CN113151004A - Novel strain of Asira zeylanica and application thereof - Google Patents

Abstract

A new strain of Asira zeylanica and application thereof belong to the technical field of microorganism application. The invention provides a new fungus with cellulase production capacity and antibacterial activity, and further improves the utilization rate of cellulose resources, the fungus is separated from forest litters collected from great XingAnling forest to obtain a new species of a strain of a fungus, the new species is named as a strain of a fungus of a great fungus of a.

Description

Novel strain of Asira zeylanica and application thereof

Technical Field

The invention belongs to the technical field of microbial application, and particularly relates to a novel strain of azonian and application thereof.

Background

Fungi are an important natural resource that can produce cellulose degrading enzymes and also produce large amounts of antibacterial compounds. For example, forest litters are rich in lignocellulose and their degradation in nature depends primarily on the production of lignocellulose degrading enzymes by microorganisms such as fungi. In the aspect of antibacterial compounds, fungi can produce famous antibiotics such as penicillin, griseofulvin, echinocandin and the like. Over 148000 fungi are currently found, most of which have not been fully exploited. Currently, the commercialized fungi of cellulose-degrading enzymes and antibacterial compounds are mainly concentrated in a few fungi of the genera Trichoderma (Trichoderma), Penicillium (Penicillium), Aspergillus (Aspergillus), and the like. Over 2000 new fungal species are reported each year and their ability to produce cellulose degrading enzymes or antibacterial compounds is worthy of screening.

Currently used cellulose degrading enzyme producing bacteria are mainly concentrated in several genera, and various methods are attempted to stimulate their potential. Even so, a large amount of cellulose-rich renewable resources are wasted each year, suggesting that research on cellulase-producing fungi is yet to be further improved. At present, a large number of fungi resources are not researched and utilized, and novel cellulase-producing fungi are searched for as alternative resources, so that the utilization rate of cellulose resources is expected to be further improved.

The fungus of the genus alismoid (Arxiella) belonging to the class of Ascomycetes (dothideomyces) was originally isolated in 1967 from forest litters and has the typical morphological characteristics of producing unique naviculiform spores with a sharp oblique tip at the end of the spores. Currently, this genus includes: three species of Arxiella terrestris (Arxiella terrestris), Arxiella palmata (Arxiella dolichiana), and Arxiella lunata (Arxiella lunata). A strain of A.terrestris (Arxiella terrestris), the first species discovered in this genus, has been reported to have pectinase activity and protease activity, but the strain has no cellulolytic activity (Jacob M. students on Diversity, Ecology and activity of the microbial enzyme associated with tissue culture of fungi Linn. and Carissa Congesta light from Goa State [ D ]. Goa University,2000.) and no antifungal activity has been found in this genus.

Disclosure of Invention

The invention provides a new strain of the alura japonica, aiming at searching for a new fungus with cellulase production capability and antibacterial activity, wherein the strain number of the alura japonica is SGSF303, the strain is named as a strain of the alura longispora (Arxiella longispora), the Arzeala japonica is classified and named as Arxiella sp.SGSF303, the strain is preserved in the China center for type culture Collection at 1 month and 18 days 2021 month in China, Wuhan university, and the preservation number is CCTCC NO: m2021101.

Further defined, the a ze is isolated from a forest litter in greater Khingan mountains.

The invention also provides application of the aliskirilowii in cellulose degradation of agricultural wastes or a composting process related to cellulose degradation and utilization.

The application of the azosemium for preventing and treating rhizoctonia solani is provided.

The application of the azonian for producing the cellulase.

Further defined, the application is to use bran to induce the aspergillus to produce cellulase.

Optimally, the composition of the culture medium formula for producing the cellulase comprises 15g of bran, 5g of peptone, 2g of monopotassium phosphate and 1L of water.

The invention also provides application of the aliskirilowii in producing antibacterial compounds.

Further defined, the application is a compound for inducing the aliskirilowii to generate the rhizoctonia solani by using nicotinamide.

