CN106947748B - A kind of super resistance to Mn2+Bacterial laccase, recombinant vector, recombinant bacterium, enzyme preparation, compound enzyme system and the preparation method and application thereof - Google Patents
A kind of super resistance to Mn2+Bacterial laccase, recombinant vector, recombinant bacterium, enzyme preparation, compound enzyme system and the preparation method and application thereof Download PDFInfo
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Abstract
The present invention relates to a kind of super resistance to Mn2+Bacterial laccase, recombinant vector, recombinant bacterium, enzyme preparation, compound enzyme system and the preparation method and application thereof, belong to field of biotechnology.The present invention derives from the bacterial laccase lac1542 of macro genome, encodes the gene nucleotide series of the laccase as shown in SEQ ID NO.1, optimal pH is 4.0 when the bacterial laccase is using ABTS as substrate；Optimal reactive temperature is 75 DEG C and high temperatures are strong；The Mn of 100mmol/L2+It remains to play activation to the enzyme；The bacterial laccase and manganese peroxidase, Coprinus cinereus peroxidase composition complex enzyme system at low concentrations can lignin degrading up to 71.5%；The bacterial laccase and the compound enzyme system of Coprinus cinereus peroxidase reach 54.8% to the percent of decolourization of methyl orange.Bacterial laccase of the invention can be used as the new enzyme source of one kind for papermaking, dyeing and weaving and environment protection field, have wide prospects for commercial application.
Description
Technical field
The present invention relates to a kind of super resistance to Mn2+Bacterial laccase, recombinant vector, recombinant bacterium, enzyme preparation, compound enzyme system and its system
Preparation Method and application, belong to field of biotechnology.
Background technique
Laccase (laccase) is a kind of polyphenol oxidase of cupric, has oxidation polyphenol, toxic arylamine class, and dyestuff is de-
The effects of color and decomposition lignin, be always the research hotspot of biology, chemistry and environmental science；Lignin degradation,
The fields such as paper pulp bleaching, organic pollutant processing have obtained extensive research and application.Before the 1980s, laccase
It is mainly directly separated from plant or fungi, yield is low, at high cost.With the development of molecular biology technology, for clone's paint
Enzyme gene realizes that heterogenous expression, industrialized production provide possibility.Fronhman in 1988 etc. utilizes rapid amplifying cDNA for the first time
Technology is cloned into laccase pox1 gene from whiterot fungi oyster cap fungus (P.ostreatus), and later researcher is mostly used RT-
Round pcr obtains laccase gene.It is considered that laccase is merely present in plant and fungi, therefore the research master about laccase
It concentrates in fungal laccase.In order to study function, heterogenous expression, generation and the regulatory mechanism of fungal laccase, has surpass at present
40 kinds or more of fungal laccase gene is crossed to be cloned and have made extensive and intensive studies.However, in recent years
For Alexandre etc. by carrying out homogeneous assay to Protein Data Bank and bacterial genomes, discovery laccase is widely present in original
In core biology.Bacterial laccase is widely present, and has been opened up new field for the research of laccase, has been increased new type and resource.
Bacterial laccase is especially interestingly compared with fungal laccase has advantage such as Cu in terms of certain zymologic properties2+It is anti-
Property, be not required to glycosylation, the advantages that thermal stability is good and the optimum pH range of enzyme is wide.
What is had now been found that has the active bacterium of laccase-like and few, therefrom separates, identifies, purifies and retouches
It is less to state its properties of laccase.Due to educable microorganism only account for microbe species less than 1%, from educable micro- life
The laccase type cloned in object is very limited.Technique of metagenome is produced as from the Anticipated transient without scram of different natural habitats
It finds new laccase gene resource and provides strong technological means.
Efficiency is not high either in terms of lignin degradation or environmental improvement for single laccase, and research has shown that by laccase
With manganese peroxidase (Manganese peroxidase Mnp) composition compound enzyme system can efficiently lignin degrading, dye
Material decoloration.Unfortunately manganese peroxidase plays activity and tends to rely on Mn2+Presence；And lot of documents reports Mn2 +There is inhibiting effect to a variety of laccase activities.Papermaking and the reaction of dye industry often carry out at high temperature in reality production, commonly
Enzyme be difficult adapt to industrial applications.High temperature and high concentration Mn can be resistant to simultaneously at present2+Bacterial laccase it is less.Full of leaves equal utilization
Macro genome means are separated to an alkaline laccase from mangrove and have carried out directed evolution to the enzyme, but the enzyme is by Mn2+Suppression
System, the Mn of low concentration2+Enzymatic activity can be made to be reduced to 70%；It is 55 DEG C by substrate optimal reactive temperature of guaiacol, the temperature
Degree is lower to handle 30min, active only residue 20%.
