction shown is substantially xylan degrading bacterium, is lacked Point is which is cumbersome, and Congo red solution, which is added, can make to mix between bacterium colony；Another way is to prepare plate When be added it is Congo red, the advantages of which be it is easy to operate, bacterium colony mixed questions are not present, the disadvantage is that in agar, murphy juice all Containing starchy material, the microorganism for generating amylase can be made false positive reaction occur, it is in addition certain that there is degradation pigment ability Microorganism can degrade in prolonged incubation Congo red and form transparent circle, be not easy and xylan degrading bacterium distinguish. The present invention carries out being separately cultured for bacterial strain in growth medium first, and double-layer agar technique is recycled to carry out the screening of bacterial strain, upper layer Azo-xylan (Birchwood) is added in culture medium as screening substrate, can solve the above problem, can quickly, it is accurate, have The screening endo-xylanase of effect produces bacterial strain.In addition, the bacterial strain Saccharothrix screened using this method Variisporea YJ is different from the bacterial strain of previously reported screening, and produced zytase can be produced with direct hydrolysis maize cob meal Xylo-oligosaccharide eliminates the step of alkali process corncob prepares xylan, to reduce environmental pollution and production cost.
Summary of the invention
For the problem, the present invention are screening substrate with Azo-xylan (Birchwood) in the prior art above, Using double-layer agar technique, can solve the above problem.Azo-xylan (Bichwood) be origin derived from birch xylan with Dyestuff Remazol brilliant blue R combines high-molecular compound (the 1 Remazol brilliant blue generated R combines 20 saccharide residues)；Zytase can degrade Azo-xylan generate low molecular weight dye composition and oligosaccharide, The dye composition of low molecular weight is soluble (pellucidity is presented in the culture medium of upper panel) in ethanol solution, And the dye composition of high molecular weight is then rendered as precipitated form in ethanol solution and (presents in the culture medium of upper panel blue Color state).The present invention adds Azo-xylan as screening substrate in upper panel, produces the bacterial strain of zytase on upper layer Transparent circle is generated in plate, the screening endo-xylanase that this method can be quick, accurate and effective produces bacterial strain.The present invention is logical Cross following technical scheme realization: a kind of efficient endo-xylanase produces screening technique and its application of bacterium, including following step It is rapid:
(1) sample source: banking up fermentation time as 3d, pig manure and the sugarcane top hybrid composting 20g that fermentation temperature is 40 DEG C, Initial C/N ratio is 25:1, moisture content 60%, and initial pH is 8.0 to obtain fermentation material；
(2) bacterial strain screening: the fermentation material in 1g step (1) is taken to be added in the sterile water of 10mL, then gradient dilution is at 10-6-10-8, take 100 μ L to be coated on growth medium, the screening and culturing medium of falling upper layer after 35 DEG C of culture 3-4d, 35 DEG C are continued to cultivate The ethyl alcohol of 2ml 95% is added in 1d, and the bacterium colony scribing line culture of hydrolysis circle is selected with transfer needle, obtains purifying bacterial strain；It is ordered Entitled Saccharothrix variisporeaYJ was preserved in Guangdong Province's Microbiological Culture Collection on September 5th, 2018 The heart, deposit number are as follows: GDMCC NO:60442, address: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100.
(3) preparation of zytase fermentation liquid: the pure culture bacterial strain that step (2) screens is seeded to 20ml grown cultures In base, in 35 DEG C, 160rpm overnight incubation, as the seed liquor for preparing zytase fermentation liquid；1ml seed liquor is taken to be seeded to In the sterile fermentation culture of 100ml, after 40 DEG C, 160rpm, shake culture 6d, 12000rpm is centrifuged 5min, takes the supernatant to be Fermentation produces the fermentation liquid of zytase；
(4) corncob that obtained zytase fermentation liquid is milled to 150 mesh to natural substrate is degraded, by 5- After the processing of 7h, reduced sugar can be obtained from corncob.
The growth medium is the preparation method comprises the following steps: NaNO32.0g, K2HPO41.0g, KCl 0.5g, MgSO40.5g, FeSO40.01g, sucrose 10g, 15g agar powder, ddH2O are settled to 1000ml, 115 DEG C of sterilizing 25min.
