CN112251374A - High-temperature-resistant high-yield cellulase bacillus subtilis and application thereof - Google Patents
High-temperature-resistant high-yield cellulase bacillus subtilis and application thereof Download PDFInfo
- Publication number
- CN112251374A CN112251374A CN202011111287.7A CN202011111287A CN112251374A CN 112251374 A CN112251374 A CN 112251374A CN 202011111287 A CN202011111287 A CN 202011111287A CN 112251374 A CN112251374 A CN 112251374A
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- cellulase
- temperature
- cmc
- enzyme activity
- 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.)
- Pending
Links
Images
Classifications
-
- 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
-
- 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
- C12N1/205—Bacterial isolates
-
- 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
- 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)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Molecular Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a high-temperature-resistant high-yield cellulase bacillus subtilis and application thereof, wherein cellulase production strains are separated from surface soil of a corn straw pile in Jiangjin region in Chongqing, and the bacillus subtilis has the preservation number as follows: CCTCC NO: M2020002; the strain Z2 with the best enzyme production performance is finally obtained through enrichment culture, CMC-Na plate experiment, lignin plate experiment and enzyme production experiment, and the strain is determined to be Bacillus subtilis Z2 by 16S rRNA sequence comparison and identification of the obtained strain. The filter paper enzyme activity under the optimal enzyme production condition is obtained by optimizing the culture medium and the culture condition, and the CMC enzyme activity and the beta-glucosidase enzyme activity are respectively 0.800U/ml, 5.20U/ml and 2.07U/ml. The cellulase produced by the strain has better activity and stability under the conditions of a buffer solution with the pH value ranging from 4.0 to 10.0 and the temperature of 30-80 ℃. The cellulase produced by the strain and commercial xylanase are mixed and then saccharified to obtain 84.27mg/ml total reducing sugar.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to high-temperature-resistant high-yield cellulase bacillus subtilis and application thereof.
Background
Lignocellulose of plant origin is a renewable natural carbohydrate polymer. The effective utilization of abundant natural biomass is a hot topic in recent years. Cellulases are a general term for a family of multicomponent enzyme systems capable of converting cellulose into fermentable sugars. Cellulases are mainly composed of exoglucanase, endoglucanase and β -glucosidase, and other accessory proteins such as cellobiose dehydrogenase and swollenin. The cellulase has good application prospect in the industries of food, fuel ethanol preparation, feed processing and the like.
The cellulase producing bacteria include bacteria, fungi, actinomycetes, etc. The bacteria can be used as an industrially important high-efficiency source for converting the cellulose biomass by using the enzyme due to the higher growth speed of the bacteria; the more complex glycoside hydrolases have a synergistic effect and a very high natural diversity, and the cellulases produced by them have a high stability. Therefore, bacteria are being widely developed as a highly efficient source of cellulases and hemicellulases.
The current practical use of cellulases is still limited by their high production costs. The higher production costs of cellulases are mainly due to two reasons: firstly, the enzyme activity produced by the production strain is low, which is also a main restriction factor limiting the large-scale production of cellulase. Secondly, the cellulase producing bacteria are mainly fungi at present, the fungus production period is long, generally more than 6 days, and the time consumption and long-acting rate are low.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides high-temperature-resistant high-yield cellulase bacillus subtilis and application thereof.
A high-temperature-resistant high-yield cellulase Bacillus subtilis is a strain for producing cellulase, named as Bacillus subtilis Z2, obtained by primary screening and secondary screening of surface soil of corn straw piles in Jiangjin area of Chongqing city, and the preservation number of the Bacillus subtilis is as follows: m2020002, and the Bacillus subtilis comprises a sequence shown as SEQ ID NO. 1.
The classification of the biomaterial is named: bacillus subtilis Z2
The preservation unit: china center for type culture Collection
And (4) storage address: university of Wuhan, China
Preservation time: year 2020, 1 month and 3 days
The preservation number is: CCTCC NO: M2020002
A preparation method of high-temperature-resistant high-yield cellulase bacillus subtilis is disclosed, wherein the bacillus subtilis is obtained from surface soil of a corn straw pile by enrichment, primary screening and secondary screening, and the preservation number of the bacillus subtilis is as follows: CCTCC NO: M2020002.
