CN113652362B - Strain HSU-6 for producing heat-resistant acidic cellulase and application thereof - Google Patents

Abstract

The invention belongs to the field of microorganisms, and in particular relates to a heat-resistant acid cellulase-producing strain HSU‑6 and its application. The strain is Bacillus cereus, which was preserved in China's typical culture on May 24, 2021. Culture Collection Center (CCTCC), the strain preservation number is: CCTCC NO: M2021599. The cellulase produced by the strain can withstand the temperature of 40-50°C, and has certain tolerance to metal ions Fe ³⁺ , Ca ²⁺ , Cu ²⁺ , Ni ²⁺ and Hg ²⁺ and organic solvents, etc. , these properties show that the cellulase is particularly suitable for use in industrial production.

Description

A heat-resistant acid cellulase producing strain HSU-6 and its application

technical field

The invention belongs to the field of microorganisms, and in particular relates to a heat-resistant acid cellulase-producing strain HSU-6 and an application thereof.

Background technique

Energy is an important factor restricting social development and human progress. In the "post-petroleum era", the energy problem in today's society cannot be ignored; therefore, it is urgent to develop renewable clean energy. Cellulose is one of the main components of plant straw, and it is also the most widely distributed and abundant polysaccharide in nature; it can produce more than 75 billion tons through plant photosynthesis every year, and is a renewable biomass energy with abundant sources; Its development and utilization has become a research hotspot in the field of energy.

Cellulose is polymerized by D-glucopyranose through β-1,4-glycosidic bonds, and its degree of polymerization can reach about 10,000, which is a kind of polysaccharide with high degree of polymerization. At the same time, there are a large number of hydroxyl groups on the cellulose sugar chain, which can form intramolecular or intermolecular hydrogen bonds, making it difficult to dissolve in water or organic solvents, affecting its development and utilization. At present, the methods for degrading cellulose are mainly divided into physical, chemical and biological methods. Physical methods mainly use superfine pulverization, steam explosion and other methods to change the natural structure of cellulose to increase its solubility and reactivity, but these methods consume a lot of energy and cost, which is not conducive to the utilization of cellulose. The chemical method mainly uses strong acid or strong base for treatment. Although it can effectively degrade cellulose, strong acid or strong base is very corrosive, which requires high reaction equipment and high process requirements, and needs to be strengthened in subsequent products. Acid or alkali treatment process is more serious to environmental pollution. Therefore, biological methods that are environmentally friendly, low in energy consumption, and capable of degrading cellulose under normal temperature and pressure have become the focus and focus of the development and utilization of cellulose resources.

Contents of the invention

Aiming at the problems existing in the prior art, the present invention provides a heat-resistant acid cellulase producing strain HSU-6 and its application. The cellulase produced by the strain can withstand the temperature of 40-50° C. ³⁺ , Ca ²⁺ , Cu ²⁺ , Ni ²⁺ , Hg ^{2 +} , and organic solvents have a certain tolerance, and these characteristics show that the cellulase is particularly suitable for use in industrial production.

The technical scheme that the present invention adopts for realizing the above object is:

A heat-resistant acid cellulase-producing strain, said strain is Bacillus cereus, was preserved in China Center for Type Culture Collection (CCTCC) on May 24, 2021, and the strain preservation number is: CCTCC NO : M2021599, deposit address: China, Wuhan, Wuhan University.

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

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

27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

1492R: 5'-TACGGCTACCTTGTTACGACTT-3'.

The invention discloses a method for preparing cellulase, which comprises culturing and fermenting the above bacterial strain, and extracting cellulase from the fermented culture solution.

A cellulase, which is prepared by the above method.

A heat-resistant acid cellulase-producing bacterial agent, the bacterial agent comprising the above strains.

Application of the above bacterial strain or cellulase in biological fermentation or production of decontamination products.

Further, the application temperature is 40-50° C., and the pH value is 4-6.

A detergent, comprising the above-mentioned cellulase, and one or more of Fe ³⁺ , K ⁺ , Ca ²⁺ , Cu ²⁺ , Ni ²⁺ or Hg ²⁺ .

