CN114774330B

CN114774330B - Novel species of cellulolytic female phytes and application thereof

Info

Publication number: CN114774330B
Application number: CN202210585141.9A
Authority: CN
Inventors: 张玉琴; 韩雪飞; 邓阳; 姜竹鸣; 苏静; 余利岩; 赵莉莉
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2022-05-27
Filing date: 2022-05-27
Publication date: 2023-07-21
Anticipated expiration: 2042-05-27
Also published as: CN114774330A

Abstract

The invention discloses a new species for understanding cellulose female phyte and application thereof. The invention provides an IMB220419 for understanding cellulose female plant bacteria (Herbiconiux cellulolyticus), which has a registration number of CGMCC No.24729 in the China general microbiological culture Collection center. The cellulose-degrading female phyta (Herbiconiux cellulolyticus) IMB220419 provided by the invention is a new species of the genus Phytophyta, has cellulose degradation capability, and has wide application prospects in cellulose degradation. Can be used for sewage treatment, environmental treatment, soil improvement, bacterial fertilizer preparation production and the like.

Description

Novel species of cellulolytic female phytes and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to a new species of cellulolytic female phytbacteria and application thereof.

Background

The genus estrus (Herbiconiux) belongs to the family Microbacteriaceae (Park, y.h., suzuki, k.i., yim, d.g., lee, k.c., kim, e., yoon, j.s., kim, s.j., kho, y.h., goodfellow, M.&Komagata, K. (1993) Suprageneric classification of peptidoglycan group B actinomycetes by nucleotide sequencing of 5S ribosomal RNA.Antonie Van Leeuwenhoek 64,307-313), the model species of the genus Panax (Herbiconiux ginsengi) was reclassified from Leifsonia ginsengi (Behrendt, U.S. Schumann, P., hamada, M., suzuki, K.,C.&ulrich, a. (2011) Reclassification of Leifsonia ginsengi (Qiu et al 2007) as Herbiconiux ginsengi gen.nov., comb.nov.and description of Herbiconiux solani sp.nov., an actinobacterium associated with the phyllosphere of Solanum tuberosum l.int J Syst Evol Microbiol, 1039-1047.). To date, the genus estrus houses a total of 4 effective descriptive species: herbiconiumginsenginegi, herbiconiux solani (Behrendt, u., schumann, p., hamada, m., suzuki, k., j/f>C.&Ulrich,A.(2011).Reclassification of Leifsonia ginsengi(Qiu etal.2007)as Herbiconiux ginsengi gen.nov.,comb.nov.and description of Herbiconiux solani sp.nov.,an actinobacterium associated with the phyllosphere of Solanum tuberosum L.Int J Syst EvolMicrobiol 61,1039-1047.)，Herbiconiux flava(Hamada,M.,Komukai,C.,Tamura,T.,Evtushenko,L.I.,Vinokurova,N.G.&Suzuki, k (2012), description of Herbiconiux flava sp.nov.and emended description of the genus herbiconiux.int J Syst Evol Microbiol, 795-799) and Herbiconiux moechotypicola (Kim, b.c., park, d.s., kim, h, oh, h.w., lee, k.h., shin, K.S.&Bae, k.s. (2012) Herbiconiux moechotypicola sp.nov., a xylanolytic bacterium isolated from the gut of hairy long-horned toad beetles, moechotypa diphysis (passee) Int Syst Evol Microbiol 62,90-95), isolated from plant and animal bodies, respectively.

The female plant bacteria strain does not generate hypha, cells are in a short rod shape, cell walls contain L-/D-Dab, gly, ala and threo-3-hydroxyglutamic acid, the female plant bacteria strain belongs to B2 gamma cell walls, main polar lipid components comprise Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and Glycolipid (GL), and the dominant respiratory quinone is MK-11 and also contains a small amount of MK-10; the dominant fatty acid contains ai-C _15:0 。

Cellulose is the most abundant renewable carbohydrate resource in nature, its structure is very complex, and microorganisms play a key role in the biomass conversion process. Microbial cellulases are a key enzyme system for cellulose degradation and conversion. Cellulase-producing bacteria are capable of directly degrading modified cellulose CMC-Na, and therefore, strain having cellulose degrading ability can be rapidly screened using CMC-Na as a substrate (Zhou, x., chen, H. & Li, z. (2004) & CMCase activity assay as a method for cellulase adsorption analysis.enzyme Microbiol Technol,35, 455-459.).