As a preferable aspect of the present invention, the method includes the steps of:
1) enrichment culture medium: 1-2.5g/L KH2PO4，1.2-1.8g/L(NH4)2SO4，0.3-0.6g/L MgSO4.7H2O， 0.1-0.4g/L CaCl2，0.001-0.005g/L FeSO4.7H2O，0.5-1.5g/L tryptone，5-10g/L CMC-Na；
2) Primary screening of culture medium:
CMC-Na plate culture medium: 1-2.5g/L K2HPO4，0.5-2.5g/L NaNO3，0.1-0.5g/L MgSO4.7H2O，0.2-1g/L KCl，0.1-0.5g/L tryptone，1-5g/L CMC-Na，15-20g/L agar；
Inorganic salt-lignin medium: 1-3g/L K2HPO4，1-5g/L NaNO3，0.3-0.5g MgSO4.7H2O，0.3-0.8 g/L KCl，0.1-0.4g tryptone，0.005-0.015g/L FeSO4.7H2O，0-5g/L lignin，15-20g/L agar；
3) Re-screening the culture medium: 0.8-2g/L K2HPO4，0.5-2g/L NH4SO4，0.5-2g/L NaCl，1-5g/L Yeast extract，0.2-0.5g/L Cysteine，0.05-0.1g/L CaCl2，0.5-1g/L MgSO4.7H2O，2-5g/L CMC-Na。
The bacillus subtilis has high cellulase production capacity, can produce enzyme by liquid fermentation with yeast extract, beef extract, corn steep liquor or ammonium chloride as nitrogen source and straw stalk, corn stalk, corncob or CMC-Na as carbon source, and has filter paper enzyme activity after culturing at 45-55 ℃ for 6-24 hours, wherein the CMC enzyme activity and the beta-glucosidase enzyme activity are respectively 0.800U/ml, 5.20U/ml and 2.07U/ml, and the fermentation period is greatly shortened.
The stability of the cellulase produced by the prepared strain in different pH value ranges and temperature ranges is analyzed, and the analysis accurately reflects the stability of the cellulase. The cellulase produced by the prepared bacillus subtilis and the commercial xylanase are mixed to hydrolyze lignocellulose, so that higher reducing sugar can be obtained.
A method for evaluating the performance of cellulase produced by high-temperature-resistant high-yield cellulase bacillus subtilis comprises the following steps:
1) heating cellulase produced by the screened strain in a buffer solution with the pH value ranging from 4.0 to 10.0 and a water bath kettle at the temperature of 30-80 ℃, and detecting the enzyme activity by taking 2-5% of CMC-Na as a matrix to obtain the optimal pH and temperature and the stability of the cellulase in the range;
2) the yield of reducing sugar was 84.27mg/ml after 48h of mixing reaction of cellulase produced by the selected strain and commercial xylanase.
Compared with the prior art, the invention has the following technical advantages:
1. the strain has high growth speed, high enzyme production efficiency and short fermentation time, and can finish fermentation within 24 hours;
2. the strain has the characteristics of easy pure culture and difficult contamination;
3. the fermentation performance is stable, the filter paper enzyme activity, the CMC enzyme activity and the beta-glucosidase activity are respectively 0.800U/ml, 5.20U/ml and 2.07U/ml;
4. the produced cellulase is high temperature resistant and has wider temperature and pH stability range;
5. the produced cellulase can be mixed with commercial xylanase for use, and has high hydrolysis efficiency.
Drawings
FIG. 1 is a diagram of a hydrolysis loop and a colony of a CMC-Na plate of different strains;
FIG. 2 is a graph showing the growth of different strains on lignin plates;
FIG. 3 is a graph showing the activities of different strains of filter paper enzyme, CMC enzyme and beta glucosidase;
FIG. 4 is a Z2 phylogenetic tree diagram;
FIG. 5 is a graph showing the effect of pH on CMC enzyme activity;
FIG. 6 is a graph showing the effect of temperature on CMC enzyme activity;
FIG. 7 is a graph of the pH stability of CMC enzyme activity;
FIG. 8 is a graph of the temperature stability of CMC enzyme activity;
FIG. 9 is a graph of the total reducing sugar released by the combined hydrolysis of pretreated rice straw with different enzymes.