Advantageous effects

According to the invention, a new strain SGSF303 of the genus Azelavia is obtained by separating litters under forests of great Khingan mountains, and experiments prove that the strain has good cellulose degradation activity and antifungal activity. In the aspect of producing cellulase, the optimal carbon source for inducing the strain to produce the cellulase is bran, and the optimal nitrogen source is peptone. When the bran is used as a fermentation substrate, the enzyme activity is improved by about 2 times, the yield of the endonuclease is relatively high, and the specific enzyme activity is as follows: the activity of the endo-enzyme reaches 0.86U/mL, the activity of the exonuclease reaches 0.58U/mL, the activity of the beta-glucosidase reaches 0.26U/mL, and the activity of the filter paper enzyme reaches 0.32U/mL. In terms of antibacterial activity, the bacterium can produce an active compound against Rhizoctonia solani under the induction of nicotinamide.

Drawings

FIG. 1 shows the effect of different culture conditions on the growth of SGSF303 colonies, wherein A in FIG. 1 is the effect of different temperatures on the growth of SGSF303 colonies; b in FIG. 1 is the effect of different pH values on the growth of SGSF303 colonies;

FIG. 2 strain SGSF303 morphological features, where A (1) in FIG. 2 is colony front; a (2) in FIG. 2 is the reverse side of the colony; b in fig. 2 is the hyphal and spore characteristics, scale: 20 μm;

FIG. 3 shows the effect of different carbon and nitrogen sources on the production of cellulase by SGSF303 strain, wherein A in FIG. 3 is the production of cellulase by SGSF303 strain cultured at 5d by adding different carbon sources (1.5%), the production of cellulase by SGSF303 strain cultured at 5d by adding different nitrogen sources (0.5%), and different letters on the bar chart indicate that the difference of cellulase induced by different culture media of the same enzyme is significant (p < 0.05).

Detailed Description

The present invention will be described in further detail with reference to the following examples and drawings, and the procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for those specifically mentioned below, and the present invention is not particularly limited thereto.

Preparing a culture medium according to the following culture medium formula for later use:

(1) potato Dextrose Agar medium (Potato Dextrose Agar, PDA): 200g of potato, 20min of boiling water leaching liquor, 20g of glucose, 20g of agar and distilled water to constant volume of 1L.

(2) Corn Meal Agar medium (Corn Meal Agar, CMA): 60g of corn flour, 20g of agar and distilled water to a constant volume of 1L.

(3) Oat medium (oat Agar, OA): 30g of oat is extracted from boiling water for 1 hour, 20g of agar and distilled water are added to a constant volume of 1L.

(4) Malt Extract Agar (MEA): 40g of malt extract powder, 20g of agar and distilled water to a constant volume of 1L.

(5) Screening a culture medium: 15g of sodium carboxymethylcellulose, 5g of sodium chloride, 1g of monopotassium phosphate, 0.2g of magnesium sulfate heptahydrate, 10g of peptone, 5g of yeast extract powder, 20g of agar and distilled water to a constant volume of 1L.

(6) Potato Dextrose Broth (PDB): 200g of potato, 20min of boiling water leaching liquor, 20g of glucose and distilled water to constant volume of 1L.

(7) Filter paper degradation experiment liquid culture medium: 1g of monopotassium phosphate, 0.1g of sodium chloride, 0.3g of magnesium sulfate heptahydrate, 2.5g of sodium nitrate, 0.01g of ferric chloride, 0.1g of anhydrous calcium chloride, a plurality of filter paper strips of 1cm multiplied by 6cm, and the volume of distilled water is up to 1L.

(8) Enzyme production basal medium: 15g of sodium carboxymethylcellulose, 5g of yeast extract powder, 2g of monopotassium phosphate and distilled water to a constant volume of 1L.

(9) Maltose Yeast extract media (Sabouraud Maltose Yeast extract Agar, SMYA): 10g of peptone, 40g of maltose, 4g of agar, 10g of yeast extract powder and distilled water to a constant volume of 1L.

(10) LB Agar medium (Luria Bertani Agar, LBA): 5g of sodium chloride, 10g of tryptone, 5g of yeast extract powder and 15g of agar, and adding water to a constant volume of 1L.

