CN114149945B

CN114149945B - High-yield cellulase bacteria and application thereof

Info

Publication number: CN114149945B
Application number: CN202111449312.7A
Authority: CN
Inventors: 金龙; 卓也; 崔成妲
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-05-06
Anticipated expiration: 2041-11-30
Also published as: CN114149945A

Abstract

The invention discloses a high-yield cellulase bacterium and application thereof, belonging to the technical field of microorganisms. The high-yield cellulase bacterium Xanthomonas sp.CL-3 is preserved in China center for type culture Collection with the preservation number of CCTCC M2021680, the preservation date of 2021, 6 months and 7 days, and the preservation address of Wuhan university in Wuhan, China. The high-yield cellulase bacteria Xanthomonas sp.CL-3 have stronger capability of producing cellulase at 30 ℃, and the produced cellulase comprises endo-beta-1, 4 glucanase, exo-beta-1, 4 glucanase and beta-glucosidase. The endo beta-1, 4 glucanase produced by the novel bacterium is obviously higher than exo beta-1, 4 glucanase and beta-glucosidase, so the novel bacterium has a good application prospect.

Description

High-yield cellulase bacteria and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a high-yield cellulase bacterium and application thereof.

Background

With the progress of industrialization, the increasing shortage of fossil energy has become a global problem, and the search for new green renewable energy is a new problem and direction we must face. Among them, the development and utilization of biomass resources are a research hotspot in recent years. Straw is taken as an important renewable biomass resource, the main component of the straw is cellulose, and the cellulose is the most abundant and renewable organic molecule on the earth. Therefore, based on the contradiction between agricultural production and environmental protection, it is very important to research the degradation and utilization of straws, and research on the degradation mechanism of cellulose is a great important factor for solving the problem of straws.

Cellulose molecules are basic units for forming cellulose, the cellulose molecules are filamentous, and the molecules are connected into cellulose micelles in a hydrogen bond mode, so that the physical and chemical properties are stable. The natural hydrolysis product of cellulose is the second fiber pool, and the final hydrolysis product is glucose. In addition, in most plant cells, cellulose is embedded with hemicellulose and lignin to form a network structure, and lignin is accumulated around cellulose bundles to form a protective layer, making it difficult to hydrolyze.

The cellulose is degraded mainly by the action of cellulase, and the main purpose of degrading the cellulose is to convert the cellulose into simple small molecules for biological reuse. The conventional methods for degrading cellulose mainly comprise: acid degradation, alkali degradation, thermal degradation, oxidative degradation, and the like. However, the acid, alkali and hydrolysate contain more toxic substances, and the degradation of cellulose can bring about certain influence on the environment. Compared with the traditional method, the microbial degradation has the advantages of mild condition, low cost, greenness, no pollution and the like.

Disclosure of Invention

In view of the above problems in the prior art, the technical problem to be solved by the present invention is to provide a high-yield cellulase bacterium Xanthomonas sp.CL-3. The invention also aims to provide application of the high-yield cellulase bacterium Xanthomonas sp.CL-3 in preparation of cellulase or degradation of cellulose. The invention finally aims to provide a microbial inoculum or cellulase prepared from the high-yield cellulase bacterium Xanthomonas sp.CL-3.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a high-yield cellulase bacterium Xanthomonas sp.CL-3 is preserved in China center for type culture Collection with the preservation number of CCTCC M2021680, the preservation date of 2021, 6 months and 7 days, and the preservation address of Wuhan university, Wuhan, China.

The application of the high-yield cellulase bacterium Xanthomonas sp.CL-3 in degrading cellulose.

The application of the high-yield cellulase bacterium Xanthomonas sp.CL-3 in producing cellulase.

Further, the cellulase is endo-beta-1, 4-glucanase, exo-beta-1, 4-glucanase and/or beta-glucosidase.

The microbial inoculum containing the high-yield cellulase bacteria Xanthomonas sp.CL-3.

Further, the microbial inoculum is fermentation liquor containing high-yield cellulase bacteria Xanthomonas sp.CL-3.

