CN102337222A - New species of Rhizophora stylosa root cellulose degrading fungus Hypoxylon sp. DPZ-SYz-36 and application thereof - Google Patents
New species of Rhizophora stylosa root cellulose degrading fungus Hypoxylon sp. DPZ-SYz-36 and application thereof Download PDFInfo
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Abstract
The invention discloses a new species of a Rhizophora stylosa root cellulose degrading fungus Hypoxylon sp.DPZ-SYz-36 and application thereof. The Hypoxylon sp. DPZ-SYz-36 was collected in China General Microbiological Culture Collection Center (CGMCC) on May 16, 2011 with the collection number of CGMCC No.4872. The Hypoxylon sp. DPZ-SYz-36 has cellulase producing activity, can be used for producing cellulase, has important value for production and utilization of the cellulase, and can be further used for preparing biological organic matter degrading bacterial fertilizers.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of Rhizophora stylosa rhizosphere cellulose degradation fungi novel species Hypoxylon sp.DPZ-SYz-36 and the application in the production of cellulose enzyme is degraded bacterial manure with the preparation biological organic matter thereof.
Background technology:
Mangrove forest is the woody plant community, xylium of NATURAL DISTRIBUTION in the torrid zone, seashore tideland, subtropics; Cover about 60～75% torrid zone and subtropics seashores; The eubiosis to safeguarding the river mouth, bay plays an important role, and is important component part (Holguin et al., 2001 of fecundity of the sea; Long Han etc., 2005), be considered to have one of ecosystem than high productivity.This ecosystem has higher organic level (global annual litter is 100Tg C), for the stretch of coastal water and the relevant coenosis habitat of adjoining provides organism (Holguin et al., 2001; Lugomela and Bergman, 2002).Because the mangrove forest settling has strong reducing property, strongly-acid, supersalinity, nutritious (Takeuchi M et al., 1998; Characteristic such as Lin Peng, 1997).Therefore, the mikrobe, enzyme and the genetic resources that have contained a large amount of uniquenesses here.In recent years, mangrove forest unique ecological environment and rich species variety have caused more ocean science workers' concern.But the microbiological research of mangrove area is started late, and research is comparatively weak.At present, the mangrove area microbiological research mainly concentrates on: the research of mangrove forest micro-flora; Microbiological research in the litter decomposition course; The research of mangrove rhizosphere actinomycetes; Mangrove area microbial contamination ecological study (Yuan KP et al., 2005; Muniswaran A.et al.1994).
Along with the shortage of fossil oil, the energy is the significant problem of face of mankind.Seek and develop Sustainable development and even human existence problem that renewable energy source and new forms of energy are related to economy.Mierocrystalline cellulose is different with fossil fuel, and it is a kind of reproducible resource.Annual photosynthesis can produce about 10,000,000,000 tons plant dry matter on the earth, and wherein over half is Mierocrystalline cellulose and semicellulose (Campbell BA et al., 1998).In addition; Also contain a large amount of cellulosic materials in the waste that mankind's activity produces, like agricultural wastes (straw, rice husk, stalk, Pericarppium arachidis hypogaeae, corn cob, cotton seed hulls, bagasse etc.), food-processing waste (pericarp, pomace etc.), timber waste (wood chip, bark) and urban waste (40%～60% solid waste is rubbish and waste paper) etc.If can effectively utilize conversion technology these cellulose resource are changed into simple sugars; Fermentation produces ethanol equal energy source material again; Not only can turn waste into wealth; But also can avoid because fossil energy shortage and even the exhausted worldwide energy dilemma of bringing can alleviated or solve simultaneously to the environmental pollution that combustion of fossil fuel brought.
