CN113652362A

CN113652362A - Strain HSU-6 for producing heat-resistant acidic cellulase and application thereof

Info

Publication number: CN113652362A
Application number: CN202110579381.3A
Authority: CN
Inventors: 柏晓辉; 潘健; 许竟成; 佘新松; 翟大才; 吕顺清; 程锦; 吕倩丽; 袁家伟; 李强; 郑青青; 张群遥
Original assignee: Huangshan University
Current assignee: Huangshan University
Priority date: 2021-05-26
Filing date: 2021-05-26
Publication date: 2021-11-16
Anticipated expiration: 2041-05-26
Also published as: CN113652362B

Abstract

The invention belongs to the field of microorganisms, and particularly relates to a strain HSU-6 for producing heat-resistant acidic cellulase and application thereof, wherein the strain is Bacillus cereus (Bacillus cereus) and is preserved in China Center for Type Culture Collection (CCTCC) at 24 months and 5 months in 2021, and the preservation number of the strain is as follows: CCTCC NO: M2021599. The cellulase produced by the strain can tolerate the temperature of 40-50 ℃ and is resistant to metal ion Fe³⁺、Ca²⁺、Cu²⁺、Ni²⁺And Hg²⁺And organic solvents and the like have certain tolerance, and the characteristics show that the cellulase is particularly suitable for industrial production.

Description

Strain HSU-6 for producing heat-resistant acidic cellulase and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a strain HSU-6 for producing heat-resistant acidic cellulase and application thereof.

Background

Energy is an important factor for restricting social development and human progress, and the energy problem in the current society of the 'post-petroleum age' is not ignored; therefore, the development of renewable clean energy is not slow. Cellulose is one of main components forming plant straws, and is polysaccharide which is most widely distributed and contains most of the cellulose in the nature; the biomass energy can generate more than 750 hundred million tons of biomass energy through plant photosynthesis every year, and is a renewable biomass energy source with rich sources; the development and utilization of the organic light emitting diode has become a research hotspot in the field of energy.

The cellulose is polymerized by D-glucopyranose through beta-1, 4-glycosidic bond, the degree of polymerization can reach about 10000, and the cellulose is a polysaccharide with high degree of polymerization. Meanwhile, a large number of hydroxyl groups exist on the cellulose sugar chain, and intramolecular or intermolecular hydrogen bonds can be formed, so that the cellulose sugar chain is difficult to dissolve in water or an organic solvent, and development and utilization of the cellulose sugar chain are influenced. At present, methods for degrading cellulose are mainly classified into physical, chemical and biological methods. Physical methods mainly adopt methods such as superfine grinding, steam explosion and the like to change the natural structure of cellulose so as to increase the solubility and the reactivity of the cellulose, but the methods have large energy consumption and high cost and are not beneficial to the utilization of the cellulose. The chemical method mainly adopts strong acid or strong base for treatment, although the strong acid or strong base can effectively degrade cellulose, the strong acid or strong base has very strong corrosivity, has high requirements on reaction equipment and a high requirement on the process, needs to strengthen the acid or alkali treatment process in subsequent products, and has serious environmental pollution. Therefore, the biological method which is environment-friendly, has low energy consumption and can degrade cellulose at normal temperature and normal pressure becomes a key point and a hotspot for developing and utilizing cellulose resources.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a strain HSU-6 for producing heat-resistant acidic cellulase and application thereof, wherein the cellulase produced by the strain can tolerate the temperature of 40-50 ℃ and is resistant to metal ion Fe³⁺、Ca²⁺、Cu²⁺、Ni²⁺And Hg² ⁺And organic solvents and the like have certain tolerance, and the characteristics show that the cellulase is particularly suitable for industrial production.

The technical scheme adopted by the invention for realizing the purpose is as follows:

a strain for producing heat-resistant acidic cellulase is Bacillus cereus (Bacillus cereus) and is preserved in China Center for Type Culture Collection (CCTCC) at 24 months and 5 months in 2021, and the preservation number of the strain is as follows: CCTCC NO: M2021599, preservation address: china, wuhan university.