A detergent, including the above cellulase, also includes one or more of methanol, ethanol, isopropanol, dimethyl sulfoxide and Triton X-100.

Beneficial effect:

Various cellulases are mainly used in biology such as exo-1,4-β-glucanase (CBH), endo-glucosidase (endo-1,4-β-glucanase, CMCase or EG) and β-glucosidase (β-glucosidase, BG) to degrade cellulose to obtain products such as cellulose oligosaccharides and cellobiose, and finally decompose them into glucose for the production of bioethanol and single-cell protein. Therefore, it is of great value to screen cellulase with high activity and wide application. The present invention uses the rumen of ruminant sheep as the experimental material, adopts Congo red selection medium to screen cellulase with high activity, and uses 16S rDNA sequencing technology and enzyme activity measurement method to carry out molecular identification and cellulose production of the screened bacterial strain respectively. The enzymatic parameters of enzymes were determined in order to provide strain resources for screening cellulase suitable for industrial applications.

Description of drawings

Figure 1 is the colony characteristics of strain HSU-6.

Figure 2 is the molecular phylogenetic tree of strain HSU-6 based on the 16S rDNA sequence.

Figure 3 is the effect of pH value on cellulase enzyme activity.

Figure 4 is the effect of temperature on cellulase enzyme activity.

Figure 5 is the effect of fermentation time on cellulase enzyme activity.

Figure 6 shows the effect of metal ions on cellulase enzyme activity.

Figure 7 is the effect of organic solvents on the enzyme activity of cellulase.

Figure 8 is the effect of organic solvents on cellulase enzyme activity.

Detailed ways

Embodiments of the present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but should not be used to limit the scope of the present invention.

1. Materials and methods

1.1 Experimental materials

1.1.1 Preliminary screening samples of bacterial strains

The sheep rumens used in this experiment for the screening of cellulase-producing strains were purchased from local farmers’ markets and stored at 4°C for later use.

1.1.2 Experimental Instruments

Biological safety cabinet (Singapore Esco, AC2-4S1 type), autoclave (Chongqing Yamato, SQ510C type), constant temperature oscillation incubator (Shanghai Minquan, MQD-B2R type), electric three-temperature zone constant temperature water tank (Shanghai Kuangbei Industry, DK-8D type), ultraviolet-visible spectrophotometer (Shanghai Precision Scientific Instrument, UV-765 type), high-speed centrifuge (Baker Coulter Company of the United States, AvantiJ-E type).

1.1.3 Experimental reagents

Carboxymethylcellulose Sodium (CMC-Na), 3,5-Dinitrosalicylic Acid, Phenol, Citric Acid, Potassium Sodium Tartrate, Trisodium Citrate, NaCl, NaOH, ZnCl ₂ , HCl, CaCl ₂ , KCl , NiCl ₂ ·6H ₂ O, etc. were purchased from Sinopharm Chemical Reagent Co., Ltd. Congo red, peptone, yeast powder, agarose, etc. were purchased from Shanghai Sangon Bioengineering Co., Ltd.; bacterial genomic DNA extraction kit (DP302) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

1.1.4 Preparation of DNS reagent and culture medium

The reducing sugar detection reagent DNS is prepared as follows: Weigh 182g of potassium sodium tartrate with an electronic balance, add 450-650mL of distilled water into a beaker, stir continuously with a glass rod to fully dissolve it, and then add 3,5-Di Stir 6.3g of nitrosalicylic acid, 21g of NaOH and 5g of phenol until there is no particulate matter, cool to room temperature and set the volume to 1000mL, filter to remove bacteria and impurities, store in a brown bottle, and keep it away from light for a week before use . The enrichment and fermentation medium preparation of cellulase-producing strains is as follows: Accurately weigh 10 g of CMC-Na, 5.0 g of yeast powder, 10 g of sodium chloride, and 10 g of peptone into a beaker and add 400 mL of distilled water to stir until completely dissolved. Transfer it to an Erlenmeyer flask to 1000mL, sterilize at 121°C, 0.1MPa for 20min. Congo red solid selection medium is prepared as follows: accurately weigh 1.88g of CMC-Na, 10g of peptone, 0.5g of K ₂ HPO4, 5g of yeast powder, 0.25g of MgSO ₄ .7H ₂ O, 14g of agar, and 2.00g of gelatin into a beaker And add 400mL of distilled water to stir until completely dissolved, transfer to a Erlenmeyer flask to constant volume to 1000mL, 121°C, 0.1MPa, sterilize for 20min. At the same time, sterilize the prepared 1mg/mL Congo red dye solution at 0.1MPa at 115°C for 30min. Finally, cool to a certain temperature, add 5 mL of Congo red dye solution to the culture medium, mix well (do not generate air bubbles), pour it onto a plate, and store it in a 4°C refrigerator after the Congo red culture medium solidifies.