Because the unique advantage of mikrobe aspect cellulose utilization sought new cellulose utilization bacterial classification and exploitation high yield cellulase strain, is the cellulose resource key for high-efficient use.Can utilize the fungi that eukaryotic microorganisms such as viride (T.virid), black mold (Aspergillus niger) and Trichodermareesei main cellulase-producings such as (Trichogerma reesei) are not only arranged with cellulolytic species; Wherein Trichoderma is to study cellulase producing bacteria the most widely; 20% cellulase is from Trichoderma and Aspergillus in the cellulase market, the world; But in recent years; The research report of new cellulose degradation strain also day by day increases (Mario CNS et al., 2008; Revankar MS et al., 2006).In addition, also have some protozoons and the bacterium also can decomposition of cellulose class material, but because bacterium excretory cellulase amount is few, and the enzyme that produces belong to intracellular enzyme or is adsorbed on the cell walls, so the industrial less production bacterial classification of making cellulase of bacterium.In addition, the animal that has such as termite, small lobsters etc. also can produce and be different from the cellulase that its endosymbiontic microorganism group is produced fully.Because the cellulose degradation fungi has its important effect aspect energy utilization, a lot of for this reason scientific workers have carried out a large amount of research and have obtained significant achievement this type fungi.
Summary of the invention:
First purpose of the present invention provides and a kind ofly from the Rhizophora stylosa plant rhizosphere settling of Sanya, Chinese Hainan Province, filters out; Has the active fungi novel species of higher cellulose degradation: Hypoxylon (Hypoxylon sp.) DPZ-SYz-36; This bacterium was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on 05 16th, 2011; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, deposit number: CGMCC No:4872.
Rhizophora stylosa rhizosphere cellulose degradation fungi novel species of the present invention: Hypoxylon (Hypoxylon sp.) DPZ-SYz-36 filters out from the Rhizophora stylosa plant rhizosphere settling of Sanya, Chinese Hainan Province.
Its bacteria characteristic is described below:
Bacterial classification is described: as shown in Figure 1, bacterial strain vegetative hyphae wall is smooth, and tool is separated, branch, wide 1.5-3.6 μ m.Do not see any kind spore.Colony growth is very fast on the wort agar substratum, following 14 days colony diameter 40-45mm of 25 ℃ of dark conditions, and white, cotton-shaped, aerial hyphae is luxuriant; To dun, do not have water-soluble pigment in the bacterium colony back side, multiple antibiosis is have obvious resistance.This inoculation to being on the flat board of main carbon source with Xylo-Mucine (CMC-Na), after 3 days, can be formed the hydrolysis transparent circle (Fig. 1 D) of diameter 19mm 30 ℃ of cultivations, show that this bacterial strain has higher biomass degradations such as Mierocrystalline cellulose activity.
Ordinary method is extracted genomic dna from the pure growth of Hypoxylon (Hypoxylon sp.) DPZ-SYz-36; The specific primer I TS1/ITS4 of utilization rDNA internal transcribed spacer district (ITS) sequence; Obtain the ITS sequence through pcr amplification and sequencing analysis, its sequence is shown in SEQ ID NO.1.(the specific primer Bt2a/Bt2b of sequence of β-tubulin) obtains β-tubulin sequence through pcr amplification and sequencing analysis to the utilization beta tubulin, and its sequence is shown in SEQ ID NO.2.The specific primer LROR/LR7 of utilization 28s rDNA sequence obtains 28s rDNA sequence through pcr amplification and sequencing analysis, and its sequence is shown in SEQ ID NO.3.Through the BLAST software among the GenBank sequence in ITS sequence, β-tubulin sequence and 28s rDNA sequence and the GenBank DB is compared; In DB, do not find on all four sequence, show the new gene order of these 3 gene orders for finding first.
In the GenBank DB, chosen part and measured the higher representative gene order of sequence similarity, with Neibor-joining method constructing system evolutionary tree (seeing Fig. 2,3,4), carried out Phylogenetic Analysis through ClustalW software and Mega software.Can find out that from phylogenetic tree Hypoxylon (Hypoxylon sp.) DPZ-SYz-36 and known fungi have bigger otherness.Its rDNA internal transcribed spacer district (ITS) sequence sequencing analysis shows: Hypoxylonmonticulosum (FM209467.1) sequence similarity nearer with its sibship reaches 99%, reaches 98% with Hypoxylon sp. (DQ631939.1) sequence similarity; But its beta tubulin (sequence of β-tubulin) is except that having 97% the similarity with Hypoxylon monticulosum (AY951737.1), with the similarity of other bacterial strains all less than 90%.In addition, strain endogenetic fungus (EF420088.1) sequence similarity nearest with its 28s rDNA sequence sibship has only 96%, and it forms a branch separately in evolutionary tree.In conjunction with its morphological feature, we are included into Hypoxylon with it and belong to (Hypoxylon), are the novel species that a strain Hypoxylon belongs to, called after: Hypoxylon (Hypoxylon sp.) DPZ-SYz-36.