Further, the 16S rDNA sequence of the strain is shown in SEQ ID NO. 1.

Further, the amplification primers of the 16S rDNA sequence of the strain are as follows:

27F:5′-AGAGTTTGATCCTGGCTCAG-3′；

1492R:5′-TACGGCTACCTTGTTACGACTT-3′。

a method for preparing cellulase comprises culturing the above strain, fermenting, and extracting cellulase from the fermented culture solution.

The cellulase is prepared by the method.

A bacterial agent for producing thermostable acidic cellulase comprises the strain.

The application of the strain or the cellulase in biological fermentation or production of decontamination products.

Furthermore, the application temperature is 40-50 ℃, and the pH value is 4-6.

A detergent comprising the cellulase and Fe³⁺、K⁺、Ca²⁺、Cu²⁺、Ni²⁺Or Hg²⁺One or more of (a).

A detergent comprising the cellulase enzyme and one or more of methanol, ethanol, isopropanol, dimethyl sulfoxide and Triton X-100.

Has the advantages that:

cellulose is biologically degraded mainly by various cellulases such as exo-glucosidase (exo-1,4- β -glucanase, CBH), endoglycosidase (endo-1,4- β -glucanase, CMCase or EG) and β -glucosidase (β -glucosidase, BG) to obtain products such as cellooligosaccharide, cellobiose, etc., and finally decomposed into glucose for the production of bioethanol, single cell protein, etc. Therefore, the method has important value in screening the cellulase with high activity and wide application. The invention takes ruminant sheep rumen as an experimental material, adopts Congo red selection culture medium to screen high-activity cellulase, and respectively uses 16S rDNA sequencing technology and enzyme activity determination method to carry out molecular identification on the screened strain and determine the enzymatic parameters of the cellulase produced by the strain, so as to provide strain resources for screening the cellulase suitable for industrial application.

Drawings

FIG. 1 shows the colony characteristics of strain HSU-6.

FIG. 2 is a molecular evolutionary tree constructed by strain HSU-6 based on 16S rDNA sequences.

FIG. 3 is a graph showing the effect of pH on cellulase activity.

FIG. 4 is a graph showing the effect of temperature on cellulase enzyme activity.

FIG. 5 is a graph showing the effect of fermentation time on cellulase enzyme activity.

FIG. 6 is a graph showing the effect of metal ions on cellulase enzyme activity.

FIG. 7 is a graph showing the effect of organic solvents on cellulase activity.

FIG. 8 is a graph of the effect of organic solvents on cellulase activity.

Detailed Description

The embodiments of the present invention will be described in further detail with reference to the drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

1. Materials and methods

1.1 Experimental materials

1.1.1 preliminary screening samples for the Strain

The rumen of sheep screened by the cellulase-producing strain in the experiment is purchased in local farmer markets and stored at 4 ℃ for later use.

1.1.2 Experimental instruments

A biological safety cabinet (Singapore Esco, model AC2-4S 1), an autoclave (Chongqing Yamautou, model SQ 510C), a constant-temperature shaking incubator (Shanghai Min spring, model MQD-B2R), an electrothermal three-temperature-zone constant-temperature water tank (Shanghai Kubei industry, model DK-8D), an ultraviolet visible spectrophotometer (Shanghai Jing Ming Jing scientific instruments, model UV-765), and a high-speed centrifuge (Beckkurt company, USA, model Avanti J-E).

1.1.3 Experimental reagents

Sodium carboxymethylcellulose (CMC-Na), 3, 5-dinitrosalicylic acid, phenol, citric acid, sodium potassium tartrate, trisodium citrate, NaCl, NaOH and ZnCl₂、HCl、CaCl₂、KCl、NiCl₂·6H₂O, et al, available from the national pharmaceutical group chemical reagents, Inc. Congo red, peptone, yeast powder, agarose and the like are purchased from Shanghai biological engineering Co., Ltd; the bacterial genomic DNA extraction kit (DP302) was purchased from Tiangen Biochemical technology (Beijing) Ltd.

