CN102373159B - New species Xylariaceae sp. DPZ-SY43 of mangrove rhizosphere cellulose degrading fungi and application thereof - Google Patents
New species Xylariaceae sp. DPZ-SY43 of mangrove rhizosphere cellulose degrading fungi and application thereof Download PDFInfo
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Abstract
The invention discloses a new species Xylariaceae sp. DPZ-SY43 of mangrove rhizosphere cellulose decomposed fungi and application thereof. Xylariaceae sp. DPZ-SY43 is preserved in the China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC No:4871 on May 16, 2011 at Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 West Beichen Road, Chaoyang District, Beijing, China. The fungus has the activity of producing cellulase, can be used for producing cellulase, and thus has important value in production and utilization of cellulase. Furthermore, the Xylariaceae sp. DPZ-SY43 can be used for preparing biological organic matter degrading bacterial manure.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of mangrove plant rhizosphere cellulose degradation new species of fungi Xylariaceae sp.DPZ-SY43 and the application in production of cellulose enzyme and preparation biological organic matter degraded bacterial manure thereof.
Background technology
Mangrove ecosystem (Mangrove ecosystems) is to be in the torrid zone, seashore tideland, subtropics, the zoocenosis, the mangrove woody plant community, xylium that comprise abundant species, the complexity of microflora and unique ecosystem, in bank nature Marine ecosystems, can keep the higher level of the productive forces, one of the important Wetlands ecosystems type of littoral zone (Holguin et al., 2001; Cao Qimin etc., 2008).Owing to be located in ocean, land intersection, the height salt dutyization of its Intertidal Habitats, the anoxic of soil, high optical radiation and periodic seawater soak and flood, and make it contain a large amount of unique microorganism, enzyme and genetic resources (Takeuchi M et al, 1998; Lyimo TJ et al., 2000).In the mangrove ecosystem, Litter, organic debris content are very abundant.Microorganism (Pointing SB et al., 1999 of containing the larger molecular organicses such as a large amount of degraded celluloses, xylogen and chitin; Yuan KP et al., 2005).But owing to the relatively evening that mangrove ecosystem is familiar with composition, distribution, the function and structure of its Microbial resources are known little about it.Although the research report about cellulose degradation strain has a lot (Andreas Ulrich et al., 2008 both at home and abroad; Moon-Jung Cho et al., 2010), but derive from the research report seldom (Yuan kang-pei et al., 2005) of the cellulose degradation microorganism of mangrove ecosystem.So should strengthen excavation and utilization to biomass resources such as cellulose degradation microorganisms in the mangrove ecosystem of native country.
Along with development economic and society, the supply of fossil oil is day by day in short supply, seeks and develop the widespread consensus that renewable energy source and new forms of energy become countries in the world.USDOE in 1993 and twice planning in 1997 all with biomass energy as prior development direction, and predict that bioenergy in 2010 will account for 50% of whole energy market.European Union proposed clean energy " Green Book " in 2000, had started again " European clever energy plan " in 2002, planned bioenergy in 2010 and reached about 12%.China has progressively begun the development and use research of biomass energy during " Eighth Five-Year Plan ", obtained certain achievement in research.Mierocrystalline cellulose is the important biomass energy of a class, and it is photosynthetic Primary product on the earth, accounts for the 35%-45% of plant dry weight, and annual global biosynthetic recyclability Mierocrystalline cellulose reaches (Lyndl R et al., 2002) more than 1,000 hundred million tons.China is large agricultural country, and can produce a large amount of stalk celluloses every year, but mainly is to utilize by simply burning mode of tradition, and energy utilization rate is extremely low, and environmental pollution is larger.How to utilize efficiently cellulose resource to become the important issue that concerns national energy security.Because the unique advantage of microorganism aspect cellulose utilization sought new cellulose utilization bacterial classification and exploitation High Cellulase Production bacterial classification, is the efficient key of utilizing of cellulose resource.Have more full enzyme system because fungus and bacterium, actinomycetes are compared, the FPA enzyme is lived, the CMC enzyme is lived, and all both are high than rear, and the microorganism that the native land is inside and outside to be used for researching and producing cellulase belongs to fungi mostly.To the course in existing more than 20 year of research of cellulose degradation fungi, the research report concentrates on several Pseudomonas (Christopher HV et al., 2003 such as whiterot fungi, flat lead fungi, bolt bacterium both at home and abroad; Lekounougou S et al., 2008), wherein Trichoderma is to study the most widely cellulase producing bacteria, 20% cellulase is from Trichoderma and Aspergillus in the cellulase market, the world.But in recent years, the research of new cellulose degradation strain report also day by day increases (Mario CNS et al., 2008; Revankar MS et al., 2006).