Detailed Description
The invention is described in further detail below with reference to the figures and the detailed description.
Example 1
Enrichment culture:
1g of soil sample was added to 50ml of enrichment medium, the enrichment medium composition being: 1-2.5g/L KH2PO4， 1.2-1.8g/L(NH4)2SO4，0.3-0.6g/L MgSO4.7H2O，0.1-0.4g/L CaCl2，0.001-0.005g/L FeSO4.7H2O, 0.5-1.5g/L tryptone and 5-10g/L CMC-Na. Culturing at 37 deg.C and 200rpm for 48h, and culturing 1ml of culture solution in fresh enrichment medium under the same conditions for three times.
Primary screening:
CMC-Na plate experiment
1ml of enrichment culture solution is taken, diluted properly and coated on a CMC-Na plate, single colony with hydrolysis loop is obtained after Congo red staining and NaCl decoloration, and enrichment culture is carried out and the single colony is spotted on a new CMC-Na plate again to obtain accurate hydrolysis loop diameter/colony diameter data, as shown in figure 1. The CMC-Na plate culture medium comprises the following components: 1-2.5g/L K2HPO4，0.5-2.5g/L NaNO3，0.1-0.5g/L MgSO4.7H2O, 0.2-1g/L KCl, 0.1-0.5g/L tryptone, 1-5g/L CMC-Na and 15-20g/L agar. As shown in Table 1, Z2 has a maximum hydrolytic cycle diameter/colony diameter value of 8.00.
Table 1 shows the ratio of the hydrolysis ring diameter of CMC-Na plate to the colony diameter of different strains
lignin plate experiment
Carrying out enrichment culture on the 8 strains of bacteria obtained by separation and sequentially diluting the 8 strains of bacteria by 10-1,10-2,10-3,10-4Taking 5 mul of the solution to be spotted on a lignin plate, wherein the inorganic salt-lignin culture medium comprises the following components: 1-3g/L K2HPO4，1-5g/L NaNO3，0.3-0.5g MgSO4.7H2O，0.3-0.8g/L KCl，0.1-0.4g tryptone，0.005-0.015g/L FeSO4.7H2O, 0-5g/L lignin and 15-20g/L agar. The culture is carried out for 48h at 37 ℃, and Z2 is observed to have better lignin tolerance. The growth of the different strains on lignin plates is shown in FIG. 2.
Re-screening:
as can be seen from the above qualitative analysis, 8 strains of bacteria separated in the laboratory have better cellulase production characteristics, and then the 8 strains of bacteria are subjected to enzyme production quantitative analysis. Respectively inoculating 8 strains of bacteria into a triangular flask with the volume of 50ml, and rescreening a culture medium with the following components: 0.8-2g/L K2HPO4，0.5-2g/L NH4SO4，0.5-2g/L NaCl，1-5g/L Yeast extract，0.2-0.5g/L Cysteine，0.05-0.1g/L CaCl2，0.5-1g/L MgSO4.7H2O, 2-5g/L CMC-Na. Culturing at 37 deg.C and 200rpm for 48 h. The activities of the filter paper enzyme, the CMC enzyme and the beta-glucosidase were detected, wherein Z2 has the highest enzyme activity, and the activities of the filter paper enzyme, the CMC enzyme and the beta-glucosidase are respectively 0.800U/ml, 5.20U/ml and 2.07U/ml, as shown in FIG. 3.
Example 2
A16S rRNA sequence amplification primer is designed, a Z216S rRNA sequence blast is subjected to evolutionary tree analysis, and the result shows that Z2 is Bacillus subtilis, as shown in FIG. 4.
Example 3
Effect of pH and temperature on enzyme activity:
the appropriate amount of enzyme solution and the same volume of buffer solution with pH ranging from 4.0 to 10.0 were incubated overnight at 50 ℃ and then the enzyme activity was measured with 2-5% CMC-Na as matrix to obtain an optimal pH of 7. Wherein the Citrate buffer (pH 3.0-5.0), Tris-HCl buffer (pH 6.0-8.0) and Glycine-NaOH buffer (pH 9.0-10.0). Adjusting pH to 7, heating in 30-80 deg.C water bath overnight, and detecting enzyme activity with 2-5% CMC-Na as matrix to obtain the optimal temperature of 50 deg.C. The effect of pH on CMC enzyme activity is shown in FIG. 5, and the effect of temperature on CMC enzyme activity is shown in FIG. 6.