1.1.4 DNS reagent and Medium preparation

The reducing sugar detection reagent DNS is prepared as follows: weighing 182g of potassium sodium tartrate by using an electronic balance, putting the weighed potassium sodium tartrate into a beaker, adding 450-650 mL of distilled water, and using glassStirring continuously to dissolve the mixture fully, then adding 6.3g of 3, 5-dinitrosalicylic acid, 21g of NaOH and 5g of phenol in sequence, stirring fully until no granular substance exists, cooling to room temperature, fixing the volume to 1000mL, filtering, sterilizing and removing impurities, storing in a brown bottle, and storing in dark place for a week. The enrichment and fermentation culture medium of the cellulase-producing strain is prepared as follows: accurately weighing 10g of CMC-Na, 5.0g of yeast powder, 10g of sodium chloride and 10g of peptone, putting into a beaker, adding 400mL of distilled water, stirring until the materials are completely dissolved, transferring the materials into a conical flask, fixing the volume to 1000mL, keeping the temperature at 121 ℃ under 0.1MPa, and sterilizing for 20 min. Congo red solid selection medium was prepared as follows: accurately weighing 1.88g of CMC-Na, 10g of peptone and K₂HPO40.5g, yeast powder 5g, MgSO₄.7H₂Putting 0.25g of O, 14g of agar and 2.00g of gelatin into a beaker, adding 400mL of distilled water, stirring until the materials are completely dissolved, transferring the mixture into a conical flask, fixing the volume to 1000mL, keeping the temperature at 121 ℃ under 0.1MPa, and sterilizing for 20 min. Meanwhile, the prepared Congo red dye solution with the concentration of 1mg/mL is sterilized for 30min under the pressure of 0.1MPa and the temperature of 115 ℃. And finally, cooling to a certain temperature, adding 5mL of Congo red dye solution into the culture medium, uniformly mixing (no bubbles are generated), pouring the mixture into a flat plate, and storing the mixture in a refrigerator at 4 ℃ after the Congo red culture medium is solidified.

1.2 Experimental methods

1.2.1 preparation of preliminary Strain screening samples

Randomly shearing several tissues from the rumen of a fresh sheep by using a sterilized surgical scissors, and putting the tissues into the same sterile beaker; cutting into pieces with surgical scissors, placing into a sterile homogenizer, adding appropriate amount of sterile water, and homogenizing. Pouring out the homogenate, standing for half an hour, and sucking 1.0mL of solution as initial bacterial liquid.

1.2.2 isolation and purification of cellulase producing strains

0.5mL of the original bacterial liquid is sucked and transferred into an enrichment medium (taking sodium carboxymethylcellulose as a unique carbon source), and the mixture is put into a constant temperature incubator at 37 ℃ for 180 r.min^-1And (5) enrichment culture. After 12h of culture, the enrichment culture solution is respectively sucked and diluted to 10^-4、10^-5And 10^-6Respectively coating the two materials on Congo red screening culture media, and culturing at the constant temperature of 37 ℃ for 2-3 days; at the same time, the steel is observed every 24hWhether a transparent hydrolysis ring is formed on the fruit red culture medium. After a stable hydrolysis ring is formed, respectively measuring the diameter (C) of a bacterial colony and the diameter (H) of the hydrolysis ring, and calculating the ratio of H/C; and (4) taking the strain with a large ratio for streak purification until a single colony is stably formed. The purified strains are numbered respectively and stored for later use.

1.2.3 molecular characterization of cellulase producing strains

Selecting a strain HSU-6 (one of the strains with a large H/C ratio) as a research material, and observing the colony characteristics of the strain; then extracting the genome DNA by using a genome DNA extraction kit and detecting the purity of the genome DNA. Synthetic bacterial Universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and

5'-TACGGCTACCTTGTTACGACTT-3', and amplifying the 16S rDNA sequence of the strain HSU-6 by using the pair of primers, and after the purity is verified, the sequencing is entrusted to Shanghai biological engineering company Limited. The 16S rDNA sequence of the strain was obtained and then subjected to sequence alignment in the 16S ribosomal RNA sequences database (NCBI database). Selecting a representative sequence with high sequence consistency from the comparison result, and carrying out homology analysis and constructing a molecular evolution tree by using software Clustal X2.1 and MEGA 6.06 so as to determine the species of the strain HSU-6.