The Chinese invention patent of Publication No. CN106434707A discloses a kind of bacterium from hay bacillus ZNXH4
Laccase gene, bacterial laccase and its application, but only it is resistant to the Mn of 10mM2+.Therefore heat-resisting, tolerance Mn is found2+Laccase be
The important channel improved the compound enzyme system degradation capability of lac/Mnp, widen laccase industrial application.
Summary of the invention
The purpose of the present invention is to provide a kind of bacterial laccase, which is resistant to high temperature and high concentration Mn2+And Cu2+,
And can be with manganese peroxidase, Coprinus cinereus peroxidase combined degradation lignin, decolourize dyestuff.
The present invention also provides the recombinant vectors of the genetic fragment comprising encoding above-mentioned bacterial laccase.
The present invention also provides the preparation methods of above-mentioned recombinant vector.
The present invention also provides the recombinant bacteriums comprising above-mentioned recombinant vector.
The present invention also provides the preparation methods of above-mentioned recombinant bacterium.
The present invention also provides the methods for preparing bacterial laccase.
The present invention also provides the enzyme preparations comprising above-mentioned bacterial laccase.
The present invention also provides the compound enzyme systems comprising above-mentioned bacterial laccase.
The present invention also provides above-mentioned bacterial laccases, the application of enzyme preparation or compound enzyme system.
To achieve the goals above, the technical scheme adopted by the invention is that:
A kind of super resistance to Mn2+Bacterial laccase, the amino acid sequence of the bacterial laccase is as shown in SEQ ID NO.2.
Above-mentioned bacterial laccase, the gene for encoding the bacterial laccase is Lac1542 gene, nucleotide sequence such as SEQ
Shown in ID NO.1.
Recombinant vector, the genetic fragment comprising encoding above-mentioned bacterial laccase.
The preparation method of above-mentioned recombinant vector, comprising: the genetic fragment for encoding the bacterial laccase is inserted into load of setting out
In body, after conversion screen to get.
Above-mentioned recombinant vector can be made using carrier construction method commonly used in the art.
The carrier that sets out of the invention is the carrier that sets out commonly used in the art, does not do strict control.
Preferably, the carrier that sets out is pet28a, pet32a or pUC118 carrier.
Recombinant bacterium includes above-mentioned recombinant vector.
The preparation method of above-mentioned recombinant bacterium, comprising: the recombinant vector is transformed into host cell, culture, screening, i.e.,
?.
Preferably, the host cell is prokaryotic cell bacillus coli DH 5 alpha, e. coli bl21 (DE3).
Above-mentioned recombinant bacterium can be made using recombinant bacterium construction method commonly used in the art.
The method for preparing bacterial laccase using above-mentioned recombinant bacterium includes the following steps: to cultivate the recombinant bacterium, inducing expression
Bacterial laccase, purification of bacterial laccase to get.
Inducing expression described in step 1) is using IPTG and CuSO4Inducing expression.
The concentration of the IPTG is 0-1.52mmol/L.Preferably, the concentration of the IPTG is 0.25mmol/L.
The CuSO4Concentration be 0-4mmol/L.Preferably, the CuSO4Concentration be 1mmol/L.
Preferably, inducing expression described in step 1) is in 30 DEG C of induction 8h.
Purifying described in step 3) is to be purified using Ni-spin coloumn (Qiagen company).
Enzyme preparation includes above-mentioned bacterial laccase.
Compound enzyme system includes the bacterial laccase, further includes in manganese peroxidase and Coprinus cinereus peroxidase
It is one or two kinds of.
Compound enzyme system includes the bacterial laccase and manganese peroxidase.
In above-mentioned compound enzyme system in lignin degrading, the bioactivity (unit U) of bacterial laccase and manganese peroxidase
Ratio is 1.5-2:10-15.
Preferably, when above-mentioned complex enzyme ties up to lignin degrading, the bioactivity of bacterial laccase and manganese peroxidase is (single
Position U) ratio be 1.8:12.
Preferably, when above-mentioned complex enzyme ties up to lignin degrading, reaction condition is to contain lignin in 4ml reaction system
The Mn of 50g/L, 0.25mM2+, the bacterial laccase of 1.8U, the manganese peroxidase of 12U, hydrogen peroxide is 20 μ L.
