CN109825485A - A kind of feruloyl esterase EpFAE1 and its encoding gene and application - Google Patents

A kind of feruloyl esterase EpFAE1 and its encoding gene and application Download PDF

Info

Publication number
CN109825485A
CN109825485A CN201910230349.7A CN201910230349A CN109825485A CN 109825485 A CN109825485 A CN 109825485A CN 201910230349 A CN201910230349 A CN 201910230349A CN 109825485 A CN109825485 A CN 109825485A
Authority
CN
China
Prior art keywords
epfae1
feruloyl esterase
acid
ferulic acid
added
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201910230349.7A
Other languages
Chinese (zh)
Inventor
龙良鲲
吴莲秋
丁少军
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Forestry University
Original Assignee
Nanjing Forestry University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Forestry University filed Critical Nanjing Forestry University
Priority to CN201910230349.7A priority Critical patent/CN109825485A/en
Publication of CN109825485A publication Critical patent/CN109825485A/en
Priority to CN201910540226.3A priority patent/CN110106156B/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01073—Feruloyl esterase (3.1.1.73)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention discloses a kind of feruloyl esterase EpFAE1 and its encoding gene and applications.The amino acid sequence of the feruloyl esterase EpFAE1 is as shown in SEQ ID NO.1, and the DNA sequence dna of encoding gene is as shown in SEQ ID NO.2.The present invention clones from fine penicillium 4-14 and obtains the encoding gene of feruloyl esterase EpFAE1, and is expressed by Pichia pastoris its recombinant vector, obtains pure enzyme.It is confirmed by test, feruloyl esterase EpFAE1 of the invention has high specific acitivity, high enzymolysis efficiency, high stability and the advantages such as certain resistance to metal ion and salt tolerance, has important application prospect in fields such as medicines and health protection, food, feed and bioenergies.

