CN104893988A - Trichoderma atroviride strain capable of producing high-temperature-resistant feruloyl esterase and high-temperature-resistant cellulase and application thereof - Google Patents

Trichoderma atroviride strain capable of producing high-temperature-resistant feruloyl esterase and high-temperature-resistant cellulase and application thereof Download PDF

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CN104893988A
CN104893988A CN201510268516.9A CN201510268516A CN104893988A CN 104893988 A CN104893988 A CN 104893988A CN 201510268516 A CN201510268516 A CN 201510268516A CN 104893988 A CN104893988 A CN 104893988A
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trichoderma atroviride
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feruloyl esterase
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高兆建
张建萍
刘恩岐
侯进慧
孙会刚
徐大伟
杨宪瑶
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Xuzhou University of Technology
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Abstract

The invention discloses a Trichoderma atroviride strain capable of producing high-temperature-resistant feruloyl esterase and high-temperature-resistant cellulase and application thereof. The collection number of the Trichoderma atroviride AWS26 strain is CGMCC No.8673. The Trichoderma atroviride strain has the advantages of low nutritional requirements, high reproduction speed and short fermentation period, and the fermentation products can be easily extracted and separated. The Trichoderma atroviride strain can be fermented to obtain the feruloyl esterase-cellulase composite enzyme. The composite enzyme has the advantages of high temperature resistance and high acid/alkali stability.