Summary of the invention:
First purpose of the present invention provides and a kind ofly filters out from littoral mangrove area Rhizophora stylosa (Rhizophora stylosa) the rhizodeposition thing in red Shahe, Sanya, Chinese Hainan Province, mangrove plant rhizosphere cellulose degradation new species of fungi with higher cellulose degradation activity: charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43, 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:4871.
Mangrove plant rhizosphere cellulose degradation new species of fungi of the present invention: charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43 filters out in littoral mangrove area Rhizophora stylosa (Rhizophora stylosa) the rhizodeposition thing in red Shahe, Sanya, Chinese Hainan Province
Its bacteria characteristic is described below:
Bacterial classification is described: as shown in Figure 1, vegetative hyphae is light brown, and wall is smooth or coarse, and tool is separated, branch, wide 1.0-2.5 μ m.Produce chlamydospore, Vandyke brown or black, out-of-shape, bunchiness is born in the middle of the mycelia or singly is born on the short branch of mycelia.Bacterial strain colony growth on the wort agar substratum is very fast, lower 14 days colony diameter 60-65mm of 25 ℃ of dark conditions, and initial stage white, the later stage beige, cotton-shaped, aerial hyphae is luxuriant; Bacterium colony back side dun without water-soluble pigment, has obvious resistance to Multiple Classes of Antibiotics.This bacterial strain can and produce significantly hydrolysis transparent circle in the growth of the flat board take Xylo-Mucine (CMC-Na) as sole carbon source, shows to have higher biomass degradation activity.
According to the pure growth extraction genomic dna of ordinary method from charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43, use the specific primer I TS1/ITS4 of rDNA the Internal Transcribed Spacer (ITS) sequence, obtain the ITS sequence by 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 by pcr amplification and sequencing analysis, and its sequence is shown in SEQ ID NO.2 to use beta tubulin.Use the specific primer cmd5/cmd6 of calmodulin (calmodulin) sequence, obtain the calmodulin sequence by pcr amplification and sequencing analysis, its sequence is shown in SEQ ID NO.3.By the BLAST software among the GenBank sequence in ITS sequence, β-tubulin sequence and calmodulin sequence and the GenBank database is compared, in database, do not find on all four sequence, show the new gene order of these 3 gene orders for finding first.
In the GenBank database, chosen the part representative gene order higher with measuring sequence similarity, with Neibor-joining method constructing system evolutionary tree (seeing Fig. 2,3,4), carry out Phylogenetic Analysis by ClustalW software and Mega software.Can find out that from phylogenetic tree charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43 and known fungi have larger otherness.Its rDNA the Internal Transcribed Spacer (ITS) sequence reaches 863bp, than the large approximately 400-500bp of other fungies, its BLAST sequence alignment fraction of coverage only has 50% approximately, and Hypoxylon multiforme (AF201717.2) sequence similarity nearer with its sibship only has 88%.(sequence of β-tubulin) and Annulohypoxylon ilanense (AY951657.1) have 97% similarity to its beta tubulin, have 92% similarity with Annulohypoxylon minutellum (AY951659.1).Its calmodulin (calmodulin) sequence alignment fraction of coverage only has 45-65% in addition, and Glomerella acutata (FJ917511.1) sequence similarity nearest with its sibship only has 78%.In conjunction with its morphological feature, we determine that it is a fungal strain novel species, are classified to Xylariaceae (Xylariaceae), called after charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43.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:4871.
