CN102363748B - New fungus Acremonium sp. DPZ-SYz-2-3 for high efficiency cellulose degradation and application thereof - Google Patents

Abstract

The invention discloses a new high-efficiency cellulose-degrading fungus Acremonium sp.DPZ-SYz-2-3 and its application. Acremonium sp (Acremonium sp) DPZ-SYz-2-3 was preserved in the General Microorganism Center (CGMCC) of China Committee for Microorganism Culture Collection (CGMCC) on May 16, 2011, address: Yard 3, No. 1 Beichen West Road, Chaoyang District, Beijing No., Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC No: 4810. The bacterium has cellulase-producing activity and can be used to produce cellulase, so it is of great value to the production and utilization of cellulase. Further, it can be used to make bio-organic matter degrading bacterial fertilizer.

Description

A new species of high-efficiency cellulose-degrading fungus Acremonium sp.DPZ-SYz-2-3 and its application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a new high-efficiency cellulose-degrading fungus Acremonium sp.DPZ-SYz-2-3 and its application in producing cellulase and preparing bio-organic matter-degrading bacterial fertilizer.

Background technique

Mangrove is a woody plant community distributed in the intertidal zone of tropical and subtropical coasts, and it is a unique forest type on the beach (LinP et al.1999). Although it only accounts for one-thousandth of the global land area, it forms four high-productivity marine ecosystems with high productivity equivalent to the Amazon rainforest, coral reef ecosystems, upwelling areas, and seagrass bed ecosystems (Longhan et al. , 2005). There are 15,122 hectares of mangroves in my country, which are mainly distributed in Hainan, Guangdong, Guangxi and other places. Among them, Hainan Province is the region with the most species of mangroves and the best growth in my country. Mangrove sediments are characterized by strong reducing properties, strong acidity, high salinity, and rich nutrients (Takeuchi M et al., 1998; Lin Peng, 1997). Therefore, a large number of unique microorganisms, enzymes and genetic resources are contained here. In recent years, the unique ecological environment and rich species diversity of mangroves have attracted the attention of more marine scientists. However, the research on microbiology in the mangrove area started late, and the research is relatively weak. Among the research reports on mangroves, the microbiological research literatures in mangrove areas account for very little share. At present, microbiological research in mangrove areas mainly focuses on: the study of mangrove microbial flora; the study of microbiology in the process of litter decomposition; the study of mangrove rhizosphere actinomycetes; al., 2005; Muniswaran A. et al. 1994). Because the biomass resources such as litter in the mangrove area are very rich, a large number of microorganisms in the mangrove area can produce substances such as cellulolytic enzymes and lignin decomposing enzymes.

Energy and environmental issues are central issues related to national security and sustainable economic and social development faced by all countries in the world today, and have become the focus of global attention. Therefore, people began to turn their attention to the sustainable development of renewable energy systems. Experts believe that the biomass resource conversion system is a technology platform leading the third world energy revolution. Lignocellulose is the most abundant renewable resource on the earth, and it is also the resource with the lowest utilization rate at present. It is the focus of new resource strategies of various countries. The available lignocellulose in my country is about 700 million tons per year. These abundant and cheap natural resources mainly come from agricultural and forestry waste, industrial waste and urban waste. Therefore, cellulose is an inevitable direction for the development of energy utilization in the future (Zhang Bailiang, et al., 2007). Lignocellulose includes cellulose, hemicellulose, and lignin. The main way to use lignocellulose to produce fuel ethanol is to first degrade the cellulose and hemicellulose in lignocellulose into monosaccharides, and then use yeast to ferment the monosaccharides to produce fuel ethanol. Among them, the low efficiency of degrading lignocellulose into monosaccharides is a factor restricting its industrial utilization. Therefore, the screening of new efficient cellulose or hemicellulose degrading strains is the key to the efficient utilization of cellulose resources. There have been more than 20 years of research on cellulose-degrading bacteria at home and abroad, and research reports have focused on several genera such as white rot fungi, Phaneroderma, and Trametes (Christopher HV et al., 2003; Lekounogou S et al., 2008), among which Trichoderma is the most widely studied cellulase producer, and 20% of the cellulase in the world cellulase market comes from Trichoderma and Aspergillus. However, in recent years, the research reports on new cellulose-degrading strains are also increasing (Mario CNS et al., 2008; Revankar MS et al., 2006).