1.2.4 drawing of glucose Standard Curve

Preparing a 1mg/mL standard glucose solution, measuring the light absorption value at the position with the wavelength of 540nm after the standard glucose solution is uniformly mixed with the DNS solution, repeating for 3 times, and drawing a glucose standard curve after averaging. The regression equation for the standard solution glucose concentration (Y) and absorbance (Z) measured herein is Z-12.868Y-0.2372, R²0.9995; the linearity is good, and the method can be used for subsequent enzyme activity quantitative experiments.

1.2.5 preparation of crude enzyme solution

Taking out the preserved HSU-6 strain from a refrigerator at the temperature of-80 ℃, and streaking and recovering on a Congo red culture medium; then selecting a monoclonal colony of a bacterial strain HSU-6 from the recovered flat plate, transferring the colony to a fermentation culture medium, and culturing for 12 hours; then inoculating into new fermentation culture medium according to 1.0% inoculation amount, and placing in 28 deg.C shaking table for 180 r.min^-1And (5) fermenting for 3 d. Taking the fermentation liquor in a refrigerated centrifuge at 4 ℃ for 12000 r.min^-1Centrifuging for 10min, and centrifugingThe supernatant was used as a crude enzyme solution.

1.2.6 optimum reaction pH value of cellulase

Taking 6 clean glass test tubes, respectively adding 2.0mL of 1% CMC-Na substrate prepared by different buffer solutions with pH values of 3, 4, 5, 6, 7 and 8, adding 1.0mL of crude enzyme solution, mixing uniformly,

then placing the mixture in a constant temperature water bath kettle at 40 ℃ for reaction for 20 min. Adding 2.0mL of DNS reagent into the reaction solution, uniformly mixing, and boiling in boiling water for 10 min; after cooling to room temperature, the volume was adjusted to 10mL, and the absorbance at 540nm was measured and repeated 3 times. Meanwhile, taking the inactivated crude enzyme solution as a control group, measuring the light absorption value under the same condition, and calculating the enzyme activity. Cellulase activity is defined as the amount of enzyme required to hydrolyze a CMC-Na substrate to produce 1. mu. mol glucose per hour per ml of crude enzyme solution under certain conditions.

1.2.7 optimum reaction temperature of cellulase

The enzymatic activity of the cellulase at different reaction temperatures of 30, 40, 50, 60 and 70 ℃ is determined by the method of step 1.2.6 with reference to the optimum reaction pH value determined at 1.2.6.

1.2.8 influence of fermentation time on enzyme activity

Referring to the method in step 1.2.5, taking fermentation liquor at

different fermentation times

2, 3, 4, 5, 6 and 7d to prepare crude enzyme liquid, and determining the enzyme activity of the fermentation liquor at the optimal reaction pH value and temperature determined in steps 1.2.6 and 1.2.7.

1.2.9 Effect of Metal ions on enzyme Activity

Adding the mixture to the reaction kettle to a final concentration of 1 mmol.L^-1In (C) is₂、HgCl₂、KCl、NiCl₂And CuCl₂And (3) carrying out plasma treatment on the metal ions, and determining the influence of different metal ions on the cellulase by referring to the method in the step 1.2.7.

1.2.10 cellulase resistance to high temperatures

And (3) respectively treating the crude enzyme liquid after fermentation for 5d at 30, 40, 50, 60, 70 ℃ and other temperatures for 0.5, 1 and 2 hours, and then determining the enzyme activity of the cellulase under different temperature treatments according to the optimal reaction pH value and temperature determined in the step 1.2.7. Meanwhile, crude enzyme liquid which is not subjected to heat-resistant treatment is used as a control group, and the tolerance capability of the cellulase to different temperatures is compared.

