CN104893988B - A kind of Trichoderma atroviride and its application for producing high temperature resistant feruloyl esterase and high temperature-resisting cellulase - Google Patents
A kind of Trichoderma atroviride and its application for producing high temperature resistant feruloyl esterase and high temperature-resisting cellulase Download PDFInfo
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Abstract
The invention discloses a kind of Trichoderma atroviride and its application for producing high temperature resistant feruloyl esterase and high temperature-resisting cellulase, the Trichoderma atroviride（Trichoderma atroviride）AWS26, deposit number is CGMCC No. 8673.The Trichoderma atroviride of the present invention has the advantages that nutritional requirement is low, breeding is fast, tunning is easy to extraction separation, fermentation period is short, and its fermentation energy accesses feruloyl esterase and cellulase complex enzyme, and the complex enzyme high temperature resistant, ph stability are good.
Description
Technical field
The invention belongs to microbial technology field, and in particular to one kind production high temperature resistant feruloyl esterase and high-temperature fibre element
The Trichoderma atroviride of enzyme and its application.
Background technology
Feruloyl esterase (E.C.3.1.1.73, ferulic acid esterase, FAE) is also known as Ferulic acid esterase, is one
Kind new enzyme preparation, it mainly using Ferulic acid methylester, oligosaccharide ferulic acid ester, polysaccharide ferulic acid ester and lignin ferulic acid ester as
Substrate, the ester bond that is connected with polysaccharide of destruction forulic acid, discharges forulic acid from plant cell wall, allows remaining polysaccharide main chain easily quilt
Degraded, effectively increases the nutritive value of the byproduct containing a large amount of lignocellulosics and forulic acid.Forulic acid has a variety of lifes
Reason function such as removes free radical, ultra-violet radiation resisting, antithrombotic, reducing blood lipid, prevention and treatment of coronary heart disease, anti-inflammation, analgesic, anti-prominent
Become, give protection against cancer, strengthening sperm motility and regulation human immunity etc..Therefore, forulic acid is interrupted using the enzyme in the food industry
With the crosslinking of polysaccharide in cell wall material such as wheat bran, stalk, efficient degradation polysaccharide simultaneously obtains trans-ferulaic acid, obtains feature food
Product base-material.Feruloyl esterase has broad application prospects in the food industry.
In feed industry, the fiber raw material of crop plants is after ferulic acid ester ferment treatment, and forulic acid is from plant
Separate out in the structure of thing cell membrane, the fine and close cytoskeletal structure of vegetable material is destroyed, and has broken lignin, half fibre
Connection between dimension element and cellulose, this loose vegetable raw material is easier to be digested and absorbed by livestock, can significantly carry
The utilization rate of high feed.
Cellulase is the O- glycosylation hydrolases of β -1,4 glycosidic bonds in a class energy degraded cellulose.In feed industry production
In, cellulase is as a kind of new feed addictive, and additive capacity is typically in 0.1%-0.3%.Due to plant cell wall master
To be made up of cellulose, hemicellulose and pectin, prevent animal to digest and assimilate nutriment in plant cell, fiber
Plain enzyme can the complicated cellulose of decomposition texture in animal body, destroy plant cell wall and simultaneously discharge nutriment, generation is easy to disappear
The glucose of change, increases the utilization rate of feed, while promoting acidity, activates pepsin, promotes to digest and assimilate；Cellulase
The quantity of Escherichia coli can also be substantially reduced in animal intestinal tract and promote beneficial microorganism to grow, improve intestinal flora and put down
Weighing apparatus.
The microbial strains of a variety of secretion feruloyl esterases and cellulase, including fungi, bacterium have been screened at present
And yeast.It was found that production feruloyl esterase microorganism mainly have aspergillus niger (Aspergillus niger), streptomycete (such as
Streptomyces avermitilis), clostridium (such as Clostridium thermocellum), bacillus (such as Bacillus
Sp.), Bacillus acidi lactici (Lactobacilli), pseudomonad (such as Pseudomonas fluorescens).But it is most
Feruloyl esterase is isolated from fungi, such as aspergillus oryzae (Aspergillus oryzae), aspergillus niger (Aspergillus
Niger), aspergillus nidulans (Aspergillus nidulans), Fusarium oxysporum (Fusarium oxysporum), thermophilic side spore
Mould (Sporotrichum thermophile), aspergillus flavipes (Aspergillus flavipes), aspergillus awamori
(Aspergillus awamori), Tabin aspergillus (Aspergillus tubingensis), mould class (Penicillium).
At present, demand of the feruloyl esterase in feed industry is very big, and mainly comes applied to the feruloyl esterase of industry
Aspergillus niger is come from, because aspergillus niger feruloyl esterase is not enough to contingent tolerances such as high temperature, acid or alkali, was used in industrial production
Cheng Zhong, feruloyl esterase enzyme activity loss is serious, largely limits the application of feruloyl esterase industrially.
The content of the invention
The purpose of the present invention is to overcome the deficiencies in the prior art and provide a kind of production high temperature resistant feruloyl esterase and high temperature resistant
The Trichoderma atroviride of cellulase and its application, the bacterial strain has that nutritional requirement is low, breeding is fast, tunning is easy to extract separation,
The short advantage of fermentation period, and its fermentation energy accesses feruloyl esterase and cellulase complex enzyme, complex enzyme high temperature resistant, the acid
Alkaline stability is good.