The cellulase activity of charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43 (the CMC enzyme is lived) be studies show that: bacterial strain is under 30 ℃, ph 7.0 conditions, and the liquid fermenting cellulase activity reaches maximum value 60.1U/L (Fig. 5) about 10 days.Carry out simultaneously the biomass degradation test of Bruguiera conjugata (Bruguiera gymnorhiza) blade with the bacterium liquid formulation that contains charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43.The result shows: the degradation treatment Bruguiera conjugata blade rate of weight loss through 10 days reaches 79.1%, and it is active to illustrate that charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43 bacterial strain has preferably a biomass degradation such as Mierocrystalline cellulose.Therefore second purpose of the present invention provides the application of charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43 in cellulase-producing.
The 3rd purpose of the present invention provides the application of charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43 in preparation biological organic matter degraded bacterial manure.
The invention provides a kind of new species of fungi: charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43, this bacterium has the cellulase-producing activity, can be used for the production of cellulose enzyme, therefore production and the utilization of cellulase is had important value.Further can be used for making biological organic matter degraded bacterial manure.
Charcoal of the present invention angle bacterium (Xylariaceae sp.) DPZ-SY43, 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:4871.
Description of drawings:
Fig. 1 is Xylariaceae sp.DPZ-SY43 growing state (A), optical microscope photograph (B, C and D) on malt extract medium;
Fig. 2 is Xylariaceae sp.DPZ-SY43 site plan in rDNA the Internal Transcribed Spacer (ITS) unrooted phylogenetic tree;
Fig. 3 is Xylariaceae sp.DPZ-SY43 at beta tubulin (site plan in the unrooted phylogenetic tree of β-tubulin);
Fig. 4 is Xylariaceae sp.DPZ-SY43 site plan in calmodulin (calmodulin) unrooted phylogenetic tree;
Fig. 5 is liquid fermenting cellulase activity (the CMC enzyme is lived) figure of Xylariaceae sp.DPZ-SY43.
Embodiment:
Following examples are to further specify of the present invention, rather than limitation of the present invention.
Embodiment 1:
One, materials and methods
1, material
11 soil sample collections
Sample picks up from littoral mangrove area Rhizophora stylosa (Rhizophora stylosa) the rhizodeposition thing in red Shahe, Sanya, Hainan Province, in the sealing polyethylene bag of the sterilization of packing into behind the sample collecting, takes back the 4 ℃ of cryopreservation in laboratory.
1.2 substratum
1.2.1 primary dcreening operation substratum (g/L):
The compound method of every liter of primary dcreening operation substratum is as follows: (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, filtrate adds water and mends to 1000ml) 800ml, sucrose, 20.0g; NaCl, 1.5g; Agar, 15g, soil extraction, 200ml; 121 ℃ of sterilizations 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 afterwards.
1.2.2 sieve again substratum (g/L):
Every liter of compound method of sieving again substratum is as follows: 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 for subsequent use.
1.2.3 storage medium (g/L):
The compound method of every liter of storage medium is as follows: (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, filtrate adds water and mends to 1000ml) 1000ml, sucrose, 20.0g; NaCI, 1.5g; Agar, 15g.121 ℃ of sterilizations are for subsequent use.
1.2.4 fermention medium (g/L):
The compound method of every liter of fermention medium is as follows: 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 for subsequent use.
1.2.5 mangrove blade biomass substratum (g/L):
The compound method of every liter of mangrove blade biomass substratum is as follows: peptone 0.5g, yeast powder 1.0g, sucrose 0.5g, Zulkovsky starch 0.5g, mangrove blade 10.0g, NaCl 2.0g, CaCO
31.0g, MgSO
41.0g, water 1000mL.121 ℃ of sterilizations are for subsequent use.