Disclosure of Invention

The invention aims to provide a novel species of cellulolytic female phyta and application thereof.

In a first aspect, the invention claims a new species of the genus Phytophyton.

The new species of the female phytoplanugo claimed by the invention is cellulose-decomposing female phytoplanugo (Herbiconiux cellulolyticus) IMB220419, and the registration number of the female phytoplankton in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No.24729.

The female cellulolytic bacteria (Herbiconiux cellulolyticus) IMB220419 are gram-positive bacteria and aerobic. After the strain is cultured on the R2A culture medium for 72 hours at the temperature of 28 ℃, wet, smooth and milky colonies can be formed. The strain has a growth tolerance range of 4-37 ℃, 0-5% NaCl and pH 6.0-11.0, and the optimum growth conditions are 28 ℃, 0-1% NaCl and pH 8.0-9.0. The strain has positive gelatin liquefaction reaction, positive cellulose hydrolysis and negative nitrate reduction reaction. The 16S rRNA sequence of the strain is shown as SEQ ID No. 1.

In a second aspect, the invention claims a culture.

The culture claimed in the present invention is the culture of the above-mentioned female cellulolytic bacteria (Herbiconiux cellulolyticus) IMB220419 of the first aspect, and is the substance obtained by culturing the female cellulolytic bacteria (Herbiconiux cellulolyticus) IMB220419 in a bacterial culture medium (all substances in a culture container).

In the above cultures, the substances include the metabolites of the Deuteromycetes (Herbiconiux cellulolyticus) IMB220419 (the cells themselves) and the Deuteromycetes (Herbiconiux cellulolyticus) IMB220419.

In the above culture, the bacterial culture medium may be a solid culture medium or a liquid culture medium.

The term "culture" refers to a collective term for a liquid or solid medium in which a population of microorganisms has grown after artificial inoculation and cultivation. I.e. the product obtained by growing and/or amplifying the microorganism, which may be a biologically pure culture of the microorganism, or may contain a certain amount of medium, metabolites or other components produced during the culture. The term "culture" also includes subcultures obtained by passaging microorganisms, which may be a culture of a certain generation or a mixture of several generations.

In a specific embodiment of the invention, the bacterial culture medium is specifically an R2A culture medium.

In a third aspect, the invention claims a metabolite.

The claimed metabolites are those of the aforementioned female cellulolytic bacteria (Herbiconiux cellulolyticus) IMB220419.

The term "metabolite" refers to a primary metabolite and/or a secondary metabolite produced during metabolism of a microorganism. Primary metabolism refers to a process in which microorganisms absorb various nutrients from the outside and produce substances and energy that maintain vital activities through catabolism and anabolism. The primary metabolite is primary metabolite such as monosaccharide or monosaccharide derivative, nucleotide, vitamin, amino acid, fatty acid, and various macromolecular polymers composed of the same, such as protein, nucleic acid, polysaccharide, and lipid. Secondary metabolism refers to the process of synthesizing substances which have no definite function on the life activities of microorganisms by taking primary metabolites as precursors in a certain growth period of microorganisms. The secondary metabolite is the secondary metabolite, and most of the secondary metabolites are compounds with relatively complex molecular structures. Depending on their action, they can be classified into antibiotics, hormones, alkaloids, toxins, etc.

In a fourth aspect, the invention claims a microbial agent.

The claimed microbial agents contain an IMB220419 of a female cellulolytic bacterium (Herbiconiux cellulolyticus) as described in the first aspect above, a culture as described in the second aspect above and/or a metabolite as described in the third aspect above.

The microbial inoculum is a microbial inoculum for hydrolyzing cellulose.