Further, the high-yield cellulase bacteria Xanthomonas sp.CL-3 are inoculated in a liquid culture medium for fermentation culture, and the microbial inoculum is obtained.

Further, the liquid medium comprises the following components: sodium carboxymethylcellulose, peptone, yeast extract powder, ammonium sulfate, potassium dihydrogen phosphate and magnesium sulfate heptahydrate.

The cellulase is produced by fermenting the high-yield cellulase bacterium Xanthomonas sp.CL-3.

The cellulase is applied to degrading cellulose.

Compared with the prior art, the invention has the beneficial effects that:

the high-yield cellulase bacteria Xanthomonas sp.CL-3 provided by the invention are separated from the surface layer humus of the forest park soil of east-Taiwan yellow sea of Jiangsu, have strong cellulase production capability at about 30 ℃, and the produced endo beta-1, 4 glucanase, exo beta-1, 4 glucanase and beta-glucosidase have high activity, thereby providing an effective means for treating cellulose and having good industrial application prospect.

Drawings

FIG. 1 is a flow chart of the isolation, screening and identification of bacterial CL-3;

FIG. 2 is a colony morphology of bacterium CL-3 (30 ℃, 3 days);

FIG. 3 is a 16S rDNA phylogenetic tree constructed using the adjacency approach;

FIG. 4 is a schematic view showing the hydrolysis effect of a CL-3 Congo red sodium carboxymethyl cellulose flat plate;

FIG. 5 is a graph showing the results of determination of cellulase production by CL-3.

Detailed Description

The invention is further described with reference to specific examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Media used in the examples:

R2A medium, comprising the following components in concentrations: 0.5g of yeast extract powder, 0.5g of peptone, 0.5g of casein hydrolysate, 0.5g of glucose, 0.5g of soluble starch, 0.3g of dipotassium phosphate, 0.024g of anhydrous magnesium sulfate, 0.3g of sodium pyruvate and 15g of agar powder, adding 1000mL of distilled water to adjust the pH of the liquid to 7.0-7.2, and sterilizing the culture medium for 20min at 121 ℃ by high-pressure steam for later use.

The CMC-Na solid medium comprises the following components in concentration: 10g of sodium carboxymethylcellulose, 3g of peptone, 0.2g of yeast extract powder, 2g of ammonium sulfate, 4g of monopotassium phosphate, 0.3g of magnesium sulfate heptahydrate and 15g of agar powder, adding 1000mL of distilled water to adjust the pH of the liquid to 7.0-7.2, and sterilizing the culture medium at 121 ℃ for 20min by high-pressure steam for later use.

CMC-Na liquid medium: 10g of sodium carboxymethylcellulose, 3g of peptone, 0.2g of yeast extract powder, 2g of ammonium sulfate, 4g of monopotassium phosphate and 0.3g of magnesium sulfate heptahydrate, adding 1000mL of distilled water to adjust the pH of the liquid to 7.0-7.2, and performing high-pressure steam sterilization on the culture medium at 121 ℃ for 20min for later use.

Example 1:

FIG. 1 shows a flow chart of the production of cellulose-degrading bacteria CL-3 of the present invention. The preparation method of the cellulose degrading bacteria CL-3 provided by the embodiment of the invention comprises the following steps: collecting humus on the surface layer of soil in the forest park of east Tai Huanghai of Jiangsu, putting the humus into a centrifugal tube filled with sterile water, fully vibrating and uniformly mixing

Diluting the prepared soil stock solution to 10 degrees by adopting a gradient dilution method^-6. And then absorbing part of the diluted soil diluent to a CMC-Na culture medium plate for coating treatment, uniformly coating, then putting the plate into an incubator at 30 ℃ for culture, carrying out Congo red dyeing after obvious bacteria grow on the plate after 2-3 days, selecting the strain with the largest transparent circle, carrying out separation, purification, culture and preservation, wherein the strain is identified as Xanthomonas sp, and is named as CL-3.