A kind of Trichoderma atroviride for producing high temperature resistant feruloyl esterase and high temperature-resisting cellulase（Trichoderma
atroviride）AWS26, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on January 2nd, 2014
Center CGMCC（Address is：City of BeiJing, China Chaoyang District North Star West Road 1 institute 3, postcode 100101）, deposit number：CGMCC
No. 8673。
Trichoderma atroviride bacterium AWS26 of the present invention screening technique comprises the following steps：
Step 1, sampling and sample treatment
From Xuzhou City of Jiangsu Province Yunlong District great Han villages be in the compost of hot fermentation in 10 parts of soil sampling.Weigh solid soil
Sample 5g, is put into the triangular flask equipped with bead and the sterilized physiological saline of 50mL, with 160r/min rotating speed shaken at room temperature
30min, is made suspension, and then this triangular flask is put into 60-70 DEG C of isothermal vibration water-bath, 100r/min concussion processing 20-
30min, obtains soil sample turbid solution.
Step 2, enrichment culture
Soil sample turbid solutions of the 2-4mL after high-temperature process is drawn, is added to the triangle that 30-60mL enriched mediums one are housed
In bottle, 45-50 DEG C is placed in, cultivate 5-7d in constant-temperature table under rotating speed 160-200r/min.Then sterile working measures 30-50mL
Enrichment culture liquid is into centrifuge tube, and 4000-6000r/min centrifugation 5-10min are outwelled supernatant, will centrifuged with enriched medium two
Precipitation afterwards is washed till in the triangular flask equipped with 30-60mL enriched mediums two, is again placed in 45-50 DEG C, rotating speed 160-200r/
5-7d is cultivated in min constant-temperature table.
Enriched medium one：Ferulic acid ethyl ester 0.2g, sodium carboxymethylcellulose 0.5g, mineral nutrition liquid 1mL supplement water
To 100mL, pH4.5.After 121 DEG C of 15min sterilizing, natural cooling, the ampicillin of filtration sterilization is added to final concentration
50mg/L。
Enriched medium two：Wheatfeed 1g, corn stalk powder 1g, ferulic acid ethyl ester 0.2g, mineral nutrition liquid 1mL are mended
It is filled with water to 100mL, pH4.5.After 121 DEG C of 15min sterilizing, natural cooling, the ampicillin of filtration sterilization is added to final concentration
50mg/L。
Mineral nutrition liquid：NaNO32.0g, K2PO41.5g, CaCl2 1.5g, MgSO40.3g, FeSO4•7H2O
0.1g, MnSO4•H2O 1.6mg, ZnSO4•H2O 0.05g, CoCl20.5mg, (NH4)2SO4 5g, deionized water 1000mL.
Step 3, primary dcreening operation
Nutrient solution after second is enriched with is diluted to 10 step by step-3、10-4、10-5、10-6, take the nutrient solution of dilutions at different levels
0.2mL is respectively coated on mould screening and culturing medium, culture dish dry in the air to surface there is no working fluid when, with preservative film by culture dish
Mouthful seal, be placed in being inverted culture 18-24h in 37-40 DEG C of constant incubator, then by flat board be placed in 40-45 DEG C it is incubated
Continue to be inverted culture 2-4d in case.
By the same position of the bacterium colony dibbling grown on flat board to two primary dcreening operation Selective agar mediums, two primary dcreening operation culture mediums point
Wei not feruloyl esterase primary dcreening operation culture medium and cellulase primary dcreening operation culture medium.40-45 DEG C of constant incubator of the culture dish being inoculated with
It is middle to be inverted culture 2-4d.Treat that obvious bacterium colony is formed, whetheing there is obvious transparent circle on observation feruloyl esterase primary dcreening operation culture medium occurs.
A certain amount of 0.1% Congo red dye liquor will be added on cellulase primary dcreening operation flat board, 1-10min is dyed, dyeing liquor is outwelled, bacterium is observed
Fall around whether the Congo's red color substantially shoals.To occur the bacterial strain of hydrolysis circle on two kinds of primary dcreening operation flat boards from feruloyl esterase
Inoculation comes out on primary dcreening operation flat board, further secondary screening.
Mould screening and culturing medium：PDA culture medium 800mL, rose-bengal 0.02g, chloramphenicol 0.1g, mineral nutrition liquid
1mL, moisturizing to 1000mL, agar powder 15g.121 DEG C, 15min sterilizings.Added after rose-bengal and chloramphenicol sterilizing.
PDA culture medium：Potato 200g, sucrose 20g, distilled water is settled to 1000mL.
Feruloyl esterase primary dcreening operation culture medium：PDA culture medium 100mL, mineral nutrition liquid 1mL, ferulic acid ethyl ester solution
1mL, agar powder 1.5g, pH4.5.121 DEG C, sterilize 20min.Fully shaken up after medium sterilization, with shake, culture medium is gradually
Become opaque, poured at 60-70 DEG C in flat board.
Ferulic acid ethyl ester solution is prepared：0.22g ferulic acid ethyl esters are weighed in centrifuge tube, 500 μ LN, N- dimethyl are added
Amide solution, adds 500 μ L sterilized waters after concussion dissolving, fully shakes up.
Cellulase primary dcreening operation culture medium：Cellulose powder 1g, peptone 0.1g, dusty yeast 0.1g, mineral nutrition liquid 1mL,
Agar 1.5, plus distilled water are settled to 100mL, pH value 4.5.121 DEG C of sterilizing 15min.