In the microbial inoculum, the microbial inoculum contains a carrier in addition to the active ingredient. The carrier may be a carrier commonly used in the pesticide arts and which is biologically inert. The carrier may be a solid carrier or a liquid carrier; the solid carrier can be mineral material, plant material or high molecular compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of corn flour, soy flour and starch; the polymer compound may be polyvinyl alcohol and/or polyglycol; the liquid carrier may be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane and/or dodecane.

Among the above-mentioned bacterial agents, the formulation of the bacterial agent can be various formulations, such as liquid, emulsion, suspending agent, powder, granule, wettable powder or water dispersible granule.

Surfactants (such as Tween 20, tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added into the microbial inoculum according to the need.

In a fifth aspect, the invention claims the use of an estrus cellulolytic bacterium (Herbiconiux cellulolyticus) IMB220419 as described in the first aspect above or a culture as described in the second aspect above or a metabolite as described in the third aspect above or a microbial agent as described in the fourth aspect above in any of the following:

(A1) Hydrolyzing cellulose;

(A2) Preparing a product for hydrolyzing cellulose;

(A3) Preparing cellulase;

(A4) Preparing a product with cellulase activity.

In a sixth aspect, the invention claims the use of a female-phytoplankton cellulolytic (Herbiconiux cellulolyticus) IMB220419 as described in the first aspect hereinbefore or a culture as described in the second aspect hereinbefore or a metabolite as described in the third aspect hereinbefore or a microbial inoculum as described in the fourth aspect hereinbefore, in any one of the following:

(B1) Treating sewage;

(B2) Environmental treatment;

(B3) Soil improvement;

(B4) And (5) producing a bacterial fertilizer preparation.

In a seventh aspect, the invention claims a product for hydrolyzing cellulose.

The product for hydrolyzing cellulose claimed in the present invention has an active ingredient of the estrus cellulolytic bacterium (Herbiconiux cellulolyticus) IMB220419 described in the first aspect, the culture described in the second aspect, the metabolite described in the third aspect, or the microbial inoculum described in the fourth aspect.

In an eighth aspect, the invention claims a product having cellulase activity.

The product with cellulase activity claimed in the present invention has the active ingredient of the estrus cellulolytic bacterium (Herbiconiux cellulolyticus) IMB220419 described in the first aspect, the culture described in the second aspect, the metabolite described in the third aspect or the microbial inoculum described in the fourth aspect.

In a ninth aspect, the invention claims a method of hydrolyzing cellulose.

The method of hydrolyzing cellulose claimed in the present invention may comprise the steps of: treatment of a sample to be treated with a female cellulolytic bacterium (Herbiconiux cellulolyticus) IMB220419 as described in the first aspect above or a culture as described in the second aspect above or a metabolite as described in the third aspect above or a bacterial agent as described in the fourth aspect above.

In a tenth aspect, the invention claims the use of an estrus cellulolytic bacterium (Herbiconiux cellulolyticus) IMB220419 as described in the first aspect above for the preparation of a culture as described in the second aspect above or a metabolite as described in the third aspect above or a microbial inoculum as described in the fourth aspect above.

Experiments prove that the cellulose-decomposing female plant bacteria (Herbiconiux cellulolyticus) IMB220419 provided by the invention has a part of obvious characteristic differences from the existing female plant bacteria species, including aspects of phenotype, physiological biochemistry, cytochemistry components and the like. Meanwhile, the phylogenetic analysis based on the gene level further shows that the strain IMB220419 can be different from each effective species of the existing female phytoplankton, and fully proves that the strain IMB220419 represents a new species of the female phytoplankton and is named as cellulolytic female phytoplankton. Meanwhile, the detection of cellulose degradation capability proves that the strain has cellulose degradation capability and has wide application prospect in cellulose degradation. Can be used for sewage treatment, environmental treatment, soil improvement, bacterial fertilizer preparation production and the like.