Description

A kind of feruloyl esterase EpFAE1 and its encoding gene and application
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of feruloyl esterase EpFAE1 and its encoding gene and Using.
Background technique
Ferulic acid (Ferulic acid, FA) is also known as Ferulic acid, is widely present in plant cell wall A kind of middle phenolic acid.In plant cell wall, ferulic acid can be in the form of ester bond and Hemicellulose Polysaccharide, or with ehter bond Form and lignin are crosslinked, and form complicated reticular structure.The presence of ferulic acid enhances the mechanical strength of cell wall, for anti- Only enzyme is degraded, the integrality of protection cell wall structure has a very important role.Due to specific molecular structure, ferulic acid The precursor that with excellent antioxygenic property, uses, and can be synthesized with new drug as antioxidant in field of medicaments.Meanwhile Ah Wei's acid has the characteristics such as antibacterial, antitumor, anti-inflammatory, whitening and uvioresistant, answers in industries such as medical and health, functional food and cosmetics Use Huge value.
Feruloyl esterase (E.C.3.1.1.73, feruloyl esterase, FAE) is a kind of Asia of carboxylic ester hydrolases Class can be catalyzed the hydrolysis of ester bond between hydroxycinnamic acid and glycan molecule in plant cell wall.Studies have shown that feruloyl esterase is The key effect enzyme of ferulic acid can be released effectively under the synergistic effect of internal cutting type xylanase etc. in release plant cell wall Put the ferulic acid in the matrix such as wheat bran.Industrially, feruloyl esterase can be used for from agricultural machining by-product (such as wheat Bran and corn bran) phenolic acid is prepared, achieve the purpose that high added value converts；As the additive of dietary fiber, promote thin The release of ferulic acid in cell wall；Applied to feed and paper industry, promote in the degradation or paper pulp of feed hemicellulose and wooden Element removal etc..
Currently, many different types of feruloyl esterases report in succession by research, but ferulic acid enzyme preparation etc. in the market Product is still insufficient.Therefore, novel feruloyl esterase gene resource is excavated, high level expression and preparation is realized, specifies its enzyme Characteristic and industrial application condition are learned, is to develop that sequence is novel, easily the stable feruloyl esterase of preparation and properties is effective Means.
Summary of the invention
Goal of the invention: for the deficiency of existing market ferulic acid enzyme preparation product, the object of the present invention is to provide one kind to come Feruloyl esterase EpFAE1 in fine penicillium 4-14, makes it have preferable catalytic capability, temperature stability and pH Stability.It is a further object of the present invention to provide the applications of above-mentioned feruloyl esterase EpFAE1.
Technical solution: in order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention are as follows:
A kind of feruloyl esterase EpFAE1, amino acid sequence is as shown in SEQ ID NO.1.
Discovery feruloyl esterase EpFAE1 is compared through albumen homology and belongs to 1 member of carbohydrate esterase family, and is belonged to In A type feruloyl esterase.Its optimum temperature is 50 DEG C, is resistant to 50 DEG C of high temperature, and optimal pH 5.5, pH has good stability, There is certain tolerance to metal ion and salt, specific activity is high.
The encoding gene of the feruloyl esterase EpFAE1, base sequence is as shown in SEQ ID NO.2.
The expression vector of encoding gene containing the feruloyl esterase EpFAE1.
The feruloyl esterase EpFAE1 prepares the application in ferulic acid in enzymatic hydrolysis wheat bran.Utilize 1.75% phosphoric acid Processing, and under the collective effect of feruloyl esterase EpFAE1 and zytase, the release rate of ferulic acid reaches as high as 83.07%.Highest can discharge 0.36g ferulic acid from 100g desizing wheat bran, and enzymatic hydrolyzation is high.
The feruloyl esterase EpFAE1 prepares the application in ferulic acid in enzymatic hydrolysis corn bran.It is handled using 1% acid, And under the collective effect of feruloyl esterase EpFAE1 and zytase, the release rate of ferulic acid reaches as high as 56.52%.Highest The ferulic acid of 1.16g can be discharged from 100g desizing corn bran, enzymatic hydrolyzation is high.
For expanding the specific primer of the feruloyl esterase EpFAE1 gene, including following two sequences:
Upstream primer: 5 '-CCGgaattcGCCGTTACGCAGGGCGTCTCTG-3 '；
Downstream primer:
5′-CTAGtctagaTCAgtgatggtgatggtgatgCCAACTGCAAGCTCCGCTCG-3′。
The utility model has the advantages that compared with prior art, the present invention clones from fine penicillium 4-14 and obtains feruloyl esterase The encoding gene of EpFAE1, and its recombinant vector is expressed by Pichia pastoris, obtain pure enzyme.It is confirmed by test, this The feruloyl esterase EpFAE1 of invention have high specific acitivity, high enzymolysis efficiency, high stability and certain resistance to metal ion and The advantages such as salt tolerance have important application prospect in fields such as medicines and health protection, food, feed and bioenergies.
Detailed description of the invention
Fig. 1 is the SDS-PAGE electrophoresis for recombinating feruloyl esterase EpFAE1；In figure, M:Marker；EpFAE1: purifying Feruloyl esterase；
Fig. 2 is the optimum temperature result figure for recombinating feruloyl esterase EpFAE1；
Fig. 3 is the temperature stability result figure for recombinating feruloyl esterase EpFAE1；
Fig. 4 is the optimal pH result figure for recombinating feruloyl esterase EpFAE1；
Fig. 5 is the pH stability result figure for recombinating feruloyl esterase EpFAE1；
Fig. 6 is to recombinate feruloyl esterase EpFAE1 to the tolerability results figure of NaCl；
