CN102363748A - New fungus Acremonium sp. DPZ-SYz-2-3 for high efficiency cellulose degradation and application thereof - Google Patents
New fungus Acremonium sp. DPZ-SYz-2-3 for high efficiency cellulose degradation and application thereof Download PDFInfo
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Abstract
The invention discloses a new fungus Acremonium sp. DPZ-SYz-2-3 for the high efficiency cellulose degradation and an application thereof. Acremonium sp. DPZ-SYz-2-3 is preserved in the China general microbiological culture collection center (CGMCC) on 05/16/2011 and has a preservation number of CGMCC No: 4810, wherein the address of the CGMCC is No.3 of No.1 Beichen Road West, Chaoyang District, Beijing, Institute of Microbiology Chinese Academy of Sciences. The fungus of the invention, which has a cellulase production activity, can be used for producing cellulase, so the fungus has important values to the production and the utilization of cellulase; and the fungus can be further used for making a biological organic matter degradation bacterial fertilizer.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of highly effective cellulose degradative fungi novel species Acremonium sp.DPZ-SYz-2-3 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 that is distributed in the torrid zone, seashore tideland, subtropics, is distinctive Forest Types on the seabeach (Lin P et al.1999).Though it only accounts for the per mille of global land area, the four Large Marine Ecosystem high productivity ecosystems (Long Han etc., 2005) have been formed with the high productivity and coral reef ecologic system, upwelling zone, the sea grass bed ecosystem that are equivalent to the Amazon tropical rain forest.15122 hectares in the existing mangrove forest of China mainly is distributed in ground such as Hainan, Guangdong, Guangxi, and wherein Hainan Province is China's mangrove most species, the best zone of growth.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 mangrove area microbiological research is started late, and research is comparatively weak.In the research report of relevant mangrove forest, the microbiological research document portion of mangrove area is few.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).Because biomass resources such as mangrove area litter are very abundant, so a large amount of mikrobe of mangrove area can produce materials such as cellulolytic enzyme, lignin decomposition enzyme.
The energy and environmental problem are the central topics that concerns the national security and the sustainable development of socio-economy that each state of the world today all faces, and have become the focus that the whole world is paid close attention to.Therefore, people begin to transfer to sight on the renewable energy source system of Sustainable development.The expert thinks that the biomass resource transformation system is the technology platform that leads world energy sources revolution for the third time.Lignocellulose is a renewable resources the abundantest on the earth, also is the minimum resource of current utilization ratio, is the emphasis of various countries' new resources strategy.The natural resources of these rich and cheap is mainly derived from eagroforestry waste, trade waste and urban waste to the available lignocellulose of China about 700,000,000 tons every year.So Mierocrystalline cellulose is the inevitable direction (Zhang Bailiang, etc., 2007) of future source of energy utilization development.Lignocellulose comprises Mierocrystalline cellulose, semicellulose, xylogen.Utilizing lignocellulose preparing fuel ethanol main path is partly to be degraded to monose to Mierocrystalline cellulose in the lignocellulose and semicellulose earlier, utilizes yeast fermentation monose to produce alcohol fuel again.Wherein, ligocellulose degradation be the efficient of monose low be the factor of its industrialized utilization of restriction.Therefore, new Mierocrystalline cellulose efficiently or the semicellulose degradation bacteria strains of screening is the cellulose resource key for high-efficient use.To the course in existing more than 20 year of research of cellulose-degrading bacteria, 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 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 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 from the rhizosphere soil of Sanya, Chinese Hainan Province torrid zone mangrove forest, filters out; Has the active mangrove plant rhizosphere fungi of higher cellulose degradation novel species: branch top spore (Acremonium sp.) DPZ-SYz-2-3; 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:4810.
Highly effective cellulose degradative fungi novel species of the present invention: branch top spore (Acremonium sp) DPZ-SYz-2-3 screens from the rhizosphere soil of the tropical mangrove forest of Sanya, Hainan Province.
Its bacteria characteristic is described below:
Bacterial classification is described: as shown in Figure 1, the bacterial strain vegetative hyphae is very thin, and tool is separated, branch, wide 1.0-2.5 μ m.No specialization conidiophore, conidiogenous cell directly is born on the mycelia; Bottle stalk formula is produced spore, produces spore bottle stalk lanceolar, and is straight or crooked, simply or idol branch is arranged, long 16.4-34.2 μ m, the wide 1.2-2.5 μ of base portion m, top less than 1.0 μ m; Conidium is oval, rare nearly column, and 2.4-4.5 * 1.0-2.0 μ m assembles agglomerating.Colony growth is slower on the wort agar substratum, following 14 days colony diameter 18-24mm of 25 ℃ of dark conditions, and white, cotton-shaped, aerial hyphae is luxuriant, and a large amount of rhizomorphs are arranged; The bacterium colony back side is light brown, does not have water-soluble pigment, and 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 25mm 30 ℃ of cultivations, show that this bacterial strain has higher biomass degradations such as Mierocrystalline cellulose activity.