Step 4, secondary screening
The bacterial strain that primary dcreening operation is obtained is placed in 40-45 DEG C of constant incubator in the flat lining out of PDA culture medium and cultivates 2-
3d, repeats line culture more than three times, thinks that bacterial strain has been purified if continuous several times observe colonial morphology unanimously.
The bacterial strain that secondary screening is obtained after purification, is inoculated on solid seed culture medium, is cultivated 3-5d at 35-40 DEG C, is cut 1
The lawn of square centimeter is inoculated into the triangular flask equipped with 30-50mL liquid seed culture mediums, under 160-180r/min rotating speeds
37-40 DEG C of progress liquid concussion and cultivate.Cultivate after 3-4d, according to 6-10%（v/v）Inoculum concentration, liquid seeds are inoculated into liquid
In body fermentation medium, 40-45 DEG C carries out liquid concussion fermented and cultured under 160-180r/min rotating speeds.Fermented and cultured 3-5d,
Zymotic fluid 1200r/min, 10min collected after centrifugation supernatant is taken to determine feruloyl esterase and cellulose enzyme activity.Choose two
Kind of enzymatic activity higher bacterial strain carries out line Secondary Culture, finally gives that one plant of producing enzyme is stable and both productions that have high enzyme to live are resistance to
High feruloyl esterase produces the producing bacterial strain of high temperature-resisting cellulase again.The inoculation of purifying is on PDA slant mediums, 4 DEG C
Preservation.
Solid seed culture medium：PDA culture medium 99mL, mineral nutrition liquid 1mL, agar powder 1.5g, pH5.0,121 DEG C,
15min sterilizes.
Liquid seed culture medium：PDA culture medium 99mL, mineral nutrition liquid 1mL.121 DEG C, 15min sterilizings.
Liquid fermentation medium：PDA culture medium 80mL, mineral nutrition liquid 1mL, peptone 1g, Tween 80 0.5mL,
(NH4) 2SO4 1g, supplement deionized water to 100mL.121 DEG C, 15min sterilizings.
Through above-mentioned screening technique obtained strains, bacterium colony radially fast-growth on PDA plate, initial stage, hyphal surface was dredged
Loose, flat, white is presented light green because producing conidium, gradually increased later, dark green.Subiculum is fine and close, compared with
Thickness, gonimoblast beam is arranged in concentric wheel stripe shape, and the bacterium colony back side is colourless.In 400 times of optical microphotograph Microscopic observation of amplification, mycelium
It is made up of the branch mycelia with tabula, sporophore is uprightly born by mycelia, and branch is complicated, approximate right angle is stretched out, to raw or mutual
It is raw, arranged in dendroid, sporophore is long 5.0-12.0 μm, middle wide 2.5-3.7 μm, the wide 1.5-3.0 of base portion.Conidium cluster
It is born in the top of conidiophore.Conidium is spherical or ellipse, and size is 3.0-4.5 × 2.5-3.8 μm, and surface is thick
It is rough, it is covered with spinule, is gathered into spherical green conidial head on stigma by mucus.Control《Fungal identification handbook》And correlation
Document determines that the bacterial strain is Trichoderma atroviride（Trichodermaa troviride）.
Described production high temperature resistant feruloyl esterase and the Trichoderma atroviride of high temperature-resisting cellulase（Trichoderma
atroviride）Applications of the AWS26 in feedstuff degraded, specific method is：By dry corn stalk powder 10g, wheat bran
5g, ammonium sulfate 1g, bean cake powder 1g, ammonium nitrate 0.4g, NaCl0.5g, potassium dihydrogen phosphate 0.2g and MgSO40.05g is mixed, by solid
Liquid mass ratio 1:1.3 add deionized water, inoculate bacterial strain（Trichoderma atroviride）AWS26,26-30 DEG C of cultures
10 days.
Described production high temperature resistant feruloyl esterase and the Trichoderma atroviride of high temperature-resisting cellulase（Trichoderma
atroviride）Applications of the AWS26 in fermenting wheat bran prepares forulic acid, comprises the following steps：
Step 1, by bacterial strain（Trichoderma atroviride）AWS26 spore suspensions are inoculated into fermentation medium
In, 30-35 DEG C of culture 3-5d, middle turning is bent 2-3 times；
Step 2, deionized water is added in step 1 gained wheat bran, stirred, be placed in concussion shaking table 100r/min, 50 DEG C of leachings
2h is carried, is filtered, 8000r/min centrifugation 20min, it is 80% (NH to take supernatant saturation degree4)2SO4Saltout, 4 DEG C stand overnight,
8000r/min centrifuges 20min, collects precipitation, is dissolved with pH4.0 phosphoric acid-citric acid solution, obtains high temperature resistant forulic acid
Esterase and high temperature-resisting cellulase mix thick enzyme；
Step 3, thick enzyme obtained by step 2 is added into desizing wheat bran, adds water, adjust pH4.0,65 DEG C of enzymolysis 12h,
Obtain forulic acid.
Production high temperature resistant feruloyl esterase and the Trichoderma atroviride of high temperature-resisting cellulase that the present invention is provided, with nutritional requirement
The advantage that low, breeding is fast, tunning is easy to extract separation, fermentation period is short, and its fermentation energy access feruloyl esterase with
Cellulase complex enzyme, the complex enzyme high temperature resistant, ph stability are good.