Influence of pH and temperature on enzyme activity stability
The appropriate amount of enzyme solution and the same volume of buffer solution with pH ranging from 4.0 to 10.0 were incubated at 50 ℃ for 30min, and then the pH stability was measured by measuring the enzyme activity using 2-5% CMC-Na as a matrix, as shown in FIG. 7. Wherein the Citrate buffer (pH 3.0-5.0), Tris-HCl buffer (pH 6.0-8.0) and Glycine-NaOH buffer (pH 9.0-10.0). Adjusting pH to 7, heating in 30-80 deg.C water bath for 30min, detecting enzyme activity with 2-5% CMC as matrix, and measuring temperature stability as shown in FIG. 8.
Example 4
Loading 10-30% (w/v) of pretreated straw stalks in a 50ml system, respectively adding 5-10FPU/g of enzyme solution produced by experimental strains, 20-50FPU/g of commercial cellulase and 20-50FPU/g of commercial xylanase in the enzyme solution of the experimental strains. The yield of reducing sugar was measured after 48h reaction at 50 ℃ and 200rpm, wherein the highest amount of reducing sugar was 84.27mg/ml when the enzyme solution of the experimental strain plus commercial xylanase was hydrolyzed for 48 h. The total amount of reducing sugars released by the hydrolysis of pretreated rice straw with the combination of different enzymes is shown in fig. 9.
Example 5
And (3) detecting the enzyme activity of the filter paper:
the enzyme activity of the filter paper enzyme is determined by a DNS (3, 5-dinitousalicylic acid) method. The cultured cell culture fluid was placed in a sterilized EP tube, centrifuged at 8000 g at 4 ℃ for 10min, and the supernatant was collected. The supernatant was collected in an amount of 100. mu.l, added to Whatman No.1 filter paper (1X 6 cm. apprxeq.50.0 mg) as a substrate, and incubated in 50mM sodium citrate-sodium citrate buffer (pH 5.0) (60 min at 50 ℃). After the incubation, the amount of the released reducing sugar was measured by DNS method at 540 nm. Enzyme activity defines the amount of enzyme required to release 1. mu. mol glucose per minute.
CMCase enzyme activity assay:
the CMC enzyme activity was determined by DNS (3, 5-dinitousalicylic acid) method. The cultured cell culture fluid was placed in a sterilized EP tube, centrifuged at 8000 g at 4 ℃ for 10min, and the supernatant was collected. The supernatant was collected in an amount of 500. mu.l, and 1% (w/v) CMC-Na was added as a substrate to incubate at 50mM sodium citrate-sodium citrate buffer (pH 5.0) (60 min at 50 ℃). After the incubation, the amount of the released reducing sugar was measured by DNS method at 540 nm. Enzyme activity defines the amount of enzyme required to release 1. mu. mol glucose per minute.
Detecting the enzyme activity of beta-glucosidase:
the enzymatic activity of beta-glucosidase is determined by pNPG (p-nitrophenyl-beta-D-glucopyranoside) method. Placing the cultured cell culture solution in sterilized EP tube, centrifuging at 8000 g and 4 deg.C for 10min,the supernatant was collected. Collecting supernatant 100-2CO3The reaction was terminated and the absorbance was measured at 400 nm. Enzyme activity defines the amount of enzyme required to release 1. mu. mol of p-nitrophenol per minute.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Sequence listing
<110> university of Chongqing
<120> high-temperature-resistant high-yield cellulase bacillus subtilis and application thereof
<160> 29
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 1
gcggctggct cctaaaaggt tacctcaccg acttcgggtg ttacaaactc 50
<210> 2
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 2
tcgtggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg 50
<210> 3
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 3
gcatgctgat ccgcgattac tagcgattcc agcttcacgc agtcgagttg 50