Preservation description

Classification naming: a cellulolytic female plant (Herbiconiux cellulolyticus);

biological materials according to: IMB220419;

preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection);

the preservation organization is abbreviated as: CGMCC;

address: beijing, chaoyang area, north Chenxi Lu No.1, 3;

preservation date: 2022, 4, 20;

accession numbers of the preservation center: CGMCC No.24729.

Drawings

FIG. 1 shows the results of detection of polar lipid components of strain IMB220419.

Fig. 2 is a cellulose degradation capacity screening picture of strain IMB220419.

FIG. 3 shows the construction of a phylogenetic tree based on the gene sequences of the strain IMB220419 and the 16S rRNA gene of related species of the family Microbacteriaceae (phylogenetic tree Brevibacterium metallicus NM E3 ^T (GenBank accession No. KM874400) as outer group).

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 isolation screening and identification of female Decelluose (Herbiconiux cellulolyticus) IMB220419

1. Isolation and screening of Strain IMB220419

The strain IMB220419 is isolated from stems of orange daphne plants collected in Yunnan. The specific separation operation is as follows: the separation medium used is an R2A medium, and comprises the following specific components: glucose 0.5g, soluble starch 0.5g,Peptone 0.5g, yeast extract 0.5g, acid casein 0.5g, sodium pyruvate 0.3g, dipotassium hydrogen phosphate 0.3g, magnesium sulfate heptahydrate 0.05g, agar 15g, ddH ₂ O1L, pH value 7.2. The strain separation operation procedure: firstly, using sodium hypochlorite (the effective chlorine content is 4.5%, v/v) and alcohol (75%, v/v) to disinfect the surfaces of plants; then washing the surface disinfectant with sterile water; transferring the plant tissue with the surface sterilized into a sterile culture dish, and airing the water on the surface of the plant tissue in an ultra-clean bench; cutting off the tail ends of the plant tissues, and crushing the rest middle parts of the plant tissues; and uniformly spreading a proper amount of crushed plant tissues on an R2A separation culture medium, and incubating at 28 ℃ for 2-3 weeks. Selecting a plate with better microorganism diversity, inoculating single colonies with complete colonies and different morphological characteristics on the R2A plate for streak culture to obtain a pure culture for subsequent study. Meanwhile, the obtained pure culture is preserved in liquid nitrogen at ultralow temperature and frozen at-80 ℃ respectively by taking 20% (v/v) glycerol as a protective agent. Obtaining a strain and Herbiconiux flava DSM 26474 ^T New species, strains with the nearest border (16S rRNA gene similarity 98.34%)Numbered IMB220419.

2. Identification of Strain IMB220419

The strain is subjected to morphological, physiological and biochemical, cytochemical and gene level researches by using an R2A culture medium at 28 ℃, and other special cases are described.

1. Cell morphology observation and physiological and biochemical characteristic detection of strain IMB220419

The growth temperature detection range of the strain IMB220419 is 4, 10, 28, 30, 32, 37, 42 and 45 ℃; 6 (0, 1, 3, 5,7, 10) concentration gradients with a salt tolerance concentration (NaCl) detection range of 0-10% (0-10 g/100 ml); the growth pH detection range is 10 (4, 5, 6, 7, 8, 9, 10, 11, 12, 13) gradients between pH 4-13. The physiological and biochemical characteristics of the strain are detected by using detection kits such as API 50CH, API ZYM, and BiOLOG GEN III. Other strain physiological characteristics, including gram stain properties, oxygen demand, contact enzyme activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity and cellulose hydrolysis activity, are described mainly in the actinomycetes systems identification handbook (Xu L. Actinomycete systems: principles, methods and practice.Beijing: science Press,2007,93-108.).

The identification result shows that: the strain IMB220419 is gram-positive bacteria and aerobic. After the strain is cultured on the R2A culture medium for 72 hours at the temperature of 28 ℃, wet, smooth and milky colonies can be formed. The strain has a growth tolerance range of 4-37 ℃, 0-5% NaCl and pH 6.0-11.0, and the optimal growth conditions are 28 ℃, 0-1% NaCl and pH 8.0-9.0.