1. Ecological identification

The strain CL-3 provided by the embodiment of the invention is inoculated on an R2A solid medium, then the plate is inverted and cultured for 2-3 days under the condition that the temperature is 30 ℃, and the growth condition of colonies on the plate is observed and recorded. The colonial phenotype of the strain CL-3 provided by the examples of the invention is shown in FIG. 2. FIG. 2 shows that the colonies are yellow, round, convex and opaque, the surfaces of the bacterial cells are smooth, the shapes of the bacterial cells are rod-shaped, the bacterial cells do not move, and the bacterial cells are gram-negative through gram staining results. The strain CL-3 can grow under the condition of 4-42 ℃, wherein the growth vigor is weaker under the conditions of 4 ℃ and 42 ℃, and the optimal growth temperature is 15-30 ℃. The optimum growth pH is 6-7, and the growth can not be carried out under the conditions of pH 5 and pH 8. Under the condition of adding 1% -4% NaCl, the strain can grow normally, but the growth vigor is weaker under the condition of 5% NaCl. Meanwhile, the growth conditions of the strain CL-3 on different culture media are also detected, the strain can normally grow on a TSA culture medium (soybean casein agar culture medium), a NA culture medium (nutrient agar culture medium) and an LB culture medium, and different nutrient components can be utilized. The strain CL-3 is negative to the action of oxidase and weak positive to the action of catalase.

2. 16S sequence analysis

In the embodiment of the invention, the DNA in the strain is extracted by adopting a traditional method. Wherein, the 30 μ L PCR reaction system comprises 1 μ L template primer, 1 μ L template DNA, 15 μ L TAq enzyme premix solution and 12 μ L sterile water. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 7 min; denaturation at 94 deg.C for 1 min; annealing at 55 deg.C for 1 min; extension at 72 ℃ for 1.5min, 35 cycles, storage at 4 ℃.

The PCR product was sequenced by Oncorks Ltd, and the sequencing result was shown by the nucleotide sequence in the attached table (SEQ ID NO. 1). BLAST similarity comparison of the obtained sequence in GenBank is carried out to obtain a related sequence with higher similarity with the strain, MEGA7.0 software is used for constructing a phylogenetic tree of the strain, and a phylogenetic tree diagram of the strain is shown in figure 3. Combining the above ecological observations with the results of 16S sequence analysis, it could be determined that the present strain CL-3 is a member of Xanthomonas, which was named CL-3. The strain is preserved in China center for type culture Collection (CCTCC for short, preservation Unit Address: Wuhan, Wuhan university, China; postal code: 430072) at 6 months and 7 days in 2021, and is separated and named: CL-3) with the preservation number of CCTCC M2021680.

Example 2: determination of cellulose degradation ability of CL-3

The embodiment of the invention provides a schematic diagram of the hydrolysis effect of a cellulose degrading strain CL-3 on a congo red sodium carboxymethyl cellulose flat plate, as shown in FIG. 4. As can be seen from FIG. 4, after 2 days at 30 ℃ the CMC-Na plate inoculated with CL-3 had a clear ring appearing on the Congo red stain, and the value of the clear ring diameter/colony diameter was measured to be 12. Therefore, it can be seen that the efficiency of producing cellulase is high according to the size and speed of growing colonies and the size of transparent circles.

Example 3: determination of cellulase production ability of CL-3

The embodiment of the invention performs the determination of the cellulase production capacity of CL-3, and the determination comprises the following contents:

1. preparation of crude enzyme solution

The CL-3 strain provided by the embodiment 1 of the invention is inoculated into a CMC-Na liquid culture medium, cultured for 3d at 30 ℃ and 150r/min, and centrifuged to filter thalli and take supernatant fluid, namely crude enzyme liquid.

2. Preparation of glucose Standard Curve

0.00mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL, 1.00mL (0.2 mL per tube) of 1mg/mL glucose standard solution was added to a graduated tube, and then 2mL of a DNS reagent (pH 10) borax-sodium hydroxide buffer solution was added thereto to make up to 1mL, followed by mixing in a boiling water bath for 30 min. Taking out, cooling to room temperature, and measuring OD with spectrophotometer₅₃₀. The optical density value is used as the abscissa and the glucose content (mg) is used as the ordinate to make a glucose standard curve.