Research shows to the cellulase activity of bacterial strain Hypoxylon sp.DPZ-SYz-36 (the CMC enzyme is lived): Hypoxylon (Hypoxylon sp.) DPZ-SYz-36 is under 30 ℃, pH 7.0 conditions; Liquid fermenting, the cellulase activity of fermented liquid reach 121.1U/L (Fig. 5) about 10 days.Carry out Rhizophora stylosa leaf degraded test with the bacterium liquid formulation that contains Hypoxylon sp.DPZ-SYz-36 simultaneously.The result shows: the degradation treatment Rhizophora stylosa leaf rate of weight loss through 10 days reaches 68.9%, and it is active to explain that thus Hypoxylon sp.DPZ-SYz-36 has preferably a biomass degradation such as Mierocrystalline cellulose.Therefore second purpose of the present invention provides the application of Hypoxylon (Hypoxylon sp.) DPZ-SYz-36 in cellulase-producing.
The 3rd purpose of the present invention provides the application of Hypoxylon (Hypoxylon sp.) DPZ-SYz-36 in preparation biological organic matter degraded bacterial manure.
The invention provides a kind of Rhizophora stylosa rhizosphere cellulose degradation fungi novel species: Hypoxylon (Hypoxylon sp.) DPZ-SYz-36; This bacterium has the cellulase-producing activity; Can be used for the production of cellulose enzyme, therefore production and the utilization to cellulase has significant values.Further can be used to make biological organic matter degraded bacterial manure.
Hypoxylon of the present invention (Hypoxylon sp.) DPZ-SYz-36 was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on 05 16th, 2011; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, deposit number: CGMCC No:4872.
Description of drawings:
Fig. 1 is that Hypoxylon sp.DPZ-SYz-36 bacterium colony is in growing state on the malt extract medium (A), optical microscope photograph (B and C) and Hypoxylon sp.DPZ-SYz-36 bacterium colony decolouring situation (D) on the Congo red flat board of CMC-;
Fig. 2 is Hypoxylon sp.DPZ-SYz-36 site plan in rDNA internal transcribed spacer district (ITS) unrooted phylogenetic tree;
Fig. 3 is Hypoxylon sp.DPZ-SYz-36 at beta tubulin (site plan in the unrooted phylogenetic tree of β-tubulin);
Fig. 4 is Hypoxylon sp.DPZ-SYz-36 site plan in 28s rDNA unrooted phylogenetic tree;
Fig. 5 is Hypoxylon sp.DPZ-SYz-36 liquid fermenting cellulase activity (the CMC enzyme is lived) figure.
Embodiment:
Following examples are to further specify of the present invention, rather than limitation of the present invention.
Embodiment 1:
1, material
1.1 soil sample collection
Pick up from littoral mangrove area Rhizophora stylosa (Rhizophora stylosa) the rhizosphere settling in red Shahe, Sanya, Hainan Province sample and in September, 2010, the 4 ℃ of cryopreservation in laboratory are taken back in sealing in the polyethylene bag of the sterilization of packing into behind the sample collecting.
1.2 substratum
1.2.1 primary dcreening operation substratum:
Every liter of primary dcreening operation culture medium preparation method is following: (peeling potatoes digs eye to potato juice, cleans section.Claim that 200g puts into the 1000ml tap water, the 30min that boils that simmers in water, double gauze filters, filtrating adds water and mends to 1000ml) 800ml, sucrose, 20.0g; NaCl, 1.5g; Agar, 15g, soil extraction, 200ml; Wait substratum to be cooled to the penbritin of 60 ℃ of 100 μ g/ml that add filtration sterilizations and each 1ml of Vetstrep of 100 μ g/ml after 121 ℃ of sterilizations.