<210> 4
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 4
cagactgcga tccgaactga gaacagattt gtgggattgg cttaacctcg 50
<210> 5
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 5
cggtttcgct gccctttgtt ctgtccattg tagcacgtgt gtagcccagg 50
<210> 6
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 6
tcataagggg catgatgatt tgacgtcatc cccaccttcc tccggtttgt 50
<210> 7
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 7
caccggcagt caccttagag tgcccaactg aatgctggca actaagatca 50
<210> 8
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 8
agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg 50
<210> 9
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 9
acgacaacca tgcaccacct gtcactctgc ccccgaaggg gacgtcctat 50
<210> 10
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 10
ctctaggatt gtcagaggat gtcaagacct ggtaaggttc ttcgcgttgc 50
<210> 11
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 11
ttcgaattaa accacatgct ccaccgcttg tgcgggcccc cgtcaattcc 50
<210> 12
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 12
tttgagtttc agtcttgcga ccgtactccc caggcggagt gcttaatgcg 50
<210> 13
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 13
ttagctgcag cactaagggg cggaaacccc ctaacactta gcactcatcg 50
<210> 14
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 14
tttacggcgt ggactaccag ggtatctaat cctgttcgct ccccacgctt 50
<210> 15
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 15
tcgctcctca gcgtcagtta cagaccagag agtcgccttc gccactggtg 50
<210> 16
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 16
ttcctccaca tctctacgca tttcaccgct acacgtggaa ttccactctc 50
<210> 17
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 17
ctcttctgca ctcaagttcc ccagtttcca atgaccctcc ccggttgagc 50
<210> 18
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 18
cgggggcttt cacatcagac ttaagaaacc gcctgcgagc cctttacgcc 50
<210> 19
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 19
caataattcc ggacaacgct tgccacctac gtattaccgc ggctgctggc 50
<210> 20
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 20
acgtagttag ccgtggcttt ctggttaggt accgtcaagg taccgcccta 50
<210> 21
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 21
ttcgaacggt acttgttctt ccctaacaac agagctttac gatccgaaaa 50
<210> 22
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 22
ccttcatcac tcacgcggcg ttgctccgtc agactttcgt ccattgcgga 50
<210> 23
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 23
agattcccta ctgctgcctc ccgtaggagt ctgggccgtg tctcagtccc 50
<210> 24
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 24
agtgtggccg atcaccctct caggtcggct acgcatcgtc gccttggtga 50
<210> 25
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 25
gccgttacct caccaactag ctaatgcgcc gcgggtccat ctgtaagtgg 50
<210> 26
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 26
tagccgaagc caccttttat gtttgaacca tgcggttcaa acaaccatcc 50
<210> 27
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 27
ggtattagcc ccggtttccc ggagttatcc cagtcttaca ggcaggttac 50
<210> 28
<211> 50
<212> DNA
<213> Bacillus subtilis
<400> 28
ccacgtgtta ctcacccgtc cgccgctaac atcagggagc aagctcccat 50
<210> 29
<211> 17
<212> DNA
<213> Bacillus subtilis
<400> 29
ctgtccgctc gacttgc 17
Claims (6)
1. The high-temperature-resistant high-yield cellulase bacillus subtilis is characterized by comprising the following preservation numbers: m2020002, and the Bacillus subtilis comprises a sequence shown as SEQ ID NO. 1.
2. The preparation method of the high-temperature-resistant high-yield cellulase bacillus subtilis is characterized in that the bacillus subtilis is obtained from surface soil of a corn straw pile by enrichment, primary screening and secondary screening, and the preservation number of the bacillus subtilis is as follows: CCTCC NO: M2020002.