1.2 Experimental method

1.2.1 Preparation of samples for primary screening of strains

Use sterilized surgical scissors to randomly cut several pieces of tissue from the rumen of fresh sheep, and put them into the same sterile beaker; cut them into pieces with surgical scissors, put them into a sterile homogenizer, add appropriate amount of sterile water homogenate. Pour out the homogenate and let it stand for half an hour, then draw 1.0mL of the solution as the initial bacterial solution.

1.2.2 Isolation and purification of cellulase-producing strains

Take 0.5 mL from the above-mentioned original bacterial solution and transfer it to the enrichment medium (with sodium carboxymethyl cellulose as the sole carbon source), and enrich the culture in a constant temperature incubator at 37°C at 180r·min ⁻¹ . After culturing for 12 hours, absorb the enriched culture solution and dilute to 10 ^-4 , 10 ^-5 and 10 ^-6 , respectively spread on the Congo red screening medium, and incubate at 37°C for 2-3 days; at the same time, observe every 24 hours Whether a transparent hydrolysis circle is formed on the Congo red medium. After a stable hydrolysis zone is formed, measure the diameter of the colony (C) and the diameter of the hydrolysis zone (H) respectively, and calculate the H/C ratio; take the strain with a larger ratio for streak purification until a single colony is formed stably. Number the purified strains respectively and save them for future use.

1.2.3 Molecular identification of cellulase-producing strains

The strain HSU-6 (one of the strains with a large H/C ratio) was selected as the research material, and its colony characteristics were observed; then its genomic DNA was extracted with a genomic DNA extraction kit and its purity was tested. Synthetic bacterial universal primer 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and

1492R: 5′-TACGGCTACCTTGTTACGACTT-3′, and use this pair of primers to amplify the 16S rDNA sequence of strain HSU-6, and after verifying the purity, entrust Shanghai Sangon Bioengineering Co., Ltd. to perform sequencing. After obtaining the 16S rDNA sequence of the strain, the sequence was compared with the 16S ribosomal RNA sequences database (a sub-library of the NCBI database). Representative sequences with high sequence identity were selected from the comparison results, and the homology analysis and molecular phylogenetic tree were constructed with the software Clustal X 2.1 and MEGA 6.06 to determine the species of strain HSU-6.

1.2.4 Drawing of glucose standard curve

Prepare a 1 mg/mL standard glucose solution, and measure the absorbance at a wavelength of 540 nm after mixing it with the DNS solution, repeat 3 times, and draw the glucose standard curve after calculating the average value. The regression equation of the standard solution glucose concentration (Y) and absorbance value (Z) measured in this paper is Z=12.868Y-0.2372, R ² =0.9995; the linearity is good, and it can be used for subsequent enzyme activity quantitative experiments.

1.2.5 Preparation of crude enzyme solution

Take out the preserved HSU-6 strain from the -80°C refrigerator, streak it on the Congo red medium for recovery; then pick a single clone colony of strain HSU-6 from the recovered plate and transfer it to the fermentation medium for 12 hours ; Then inoculate in new fermentation medium according to 1.0% inoculum size, and place in a shaker at 28°C for 180r·min ^-1 fermentation for 3 days. The fermentation broth was centrifuged at 12 000r·min ⁻¹ in a refrigerated centrifuge at 4°C for 10 minutes, and the supernatant obtained by centrifugation was used as the crude enzyme solution.