Description

A kind of Trichoderma atroviride and application thereof of producing high temperature resistant feruloyl esterase and high temperature-resisting cellulase
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of Trichoderma atroviride and application thereof of producing high temperature resistant feruloyl esterase and high temperature-resisting cellulase.
Background technology
Feruloyl esterase (E.C.3.1.1.73, ferulicacid esterase, FAE) also known as Ferulic acid esterase, it is a kind of novel enzyme preparation, it mainly with Ferulic acid methylester, oligosaccharide ferulic acid ester, polysaccharide ferulic acid ester and xylogen ferulic acid ester for substrate, destroy the ester bond that is connected with polysaccharide of forulic acid, from plant cell wall, discharge forulic acid, allow remaining polysaccharide main chain easily be degraded, effectively improve the nutritive value of the byproduct containing a large amount of lignocellulose and forulic acid.Forulic acid have different physiological roles as scavenging free radicals, ultra-violet radiation resisting, antithrombotic, reducing blood-fat, prevent and treat coronary heart disease, antisepsis and anti-inflammation, pain relieving, anti-mutation, anti-cancer, enhancing motility of sperm and regulate human immunity etc.Therefore, this enzyme can be utilized in the food industry to interrupt forulic acid and cell wall material being cross-linked as polysaccharide in wheat bran, stalk, and efficient degradation polysaccharide also obtains trans-ferulaic acid, obtains functional food ingredient.Feruloyl esterase has broad application prospects in the food industry.
In fodder industry, the fiber raw material of crop plants is after ferulic acid ester ferment treatment, forulic acid is dissociated out from the structure of plant cell wall, the cytoskeletal structure of vegetable material densification is damaged, break the connection between xylogen, hemicellulose and Mierocrystalline cellulose, this loose vegetable raw material is is more easily digested and absorbed by livestock, can significantly improve the utilization ratio of feed.
Cellulase is the O-glycosylation lytic enzyme of β-Isosorbide-5-Nitrae glycosidic link in a class energy degraded cellulose.In feed industry is produced, cellulase is as a kind of novel fodder additives, and additive capacity is generally at 0.1%-0.3%.Because plant cell wall forms primarily of Mierocrystalline cellulose, hemicellulose and pectin, prevent animal digesting and assimilating nutritive substance in vegetable cell, cellulase can the Mierocrystalline cellulose of decomposition texture complexity in animal body, destroy plant cell wall and releasing nutrients material, generate the glucose being easy to digest, the utilization ratio of feed is increased, promotes acidity simultaneously, activate stomach en-, promoting digestion absorbs; Cellulase significantly can also reduce colibacillary quantity in animal intestinal and promote that beneficial microorganism grows, and improves intestinal flora balance.
Screen the microorganism strains of multiple secretion feruloyl esterase and cellulase at present, comprise fungi, bacterium and yeast.The product feruloyl esterase microorganism found mainly contains aspergillus niger (Aaspergillusniger), streptomycete (as Streptomycesavermitilis), clostridium (as Clostridiumthermocellum), bacillus (as Bacillussp.), lactobacillus (Lactobacilli), pseudomonas (as Pseudomonasfluorescens) etc.But most feruloyl esterase is separated and obtains from fungi, as aspergillus oryzae (Aspergillusoryzae), aspergillus niger (Aspergillusniger), Aspergillus nidulans (Aspergillusnidulans), Fusarium oxysporum (Fusariumoxysporum), sporotrichum thermophile (Sporotrichumthermophile), aspergillus flavipes (Aspergillusflavipes), Aspergillus awamori (Aspergillusawamori), Tabin aspergillus (Aspergillustubingensis), mould class (Penicillium).
At present, the demand of feruloyl esterase in fodder industry is very large, and the feruloyl esterase being applied to industry is mainly derived from aspergillus niger, because aspergillus niger feruloyl esterase is not enough to contingent tolerances such as high temperature, acid or alkali, in industrial production use procedure, feruloyl esterase enzyme lives loss seriously, limits feruloyl esterase application industrially to a great extent.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art and a kind of Trichoderma atroviride and application thereof of producing high temperature resistant feruloyl esterase and high temperature-resisting cellulase are provided, this bacterial strain has the advantage that nutritional requirement is low, breeding is fast, tunning is easy to extraction and isolation, fermentation period is short, and its fermentation energy accesses feruloyl esterase and cellulase prozyme, this prozyme is high temperature resistant, ph stability good.
A kind of Trichoderma atroviride (Trichoderma atroviride) AWS26 producing high temperature resistant feruloyl esterase and high temperature-resisting cellulase, (address is: No. 3, No. 1, North Star West Road, Chaoyang District, city of BeiJing, China institute to be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC on January 2nd, 2014, postcode 100101), deposit number: CGMCC No.8673.
The screening method of Trichoderma atroviride bacterium AWS26 of the present invention comprises the following steps:
Step 1, sampling and sample preparation
Soil sampling 10 parts the compost of thermophilic fermentation is in from great Han village, Yunlong District, Xuzhou City of Jiangsu Province.Take solid soil sample 5g, put into the triangular flask that granulated glass sphere and the sterilized physiological saline of 50mL are housed, with the rotating speed shaken at room temperature 30min of 160r/min, make suspension, then this triangular flask is put into 60-70 DEG C of isothermal vibration water-bath, 100r/min concussion process 20-30min, obtains soil sample turbid solution.
Step 2, enrichment culture
Draw the soil sample turbid solution of 2-4mL after pyroprocessing, be added in the triangular flask that 30-60mL enrichment medium one is housed, be placed in 45-50 DEG C, constant-temperature table cultivates 5-7d under rotating speed 160-200r/min.Then aseptic technique measures 30-50mL enrichment culture liquid in centrifuge tube, the centrifugal 5-10min of 4000-6000r/min, outwell supernatant, with enrichment medium two, the precipitation after centrifugal is washed till in the triangular flask that 30-60mL enrichment medium two is housed, is again placed in 45-50 DEG C, the constant-temperature table of rotating speed 160-200r/min cultivates 5-7d.