Invention content:

The first object of the present invention is to provide a new species of mangrove rhizosphere fungus with higher cellulose degrading activity: Acremonium acremonium, screened from the rhizosphere soil of tropical mangroves in Sanya City, Hainan Province, China (Acremonium sp.) DPZ-SYz-2-3, which was preserved in the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microbial Cultures on May 16, 2011, address: No. 1, Beichen West Road, Chaoyang District, Beijing No. 3, Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC No: 4810.

The new high-efficiency cellulose-degrading fungus species of the present invention: Acremonium sp DPZ-SYz-2-3 is screened from rhizosphere soil of tropical mangroves in Sanya City, Hainan Province.

Its bacteriological characteristics are described as follows:

Description of the strain: As shown in Figure 1, the vegetative hyphae of the strain are slender, separated, branched, and 1.0-2.5 μm wide. No specialized conidiophores, spore-forming cells grow directly on the hyphae; phialides, phialides lanceolate, straight or curved, simple or occasionally branched, 16.4-34.2μm long, base 1.2-2.5 μm wide, less than 1.0 μm at the top; conidia are oval, rarely nearly columnar, 2.4-4.5×1.0-2.0 μm, aggregated into clusters. The colony on the malt juice agar medium grows slowly, and the diameter of the colony is 18-24mm at 25°C in the dark for 14 days, white, flocculent, with lush aerial hyphae and a large number of mycelium bundles; the back of the colony is light brown, without water-soluble pigments , have obvious resistance to various antibiotics. The strain was inoculated on a plate with carboxymethylcellulose sodium (CMC-Na) as the main carbon source, and after culturing at 30°C for 3 days, a hydrolysis transparent circle with a diameter of 25 mm could be formed (Fig. 1D), indicating that the strain had relatively High biodegradation activity such as cellulose.

Genomic DNA was extracted from the pure culture of the strain Acremonium sp.DPZ-SYz-2-3, and the ITS sequence was obtained by PCR amplification and sequencing analysis using rDNA Internal Transcribed Spacer (ITS) sequence-specific primers ITS1/ITS4. The sequence is shown in SEQ ID NO.1. Using β-tubulin (β-tubulin) sequence-specific primers Bt2a/Bt2b, the β-tubulin sequence was obtained through PCR amplification and sequencing analysis, and its sequence is shown in SEQ ID NO.2. Using primers cmd5/cmd6 specific to the sequence of calmodulin, the sequence of calmodulin was obtained by PCR amplification and sequencing analysis, and its sequence is shown in SEQID NO.3. The ITS sequence, β-tubulin sequence, and calmodulin sequence were compared with the sequences in the GenBank database through the BLAST software in GenBank, and no completely similar sequences were found in the database, indicating that these three gene sequences were new gene sequences discovered for the first time .

In the GenBank database, some representative gene sequences with high similarity to the determined sequence were selected, and the phylogenetic tree was constructed by the Neibor-joining method through ClustalW software and Mega software (see Figures 2, 3, 4) for phylogenetic analysis. It can be seen from the phylogenetic tree that the strain Acremonium sp.DPZ-SYz-2-3 is quite different from the known fungi. Sequencing analysis of its rDNA internal transcriptional spacer (ITS) sequence showed that its close relatives were two strains of Ascomycetes (AF502849.1 and AF502848.1) isolated from rotten leaves, and their similarity reached 98%. But the author did not further classify it; the closest relative to it is Acremonium sp. AB540578.1, which has only 90% similarity with Acremoniumsp.DPZ-SYz-2-3. The Blast analysis of its β-tubulin sequence showed that Acremonium sp.DPZ-SYz-2-3 has only about 75%-85% similarity with the β-tubulin region of all known strains. In addition, its calmodulin sequence is about 300bp larger than other strains, and its alignment coverage is only about 41%, and its closest relative, Fusarium cf.solani PUF008 (HQ412318.1), has a sequence similarity of only 79%. Combined with its morphological characteristics, we determined that it was a new species of fungus, classified it into the genus Acremonium, and named it Acremonium sp. DPZ-SYz-2-3. It was deposited in the General Microbiology Center (CGMCC) of China Committee for Culture Collection of Microbial Cultures (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and deposit number: CGMCC No: 4810.

The research on the cellulase activity (CMC enzyme activity) of the strain Acremonium sp.DPZ-SYz-2-3 showed that the cellulase activity of the strain in liquid fermentation reached 145.6U/L in about 10 days at 30°C and pH 7.0 (Figure 5). At the same time, the filter paper degradation test was carried out with the bacterial liquid preparation containing Acremoniumsp.DPZ-SYz-2-3. The results showed that the weight loss rate of the filter paper reached 71.5% after 10 days of degradation treatment, which indicated that the Acremonium sp.DPZ-SYz-2-3 strain had better biodegradation activity such as cellulose. Therefore the second object of the present invention is to provide the application of Acremonium sp. DPZ-SYz-2-3 in producing cellulase.