(11) PDB + Nicotinamide (PDB plus Nicotinamide, P + Nic): 200g of potato, 20min of boiling water extract, 20g of glucose, 0.2g of nicotinamide and distilled water to constant volume of 1L.

Example 1 isolation and characterization of A. Azela SGSF303

(1) Isolation of Ashbya SGSF303

The strain separation is carried out by taking forest litters collected from great Khingan forest as a separation source. Firstly, drying an acquired litter sample, crushing the dried litter sample into particles of 100-; adding 50 μ L of particle suspension with different concentrations into 1/4PDA culture medium (other components except agar are diluted to 1/4) by dilution coating plate method, and uniformly coating the particles on the culture medium by using a coater; culturing at 25 deg.C, observing every 12h during culture period, picking colony to PDA plate, purifying and storing in slant. Purifying and culturing to obtain a fungus, and numbering the fungus as SGSF 303.

(2) Testing the influence of different culture media, temperatures and pH values on the growth of SGSF303 bacterial colony

Inoculating the strain SGSF303 on 4 different culture media (PDA, CMA, OA and MEA) to measure the growth rate of colonies by the different culture media; inoculating the SGSF303 strain on a PDA culture medium, culturing at 15 ℃,20 ℃, 25 ℃, 30 ℃ and 35 ℃ respectively, and measuring the growth rate of a bacterial colony; the strain SGSF303 was inoculated on PDA medium at pH4, 5, 6, 7, 8, 9 and 10, respectively, and the colony growth rate was measured. The colony growth rate was measured by the cross method (diameter 6mm excluding the inoculated cake).

The test results are shown in FIG. 1, which shows that the optimum temperature for the growth of SGSF303 strain on PDA medium is 20-25 deg.C, the growth rate is reduced significantly when the culture temperature is 30 deg.C or higher, and the growth rate is reduced but better than 30 deg.C at 15 deg.C. Compared with the influence of temperature, the influence of different pH values on the growth of colonies is small, the relative growth is fast when the pH value is 5-10, and the growth speed is obviously reduced when the pH value is 4.

(3) Morphological identification of Ashlaa SGSF303

Inoculating the strain SGSF303 on a PDA culture medium, culturing at 25 ℃ in the dark, observing a fungus colony by using a stereoscopic microscope, and shooting structures such as sporulation cells, spores and the like by using an optical microscope.

The colony morphology of the strain SGSF303 on the PDA culture medium is shown as A in figure 2, the morphology of the strain is shown as B in figure 2 shot by an optical microscope, and the observation shows that the colony culture characteristics are that the initial colony is white, the later colony is annular brown or dark grey brown, the outer edge is off-white, and the colony is full-edge or has ripples; the back of the colony is dark brown and has wrinkles. The hyphae and spores are characterized in that: the primary hyphae are transparent, loose and branched, and have diameter of 1.5-3 μm, and the old hyphae are brown, have septa, thick wall and partially swell, and have diameter of 2-5 μm. The spore-forming cells are spherical or egg-spherical, the spores are transparent and navicular, are connected occasionally, have 0-1 partition in the middle, have a size of (14-22) mum x (3-4) mum, a length of more than 20 μm and a maximum length of 24 μm, and have an average aspect ratio of more than 5 μm. No chlamydospores were found.

According to the characteristics of boat-shaped conidium and the like generated by the strain SGSF303, the strain belongs to the Arziella sp fungi. But its spores are larger than all known a zirella sp.

(4) Molecular biological identification of Ashbya SGSF303

The purified strain SGSF303 was first subjected to total DNA extraction by CTAB method, and then the Internal Transcribed Spacer (ITS) was subjected to PCR amplification using the fungal universal primers ITS1 and ITS4 for molecular biological identification. And (4) detecting the PCR product by electrophoresis, and sending the PCR product to a sequencing company for bidirectional sequencing. The credible sequence obtained after sequencing is compared and analyzed by using BLAST (Basic Local Alignment Search Tool, https:// BLAST. NCBI. nlm. nih. gov /) of NCBI website, and credible sequence splicing and phylogenetic analysis are carried out by using Mega7.0 software.