From the pure growth of strains A cremonium sp.DPZ-SYz-2-3, extract genomic dna; 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 cmd5/cmd6 of utilization calmodulin (calmodulin) sequence obtains the calmodulin sequence through pcr amplification and sequencing analysis, and its sequence is shown in SEQID NO.3.Through the BLAST software among the GenBank sequence in ITS sequence, β-tubulin sequence and calmodulin 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 strains A cremonium sp.DPZ-SYz-2-3 and known fungi have bigger otherness.Through its rDNA internal transcribed spacer district (ITS) sequence sequencing analysis is shown; With the nearer isolating ascomycetess (AF502849.1 and AF502848.1) that are two strains from the leaf that rots of its sibship; Its similarity reaches 98%, but the author does not further classify to it; And with the nearest Acremonium bacterial strain of its sibship be AB540578.1, it and Acremonium sp.DPZ-SYz-2-3 have only 90% similarity.(Blast of the sequence of β-tubulin) analyzes and shows its beta tubulin: only the have an appointment similarity of 75%-85% of the beta tubulin district of Acremonium sp.DPZ-SYz-2-3 and all known bacterial classifications.Its calmodulin (calmodulin) sequence is than the big approximately 300bp of other bacterial classifications in addition, and its comparison fraction of coverage has only 41% approximately, and Fusarium cf.solani PUF008 (HQ412318.1) sequence similarity nearest with its sibship has only 79%.In conjunction with its morphological feature; We confirm that it is a fungal strain novel species, and it is included into Acremonium (Acremonium), called after branch top spore (Acremonium sp.) DPZ-SYz-2-3; Be 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:4810.
Research shows to the cellulase activity of strains A cremonium sp.DPZ-SYz-2-3 (the CMC enzyme is lived): bacterial strain is under 30 ℃, pH 7.0 conditions, and the liquid fermenting cellulase activity reaches 145.6U/L (Fig. 5) about 10 days.Carry out filter paper degraded test with the bacterium liquid formulation that contains Acremonium sp.DPZ-SYz-2-3 simultaneously.The result shows: the degradation treatment filter paper rate of weight loss through 10 days reaches 71.5%, and it is active to explain that Acremonium sp.DPZ-SYz-2-3 bacterial strain has preferably a biomass degradation such as Mierocrystalline cellulose.Therefore second purpose of the present invention provides the application of a top spore (Acremonium sp.) DPZ-SYz-2-3 in cellulase-producing.
The 3rd purpose of the present invention provides the application of a top spore (Acremonium sp.) DPZ-SYz-2-3 in preparation biological organic matter degraded bacterial manure.
The invention provides a kind of fungi novel species: branch top spore (Acremonium sp.) DPZ-SYz-2-3, this bacterium has the cellulase-producing activity, can be used for the production of cellulose enzyme, and therefore production and the utilization to cellulase has significant values.Further can be used to make biological organic matter degraded bacterial manure.
Branch top spore (Acremonium sp.) DPZ-SYz-2-3 of the present invention 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:4810.
Description of drawings:
Fig. 1 is that Acremonium sp.DPZ-SYz-2-3 bacterium colony is in growing state on the malt extract medium (A), optical microscope photograph (B and C) and Acremonium sp.DPZ-SYz-2-3 bacterium colony decolouring situation (D) on the Congo red flat board of CMC-;
Fig. 2 is Acremonium sp.DPZ-SYz-2-3 site plan in rDNA internal transcribed spacer district (ITS) unrooted phylogenetic tree;
Fig. 3 is Acremonium sp.DPZ-SYz-2-3 at beta tubulin (site plan in the unrooted phylogenetic tree of β-tubulin);
Fig. 4 is Acremonium sp.DPZ-SYz-2-3 site plan in calmodulin (calmodulin) unrooted phylogenetic tree;
Fig. 5 is Acremonium sp.DPZ-SYz-2-3 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:
One, materials and methods
1, material
11 soil sample collections
Sample picks up from littoral mangrove area Rhizophora stylosa (Rhizophora stylosa) the rhizosphere settling in red Shahe, Sanya, Hainan Province in September, 2010, and 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 isolation medium (g/L):
The compound method of every liter of isolation 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) 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 (g/L):
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.
1.2.4 fermention medium (g/L):
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.
1.2.5 filter paper degradation experiment substratum (g/L):
Every liter of filter paper degradation experiment culture medium preparation method is following: peptone 0.5g, yeast powder 1.0g, sucrose 0.5g, Zulkovsky starch 0.5g, filter paper 3.3g, NaCl 2.0g, CaCO
31.0g, MgSO
41.0g, water 1000mL.
2, method
2.1 the separation screening of bacterial strain
From appearance choose the eugonic Rhizophora stylosa plant of three strains (Rhizophora stylosa); Gather their rhizosphere soil sample 50g; Place the triangular flask that contains the 50ml sterile purified water with getting about 5g behind the three duplicate samples mixings; 37 ℃, 150r/min concussion shake up 15min, leave standstill the bacterium liquid gradient dilution to 10 of getting 1ml behind the 30sec
-3, 10
-4, 10
-5, inoculum size is 0.05mL, coats on the isolation medium, 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 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, obtains a strain bacterial strain pure growth therefrom; Called after DPZ-SYz-2-3 is stored in the storage medium.