1.2.6 Optimum reaction pH value of cellulase

Take 6 clean glass test tubes, add 2.0mL of 1% CMC-Na substrate prepared by buffer solutions with different pH values of 3, 4, 5, 6, 7 and 8 respectively, then add 1.0mL of crude enzyme solution and mix well,

Then placed in a constant temperature water bath at 40°C for 20 min. Add 2.0mL DNS reagent to the reaction solution, mix well and boil in boiling water for 10min; after cooling to room temperature, dilute to 10mL, and measure the absorbance value at 540nm, repeat 3 times. At the same time, take the inactivated crude enzyme solution as a control group, measure the absorbance value under the same conditions, and calculate the enzyme activity. Cellulase activity is defined as the amount of enzyme needed to hydrolyze CMC-Na substrate per milliliter of crude enzyme solution per hour to produce 1 μmol of glucose under certain conditions.

1.2.7 Optimum reaction temperature of cellulase

Under the optimal reaction pH value determined in 1.2.6, the enzyme activity of the cellulase at different reaction temperatures of 30, 40, 50, 60 and 70° C. was measured by the method of step 1.2.6.

1.2.8 Effect of fermentation time on enzyme activity

Referring to the method in step 1.2.5, take the fermented liquid at different fermentation times 2, 3, 4, 5, 6 and 7 days to prepare crude enzyme liquid, and determine the optimal reaction pH value in steps 1.2.6 and 1.2.7 and temperature, the enzyme activity of the fermentation broth was determined.

1.2.9 Effect of metal ions on enzyme activity

Add metal ions such as CaCl ₂ , HgCl ₂ , KCl, NiCl ₂ and CuCl ₂ at a final concentration of 1 mmol·L ^-1 for treatment, and refer to step 1.2.7 to measure the influence of different metal ions on the cellulase.

1.2.10 Cellulase tolerance to high temperature

Put the crude enzyme solution after 5 days of fermentation at 30, 40, 50, 60 and 70°C for 0.5, 1 and 2 hours, and then refer to the optimal reaction pH and temperature determined in step 1.2.7 to measure the fiber Enzyme activity of the enzyme under different temperature treatments. At the same time, the crude enzyme solution without heat-resistant treatment was used as a control group to compare the tolerance of the cellulase to different temperatures.

1.2.11 Cellulase tolerance to organic solvents

Add organic reagents such as methanol, isopropanol, ethanol, Triton X-100 and dimethyl sulfoxide at final concentrations of 1% and 15% (v/v) to the cellulase activity assay system, refer to step 1.2. Method 7 was used to measure its enzyme activity; at the same time, the cellulase treated with an equal amount of reaction buffer was used as a control group to compare the tolerance of the cellulase to different organic reagents.

2. Results and Analysis

2.1 Molecular identification of cellulase-producing strain HSU-6

A strain numbered HSU-6 was screened from sheep rumen using Congo red screening medium, which can produce cellulase (Figure 1). As can be seen from the results in Figure 1, strain HSU-6 can produce stable and obvious hydrolysis transparent circles on the Congo red solid medium with CMC-Na as the only carbon source; the diameter (H) of the transparent circles produced by it is 44.5mm, and the bacterial strain The colony diameter (C) of HSU-6 was 8.5mm, and the ratio H/C was about 5.2; the strain was found to be a Gram-positive bacillus by Gram staining.

The 16S rDNA sequence on the genome of the extracted strain HSU-6 was amplified and sequenced with the general primers 27F and 1492R for bacterial identification, and the sequenced results were compared and analyzed in the Nucleotide BLAST tool of the NCBI database, and selected from the comparison results The molecular phylogenetic tree of the strain HSU-6 was constructed with sequences with more than 99% sequence identity (Fig. 2). From the results of the constructed phylogenetic tree, the strain HSU-6 is located on the same branch of the phylogenetic tree as Bacillus cereus strain NBRC 15305 and other Bacillus cereus strains; based on the results of colony characteristics, Gram staining and molecular phylogenetic tree analysis, the strain HSU -6 identified as Bacillus cereus strain HSU-6 (Bacillus cereus strain HSU-6).