The third object of the present invention is to provide the application of Acremonium sp. DPZ-SYz-2-3 in the preparation of biological organic matter degradation bacterial fertilizer.

The invention provides a new species of fungus: Acremonium sp. DPZ-SYz-2-3, which has cellulase-producing activity and can be used to produce cellulase, so it has great influence on the production and production of cellulase Utilization has significant value. Further, it can be used to make bio-organic matter degrading bacterial fertilizer.

Acremonium sp. of the present invention (Acremonium sp.) DPZ-SYz-2-3 was preserved on May 16, 2011 in the General Microbiology Center (CGMCC) of China Microbiological Culture Collection Management Committee, address: Beichen West Road, Chaoyang District, Beijing No. 1, No. 3, Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC No: 4810.

Description of drawings:

Figure 1 is the growth of Acremonium sp.DPZ-SYz-2-3 colonies on wort medium (A), optical microscope photos (B and C) and Acremonium sp.DPZ-SYz-2-3 colonies in CMC-Congo Decolorization on the red plate (D);

Fig. 2 is a position map of Acremonium sp.DPZ-SYz-2-3 in the rootless phylogenetic tree of rDNA internal transcriptional spacer (ITS);

Fig. 3 is the position diagram of Acremonium sp.DPZ-SYz-2-3 in the unrooted phylogenetic tree of β-tubulin (β-tubulin);

Fig. 4 is a position map of Acremonium sp.DPZ-SYz-2-3 in the unrooted phylogenetic tree of calmodulin (calmodulin);

Fig. 5 is a graph of Acremonium sp.DPZ-SYz-2-3 liquid fermentation cellulase activity (CMC enzyme activity).

Detailed ways:

The following examples are to further illustrate the present invention, rather than limit the present invention.

Example 1:

1. Materials and methods

1. Materials

11 Soil sample collection

The samples were collected from the rhizosphere deposits of Rhizophora stylosa in the mangrove area along the Hongsha River in Sanya City, Hainan Province in September 2010. After the samples were collected, they were put into sterilized sealed polyethylene bags and brought back to the laboratory at a low temperature of 4°C. save.

1.2 Medium

1.2.1 Separation medium (g/L):

The preparation method of every liter of separation medium is as follows: Potato juice (peel the potato, dig out the bud eyes, wash, slice. Weigh 200g and put it into 1000ml tap water, boil with a slow fire for 30min, filter with double gauze, add water to the filtrate to make up to 1000ml) 800ml , sucrose, 20.0g; NaCl, 1.5g; agar, 15g, soil extract, 200ml; after sterilizing at 121°C, wait for the medium to cool to 60°C, add filter-sterilized 100μg/ml ampicillin and 100μg/ml 1ml each of streptomycin sulfate.

1.2.2 Rescreening medium (g/L):

_The preparation method of double sieving medium per liter is as follows: CMC- _Na 10.0g; peptone 0.5g; yeast extract 0.5g; sucrose 0.5g; KNO ₃ 1.0g; K ₂ HPO ₄ 0.5g; g; NaCl 1.5g; agar 15g, water 1000mL. Sterilized at 121°C for use

1.2.3 Preservation medium (g/L):

The preparation method of each liter of preservation medium is as follows: Potato juice (peel the potato, dig out the bud eyes, wash, slice. Weigh 200g and put it into 1000ml tap water, boil with a slow fire for 30min, filter with double gauze, add water to the filtrate to make up to 1000ml) 1000ml , sucrose, 20.0g; NaCI, 1.5g; agar, 15g.

1.2.4 Fermentation medium (g/L):

The preparation method per liter of fermentation medium is as follows: CMC-Na 5.0g; peptone 1.0g; yeast extract 1.0g; sucrose 1.0g; KNO ₃ 1.0g; K ₂ HPO ₄ 0.5g; MgSO ₄ .7H ₂ O 0.5g ; NaCl 1.5g; water 1000mL.

1.2.5 Medium for filter paper degradation experiment (g/L):

The preparation method of each liter of filter paper degradation experiment medium is as follows: peptone 0.5g, yeast powder 1.0g, sucrose 0.5g, soluble starch 0.5g, filter paper 3.3g, NaCl 2.0g, CaCO ₃ 1.0g, MgSO ₄ 1.0g, water 1000mL .