2.2 strain morphology is learned characteristic
The morphological feature of bacterial strain and preliminary appraisal basis " fungi identification handbook ".
The DPZ-SYz-2-3 bacterial strain that above-mentioned steps screens, its bacterial classification morphology is described below: as shown in Figure 1, the bacterial strain vegetative hyphae is very thin, and tool is separated, branch, wide 1.0-2.5 μ m.No specialization conidiophore, conidiogenous cell directly is born on the mycelia; Bottle stalk formula is produced spore, produces spore bottle stalk lanceolar, and is straight or crooked, simply or idol branch is arranged, long 16.4-34.2 μ m, the wide 1.2-2.5 μ of base portion m, top less than 1.0 μ m; Conidium is oval, rare nearly column, and 2.4-4.5 * 1.0-2.0 μ m assembles agglomerating.Colony growth is slower on the wort agar substratum, following 14 days colony diameter 18-24mm of 25 ℃ of dark conditions, and white, cotton-shaped, aerial hyphae is luxuriant, and a large amount of rhizomorphs are arranged; The bacterium colony back side is light brown, does not have water-soluble pigment, and 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 25mm 30 ℃ of cultivations, show that this bacterial strain has higher biomass degradations such as Mierocrystalline cellulose 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-SYz-2-3 that obtains extracts DNA, is used for the amplification of rRNA 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
ITS?1：5′--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′。
Universal primer cmd5/cmd6 (Seung-Beom Hong, Hye-Sun Cho, et al2006) is adopted in the amplification of calmodulin (calmodulin) sequence
cmd5：5′-CCGAGTACAAGGAGGCCTTC-3′，
cmd6：5′-CCGATAGAGGTCATAACGTGG-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 cmd5/cmd6 of utilization calmodulin (calmodulin) sequence carries out pcr amplification, obtains the calmodulin 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 calmodulin 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 strains A cremonium sp.DPZ-SYz-2-3 and known fungi have bigger otherness.Through its rDNA internal transcribed spacer district (ITS) sequence sequencing analysis is shown; With the nearer isolating ascomycetess (AF502849.1 and AF502848.1) that are two strains from the leaf that rots of its sibship; Its similarity reaches 98%, but the author does not further classify to it; And with the nearest Acremonium bacterial strain of its sibship be AB540578.1, it and Acremonium sp.DPZ-SYz-2-3 have only 90% similarity.(Blast of the sequence of β-tubulin) analyzes and shows its beta tubulin: only the have an appointment similarity of 75%-85% of the beta tubulin district of Acremonium sp.DPZ-SYz-2-3 and all known bacterial classifications.Its calmodulin (calmodulin) sequence is than the big approximately 300bp of other bacterial classifications in addition, and its comparison fraction of coverage has only 41% approximately, and Fusarium cf.solaniPUF008 (HQ412318.1) sequence similarity nearest with its sibship has only 79%.In conjunction with its morphological feature, we confirm that it is a fungal strain novel species, and it is included into Acremonium (Acremonium), called after branch top spore (Acremonium sp.) DPZ-SYz-2-3.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:4810.
2.4 the mensuration of cellulase activity (the CMC enzyme is lived)
DPZ-SYz-2-3 is inoculated in the fermention medium with branch top spore (Acremonium 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 secondary fermentation liquid reaches 145.6U/L.Show that thus branch top spore (Acremonium sp.) DPZ-SYz-2-3 of the present invention 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 bacterial strain preparation filter paper degraded test
DPZ-SYz-2-3 is inoculated on the solid PDA flat board with branch top spore (Acremonium sp.); Get with the 6cm punch tool to a certain size back Deng bacterium colony length and to leave standstill cultivation 10 days in the filter paper degradation experiment substratum that three ferfas tongues are inoculated in three 250ml respectively; After 10 days, the filter paper rate of weight loss in the substratum is 71.5%.
Claims (3)
1. a branch top spore (Acremonium sp.) DPZ-SYz-2-3, its deposit number is: CGMCC No:4810.
2. the application of described top spore of claim 1 (Acremonium sp.) DPZ-SYz-2-3 in cellulase-producing.
3. the application of described top spore of claim 1 (Acremonium sp.) DPZ-SYz-2-3 in preparation biological organic matter degraded bacterial manure.
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| CN105420218B (en) * | 2015-12-23 | 2019-02-05 | 天津科技大学 | A kind of xyloside of low temperature/arabinofuranosidase glucosides bifunctional enzyme AX543 and its gene and application |
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| CN113151004B (en) * | 2021-03-16 | 2022-11-04 | 黑龙江大学 | Novel strain of Asira zeylanica and application thereof |
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| CN115369042B (en) * | 2021-05-18 | 2024-04-05 | 中国科学院微生物研究所 | Acremonium 320 and application thereof |
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