2.2 Effect of pH value on cellulase enzyme activity

In order to explore the optimal reaction pH value of the cellulase, the enzyme activity of the cellulase was tested with buffers of different pH values ( FIG. 3 ). It can be seen from the experimental results that the cellulase produced by strain HSU-6 has an enzyme activity of about 3.3U·mL ^-1 at pH 3.0; as the pH rises to 4.0, its enzyme activity also rapidly increases to 6.3U·mL -1 . mL ^-1 ; when the pH value continued to rise, the enzyme activity did not continue to increase, but decreased slowly; when the pH value rose to 7.0 or 8.0, the enzyme activity decreased to 4.1U·mL ^-1 and below. The above results indicated that the optimum reaction pH value of the cellulase was 4.0.

2.3 Effect of temperature on cellulase enzyme activity

When the optimal reaction pH value determined above was 4.0, the enzyme activity of the cellulase produced by the strain HSU-6 at a temperature of 30-70° C. was measured ( FIG. 4 ). It can be seen from the experimental results that when the reaction temperature is 30-40°C, the activity of the cellulase increases with the increase of temperature, and reaches the maximum value of 7.5U·mL ^-1 at 40°C; when the reaction temperature continues to rise, its The enzyme activity did not continue to increase but decreased, and reached the lowest value of 4.8U·mL ^-1 at 70°C. The experimental results show that the optimum reaction temperature of the cellulase is 40°C.

2.4 Effect of fermentation time on cellulase enzyme activity

Under the optimal reaction pH value and temperature determined above, the enzyme activity of cellulase produced at different fermentation times was measured ( FIG. 5 ). According to the experimental results, the cellulase activity in the fermentation broth can reach 8.3 U·mL ^-1 in 2 days of fermentation; with the increase of fermentation time, the cellulase activity in the fermentation broth also increases slowly, and in the fermentation broth The maximum enzyme activity was 9.1U·mL ^-1 at 5 days; when the fermentation time continued to increase to 6 and 7 days, the cellulase activity in the fermentation broth decreased to 7.5U·mL ^-1 and remained relatively stable.

2.5 Effect of metal ions on cellulase enzyme activity

When the optimal reaction pH value was 4.0 and the optimal reaction temperature was 40° C., the effects of different metal ions on the cellulase enzyme activity were determined ( FIG. 6 ). From the measured experimental data, Fe ³⁺ ions can slightly increase the enzyme activity of the cellulase; K ⁺ and Ca ²⁺ ions can slightly reduce the enzyme activity of the cellulase to 93.9% and 92.5%; Cu ²⁺ , Ni ²⁺ and Hg ²⁺ ions can significantly reduce the enzyme activity to 80.2%, 79.8% and 76.1%. The experimental results show that the cellulase has a certain tolerance to metal ions.

2.6 Cellulase tolerance to high temperature

The crude cellulase enzyme solution was placed at a temperature of 30-70° C. for different periods of time, and its tolerance to different temperatures was determined ( FIG. 7 ). According to the experimental results, in the temperature range of 30-50°C, treatment for 1 or 2 hours is beneficial to maintain the enzyme activity of the cellulase; while at 60 or 70°C, treatment for 1 or 2 hours will rapidly reduce the enzyme activity, And make it down to about 10% of normal enzyme activity. Therefore, the cellulase can withstand the temperature of 40-50°C, and is a kind of heat-resistant cellulase.

2.7 Cellulase tolerance to organic solvents

The cellulase activity determined under the optimal pH and temperature conditions is 100%, and the effect of adding 1% and 15% (v/v) organic solvents at two concentrations on the cellulase enzyme activity is compared (Figure 8) . Know from Fig. 8 result, when organic solvent concentration is 1%, methanol, isopropanol and dimethyl sulfoxide (DMSO) have little effect on the enzyme activity of this cellulase, and ethanol can slightly reduce enzyme activity, but Triton X-100 will slightly increase the enzyme activity. When the concentration of organic solvent increased to 15%, methanol, ethanol, isopropanol and dimethyl sulfoxide would slightly reduce the enzyme activity of the cellulase, but the enzyme activity was above 85%; while Triton X-100 would The enzyme activity was significantly increased, which indicated that the cellulase had a certain tolerance to organic solvents.