Brief description of the drawings
Fig. 1 is Trichoderma atroviride primary dcreening operation Enzyme activity assay lithograph of the invention；
Fig. 2 is Trichoderma atroviride secondary screening Enzyme activity assay lithograph of the invention；
Fig. 3 is temperature in embodiment 3 to feruloyl esterase and the influence curve of cellulose enzyme activity；
Fig. 4 is pH value in embodiment 3 to feruloyl esterase and the influence curve of cellulose enzyme activity；
Fig. 5 is the heat endurance curve of feruloyl esterase and cellulose enzyme in complex enzyme formulation in embodiment 3；
Fig. 6 is feruloyl esterase and cellulase pH stability curves in complex enzyme formulation in embodiment 3.
Embodiment
Embodiment 1
This example demonstrates that Trichoderma atroviride AWS26 screening technique, is comprised the following steps that：
Step 1, sampling and sample treatment：
From Xuzhou City of Jiangsu Province Yunlong District great Han villages be in the compost of hot fermentation in 10 parts of soil sampling.Weigh solid soil
Sample 5g, is put into equipped with bead and the 50mL triangular flask of sterile saline, suspension is made in 160r/min vibration 30min,
Then this triangular flask is put into 60-70 DEG C of isothermal vibration water-bath, 100r/min concussion processing 20-30min obtain soil sample muddy
Liquid.
Step 2, enrichment culture：
2-4mL step 1 gained soil sample turbid solutions are drawn, the capacity equipped with 30-60mL enriched mediums one are added to for 250mL
Triangular flask in, cultivate 5-7d in 45-50 DEG C, rotating speed 160-200r/min constant-temperature table, obtain enrichment culture liquid.Sterile behaviour
Measure 30-50mL enrichment culture liquid, 4000-6000r/min centrifugation 5-10min outwell supernatant, will be from enriched medium two
Precipitation after the heart is washed till in the 250mL triangular flasks equipped with 30-60mL enriched mediums two, in 45-50 DEG C, rotating speed 160-200r/
5-7d is cultivated in min constant-temperature table.
Step 3, primary dcreening operation：
Nutrient solution after second is enriched with is diluted to 10 step by step-3、10-4、10-5、10-6, take the nutrient solution of dilutions at different levels
0.2mL is respectively coated on mould screening and culturing medium, culture dish dry in the air to surface there is no working fluid when, with preservative film by culture dish
Mouthful seal, be placed in being inverted culture 18-24h in 37-40 DEG C of constant incubator, then by flat board be placed in 40-45 DEG C it is incubated
Continue to be inverted culture 2-4d in case.
By the same position of the bacterium colony dibbling grown on flat board to two primary dcreening operation Selective agar mediums, two primary dcreening operation culture mediums point
Wei not feruloyl esterase primary dcreening operation culture medium and cellulase primary dcreening operation culture medium, 40-45 DEG C of constant incubator of the culture dish being inoculated with
It is middle to be inverted culture 2-4d.Treat that obvious bacterium colony is formed, whetheing there is obvious transparent circle on observation feruloyl esterase primary dcreening operation culture medium occurs.
A certain amount of 0.1% Congo red dye liquor will be added on cellulase primary dcreening operation flat board, 1-10min is dyed, dyeing liquor is outwelled, bacterium is observed
Fall around whether the Congo's red color substantially shoals.To occur the bacterial strain of hydrolysis circle on two kinds of primary dcreening operation flat boards from feruloyl esterase
Inoculation comes out on primary dcreening operation culture medium flat plate, further secondary screening.Feruloyl esterase and cellulase primary dcreening operation flat board testing result difference
As shown in Table 1 and Table 2.
Step 4, secondary screening：
The bacterial strain that step 3 primary dcreening operation is obtained is placed in 40-45 DEG C of constant incubator and trained in the flat lining out of PDA culture medium
2-3d is supported, line culture more than three times is repeated, thinks that bacterial strain has been purified if continuous several times observe colonial morphology unanimously, it is described
Bacterial strain primary dcreening operation Enzyme activity assay lithograph is as shown in Figure 1.
Step 5, culture presevation：
The bacterial strain that secondary screening is obtained after purification, is inoculated on solid seed culture medium, is cultivated 3-5d at 35-40 DEG C, is cut 1
The lawn of square centimeter is inoculated into the 250mL triangular flasks equipped with 30-50mL liquid seed culture mediums, is turned in 160-180r/min
The lower 37-40 DEG C of progress liquid concussion and cultivate of speed.Cultivate after 3-4d, according to 6-10%（v/v）Inoculum concentration, liquid seeds are inoculated with
Into liquid fermentation and culture, 40-45 DEG C carries out liquid concussion fermented and cultured under 160-180r/min rotating speeds.Fermented and cultured 3-
5d, takes zymotic fluid 1200r/min, 10min collected after centrifugation supernatant to determine feruloyl esterase and cellulose enzyme activity.Choose
Two kinds of enzymatic activitys higher bacterial strain carries out line Secondary Culture, finally gives both productions that one plant of producing enzyme is stable and has high enzyme to live
Resistance to high feruloyl esterase produces the producing bacterial strain of high temperature-resisting cellulase again.The inoculation of purifying is on PDA slant mediums, and 4
DEG C preservation.Secondary screening result as shown in table 3, considers the enzyme activity situation of feruloyl esterase and cellulase, selection strains A WS26
It is used as purpose bacterial strain.Secondary screening obtained strains AWS26 ferulaic acid esterase activities detect flat board as shown in Fig. 2
Wherein, used culture medium prescription is as follows：
Enriched medium one：Ferulic acid ethyl ester 0.2g, sodium carboxymethylcellulose 0.5g, mineral nutrition liquid 1mL supplement water
Sterilized to 100mL, pH4.5,121 DEG C, 15min, after natural cooling, add the ampicillin of filtration sterilization to final concentration
50mg/L。
Enriched medium two：Wheatfeed 1g, corn stalk powder 1g, ferulic acid ethyl ester 0.2g, mineral nutrition liquid 1mL are mended
100mL, pH4.5,121 DEG C, 15min sterilizings are filled with water to, after natural cooling, the ampicillin of filtration sterilization is added to final concentration
50mg/L。
Mould screening and culturing medium：PDA culture medium 800mL, rose-bengal 0.02g, chloramphenicol 0.1g, mineral nutrition liquid
1mL, moisturizing to 1000mL, agar powder 15g.121 DEG C, 15min sterilizings.Added after rose-bengal and chloramphenicol sterilizing.