3. Determination of enzyme Activity

(1) Determination of endo-beta-1, 4-glucanase activity

Adding sodium carboxymethylcellulose into sodium citrate buffer solution with pH of 4.5 to prepare 1% substrate solution, taking 2mL of the substrate solution, adding 1mL of the prepared crude enzyme solution, reacting in a water bath kettle at 50 ℃ for 30min, taking 1mL of reaction solution after reaction is stopped, adding 2mL of DNS reagent and 2mL of borax-sodium hydroxide buffer solution with pH of 10, shaking all the tubes uniformly, carrying out boiling water bath for 30min, taking out, cooling to room temperature, and measuring the OD value of the solution at the wavelength of 530 nm. The blank group was not subjected to a 50 ℃ water bath, and DNS was added first to inactivate the enzyme activity, and the others were the same as those in the test group. The enzyme activity unit is the generation of 1 mu mol of glucose per minute.

(2) Determination of exo-beta-1, 4-glucanase activity

Adding microcrystalline cellulose into sodium citrate buffer solution with pH 4.5 to prepare 1% substrate solution, taking 2mL of the substrate solution, adding 1mL of the prepared crude enzyme solution, reacting in a water bath kettle at 50 ℃ for 30min, taking 1mL of reaction solution after reaction is stopped, adding 2mL of DNS reagent and 2mL of borax-sodium hydroxide buffer solution with pH 10, shaking all the tubes uniformly, carrying out boiling water bath for 30min, taking out, cooling to room temperature, and measuring the OD value of the solution at the wavelength of 530 nm. The blank group was not subjected to a 50 ℃ water bath, and DNS was added first to inactivate the enzyme activity, and the others were the same as those in the test group. The enzyme activity unit is the generation of 1 mu mol of glucose per minute.

(3) Determination of beta-glucosidase Activity

Adding saligenin into citric acid buffer solution with pH 4.5 to prepare 1% substrate solution, taking 2mL of the substrate solution, adding 1mL of the prepared crude enzyme solution, reacting in a water bath kettle at 50 ℃ for 30min, taking 1mL of reaction solution after stopping the reaction, adding 2mL of DNS reagent and 2mL of borax-sodium hydroxide buffer solution with pH 10, shaking all tubes uniformly, carrying out boiling water bath for 30min, taking out, cooling to room temperature, and measuring the OD value at the wavelength of 530 nm. The blank group was not subjected to a 50 ℃ water bath, and DNS was added first to inactivate the enzyme activity, and the others were the same as those in the test group. The enzyme activity unit is the generation of 1 mu mol of glucose per minute.

(4) Filter paper enzyme activity assay

Cutting 1g of filter paper, adding into 2mL of citric acid buffer solution with pH 4.5, adding 1mL of the crude enzyme solution, reacting in a water bath kettle at 50 ℃ for 30min, taking 1mL of the reaction solution after the reaction is stopped, adding 2mL of DNS reagent and 2mL of borax-sodium hydroxide buffer solution with pH 10, shaking the tubes, carrying out boiling water bath for 30min, taking out, cooling to room temperature, and measuring the OD value at the wavelength of 530 nm. The blank group was not subjected to a 50 ℃ water bath, and DNS was added first to inactivate the enzyme activity, and the others were the same as those in the test group. The enzyme activity unit is the generation of 1 mu mol of glucose per minute.

The invention provides a cellulase production determination result chart of a cellulose degrading strain CL-3, which comprises endo-beta-1, 4 glucanase, exo-beta-1, 4 glucanase, beta-glucosidase and filter paper enzyme activity. The results are shown in FIG. 5.