Enrichment medium one: Ferulic acid ethylester 0.2g, Xylo-Mucine 0.5g, mineral nutrition liquid 1mL, make up water is to 100mL, pH4.5.121 DEG C of 15min sterilizings, after naturally cooling, add the penbritin of filtration sterilization to final concentration 50mg/L.
Enrichment medium two: bran powder 1g, corn stalk powder 1g, Ferulic acid ethylester 0.2g, mineral nutrition liquid 1mL, make up water is to 100mL, pH4.5.121 DEG C of 15min sterilizings, after naturally cooling, add the penbritin of filtration sterilization to final concentration 50mg/L.
Mineral nutrition liquid: NaNO 32.0g, K 2pO 41.5g, CaCl 21.5g, MgSO 40.3g, FeSO 47H 2o 0.1g, MnSO 4h 2o 1.6mg, ZnSO 4h 2o 0.05g, CoCl 20.5mg, (NH 4) 2sO 45g, deionized water 1000mL.
Step 3, primary dcreening operation
By the nutrient solution stepwise dilution after second time enrichment to 10 -3, 10 -4, 10 -5, 10 -6the nutrient solution 0.2mL getting dilution at different levels coats in mould screening culture medium respectively, when culture dish dries in the air and not to have fluxion to surface, with preservative film, culture dish mouth is sealed, and be placed in 37-40 DEG C of constant incubator inversion cultivation 18-24h, then flat board is placed in 40-45 DEG C of constant incubator and continues to be inverted cultivation 2-4d.
By bacterium colony dibbling that flat board the grows same position to two primary dcreening operation Selective agar medium, two primary dcreening operation substratum are respectively feruloyl esterase primary dcreening operation substratum and cellulase primary dcreening operation substratum.Be inverted in the culture dish 40-45 DEG C constant incubator inoculated and cultivate 2-4d.Treat that obvious bacterium colony is formed, observed on feruloyl esterase primary dcreening operation substratum and occur with or without obvious transparent circle.Whether cellulase primary dcreening operation flat board will add the Congo red dye liquor of a certain amount of 0.1%, dyeing 1-10min, outwells staining fluid, observe the Congo red color of periphery of bacterial colonies and obviously shoal.To all occur that on two kinds of primary dcreening operation flat boards the bacterial strain of hydrolysis circle is inoculated out from feruloyl esterase primary dcreening operation flat board, multiple sieve further.
Mould screening culture medium: PDA substratum 800mL, rose-bengal 0.02g, paraxin 0.1g, mineral nutrition liquid 1mL, moisturizing to 1000mL, agar powder 15g.121 DEG C, 15min sterilizing.Add after rose-bengal and paraxin sterilizing.
PDA substratum: potato 200g, sucrose 20g, distilled water is settled to 1000mL.
Feruloyl esterase primary dcreening operation substratum: PDA substratum 100mL, mineral nutrition liquid 1mL, Ferulic acid ethylester solution 1mL, agar powder 1.5g, pH4.5.121 DEG C, sterilizing 20min.Fully shake up after medium sterilization, along with shake, substratum becomes opaque gradually, pours in flat board at 60-70 DEG C.
Ferulic acid ethylester solution preparation: take 0.22g Ferulic acid ethylester in centrifuge tube, adds 500 μ LN, N-DMF solution, and concussion adds 500 μ L sterilized waters after dissolving again, fully shakes up.
Cellulase primary dcreening operation substratum: cellulose powder 1g, peptone 0.1g, yeast powder 0.1g, mineral nutrition liquid 1mL, agar 1.5, adding distil water is settled to 100mL, pH value 4.5.121 DEG C of sterilizing 15min.
Step 4, multiple sieve
The bacterial strain obtained by primary dcreening operation is rule on PDA culture medium flat plate, is placed in 40-45 DEG C of constant incubator and cultivates 2-3d, repeat streak culture more than three times, observes colonial morphology unanimously then think bacterial strain purifying as continuous several times.
After the bacterial strain purifying that multiple sieve is obtained, be inoculated on solid seed culture medium, cultivate 3-5d at 35-40 DEG C, the lawn cutting 1 square centimeter is inoculated in the triangular flask that 30-50mL liquid seed culture medium is housed, and under 160-180r/min rotating speed, 37-40 DEG C is carried out liquid concussion cultivation.After cultivating 3-4d, according to the inoculum size of 6-10% (v/v), be inoculated into by liquid seeds in liquid fermentation medium, under 160-180r/min rotating speed, 40-45 DEG C is carried out liquid concussion fermentation culture.Fermentation culture 3-5d, gets fermented liquid 1200r/min, and 10min collected after centrifugation supernatant liquor measures feruloyl esterase and cellulose enzyme is active.The bacterial strain choosing two kinds of enzymic activitys all higher carries out line Secondary Culture, finally obtain a strain produce enzyme stablize and have high enzyme to live not only produce resistance to high feruloyl esterase but also produce the producing bacterial strain of high temperature-resisting cellulase.The inoculation of purifying on PDA slant medium, 4 DEG C of preservations.
Solid seed culture medium: PDA substratum 99mL, mineral nutrition liquid 1mL, agar powder 1.5g, pH5.0,121 DEG C, 15min sterilizing.
Liquid seed culture medium: PDA substratum 99mL, mineral nutrition liquid 1mL.121 DEG C, 15min sterilizing.
Liquid fermentation medium: PDA substratum 80mL, mineral nutrition liquid 1mL, peptone 1g, tween 80 0.5mL, (NH4) 2SO4 1g, supplements deionized water to 100mL.121 DEG C, 15min sterilizing.
Through above-mentioned screening method obtained strains, bacterium colony grows fast radially on PDA flat board, and initial stage hyphal surface is loose, smooth, and white presenting light green because producing conidium, increasing gradually, in deep green later.Subiculum is fine and close, thicker, and gonimoblast bundle is arranged in concentric wheel stripe shape, and the bacterium colony back side is colourless.At the optical microphotograph Microscopic observation of amplification 400 times, mycelium is made up of the branch mycelia with tabula, sporophore is uprightly born by mycelia, branch is complicated, and approximate right angle is stretched out, to raw or alternate, arrange in dendroid, the long 5.0-12.0 μm of sporophore, middle wide 2.5-3.7 μm, the wide 1.5-3.0 of base portion.Conidium cluster the top being born in conidiophore.Conidium is spherical or oval, and size is 3.0-4.5 × 2.5-3.8 μm, and surface irregularity, is covered with spinule, is gathered into the conidial head of spherical green by mucus on stigma.Contrast " Fungal identification handbook " and pertinent literature determine that this bacterial strain is Trichoderma atroviride (Trichodermaatroviride).
The application of Trichoderma atroviride (Trichoderma atroviride) AWS26 in feedstuff raw material degraded of described product is high temperature resistant feruloyl esterase and high temperature-resisting cellulase, concrete grammar is: by the corn stalk powder 10g of drying, wheat bran 5g, ammonium sulfate 1g, bean cake powder 1g, ammonium nitrate 0.4g, NaCl0.5g, potassium primary phosphate 0.2g and MgSO 40.05g mixes, and adds deionized water by solid-liquid mass ratio 1:1.3, inoculates bacterial strain (Trichoderma atroviride) AWS26,26-30 DEG C and cultivates 10 days.
Trichoderma atroviride (Trichoderma atroviride) AWS26 of described product is high temperature resistant feruloyl esterase and high temperature-resisting cellulase prepares the application in forulic acid at fermenting wheat bran, comprise the following steps:
Step 1, is inoculated in fermention medium by bacterial strain (Trichoderma atroviride) AWS26 spore suspension, cultivates 3-5d for 30-35 DEG C, bent 2-3 time of middle turning;
Step 2, adds deionized water in step 1 gained wheat bran, stirs, and be placed in concussion shaking table 100r/min, 50 DEG C of lixiviate 2h, filter, the centrifugal 20min of 8000r/min, getting supernatant liquor saturation ratio is 80% (NH 4) 2sO 4saltout, 4 DEG C of hold over night, the centrifugal 20min of 8000r/min, collecting precipitation, dissolves with the phosphoric acid of pH4.0-citric acid solution, obtains high temperature resistant feruloyl esterase and high temperature-resisting cellulase mixes thick enzyme;
Step 3, adds destarching wheat bran by thick for step 2 gained enzyme, then adds water, and regulate pH4.0,65 DEG C of enzymolysis 12h, obtain forulic acid.
The Trichoderma atroviride of product provided by the invention is high temperature resistant feruloyl esterase and high temperature-resisting cellulase, there is the advantage that nutritional requirement is low, breeding is fast, tunning is easy to extraction and isolation, fermentation period is short, and its fermentation energy accesses feruloyl esterase and cellulase prozyme, this prozyme is high temperature resistant, ph stability good.
Accompanying drawing explanation
Fig. 1 is Trichoderma atroviride primary dcreening operation Enzyme activity assay lithograph of the present invention;
Fig. 2 is that Trichoderma atroviride of the present invention sieves Enzyme activity assay lithograph again;
Fig. 3 be in embodiment 3 temperature to the influence curve of feruloyl esterase and cellulose enzyme activity;
Fig. 4 be in embodiment 3 pH value to the influence curve of feruloyl esterase and cellulose enzyme activity;
Fig. 5 is the thermostability curve of feruloyl esterase and cellulose enzyme in compound enzymic preparation in embodiment 3;
Fig. 6 is feruloyl esterase and cellulase pH beta stability line in compound enzymic preparation in embodiment 3.
Embodiment
Embodiment 1
The present embodiment illustrates the screening method of Trichoderma atroviride AWS26, and concrete steps are as follows:
Step 1, sampling and sample preparation:
Soil sampling 10 parts the compost of thermophilic fermentation is in from great Han village, Yunlong District, Xuzhou City of Jiangsu Province.Take solid soil sample 5g, put into the triangular flask that granulated glass sphere and 50mL sterile saline is housed, 160r/min vibrates 30min, make suspension, then this triangular flask is put into 60-70 DEG C of isothermal vibration water-bath, 100r/min concussion process 20-30min, obtains soil sample turbid solution.
Step 2, enrichment culture:
Draw 2-4mL step 1 gained soil sample turbid solution, adding to the capacity that 30-60mL enrichment medium one is housed is in the triangular flask of 250mL, in 45-50 DEG C, cultivate 5-7d in the constant-temperature table of rotating speed 160-200r/min, obtain enrichment culture liquid.Aseptic technique measures 30-50mL enrichment culture liquid, the centrifugal 5-10min of 4000-6000r/min, outwell supernatant, precipitation after centrifugal being washed till with enrichment medium two is equipped with in the 250mL triangular flask of 30-60mL enrichment medium two, in 45-50 DEG C, cultivate 5-7d in the constant-temperature table of rotating speed 160-200r/min.
Step 3, primary dcreening operation:
By the nutrient solution stepwise dilution after second time enrichment to 10 -3, 10 -4, 10 -5, 10 -6the nutrient solution 0.2mL getting dilution at different levels coats in mould screening culture medium respectively, when culture dish dries in the air and not to have fluxion to surface, with preservative film, culture dish mouth is sealed, and be placed in 37-40 DEG C of constant incubator inversion cultivation 18-24h, then flat board is placed in 40-45 DEG C of constant incubator and continues to be inverted cultivation 2-4d.
By bacterium colony dibbling that flat board the grows same position to two primary dcreening operation Selective agar medium, two primary dcreening operation substratum are respectively feruloyl esterase primary dcreening operation substratum and cellulase primary dcreening operation substratum, be inverted and cultivate 2-4d in the culture dish 40-45 DEG C constant incubator inoculated.Treat that obvious bacterium colony is formed, observed on feruloyl esterase primary dcreening operation substratum and occur with or without obvious transparent circle.Whether cellulase primary dcreening operation flat board will add the Congo red dye liquor of a certain amount of 0.1%, dyeing 1-10min, outwells staining fluid, observe the Congo red color of periphery of bacterial colonies and obviously shoal.To all occur that on two kinds of primary dcreening operation flat boards the bacterial strain of hydrolysis circle is inoculated out from feruloyl esterase primary dcreening operation culture medium flat plate, multiple sieve further.Feruloyl esterase and the dull and stereotyped detected result of cellulase primary dcreening operation are respectively as shown in Table 1 and Table 2.
Step 4, multiple sieve:
The bacterial strain obtained by step 3 primary dcreening operation is rule on PDA culture medium flat plate, be placed in 40-45 DEG C of constant incubator and cultivate 2-3d, repeat streak culture more than three times, observe colonial morphology as continuous several times and unanimously then think bacterial strain purifying, described bacterial strain primary dcreening operation Enzyme activity assay lithograph as shown in Figure 1.
Step 5, culture presevation:
After the bacterial strain purifying that multiple sieve is obtained, be inoculated on solid seed culture medium, cultivate 3-5d at 35-40 DEG C, the lawn cutting 1 square centimeter is inoculated into and is equipped with in the 250mL triangular flask of 30-50mL liquid seed culture medium, and under 160-180r/min rotating speed, 37-40 DEG C is carried out liquid concussion and cultivate.After cultivating 3-4d, according to the inoculum size of 6-10% (v/v), be inoculated into by liquid seeds in liquid fermentation and culture, under 160-180r/min rotating speed, 40-45 DEG C is carried out liquid concussion fermentation culture.Fermentation culture 3-5d, gets fermented liquid 1200r/min, and 10min collected after centrifugation supernatant liquor measures feruloyl esterase and cellulose enzyme is active.The bacterial strain choosing two kinds of enzymic activitys all higher carries out line Secondary Culture, finally obtain a strain produce enzyme stablize and have high enzyme to live not only produce resistance to high feruloyl esterase but also produce the producing bacterial strain of high temperature-resisting cellulase.The inoculation of purifying on PDA slant medium, 4 DEG C of preservations.Sieve result again as shown in table 3, consider the enzyme situation alive of feruloyl esterase and cellulase, select strains A WS26 as object bacterial strain.Sieve obtained strains AWS26 ferulaic acid esterase activity again and detect flat board as shown in Figure 2,
Wherein, the culture medium prescription used is as follows:
Enrichment medium one: Ferulic acid ethylester 0.2g, Xylo-Mucine 0.5g, mineral nutrition liquid 1mL, make up water is to 100mL, pH4.5, and 121 DEG C, 15min sterilizing, after naturally cooling, add the penbritin of filtration sterilization to final concentration 50mg/L.
Enrichment medium two: bran powder 1g, corn stalk powder 1g, Ferulic acid ethylester 0.2g, mineral nutrition liquid 1mL, make up water is to 100mL, pH4.5, and 121 DEG C, 15min sterilizing, after naturally cooling, add the penbritin of filtration sterilization to final concentration 50mg/L.
Mould screening culture medium: PDA substratum 800mL, rose-bengal 0.02g, paraxin 0.1g, mineral nutrition liquid 1mL, moisturizing to 1000mL, agar powder 15g.121 DEG C, 15min sterilizing.Add after rose-bengal and paraxin sterilizing.
Feruloyl esterase primary dcreening operation substratum: PDA substratum 100mL, mineral nutrition liquid 1mL, Ferulic acid ethylester solution 1mL, agar powder 1.5g, pH4.5,121 DEG C of sterilizing 20min.Fully shake up after medium sterilization, along with shake, substratum becomes opaque gradually, pours in flat board at 60-70 DEG C.Wherein, Ferulic acid ethylester solution: take 0.22g Ferulic acid ethylester in centrifuge tube, adds 500 μ L N, N-DMF solution, and concussion adds 500 μ L sterilized waters after dissolving again, fully shakes up.
Cellulase primary dcreening operation substratum: cellulose powder 1g, peptone 0.1g, yeast powder 0.1g, mineral nutrition liquid 1mL, agar 1.5, adding distil water is settled to 100mL, pH value 4.5.121 DEG C of sterilizing 15min.
Solid seed culture medium: PDA substratum 99mL, mineral nutrition liquid 1mL, agar powder 1.5g, pH5.0,121 DEG C, 15min sterilizing.
Liquid seed culture medium: PDA substratum 99mL, mineral nutrition liquid 1mL.121 DEG C, 15min sterilizing.
Liquid fermentation medium: PDA substratum 80mL, mineral nutrition liquid 1mL, peptone 1g, tween 80 0.5mL, (NH 4) 2sO 41g, supplements deionized water to 100mL.121 DEG C, 15min sterilizing.
In above-mentioned substratum, mineral nutrition liquid: NaNO 32.0g, K 2pO 41.5g, CaCl 21.5g, MgSO 40.3g, FeSO 47H 2o 0.1g, MnSO 4h 2o 1.6mg, ZnSO 4h 2o 0.05g, CoCl 20.5mg, (NH 4) 2sO 45g, deionized water 1000mL.
PDA substratum: potato 200g, sucrose 20g, distilled water is settled to 1000mL.Take 200g potato, clean peeling chopping, the 1000mL that adds water boils half hour, filtered through gauze, and add sucrose 20g, make up water is to 1000mL.
Table 1 produces the dull and stereotyped primary dcreening operation result of feruloyl esterase mould
The dull and stereotyped primary dcreening operation result of table 2 cellulase-producing mould
Table 3 each bacterial strain crude enzyme liquid enzyme is lived
Obtained strains AWS26 of the present invention, bacterium colony grows fast radially on PDA flat board, and initial stage hyphal surface is loose, smooth, and white presenting light green because producing conidium, increasing gradually, in deep green later.Subiculum is fine and close, thicker, and gonimoblast bundle is arranged in concentric wheel stripe shape, and the bacterium colony back side is colourless.At the optical microphotograph Microscopic observation of amplification 400 times, mycelium is made up of the branch mycelia with tabula, sporophore is uprightly born by mycelia, branch is complicated, and approximate right angle is stretched out, to raw or alternate, arrange in dendroid, the long 5.0-12.0 μm of sporophore, middle wide 2.5-3.7 μm, the wide 1.5-3.0 of base portion.Conidium cluster the top being born in conidiophore.Conidium is spherical or oval, and size is 3.0-4.5 × 2.5-3.8 μm, and surface irregularity, is covered with spinule, is gathered into the conidial head of spherical green by mucus on stigma.Contrast " Fungal identification handbook " and pertinent literature determine that this bacterial strain is Trichoderma atroviride (Trichodermaatroviride).
Extract strains A WS26 genome of the present invention, according to primers the most conservative in fungi 18SrDNA, amplification adopts upstream primer: 5'-CCGTAGGTGAACCTGCGG-3'(as shown in SEQ ID NO.2), downstream primer: 5'-TCCTCCGCTTATTGATATGC-3'(is as shown in SEQ ID NO.30.).Reaction conditions is: 95 DEG C of denaturation 5min, 94 DEG C of sex change 40S, 52 DEG C of annealing 40S, and 72 DEG C extend lmin, totally 30 circulations, and last 72 DEG C extend 10min, 4 DEG C of preservations.Amplified production connects pMD18-T carrier.PCR reaction system is: 10 × Buffer2 μ L, dNTP (2.5mmol/L) 2 μ L, upstream primer 1 μ L, downstream primer 1 μ L, template10 ~ 50ng, Taq enzyme (2.5U) 0.3 μ l, adds water and supply 20 μ L.Its 18S rDNA fragment 712bp is obtained through pcr amplification.This amplified fragments gel is reclaimed and checks order, GenBank is submitted to carry out nucleic acid homology analysis, record sequence as shown in SEQ ID NO.1, the 18SrDNA sequence of the close mould obtained with comparison from NCBI adopts ClustalX1.83 to carry out Multiple Sequence Alignment, then MEGA4.0 biological software constructing system evolutionary tree is adopted, the 18S rDNA nucleotide sequence homology of the result display 18SrDNA nucleotide sequence of strains A WS26 and Trichodermaatroviride (JX462604) is the highest, and maximum comparability (maxident) reaches 99%.Comprehensively observe in conjunction with its morphological specificity, cultural characteristic, strains A WS26 is accredited as Trichoderma atroviride (Trichodermaatroviride).
The Trichoderma atroviride that embodiment 2 produces high temperature resistant feruloyl esterase and high temperature-resisting cellulase is applied to feedstuff raw material degraded
Dried corn stalk powder 10g, wheat bran 5g, ammonium sulfate 1g, bean cake powder 1g, ammonium nitrate 0.4g, NaCl 0.5g, potassium primary phosphate 0.2g, MgSO4 0.05g, add deionized water by solid-liquid mass ratio 1:1.3, above feed is placed in triangular flask, after disinfection inoculation, put into fixed temperature and humidity incubator, regulate temperature 26-30 DEG C, pH6.0.Inoculation bacterial classification is the bacterial classification AWS26 that activation of the present invention is good.In fermenting process, every day stirs triangular flask, after fermentation 10d, and the content of detection fibers element, hemicellulose and xylogen.Detected result shows, and in fermentation raw material, the degradation rate of xylogen is 77.4%, and cellulosic degradation rate is 67.6%, and the degradation rate of hemicellulose is 47.9%, and conversion rate of reducing sugar is 0.697g/g stalk.The feedstuff raw material such as the high temperature resistant feruloyl esterase of product that employing the present invention screens and high temperature-resisting cellulase bacterial strain process maize straw, wheat bran is described, the degradation rate of stodgy macromolecular carbohydrate can be improved significantly, provide possibility for it is applied to fodder industry.
The Trichoderma atroviride that embodiment 3 produces high temperature resistant feruloyl esterase and high temperature-resisting cellulase is applied to fermenting wheat bran and prepares forulic acid
Wheat bran 50g is added, ammonium sulfate 1g, peptone 2g, potassium primary phosphate 0.2g, magnesium sulfate 0.05g, 75mL water mixing sterilizing in 500mL triangular flask.After PDA medium slant cultivates inoculation Trichoderma atroviride bacterial classification of the present invention, cultivate 3-4d for 30-35 DEG C, then add sterilized water 10ml in inclined-plane and the spore that fully suspends, 2ml spore suspension is added in substratum after sterilizing, after fully mixing, cultivate 3-5d in 30-35 DEG C, bent 2-3 time of middle turning.In the wheat bran fermented, add deionized water, fully stir and wheat bran is disperseed, be positioned on concussion shaking table under 100r/min rotating speed, 50 DEG C of lixiviate 2h.Filtration, the centrifugal 20min of 8000r/min, getting supernatant liquor saturation ratio is 80% (NH 4) 2sO 4saltout, 4 DEG C of hold over night, the centrifugal 20min of 8000r/min, collecting precipitation, phosphoric acid-citric acid solution dissolving of pH4.0, obtains high temperature resistant feruloyl esterase and high temperature-resisting cellulase mixes thick enzyme.
In 250mL triangular flask, add destarching wheat bran 5g, add Trichoderma atroviride of the present invention fermentation self-control mixed enzyme 5mL, water 115mL, regulate pH4.0, temperature is 65 DEG C, enzymolysis time 12h.Boiling water goes out enzyme, and centrifuging and taking supernatant measures the mass concentration of forulic acid and reducing sugar.Forulic acid burst size is up to 9.12mg/g wheat bran with this understanding.Utilize strain fermentation of the present invention produce mixed enzyme production forulic acid reaction temperature and, easily control, can not brown stain be caused, be best extracting method, be applicable to industrial production forulic acid.
The enzyme of embodiment 3 is high temperature resistant feruloyl esterase and high temperature-resisting cellulase is lived and enzymatic property measures
Ferment screening the Trichoderma atroviride AWS26 bacterial strain obtained, fermentative medium formula is: PDA substratum 80mL, mineral nutrition liquid 1mL, peptone 1g, tween 80 0.5mL, (NH 4) 2sO 41g, supplements deionized water to 100mL.121 DEG C, 15min sterilizing.Fermentation condition is: fermentation period 2-3d, leavening temperature 35-40 DEG C, inoculum size 4-8%, shaking speed 160-200r/min.
After fermentation ends, centrifugal for fermented liquid 8000r/min 20min is removed thalline, and filters remaining thalline and insoluble substance with 0.22 μm, collect filtrate.According to ammonium sulfate saturation ratio 75%, to to add ammonium sulfate in top fermentation filtrate, after fully dissolving, 4 DEG C of hold over night, the centrifugal 20min of 8000r/min, collecting precipitation, dissolves with 20mmol/L, pH6.0 phosphate buffer solution, the desalination of loading Sephedex G25 molecular sieve gel post, chromatography column is to carry out wash-out with identical buffered soln after the phosphoric acid buffer of pH6.5 balance, and elution rate is 15 ~ 20ml/h, obtains crude enzyme liquid.The work of gained fermented liquid feruloyl esterase enzyme is: 262U/L; Cellulose enzyme activity is: 915U/mL.
Activity determination method is:
The ferulic acid ester enzyme activity determination of fermented liquid
In 2mL centrifuge tube, add the enzyme liquid of 0.5mL after 1-5 doubly dilutes, and then add 0.5mL Ferulic acid methylester solution, fully mix, 65 DEG C of insulation 20min.Adding 1mL volume fraction is again that the glacial acetic acid of 10% is with termination reaction.Sample is in the centrifugal 10min of 12000r/min.Retain supernatant liquor, after dilution 2-4 times, with the ferulaic acid content in high performance liquid chromatography (HPLC) method working sample.To boil the enzyme liquid of deactivation as a control group, treatment process is the same.
The enzyme of feruloyl esterase is lived and is defined: at 50 DEG C, pH value is under the condition of 6.5, per minute esterlysis Ferulic acid methylester, generates the enzyme amount needed for 1 μm of ol forulic acid.Forulic acid adopts high effective liquid chromatography for measuring.
The measuring method of cellulase filter paper enzyme activity: it is 1 × 6cm that Xinhua's qualitative filter paper is cut into size, is placed in centrifuge tube, adds pH4.0 Sodium phosphate dibasic-citrate buffer solution 1mL, preheating in the water-bath of 55 DEG C of temperature.Add enzyme liquid 500 μ L, insulation reaction 60min.Add the DNS reagent of 3mL, boiling water bath 5min develops the color, and places in cold water and is cooled to room temperature.Draw 200 μ L and react supernatant liquor, at the light absorption value of wavelength 540nm place working sample, calculate the glucose concn in reaction system according to the glucose concn typical curve drawn.To boil the enzyme liquid of deactivation as a control group, treatment process is the same.
The unit of activity (U) of cellulase is defined as: under these conditions, and it is 1 enzyme activity unit (U) that per minute decomposes the Xylo-Mucine enzyme amount generated required for 1 μm of ol reducing sugar.
1. enzyme reaction optimum temperuture measures
Reaction system is placed in respectively the water-bath of 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C, after appropriateness dilution, measure feruloyl esterase enzyme and cellulase activity respectively, research enzymatic reaction optimum temperuture.With enzyme under optimum temperuture survey enzyme live as 100%, the lower institute of other temperature surveys as to its relative enzyme work.As shown in Figure 1, feruloyl esterase is substantially identical with the variation tendency of temperature rising enzyme with cellulase for result.The feruloyl esterase from 20 DEG C to 65 DEG C, increases gradually along with temperature raises enzymic activity, and when temperature reaches 65 DEG C, enzyme is lived and reached maximum value.Later along with temperature raises, enzyme is lived and is reduced gradually, therefore the feruloyl esterase optimum temperature of enzyme is 65 DEG C.And the temperature trip point of cellulase is 55 DEG C, namely the most applicable operative temperature of cellulase is 55 DEG C.Entirety sees that two kinds of enzymes are all high temperature enzyme, and between 45 DEG C-70 DEG C, the enzyme of two kinds of enzymes is lived all more than 80%, therefore compound enzymic preparation all can play efficient enzymolysis in this temperature range.
2. enzyme effect optimal pH measures
Zymin carries out enzymatic reaction under different pH (3.0 ~ 9.0), measures feruloyl esterase and the suitableeest action pH of cellulase.Damping fluid used is: the glycine-HCI buffered soln of pH2.0 ~ 3.0, pH3.0 ~ 4.0 citrate phosphate buffer, the acetic acid-sodium acetate buffer solution of pH4.0 ~ 5.0, pH5.0 ~ 7.0 Sodium phosphate dibasic-potassium phosphate buffer, pH7.0 ~ 9.0Tris-HCl buffered soln.With enzyme under optimum pH survey enzyme live as 100%, the lower institute of other pH value surveys as to its relative enzyme work.Zymin is in above different pH buffer system, and at 65 DEG C and 55 DEG C, measure feruloyl esterase and cellulose enzyme activity respectively, as shown in Figure 2, enzymic activity is more than 80% between pH3.5 ~ 7.0 for feruloyl esterase, and the suitableeest action pH is 4.5 for result.Cellulase between pH4.5-7.0 survey enzyme and live all more than 90%, the suitableeest enzyme work 5.5.Entirety sees zymin two kinds of enzymes, and between pH4.5-7.0, enzymic activity is all more than 80%, therefore zymin is applicable to enzyme digestion reaction in acid condition.
3. the thermostability experiment of enzyme
After the zymin being dissolved in pH6.5 phosphoric acid buffer is incubated 4h under the differing temps of 40 DEG C-80 DEG C, ice bath immediately, after appropriateness dilution, measures feruloyl esterase and cellulose enzyme vigor respectively.With enzyme under most equilibrium temperature survey enzyme live as 100%, the lower institute of other temperature surveys as to its relative enzyme work.The results are shown in Figure shown in 3, two kinds of enzymes reduce gradually with the rising enzyme activity of temperature substantially, and when temperature is higher than 60 DEG C, feruloyl esterase enzymic activity declines significantly.Two kinds of enzymes are below 60 DEG C, and enzyme lives storage rate all more than 80%; Less than 55 DEG C, residual enzyme is lived all more than 90%.Compound enzymic preparation is high temperature resistant type.
4.pH value is on the impact of enzyme stability
Zymin, in the damping fluid of different pH, respectively at 40 DEG C of insulation 24h, then measures feruloyl esterase and cellulose enzyme vigor respectively, lives as 100% with enzyme institute's surveys enzyme under the most stable pH, other pH lower survey as to its relative enzyme work.The results are shown in Figure shown in 4,40 DEG C, within the scope of pH3.0-8.0,24h feruloyl esterase enzymic activity does not change substantially, activity stabilized, and relative enzyme is lived all more than 95%.Cellulase enzymic activity between pH4.0-9.0 does not decline substantially.Entirety sees that compound enzymic preparation has wider ph stability, and character is more stable in acid condition.
5. the shelf time of zymin is on the impact of enzymic activity
The solid polypeptide formulation prepared is preserved respectively under 4 DEG C and room temperature, detects an enzyme activity each moon, detect 6 months altogether, check enzyme reservation situation alive.Result is as shown in the table:
Within six months, at 4 DEG C, feruloyl esterase and cellulose enzyme activity storage rate are all more than 90%, and under room temperature condition, enzyme is lived also more than 80%, can find out the good stability of enzyme.
SEQUENCE LISTING
 
<110> Xuzhou Engineering Institute
 
<120> mono-kind produces Trichoderma atroviride and the application thereof of high temperature resistant feruloyl esterase and high temperature-resisting cellulase
 
<130> 001
 
<160> 3
 
<170> PatentIn version 3.3
 
<210> 1
<211> 631
<212> DNA
<213> Trichoderma atroviride (Trichoderma atroviride)
 
<400> 1
tcatttagag gaagtaaaag tcgtaacaag gtctccgttg gtgaaccagc ggagggatca 60
 
ttaccgagtt tacaactccc aaacccaatg tgaaccatac caaactgttg cctcggcggg 120
 
gtcacgcccc gggtgcgtcg cagccccgga accaggcgcc cgccggaggg accaaccaaa 180
 
ctcttttctg tagtcccatc gcggacgtta tttcttacag ctctgagcaa aaattcaaaa 240
 
tgaatcaaaa ctttcaacaa cggatctctt ggttctggca tcgatgaaga acgcagcgaa 300
 
atgcgataag taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacatt 360
 
gcgcccgcca gtattctggc gggcatgcct gtccgagcgt catttcaacc ctcgaacccc 420
 
tccggggggt cggcgttggg gacctcggga gcccctaaga cgggatcccg gccccgaaat 480
 
acagtggcgg tctcgccgca gcctctcctg cgcagtagtt tgcacaactc gcaccgggag 540
 
cgcggcgcgt ccacgtccgt aaaacaccca acttctgaaa tgttgacctc ggatcaggta 600
 
ggaatacccg ctgcacttaa gcatatcact a 631
 
 
<210> 2
<211> 18
<212> DNA
<213> upstream primer
 
<400> 2
ccgtaggtga acctgcgg 18
 
 
<210> 3
<211> 20
<212> DNA
<213> downstream primer
 
<400> 3
tcctccgctt attgatatgc 20
 
 

Claims (5)

1. the Trichoderma atroviride producing high temperature resistant feruloyl esterase and high temperature-resisting cellulase ( trichoderma atroviride) AWS26, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, deposit number: CGMCC No. 8673.
2. the high temperature resistant feruloyl esterase of product according to claim 1 and high temperature-resisting cellulase Trichoderma atroviride ( trichoderma atroviride) application of AWS26 in feedstuff raw material degraded.
3. the high temperature resistant feruloyl esterase of product according to claim 1 and high temperature-resisting cellulase Trichoderma atroviride ( trichoderma atroviride) AWS26 prepares application in forulic acid at fermenting wheat bran.
4. the high temperature resistant feruloyl esterase of product according to claim 2 and high temperature-resisting cellulase Trichoderma atroviride ( trichoderma atroviride) application of AWS26 in feedstuff raw material degraded, it is characterized in that: comprise the following steps: by the corn stalk powder 10g of drying, wheat bran 5g, ammonium sulfate 1g, bean cake powder 1g, ammonium nitrate 0.4g, NaCl0.5g, potassium primary phosphate 0.2g and MgSO 40.05g mix, add deionized water by solid-liquid mass ratio 1:1.3, inoculate bacterial strain ( trichoderma atroviride) AWS26,26-30 DEG C cultivate 10 days.
5. the high temperature resistant feruloyl esterase of product according to claim 3 and high temperature-resisting cellulase Trichoderma atroviride ( trichoderma atroviride) AWS26 prepares application in forulic acid at fermenting wheat bran, it is characterized in that: comprise the following steps:
Step 1, by bacterial strain ( trichoderma atroviride) AWS26 spore suspension is inoculated in fermention medium, cultivates 3-5d for 30-35 DEG C, bent 2-3 time of middle turning;
Step 2, adds deionized water in step 1 gained wheat bran, stirs, and be placed in concussion shaking table 100r/min, 50 DEG C of lixiviate 2h, filter, the centrifugal 20min of 8000r/min, getting supernatant liquor saturation ratio is 80% (NH 4) 2sO 4saltout, 4 DEG C of hold over night, the centrifugal 20min of 8000r/min, collecting precipitation, dissolves with the phosphoric acid of pH4.0-citric acid solution, obtains high temperature resistant feruloyl esterase and high temperature-resisting cellulase mixes thick enzyme;
Step 3, adds destarching wheat bran by thick for step 2 gained enzyme, then adds water, and regulate pH4.0,65 DEG C of enzymolysis 12h, obtain forulic acid.
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CN110591927B (en) * 2019-09-23 2022-04-19 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) Application of trichoderma strain HB20111 in digesting phenolic acid autotoxic substances of crop continuous cropping obstacle factors and microbial inoculum
CN117004495A (en) * 2023-08-04 2023-11-07 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) Trichoderma atroviride T280, screening method and application thereof

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