Feruloyl esterase primary dcreening operation culture medium：PDA culture medium 100mL, mineral nutrition liquid 1mL, ferulic acid ethyl ester solution
1mL, 1.5g, pH4.5,121 DEG C of sterilizing 20min of agar powder.Fully shaken up after medium sterilization, with shake, culture medium is gradually
Become opaque, poured at 60-70 DEG C in flat board.Wherein, ferulic acid ethyl ester solution：Weigh 0.22g ferulic acid ethyl esters in from
In heart pipe, 500 μ L N, N- DMF solutions are added, 500 μ L sterilized waters is added after concussion dissolving, fully shakes up.
Cellulase primary dcreening operation culture medium：Cellulose powder 1g, peptone 0.1g, dusty yeast 0.1g, mineral nutrition liquid 1mL,
Agar 1.5, plus distilled water are settled to 100mL, pH value 4.5.121 DEG C of sterilizing 15min.
Solid seed culture medium：PDA culture medium 99mL, mineral nutrition liquid 1mL, agar powder 1.5g, pH5.0,121 DEG C,
15min sterilizes.
Liquid seed culture medium：PDA culture medium 99mL, mineral nutrition liquid 1mL.121 DEG C, 15min sterilizings.
Liquid fermentation medium：PDA culture medium 80mL, mineral nutrition liquid 1mL, peptone 1g, Tween 80 0.5mL,
(NH4)2SO41g, supplement deionized water to 100mL.121 DEG C, 15min sterilizings.
In above-mentioned culture medium, mineral nutrition liquid：NaNO32.0g, K2PO41.5g, CaCl21.5g, MgSO40.3g,
FeSO4·7H2O 0.1g, MnSO4·H2O 1.6mg, ZnSO4·H2O 0.05g, CoCl20.5mg, (NH4)2SO45g, go from
Sub- water 1000mL.
PDA culture medium：Potato 200g, sucrose 20g, distilled water is settled to 1000mL.200g potatos are weighed, cleans and goes
Skin is shredded, and the 1000mL that adds water boils half an hour, filtered through gauze, adds sucrose 20g, and benefit is filled with water to 1000mL.
Table 1 produces feruloyl esterase mould flat board primary dcreening operation result
The cellulase-producing mould flat board primary dcreening operation result of table 2
Each bacterial strain crude enzyme liquid enzyme activity of table 3
Obtained strains AWS26 of the present invention, bacterium colony radially fast-growth on PDA plate, initial stage hyphal surface it is loose,
Flat, white is presented light green because producing conidium, gradually increased later, dark green.Subiculum is fine and close, thicker, production
Spore Cong Shu is arranged in concentric wheel stripe shape, and the bacterium colony back side is colourless.In 400 times of optical microphotograph Microscopic observation of amplification, mycelium is by having
The branch mycelia for having tabula is constituted, and sporophore is uprightly born by mycelia, and branch is complicated, and approximate right angle is stretched out, and to raw or alternate, is in
Dendroid is arranged, and sporophore is long 5.0-12.0 μm, middle wide 2.5-3.7 μm, the wide 1.5-3.0 of base portion.Conidium cluster raw
In the top of conidiophore.Conidium is spherical or ellipse, and size is 3.0-4.5 × 2.5-3.8 μm, rough surface, cloth
Full spinule, spherical green conidial head is gathered into by mucus on stigma.Control《Fungal identification handbook》And pertinent literature
It is Trichoderma atroviride to determine the bacterial strain（Trichodermaa troviride）.