Cellulose is a kind of renewable biomass energy with rich sources, which can be used in energy, feed and other industries, and has great development value; and cellulase plays a pivotal role in the development and utilization of cellulose, so it can be obtained from the intestinal tract of plants and animals. Screening strains with high cellulase production, rumen of ruminants, etc. has always been a hot spot in the field of microbiology.

There are microbial communities such as protozoa, bacteria and archaea in the rumen of ruminants, which play an important role in the process of cellulose decomposition and are an important source for screening high-producing cellulase strains. The invention uses the rumen of sheep raised locally in Huangshan as materials, and screens out a cellulase-producing strain HSU-6, which is identified as bacillus cereus by molecular identification. Subsequent measurement of enzyme activity parameters found that the optimal pH value of cellulase produced by strain HSU-6 is 4.0, and maintains a relatively high enzyme activity in the range of pH 4.0-6.0, which is an acid cellulase; acid fiber Sulfase has important application value in animal husbandry, energy and environmental protection industries, so the cellulase screened by the present invention has important application value. At the same time, the present invention also found that the cellulase can withstand the temperature of 40-50°C, and has a certain resistance to metal ions Fe ³⁺ , Ca ²⁺ , Cu ²⁺ , Ni ²⁺ and Hg ²⁺ and organic solvents. These characteristics show that the cellulase is particularly suitable for use in industrial production, so the strain HSU-6 is worth developing and utilizing. In the follow-up, modern molecular biology techniques can be used to study and analyze the coding gene and protein amino acid sequence of the cellulase, laying the foundation for the construction of the cellulase high-yielding Escherichia coli strain.

The present invention screens a heat-resistant acid cellulose-producing B. cereus strain HSU-6 from the rumen of sheep. The optimum pH value and temperature for producing cellulase are 4.0 and 40°C respectively, and it is a heat-resistant acidic fiber Sulfase; and also has a certain tolerance to organic solvents. The enzyme activity of the crude cellulase solution produced by the strain can reach 9.1U·mL ^-1 under optimal conditions, which can provide new cellulase resources for in vitro degradation of cellulose.

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

>HSU-6

TGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACG GCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCA CCTTGACGGTACCTAACCAGAAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGTTTCTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGA AATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTC AAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGC CATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTA GTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACC.

sequence listing

<110> Huangshan College

<120> A thermostable acid cellulase producing strain HSU-6 and its application

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<213> Artificial Sequence (Artificial Sequence)

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tggattaaga gcttgctctt atgaagttag cggcggacgg gtgagtaaca cgtgggtaac 60

ctgcccataa gactgggata actccgggaa accggggcta ataccggata aattttgaa 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 (5)

1. The application of a heat-resistant acid cellulase strain in biological fermentation or in the production of decontamination products, characterized in that the strain is Bacillus cereus ( Bacillus cereus ) HSU-6, released on May 24, 2021 It was preserved in the China Center for Type Culture Collection (CCTCC), and the strain preservation number is: CCTCC NO: M 2021599. 2. The application according to claim 1, characterized in that, the bacterial strain according to claim 1 is cultivated and fermented, and cellulase is extracted in the fermented culture fluid. 3. The application according to claim 1, characterized in that the application temperature is 40-50°C, and the pH value is 4-6. 4. The application according to claim 1, characterized in that the reaction system during decontamination includes one or more of Fe ³⁺ , K ⁺ , Ca ²⁺ , Cu ^{2 +} , Ni ²⁺ or Hg ²⁺ kind. 5. application according to claim 1, is characterized in that, the reaction system during decontamination comprises one or more in methanol, ethanol, Virahol, dimethyl sulfoxide and Triton X-100.

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