The upper layer screening and culturing medium is the preparation method comprises the following steps: agar powder 1g, Azo-xylan 20ml, phosphate buffer (20mM, pH8.0) is settled to 100ml；
The fermentation culture is the preparation method comprises the following steps: NaNO32.0g, K2HPO41.0g, KCl 0.5g, MgSO40.5g, FeSO40.01g, corncob 4.0g are sieved by 60 mesh, are settled to 1000ml, 115 DEG C of sterilizing 25min.
The identification of Saccharothrix variisporeaYJ bacterial strain:
Genome, 16S rDNA gene PCR amplification: 50 μ l systems, 25 μ l are extracted using bacterium extracts kit PrimerSTAR Max archaeal dna polymerase (2 ×), 1 μ l template, primers F (5'-AAATTTTTTTTCCCCCCCGG-3') and R (3 ' GGGCCCCCTTTAAAAAAAAAC-5') each 1 μ l, 22 μ l ddH2O；Response procedures: 98 DEG C of 4min, 98 DEG C of 10sec, 55 DEG C 10sec, 72 DEG C of 1min 30 circulations, 72 DEG C of extension 10min, PCR product send Hua Da gene sequencing.
Extracted through DNA, PCR amplification, sequence and comparison, find the 16S rDNA sequence of the bacterial strain with The homology highest of Saccharothrix variisporea is 99.33%.The bacterial strain is initially identified as Saccharothrix variisporea is named as Saccharothrix variisporea YJ and (is preserved in Guangdong Save Culture Collection, deposit number are as follows: 60442).Sequence is as follows:
GGGCTTCGGGTGTTACCGACTTTCGTGACG TGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCA CCGCAG CGTTGCTGATCTGCGATTACTAGCGACTCCGACTTCACGGGGTC GAGTTGCAGACCCCGATCCGAACT GAGACCGGCTTTGTGGGAT TCGCTCCACCTCACGGCTTAGCAGCCCTCTGTACCGGCCATTGT AGCATGTGTGA AGCCCTGGACATAAGGGGCATGATGACTTGAC GTCATCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCCATG AGTCCCCGCCATAACGCGCTGGCAACATGGAACGAGGGTTGCG CTCGTTGCGGGACTTAACCCAACATCTCACGA CACGAGCTGAC GACAGCCATGCACCACCTGTACACCAGTCCGAAGAGGCCTACA TCTCTGCAGGTTTCCGGTGC ATGTCAAGCCCAGGTAAGGTTCTT CGCGTTGCATCGAATTAATCCACATGCTCCGCCGCTTGTGCGGG CCCCCG TCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCC CAGGCGGGGTGCTTAATGCGTTAGCTGCGGCACGGAG AACGTG GAAGTCCCCCACACCTAGCACCCACCGTTTACGGCGTGGACTA CCAGGGTATCTAATCCTGTTCGCTC CCCACGCTTTCGCTCCTCA GCGTCAGTATCGGCCCAGAGACCCGCCTTCGCCACCGGTGTTC CTCCTGATATCT GCGCATTTCACCGCTACACCAGGAATTCCAGT CTCCCCTGCCGAACTCAAGTCTGCCCGTATCGACCGCAGGCTCC ACGTTAAGCGTGAAGTTTTCACGGCCGACGCAACAAACCGCCT ACGAGCTCTTTACGCCCAATAATTCCGGACAA CGCTCGCACCCT ACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTCTT CTGCAGGTACCGTCACTC ACGCTTCGTCCCTGCTGAAAGAGGT TTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATC AGGCT TTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTA GGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCG GTCACCCT CTCAGGCCGGCTACCCGTCGTCGCCTTGGTAGGCCATCACCCCA CCAACAAGCTGATAGGCCGCGG GTCCATCCCGTACCGCCGGAA CTTTCCACCACCACGGATGCCTGAAGTGGTCATATCCGGTATTA GACCTAGTT TCCCAGGCTTATCCCAGAGTACAGGGCAGGTTACC CACGTGTTACTCACCCGTTCGCCGCTCGTGTACCCCGAAG GGCC。
Beneficial effects of the present invention
(1) present invention carries out being separately cultured for bacterial strain in growth medium first, and double-layer agar technique is recycled to carry out bacterial strain Screening, as screening substrate, this method, which can solve, is first cultivated for addition Azo-xylan (Birchwood) in the culture medium of upper layer Microorganism adds Congo red solution and carries out the problem of mixing during color reaction between bacterium colony, can also solve It is added Congo red when preparing plate, then carries out the false positive issue occurred in microbial cultivation process.
(2) bacterium of bacterial strain the Saccharothrix variisporea YJ and previously reported screening screened with this method Strain is different, and produced zytase can produce xylo-oligosaccharide with direct hydrolysis maize cob meal, eliminates the preparation of alkali process corncob The step of xylan, to reduce environmental pollution and production cost.
(3) bacterial strain Saccharothrix variisporea YJ fermentation liquid can be to the corncob of 150 mesh of natural substrate It degrades, after the processing of 6h, (0.88mg) reduced sugar can be obtained from corncob, be to extract corn through alkali process method Core xylan carries out 22 times (0.04mg) of gained xylo-oligosaccharide after fermentation liquor treatment 6h again.
Detailed description of the invention
Fig. 1 is the screening for producing endo-xylanase bacterial strain；
Fig. 2 is influence of the temperature to xylanase activity and stability；
Fig. 3 is influence of the pH to enzymatic activity.
Specific embodiment
Embodiment 1
Produce the screening of zytase bacterial strain
It from the sample of pig manure and sugarcane top hybrid composting, is coated in growth medium after diluting, 35 DEG C of cultures The screening and culturing medium of falling upper layer after 3d, as shown in Figure of description 1A, can be formed by producing by one around the bacterial strain of endo-xylanase Transparent circle.It is crossed with aseptic inoculation needle picking colony, carries out secondary screening after 35 DEG C of culture 3d, as a result in bacterium shown in Figure of description 1B There is apparent hydrolysis circle on the periphery fallen.