Compound enzyme system includes the bacterial laccase and Coprinus cinereus peroxidase.
When above-mentioned complex enzyme ties up to decoloration methyl orange, the bioactivity of bacterial laccase and Coprinus cinereus peroxidase is (single
Position U) ratio be 1.2-1.5:8-12.
Preferably, when above-mentioned complex enzyme ties up to decoloration methyl orange, the biology of bacterial laccase and Coprinus cinereus peroxidase
Active (unit U) ratio is 1.47:10.
Compound enzyme system includes the bacterial laccase, manganese peroxidase and Coprinus cinereus peroxidase.
When above-mentioned complex enzyme ties up to lignin degrading, bacterial laccase, manganese peroxidase and Coprinus cinereus peroxidase
Bioactivity (unit U) ratio be 1.5-2:4.5-5:7-7.5.
Preferably, when above-mentioned complex enzyme ties up to lignin degrading, bacterial laccase, manganese peroxidase and Coprinus cinereus peroxide
Bioactivity (unit U) ratio of compound enzyme is 1.8:4.8:7.2.
Above-mentioned bacterial laccase, enzyme preparation or complex enzyme ties up to the application of paper-making industry, dyeing and weaving industry, environmental protection aspect.
Above-mentioned bacterial laccase, enzyme preparation or complex enzyme ties up to the application in paper-making industry in terms of lignin degrading.
Above-mentioned bacterial laccase, enzyme preparation or complex enzyme ties up to the application of dye decolored aspect in dyeing and weaving industry.
Above-mentioned bacterial laccase, enzyme preparation or complex enzyme ties up to the application of organic pollutant processing aspect in environmental protection.
The functional screening technology of metagenomics can directly be sieved to the DNA sequence with specific function from soil genome
Column are conducive to find novel gene and novel enzyme, and functional gene can be made independently of host cell table by construction of expression vector
Up to corresponding functional protein out.Develop in high-flux sequence swift and violent today, macro gene function screening technique still has it that can not replace
The advantage in generation.
The present invention is macro constructed by the paper mill sewage draining exit mud sample from Henan Province's Xuchang City by technique of metagenome
Obtained in genomic library one it is new there has been no the DNA sequence dna Lac1542 of functional study, then use technique for gene engineering pair
The DNA sequence dna has carried out functional study, as a result, it has been found that recombinant protein expressed by the DNA sequence dna has the characteristics that bacterial laccase,
And Mn2+And Cu2+The activity of the recombinant protein can be significantly improved, therefore can be with dependence Mn2+Mnp form compound enzyme system and use
In the fields such as paper industry lignin degradation, dyeing and weaving industry be dye decolored.The nucleotide sequence of the DNA sequence dna such as SEQ ID
Shown in NO.1, the amino acid sequence of the DNA sequence encoding is as shown in SEQ ID NO.2, i.e., resistance to Mn2+Bacterial laccase lac1542.
DNA sequence dna shown in SEQ ID NO.1 is cloned on pet28a carrier by the present invention, then electroporated Escherichia coli
DH5 α competent cell, by fermenting to positive transformant, inducing expression obtains recombinant protein, i.e., bacterial laccase of the invention.
Then zymologic property research has been carried out to the recombinant protein, as a result as follows:
(1) recombinant protein has the characteristics that highly soluble expression in Escherichia coli, and inclusion body forms less；
(2) molecular weight of the recombinant protein is about 70KDa；
(3) for the recombinant protein when being reacted using ABTS as substrate, optimal reaction pH is 4.0, in pH 3.0-pH6.5
Between have 80% or more opposite enzyme activity, stablize between pH3.0-8.0, remnant enzyme activity is more than 60% after handling 2h；
(4) the recombinant protein optimum temperature be 75 DEG C, 85 DEG C still have 60% or more activity, still protected after 70 DEG C of processing 2h
Stay 80% or more opposite enzyme activity；
(5) the most suitable substrate of the enzyme be ABTS, the sequence of other most suitable substrates be followed successively by SGZ > catechol > 2,6-DMP >
Guaiacol；
(6) high concentration Mn is being added in the recombinant protein2+And Cu2+After remain to play a driving role to enzyme activity.
Can be seen that bacterial laccase expressed by sequence shown in SEQ ID NO.1 by the above result of study has preferably
High temperature resistant, resistance to Mn2+Laccase activity, the expression product of the DNA sequence dna can be used as a kind of new laccase and Mnp forms complex enzyme
System is used for papermaking, dyeing and weaving field.