Fig. 7 is the zymetology aerodynamic measurement result figure for recombinating feruloyl esterase EpFAE1；Substrate is Ferulic acid methylester.
Fig. 8 is the knot for recombinating feruloyl esterase EpFAE1 collaboration zytase degradation desizing wheat bran release ferulic acid Fruit figure；Supernatant enzymatic hydrolysis, is digested with the EpFAE1 of 1U/mL；Matrix enzymatic hydrolysis, with the source EpFAE1 and 200U/g of 1U/g matrix In the internal cutting type xylanase EpXYN1 Synergistic degradation of fine penicillium.
Fig. 9 is the knot for recombinating feruloyl esterase EpFAE1 collaboration zytase degradation desizing corn bran release ferulic acid Fruit figure；Supernatant enzymatic hydrolysis, is digested with the EpFAE1 of 1U/mL；Matrix enzymatic hydrolysis, with the source EpFAE1 and 200U/g of 1U/g matrix In the internal cutting type xylanase EpXYN1 Synergistic degradation of fine penicillium.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below, but these examples are not intended to limit the invention.It is following Experimental method used in embodiment is conventional method unless otherwise specified.Material as used in the following examples, reagent Deng being commercially available unless otherwise specified.
Material and reagent used in following embodiment are as follows:
Bacterial strain and carrier: fine penicillium 4-14 (Eupenicillium parvum 4-14) is preserved in Chinese Typical Representative training Object collection is supported, deposit number is CCTCC No:M2015404.Pichia pastoris yeast (Pichia pastoris GS115) And expression vector pPICZ α A is purchased from Invitrogen company, Escherichia coli Top10 and genetic manipulation plasmid pEASY-Blunt purchase From Beijing Quanshijin Biotechnology Co., Ltd (TransGen Biotech).
Enzyme and other biochemical reagents: restriction enzyme, archaeal dna polymerase, ligase and dNTP are purchased from TaKaRa company； Ferulic acid methylester is purchased from Aladdin company；It is other all (to be bought from common biochemical Reagent Company for domestic analytical reagents To).
LB culture medium: Peptone 10g, Yeast extract 5g, NaCl 10g adds distilled water to 1000mL, and pH is certainly So (about 7).Solid medium adds 1.5% (w/v) agar on this basis.
PDA culture medium: potato 200g, glucose 20g, agar powder 15g, autoclave sterilization,
YPD culture medium: Yeast Extract 10g, peptone 20g, glucose 20g are dissolved in 900mL water, add distillation Water is settled to 1000mL, 6.5,115 DEG C of pH, 30min sterilizing.
BMGY culture medium: Yeast Extract 10g, peptone 20g are dissolved in 700mL water, and autoclave sterilization is cooling The kaliumphosphate buffer (autoclave sterilization) of the 1M of 100mLpH=6 is added after to room temperature, 10 × YNB of 100mL (is crossed and filtered out Bacterium), 10 × GY of 100mL (100mL glycerol is dissolved in 900mL water autoclave sterilization), 4 DEG C of preservations after mixing.
BMMY culture medium: Yeast Extract 10g, peptone 20g are dissolved in 700mL water, and autoclave sterilization is cooling It is added after to room temperature, the kaliumphosphate buffer (autoclave sterilization) of the 1M of 100mL pH 6,10 × YNB of 100mL (is crossed and filtered out Bacterium), 10 × M of 100mL (5% methanol), 4 DEG C of preservations after mixing.
Do not make the experimental methods of molecular biology illustrated in following embodiment, referring to " Molecular Cloning:A Laboratory guide " Listed specific method carries out in one book of (third edition) J. Pehanorm Brooker, or carries out according to kit and product description.
The clone of 1 feruloyl esterase EpFAE1 encoding gene of embodiment
Fungal culture and Total RNAs extraction: the thallospore mixture of the fine penicillium 4-14 bacterial strain of about 10mg is taken to access In 50mL PDA liquid medium, 37 DEG C, 180rpm culture 4 days.1mL culture is taken to access one bottle of solid-state fermentation culture medium (L.Long, D.Ding, Z.Han, H.Zhao, Q.Lin, S.Ding, Thermotolerant hemicellulolytic and cellulolytic enzymes from Eupenicillium parvum 4-14 display high efficiency Upon release of ferulic acid from wheat bran, J.Appl.Microbiol.121 (2016) 422- 434.) it in, shakes up and is placed on 37 DEG C, stationary culture 3 days under 70% damp condition.Extracting waste mycelium is clean with rinsed with sterile water Afterwards, filter paper suck dry moisture, liquid nitrogen flash freezer are placed on -70 DEG C of preservations.With TransZolTM Plant kit (TransGen, north Capital) extract thallus total serum IgE.
Gene cloning: take appropriate total serum IgE with EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen, Beijing) and oligo (dT) are that primer carries out reverse transcription reaction acquisition cDNA.To obtain cDNA as template, with primer fae1_f1 (5 '-CGTTGAACCATTGTCCATCCA-3 ') and fae1_r1 (5 '- TGAATCGCCTCTGACTACCAA-3 ') routine PCR reaction is carried out, obtain target gene segment.Further, by target gene piece Section is cloned into carrier pEASY-Blunt (TransGen, Beijing), and completes sequence by Suzhou Jin Weizhi Bioisystech Co., Ltd Analysis.
Obtain the segment of the full length gene containing feruloyl esterase EpFAE1.Sequencing result shows, the feruloyl esterase EpFAE1 full length gene 843bp, DNA sequence dna are shown in SEQ ID NO.2, and albumen (feruloyl esterase EpFAE1) sequence of expression is shown in SEQ ID NO.1, reading frame includes 280 amino acid, wherein preceding 20 amino acid is signal peptide.It is compared through albumen homology It was found that it belongs to 1 member of carbohydrate esterase family, and belongs to A type feruloyl esterase, the theoretical molecular weight of maturation protein is 28.22kDa, theoretical isoelectric point (pI) are 4.97.
The Pichia anomala expression of 2 feruloyl esterase EpFAE1 of embodiment and purifying
The specific primer fae1_f2 and fae1_r2 of synthesis expression feruloyl esterase EpFAE1 is separately designed, as follows:
Fae1_f2:5 '-CCGgaattcGCCGTTACGCAGGGCGTCTCTG-3 '；