3. The method for preparing the high-temperature-resistant high-yield cellulase bacillus subtilis according to claim 2, which comprises the following steps:
1) enrichment culture medium: 1-2.5g/L KH2PO4，1.2-1.8g/L(NH4)2SO4，0.3-0.6g/L MgSO4.7H2O， 0.1-0.4g/L CaCl2，0.001-0.005g/L FeSO4.7H2O，0.5-1.5g/L tryptone，5-10g/L CMC-Na；
2) Primary screening of culture medium:
CMC-Na plate culture medium: 1-2.5g/L K2HPO4，0.5-2.5g/L NaNO3，0.1-0.5g/L MgSO4.7H2O，0.2-1g/L KCl，0.1-0.5g/L tryptone，1-5g/L CMC-Na，15-20g/L agar；
Inorganic salt-lignin medium: 1-3g/L K2HPO4，1-5g/L NaNO3，0.3-0.5g MgSO4.7H2O，0.3-0.8g/L KCl，0.1-0.4g tryptone，0.005-0.015g/L FeSO4.7H2O，0-5g/L lignin，15-20g/L agar；
3) Re-screening the culture medium: 0.8-2g/L K2HPO4，0.5-2g/L NH4SO4，0.5-2g/L NaCl，1-5g/L Yeast extract，0.2-0.5g/L Cysteine，0.05-0.1g/L CaCl2，0.5-1g/L MgSO4.7H2O，2-5g/L CMC-Na。
4. The application of the high-temperature-resistant high-yield cellulase bacillus subtilis is characterized in that the bacillus subtilis of claim 1 is adopted, yeast extract, beef extract, corn steep liquor or ammonium chloride is used as a nitrogen source, straw stalk, corn stalk, corncob or CMC-Na is used as a carbon source to carry out liquid fermentation to produce enzyme, and after the bacillus subtilis is cultured for 6 to 24 hours at the temperature of between 45 and 55 ℃, the filter paper enzyme activity is carried out, and the CMC enzyme activity and the beta-glucosidase enzyme activity are respectively 0.800U/ml, 5.20U/ml and 2.07U/ml.
5. Use of a high-temperature-resistant high-yield cellulase-producing bacillus subtilis, wherein the cellulase produced by the bacillus subtilis of claim 1 and a commercial xylanase are mixed to hydrolyze lignocellulose and obtain higher reducing sugar.
6. A method for evaluating the performance of cellulase produced by high-temperature resistant high-yield cellulase bacillus subtilis is characterized in that the preservation number of the bacillus subtilis is as follows: m2020002, the method comprises the following steps:
1) heating cellulase produced by the screened strain in a buffer solution with the pH value ranging from 4.0 to 10.0 and a water bath kettle at the temperature of 30-80 ℃, and detecting the enzyme activity by taking 2-5% of CMC-Na as a matrix to obtain the optimal pH and temperature and the stability of the cellulase in the range;
2) the yield of reducing sugar was 84.27mg/ml after 48h of mixing reaction of cellulase produced by the selected strain and commercial xylanase.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2020103836218 | 2020-05-08 | ||
| CN202010383621 | 2020-05-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112251374A true CN112251374A (en) | 2021-01-22 |
Family
ID=74244672
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011111287.7A Pending CN112251374A (en) | 2020-05-08 | 2020-10-16 | High-temperature-resistant high-yield cellulase bacillus subtilis and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112251374A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109517754A (en) * | 2018-11-20 | 2019-03-26 | 上海交通大学 | A method of high temperature bacterial strain is isolated and purified using common biochemical equipment |
| CN112920973A (en) * | 2021-03-22 | 2021-06-08 | 广西壮族自治区兽医研究所 | Bacillus subtilis GL-4 for producing cellulase and application thereof |
| CN113136358A (en) * | 2021-06-01 | 2021-07-20 | 重庆大学 | Aerobic co-culture probiotic fermentation process for increasing ginsenoside yield |
| CN113215201A (en) * | 2021-06-09 | 2021-08-06 | 重庆大学 | Coupling process for mixing pig manure with rice straw solid-state biogas fermentation and biogas residue aerobic composting |
| CN114134077A (en) * | 2021-11-19 | 2022-03-04 | 江苏科技大学 | Silkworm excrement-derived cellulose degrading bacterium DC11 and screening method and application thereof |
| CN114395506A (en) * | 2022-01-12 | 2022-04-26 | 福建省农业科学院农业生物资源研究所 | High-temperature-resistant cellulase-producing bacillus subtilis and culture method and application thereof |