The present invention provides a kind of tolerance Mn2+、Cu2+, bacterial laccase lac1542 resistant to high temperature, the laccase is using ABTS as substrate
Optimal pH 4.0, it is all stable at pH3.0-8.0；Optimal reactive temperature is 75 DEG C, 70 DEG C of heat preservation 2h still 80% activity of remnants, high
Warm stability inferior is strong；The Mn of 100mmol/L2+Or Cu2+It remains to play activation to the enzyme；The bacterial laccase and manganese peroxidase
Enzyme independent role lignin degrading respectively reaches 16% and 19%, and the two forms the compound degradable lignin of enzyme system up to 47.5%,
Bacterial laccase and manganese peroxidase, Coprinus cinereus peroxidase composition complex enzyme system at low concentrations can lignin degradings
Up to 71.5%, degradation rate is increased substantially；Bacterial laccase and Coprinus cinereus peroxidase form compound enzyme system to methyl orange
Percent of decolourization reaches 54.8%.Bacterial laccase of the invention can be used as the new enzyme source of one kind for papermaking, dyeing and weaving and environment protection field, tool
There is wide prospects for commercial application.
Detailed description of the invention
Fig. 1 is the guaiacol development process preliminary screening library schematic diagram in embodiment 1；
Fig. 2 is ABTS the and SGZ development process secondary screening library schematic diagram in embodiment 1；
Fig. 3 is the SDS-PAGE electrophoresis of recombinant protein bacterial laccase in test example；
Fig. 4 be in test example using ABTS as substrate when pH to the active influence diagram of bacterial laccase；
Fig. 5 be in test example using ABTS as substrate when temperature to the active influence diagram of bacterial laccase；
Fig. 6 is bacterial laccase in test example and compound enzyme system to lignin degradation experiment effect figure；
Fig. 7 is the Mnp and CIP of ratio different in compound enzyme system in test example to lignin degradation efficiency chart；
Fig. 8 be in test example single enzyme and compound enzyme system to the decolorizing efficiency figure of methyl orange.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.Unless stated otherwise, agents useful for same and material of the present invention are commercially available.In following embodiment and comparative example
The entitled lac1542 of the bacterial laccase.
Embodiment 1
Bacterial laccase gene is included the following steps: in the present embodiment
(1) in pedotheque macro genome DNA acquisition: sample acquisition from Henan Province's Xuchang City paper mill sewage draining exit become silted up
Mud, specific acquisition step are as follows: the centrifuge tube for taking two 50mL weighs papermaking sewage draining exit mud, every pipe 8g, respectively plus DNA
Extraction buffer 18mL (contains 100mM EDTA, 100mM sodium phosphate, 1.5M NaCl, 1%CTAB and 100mM
Tris-Hcl, pH 8.0), the concussion that is vortexed mixes, shaking table 220rpm, 37 DEG C of processing 30min, then adds 2mL's 20% to each pipe
SDS makes its final concentration reach 2% (w/v).Then the water-bath 2h in 65 DEG C of water-baths, gently overturns several every 15min or more
Secondary mixing.Room temperature 6,000g are centrifuged 10min, after collecting supernatant, are transferred to clean centrifuge tube, isometric chloroform: isoamyl is added
Alcohol (24:1, V/V), is gently mixed by inversion up and down.Then 11,000g centrifugation 20min collects supernatant at 4 DEG C, and 0.6 times of body is added
Long-pending isopropanol turns upside down mixing, is stored at room temperature 2h or more, and 11,000 g is centrifuged 20min at 4 DEG C, collects precipitating, pre- with 4 DEG C
75% cold ethanol washing 1 time is resuspended in the ddH of 65 DEG C of preheatings2It is macro to obtain the paper waste deposit that final volume is 600 μ L by O
Genomic DNA.
(2) acquisition of the building of the macro genomic library of paper waste deposit and DNA sequence dna:
Specific steps are as follows:
1, the building, preservation of the macro genomic library of paper waste deposit
With the resulting paper waste deposit macro genome DNA of the partially digested embodiment 1 of Bam HI enzyme, and electrophoresis recycles
The endonuclease bamhi of 2.5~7.5kb.The DNA fragmentation being recovered to is carried with through the pUC118 of Bam HI digestion and dephosphorylation process
Body (Takara company) connection, electroporated E.coli DH5 α competent cell, and be coated with and contain 100 μ g/mL ampicillins
(Amp), thus the LB solid plate of 40 μ g/mL X-gal and 0.5mM IPTG constructs a storage capacity and converts up to 14200
The good macro gene library of son, diversity.All white colony clones of picking are seeded in 37 DEG C of 384 orifice plate trainings of the culture medium containing library
Feeding shaking table is incubated overnight, -80 DEG C of preservations.