The GenBank accession number of the ITS sequence of strain SGSF303 is MT 921658. The most closely related classification unit of ITS ITS sequences is A. terrestris (Arxiella terrestis), and the similarity between their sequences is less than 98%. Furthermore, the ITS sequences of SGSF303 share less than 90% sequence similarity with both A.palmata (Arxiella dolichiana) and A.lunata (Arxiella lunata). Phylogenetic tree analysis revealed that the SGSF303 strain forms a single population with a strain of A.terrestris (Arxiella terrestris) or A.palmettii (Arxiella dolichiana).

The genus alismoid (Arxiella sp.) currently contains 3 species, which are classified according to their conidia size and DNA sequence into alismoid terrestris (Arxiella terrestris), alismoid lunata (Arxiella lunata) and alismoid palmata (Arxiella dolichardrae). The morphological characteristics and molecular biological characteristics of the strain SGSF303 are compared with those of similar bacteria to find that: the spores of the strain of alzeria were both navicular (flat bottom) or moon (curved bottom), but the spores of SGSF303 were larger than these 3 known species and the spores were much longer (average aspect ratio greater than 5). While the navicular spores of the Ashbya terrestris are (6-16) μm x (3-4.5) μm in size and the aspect ratio is about 2.9; the spores of A.lunata have a crescent spore size of (10-17) μm x (3-4) μm and an aspect ratio of about 3.9; the navicula size of a. palmata is (10-11) μm × (2.5-3) μm, and the aspect ratio is about 3.8(Rusco Q W1970, Crous PW 2014). From the above ITS sequence analysis of SGSF303, it is clear that the most similar strain is A.terrestris, but the average size of the spores of SGSF303 is significantly larger than A.terrestris, and the colonies of SGSF303 appear dark brown on PDA medium and different from dark green of A.terrestris. Furthermore, A.terrestris was tested to have no cellulolytic Activity (Papendorf M C, Two new genes of soil from South Africa [ J ]. Transactions of the British Mycological Society,1967,50(1):69, IN6-75, IN6.), (11.Jacob M. students on Diversity, Ecology and Activity of the Microfungi Associated with sight glass of corn State [ D ]. Goa University,2000.) but SGSF303 has cellulolytic Activity. The strain SGSF303 was therefore identified as a new species of A.alzeylanica, named A.alzeylanica (Arxiella longispora) and deposited in the center of the China center for type culture Collection, located in Wuhan, under the accession number CCTCC M2021101.

Example 2 application of Ashbya SGSF303 in cellulase production

(1) Qualitative test of cellulose degradation Activity

Congo red dyeing test

Firstly, obtaining a bacterial cake at the edge of a bacterial colony by using a puncher, inoculating the bacterial cake into a screening culture medium (15 g of sodium carboxymethylcellulose, 5g of sodium chloride, 1g of monopotassium phosphate, 0.2g of magnesium sulfate heptahydrate, 10g of peptone, 5g of yeast extract powder, 20g of agar and constant volume of distilled water to 1L), and repeating for three times. Culturing in a constant temperature incubator at 25 ℃ for 4 days, adding 1 mg/mL Congo red solution to stain the screening culture medium, pouring out the dye solution after 1h, washing for 1-2 times by running water, and adding 1mol/L NaCl solution to immerse and decolor for 30 min. Observing the growth condition of the bacterial colony, the size and the transparency degree of the degradation ring, recording the diameter (D/cm) of the degradation ring and the diameter (D/cm) of the bacterial colony, and determining the cellulose degradation activity of the bacterial colony.

The result shows that the degradation ring formed by the bacterial strain SGSF303 is clearly visible, the diameter of the degradation ring is 1.54 +/-0.27 cm, the diameter of a bacterial colony is 0.86 +/-0.06 cm, and the ratio of the degradation ring to the diameter of the bacterial colony is 1.79 +/-0.22, namely on a culture medium with sodium carboxymethyl cellulose, the bacterial strain can break long-chain cellulose, and the bacterial strain SGSF303 has cellulose degradation activity.