Fae1_r2:
5′-CTAGtctagaTCAgtgatggtgatggtgatgCCAACTGCAAGCTCCGCTCG-3′。
Using this, to primer, from the plasmid containing feruloyl esterase EpFAE1 gene amplification obtains it without signal peptide Feruloyl esterase EpFAE1 genetic fragment.It is double digested that the target gene segment of amplification is subjected to EcoRI-XbaI, and with warp The plasmid pPICZ α A of EcoRI-XbaI digestion is attached, and obtains gene expression plasmid pPIC-EpFae1.Recombinant plasmid pPIC- EpFae1 through restriction enzyme SacI linearisation after electric shock be transferred to Pichia pastoris GS115 after, using contain the mountain 1M The YPD plate of pure and mild 100 μ g/mL Zeocin (Invitrogen) antibiotic of pears carries out the screening of positive colony.By what is screened Positive colony access is equipped with 28 DEG C in the test tube of 5mLYPD and 100 μ g/mL antibiotic Zeocin, 200rpm shaking table culture, after 20h Bacterium solution is transferred to in 50mLBMGY culture solution 28 DEG C respectively, 200rpm shaking table culture to its OD600After reaching 3.0, it is collected by centrifugation Thallus, then be transferred in 25mL BMMY culture solution 28 DEG C, 200rpm shaking table culture, every adding methanol for 24 hours, methanol concentration is Crude enzyme liquid is collected by centrifugation after 0.8-1.0% (v/v), continuous culture 5-7d.Crude enzyme liquid is initially charged in bag filter, 4 DEG C in pH Gentle agitation is dialysed for 24 hours in 8.0 phosphate buffer.Method of the purifying of enzyme solution referring to Ni-NTA Agarose (Qiagen). Protein quantification is completed using BCA detection kit (Thermo Tech, USA).The albumen that purifying is obtained utilizes SDS- PAGE is detected.As a result as shown in Figure 1, it is seen that recombination feruloyl esterase EpFAE1 is expressed in Pichia pastoris, is passed through It is after purification single band, molecular weight is close to 40kDa.Actual molecular weight size is bigger than theoretical molecular weight, thus it is speculated that is by glycosylating It is caused.
1, the activity determination method of feruloyl esterase EpFAE1
The activity of feruloyl esterase EpFAE1 is measured using Ferulic acid methylester as substrate.First with level-one Chromatographic Pure Methanol dissolution Ah Wei's acid methyl esters, is configured to 50mM concentration.800 μ L citric acid-sodium citrate (0.1M, pH 6.0) buffers are taken, are added 100 μ L's Ferulic acid methylester methanol solution is placed in 40 DEG C of preheating 5min, and the enzyme solution that 100 μ L are diluted to debita spissitudo is added.By mixture in 30min is reacted at 40 DEG C, and is handled 10min at 99 DEG C and terminated reaction.Reaction mixture is cooling in room temperature, and to inactivate enzyme solution It compares.
According to the improved HPLC analysis method such as Andersen (Andersen A, Svendsen A, et al.Studies on ferulic acid esterase activity in fungal lipases and cutinases.Colloids And Surfaces B:Biointerfaces, 2002,26:47-55) in measurement reaction solution ferulic acid yield.It uses Column model be ZORBAX Eclipse Plus C18,3.5 μm of filler diameter, size 4.6mm × 100mm.Mobile phase is first Alcohol: 0.1% acetic acid=35: 65, flow velocity 0.8mL/min, 30 DEG C of column temperature, sample volume 5 μ L, Detection wavelength 320nm.Ferulic acid The definition of esterase enzyme activity: under the conditions of 40 DEG C, 6.0 pH, substrate Ferulic acid methylester is hydrolyzed per minute and is generated needed for 1 μm of ol ferulic acid Enzyme amount be 1 unit of activity (U).The production of ferulic acid standard curve: preparing a series of ferulic acid methanol solution of concentration, 0.04mM, 0.2mM, 1mM, 2.5mM and 5mM pass through HPLC detection assay 320nm wavelength respectively with after 0.22 μm of membrane filtration Under each concentration peak area, using the peak area measured as ordinate, material concentration is that abscissa draws standard curve, and by soft Part carries out linear fit and obtains equation of linear regression.Ferulic acid ester enzyme activity in crude enzyme liquid can be calculated according to standard curve (U/mL)。
Enzymatic activity calculation formula is (U/mL)=(v × x)/(c × T) × n, wherein
The final volume (mL) of v- enzymatic reaction system；
X- is according to the calculated ferulaic acid content of ferulic acid standard curve (μm ol/mL)；
The amount (mL) of enzyme solution added by c-；
T- time of enzymatic reacting (min)；
The extension rate of n- enzyme solution.
2, the measurement of feruloyl esterase EpFAE1 enzymatic property
1) the most suitable catalytic temperature and temperature stability of feruloyl esterase EpFAE1
Most suitable catalytic temperature: Ferulic acid methylester level-one Chromatographic Pure Methanol is dissolved and is configured to 50mM concentration.Take 800 μ L The citric acid-sodium citrate buffer solution (pH 6.0) of 0.1M is added the substrate solution of 100 μ L, is respectively placed in 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, preheat 5min in 60 DEG C and 70 DEG C of water-baths, the enzyme solution after 100 μ L dilution is added.After reacting 30min, boiling Water inactivates 10min and terminates reaction.Reaction mixture is cooling in room temperature, detects enzyme activity by the method for HPLC, and with the enzyme of inactivation Liquid compares.As a result as shown in Fig. 2, the optimal reactive temperature of feruloyl esterase EpFAE1 is 50 DEG C.
Temperature stability: the feruloyl esterase EpFAE1 of purifying is respectively placed in 45 DEG C, 50 DEG C and 55 DEG C and is incubated for respectively Remaining enzyme activity is detected according to standard method after 0.5-6h, is the opposite enzyme activity of 100% calculating with the activity of untreated enzyme.As a result As shown in figure 3, placing the activity that 6h is still able to maintain about 70% in 50 DEG C, but the enzyme is unstable at 55 DEG C, and it is complete to place 6h It loses activity.