2, the screening of target gene and sequence analysis
96 hole depth plates of the inoculation library to every hole containing 500 μ l LB (containing 50 μ g/mL Amp and 10 μ g/mL IPTG), 37 DEG C
Overnight incubation, 4000rpm are centrifuged 8min, take cell, and the BugBuster Protein of 100 μ l Novagen companies is added in every hole
Extraction Reagent lytic cell 20-30min at room temperature.Be added final concentration 0.5mmol/L guaiacol and
The CuSO4 of 0.1mmol/L selects the bacterium colony to redden after reaction.The result of reaction as shown in Figure 1: in reaction plate B group from left number
One is positive strain, the color reaction with Chinese red.Using same method with 2,2'- azine group-it is bis--(3- ethylo benzene
And thiazoline quinoline -6- sulfonic acid) (ABTS) and syringaldehyde connection nitrogen (SGZ) be substrate secondary screening, ABTS reaction after be blue-green, SGZ
It is the conduct positive clone molecule of purple powder after reaction, delivers sequencing.ABTS, SGZ development process result are as shown in Fig. 2, and control group
CK is compared, positive strain reacted with ABTS after for blue-green reaction color, be purple powder after react with SGZ.
The positive clone molecule that sequencing result shows contains the exogenous sequences that length is 4.5kb, using on the website NCBI
ORF Finder analysis in Sequence Analysis, finds the ORF sequence (1542bp) an of laccase gene, nucleosides
Acid sequence is as shown in SEQ ID NO.1, i.e., the resistance to Mn of high temperature resistant of the present invention2+Laccase gene；The gene encodes 513 amino
Acid is named as Lac1542, and amino acid sequence is as shown in SEQ ID NO.2.
Embodiment 2
Bacterial laccase recombinant vector is included the following steps: in the present embodiment
1, the positive clone molecule that embodiment 1 obtains is inoculated in 5mL LB culture medium (100 μ g/mL Amp), 37 DEG C 220
Rpm shaking table is incubated overnight, and extracts plasmid.
2, design primer LacF and LacR, primer both ends introduce can be inserted on coli expression carrier pet28a respectively
EcoR I and Hind III digestion site, eliminates terminator codon.
The nucleotide sequence of primer LacF (upstream primer) is as shown in SEQ ID NO.3；
The nucleotide sequence of primer LacR (downstream primer) is as shown in SEQ ID NO.4.
3, using the plasmid of extraction as template, PCR amplification: 30 μ of PCR system total volume is carried out using primer LacF and LacR
15 μ L of L, mix containing Taq (Tiangeng company), 1 μ L of each 1 μ L of two kinds of primers (10 μM), 12 μ L of sterile purified water and template DNA
(1ng)；PCR amplification condition: 95 DEG C of initial denaturation 4min；95 DEG C of 30sec, 52 DEG C of 45sec, 72 DEG C of 1min react 30 circulations, and 72
DEG C extend 5min.
4, gained PCR product length is 1.5kb or so, recycles target fragment, EcoR I and Hind using conventional method
It is connected after III double digestion in pet28a plasmid to get the recombinant vector.
Embodiment 3
The acquisition of recombinant bacterium includes: that the recombinant vector for obtaining embodiment 2 converts host strain Escherichia coli in the present embodiment
BL21 (DE3), conversion fluid coating contain the LB culture medium flat plate of 100 μ g/mL kanamycins (Kam), and 37 DEG C are incubated overnight, and extract
Positive bacterium colony plasmid carries out PCR identification using primer LacF and LacR, and what it is comprising above-mentioned bacterial laccase gene is to obtain just
True recombinant bacterium.
Embodiment 4
The preparation of the enzyme preparation of bacterial laccase in the present embodiment includes:
Recombinant bacterium transformant identifying, containing DNA sequence dna shown in SEQ ID NO.1 is induced in embodiment 3
Expression study.Scribing line activated spawn, picking single colonie are seeded in LB liquid medium of the 5mL containing 50 μ g/mL Kam and are incubated overnight,
With 1:100 amount inoculation be incubated overnight bacterium solution into 500mL triangular flask, in bottle contain 100mL LB liquid medium, 37 DEG C
200rpm is cultivated to OD600=0.8, and IPTG the and 1mmol/L CuSO4 of 0.25mmol/L is added in 30 DEG C of induction 8h.At 4 DEG C
5000g is centrifuged 10min and harvests cell.Appropriate NPI10 is added to cell precipitation to be resuspended, addition lysozyme to final concentration 1mg/mL,
30min (ultrasonic disruption can also be added) is placed on ice and cracks thallus, crude enzyme liquid is obtained, then according to Ni-spin
Coloumn (Qiagen company) specification carries out purification process, obtains recombinant protein after purification, as SEQ ID NO.1 institute
Show the enzyme preparation of the recombinant protein bacterial laccase of DNA sequence dna expression.