The preparation of zytase fermentation liquid
(1) the pure culture bacterial strain that picking screens, is seeded in 20ml sterile growth media, and 35 DEG C, 160rpm culture Overnight, as the seed liquor for preparing zytase fermentation liquid；
(2) 1ml seed liquor is taken to be seeded in the sterile fermentation culture of 100ml, after 40 DEG C, 160 rpm, shake culture 6d, 12000rpm is centrifuged 5min, and taking supernatant is the fermentation liquid for fermenting and producing zytase.
The zytase zymologic property of fermentation liquid
100 μ l fermentation liquids are taken, 100 μ l 1%AZO-xylan (Birchwood), 55 DEG C of water-baths 2min, 8000rpm are added, It is centrifuged 5min, the ethyl alcohol of 500 μ l 95% is added, is placed at room temperature for 5min after mixing, supernatant is taken to measure absorbance value in 590nm.Slightly Negative control is used as after 100 DEG C of processing 10min of enzyme solution, 3, enzyme solution sample parallel.
(1) optimal reactive temperature: its enzymatic activity is measured according to the above method under conditions of reaction temperature is 30-65 DEG C, is obtained To its optimal reactive temperature (be denoted as 100% when enzyme activity highest).
(2) optimal reaction pH: fermentation liquid pH is respectively to pH 6.0-8.0 (100mM phosphate buffer), pH 7.5- for adjustment 9.0 (100mM Tris-HCl buffers), pH 8.5-10.0 (100 mM glycine-NaOH buffer), then most suitable anti- The xylanase activity of crude enzyme liquid is measured at a temperature of answering (be denoted as 100% when enzyme activity highest).
(3) temperature stability: fermentation liquid is kept the temperature under the conditions of 30,35,40,45,50,55,60,65 and 75 DEG C respectively Then 3h measures remaining enzyme activity under the conditions of optimal reactive temperature and pH, obtains the thermal stability of enzyme, to save under the conditions of 4 DEG C The enzymatic activity of enzyme solution be defined as 100%, see Figure of description 2.
(4) pH stability: by fermentation liquid respectively in the buffer of pH4.0,5.0,6.0,7.0,8.0,9.0 and 10.0 30min is placed, remaining enzyme activity is then measured under the conditions of optimal reactive temperature and pH, the pH stability of enzyme is obtained, with 4 DEG C of conditions The enzymatic activity of the enzyme solution of lower preservation is defined as 100%, is specifically shown in Figure of description 3.
(5) degradation of the bacterial strain fermentation liquor to corncob: the maize cob meal of 150 mesh of 0.05g sieving is weighed, is added 1.0mL's Fermentation liquid under the conditions of 45 DEG C after 6h, takes 200 μ L reaction solutions, and 200 μ L DNS, 100 DEG C of water-bath 10min are added and use spectrophotometer OD value is surveyed at 520nm.
Degradation of xylan enzyme of the fermentation liquid to corncob: fermentation liquid can drop the corncob of 150 mesh of natural substrate Solution, after the processing of 6h, can obtain the reduced sugar of 0.88mg from corncob.
Embodiment 2
The maize cob meal for weighing the sieving of 150 mesh of 0.05g is taken using conventional alkali carries and is handled corncob, remaining is same Embodiment 1, obtaining reduced sugar is 0.04mg.
In conclusion the double-layer agar technique that the present invention uses (adds Azo-xylan (Birchwood) in the culture medium of upper layer As screening substrate) zytase production bacterial strain is screened, this method both can solve and first cultivate microorganism, adds Congo red molten Liquid carries out the problem of mixing between bacterium colony during color reaction, can also solve to be added when preparing plate it is Congo red, The false positive issue occurred in microbial cultivation process is carried out again；In addition, the bacterial strain Saccharothrix screened with this method Variisporea YJ is different from the bacterial strain of previously reported screening, and produced zytase can be produced with direct hydrolysis maize cob meal Xylo-oligosaccharide eliminates the step of alkali process corncob prepares xylan, to reduce environmental pollution and production cost；And And the bacterial strain fermentation liquor can degrade to the corncob of 150 mesh of natural substrate, it, can be from corncob after the processing of 6h The reduced sugar for obtaining 0.88mg is to extract the oligomeric wood of gained after Corncob Xylan carries out fermentation liquor treatment 6h again through alkali process method 22 times (0.04mg) of sugar.