② filter paper strip degradation test

Adopting an improved filter paper strip degradation method, cutting a colony in PDA into a plurality of small blocks of 5mm multiplied by 5mm by using a scalpel, inoculating the small blocks into a PDB liquid culture medium, culturing for 4 days at 25 ℃ and 180r/min, absorbing 3mL of the small blocks, inoculating the small blocks into 50mL of starch-free filter paper degradation culture medium, and performing constant temperature shaking culture for 10 days at 25 ℃ and 180 r/min. A blank control with the same amount of sterile water was set and the effect of breaking the filter paper strips was observed every day.

The filter paper strip degradation test result shows that the filter paper strip is degraded into round small pieces after 4 days of inoculation of the bacterial strain SGSF303 and is in an amorphous shape, and the filter paper strip is pasty after 10 days and has small fibers which are dispersed and suspended in a liquid culture medium, so that the filter paper strip has strong potential for degrading cellulose.

(2) Influence of different carbon and nitrogen sources on cellulase production of SGSF303 strain

Preparing a crude enzyme solution by adopting liquid culture: sodium carboxymethylcellulose, microcrystalline cellulose, corncob powder, bran, straw powder and corn straw powder are respectively used as carbon sources, and the addition amount is 1.5%. In addition to the carbon source, 0.5% yeast extract powder (nitrogen source) and 0.2% potassium dihydrogen phosphate were added to prepare a culture medium. The PDB seed suspension of the SGSF303 strain was inoculated into 50mL of 6 different carbon source enzyme production media at 6% inoculum size and fermented in 250mL Erlenmeyer flasks. Culturing the fermented liquid at 25 deg.C and 180r/min for 5d, centrifuging the fermented liquid at 4 deg.C and 12000r/min for 10min, collecting supernatant to obtain crude enzyme solution, and measuring enzyme activity by the following method. After obtaining the optimal carbon source, five nitrogen sources of yeast extract powder, peptone, sodium nitrate, ammonium sulfate and ammonium nitrate are utilized, and the enzyme activity of the crude enzyme solution is measured when the addition amount of the five nitrogen sources is 0.5%.

The method for determining the enzyme activity of the endonuclease, the exonuclease, the beta-glucosidase and the filter paper enzyme adopts a method for determining reducing sugar by using 3, 5-dinitrosalicylic acid (DNS), and comprises the following specific steps: respectively using 1mL of 1% sodium carboxymethylcellulose solution, 1% microcrystalline cellulose solution and 1% salicin solution as reaction substrates, adding 0.5mL of 0.05mol/L citrate buffer solution (0.05 mol/L citric acid and sodium citrate solution are respectively prepared and mixed according to a proportion, the final pH value is 4.8 +/-0.05), adding 0.5mL of properly diluted enzyme solution, immediately adding 1.5mL of DNS reagent after water bath at 50 ℃ for 30min, carrying out boiling water bath for 5min, carrying out cold water bath to room temperature, then using distilled water to fix the volume, and using the inactivated enzyme solution with the same volume to participate in the reaction as a blank control to measure the enzyme activity. The absorbance was measured at 540nm using a spectrophotometer and recorded. And (3) measuring the enzyme activity of the filter paper, taking a 1cm multiplied by 6cm Xinhua I filter paper strip and 1.5mL of citrate buffer solution as reaction substrates, adding the filter paper enzyme for reaction for 60min, and carrying out the rest operations like the enzyme measurement. According to the trade standard QB-T2583-. Enzyme activity is defined as: hydrolysis of the substrate at 50 ℃ and pH 4.8. + -. 0.05 yielded an amount of 1mg reducing sugars (calculated as glucose) per 1mL enzyme solution per 1h, expressed as U/mL.