1.2.2 sieve substratum (g/L) again:
Every liter to sieve the culture medium preparation method again following: CMC-Na 10.0g; Peptone 0.5g; Yeast extract 0.5g; Sucrose 0.5g; KNO
31.0g; K
2HPO
40.5g; MgSO
4.7H
2O 0.5g; NaCl 1.5g; Agar 15g, water 1000mL.121 ℃ of sterilizations are subsequent use.
1.2.3 storage medium:
The compound method of every liter of storage medium is following: (peeling potatoes digs eye to potato juice, cleans section.Claim that 200g puts into the 1000ml tap water, the 30min that boils that simmers in water, double gauze filters, filtrating adds water and mends to 1000ml) 1000ml, sucrose, 20.0g; NaCI, 1.5g; Agar, 15g.121 ℃ of sterilizations are subsequent use.
1.2.4 fermention medium:
The compound method of every liter of fermention medium is following: CMC-Na 5.0g; Peptone 1.0g; Yeast extract 1.0g; Sucrose 1.0g; KNO
31.0g; K
2HPO
40.5g; MgSO
4.7H
2O 0.5g; NaCl 1.5g; Water 1000mL.121 ℃ of sterilizations are subsequent use.
1.2.5 Rhizophora stylosa leaf degradation experiment substratum:
Every liter of Rhizophora stylosa leaf degradation experiment culture medium preparation method is following: peptone 0.5g, yeast powder 1.0g, sucrose 0.5g, Zulkovsky starch 0.5g, Rhizophora stylosa leaf 3.3g, NaCl 2.0g, CaCO
31.0g, MgSO
41.0g, water 1000mL.121 ℃ of sterilizations are subsequent use.
2, method
2.1 the separation screening of bacterial strain
From appearance choose three strain Rhizophora stylosas (Rhizophora stylosa); Gather their rhizosphere sediment sample 50g; Place 37 ℃ of triangular flasks containing the 50ml sterile purified water, 150r/min concussion to shake up 15min with getting about 5g behind the three duplicate samples mixings, leave standstill the liquid gradient of getting 1ml behind the 30sec and be diluted to 10
-3, 10
-4, 10
-5, inoculum size is 0.05mL, coats on the primary dcreening operation substratum, 8 repetitions are established in each processing.Cultivated 3-5 days for 37 ℃, the typical single bacterium colony of picking form is put and is connect purifying and obtain pure growth 2-3 time.To obtain pure growth and be inoculated on the multiple sieve substratum, and be inverted for 30 ℃ and cultivated 3 days, grow on the substratum of bacterium colony; After covering quality concentration is Congo red solution 10～15min of 1mg/mL; Remove Congo red solution, adding concentration again is the NaCl solution of 1mol/L, outwells NaCl solution behind the 15min; At this moment, transparent circle will appear in the periphery of bacterial colonies of generation cellulase.Distinguish the bacterial strain with cellulase-producing according to preliminary decolorizing effect, measure transparent circle diameter and colony diameter, screening effect bacterial strain preferably carries out next step research.Obtain a strain bacterial strain pure growth therefrom, called after DPZ-SYz-36 is stored in it in storage medium.
2.2 strain morphology is learned characterized
The morphological feature of bacterial strain and preliminary appraisal basis " fungi identification handbook ".
The bacterial strain DPZ-SYz-36 that last step screening obtains, its bacterial classification morphological feature is described below: as shown in Figure 1, bacterial strain vegetative hyphae wall is smooth, and tool is separated, branch, wide 1.5-3.6 μ m.Do not see any kind spore.Colony growth is very fast on the wort agar substratum, following 14 days colony diameter 40-45mm of 25 ℃ of dark conditions, and white, cotton-shaped, aerial hyphae is luxuriant; To dun, do not have water-soluble pigment in the bacterium colony back side, multiple antibiosis is have obvious resistance.This inoculation to being on the flat board of main carbon source with Xylo-Mucine (CMC-Na), after 3 days, can be formed the hydrolysis transparent circle (Fig. 1 D) of diameter 19mm 30 ℃ of cultivations, show that this bacterial strain has higher biomass degradations such as Mierocrystalline cellulose activity.