2. Method

2.1 Isolation and screening of strains

Select three vigorously growing red sea olive plants (Rhizophora stylosa) from the sample plot, collect 50 g of their rhizosphere soil samples, mix the three samples, get about 5 g and place them in a triangular flask containing 50 ml of sterilized distilled water, at 37 ° C, Shake at 150r/min for 15min, let it stand for 30sec, take 1ml of the bacterial solution and dilute it to 10 ^-3 , 10 ^-4 , 10 ^-5 , inoculum size is 0.05mL, spread on the separation medium, each treatment set 8 repetitions. Cultivate at 37°C for 3-5 days, pick a single colony with a typical morphology and perform spot purification 2-3 times to obtain a pure culture. Inoculate the obtained pure culture on the re-screening medium, and culture it upside down at 30°C for 3 days, cover the medium with a mass concentration of 1 mg/mL Congo red solution for 10-15 minutes on the medium where colonies grow, and pour off the Congo red solution , and then add a NaCl solution with a concentration of 1mol/L, and pour off the NaCl solution after 15 minutes. At this time, a transparent circle will appear around the colony producing cellulase. According to the preliminary decolorization effect, the cellulase-producing strains were identified, the diameter of the transparent circle and the colony diameter were measured, and the strains with better effects were screened for further research, and a pure culture of the strain was obtained, named DPZ-SYz-2 -3, preserved in preservation medium.

2.2 Morphological characteristics of strains

The morphological characteristics and preliminary identification of the strain were based on the "Handbook of Fungal Identification".

The morphological description of the DPZ-SYz-2-3 strain screened in the above steps is as follows: As shown in Figure 1, the vegetative hyphae of the strain are slender, separated, branched, and 1.0-2.5 μm wide. No specialized conidiophores, spore-forming cells grow directly on the hyphae; phialides, phialides lanceolate, straight or curved, simple or occasionally branched, 16.4-34.2μm long, base 1.2-2.5 μm wide, less than 1.0 μm at the top; conidia are oval, rarely nearly columnar, 2.4-4.5×1.0-2.0 μm, aggregated into clusters. The colony on the malt juice agar medium grows slowly, and the diameter of the colony is 18-24mm at 25°C in the dark for 14 days, white, flocculent, with lush aerial hyphae and a large number of mycelium bundles; the back of the colony is light brown, without water-soluble pigments , have obvious resistance to various antibiotics. The strain was inoculated on a plate with carboxymethylcellulose sodium (CMC-Na) as the main carbon source, and after culturing at 30°C for 3 days, a hydrolysis transparent circle with a diameter of 25 mm could be formed (Fig. 1D), indicating that the strain had relatively High biodegradation activity such as cellulose.

2.3 Extraction of strain genomic DNA and molecular biology identification

The obtained pure culture strain DPZ-SYz-2-3 was extracted with Genome Extraction Kit of Shanghai Sangon Company, and then used for rRNA gene amplification.

The partial sequence of fungal ITS region was amplified using universal primers ITS1/ITS4 (T.J.White, T.Bruns, et al 1990)

ITS 1: 5′--TCCGTAGGTGAACCTGCGG--3′,

ITS4: 5'--TCCTCCGCTTAT TGATATGC--3'.

The amplification of β-tubulin (β-tubulin) sequence uses universal primers Bt2a/Bt2b (Glass & Donaldson, 1995)

Bt2a: 5'--GGTAACCAAATCGGTGCTGCTTTC--3'

Bt2b: 5'--ACCCTCAGTGTAGTGACCCTTGGC--3'.

Amplification of calmodulin sequence using universal primers cmd5/cmd6 (Seung-BeomHong, Hye-Sun Cho, et al2006)

cmd5: 5'-CCGAGTACAAGGAGGCCTTC-3',

cmd6: 5'-CCGATAGAGGTCATAACGTGG-3'.