Claims (4)

1. screening technique and its application of a kind of efficient endo-xylanase production bacterium, it is characterised in that: the following steps are included:
(1) sample source: banking up fermentation time as 3d, pig manure and the sugarcane top hybrid composting 20g that fermentation temperature is 40 DEG C, initially C/N ratio is 25:1, and moisture content 60%, initial pH is 8.0 to obtain fermentation material；
(2) bacterial strain screening: the fermentation material in 1g step (1) is taken to be added in the sterile water of 10mL, then gradient dilution is at 10-6-10-8, take 100 μ L to be coated on growth medium, the screening and culturing medium of falling upper layer after 35 DEG C of culture 3-4d, 35 DEG C are continued to cultivate 1d, are added The ethyl alcohol for entering 2ml 95% selects the bacterium colony scribing line culture of hydrolysis circle with transfer needle, repeats step (2) until obtaining purifying bacterium Strain；It is named as Saccharothrix variisporea YJ and is now preserved in Guangdong Province's Culture Collection, protected Hiding number are as follows: GDMCC NO:60442；
(3) preparation of zytase fermentation liquid: the pure culture bacterial strain that step (2) screens is seeded in 20ml growth medium, In 35 DEG C, 160rpm overnight incubation, as the seed liquor for preparing zytase fermentation liquid；Take 1ml seed liquor be seeded to 100ml without In bacterium fermentation culture, after 40 DEG C, 160rpm, shake culture 6d, 12000rpm is centrifuged 5min, takes supernatant, must both ferment production The fermentation liquid of zytase；
(4) corncob that obtained zytase fermentation liquid is milled to 150 mesh to natural substrate is degraded, by 5-7h's After processing, reduced sugar can be obtained from corncob.
2. screening technique and its application of a kind of efficient endo-xylanase production bacterium according to claim 1, the life Long culture medium is the preparation method comprises the following steps: NaNO32.0g, K2HPO41.0g, KCl 0.5g, MgSO40.5g, FeSO40.01g, sucrose 10g, 15g agar powder, ddH2O is settled to 1000ml, 115 DEG C of sterilizing 25min.
3. screening technique and its application of a kind of efficient endo-xylanase production bacterium according to claim 1, it is described on Layer screening and culturing medium is the preparation method comprises the following steps: agar powder 1g, Azo-xylan 20ml, phosphate buffer (20mM, pH8.0) are settled to 100ml。
4. screening technique and its application of a kind of efficient endo-xylanase production bacterium according to claim 1, the hair Ferment culture solution is the preparation method comprises the following steps: NaNO32.0g, K2HPO41.0g, KCl 0.5g, MgSO40.5g, FeSO40.01g, corn Core 4.0g is sieved by 60 mesh, is settled to 1000ml, 115 DEG C of sterilizing 25min.
CN201811600816.2A 2018-12-26 2018-12-26 Screening method and application of efficient endoxylanase producing strain Active CN109679866B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811600816.2A CN109679866B (en) 2018-12-26 2018-12-26 Screening method and application of efficient endoxylanase producing strain