| CN116463238A (en) * | 2022-11-18 | 2023-07-21 | 西北农林科技大学 | Bacillus subtilis capable of degrading cellulose and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101696400A (en) * | 2009-11-05 | 2010-04-21 | 南开大学 | Enzyme composition for improving degradation rate of hemicellulose xylan |
| CN102220261A (en) * | 2011-04-29 | 2011-10-19 | 济南海华生物科技有限公司 | Preparation and use of bacillus subtilis and clostridium butyricum composite bacterial preparation |
| CN103642774A (en) * | 2013-11-13 | 2014-03-19 | 宁夏夏盛实业集团有限公司 | Mixed neutral cellulase, preparation method thereof and application thereof to papermaking beating |
| CN104988077A (en) * | 2015-07-29 | 2015-10-21 | 南京林业大学 | Eupenicillium parvum producing high temperature cellulase and xylanase and application thereof |
| CN106434417A (en) * | 2016-08-01 | 2017-02-22 | 奥为（天津）环保科技有限公司 | High-temperature-resistant cellulase producing bacterium and application thereof |
| CN110004093A (en) * | 2019-04-24 | 2019-07-12 | 淮阴师范学院 | A kind of bacillus subtilis culture medium raw material and its preparation method and application, the culture medium for improving bacillomycin D yield |
-
2020
- 2020-10-16 CN CN202011111287.7A patent/CN112251374A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101696400A (en) * | 2009-11-05 | 2010-04-21 | 南开大学 | Enzyme composition for improving degradation rate of hemicellulose xylan |
| CN102220261A (en) * | 2011-04-29 | 2011-10-19 | 济南海华生物科技有限公司 | Preparation and use of bacillus subtilis and clostridium butyricum composite bacterial preparation |
| CN103642774A (en) * | 2013-11-13 | 2014-03-19 | 宁夏夏盛实业集团有限公司 | Mixed neutral cellulase, preparation method thereof and application thereof to papermaking beating |
| CN104988077A (en) * | 2015-07-29 | 2015-10-21 | 南京林业大学 | Eupenicillium parvum producing high temperature cellulase and xylanase and application thereof |
| CN106434417A (en) * | 2016-08-01 | 2017-02-22 | 奥为（天津）环保科技有限公司 | High-temperature-resistant cellulase producing bacterium and application thereof |
| CN110004093A (en) * | 2019-04-24 | 2019-07-12 | 淮阴师范学院 | A kind of bacillus subtilis culture medium raw material and its preparation method and application, the culture medium for improving bacillomycin D yield |
Non-Patent Citations (8)
| Title |
|---|
| RAMESHWAR TIWARI ET AL.: "Bioprospecting of novel thermostable β-glucosidase from Bacillus subtilis RA10 and its application in biomass hydrolysis", 《BIOTECHNOL BIOFUELS》 * |
| THI LAN THANH BIEN ET AL.: "Secretion of heterologous thermostable cellulases in Bacillus subtilis", 《J. GEN. APPL. MICROBIOL.》 * |
| 孙赫等: "纤维素酶与不同来源的木聚糖酶之间协同效果比较", 《第三届全国酶制剂在饲料工业中的应用学术研讨会论文集》 * |
| 张群等: "纤维素酶和木聚糖酶在降解竹粉中的作用", 《化工新型材料第》 * |
| 景宜等: "纤维素酶系组成对二次纤维形态与表面结构的影响", 《林业化学与工业》 * |
| 王义甫等: "饲用纤维素酶测定方法的探讨", 《当代畜牧养殖业》 * |
| 王福荣主编: "《生物工程分析与检验》", 30 June 2005, 《中国轻工业出版社》 * |
| 索江华等: "纤维素酶和木聚糖酶协同降解葡萄皮渣条件优化", 《中国饲料》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109517754A (en) * | 2018-11-20 | 2019-03-26 | 上海交通大学 | A method of high temperature bacterial strain is isolated and purified using common biochemical equipment |
| CN112920973A (en) * | 2021-03-22 | 2021-06-08 | 广西壮族自治区兽医研究所 | Bacillus subtilis GL-4 for producing cellulase and application thereof |
| CN112920973B (en) * | 2021-03-22 | 2022-10-28 | 广西壮族自治区兽医研究所 | Bacillus subtilis GL-4 for producing cellulase and application thereof |
| CN113136358A (en) * | 2021-06-01 | 2021-07-20 | 重庆大学 | Aerobic co-culture probiotic fermentation process for increasing ginsenoside yield |
| CN113136358B (en) * | 2021-06-01 | 2023-08-04 | 重庆大学 | Aerobic co-culture probiotics fermentation process for improving ginsenoside yield |
| CN113215201A (en) * | 2021-06-09 | 2021-08-06 | 重庆大学 | Coupling process for mixing pig manure with rice straw solid-state biogas fermentation and biogas residue aerobic composting |
| CN113215201B (en) * | 2021-06-09 | 2023-03-21 | 重庆大学 | Coupling process for mixing pig manure with rice straw solid-state biogas fermentation and biogas residue aerobic composting |