Gelatin liquefaction reaction of strain IMB220419 is positive, cellulose hydrolysis is positive, nitrate reduction reaction is negative, and the strain and related strain represent strain H.ginseng DSM 19088 ^T 、H.solani DSM 19813 ^T 、H.flava DSM 26474 ^T 、H.moechotypicolaDSM 25800 ^T The difference in the difference characteristics of (2) is shown in Table 1.

Table 1, differential characteristics of Strain IMB220419 and other species of the genus Phytophyton which are effectively described

Note that: positive, -, negative, w, weak positive, ND, no relevant data was searched.

As can be seen from the results shown in Table 1, the strain IMB220419 of the present invention is significantly different from the published strain belonging to the genus Phytophthora in terms of part of physiological and biochemical characteristics, cytochemical characteristics, gene composition and the like.

2. Cytochemical characterization of strain IMB220419

The cell chemistry components of fatty acids, quinone types, polar lipids, etc. of strain IMB220419 were detected by GC gas chromatography, HPLC liquid chromatography and TLC thin layer chromatography (Sasser M.identification of bacteria by gas ghromatography of cellular fatty acids, MIDI Technical Note 101.Newark,DE:MIDI inc;1990.Minnikin DE,O'Donnell AG,Goodfellow M,Alderson G,Athalye M et al.An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids.J Microbiol Methods 1984;2:233-241.).

Experimental results show that the main fatty acid component of the strain IMB220419 is ai-C _15:0 (51.0％),i-C _16:0 (16.2％),ai-C _15:1 A (10.6%), the major polar lipid components include dipeptidyl glycerol (DPG), phosphatidylglycerol (PG), glycolipid (GL) and small amounts of other polar lipid components (PL), as shown in fig. 1.

3. Detection of cellulase Activity of Strain

Plate screening was performed using sodium hydroxymethyl cellulose CMC-Na as the sole C source in the plates, and the cellulase activity of strain IMB220419 was detected (Reinhold-Hurek, b., hurek.t., classisens, m., van Montagu, m. (1993) Cloning, expression in Escherichia coli, and characterization of cellulolytic enzymes of Azoarcus sp., a root-planning diazotroph.J. bacterial 175,7056-7065. The method comprises the following specific steps: CMC-Na screening culture medium ((NH) was prepared ₄ ) ₂ SO ₄ 4g，NaCl 0.1g，MgSO ₄ ·7H ₂ 0 0.1g，CaCl ₂ 0.1g, yeast extract 0.5g, fe (III) EDTA 0.033g, CMC-Na 2g, agar 15g,1L ddH ₂ O, pH 7.0, selecting single colony of strain IMB220419 in logarithmic phase, inoculating on CMC-Na screening culture medium, culturing for 3 days, and detecting cellulase activity of the strain by Congo red dye liquor. As a result, as shown in FIG. 2, a clear transparent ring was produced around strain IMB220419, indicating that strain IMB220419 has better cellulolytic activity. And re-screening was performed using the same experimental method to find the same experimental results as the primary screening (Teather, R.M., wood, P.J. (1982)) Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rule.appl Environ Microbiol, 777-780.

4. Determination of the phylogenetic status of strains

Genomic DNA of strain IMB220419 was extracted and the 16S rRNA gene sequence (SEQ ID No. 1) was sequenced and aligned on-line in the International authoritative bacterial taxonomic analysis database (http:// www.ezbiocloud.net /) (Kim, O.S., cho, Y.J., lee, K., yoon SH, kim M, na H, park SC, jeon YS, lee JH, yi H, won S, chun J. (2012): introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species.Int J Syst Evol Microbiol,62:716-721.). The results show that the strain IMB220419 of the invention has the highest similarity with the strains of the genus Phytophthora. The 16S rRNA gene sequence of the strain IMB220419 of the invention and the known strain (H.flava DSM 26474 ^T ) The highest similarity of (a) is 98.34%, which is below 98.75% (threshold for partitioning bacterial species). According to microbiological Today,33:152-155 from the reference "Stackebrandt E, ebers J.2006, taxonomic parameters revisited: tarnished gold standards. The strain IMB220419 is described as 1 new species of the genus Phytophyton.