Strains A WS26 genomes of the present invention are extracted, according to primers most conservative in fungi 18SrDNA, amplification
Using sense primer：5'-CCGTAGGTGAACCTGCGG-3'（As shown in SEQ ID NO.2）, anti-sense primer：5'-
TCCTCCGCTTATTGATATGC-3'（As shown in SEQ ID NO.30.）.Reaction condition is：95 DEG C of pre-degeneration 5min, 94 DEG C
It is denatured 40S, 52 DEG C of annealing 40S, 72 DEG C of extension lmin, totally 30 circulations, last 72 DEG C of extensions 10min, 4 DEG C of preservations.Amplification production
Thing connects pMD18-T carriers.PCR reaction systems are：10 × Buffer2 μ L, dNTP (2.5mmol/L）2 μ L, the μ L of sense primer 1,
Anti-sense primer 1 μ L, template10~50ng, the μ L of Taq enzyme (2.5U) 0.3, add water and supply 20 μ L.It is obtained through PCR amplifications
18S rDNA fragments 712bp.The amplified fragments gel is reclaimed and is sequenced, submits GenBank to carry out nucleic acid homology analysis, is surveyed
Sequence is obtained as shown in SEQ ID NO.1, the 18SrDNA sequences of the close mould obtained with being compared from NCBI are used
ClustalX1.83 carries out Multiple Sequence Alignment, then using MEGA4.0 biological software constructing system chadograms, as a result shows bacterium
Strain AWS26 18SrDNA nucleotide sequences and Trichoderma atroviride（JX462604）18S rDNA nucleotides
Sequence homology highest, maximum comparability (maxident) reaches 99%.It is comprehensive to be observed with reference to its morphological feature, cultural characteristic, will
Strains A WS26 is accredited as Trichoderma atroviride（Trichoderma atroviride）.
Embodiment 2 produces high temperature resistant feruloyl esterase and the Trichoderma atroviride of high temperature-resisting cellulase is degraded applied to feedstuff
Dried corn stalk powder 10g, wheat bran 5g, ammonium sulfate 1g, bean cake powder 1g, ammonium nitrate 0.4g, NaCl 0.5g,
Potassium dihydrogen phosphate 0.2g, MgSO4 0.05g, by solid-liquid mass ratio 1:1.3 add deionized water, and above feed is placed in into triangular flask
In, constant temperature and humidity incubator is put into after disinfection inoculation, 26-30 DEG C of temperature, pH6.0 is adjusted.It is inoculated with activation of the strain for the present invention
Good strain AWS26.In fermentation process, stir daily after triangular flask, fermentation 10d, detection cellulose, hemicellulose and lignin
Changes of contents.Testing result is shown, in fermentation raw material, and the degradation rate of lignin is 77.4%, and the degradation rate of cellulose is
67.6%, the degradation rate of hemicellulose is 47.9%, and conversion rate of reducing sugar is 0.697g/g stalks.Illustrate what is screened using the present invention
The feedstuff such as high temperature resistant feruloyl esterase and high temperature-resisting cellulase bacterial strain processing maize straw, wheat bran is produced, can be significantly
The degradation rate of stodgy macromolecular carbohydrate is improved, possibility is provided applied to feed industry for it.
Embodiment 3 produces high temperature resistant feruloyl esterase and the Trichoderma atroviride of high temperature-resisting cellulase is applied to fermenting wheat bran system
Standby forulic acid
Wheat bran 50g, ammonium sulfate 1g, peptone 2g, potassium dihydrogen phosphate 0.2g, magnesium sulfate are added in 500mL triangular flasks
0.05g, 75mL water mix sterilizing.PDA culture medium inclined-plane culture is inoculated with after Trichoderma atroviride strain of the present invention, 30-35 DEG C of culture 3-
4d, then adds in inclined-plane and 2ml spore suspensions is added in sterilized water 10ml and the spore that fully suspends, the culture medium after sterilizing, fill
Divide after being well mixed, 3-5d is cultivated in 30-35 DEG C, middle turning is bent 2-3 times.Deionized water is added into the wheat bran fermented, is filled
Divide stirring to disperse wheat bran, be positioned on concussion shaking table under 100r/min rotating speeds, 50 DEG C of extraction 2h.Filtering, 8000r/min centrifugations
20min, it is 80% (NH to take supernatant saturation degree4)2SO4Saltout, 4 DEG C stand overnight, 8000r/min centrifugation 20min are collected
Precipitation, pH4.0 phosphoric acid-citric acid solution dissolving, obtains high temperature resistant feruloyl esterase and high temperature-resisting cellulase mixing
Thick enzyme.
Desizing wheat bran 5g is added in 250mL triangular flasks, Trichoderma atroviride fermentation self-control mixed enzyme 5mL of the present invention is added,
Water 115mL, adjusts pH4.0, and temperature is 65 DEG C, enzymolysis time 12h.Boiling water goes out enzyme, and centrifuging and taking supernatant determines forulic acid and reduction
The mass concentration of sugar.Forulic acid burst size is up to 9.12mg/g wheat bran on this condition.Produced using strain fermentation of the present invention
Mixed enzyme production asafoetide acid reaction is gentle, be easily controlled, and will not cause brown stain, be optimal extracting method, it is adaptable to which industry is raw
Produce forulic acid.
The enzyme activity and enzymatic property of the high temperature resistant feruloyl esterase of embodiment 3 and high temperature-resisting cellulase are determined
The Trichoderma atroviride AWS26 bacterial strains that screening is obtained are fermented, and fermentative medium formula is：PDA culture medium 80mL,
Mineral nutrition liquid 1mL, peptone 1g, Tween 80 0.5mL, (NH4)2SO41g, supplement deionized water to 100mL.121 DEG C,
15min sterilizes.Fermentation condition is：Fermentation period 2-3d, 35-40 DEG C of fermentation temperature, inoculum concentration 4-8%, shaking speed 160-
200r/min。
After fermentation ends, zymotic fluid 8000r/min centrifugations 20min is removed into thalline, and remaining bacterium is filtered out with 0.22 μm
Body and insoluble matter, collect filter liquor.According to ammonium sulfate saturation degree 75%, ammonium sulfate is added in top fermentation filter liquor, fully
After dissolving, 4 DEG C stand overnight, 8000r/min centrifugation 20min, collect precipitation, with 20mmol/L, pH6.0 phosphate buffer solutions are molten
Solution, loading Sephedex G25 molecular sieve gel post desalinations, chromatographic column balanced with pH6.5 phosphate buffer after with identical buffering
Solution is eluted, and elution rate is 15~20ml/h, obtains crude enzyme liquid.Gained zymotic fluid feruloyl esterase enzyme activity is：262U/
L；Cellulose enzyme activity is：915U/mL.