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811600816.2A CN109679866B (en) 2018-12-26 2018-12-26 Screening method and application of efficient endoxylanase producing strain

Publications (2)

Publication Number Publication Date
CN109679866A true CN109679866A (en) 2019-04-26
CN109679866B CN109679866B (en) 2022-06-10

Family

ID=66189631

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811600816.2A Active CN109679866B (en) 2018-12-26 2018-12-26 Screening method and application of efficient endoxylanase producing strain

Country Status (1)

Country Link
CN (1) CN109679866B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1261913A (en) * 1997-07-04 2000-08-02 诺沃挪第克公司 Endo-beta-1,4-glucanases from saccharothrix
CN102002471A (en) * 2010-11-05 2011-04-06 广东溢多利生物科技股份有限公司 Novel xylanase V-XYL as well as gene, high-efficiency expression method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1261913A (en) * 1997-07-04 2000-08-02 诺沃挪第克公司 Endo-beta-1,4-glucanases from saccharothrix
CN102002471A (en) * 2010-11-05 2011-04-06 广东溢多利生物科技股份有限公司 Novel xylanase V-XYL as well as gene, high-efficiency expression method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
TREVIZANO LM等: "Thermostability improvement of Orpinomyces sp xylanase by directed evolution", 《JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC》 *
林小洪等: "产木聚糖酶菌株的筛选、鉴定及酶学性质研究", 《中国食品学报》 *

Also Published As

Publication number Publication date
CN109679866B (en) 2022-06-10

Similar Documents

Publication Publication Date Title
Mihajlovski et al. From agricultural waste to biofuel: enzymatic potential of a bacterial isolate Streptomyces fulvissimus CKS7 for bioethanol production
Kolasa et al. Co-cultivation of Trichoderma reesei RutC30 with three black Aspergillus strains facilitates efficient hydrolysis of pretreated wheat straw and shows promises for on-site enzyme production
Karnwal et al. Production of amylase enzyme by isolated microorganisms and it's application
CN113046278A (en) Composite microbial agent for fermenting and degrading cellulose in straws
KR101771960B1 (en) Paenibacillus jamilae BRC15-1 strain producing cellulase and use thereof
EU et al. Screening, isolation and characterization of amylase producing bacteria and optimization for production of amylase
JP4923739B2 (en) Acid cellulase-producing bacteria
CN113652379B (en) Bacillus cell wall depolymerization GIEC and application thereof
US20150118718A1 (en) Strain of bacillus subtilis and applications thereof
CN103937691B (en) One plant production β fructosidases aspergillus oryzae strain and its cultural method and application
CN103421851B (en) A kind of method preparing sugar and ethanol with sweet potato waste
CN104357364A (en) Streptomycete strain and method for preparing alkali-resistant salt-resistant xylanase by using same
CN108795819A (en) A kind of complex microorganism culture and its application in producing carotenoid
CN104893988B (en) A kind of Trichoderma atroviride and its application for producing high temperature resistant feruloyl esterase and high temperature-resisting cellulase
CN104131042B (en) Method for production of L-lactic acid by control of growth form of rhizopus oryzae
RU2316584C1 (en) Method for technological preparing fermented sugars from lignocellulose material
CN1298838C (en) Aspergillus oryzae fungus and its application in production of Pu'er tea
Sirisena et al. Isolation and characterization of cellulolytic bacteria from decomposing rice straw
CN109679866A (en) A kind of efficient endo-xylanase produces screening technique and its application of bacterium
CN102433270B (en) Organic solvent resistant bacillus licheniformis for producing glucosidase and application of bacillus licheniformis
CN115851501A (en) Bacillus, microbial preparation, culture method and application
CN104726388A (en) Pullulanase enzyme-producing strain and method for improving enzyme-producing capacity thereof
AU2021101266A4 (en) A screening method for efficiently isolating endoxylanase producing microorganism and its application
Chen et al. Biotechnology principles of solid state fermentation
PURWOKO et al. Bioethanol production from rice and corn husks after enzymatic and microbes hydrolysis and yeast fermentation

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant