CN109777789A - A kind of Type B dyestuff-decoloration peroxidase and its mutant and application - Google Patents
A kind of Type B dyestuff-decoloration peroxidase and its mutant and application Download PDFInfo
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- CN109777789A CN109777789A CN201910016401.9A CN201910016401A CN109777789A CN 109777789 A CN109777789 A CN 109777789A CN 201910016401 A CN201910016401 A CN 201910016401A CN 109777789 A CN109777789 A CN 109777789A
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- decoloration
- dyestuff
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The present invention relates to a kind of Type B dyestuff-decoloration peroxidase and its mutant and applications.More particularly to a kind of Type B dyestuff from pseudomonad Pseudomonas sp.Q18-decoloration peroxidase gene dyp, to the gene constructed recombination engineering of dyp, and inducing expression, isolates and purifies and obtain Type B dyestuff-decoloration peroxidase PmDyP.The present invention is mutated the amino acid residue of the 125th, 142 and No. 188 position on the basis of PmDyP amino acid sequence, obtains the PmDyP-5c mutant that catalytic efficiency improves.Mutant of the invention has good degradation effect to lignin raw material, has application and promotional value in the utilization process of biomass resource.
Description
Technical field
The present invention relates to dyestuff-decoloration peroxidase, and in particular to a kind of novel bacterium Type B dyestuff-decoloration peroxide
The preparation and application of compound enzyme and its mutant.
Background technique
Dyestuff-decoloration peroxidase (abbreviation DyPs, EC 1.11.1.19) is a kind of peroxide containing ferroheme
Enzyme family is widely present in fungi and bacterium.The heme peroxidases family of they and " classics " does not have in sequence
Homology, and space structure is widely different, but has special oxidation, therefore this to polycyclic dyes and phenolic compound
Fermentoid is considered as a kind of special peroxidase and is separately classified as DyP type enzyme.According to the homologous similitude of primary structure
Four subfamilies (A-D) can be divided into.
The DyPs found in bacterium has oxidation to substrates such as aromatic compound and lignin model compounds, its quilt
The substitution enzyme for having similar to fungi LiP function is considered in bacterium.
Summary of the invention
Inventor's hair from one plant of ligninolytic bacteria pseudomonad (Pseudomonas sp.Q18, KX822686)
A kind of novel bacterium DyP enzyme is showed, which belongs to Type B DyP enzyme (being named as PmDyP), the Type B dyestuff-decoloration peroxide
The gene order of enzyme is selected from a kind of following sequence:
(1) sequence shown in SEQ ID No.1；
(2) there is the gene order of 90% or more homology with sequence shown in SEQ ID No.1.
The present invention also provides the recombinant expression carriers comprising said gene sequence.
Further, the present invention also provides the recombination engineerings comprising above-mentioned recombinant expression carrier.
In addition, Type B dyestuff-decoloration peroxidase amino acid sequence of the invention is selected from a kind of following sequence:
(1) sequence shown in SEQ ID No.2；
(2) there is the amino acid sequence of 90% homology with sequence shown in SEQ ID No.2.
By zymologic property the study found that PmDyP enzyme of the invention has extensive degradation of substrates range, to ABTS,
Guaiacol, 2,6-DMP and MnCl2Substrate can show catalytic activity.
Type B dyestuff-decoloration peroxidase of the invention has good decolorizing effect to waste water from dyestuff.
In order to make more preferably meeting the requirement of industrial applications, the catalytic efficiency of PmDyP enzyme is improved, inventor is to PmDyP
Three amino acid residues of enzyme are mutated, and the mutant enzyme PmDyP-5c that catalytic efficiency improves, the amino acid of the mutant are obtained
Sequence are as follows: the amino acid of No. 125 position of the amino acid sequence of above-mentioned Type B dyestuff-decoloration peroxidase is Y, No. 142 position
Amino acid be V and No. 188 position amino acid be K amino acid sequence.
Preferably, Type B of the invention dyestuff-decoloration peroxidase variant amino acid sequence such as SEQ ID No.4 institute
Show.
Mutant enzyme PmDyP-5c of the present invention has lignin model compound, alkali lignin and lignocellulosic material good
Good degradation, can be used for the development and utilization process of biomass resource, have great application value.The lignin
Model compound concretely guaiacol base glycerol-β-guaiacyl ether (abbreviation GGE) etc..The lignocellulosic material
It may include specifically wheat stalk, corn stover and switchgrass etc..
Detailed description of the invention
Fig. 1 enzyme PCR amplification electropherogram (M:Maker) of the present invention, (A) are dyp gene fragment amplification result；It (B) is recombination
Engineering bacteria BL21-DyP bacterium colony PCR verification result；
(swimming lane 1: the cell after ultrasound is heavy for the result that isolates and purifies of the enzyme PmDyP of the present invention of Fig. 2 engineering bacteria heterogenous expression
It forms sediment, the dissolved bacterial sediment of swimming lane 2:8M urea, swimming lane 3: PmDyP after purification, M: protein standard molecular weight Maker)；
Fig. 3 enzyme PmDyP Tertiary structure predictions illustraton of model of the present invention；
Fig. 4 recombinates the optimal pH (A) and pH stability (B) of enzyme PmDyP enzyme activity of the present invention；
Fig. 5 recombinates the optimum temperature (A) and thermal stability (B) of enzyme PmDyP enzyme activity of the present invention；
Lineweaver-Burk double reciprocal plot of Fig. 6 concentration of substrate to reaction rate；
Fig. 7 recombinates the result that enzyme of the present invention decolourizes to azure dye；
Fig. 8 mutant enzyme recombination engineering bacterium colony PCR verification result of the present invention；
The mutant enzyme PmDyP-5c's of the present invention of Fig. 9 engineering bacteria heterogenous expression isolates and purifies result (swimming lane 1: after ultrasound
Cell precipitation, the dissolved bacterial sediment of swimming lane 2:8M urea, swimming lane 3: PmDyP-5c after purification)；
Figure 10 mutant enzyme PmDyP-5c of the present invention analyzes lignin model compound GGE catabolite；
For Figure 11 mutant enzyme PmDyP-5c of the present invention to the degradation results of alkali lignin, (A) is alkali lignin degradation pictorial diagram；(B)
For alkali lignin degradation rate measurement result；
Figure 12 mutant enzyme lignocellulose degradation raw material of the present invention as a result, (A) be degradation pictorial diagram；(B) lignin drops
Solution rate measurement result.
Specific embodiment
The embodiment of the present invention is described in detail below, the present embodiment under the premise of the technical scheme of the present invention into
Row is implemented, and the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following realities
Apply example.
Embodiment 1: clone, expression and the purifying preparation of PmDyP enzyme of the present invention
(1) amplification of PmDyP enzyme gene dyp
Bacterium Pseudomonas sp.Q18 (KX822686) is screened from the rotten timber in Part of The Qinling Mountains In Shaanxi Province mountain range
It obtains.The Selective agar medium using alkali lignin as sole carbon source is used to carry out screening, separation and the purifying of bacterium.Screening and culturing
Based component (g/L) is as follows: 2.5g NaNO3, 1.0g KH2PO4, 1.0g K2HPO4, 1.0g MgSO4, 1.0g NaCl, 0.5g
CaCl2, 1.0g NH4Cl, 3.0g alkali lignin, pH7.0.
Using Ezup pillar bacterial genomes DNA extraction agent box (Sangon Biotech (Shanghai) Co., Ltd.)
The genomic DNA for extracting bacterium, is inquired according to ncbi database, designs upstream and downstream primer MY10-F, MY10-R to target gene
It is expanded, primer entrusts Beijing AudioCodes Bioisystech Co., Ltd to synthesize, and sequence is as follows:
MY10-F:5 '-AATTCCATGGGCATGCCGTTCCAGCAAGGTCTGCTTGCC -3 ' (is Nco I enzyme at scribing line
Enzyme site)
MY10-R:5 '-AATGCTCGAGGGCCCGCAGCAAGGAGCTCAGAT-3 ' (being Xho I restriction enzyme site at scribing line)
PCR reaction system and condition: including 2 × TaqMaster Mix, 12.5 μ L, upstream and downstream primer each 1 in 25 μ L systems
μ L, DNA profiling 1 μ L, ddH2O is mended to 25 μ L.95 DEG C of initial denaturation 3min；95 DEG C of denaturation 30s；63 DEG C of annealing 30s；72 DEG C of extensions
90s；Extend 7min eventually for 72 DEG C after 35 circulations, is down to 4 DEG C of preservations.It is produced with the agarose gel electrophoresis detection PCR of 1% (g/mL)
Object, as shown in Figure 1A, open reading frame 864bp includes 288 amino acid, and gene order and amino acid sequence are after sequencing
SEQ ID No.1 and SEQ ID No.2.
(2) building and identification of Escherichia coli recombinant plasmid
Using Novagen company plasmid pET-28a (+) as clone and expression vector, Nco I selected above and
Xho I restriction enzyme site matches with expression vector.Using Nco I and Xho I enzyme to after purification dyp genetic fragment and empty plasmid
Carry out double digestion, under the action of T4 ligase, by after digestion dyp gene and pET-28a (+) connection, obtain pET28a-
Dyp recombinant plasmid.By 42 DEG C of thermal shock 90s, recombinant plasmid pET28a-dyp is transformed into E.coli DH5 α, passes through bacterium colony PCR
The positive recombinant verified, and be converted into function carries out glycerol stocks, and -80 DEG C save for use.Extract pET28a-dyp matter
Grain, is transformed into e. coli bl21 (DE3), bacterium colony PCR (such as Figure 1B) obtains positive restructuring engineering bacteria BL21-DyP afterwards, and protects
It deposits stand-by.
(3) inducing expression of the recombination pET28a-dyp in Escherichia coli
After recombination engineering BL21-DyP activation, it is seeded in 5mL LB containing kanamycin (LB/Kan) culture medium
It is incubated overnight.It is forwarded in the LB/Kan culture medium of 500mL according to 1% inoculum concentration, 180r/min shaken cultivation is extremely at 37 DEG C
OD600 is 0.6 or so, the IPTG of 0.2mM is added, 150r/min shaken overnight inducing expression at 28 DEG C.Test result discovery
There is (such as Fig. 2) in the form of inclusion body in PmDyP, therefore dissolved, purified and renaturation process to inclusion body.
(4) dissolution of recombinase PmDyP
Bacteria suspension is centrifuged 20min at 8,000 xg, thallus is collected, with re-suspension liquid (20mM Tris-HCl, 150mM
NaCl, 10% glycerol, pH8.0) it cleans twice, then cell is resuspended, cell is thoroughly crushed with cell Ultrasonic Cell Disruptor.After ultrasound
It is centrifuged 30min at 8,000 xg, retains bacterial sediment.With inclusion body Wash Solution (20mM Tris-HCl, 4M urea,
5mM EDTA, pH8.0) washing thalline precipitating is primary, the inclusion body Extracting Solution of suitable urea containing 8M is added
(20mM Tris-HCl, 150mM NaCl, 8M urea, 20mM imidazoles, pH8.0), with magnetic stirrer 3h, fills thallus
Divide and be dissolved in extracting solution, is put into 4 DEG C of refrigerators and stands overnight.After precipitating dissolution, it is centrifuged (8000 × g, 10min), it will be upper
Clear liquid crosses 0.22 μm of water system filter membrane, for use.
(5) recombinase PmDyP is isolated and purified
The processing of dress Ni-NTA affinity chromatography gravity column manually, with inclusion body Extracting Solution to chromatographic column
It is balanced.By filtered solution loading to nickel column, with chromatography Wash buffer (20mM Tris-HCl, 150mM NaCl,
20mM imidazoles, 3% glycerol, pH 8.0) washing lotion removes foreigh protein removing, suitable chromatography Elution Buffer (20mM is added
Tris-HCl, 150mM NaCl, 8M urea, 500mM imidazoles, 5% glycerol, pH 8.0), destination protein PmDyP is dissolved out, is merged
Collecting protein liquid.With the purity of SDS-PAGE detection purification process destination protein, adjustment purifying parameter.
(6) renaturation of recombinase PmDyP
It is about 0.5mg/mL that PmDyP solution after purification, which is diluted to protein concentration, and loading molecular cut off is 8000-
In the bag filter of 14000Da, it is placed in chromatography cabinet, is dialysed with magnetic stirrer.Dialyzate is added to different dense when preparing
The urea of degree carries out gradient dialysis, and from high concentration to low concentration, first with the renaturation solution dialysis 8h containing 4M, it is multiple to be changed to 2M urea
Property liquid, is successively replaced, dialyse 8h every time.Finally dialysed again with 20mM Tris-HCl.
For purification result as shown in the SDS-PAGE electrophoresis result of Fig. 2, albumen size is about 31kDa, obtains albumen after purification
Concentration is the recombinase of 0.271mg/mL.Protein structure domain is inquired using InterPro, the enzyme belongs to as the result is shown
DyP-type peroxidase (IPR006314) protein family；Three-dimensional structure is predicted using threading method I-TASSER
(such as Fig. 3), C are worth being divided into 1.16, and model is reasonable.
Embodiment 2: the zymologic property measurement of PmDyP of the present invention
Inventor is with ABTS, guaiacol, 2,6-DMP and MnCl2For substrate, zymetology has been carried out to PmDyP after purification
Property measurement.
The optimum pH of specific measurement recombinase PmDyP and its tolerance at various ph values, optimum temperature and its right
The tolerance of temperature, and its kinetic parameter is characterized.
Light absorption value during monitoring recombinase PmDyP and substrate reactions under corresponding wavelength using multi-function microplate reader
Variation.Enzyme activity unit (U) is defined as: enzyme amount needed for 1.0 μM of substrates of catalysis per minute.Enzyme activity calculation formula are as follows:
In formula: Δ A is the variable quantity of light absorption value；ε is molar extinction coefficient (M-1cm-1)；B is the thickness (cm) of cuvette；
T is enzyme and substrate reactions time (min)；V1It is the total volume (L) of enzyme activity reaction system；V2It is the volume (L) of enzyme solution in system；n
For enzyme solution extension rate.
(1) influence of the pH value to PmDyP enzyme activity and its stability
PH is selected to be measured under the conditions of 2.0-12.0, is measured under corresponding pH, enzyme activity when PmDyP is initially added, puts
After setting 1h, its enzyme activity is measured again, calculates the enzyme activity stability under corresponding pH.As a result such as Fig. 4, with ABTS, guaiacol, 2,6-
DMP and MnCl2For substrate, the relative activity of maximum enzyme activity is 100%.Recombinase PmDyP is for different substrate and different
The enzyme activity difference that pH value is shown is clearly.
Using ABTS as substrate, when pH value is 3.0, opposite enzyme activity is maximum, and with the increase of pH, enzyme activity is gradually reduced, pH
Environment entirety slant acidity is adapted to, under alkaline condition, is unfavorable for PmDyP and plays oxidation；And for enzyme activity stability, in pH value
Under being 3.0 to ABTS act on 1h after, remain to retain about 50% enzyme activity, but when pH value be more than 5.0, PmDyP tolerance by
Very big influence, enzyme activity are extremely low.
With guaiacol and 2, when 6-DMP is as substrate, the two result is more similar, may be fragrance with two substrates
Compound monomer is related, the enzyme activity highest of PmDyP when pH value is 6.0, and enzyme activity stability under conditions of pH6.0
Most preferably, the enzyme activity of 50%-60% can be retained after 1h.For MnCl2For substrate, pH enzyme activity at 6.0 is best, in pH5.0-6.0
In range, the stability of enzyme activity is higher.
(2) influence of the temperature to PmDyP enzymatic activity and stability
It chooses within the scope of 20 DEG C -90 DEG C, every 10 DEG C of raising, the influence to enzyme activity, after placing 1h under relevant temperature, measures enzyme
Residual volume living, investigate stability, with ABTS (pH 3.0), guaiacol (pH 6.0), 2,6-DMP (pH 6.0) and
MnCl2(pH 6.0) is substrate, is 100% calculating with the relative activity of maximum enzyme activity.
As shown in Figure 5, as temperature increases, PmDyP enzyme activity increases, and best enzyme activity is in 50-60 DEG C, when temperature is more than 60
After DEG C, enzyme activity starts sharply to decline, and 80 DEG C or more, PmDyP enzyme activity is nearly no detectable.In terms of enzyme activity stability, at 20-50 DEG C
In range, PmDyP enzyme activity stability is preferable, can retain 60% or more, but working as temperature is more than 50 DEG C, the stabilization of PmDyP enzyme
Property gradually decreases.
(3) the enzyme kinetic analysis research of recombinase PmDyP
With ABTS (pH3.0,50 DEG C), guaiacol (pH6.0,60 DEG C), 2,6-DMP (pH6.0,60 DEG C) and MnCl2
(pH6.0,60 DEG C) is substrate, measures enzyme activity situation when different concentration of substrate.
The concentration that ABTS is arranged is respectively as follows: 0.05,0.1,0.2,0.5,1.0,2.0,3.3,5.0,8.3 and 10.0mM；More
The concentration for creating the wooden phenol is respectively as follows: 0.05,0.1,0.2,0.5,1.0,2.0,3.3,4.0 and 5.0mM, 2,6-DMP concentration difference
Are as follows: 0.05,0.1,0.2,0.5,1.0,2.0,3.3,4.0 and 5.0mM.
It is corresponding to calculate different substrates using Lineweaver-Burk double-reciprocal plot (such as Fig. 6) according to Michaelis-Menten equation
Kinetic constant.As a result such as subordinate list 1, PmDyP enzyme is recombinated to ABTS, guaiacol and 2, the maximum reaction rate of 6-DMP substrate
In 4.0U/mg or so, and for substrate MnCl2, PmDyP enzyme maximum reaction rate is the former 10 times or so, is reached
46.15U/mg, and by KmAnd kcatValue is it is found that MnCl2KmValue is less than 2,6-DMP, but kcatValue is other three kinds of substrates
20 times or more, illustrate to recombinate PmDyP enzyme for MnCl2Although affinity less than 2,6-DMP, catalytic efficiency is much larger than it
Its three kinds of substrate.
The prior art is compared, about TfuDyP zymology Quality Research in Thermobifidafusca bacterium, measurement result hair
Now it is to Mn2+With guaiacol without activity, and PmDyP of the invention can measure enzyme activity to above four kinds of substrates, equally
It is measured using ABTS as substrate, the k of PmDyPcat/KmValue is 7000M-1s-1, k much higher than TfuDyPcat/KmValue.In addition, for
Substrate guaiacol and Mn2+, the k of PmDyPcat/KmValue also above the Dyp1B from Pseudomonas fluorescens Pf-5, Dyp2B and
DyPA.PmDyP enzyme kinetic analysis is recombinated studies have shown that PmDyP is the peroxide of a great potential in terms of biotechnology applications
Compound enzyme.
The kinetic parameter of the PmDyP enzyme of the present invention of table 1
Application of the 3 recombinase PmDyP of embodiment in decoloring dye waste water
The PmDyP enzyme solution of purifying is added in the LB solid medium tablets containing 0.15g/L azure dye, at 30 DEG C
4h is stood in incubator, blank control adds isometric buffer, determines whether according to whether there is or not decoloration circles on aniline blue plate
Oxidative decoloration ability.It can be seen that recombinase PmDyP after cultivating 4h from Fig. 7 result, occur apparent decoloration around enzyme solution
Circle has good oxidative decoloration ability, and it is biggish to illustrate that PmDyP has in terms of the decolorization of waste water from dyestuff in dyeing and weaving industry
Application potential.
The preparation of the mutant enzyme of the present invention of embodiment 4
Using the gene order of wild enzyme PmDyP as template, three positions in genetic fragment are realized using overlap extension pcr
The rite-directed mutagenesis of point, is that the amino acid H (histidine CAU) of No. 125 position is become Y (tyrosine UAU) respectively, by No. 142
The amino acid A (alanine GCC) of position becomes V (valine GUC), and the amino acid E (glutamic acid GAA) of No. 188 position is become K
(lysine AAA).
Primer sequence shown in table 2 is designed, wherein MY10-F and MY10-R is the primer for expanding PmDyP full length gene sequence
(draw a horizontal line be Nco I, Xho I restriction enzyme site and mutational site).
The primer sequence of 2 rite-directed mutagenesis of table
Using PmDyP gene as template, MY10-F, H125Y-R, H125Y-F and MY10-R contain respectively as primer, amplification
The gene order in the mutational site H125Y is recycled and is purified to two sections of target gene using plastic recovery kit, with pET-
28a (+) is used as carrier, and the gene order in the mutational site containing H125Y is connect with carrier, constructs pET28a-1 recombinant vector.
By 42 DEG C of thermal shock 90s, recombinant plasmid pET28a-1 is transformed into E.coli DH5 α, is tested by bacterium colony PCR
Card, and it is converted into the positive recombinant of function.On the basis of this objective gene sequence, above step is repeated, carries out A142V respectively
It is final to obtain the recombinant vector pET28a-3 for containing three mutational sites, and mesh after sequence verification mutation with the mutation in the site E188K
Gene order and amino acid sequence be SEQ ID No.3 and SEQ ID No.4.
PET28a-3 plasmid is extracted, is transformed into e. coli bl21 (DE3), bacterium colony PCR verifying (such as Fig. 8) is simultaneously sequenced
Afterwards, positive restructuring engineering bacteria is picked out, mutant is obtained, is named as PmDyP-5c, and is saved stand-by.
The gene order and amino acid sequence of mutant enzyme PmDyP-5c is shown in SEQ ID No.3 and SEQ ID No.4 respectively.
Inducing expression is carried out to mutant PmDyP-5c, mutant enzyme is isolated and purified, step of the method with embodiment 1
Suddenly.For purification result as shown in the SDS-PAGE electrophoresis result of Fig. 9, albumen size is about 31kDa, measurement mutant enzyme PmDyP-5c's
Protein concentration is 0.134mg/mL.
Embodiment 5: the enzyme kinetics measurement of mutant enzyme PmDyP-5c of the present invention
With ABTS, guaiacol, 2,6-DMP and MnCl2For substrate, different concentration of substrate are mainly investigated to the shadow of enzyme activity
It rings, according to Michaelis-Menten equation, calculates the corresponding kinetic constant of different substrates, method is the same as embodiment 2.
The Determination of Kinetic Parameters result (subordinate list 1 and subordinate list 3) for comparing wild enzyme PmDyP and mutant enzyme PmDyP-5c can
Know, the enzyme activity catalytic efficiency of mutant enzyme PmDyP-5c is significantly improved, to the k of different substrates catalysiscat/KmValue improves
3-5 times.
From maximum reaction rate VmaxVariation for, the PmDyP-5c enzyme activity reaction rate under all substrates improves
2-3 times, especially for substrate MnCl2Oxidation rate, improve it is nearly 3 times, reached 113.07 ± 0.76U/mg；
KmThe results show that compared with wild enzyme PmDyP, mutant enzyme PmDyP-5c and Mn2+Affinity it is stronger；
Kcat value improves 2.5 times, illustrates PmDyP-5c to Mn2+Catalytic efficiency improve a lot.
For substrate guaiacol and 2,6-DMP, the catalysis of the affinity and enzyme of mutant enzyme and substrate is imitated as the result is shown
Rate has different degrees of raising.
The kinetic parameter of 3 mutant enzyme PmDyP-5c of table
Embodiment 6: degradation of the mutant enzyme PmDyP-5c of the present invention to lignin model compound
Model compound GGE is a kind of typical lignin dimer, it is that two phenol ring monomers have been keyed by β-O-4
It is formed.In the network structure of nature lignin complexity, the connection type of β-O-4 key is most commonly seen, accounts for 50-70%.Therefore,
The degradation of this class formation has vital meaning to the destruction of lignin complex network structures.Therefore, the present invention selects GGE
As model compound, recombinase is studied to the degradation capability of lignin.
It is isolated and purified according to the method for embodiment 4 and obtains mutant enzyme PmDyP-5c, be dissolved into methanol, be configured to
The mother liquor of 10mM concentration.It takes suitable substrate that enzyme solution is added, constructs the reaction system of recombinase: 2.0mM GGE, 20mM ammonium carbonate
Buffer, 1.0mMH2O2, 0.1mg/mL recombinase PmDyP-5c, system pH are 6.0.The system of recombinase PmDyP-5c is not added
As blank control.Recombinase and the mixed reaction system of GGE are placed in constant temperature and humidity incubator, are arranged 40 DEG C, reaction
8h.Catabolite is analyzed using GC-MS.To the preprocess method of catabolite, enzymatic hydrolysis system is extracted with ethyl acetate
In organic compound, then silylation is carried out to product with derivatization reagent BSTFA+TMCS (99:1).After pre-processing
Sample examination with computer, finally carry out mass spectrum compare analysis of compounds.
Rear result such as Figure 10 is analyzed and identified to catabolite, after two recombination enzyme effects, in the ion flow graph of product
There are multiple peaks, wherein retention time be 12.5min at have found lignin monomer guaiacol, be in retention time
Lignin cellular construction vanillic acid has been identified at 20.0min, has additionally identified a large amount of small molecule aldehyde and acids object
Matter.The appearance of guaiacol and vanillic acid, lignin model compound GGE can be degraded by directly demonstrating mutant enzyme, make GGE
Dimer depolymerization is two aromatic compound monomers.
Embodiment 7: degradation of the mutant enzyme PmDyP-5c of the present invention to alkali lignin
Mutant enzyme PmDyP-5c is added in the alkali lignin of various concentration, degradation effect of the observation enzyme to alkali lignin.Drop
Enzymatic hydrolysis system are as follows: the alkali lignin of 0.015,0.05,0.1,0.2,0.3,0.4 and 0.5mM, 20mM ammonium carbonate buffer,
1.0mMH2O2, 0.1mg/mL recombinase PmDyP-5c.The system of recombinase PmDyP-5c is not added as blank control.It will recombination
The reaction system of enzyme and alkali lignin is placed in constant temperature and humidity incubator, is arranged 40 DEG C, is reacted 2 days.Alkali lignin after degradation is molten
Liquid is centrifuged 10min at 8000 × g, takes supernatant, measures the light absorption value under its 280nm using ultraviolet specrophotometer, calculates
The degradation rate of alkali lignin.
As a result such as Figure 11, wherein (A) figure is degradation pictorial diagram, there are different journeys in the alkali lignin solution bottom that enzyme solution is added
The precipitating of degree, this may be when acting on alkali lignin due to recombinase, and depolymerization occurs in alkali lignin, its structure is made to change, and produce
Some low molecular weight insoluble substances are given birth to.
By the alkali lignin solution centrifugation after degradation, the degradation rate of alkali lignin is calculated, as a result such as Figure 11 (B).Recombinase is not to
Alkali lignin with gradient all has degradation capability, and when concentration of substrate is lower, degradation effect is obvious, degradation rate 90% or so,
But with the raising of concentration of substrate, when concentration is more than 0.2mM, degradation rate is remarkably decreased, this is because substrate is sufficient, each
The enzyme activity of unit is constant, therefore causes substrate remaining, cannot thoroughly be degraded.On the whole, mutant enzyme PmDyP-5c is to alkali wood
It is known as apparent degradation effect, can be applied to the degradation of soluble lignin.
Embodiment 8: degradation of the mutant enzyme PmDyP-5c of the present invention to lignocellulosic
It selects ligno-cellulosic materials wheat, corn and the switchgrass of nature as raw material, smashes it through 40 meshes；
Three kinds of raw materials for weighing 1.5g respectively are added 20mg/mL's according to the ratio that solid-liquid ratio is 15% (w/v)
1.0mMH is added in PmDyP-5c enzyme solution2O2, system pH is adjusted to 6.0.
Degradation system is placed in 40 DEG C of constant incubator, is degraded 3 days.It is right according to Klason lignin measuring method
The content of lignin measurement of degradation front and back, calculates the degradation rate of lignin, not to be inoculated with the sample of recombinase as blank control.
Figure 12 (A) is the pictorial diagram after degradation for 24 hours, the as former state blank control to be not added with enzyme solution.Raw material after degradation
Apparent variation has occurred in mode of appearance, such as the granularity of ligno-cellulosic materials becomes finer and smoother, and appearance is by hard bar
Shape becomes the cotton-shaped of suede suede；A large amount of material floats on above degradation solution, this may be due to the degradation of recombinase make it is wooden
Cellulosic material structure is destroyed, and inner material is dissolved out, and density becomes smaller and floats on above liquid；In addition, compared with the control,
Enzymolysis liquid color burn after three kinds of feed degradations in system, and do not occur this phenomenon in blank sample.To Lignin degradation rate
It measures (Figure 12 B), after mutant enzyme PmDyP-5c degradation, the weight-loss ratio of raw material is 7-10%, the Lignin degradation rate of three kinds of raw materials
For 3-5%, illustrate that recombinase has preferable degradation to lignocellulosic material.
。
Nucleotides sequence list electronic document
<110>Xibei Univ. of Agricultural & Forest Science & Technology
<120>a kind of Type B dyestuff-decoloration peroxidase and its mutant and application
<141>
<160>
<210>1
<211>864
<212>DNA
<213>Type B dyestuff-decoloration peroxidase gene order
<220>
<223>
<400>1
ATGCCGTTCCAGCAAGGTCTGCTTGCCACCCCGGTGCCGGCCCACGC
CCGTCACCTGTTCTTCACCCTGCAGTCGCCCGAGGCGTTGCCTGCCGC
GCTGGATGCCTTGCTGCCGCAGGTCGATGGCGAACAACTGTTGCTCG
GCATAGGCGCCCCCTTGGTCAAGGCTCTGGGCCGTGAAGTACCAGGC
CTGCGCGCTTTCCCGCTGCTGGATACCGCAGTGGAGAACCCCAGCAC
CCAACACGCCCTGTGGCTGTGGCTGCGTGGCGACGAACGTGGCGACC
TGCTGCTGCGCGCCCAGGCCCTGGAGCAGGCGCTGGCACCCGCCCTG
CAGTTGGCCGACAGCGTTGATGGCTTCCTGCGCCGCGGTGGCCATGA
CCTTACCGGTTACGAAGACGGCACCGAAAACCCGGTGGACGAAGATG
CCGTACAGGCGGCCATTGCCGCCGATGGTTCCAGCTTTGCCGCGTTCC
AGCTGTGGAAGCACGACCTGGAATACTTCAAGTCGCTGCCCCAGGCT
GACCAGGACAATATCATCGGCCGCCGCCTGAGCGACAACGAAGAGCT
CGGCGATGCCCCCGAGTCCGCGCACGTCAAGCGCACTGCCCAGGAAA
GCTTTGAACCCGAAGCGTTCATGGTCCGTCGCTCGGTAGCCTGGGCC
GACCAGCGCGGCGCCGGCCTGGCCTTCGTCGCCTTGGGCAAGAGTTT
CGATGCATTCGGAGTGCAATTGCGGCGCATGAGTGGCCTGGAAGACG
GCATCATCGACGGATTGTACCGCTTTAGCCGCCCGCTGACGGGTGGCT
ACTACTGGTGCCCGCCGATGGGCGAGACGGGGGTTGATCTGAGCTCC
TTGCTGCGGGCCTGA
<210>2
<211>287
<212>amino acid
<213>Type B dyestuff-decoloration peroxidase amino acid sequence
<220>
<223>
<400>2
MPFQQGLLATPVPAHARHLFFTLQSPEALPAALDALLPQVDGEQLL
LGIGAPLVKALGREVPGLRAFPLLDTAVENPSTQHALWLWLRGDERGDL
LLRAQALEQALAPALQLADSVDGFLRRGGHDLTGYEDGTENPVDEDAV
QAAIAADGSSFAAFQLWKHDLEYFKSLPQADQDNIIGRRLSDNEELGDA
PESAHVKRTAQESFEPEAFMVRRSVAWADQRGAGLAFVALGKSFDAFGV
QLRRMSGLEDGIIDGLYRFSRPLTGGYYWCPPMGETGVDLSSLLRA
<210>3
<211>864
<212>DNA
<213>Type B dyestuff-decoloration peroxide enzyme mutant gene order
<220>
<223>
<400>3
ATGCCGTTCCAGCAAGGTCTGCTTGCCACCCCGGTGCCGGCCCA
CGCCCGTCACCTGTTCTTCACCCTGCAGTCGCCCGAGGCGTTGCCTGC
CGCGCTGGATGCCTTGCTGCCGCAGGTCGATGGCGAACAACTGTTGC
TCGGCATAGGCGCCCCCTTGGTCAAGGCTCTGGGCCGTGAAGTACCA
GGCCTGCGCGCTTTCCCGCTGCTGGATACCGCAGTGGAGAACCCCAG
CACCCAACACGCCCTGTGGCTGTGGCTGCGTGGCGACGAACGTGGCG
ACCTGCTGCTGCGCGCCCAGGCCCTGGAGCAGGCGCTGGCACCCGCC
CTGCAGTTGGCCGACAGCGTTGATGGCTTCCTGCGCCGCGGTGGCTAT
GACCTTACCGGTTACGAAGACGGCACCGAAAACCCGGTGGACGAAG
ATGTCGTACAGGCGGCCATTGCCGCCGATGGTTCCAGCTTTGCCGCGT
TCCAGCTGTGGAAGCACGACCTGGAATACTTCAAGTCGCTGCCCCAG
GCTGACCAGGACAATATCATCGGCCGCCGCCTGAGCGACAACGAAAA
GCTCGGCGATGCCCCCGAGTCCGCGCACGTCAAGCGCACTGCCCAGG
AAAGCTTTGAACCCGAAGCGTTCATGGTCCGTCGCTCGGTAGCCTGG
GCCGACCAGCGCGGCGCCGGCCTGGCCTTCGTCGCCTTGGGCAAGAG
TTTCGATGCATTCGGAGTGCAATTGCGGCGCATGAGTGGCCTGGAAGA
CGGCATCATCGACGGATTGTACCGCTTTAGCCGCCCGCTGACGGGTGG
CTACTACTGGTGCCCGCCGATGGGCGAGACGGGGGTTGATCTGAGCT
CCTTGCTGCGGGCCTGA
<210>4
<211>287
<212>amino acid
<213>Type B dyestuff-decoloration peroxide enzyme mutant amino acid sequence
<220>
<223>
<400>4
MPFQQGLLATPVPAHARHLFFTLQSPEALPAALDALLPQVDGEQLL
LGIGAPLVKALGREVPGLRAFPLLDTAVENPSTQHALWLWLRGDERGDLLLRAQALEQALAPALQLADSVDGF
LRRGGYDLTGYEDGTENPVDEDVV
QAAIAADGSSFAAFQLWKHDLEYFKSLPQADQDNIIGRRLSDNEKLGDA
PESAHVKRTAQESFEPEAFMVRRSVAWADQRGAGLAFVALGKSFDAFGV
QLRRMSGLEDGIIDGLYRFSRPLTGGYYWCPPMGETGVDLSSLLRA
Claims (10)
1. a kind of Type B dyestuff-decoloration peroxidase, which is characterized in that Type B dyestuff-decoloration peroxidase gene sequence
Column selection is from a kind of following sequence:
(1) sequence shown in SEQ ID No.1；
(2) there is the gene order of 90% or more homology with sequence shown in SEQ ID No.1.