Activity determination method is：
The ferulic acid ester enzyme activity determination of zymotic fluid
Enzyme liquids of the 0.5mL after 1-5 times dilutes is added into 2mL centrifuge tubes, 0.5mL Ferulic acid methylesters are then added
Solution, is fully mixed, 65 DEG C of insulation 20min.Add 1mL volume fractions be 10% glacial acetic acid with terminating reaction.Sample in
12000r/min centrifuges 10min.Retain supernatant, after 2-4 times of dilution, with high performance liquid chromatography (HPLC) method determination sample
Ferulaic acid content.To boil the enzyme liquid of inactivation as a control group, processing method is ibid.
The enzyme activity definition of feruloyl esterase：At 50 DEG C, pH value is that under conditions of 6.5, esterlysis Ferulic acid methylester per minute is raw
Enzyme amount into needed for 1 μm of ol forulic acid.Forulic acid uses high effective liquid chromatography for measuring.
The assay method of cellulase filter paper enzyme activity：Xinhua's qualitative filter paper is cut into size for 1 × 6cm, centrifugation is placed in
Guan Zhong, adds pH4.0 disodium hydrogen phosphates-citrate buffer solution 1mL, is preheated in the water-bath of 55 DEG C of temperature.Add enzyme liquid 500
μ L, insulation reaction 60min.3mL DNS reagents are added, boiling water bath 5min colour developings are placed in cold water and are cooled to room temperature.Draw 200
μ L react supernatant, and the light absorption value of determination sample at wavelength 540nm is calculated according to the concentration of glucose standard curve of drafting
Concentration of glucose in reaction system.To boil the enzyme liquid of inactivation as a control group, processing method is ibid.
The unit of activity of cellulase（U）It is defined as：Under these conditions, decomposition sodium carboxymethylcellulose generation per minute
Enzyme amount required for 1 μm of ol reduced sugar is 1 enzyme activity unit (U).
1. enzyme reaction optimum temperature is determined
By reaction system be respectively placed in 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 of water-bath, after appropriateness dilution, determine feruloyl esterase enzyme and cellulase activity, research enzymatic is anti-respectively
Answer optimum temperature.Using enzyme under optimum temperature survey enzyme activity as 100%, other temperature are lower surveys for it with respect to enzyme activity.As a result such as
Shown in Fig. 3, the variation tendency that feruloyl esterase and cellulase rise enzyme with temperature is essentially identical.The forulic acid from 20 DEG C to 65 DEG C
Esterase, as temperature rise enzymatic activity gradually increases, when temperature reaches 65 DEG C, enzyme activity reaches maximum.Later with temperature liter
Height, enzyme activity is gradually reduced, therefore the feruloyl esterase optimum temperature of enzyme is 65 DEG C.And the temperature trip point of cellulase is 55
DEG C, i.e. the most suitable operative temperature of cellulase is 55 DEG C.Entirety sees that two kinds of enzymes are all high temperature enzymes, two between 45 DEG C -70 DEG C
The enzyme activity of enzyme is planted all more than 80%, therefore complex enzyme formulation can all play efficient enzymolysis in this temperature range.
2. enzyme effect optimal pH is determined
Enzyme preparation carries out enzymatic reaction under different pH (3.0~9.0), determines the most suitable work of feruloyl esterase and cellulase
Use pH.Buffer solution used is：The glycine-HCI cushioning liquid of pH2.0~3.0, the citrate phosphate buffer of pH3.0~4.0,
The acetic acid-sodium acetate buffer solution of pH4.0~5.0, pH5.0~7.0 disodium hydrogen phosphates-potassium phosphate buffer, pH7.0~
9.0 Tris-HCl cushioning liquid.Using enzyme under optimum pH survey enzyme activity as 100%, other pH value are lower to be surveyed to be relative to its
Enzyme activity.Enzyme preparation in difference pH buffer systems, determines feruloyl esterase and cellulase at 65 DEG C and 55 DEG C respectively more than
Enzymatic activity, as a result as shown in figure 4, feruloyl esterase enzymatic activity is more than 80% between pH3.5~7.0, most suitable action pH is
4.5.Cellulase surveys enzyme activity more than 90% between pH4.5-7.0, most suitable enzyme activity 5.5.Entirety sees two kinds of enzymes of enzyme preparation
Enzymatic activity is all more than 80% between pH4.5-7.0, therefore enzyme preparation is adapted to enzyme digestion reaction in acid condition.
3. the heat endurance experiment of enzyme
The enzyme preparation for being dissolved in pH6.5 phosphate buffers is incubated after 4h under 40 DEG C -80 DEG C of different temperatures, immediately ice
Bath, after appropriateness dilution, determines feruloyl esterase and cellulose enzyme vigor respectively.Using enzyme under most equilibrium temperature survey enzyme activity as
100%, other temperature are lower to be surveyed as to its relative enzyme activity.As a result as shown in Figure 5, two kinds of basic rising enzyme activities with temperature of enzyme
Gradually reduce, when temperature is higher than 60 DEG C, feruloyl esterase enzymatic activity declines notable.Two kinds of enzymes are below 60 DEG C, and enzyme activity is preserved
Rate is all more than 80%；Less than 55 DEG C, remaining enzyme activity is all more than 90%.Complex enzyme formulation is high temperature resistant type.