As can be seen from FIG. 5, the strain CL-3 has stronger enzyme production capability at 30 ℃, and after 3 days of fermentation culture, the endo-beta-1, 4-glucanase, the exo-beta-1, 4-glucanase, the beta-glucosidase and the filter paper enzyme all have higher enzyme activity which are 0.3133U/mL, 0.1104U/mL, 0.0278U/mL and 0.0183U/mL respectively, so that the strain CL-3 has good industrial application prospect.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Nanjing university of forestry

<120> high-yield cellulase bacteria and application thereof

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<170> SIPOSequenceListing 1.0

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tgggtggcga gtggcggacg ggtgaggaat acatcggaat ctactctttc gtgggggata 60

acgtagggaa acttacgcta ataccgcata cgacctacgg gtgaaagcgg aggaccttcg 120

ggcttcgcgc gattgaatga gccgatgtcg gattagctag ttggcggggt aaaggcccac 180

caaggcgacg atccgtagct ggtctgagag gatgatcagc cacactggaa ctgagacacg 240

gtccagactc ctacgggagg cagcagtggg gaatattgga caatgggcgc aagcctgatc 300

cagccatgcc gcgtgggtga agaaggcctt cgggttgtaa agcccttttg ttgggaaaga 360

aaagcagtcg gttaataccc gattgttctg acggtaccca aagaataagc accggctaac 420

ttcgtgccag cagccgcggt aatacgaagg gtgcaagcgt tactcggaat tactgggcgt 480

aaagcgtgcg taggtggtgg tttaagtctg ttgtgaaagc cctgggctca acctgggaat 540

tgcagtggat actgggtcac tagagtgtgg tagagggtag cggaattccc ggtgtagcag 600

tgaaatgcgt agagatcggg aggaacatcc gtggcgaagg cggctacctg gaccaacact 660

gacactgagg cacgaaagcg tggggagcaa acaggattag atacccctgg tagtccacgc 720

cctaaacgat gcgaactgga tgttgggtgc aatttggcac gcagtatcga agctaacgcg 780

ttaagttcgc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg 840

cccgcacaag cggtggagta tgtggtttaa ttcgatgcaa cgcgaagaac cttacctggt 900

cttgacatcc acggaacttt ccagagatgg attggtgcct tcgggaaccg tgagacaggt 960

gctgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1020

aacccttgtc cttagttgcc agcacgtaat ggtgggaact ctaaggagac cgccggtgac 1080

aaaccggagg aaggtgggga tgacgtcaag tcatcatggc ccttacgacc agggctacac 1140

acgtactaca atggtaggga cagagggctg caaacccgcg agggtaagcc aatcccagaa 1200

accctatctc agtccggatt ggagtctgca actcgactcc atgaagtcgg aatcgctagt 1260

aatcgcagat cagcattgct gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1320

acaccatggg agtttgttgc accagaagca ggtagcttaa ccttcgggag ggcgc 1375

Claims

1. A high-yield cellulase bacterium Xanthomonas sp.CL-3 is preserved in China center for type culture Collection with the preservation number of CCTCC M2021680, the preservation date of 2021, 6 months and 7 days, and the preservation address of Wuhan university, Wuhan, China.

2. Use of the high-yielding cellulase bacterium Xanthomonas sp.CL-3 according to claim 1 for degrading cellulose.

3. Use of the high-yielding cellulase bacterium Xanthomonas sp.CL-3 according to claim 1 for producing cellulase.

4. The use according to claim 3, wherein the cellulase enzymes comprise endo-beta-1, 4-glucanase, exo-beta-1, 4-glucanase and beta-glucosidase.

5. A microbial agent comprising the high-yielding cellulase bacterium Xanthomonas sp.cl-3 according to claim 1.

6. The microbial inoculum containing the high-yield cellulase bacteria Xanthomonas sp.CL-3 as claimed in claim 5, which is fermentation broth containing the high-yield cellulase bacteria Xanthomonas sp.CL-3.

7. The microbial inoculum containing the high-yield cellulase bacteria Xanthomonas sp.CL-3 as claimed in claim 6, which is characterized in that the microbial inoculum is obtained by inoculating the high-yield cellulase bacteria Xanthomonas sp.CL-3 into a liquid culture medium for fermentation culture.

8. The microbial inoculum containing the high-yielding cellulase bacterium Xanthomonas sp.CL-3 as claimed in claim 7, wherein the liquid culture medium comprises the following components: sodium carboxymethylcellulose, peptone, yeast extract powder, ammonium sulfate, potassium dihydrogen phosphate and magnesium sulfate heptahydrate.