Embodiment 5
Compound enzyme system in the present embodiment includes in bacterial laccase, manganese peroxidase and Coprinus cinereus peroxidase
It is one or two kinds of or three kinds.
Complex enzyme reaction system in the present embodiment, the Mn comprising 0.25mM2+, the lac1542 (1.8U) of 15 μ L, 6.84 μ L
Mnp (manganese peroxidase, 12U), hydrogen peroxide be 20 μ L, be settled to 4mL with water.
Or the Mn comprising 0.25mM2+, the Mnp (manganese peroxidase) of the lac1542 of 1.5U, 10U, hydrogen peroxide is 20 μ
L is settled to 4mL with water.
Or the Mn comprising 0.25mM2+, the Mnp (manganese peroxidase) of the lac1542 of 2U, 15U, hydrogen peroxide is 20 μ L,
4mL is settled to water.
Or include 6U Mnp and 6U CIP (Coprinus cinereus peroxidase), the Mn of 0.25mM2+, hydrogen peroxide is 20 μ L,
4mL is settled to water.
Or comprising lac1542 15 μ L (1.8U), the Mnp of the CIP and 7.2U of 4.8U, the Mn of 0.25mM2+, fixed with water
Hold to 4mL.
Or including hydrogen peroxide 13mmol/L, lac1542 1.45mg (1.47U), CIP 10U, 0.1mM CuSO4,
1.5mM ABTS is settled to 200 μ L.
Test example
(1) bacterial laccase Quality Research obtained by each step in embodiment 4
By the precipitating after cell cracking in embodiment 4, the crude enzyme liquid in purification process and recombinant protein bacterium after purification
Laccase carries out SDS-PAGE electrophoresis, as a result as shown in Figure 3.M is molecular weight protein marker in Fig. 3；1 is supernatant egg after cracking
It is white；2 be protein precipitation；3 be albumen after purification, from figure 3, it can be seen that supernatant has one at corresponding target protein position after cracking
Obvious destination protein band.It can be seen that the recombinant protein has obtained high efficient expression in Escherichia coli, and with solvable
Property expression based on.
In addition, having carried out molecule quantifier elimination to recombinant protein after purification using SDS-PAGE electrophoresis.SDS-PAGE electricity
The molecular weight of the swimming recombinant protein as the result is shown is about 70KDa, this reason calculated with us according to the amino acid sequence of target protein
It is consistent by molecular weight plus tag size.
(2) different substrates, pH, temperature, metal ion are to the active shadow of resulting bacterial laccase after purification in embodiment 4
It rings
1, active influence of the different substrates on bacterial laccase
This test example has selected 5 kinds of ABTS, 2,6-DMP, SGZ, catechol and guaiacol laccases often to use substrate, to reality
It applies the resulting recombinant protein of example 3 and has carried out relevant zymologic property research.Using the NaAC of pH4.0 as buffer, surveyed not at 75 DEG C
The corresponding enzymatic activity with substrates various under concentration, unit of enzyme activity are defined as enzyme amount required for 1 μm of ol substrate of conversion in 1min
For 1U.
Recombinant protein is 99.64U/mg to the specific enzyme activity of ABTS, shows activity to the laccase substrates of several classics, specifically
Are as follows: SGZ (0.15U/mg), catechol (0.549U/mg), guaiacol (0.014U/mg) and 2,6-DMp (0.061U/
mg)。
In general, the kinetic parameter kcat/Km (catalytic efficiency) of enzyme is taken as measuring enzyme to the specificity size of substrate
One index.Present case is fitted the kinetic parameter (as shown in table 1) of each substrate using graphpad prism 6.0.From table 1
It can be seen that catalytic efficiency highest (0.516s-1μM-1) ABTS be laccase best substrate, the sequence of remaining best substrate according to
It is secondary are as follows: SGZ > catechol > 2,6-DMP > guaiacol.