4. influence of the pH value to enzyme stability
Enzyme preparation is incubated 24h in different pH buffer solution respectively at 40 DEG C, and feruloyl esterase and fibre are then determined respectively
The plain enzyme enzyme activity of dimension, using enzyme, institute's surveys enzyme activity is 100% under most stable pH, and it is to its enzyme activity relatively that other pH are lower, which to be surveyed,.As a result see
Shown in Fig. 6,40 DEG C, 24h feruloyl esterases enzymatic activity does not change substantially in the range of pH3.0-8.0, activity stabilized, with respect to enzyme
Live more than 95%.Cellulase enzymatic activity between pH4.0-9.0 does not decline substantially.Entirety sees that complex enzyme formulation has
Wider ph stability, property is more stable in acid condition.
5. influence of the holding time of enzyme preparation to enzymatic activity
The solid polypeptide formulation prepared is preserved at 4 DEG C and at room temperature respectively, an enzyme activity is detected each moon, altogether inspection
Survey 6 months, check that enzyme activity retains situation.As a result it is as shown in the table：
Within six months, feruloyl esterase and cellulose enzyme activity storage rate are all more than 90% at 4 DEG C, room temperature condition
Lower enzyme activity is also more than 80%, it can be seen that the stability of enzyme is good.
SEQUENCE LISTING
<110>Xuzhou Engineering Institute
<120>A kind of Trichoderma atroviride and its application for producing 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>Sense primer
<400> 2
ccgtaggtga acctgcgg 18
<210> 3
<211> 20
<212> DNA
<213>Anti-sense primer
<400> 3
tcctccgctt attgatatgc 20
Claims (5)
1. a kind of Trichoderma atroviride for producing high temperature resistant feruloyl esterase and high temperature-resisting cellulase（Trichoderma
atroviride）AWS26, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, preservation
Numbering：CGMCC No. 8673.
2. the Trichoderma atroviride of the production high temperature resistant feruloyl esterase and high temperature-resisting cellulase described in claim 1
（Trichoderma atroviride）Applications of the AWS26 in feedstuff degraded.
3. the Trichoderma atroviride of the production high temperature resistant feruloyl esterase and high temperature-resisting cellulase described in claim 1
（Trichoderma atroviride）Applications of the AWS26 in fermenting wheat bran prepares forulic acid.
4. the Trichoderma atroviride of production high temperature resistant feruloyl esterase according to claim 2 and high temperature-resisting cellulase
（Trichoderma atroviride）Applications of the AWS26 in feedstuff degraded, it is characterised in that：Comprise the following steps：
By dry corn stalk powder 10g, wheat bran 5g, ammonium sulfate 1g, bean cake powder 1g, ammonium nitrate 0.4g, NaCl0.5g, potassium dihydrogen phosphate
0.2g and MgSO40.05g is mixed, by solid-liquid mass ratio 1:1.3 add deionized water, inoculate Trichoderma atroviride（Trichoderma
atroviride）AWS26,26-30 DEG C are cultivated 10 days.
5. the Trichoderma atroviride of production high temperature resistant feruloyl esterase according to claim 3 and high temperature-resisting cellulase
（Trichoderma atroviride）Applications of the AWS26 in fermenting wheat bran prepares forulic acid, it is characterised in that：Including following
Step：
Step 1, by Trichoderma atroviride（Trichoderma atroviride）AWS26 spore suspensions are inoculated into fermentation medium
In, 30-35 DEG C of culture 3-5d, middle turning is bent 2-3 times；
Step 2, deionized water is added in step 1 gained wheat bran, stirred, be placed in concussion shaking table 100r/min, 50 DEG C of extraction 2h,
Filtering, 8000r/min centrifugation 20min, it is 80% (NH to take supernatant saturation degree4)2SO4Saltout, 4 DEG C stand overnight, 8000r/
Min centrifuges 20min, collects precipitation, is dissolved with pH4.0 phosphoric acid-citric acid solution, obtain high temperature resistant feruloyl esterase and
High temperature-resisting cellulase mixes thick enzyme；
Step 3, thick enzyme obtained by step 2 is added into desizing wheat bran, adds water, adjusted pH4.0,65 DEG C of enzymolysis 12h, obtain Ah
Wei's acid.
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| CN102925365A (en) * | 2012-10-24 | 2013-02-13 | 郑州大学 | Trichoderma atroviride strain and application thereof in preparation of cellulase |
| CN104204215A (en) * | 2012-03-26 | 2014-12-10 | 诺维信北美公司 | Methods of preconditioning cellulosic material |
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| CN101492700A (en) * | 2009-03-05 | 2009-07-29 | 王建设 | Intensive processing method for stalk articles or agricultural castoff |
| CN104204215A (en) * | 2012-03-26 | 2014-12-10 | 诺维信北美公司 | Methods of preconditioning cellulosic material |
| CN102925365A (en) * | 2012-10-24 | 2013-02-13 | 郑州大学 | Trichoderma atroviride strain and application thereof in preparation of cellulase |
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