2.3 bacterial strain molecular biology identification
Using Shanghai to give birth to worker company genome extraction agent box to the pure growth bacterial strain DPZ-SYz-36 that obtains extracts DNA, is used for the amplification of goal gene then.
Universal primer ITS1/ITS4 (T.J.White, T.Bruns, et al 1990) is adopted in the amplification of fungi ITS district partial sequence
ITS1：5′--TCCGTAGGTGAACCTGCGG--3′，
ITS4：5′--TCCTCCGCTTAT?TGATATGC--3′。
(β-tubulin) universal primer Bt2a/Bt2b (Glass&Donaldson, 1995) is adopted in the amplification of sequence to 'beta '-tubulin
Bt2a：5′--GGTAACCAAATCGGTGCTGCTTTC--3′，
Bt2b：5′--ACCCTCAGTGTAGTGACCCTTGGC--3′；
The amplification employing universal primer LROR/LR7 of 28s rDNA sequence (Vilgalys, R.&M.Hester.1990.)
LROR：5′-ACCCGCTGAACTTAAGC-3′，
LR7：5′-TACTACCACCAA?GATCT-3′。
PCR reaction system and reaction conditions are following:
The PCR reaction system comprises: the PCR response procedures is:
Get 2 μ L PCR products, 1% agarose electrophoresis is carried out product and is detected.After amplified production is purified, deliver the order-checking of order-checking company.The specific primer I TS1/ITS4 of utilization rDNA internal transcribed spacer district (ITS) sequence obtains the ITS sequence through pcr amplification and sequencing analysis, and its sequence is shown in SEQ ID NO.1.(the specific primer Bt2a/Bt2b of sequence of β-tubulin) obtains β-tubulin sequence through pcr amplification and sequencing analysis to the utilization beta tubulin, and its sequence is shown in SEQ ID NO.2.The specific primer LROR/LR7 of utilization 28s rDNA sequence obtains 28s rDNA sequence through pcr amplification and sequencing analysis, and its sequence is shown in SEQ ID NO.3.Through the BLAST software among the GenBank sequence in ITS sequence, β-tubulin sequence and 28s rDNA sequence and the GenBank DB is compared; In DB, do not find on all four sequence, show the new gene order of these 3 gene orders for finding first.
In the GenBank DB, chosen part and measured the higher representative gene order of sequence similarity, with Neibor-joining method constructing system evolutionary tree (seeing Fig. 2,3,4), carried out Phylogenetic Analysis through ClustalW software and Mega software.Can find out that from phylogenetic tree Hypoxylon (Hypoxylon sp.) DPZ-SYz-36 and known fungi have bigger otherness.Its rDNA internal transcribed spacer district (ITS) sequence sequencing analysis shows: Hypoxylon monticulosum (FM209467.1) sequence similarity nearer with its sibship reaches 99%, reaches 98% with Hypoxylon sp. (DQ631939.1) sequence similarity; But its beta tubulin (sequence of β-tubulin) is except that having 97% the similarity with Hypoxylon monticulosum (AY951737.1), with the similarity of other bacterial strains all less than 90%.In addition, strain endogenetic fungus (EF420088.1) sequence similarity nearest with its 28s rDNA sequence sibship has only 96%, and it forms a branch separately in evolutionary tree.In conjunction with its morphological feature, we are included into Hypoxylon with it and belong to (Hypoxylon), are the novel species that a strain Hypoxylon belongs to, called after: Hypoxylon (Hypoxylon sp.) DPZ-SYz-36.