1.2.11 tolerance of cellulase to organic solvents

Adding 1% and 15% (v/v) of organic reagents such as methanol, isopropanol, ethanol, Triton X-100 and dimethyl sulfoxide into a cellulase activity determination system respectively, and determining the enzyme activity by referring to the method of the step 1.2.7; meanwhile, cellulase treated by adding the same amount of reaction buffer is taken as a control group, and the tolerance capability of the cellulase to different organic reagents is compared.

2. Results and analysis

2.1 molecular characterization of cellulase producing Strain HSU-6

A bacterial strain with the number of HSU-6 is screened from the sheep rumen by using Congo red screening culture medium, and can produce cellulase (figure 1). As can be seen from the results in FIG. 1, the strain HSU-6 can produce a stable and obvious hydrolysis transparent ring on a Congo red solid culture medium which takes CMC-Na as a unique carbon source; the diameter (H) of the generated transparent ring is 44.5mm, the diameter (C) of a bacterial colony of the bacterial strain HSU-6 is 8.5mm, and the ratio H/C of the two is about 5.2; the strain was found to be a gram-positive bacilli by gram-staining.

Amplifying and sequencing the 16S rDNA sequence on the extracted strain HSU-6 genome by using the bacterial identification universal primers 27F and 1492R, comparing and analyzing the sequenced result in a Nucleotide BLAST tool of an NCBI database, and selecting a sequence with more than 99 percent of sequence identity from the compared result to construct a molecular evolution tree of the strain HSU-6 (figure 2). From the constructed evolutionary tree results, the strain HSU-6 and Bacillus cereus strain NBRC 15305 and other Bacillus cereus are located on the same evolutionary tree branch; by combining the colony characteristics, gram stain and molecular evolution tree analysis results, the strain HSU-6 can be identified to be Bacillus cereus strain HSU-6(Bacillus cereus strain HSU-6).

2.2 influence of pH on cellulase Activity

To explore the pH of the cellulase for optimal reactions, the enzymatic activity of the cellulase was tested with buffers of different pH values (FIG. 3). The experimental results show that the enzyme activity of the cellulase produced by the strain HSU-6 is 3.0 at the pH valueAbout 3.3 U.mL^-1(ii) a With the increase of pH to 4.0, the enzyme activity thereof also rapidly increases to 6.3 U.mL^-1(ii) a When the pH value continues to rise, the enzyme activity does not continue to rise, but slowly drops; when the pH value is increased to 7.0 and 8.0, the enzyme activity is reduced to 4.1 U.mL^-1And the following. The above results indicate that the optimum reaction pH of the cellulase was 4.0.

2.3 Effect of temperature on cellulase Activity

And when the determined optimal reaction pH value is 4.0, measuring the enzyme activity of the cellulase produced by the strain HSU-6 at the temperature of 30-70 ℃ (figure 4). According to experimental results, when the reaction temperature is 30-40 ℃, the enzyme activity of the cellulase is increased along with the temperature increase, and reaches a maximum value of 7.5 U.mL at 40 DEG C^-1(ii) a When the reaction temperature continues to rise, the enzyme activity does not continue to rise but decreases, and reaches a minimum value of 4.8 U.mL at 70 DEG C^-1. The experimental result shows that the optimum reaction temperature of the cellulase is 40 ℃.

2.4 Effect of fermentation time on cellulase Activity

The enzyme activities of the cellulase production at different fermentation times were measured at the optimum reaction pH and temperature determined above (FIG. 5). The experimental determination result shows that the cellulase activity of the fermentation liquor can reach 8.3 U.mL when the fermentation is carried out for 2d^-1(ii) a Along with the increase of the fermentation time, the enzyme activity of the cellulase in the fermentation liquid is slowly increased and reaches the maximum enzyme activity of 9.1 U.mL at the 5 th day of fermentation^-1(ii) a When the fermentation time is continuously increased to 6 d and 7d, the cellulase activity in the fermentation liquor is reduced to 7.5 U.mL^-1And remains relatively stable.