2) the most suitable catalytic pH and pH stability of feruloyl esterase EpFAE1
Most suitable catalytic pH: Ferulic acid methylester level-one Chromatographic Pure Methanol is dissolved and is configured to 50mM concentration.Prepare pH points Not Wei 3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0 and 7.5 and concentration be 0.1M citric acid-sodium citrate buffering Liquid, respectively takes 800 μ L buffers that the substrate of 100 μ L is added, and appropriate diluted 100 μ L enzyme solution is added in 40 DEG C of preheating 5min.It will mixing After object reacts 30min at 40 DEG C, boiling water inactivates 10min and terminates reaction.The enzyme of inactivation is set as control.Reactant is in room temperature After lower cooling, the enzymatic activity under each pH is measured by HPLC method, and the enzymatic activity not deal with calculates opposite for 100% Enzyme activity.As a result as shown in figure 4, the optimal pH of feruloyl esterase EpFAE1 is 5.5, enzyme can all be significantly reduced by reducing or increase pH It is living.
PH stability: pure enzyme and the pH citric acid-sodium citrate buffer solution for being respectively the 0.1M of 3-8 are mixed in equal volume, And after placing 16 hours at 4 DEG C, remaining enzyme activity is detected by HPLC.With untreated enzyme activity for 100%, opposite enzyme is calculated It is living.As a result as shown in figure 5, EpFAE1 places the activity that 16h is able to maintain 80% or more in the buffer of pH 3-8, wherein pH Under conditions of 5.0, residual activity reaches 90% or more.
3) metal ion or chemical reagent are on the active influence of feruloyl esterase EpFAE1
In 50mM citric acid-sodium citrate buffer solution (pH5.5), the MnCl of 1 or 5mM is added respectively2Or NH4C1 or CaCl2Or CuCl2Or ZnCl2Or FeCl3Or NiSO4Or MgCl2Or EDTA, using Ferulic acid methylester as substrate, in optimal reaction item The activity of feruloyl esterase EpFAE1 is measured under part.Using untreated fish group as control, the opposite enzyme activity of each processing is calculated.As a result such as Shown in table 1, Mn2+、Ca2+、Zn2+、Ni2+Or Mg2+To enzymatic activity, there are small size influences at 1mM and 5mM.Cu2+And Fe3+Low Under concentration (1mM) can obvious inhibitory enzyme activity, high concentration (5mM) then completely inhibits enzymatic activity.Low concentration (1mM) EDTA's adds Add so that enzymatic activity reduces about 8.83%, high concentration (5mM) EDTA then influences enzymatic activity not significant.
1 metal ion of table or chemical reagent are on the active influence of feruloyl esterase EpFAE1
4) salt is on the active influence of feruloyl esterase EpFAE1
Prepare the 0.1M citric acid-sodium citrate buffer solution (pH containing 0M, 0.25M, 0.5M, 1M, 2M and 3M NaCl respectively 5.5).It takes the various buffers of 800 μ L that 100 μ L concentration 50mM Ferulic acid methylester solution are added, is placed in 50 DEG C of water-baths and preheats The 100 diluted enzyme solutions of μ l are added in 5min.According to the measurement of method described in embodiment 3 enzymatic activity (temperature is 50 DEG C).As a result As shown in fig. 6, the NaCl of 0.25-0.5M has certain facilitation to EpFAE1 enzymatic activity；Shadow of the NaCl of 1M to enzyme activity Sound is unobvious；As salinity continues to increase, enzyme activity is in the trend being gradually reduced；When salinity reaches 3M, target enzyme activity drop To the 55% of untreated fish group.Feruloyl esterase EpFAE1 has certain salt tolerance.
5) Rate activity of feruloyl esterase EpFAE1 and kinetic constant measurement
With level-one Chromatographic Pure Methanol dissolution Ferulic acid methylester be each configured to 25mM, 50mM, 75mM, 100mM, 125mM, The substrate solution of 150mM, 175mM, 200mM, 225mM and 250mM.Take the citric acid-sodium citrate (pH of 800 μ L 0.1M 5.5) buffer is separately added into the substrate solution of 100 μ L various concentrations, and 100 μ L are added after preheating 5min in 50 DEG C of water-baths Enzyme solution (0.1U/mg), reacts 30min, and boiling water inactivates 10min and terminates reaction.Reaction mixture is cooling in room temperature, passes through above-mentioned enzyme Measuring method living detects enzyme activity.
The data of Enzyme kinetic parameter carry out nonlinear regression analysis calculating using 5.0 software of Graphpad Prism should The kinetic constant of enzyme.It is obtained according to the data in Fig. 7: the V of EpFAE1maxFor 28.46U/mg；KmFor 73.19mM；KcatFor 13.42min-1。
The application of ferulic acid in 3 feruloyl esterase EpFAE1 of embodiment collaboration release cereal bran
1) preparation of the desizing processing and enzyme of cereal bran
By the wheat bran and corn bran bought from market pulverize and sieve and according to literature method (Long et al., Thermotolerant hemicellulolytic and cellulolytic enzymes from Eupenicillium parvum 4-14 display high efficiency upon release of ferulic acid from wheat Bran.Journal of Applied Microbiology, 2016,121:422-434) desizing processing is carried out, acquisition goes to form sediment Powder wheat bran and desizing corn bran, and used after carrying out 80 mesh screen sievings.By in embodiment 2 method preparation Ah Wei acid esterase EpFAE1, and zytase EpXYN1 (Long et al., Characterization of is prepared by literature method two new endo-β-1,4-xylanases from Eupenicillium parvum 4-14 and their applications for production of femloyl oligosaccharides.Applied Biochemistry And Biotechnology, 2018,186:816-833.
2) alkaline process measures the total content of ferulic acid in desizing cereal bran