The PCR reaction system and reaction conditions are as follows:

The PCR reaction system includes: The PCR reaction procedure is:

Take 2 μL of PCR product and perform product detection by 1% agarose electrophoresis. After the amplified product was purified, it was sent to a sequencing company for sequencing, and the ITS sequence was obtained by PCR amplification and sequencing analysis using rDNA internal transcriptional spacer (ITS) sequence-specific primers ITS1/ITS4, the sequence of which is shown in SEQ ID NO.1 Show. Using β-tubulin (β-tubulin) sequence-specific primers Bt2a/Bt2b, the β-tubulin sequence was obtained through PCR amplification and sequencing analysis, and its sequence is shown in SEQ ID NO.2. The primer cmd5/cmd6 specific to the sequence of calmodulin was used for PCR amplification, and the calmodulin sequence was obtained through PCR amplification and sequencing analysis, and its sequence is shown in SEQ ID NO.3. The ITS sequence, β-tubulin sequence, and calmodulin sequence were compared with the sequences in the GenBank database through the BLAST software in GenBank, and no completely similar sequences were found in the database, indicating that these three gene sequences were new gene sequences discovered for the first time .

In the GenBank database, some representative gene sequences with high similarity to the determined sequence were selected, and the phylogenetic tree was constructed by the Neibor-joining method through ClustalW software and Mega software (see Figures 2, 3, 4) for phylogenetic analysis. It can be seen from the phylogenetic tree that the strain Acremonium sp.DPZ-SYz-2-3 is quite different from the known fungi. Sequencing analysis of its rDNA internal transcriptional spacer (ITS) sequence showed that its close relatives were two strains of Ascomycetes (AF502849.1 and AF502848.1) isolated from rotten leaves, and their similarity reached 98%. But the author did not further classify it; the closest relative to it is Acremonium sp. strain AB540578.1, which has only 90% similarity with Acremonium sp.DPZ-SYz-2-3. The Blast analysis of its β-tubulin sequence showed that Acremonium sp.DPZ-SYz-2-3 has only about 75%-85% similarity with the β-tubulin region of all known strains. In addition, its calmodulin sequence is about 300bp larger than that of other strains, and its alignment coverage is only about 41%. The sequence similarity with its closest relative Fusarium cf.solaniPUF008 (HQ412318.1) is only 79%. Combined with its morphological characteristics, we determined that it was a new species of fungus, classified it into the genus Acremonium, and named it Acremonium sp. DPZ-SYz-2-3. The bacterium was preserved in the General Microbiology Center (CGMCC) of the China Committee for the Collection of Microbial Cultures on May 16, 2011, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, preservation number: CGMCC No: 4810.

2.4 Determination of cellulase activity (CMC enzyme activity)

Acremonium sp. DPZ-SYz-2-3 was inoculated in the fermentation medium, and under the conditions of 30°C and pH 7.0, a small amount of fermentation broth was taken every day to measure the cellulase activity of the fermentation broth. The fermentation broth was centrifuged at 6000r/min for 10min, and the supernatant was used as crude enzyme solution. Add 0.5mL of appropriately diluted enzyme solution to a 25ml graduated glass test tube with a stopper, preheat it in a constant temperature water bath at 50°C for 2 minutes, then add 2.0mL of 1% sodium carboxymethylcellulose solution prepared with pH 4.8 acetate buffer, at 50°C Enzyme hydrolysis in constant temperature water bath for 30min, then add 2.5mL of DNS in boiling water bath for 5min, after cooling in running water, dilute to 25ml and mix well, measure the absorbance value at 520nm, and calculate the enzyme activity after comparing with the standard curve. The enzyme activity of fermented liquid is as shown in Figure 5, as can be seen from Figure 5, fermented liquid has cellulase activity, and along with prolonging of fermentation time, its cellulase activity increases gradually, and the cellulase activity of fermented liquid reaches 10 days later. 145.6U/L. This shows that Acremonium sp. DPZ-SYz-2-3 of the present invention has cellulase-producing activity.

Enzyme activity is defined according to the International Units: the amount of enzyme required to catalyze the hydrolysis of cellulose to produce 1 μmol of glucose per minute is 1 enzyme activity unit U.

2.5 Degradation test of strain preparation filter paper

Inoculate Acremonium sp. DPZ-SYz-2-3 onto a solid PDA plate, and after the colony grows to a certain size, use a 6cm hole punch to inoculate three pieces of bacterial lawn into three 250ml filter paper degradation experiments The medium was cultured statically for 10 days, and after 10 days, the weight loss rate of the filter paper in the medium was 71.5%.

Claims (3)

1. Acremonium sp. DPZ-SYz-2-3, whose preservation number is: CGMCC No: 4810. 2. the application of acremonium sp. (Acremonium sp.) DPZ-SYz-2-3 described in claim 1 in producing cellulase. 3. the application of acremonium sp. (Acremonium sp.) DPZ-SYz-2-3 described in claim 1 in the preparation of bio-organic matter degradable bacterial fertilizer.

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