2.5 Effect of Metal ions on cellulase Activity

The influence of different metal ions on the enzymatic activity of the cellulase was determined at the optimum reaction pH of 4.0 and the optimum reaction temperature of 40 ℃ (FIG. 6). From the experimental data determined, Fe³⁺The enzyme activity of the cellulase is slightly improved by ions; k⁺And Ca²⁺Ions can slightly reduce the enzymatic activity of the cellulase to 93.9 percent and 92.5 percent; cu²⁺、Ni²⁺And Hg²⁺Ions can significantly reduce the enzymeViability was 80.2%, 79.8% and 76.1%. The experimental result shows that the cellulase has certain tolerance capability to metal ions.

2.6 cellulase resistance to high temperatures

The crude cellulase liquid is respectively treated at the temperature of 30-70 ℃ for different time, and the tolerance capacity of the crude cellulase liquid to different temperatures is measured (figure 7). According to the experimental results of the determination, the enzyme activity of the cellulase can be maintained after 1 or 2 hours of treatment at the temperature of 30-50 ℃; and at 60 or 70 ℃, the enzyme activity of the enzyme can be rapidly reduced after 1 or 2 hours of treatment, and the enzyme activity is reduced to about 10 percent of the normal enzyme activity. Therefore, the cellulase can tolerate the temperature of 40-50 ℃ and is heat-resistant cellulase.

2.7 tolerance of cellulase to organic solvents

The cellulase activity measured under the conditions of optimum pH and temperature values was 100%, and the influence of the addition of organic solvents at two concentrations of 1% and 15% (v/v) on the cellulase activity was compared (FIG. 8). From the results of fig. 8, it is known that when the concentration of the organic solvent is 1%, methanol, isopropanol and dimethyl sulfoxide (DMSO) have little influence on the enzymatic activity of the cellulase, ethanol slightly decreases the enzymatic activity, but Triton X-100 slightly increases the enzymatic activity. When the concentration of the organic solvent is increased to 15%, the enzyme activity of the cellulase is reduced to a small extent by using methanol, ethanol, isopropanol and dimethyl sulfoxide, but the enzyme activity is all over 85%; triton X-100 can obviously increase the enzyme activity, which indicates that the cellulase has certain tolerance to organic solvent.

Cellulose is a renewable biomass energy source with rich sources, can be used in industries such as energy sources and feeds, and has great development value; the cellulase plays a very important role in the development and utilization of cellulose, so that the screening of cellulase high-producing strains from plants, animal intestines, ruminant rumens and the like is a hotspot of research in the field of microorganisms.

Microbial communities such as protozoa, bacteria and archaea exist in the rumen of the ruminant, play an important role in the cellulose decomposition process, and are an important source for screening high-yield cellulase strains. The invention uses Huangshan local breedingThe sheep rumen is taken as a material, a strain HSU-6 which produces cellulase is screened out from the material, and the strain is identified as bacillus cereus by molecules. Subsequently, through enzyme activity parameter measurement, the optimum pH value of the bacterial strain HSU-6 for producing the cellulase is found to be 4.0, and the bacterial strain HSU-6 can maintain higher enzyme activity within the pH value range of 4.0-6.0, and is an acid cellulase; the acid cellulase has important application value in animal husbandry, energy and environment protection industries and the like, so the cellulase screened by the invention has important application value. Meanwhile, the invention also discovers that the cellulase can tolerate the temperature of 40-50 ℃ and can resist metal ions Fe³⁺、Ca²⁺、Cu²⁺、Ni²⁺And Hg²⁺And organic solvent and the like have certain tolerance, and the characteristics show that the cellulase is particularly suitable for industrial production, so the strain HSU-6 is worthy of development and utilization. The modern molecular biology technology can be subsequently adopted to research and analyze the coding gene and the protein amino acid sequence of the cellulase, and a foundation is laid for constructing the high-yield escherichia coli strain of the cellulase.