The desizing wheat bran or desizing corn bran for weighing dry weight 5.0g are placed in 250mL conical flask, are added 100mL contains 8%NaOH and 1%Na2SO3Solution, mix well；It is placed in shaking bath, is shaken under conditions of 70 DEG C with 180rpm Swing alkaline hydrolysis 10h；Centrifuging and taking supernatant after the completion of alkaline hydrolysis, with salt acid for adjusting pH to 2-3, centrifuging and taking supernatant again；It is added same with supernatant The ethyl acetate of volume extracts three times repeatedly；Rotary Evaporators concentrating sample, methanol solution modeling object, and constant volume sample are to whole body Product is 10mL.By the total content of ferulic acid in the method measurement each sample in embodiment 2.After measured, in desizing wheat bran Ferulic acid total content is 0.43%, and ferulaic acid content is 2.06% in desizing corn bran.
3) dilute acid pretreatment combination enzymatic isolation method prepares the ferulic acid in desizing cereal bran
Remove starch wheat bran or each 1g of desizing corn bran, respectively with the phosphoric acid of 20mL various concentration (0.5%, 1.0%, it 1.25%, 1.5%, 1.75%, 2% and 2.25%) mixes, and final concentration of 1% sodium sulfite is added (as guarantor Protect agent), using water as blank control, it is placed in 99 DEG C of water-baths after handling 5h, filtration method is separated by solid-liquid separation.Filtrate is with 1M's The EpFAE1 of 1U/mL is added after NaOH tune pH to 5 in 50 DEG C of enzymatic hydrolysis 4h.Remaining solid state substrate is rinsed well through tap water, in 60 It weighs after being dried at DEG C, and carries out coordinated enzyme by the amount addition EpXYN1 of the amount of 1U/g matrix addition EpFAE1 and 200U/g matrix Solution.The enzymolysis liquid of supernatant matrix is filtered through 0.22 μm of water system miillpore filter, obtains asafoetide acid solution.By the side in embodiment 2 Method measures ferulaic acid content in sample, calculates separately the yield of ferulic acid.
As shown in Figure 8, it is 61.60% without low-kappa number wheat bran ferulic acid release rate sum, 1-2.25% is added After the phosphoric acid of concentration is pre-processed, the ferulic acid total amount for digesting acquisition is dramatically increased.It is pre-processed with 1.75% phosphoric acid Afterwards, the yield highest of ferulic acid, can reach 83.07%, and the ferulic acid of total amount 79.31% is wherein obtained in supernatant.By going to form sediment Ferulic acid total content is 0.43% calculating in powder wheat bran, can be from 100g desizing wheat bran using sour processing-enzymatic isolation method Obtain 0.36g ferulic acid.
As shown in Figure 9, the corn bran ferulic acid release rate without low-kappa number is only 3.76%；Through 0.5%-2%'s After phosphoric acid processing, the ferulic acid total amount for digesting release is significantly improved；After being pre-processed with 1% phosphoric acid, the ferulic acid of acquisition Total amount highest, yield is up to 56.52%.It is 2.06% calculating by ferulaic acid content in desizing corn bran, utilizes acid processing- Enzymatic isolation method can obtain 1.16g ferulic acid from 100g desizing corn bran.
Sequence table
<110>Nanjing Forestry University
<120>a kind of feruloyl esterase EpFAE1 and its encoding gene and application
<130> 0
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 280
<212> PRT
<213> Eupenicillium parvum 4-14
<400> 1
Met Lys Ala Phe Ala Thr Arg Ala Leu Ala Phe Ser Val Ala Ala Gly
1 5 10 15
Gln Ala Leu Ala Ala Val Thr Gln Gly Val Ser Asp Asn Thr Tyr Asn
20 25 30
Arg Leu Val Glu Met Ala Thr Ile Ser Gln Ala Ala Tyr Ala Asn Leu
35 40 45
Cys Asn Ile Pro Ala Thr Ile Gln Thr Val Glu Lys Ile Tyr Asn Ala
50 55 60
Gln Thr Asp Ile Asn Gly Trp Val Leu Arg Asp Asp Ser Arg Gln Glu
65 70 75 80
Ile Ile Thr Val Phe Arg Gly Thr Gly Ser Asp Thr Asn Leu Gln Leu
85 90 95
Asp Thr Asn Tyr Thr Leu Ala Pro Phe Asp Thr Leu Pro Gln Cys Val
100 105 110
Gly Cys Ala Val His Gly Gly Tyr Tyr Leu Gly Trp Leu Ser Val Gln
115 120 125
Asp Gln Val Gln Ser Leu Val Gln Gln Gln Ala Ser Gln Tyr Arg Gly
130 135 140
Tyr Ala Val Thr Val Thr Gly His Ser Leu Gly Ala Ser Met Ala Ala
145 150 155 160
Ile Thr Ala Ala Gln Leu Ser Ala Thr Tyr Asp Asn Val Asn Leu Tyr
165 170 175
Thr Phe Gly Glu Pro Arg Thr Gly Asn Gln Ala Tyr Ala Ser Tyr Met
180 185 190
Asn Glu Ala Phe Asp Ser Ala Ser Pro Glu Thr Thr Arg Tyr Phe Arg
195 200 205
Val Thr His Ala Asp Asp Gly Ile Pro Asn Val Pro Pro Ala Glu Gln
210 215 220
Gly Tyr Val His Ser Gly Val Glu Tyr Trp Ser Val Glu Pro His Ser
225 230 235 240
Pro Gln Asn Thr Tyr Ile Cys Thr Gly Asp Glu Ile Gln Cys Cys Glu
245 250 255
Ala Gln Gly Gly Gln Gly Val Asn Ala Ala His Val Thr Tyr Phe Gly
260 265 270
Met Thr Ser Gly Ala Cys Ser Trp
275 280
<210> 2
<211> 843
<212> DNA
<213> Eupenicillium parvum 4-14
<400> 2
atgaaagcct ttgcaacacg cgctctcgct ttttccgttg ctgcaggaca agctctagct 60
gccgttacgc agggcgtctc tgacaacacc tacaaccgtc tggttgagat ggccaccatc 120
tcccaagctg cctatgcaaa cttgtgcaac attccggcga ccatacaaac ggtggagaaa 180
atatacaacg cccaaaccga tatcaacgga tgggtcctcc gcgacgatag tcgtcaagaa 240
atcatcaccg tctttcgcgg cactggcagt gatacgaact tgcagctcga taccaactac 300
actctcgctc cttttgacac ccttcctcaa tgcgtcggtt gtgccgtgca tggcggatac 360
tatcttggat ggctctctgt ccaagatcaa gtccagtcac ttgttcaaca gcaggccagc 420
cagtatcggg ggtatgcagt aacggtcaca ggtcacagtc tgggtgcctc gatggcagca 480
ataactgccg ctcagctgtc cgctacatac gacaatgtaa acttgtacac atttggcgaa 540
ccgcgaaccg gtaaccaggc ctacgcgtcg tatatgaatg aggctttcga ctcggctagc 600
cccgagacta cccgatattt ccgcgtcact catgccgacg atggcatccc aaatgtgccc 660
ccggctgaac agggatatgt ccattccggc gttgaatact ggagtgttga gccccatagc 720
cctcagaaca cgtatatctg tactggggat gagatccagt gctgtgaggc tcagggagga 780
cagggggtga atgctgctca tgtcacttat tttgggatga cgagcggagc ttgcagttgg 840
tag 843
<210> 3
<211> 21
<212> DNA
<213> fae1_f1(Artificial)
<400> 3
cgttgaacca ttgtccatcc a 21
<210> 4
<211> 21
<212> DNA
<213> fae1_r1(Artificial)
<400> 4
tgaatcgcct ctgactacca a 21
<210> 5
<211> 31
<212> DNA
<213> fae1_f2(Artificial)
<400> 5
ccggaattcg ccgttacgca gggcgtctct g 31
<210> 6
<211> 51
<212> DNA
<213> fae1_r2(Artificial)
<400> 6
ctagtctaga tcagtgatgg tgatggtgat gccaactgca agctccgctc g 51