2. a kind of recombinant expression carrier comprising gene order described in claim 1.
3. a kind of recombination engineering comprising recombinant expression carrier described in claim 2.
4. a kind of Type B dyestuff-decoloration peroxidase, which is characterized in that Type B dyestuff-decoloration peroxidase amino acid
Sequence is selected from a kind of following sequence:
(1) sequence shown in SEQ ID No.2；
(2) there is the amino acid sequence of 90% or more homology with sequence shown in SEQ ID No.2.
5. the Type B dyestuff of claim 1 or 4-application of the decoloration peroxidase in decoloring dye waste water.
6. a kind of Type B dyestuff-decoloration peroxide enzyme mutant, which is characterized in that the amino acid sequence of the mutant are as follows: right
It is required that the amino acid of No. 125 position of the amino acid sequence of the 4 Type B dyestuff-decoloration peroxidase is Y, No. 142 position
Amino acid is the amino acid sequence that the amino acid of V and No. 188 position is K.
7. the dyestuff of Type B described in claim 6-decoloration peroxide enzyme mutant, amino acid sequence such as SEQ ID No.4 institute
Show.
8. the application of the dyestuff of Type B described in claim 6-decoloration peroxide enzyme mutant lignin degrading model compound.
9. the application of the dyestuff of Type B described in claim 6-decoloration peroxide enzyme mutant degradation alkali lignin.
10. the application of the dyestuff of Type B described in claim 6-decoloration peroxide enzyme mutant lignocellulose degradation raw material.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115287272A (en) * | 2022-05-23 | 2022-11-04 | 浙江省农业科学院 | Novel dye decoloring method for clone and recombinase of DyP-type peroxidase gene |
| CN116574744A (en) * | 2023-05-06 | 2023-08-11 | 西北农林科技大学 | Gene related to lignin degradation and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006061063A (en) * | 2004-08-26 | 2006-03-09 | Shimane Univ | Peroxidase having decoloring activity, dna encoding the same peroxidase, microorganism in which the same peroxidase is transduced in experssible form and method for producing dcoloring agent |
| US20170247725A1 (en) * | 2012-10-09 | 2017-08-31 | California Institute Of Technology | In vivo and in vitro olefin cyclopropanation catalyzed by heme enzymes |
| CN108676783A (en) * | 2018-05-28 | 2018-10-19 | 南华大学 | A kind of decoloration peroxidase and the preparation method and application thereof |
-
2019
- 2019-01-08 CN CN201910016401.9A patent/CN109777789A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006061063A (en) * | 2004-08-26 | 2006-03-09 | Shimane Univ | Peroxidase having decoloring activity, dna encoding the same peroxidase, microorganism in which the same peroxidase is transduced in experssible form and method for producing dcoloring agent |
| US20170247725A1 (en) * | 2012-10-09 | 2017-08-31 | California Institute Of Technology | In vivo and in vitro olefin cyclopropanation catalyzed by heme enzymes |
| CN108676783A (en) * | 2018-05-28 | 2018-10-19 | 南华大学 | A kind of decoloration peroxidase and the preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| C.-X. YANG ET AL.: "Isolation, identification and characterization of lignin-degrading bacteria from Qinling, China", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| CHENXIAN YANG ET AL.: "Biodegradation of lignin by Pseudomonas sp. Q18 and the characterization of a novel bacterial DyP-type peroxidase", 《JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY》 * |
| VANIA BRISSOS ET AL.: "Engineering a Bacterial DyP-Type Peroxidase for Enhanced Oxidation of Lignin-Related Phenolics at Alkaline pH", 《ACS CATALYSIS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115287272A (en) * | 2022-05-23 | 2022-11-04 | 浙江省农业科学院 | Novel dye decoloring method for clone and recombinase of DyP-type peroxidase gene |
| CN116574744A (en) * | 2023-05-06 | 2023-08-11 | 西北农林科技大学 | Gene related to lignin degradation and application |
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