The 16S rRNA gene sequences of all the effective species strains of the genus Esculenta and part of the species strains of the genus Esculenta in the family Microbacteriaceae were selected to construct a phylogenetic tree (FIG. 3).

5. Strain whole genome information

The whole genome sequence of strain IMB220419 contained 5.3Mbp with a genomic G+C content of 68.8%. Strain IMB220419 genome and its closest bacterium H.flava DSM 26474 ^T The average nucleotide similarity (ANI) of the genomes of (a) is 80.3% far below the threshold value 95% for distinguishing bacterial species (Kim, M., oh, H.S., park, S.C., and Chun, J. (2014) Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes.Int J Syst Evol Microbiol 64,346-351.Doi: 10.1099/ijs.0.059774-0). Thus, the whole genome sequence information of strain IMB220419 supports the taxonomic status of its new species.

In conclusion, the strain IMB220419 of the invention has a part of obvious characteristic differences from the existing female phytophthora strains, including aspects of phenotype, physiology, biochemistry, cytochemistry and the like. Meanwhile, the phylogenetic analysis based on the gene level further shows that the strain IMB220419 can be different from each effective species of the existing female phytoplankton, and fully proves that the strain IMB220419 represents a new species of the female phytoplankton and is named as cellulolytic female phytoplankton. Meanwhile, the detection of cellulose degradation capability proves that the strain has cellulose degradation capability and has wide application prospect in cellulose degradation. Can be used for sewage treatment, environmental treatment, soil improvement, bacterial fertilizer preparation production and the like.

The strain IMB220419 is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 24729 and a suggested classification of the strain is named as cellulolytic female fungus (Herbiconiux cellulolyticus) in the year 4 and 20 of 2022.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

<110> institute of medical biotechnology of the national academy of medical science

<120> novel species of cellulolytic female phytes and use thereof

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Claims

1. Cellulose-decomposing female plant bacteriaHerbiconiux cellulolyticus) IMB220419, which has a registration number of CGMCC No.24729 in the China general microbiological culture Collection center.

2. The microbial inoculum is characterized in that: the bacterial agent contains the cellulolytic female phytbacteria of claim 1Herbiconiux cellulolyticus）IMB220419。

3. The microbial agent of claim 2, wherein: the microbial inoculum is a microbial inoculum for hydrolyzing cellulose.

4. The cellulolytic bacteria of claim 1Herbiconiux cellulolyticus) Use of IMB220419 or the microbial agent of claim 2 or 3 in any of the following:

(A1) Hydrolyzing cellulose;

(A2) Preparing a product for hydrolyzing cellulose;

(A3) Preparing cellulase;

(A4) Preparing a product with cellulase activity.

5. The cellulolytic bacteria of claim 1Herbiconiux cellulolyticus) Use of IMB220419 or the microbial agent of claim 2 or 3 in any of the following:

(B1) Treating sewage;

(B2) Soil improvement;

(B3) And (5) producing a bacterial fertilizer preparation.

6. A product for hydrolyzing cellulose, which comprises the gynostemma pentaphylla according to claim 1 as active ingredientHerbiconiux cellulolyticus) An IMB220419 or the microbial agent of claim 2 or 3.

7. A product with cellulase activity, the active ingredient of which is the gynostemma pentaphylla according to claim 1Herbiconiux cellulolyticus) An IMB220419 or the microbial agent of claim 2 or 3.

8. A method of hydrolyzing cellulose comprising the steps of: the method of using the cellulolytic female fungus of claim 1Herbiconiux cellulolyticus) The method for treating a sample to be treated comprising the steps of IMB220419 or the microbial inoculum of claim 2 or 3.

9. The cellulolytic bacteria of claim 1Herbiconiux cellulolyticus) Use of IMB220419 in the preparation of a microbial inoculum according to claim 2 or 3.