Claims (9)

1. a kind of feruloyl esterase EpFAE1, amino acid sequence is as shown in SEQ ID NO.1.
2. the encoding gene of feruloyl esterase EpFAE1 described in claim 1, base sequence is as shown in SEQ ID NO.2.
3. the expression vector of the encoding gene containing feruloyl esterase EpFAE1 as claimed in claim 2.
4. feruloyl esterase EpFAE1 described in claim 1 is preparing the application in ferulic acid.
5. application according to claim 4, which is characterized in that described is prepared as digesting using feruloyl esterase EpFAE1 Cereal bran discharges ferulic acid.
6. application according to claim 5, which is characterized in that the cereal bran is wheat bran or corn bran.
7. application as claimed in claim 4, which comprises the following steps:
1) wheat bran is pre-processed；
2) preparation of feruloyl esterase EpFAE1；
3) pretreated wheat bran 1g is taken, the phosphoric acid of final concentration 1.75% and the sodium sulfite of final concentration 1% is added, is placed in After handling 5h in 99 DEG C of water-baths, it is separated by solid-liquid separation；Filtrate with after the NaOH tune pH to 5 of 1M be added 1U/mL EpFAE1 in 50 DEG C of enzymatic hydrolysis 4h；Remaining solid state substrate is rinsed well through tap water, is weighed after drying at 60 DEG C, and add by the amount of 1U/g matrix Add the amount of EpFAE1 and 200U/g matrix to add zytase EpXYN1 and carries out coordinated enzymatic hydrolysis.Enzymolysis liquid is through 0.22 μm of water system micropore After membrane filtration, asafoetide aqueous acid is obtained.
8. application as claimed in claim 4, which comprises the following steps:
1) corn bran is pre-processed；
2) preparation of feruloyl esterase EpFAE1；
3) pretreated corn bran 1g is taken, the phosphoric acid of final concentration 1% and the sodium sulfite of final concentration 1% is added, is placed in 99 After handling 5h in DEG C water-bath, it is separated by solid-liquid separation；Filtrate is to be added the EpFAE1 of 1U/mL in 50 after the NaOH tune pH to 5 of 1M DEG C enzymatic hydrolysis 4h；Remaining solid state substrate is rinsed well through tap water, is weighed after drying at 60 DEG C, and is added by the amount of 1U/g matrix The amount addition zytase EpXYN1 of EpFAE1 and 200U/g matrix carries out coordinated enzymatic hydrolysis.Enzymolysis liquid is filtered through 0.22 μm of water system micropore After film filtering, asafoetide aqueous acid is obtained.
9. the specific primer for expanding feruloyl esterase EpFAE1 gene as claimed in claim 4, which is characterized in that including Following two sequences:
Upstream primer: 5 '-CCGgaattcGCCGTTACGCAGGGCGTCTCTG-3 '；
Downstream primer:
5′-CTAGtctagaTCAgtgatggtgatggtgatgCCAACTGCAAGCTCCGCTCG-3′。
CN201910230349.7A 2019-03-25 2019-03-25 A kind of feruloyl esterase EpFAE1 and its encoding gene and application Pending CN109825485A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201910230349.7A CN109825485A (en) 2019-03-25 2019-03-25 A kind of feruloyl esterase EpFAE1 and its encoding gene and application
CN201910540226.3A CN110106156B (en) 2019-03-25 2019-06-20 Feruloyl esterase EpFAE1 and coding gene and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910230349.7A CN109825485A (en) 2019-03-25 2019-03-25 A kind of feruloyl esterase EpFAE1 and its encoding gene and application