2, method
2.1 the separation screening of bacterial strain
From sample choose three strain Rhizophora stylosas (Rhizophora stylosa), gather their rhizosphere soil sample 50g, place 37 ℃ of the triangular flasks, the 150r/min concussion that contain the 50ml sterile purified water to shake up 15min with getting about 5g behind the three duplicate samples mixings, get the bacterium liquid gradient dilution of 1ml to 10-3,10-4,10-5 after leaving standstill 30sec, inoculum size is 0.05mL, coat 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 is inoculated on the multiple sieve substratum 30 ℃ and is inverted and cultivated 3 days, growing 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 is the NaCl solution of 1mol/L again, outwell NaCl solution behind the 15min, at this moment, transparent circle will appear in the periphery of bacterial colonies that produces cellulase.Distinguish the bacterial strain with cellulase-producing according to preliminary decolorizing effect, measure transparent circle diameter and colony diameter, screening effect preferably bacterial strain carries out next step research, obtains therefrom a strain bacterial strain pure growth, called after DPZ-SY43 is stored in the storage medium.
2.2 strain morphology is learned feature
The morphological feature of bacterial strain and preliminary appraisal basis " fungi identification handbook ".
The DPZ-SY43 bacterial strain that above-mentioned steps screens, its bacterial classification morphology is described below:
As shown in Figure 1, vegetative hyphae is light brown, and wall is smooth or coarse, and tool is separated, branch, wide 1.0-2.5 μ m.Produce chlamydospore, Vandyke brown or black, out-of-shape, bunchiness is born in the middle of the mycelia or singly is born on the short branch of mycelia.Bacterial strain colony growth on the wort agar substratum is very fast, lower 14 days colony diameter 60-65mm of 25 ℃ of dark conditions, and initial stage white, the later stage beige, cotton-shaped, aerial hyphae is luxuriant; Bacterium colony back side dun without water-soluble pigment, has obvious resistance to Multiple Classes of Antibiotics.This bacterial strain can and produce significantly hydrolysis transparent circle in the growth of the flat board take Xylo-Mucine (CMC-Na) as sole carbon source, shows to have higher biomass degradation activity.
2.3 the extraction of strain gene group DNA and molecular biology identification
Using Shanghai to give birth to worker company genome extraction agent box to the pure growth bacterial strain DPZ-SY43 that obtains extracts DNA, then is used for the amplification of rRNA gene.
Universal primer ITS1/ITS4 (T.J.White, T.Bruns, et al 1990) is adopted in the amplification of fungi ITS district partial sequence
ITS-1：5′--TCCGTAGGTGAACCTGCGG--3′，
ITS-4：5′--TCCTCCGCTTAT?TGATATGC--3′。
(β-tubulin) universal primer Bt2a/Bt2b (Glass﹠amp is adopted in the amplification of sequence to 'beta '-tubulin; Donaldson, 1995)
Bt2a：5′--GGTAACCAAATCGGTGCTGCTTTC--3′
Bt2b：5′--ACCCTCAGTGTAGTGACCCTTGGC--3′。
Universal primer cmd5/cmd6 (Seung-Beom Hong, Hye-Sun Cho, et al 2006) is adopted in the amplification of calmodulin (calmodulin) sequence
cmd5：5′-CCGAGTACAAGGAGGCCTTC-3′，
cmd6：5′-CCGATAGAGGTCATAACGTGG-3′。
The PCR reaction system is as follows: premix Taq 25 μ l, each 0.5 μ l of upstream and downstream primer (1mM), template DNA 2.0 μ l, 2%DMSO 1.5 μ l, sterilized water 21 μ l.Amplification program is as follows: 95 ℃ of 5min; 94 ℃ of 45sec, 55 ℃ of 45sec, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min.
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.Use the specific primer I TS1/ITS4 of rDNA the Internal Transcribed Spacer (ITS) sequence, obtain the ITS sequence by 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 by pcr amplification and sequencing analysis, and its sequence is shown in SEQ ID NO.2 to use beta tubulin.Use the specific primer cmd5/cmd6 of calmodulin (calmodulin) sequence, obtain the calmodulin sequence by pcr amplification and sequencing analysis, its sequence is shown in SEQ ID NO.3.By the BLAST software among the GenBank sequence in ITS sequence, β-tubulin sequence and calmodulin sequence and the GenBank database is compared, in database, do not find on all four sequence, show the new gene order of these 3 gene orders for finding first.