1 bacterial laccase kinetic parameter of table
| substrate | Km(μmol-1) | Kcat(s-1) | Kcat/Km(LS-1μmol-1) | activity(U/mg) |
| ABTS | 194.9±3.219 | 100.4±3.27 | 0.516 | 99.64 |
| 2,6-DMP | 4077±11.32 | 0.1126±0.017 | 3.0×10-5 | 0.061 |
| SGZ | 4744±52.76 | 0.9017±0.9017 | 1.9×10-4 | 0.15 |
| Catechol | 6202±76.73 | 0.6445±0.021 | 1.04×10-4 | 0.549 |
| guaiacol | 1395±22.46 | 0.01432±0.001 | 1.03×10-5 | 0.014 |
2, active influence of the difference pH on bacterial laccase
1) determination of activity is carried out with ABTS substrate.
2) recombinant protein optimal pH research in, with citrate phosphate buffer configure reaction system, pH3.0~
In 8.0 ranges, pH value is every to increase by 0.5 as a gradient, measures relative activity of the recombinant protein at these pH.In recombination egg
In white pH stability study, first enzyme in each pH buffer 4 DEG C put be incubated for 2h, adjust pH to optimum condition under survey remnants
Enzymatic activity, testing result are as shown in Figure 4.
Result of study shows that the optimal pH of the recombination laccase maintains 80% or more activity between 4.0, pH3.5-4.5.This
Outside, enzyme enzyme activity within the scope of pH 3.0-8.0 is stablized, and remnant enzyme activity is more than 60% (as shown in Figure 4) after handling 2h.
The above result of study shows that the recombinant protein, Lac1542 are all stable enzymes of a kind of couple of wide scope pH, is relatively suitble to
Industrial applications.
3, active influence of the different temperatures on bacterial laccase
In the optimum temperature and temperature stability research of recombinant protein, reactant is configured with the NaAC buffer of pH 4.0
System, setting temperature range are 30-90 DEG C, every 5 DEG C of gradients；Measure the relative activity of recombinant protein at these tem-peratures.Inspection
It is as shown in Fig. 5 to survey result.
Still retain 80% activity when optimum temperature is 75 DEG C, 90 DEG C when the bacterial laccase is using ABTS as substrate, enzyme is at 70 DEG C
Be below it is stable, heat preservation 2h still has 80% or more activity, 75 DEG C of heat preservation 2h enzyme activity still remaining 50%.It can be seen that the enzyme is one non-
The laccase of Chang Naire.
4, active influence of the metal ion on bacterial laccase
In this test example, metal ion is to recombinant protein activity influence measuring method are as follows:
4 DEG C of pretreatment 15min, addition 1 in the NaAC buffer of pH4.0 by suitable enzyme and different metal ions
mmol/LCuSO4With 0.5mmol/L ABTS, enzyme activity is surveyed under optimum condition, calculates relative activity, as shown in table 2.From table 2
As can be seen that the influence active on recombinant protein of these metal ions is completely different.
When the salt of 1mM is added, Fe2+The activity of strong inhibition enzyme can make enzymatic activity be down to 5.59%；K+、Na+And Ca2+
The slight activity for inhibiting enzyme；Mn2+(NH4)+It then can promote the activity of recombination laccase.
The Mg of 10mM is added2+、Na+、K+And Fe2+Enzyme activity can be inhibited to half；Fe3+And Ca2+Enzyme can slightly be inhibited
Activity；And NH4 +、Mn2+And Cu2+The activity of enzyme can then be improved.
When metal ion is added with high concentration (100mM), (NH4)+It is slight to inhibit enzyme activity；Mg2+、Na+、K+And Ca2+'s
Serious inhibitory enzyme activity is added to 10% or so；Fe2+Inhibitory effect decline instead as concentration increases；Interestingly
Mn2+And Cu2+Remain to promote the activity (as shown in table 2) of enzyme in 100mM concentration.This is for the enzyme later and the manganese for relying on manganese
Peroxidase is combined into compound enzyme system and have when lignin degradation, noxious material processing, sewage treatment containing heavy industries
Incomparable advantage.
2 different metal ions of table influence enzymatic activity
(3) activity research of single bacterial laccase and compound enzyme system lignin degrading
1, the bacterial laccase lignin degrading of embodiment 4 after purification
The detection of lignin degradation ability is carried out to bacterial laccase lac1542, reaction system 4mL is wooden containing 50g/L
Element, with 50mM sodium succinate (pH5.5) for buffer, 0.1mM CuSO4, 0.025mM ABTS, 2U enzyme, 30 DEG C of reaction 8h,
The content that residual lignin is surveyed under 280nm, measures the degradation rate 16% of lignin, as a result as shown in Figure 6.Lignin degradation rate is inclined
It is low may be related with reaction temperature, it is also possible to it is related with redox potential.