The calculation formula is as follows:

as shown in a in fig. 3, different matrixes sodium carboxymethyl cellulose, microcrystalline cellulose, corncob meal, bran, straw meal and corn straw meal have significant influence on enzyme production of SGSF303, wherein the bran can significantly induce SGSF303 to produce more cellulase. When bran is used as a fermentation substrate, the activity of the endonuclease generated by the bacterial strain SGSF303 reaches 0.86U/mL (is improved by 2-4 times compared with other matrixes), the activity of the exonuclease reaches 0.58U/mL, the activity of the beta-glucosidase reaches 0.26U/mL, and the activity of the filter paper enzyme is 0.32U/mL. Therefore, in the screened carbon source, the bran can effectively improve the capability of producing cellulase by fungi. The enzyme production effect of different nitrogen sources on the SGSF303 strain was tested by using bran as a carbon source, and as can be seen from B in FIG. 3, except for beta-glucosidase (NaNO)₃Most preferably), the enzyme production effect by peptone is better than that by other nitrogen sources. Thus, a cellulase production medium formulation was obtained, which was prepared from the following composition: 15g of bran, 5g of peptone, 2g of monopotassium phosphate and 1L of water.

Example 3 use of Ashbya SGSF303 for the production of antibacterial Compounds

Determination of antibacterial Activity

The preparation method of the extract comprises the following steps: inoculating the strain cake into SMYA culture medium (seed fermentation culture medium), and culturing at 25 deg.C and rotation speed of 180r/min for 3 d. 0.5mL of seed fermentation broth was added to the liquid culture medium PDB or P + Nic, respectively, for culture and fermentation 14 d. After fermentation, adding ethyl acetate with the volume twice that of the fermentation amount, soaking for 24h, and concentrating the filtered extracting solution under reduced pressure to obtain a crude extract. The crude extract was dissolved in 10% dimethyl sulfoxide (DMSO) and stored in a refrigerator at 4 ℃.

The antibacterial activity determination method comprises the following steps: using a punch-out chemical diffusion method. LBA medium containing the test bacteria was poured into petri dishes and after it had solidified, 5 wells were punched with a sterile punch. Or inoculating the fungi to the center of the PDA plate, and uniformly punching 5 holes on the periphery of the plate. 6mg/mL fungal crude extract solution, 10% DMSO as solvent control, and 6mg/mL amphotericin solution as positive control were added to the wells, respectively. Three replicates were set for each set of experiments. The mixture is placed in an incubator at 25 ℃ and observed and recorded every day.

TABLE 3 antibacterial Activity of Strain SGSF303

Note: "-" indicates no inhibitory effect; "+" indicates a bacteriostatic effect; PDB represents PDB culture medium fermentation crude extract; p + Nic represents PDB addition of niacinamide to ferment the crude extract.

The primary screening results of the antibacterial activity of the SGSF303 strain are shown in Table 3, the fermentation products of two culture media of the SGSF303 strain have no inhibition capacity on test bacteria, meanwhile, the P + Nic culture medium fermentation crude extract has a certain inhibition effect on the rhizoctonia solani, when the concentration is 3 mg/mL-6 mg/mL, the P + Nic culture medium fermentation crude extract has obvious antibacterial activity, the PDB culture medium fermentation crude extract has no antibacterial activity, and the P + Nic crude extracts of other strains screened in the same batch have no antibacterial activity. It is shown that niacinamide can activate strain SGSF303 to produce secondary metabolites with antibacterial activity, i.e. strain SGSF303 can produce antibacterial compounds.

Compared with the antibacterial activity of the currently known fungus of the genus alismoid, the strain SGSF303 is found to be the first strain with the antifungal activity in the genus, and other alismoid have no report of the antifungal activity.

Claims (9)

1. An Ashbya, characterized in that said Ashbya is:

the strain number is SGSF303 Alexandrium alzeylanicum (Arxiella longispora), and the preservation number is CCTCC M2021101.

2. The alzeyla sp.

3. Use of the a zeylaria of claim 1 in a composting process associated with cellulose degradation or utilization of cellulose degradation in agricultural waste.

4. Use of the a ze of claim 1 for controlling rhizoctonia solani.

5. Use of the a ze of claim 1 for the production of cellulase enzymes.

6. The use according to claim 5, characterized in that said A.zearalans is induced to produce cellulases by bran.

7. The use according to claim 6, wherein the cellulase production medium is formulated with bran 15g, peptone 5g, potassium dihydrogen phosphate 2g and water 1L.

8. Use of the alzeylaria of claim 1 for the production of antibacterial compounds.

9. The use according to claim 8, characterized in that said A.zeae is induced to produce a compound against Rhizoctonia solani using nicotinamide.

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