Publications (1)

Publication Number Publication Date
CN109825485A true CN109825485A (en) 2019-05-31

Family

ID=66872159

Family Applications (2)

Application Number Title Priority Date Filing Date
CN201910230349.7A Pending CN109825485A (en) 2019-03-25 2019-03-25 A kind of feruloyl esterase EpFAE1 and its encoding gene and application
CN201910540226.3A Active CN110106156B (en) 2019-03-25 2019-06-20 Feruloyl esterase EpFAE1 and coding gene and application thereof

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN201910540226.3A Active CN110106156B (en) 2019-03-25 2019-06-20 Feruloyl esterase EpFAE1 and coding gene and application thereof

Country Status (1)

Country Link
CN (2) CN109825485A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115025016A (en) * 2022-06-17 2022-09-09 齐鲁工业大学 Ligusticum wallichii enzymatic hydrolysate and application thereof
CN115025017A (en) * 2022-06-17 2022-09-09 齐鲁工业大学 Ligusticum wallichii and cimicifugae foetidae mixed enzymatic hydrolysate and application thereof
CN116337791A (en) * 2023-05-31 2023-06-27 北京挑战生物技术有限公司 In-vitro detection method for release rate of phosphorus phytate in feed raw material

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111471664B (en) * 2020-04-22 2021-11-30 北京工商大学 Feruloyl esterase BpFae, and coding gene and application thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8361764B1 (en) * 2009-09-30 2013-01-29 The United States Of America, As Represented By The Secretary Of Agriculture Genes and enzymes for degradation of ferulic acid crosslinks
EP3609347A1 (en) * 2017-04-10 2020-02-19 Société des Produits Nestlé S.A. Method of preparing a composition comprising ferulic acid
CN107586767B (en) * 2017-11-08 2020-03-31 南京林业大学 Heat-resistant endo-xylanase EpXYN1, and coding gene and application thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115025016A (en) * 2022-06-17 2022-09-09 齐鲁工业大学 Ligusticum wallichii enzymatic hydrolysate and application thereof
CN115025017A (en) * 2022-06-17 2022-09-09 齐鲁工业大学 Ligusticum wallichii and cimicifugae foetidae mixed enzymatic hydrolysate and application thereof
CN115025017B (en) * 2022-06-17 2023-11-14 齐鲁工业大学 Ligusticum wallichii and cimicifuga foetida mixed enzymolysis liquid and application thereof
CN116337791A (en) * 2023-05-31 2023-06-27 北京挑战生物技术有限公司 In-vitro detection method for release rate of phosphorus phytate in feed raw material
CN116337791B (en) * 2023-05-31 2023-08-15 北京挑战生物技术有限公司 In-vitro detection method for release rate of phosphorus phytate in feed raw material

Also Published As

Publication number Publication date
CN110106156B (en) 2021-10-29
CN110106156A (en) 2019-08-09

Similar Documents

Publication Publication Date Title
CN109825485A (en) A kind of feruloyl esterase EpFAE1 and its encoding gene and application
Deng et al. Heterologous expression and characterization of an antifungal chitinase (Chit46) from Trichoderma harzianum GIM 3.442 and its application in colloidal chitin conversion
Dhillon et al. Green synthesis approach: extraction of chitosan from fungus mycelia
Chi et al. Production, characterization and gene cloning of the extracellular enzymes from the marine-derived yeasts and their potential applications
Karthik et al. Production, purification and properties of fungal chitinases—a review
Somashekar et al. Chitosanases—properties and applications: a review
DK179334B1 (en) Recombinant Beta-1,3-glucanase, polynucleotide, recombinant vector, transformant, production method for beta-1,3-glucanase, enzyme preparation, and production method for paramylon having reduced molecular weight
CN109652392A (en) A kind of feruloyl esterase and its preparation method and application
Zhao et al. Degradation of ochratoxin A by supernatant and ochratoxinase of Aspergillus niger W-35 isolated from cereals
CN108699578A (en) The enzymatic activity of cracking performance polysaccharide monooxygenase
CN105713889A (en) Alginate lyase Alga and coding gene and application thereof
Álvarez-Cervantes et al. Purification and characterization of xylanase SRXL1 from Sporisorium reilianum grown in submerged and solid-state fermentation
CN105018448A (en) Heat-resisting acidic cellulase of fungus source and gene and application thereof
Pachauri et al. Kinetic study and characterization of cellulase enzyme from isolated Aspergillus niger subsp. awamori for cellulosic biofuels
CN102363774B (en) Beta-mannaseBA-Man5A with wide pH range, gene thereof and application of gene
Rajput et al. Myco-chitinases as versatile biocatalysts for translation of coastal residual resources to eco-competent chito-bioactives
CN101724573A (en) Trichoderma biocontrol recombinant engineering bacteria for efficiently expressing chitinase coding gene and Beta-1,3-glucanase coding gene as well as application thereof
Balasubramanian et al. Fusant Trichoderma HF9 with enhanced extracellular chitinase and protein content
CN104593279A (en) Genetically engineered bacterium and applications thereof
CN107987183A (en) A kind of method that chitosan oligosaccharide is extracted from filamentous fungi
Prasad et al. Advances in production and characteristic features of microbial tannases: an overview
CN111100853B (en) Xylanase xyn11A, and coding gene and application thereof
CN102586208A (en) Protein with Beta-N-acetamido glucosaminidase activity as well as encoding gene and application thereof
Graça et al. Cloning, overexpression and functional characterization of a class III chitinase from Casuarina glauca nodules
Wagh Bioconversion of tannic acid to gallic acid by using fungal tannase

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20190531