In the GenBank database, chosen the part representative gene order higher with measuring sequence similarity, with Neibor-joining method constructing system evolutionary tree (seeing Fig. 2,3,4), carry out Phylogenetic Analysis by ClustalW software and Mega software.Can find out that from phylogenetic tree charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43 and known fungi have larger otherness.Its rDNA the Internal Transcribed Spacer (ITS) sequence reaches 863bp, than the large approximately 400-500bp of other fungies, its BLAST sequence alignment fraction of coverage only has 50% approximately, and Hypoxylon multiforme (AF201717.2) sequence similarity nearer with its sibship only has 88%.(sequence of β-tubulin) and Annulohypoxylon ilanense (AY951657.1) have 97% similarity to its beta tubulin, have 92% similarity with Annulohypoxylon minutellum (AY951659.1).Its calmodulin (calmodulin) sequence alignment fraction of coverage only has 45-65% in addition, and Glomerella acutata (FJ917511.1) sequence similarity nearest with its sibship only has 78%.In conjunction with its morphological feature, we determine that he is a fungal strain novel species, are classified to Xylariaceae (Xylariaceae), called after charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43.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:4871.
2.4 the mensuration of cellulase activity (the CMC enzyme is lived)
Charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43 is inoculated in the fermention medium, 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 liquor 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 that 2.0mL prepares with pH 4.8 acetate buffer solutions, 50 ℃ of water bath with thermostatic control enzymolysis 30min, then the DNS 5min in boiling water bath that adds 2.5mL, 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, and as shown in Figure 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 secondary fermentation liquid reaches 60.1U/L.Show that thus charcoal of the present invention angle bacterium (Xylariaceae sp.) DPZ-SY43 has the cellulase-producing activity.
Enzyme activity according to the international unit stipulative definition is: it is 1 enzyme activity unit U that the per minute catalyzing cellulose hydrolysis generates the required enzyme amount of 1 μ mol glucose.
2.5 bacterial strain preparation filter paper Degrading experiment
Charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43 is inoculated on the solid PDA flat board, get with the 6cm punch tool after a certain size and leave standstill cultivation 10 days in mangrove blade biomass substratum that three ferfas tongues are inoculated in respectively three 250ml Deng bacterium colony length, after 10 days, the mangrove blade rate of weight loss in the substratum is 79.1%.
Claims (3)
1. a charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43, its deposit number is: CGMCC No:4871.
2. the application of charcoal angle bacterium (Xylariaceae sp.) DPZ-SY43 claimed in claim 1 in cellulase-producing.
3. the application of charcoal angle bacterium claimed in claim 1 (Xylariaceae sp.) DPZ-SY43 in preparation biological organic matter degraded bacterial manure.
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| CN101260370A (en) * | 2008-04-25 | 2008-09-10 | 仲恺农业技术学院 | Epiphyte Xylaria sp.FDYS-1, biological agent prepared from the same and application thereof in preventing and curing bursaphenchus xylophilus nickle |
| CN101591618A (en) * | 2008-05-28 | 2009-12-02 | 中国科学院微生物研究所 | A kind of Xylaria gracillima strain and liquid fermentation culturing method thereof and application |
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| CN101260370A (en) * | 2008-04-25 | 2008-09-10 | 仲恺农业技术学院 | Epiphyte Xylaria sp.FDYS-1, biological agent prepared from the same and application thereof in preventing and curing bursaphenchus xylophilus nickle |
| CN101591618A (en) * | 2008-05-28 | 2009-12-02 | 中国科学院微生物研究所 | A kind of Xylaria gracillima strain and liquid fermentation culturing method thereof and application |
Non-Patent Citations (1)
| Title |
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| I-Son Ng et al.Novel Cellulase Screening and Optimal Production from the Wood Decaying Xylariaceae: Daldinia Species.《Appl Biochem Biotechnol》.2010,表1，摘要. * |
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