The invention screens out a bacterial strain HSU-6 for producing heat-resistant acidic cellulose B.cereus from sheep rumen, wherein the optimum pH value and temperature for producing cellulase are 4.0 and 40 ℃ respectively, and the cellulase is heat-resistant acidic cellulase; and has certain tolerance to organic solvents. The enzyme activity of the crude enzyme solution of the strain for producing the cellulase under the optimal condition can reach 9.1 U.mL^-1Can provide a new cellulase resource for the in vitro degradation of cellulose.

The 16S rDNA sequence of the strain HSU-6 is shown in SEQ ID NO. 1.

>HSU-6

TGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACC。

Sequence listing

<110> Huangshan college

<120> strain HSU-6 for producing heat-resistant acidic cellulase and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1388

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tggattaaga gcttgctctt atgaagttag cggcggacgg gtgagtaaca cgtgggtaac 60

ctgcccataa gactgggata actccgggaa accggggcta ataccggata acattttgaa 120

ccgcatggtt cgaaattgaa aggcggcttc ggctgtcact tatggatgga cccgcgtcgc 180

attagctagt tggtgaggta acggctcacc aaggcaacga tgcgtagccg acctgagagg 240

gtgatcggcc acactgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg 300

aatcttccgc aatggacgaa agtctgacgg agcaacgccg cgtgagtgat gaaggctttc 360

gggtcgtaaa actctgttgt tagggaagaa caagtgctag ttgaataagc tggcaccttg 420

acggtaccta accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg 480

tggcaagcgt tatccggaat tattgggcgt aaagcgcgcg caggtggttt cttaagtctg 540

atgtgaaagc ccacggctca accgtggagg gtcattggaa actgggagac ttgagtgcag 600

aagaggaaag tggaattcca tgtgtagcgg tgaaatgcgt agagatatgg aggaacacca 660

gtggcgaagg cgactttctg gtctgtaact gacactgagg cgcgaaagcg tggggagcaa 720

acaggattag ataccctggt agtccacgcc gtaaacgatg agtgctaagt gttagagggt 780

ttccgccctt tagtgctgaa gttaacgcat taagcactcc gcctggggag tacggccgca 840

aggctgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 900

tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ctgaaaaccc tagagatagg 960

gcttctcctt cgggagcaga gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1020

gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca tcattaagtt 1080

gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca 1140

tcatgcccct tatgacctgg gctacacacg tgctacaatg gacggtacaa agagctgcaa 1200

gaccgcgagg tggagctaat ctcataaaac cgttctcagt tcggattgta ggctgcaact 1260

cgcctacatg aagctggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt 1320

cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc gaagtcggtg 1380

gggtaacc 1388

Claims

1. The strain for producing the heat-resistant acidic cellulase is characterized in that the strain is Bacillus cereus (Bacillus cereus) and is preserved in China Center for Type Culture Collection (CCTCC) at 2021 year, 5 months and 24 days, and the preservation number of the strain is as follows: CCTCC NO: M2021599.

2. The thermogenic acidic cellulase strain according to claim 1, wherein the 16S rDNA sequence of the strain is as shown in SEQ ID No. 1.

3. The thermoacidic cellulase-producing strain according to claim 1, wherein the amplification primers for the 16S rDNA sequence of the strain are:

27F:5′-AGAGTTTGATCCTGGCTCAG-3′；

1492R:5′-TACGGCTACCTTGTTACGACTT-3′。

4. a method for producing cellulase, characterized by culturing and fermenting the strain according to claim 1, and extracting cellulase from the culture solution after the fermentation.

5. A cellulase prepared by the method of claim 4.

6. A thermoacidic cellulase inoculant comprising the strain according to claim 1.

7. Use of the strain of claim 1 or the cellulase of claim 5 in biofermentation or in the production of a detergent product.

8. The use according to claim 7, wherein the temperature is 40-50 ℃ and the pH is 4-6.

9. A detergent comprising the cellulase of claim 5 and Fe³⁺、K⁺、Ca²⁺、Cu²⁺、Ni²⁺Or Hg²⁺One or more of (a).

10. A detergent comprising the cellulase of claim 5 and one or more of methanol, ethanol, isopropanol, dimethyl sulfoxide, and Triton X-100.