| CN114134077A (en) * | 2021-11-19 | 2022-03-04 | 江苏科技大学 | Silkworm excrement-derived cellulose degrading bacterium DC11 and screening method and application thereof |
| CN114395506A (en) * | 2022-01-12 | 2022-04-26 | 福建省农业科学院农业生物资源研究所 | High-temperature-resistant cellulase-producing bacillus subtilis and culture method and application thereof |
| CN114395506B (en) * | 2022-01-12 | 2023-05-02 | 福建省农业科学院农业生物资源研究所 | High-temperature-resistant cellulase-producing bacillus subtilis and culture method and application thereof |
| CN116463238A (en) * | 2022-11-18 | 2023-07-21 | 西北农林科技大学 | Bacillus subtilis capable of degrading cellulose and application thereof |
| CN116463238B (en) * | 2022-11-18 | 2023-10-13 | 西北农林科技大学 | Bacillus subtilis capable of degrading cellulose and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112251374A (en) | High-temperature-resistant high-yield cellulase bacillus subtilis and application thereof | |
| Mamma et al. | Bioethanol from sweet sorghum: simultaneous saccharification and fermentation of carbohydrates by a mixed microbial culture | |
| Rajoka et al. | Cellulase production by Cellulomonas biazotea cultured in media containing different cellulosic substrates | |
| Lo et al. | Isolation of cellulose-hydrolytic bacteria and applications of the cellulolytic enzymes for cellulosic biohydrogen production | |
| JP2011529345A (en) | Methods and compositions for improving production of products in microorganisms | |
| CN112111428B (en) | Geobacillus thermoglucosidasius obtained through artificial mutagenesis and application of geobacillus thermoglucosidasius in cellulose degradation | |
| Zhang et al. | Lignocellulosic hydrogen production using dark fermentation by Clostridium lentocellum strain Cel10 newly isolated from Ailuropoda melanoleuca excrement | |
| Islam et al. | Isolation and characterization of cellulase-producing bacteria from sugar industry waste | |
| Waldron et al. | Isolation and characterization of a cellulolytic actinomycete Microbispora bispora | |
| WO2009108081A1 (en) | Penicillium verruculosum filamentous fungus strain producer of a highly active complex of cellulases and accessory enzymes and a method of production of biocatalyst for cellulose and hemicellulose hydrolysis | |
| KR20110119961A (en) | Cellulase producing nectria cinnabarina and its use for saccharification | |
| US20170218354A1 (en) | Highly potent cellulolytic enzyme preparations and processes for producing same | |
| US11407974B2 (en) | Method for preparation and screening of fungal mutant with high hydrolytic activity and catabolite derepressed character | |
| US11242507B2 (en) | Mutant strain Aspergillus aculeatus for producing cellulase and xylanase and preparation method thereof | |
| CN113512501A (en) | Penicillium oxalicum XZH-2 and application thereof | |
| Afzal et al. | Isolation and characterization of cellulase producing Bacillus cereus MRLB1 from soil | |
| Garcia-Kirchner et al. | Mixed submerged fermentation with two filamentous fungi for cellulolytic and xylanolytic enzyme production | |
| Hajiabadi et al. | Isolation and molecular identification of cellulolytic bacteria from Dig Rostam hot spring and study of their cellulase activity | |
| US11149261B2 (en) | Provident method of cellulases enzymes production by Penicillium funiculosum MRJ-16 using low cost media components | |
| Jagavati et al. | Cellulase production by coculture of Trichoderma sp. and Aspergillus sp. under submerged fermentation | |
| Tabassum et al. | Production of cellulases and hemicellulases by an anaerobic mixed culture from lignocellulosic biomass | |
| CN115820428B (en) | Fungus Talaromyces endophyticus and application thereof | |
| Shiang et al. | Cellulase production by Acidothermus cellulolyticus | |
| CN114657073B (en) | Penicillium citrinum strain for high cellobiase production and application thereof | |
| Zheng et al. | Screening and performance of L-14, a novel, highly efficient and low temperature-resistant cellulose-degrading strain |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210122 |