2.4 the mensuration of cellulase activity (the CMC enzyme is lived)
DPZ-SYz-36 is inoculated in the 250ml fermention medium with Hypoxylon (Hypoxylon sp.), and under 30 ℃, ph 7.0 conditions, the fermented liquid that takes a morsel every day is measured the cellulase activity of fermented liquid.Fermented liquid is through the centrifugal 10min of 6000r/min, and supernatant is as crude enzyme liquid.In the tool plug scale glass test tube of 25ml, add the suitably enzyme liquid of dilution of 0.5mL; Behind 50 ℃ of water bath with thermostatic control preheating 2min, add 1% carboxymethylcellulose sodium solution of 2.0mL, 50 ℃ of water bath with thermostatic control enzymolysis 30min with the preparation of pH 4.8 acetate buffer solutions; The DNS 5min in boiling water bath that adds 2.5mL then; Be settled to the 25ml mixing after the flowing water cooling, under 520nm, measure its light absorption value, calculate enzyme activity behind the reference standard curve.The enzyme of fermented liquid is lived as shown in Figure 5, can know that by Fig. 5 fermented liquid has cellulase activity, and along with the prolongation of fermentation time, its cellulase activity raises gradually, and the cellulase activity of 10 days left and right sides fermented liquids reaches 121.1U/L.Show that thus Hypoxylon of the present invention (Hypoxylon sp.) DPZ-SYz-36 has the cellulase-producing activity.
Enzyme activity according to the iu stipulative definition is: it is 1 enzyme activity unit U that the PM catalyzing cellulose hydrolysis generates the required enzyme amount of 1 μ mol glucose.
2.5 the Rhizophora stylosa leaf of Hypoxylon (Hypoxylon sp.) DPZ-SYz-36 degraded test
DPZ-SYz-36 is inoculated on the solid PDA flat board with Hypoxylon (Hypoxylon sp.); Getting three ferfas tongues to a certain size back with the 6cm punch tool Deng bacterium colony length inoculates and to be inoculated into respectively in three bottles of Rhizophora stylosa leaf degradation experiment substratum that contain 250ml; Leave standstill and cultivated 10 days; Rhizophora stylosa leaf rate of weight loss reaches 68.9%, and it is active to explain that thus Hypoxylon (Hypoxylon sp.) DPZ-SYz-36 bacterial strain has preferably a biomass degradation such as Mierocrystalline cellulose.
Claims (3)
1. a Hypoxylon (Hypoxylon sp.) DPZ-SYz-36, its deposit number is: CGMCC No:4872.
2. the application of the described Hypoxylon of claim 1 (Hypoxylon sp.) DPZ-SYz-36 in cellulase-producing.
3. the application of the described Hypoxylon of claim 1 (Hypoxylon sp.) DPZ-SYz-36 in preparation biological organic matter degraded bacterial manure.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108913731A (en) * | 2018-07-26 | 2018-11-30 | 海南大学 | A kind of pyran compounds and its preparation method and application with immunosuppressive activity |
| CN114933976A (en) * | 2022-06-17 | 2022-08-23 | 广州医科大学 | Mountain Monascus montmorifolium and application thereof |
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| CN101897345A (en) * | 2009-06-01 | 2010-12-01 | 东北林业大学 | Preparation method of preparation containing hypoxylon bacteriostatic active ingredients for preventing and treating sphaeropsis sapinea |
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| CN101897345A (en) * | 2009-06-01 | 2010-12-01 | 东北林业大学 | Preparation method of preparation containing hypoxylon bacteriostatic active ingredients for preventing and treating sphaeropsis sapinea |
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| STEPHEN B. POINTING, ET AL.: "Production of wood-decay enzymes, mass loss and lignin solubilization in wood by tropical Xylariaceae", 《MYCOL. RES.》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913731A (en) * | 2018-07-26 | 2018-11-30 | 海南大学 | A kind of pyran compounds and its preparation method and application with immunosuppressive activity |
| CN108913731B (en) * | 2018-07-26 | 2021-11-16 | 海南大学 | Pyran compound with immunosuppressive activity and preparation method and application thereof |
| CN114933976A (en) * | 2022-06-17 | 2022-08-23 | 广州医科大学 | Mountain Monascus montmorifolium and application thereof |
| CN114933976B (en) * | 2022-06-17 | 2023-09-19 | 广州医科大学 | Monilinia mountain Monilis and application thereof |
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