2, manganese peroxidase lignin degrading
Reaction system 4ml, content of lignin 50g/L wherein H2O220 μ L, MnSO450mM, enzyme 16u, with 50mM succinic acid
Sodium is as buffer (pH5.5), after 30 DEG C are reacted 8 hours, the content of residual lignin is surveyed at 280nm, measures lignin
Degradation rate 19%, as a result as shown in Figure 6.
3, the compound enzyme system lignin degrading of bacterial laccase and manganese peroxidase composition
Existing document report laccase and manganese peroxidase (Mnp) are the key enzymes of lignin degradation, will in this test example
Lac1542 and Mnp is formed compound enzyme system and lignin degradation is optimized using response phase method.It is tested first with PB from numerous
Finding in factor influences significant factor to lignin degradation, (as shown in table 3) by analysis of variance table it can be seen that pH, dioxygen
The amount of water concentration and Mnp influence lignin degradation significant.Next pH, hydrogen peroxide concentration and Mnp content are carried out further
Optimization.With design expert software design center combination design, analyzed (such as from the binomial variance of center combination design
Shown in table 4) it can be seen that pH, hydrogen peroxide concentration, Mnp content, the interaction between three influences significantly lignin degradation,
One is obtained after software is analyzed about A (pH), B (Mnp) and C (H2O2) binomial equation: Y=13.81+3.96*A-
6.31*B+0.98*C+8.17*AB+4.16*BC.Equation model p < 0.0001 illustrates that model is extremely significant, R2=0.98 explanation
98% experimental result can be explained with this model.The optimal reaction condition of software prediction is to contain in 4ml reaction system
The Mn of lignin 50g/L, 0.25mM2+, the lac1542 of 15 μ L (1.8u), Mnp are 6.84 μ L (12u), and hydrogen peroxide is 20 μ L, most
Big degradation rate is 47.8%.It is verified under the optimum condition of software prediction, the degradation rate as the result is shown of experiment is average
47.5% and theoretical value it is close, explanation can replace experimental result to be predicted with theoretical value.
The analysis of table 3PB experimental variance
| Impact factor | Variance summarizes | Freedom degree | Mean of variance | F value | P value |
| Model | 337.15 | 6 | 56.19 | 7.01 | 0.0248 |
| A-PH | 148.34 | 1 | 148.34 | 8.49 | 0.0077 |
| B- temperature | 36.54 | 1 | 35.41 | 2.71 | 0.2418 |
| C-Mnp | 103.1 | 1 | 103.1 | 12.5 | 0.0158 |
| D-lac | 2.02 | 1 | 2.02 | 0.23 | 0.6373 |
| E- lignin | 13.27 | 1 | 13.27 | 1.65 | 0.2548 |
| F- hydrogen peroxide | 47.71 | 1 | 47.71 | 5.95 | 0.0487 |
| G-Mn2+ | 22.71 | 1 | 22.71 | 2.83 | 0.1532 |
The analysis of variance table of 4 response surface binomial model of table
It is from the above it is found that bacterial laccase and manganese peroxidase composition complex enzyme degradation lignin is excellent through response surface
Change, optimal reaction condition is 50g/L containing lignin, the Mn of 0.25mM in 4ml reaction system2+, the lac1542 of 15 μ L
(1.8u), the Mnp (12u) of 6.84 μ L, hydrogen peroxide are 20 μ L；The degradation rate as the result is shown of experiment is average 47.5%, as a result such as
Shown in Fig. 6.
4, the compound enzyme system lignin degrading of manganese peroxidase and Coprinus cinereus peroxidase (CIP) composition
Total enzyme activity is 12U in fixed compound enzyme system, Mnp and CIP is mixed with different bioactivity (U) ratios (1:4,
1:2,1:1,2:1,4:1), it is used for lignin degrading.As a result as shown in fig. 7, in the case that the two bioactivity ratio is 1:1
Degradation rate highest, up to 48.9%.
5, the compound enzyme system lignin degrading of bacterial laccase, manganese peroxidase and Coprinus cinereus peroxidase composition
Find the dosage of lac1542 in complex enzyme in the case where above-mentioned Lac1542/Mnp compound enzyme system lignin degrading
It is not major influence factors in system, so fixed 15 μ L (1.8u) of lac dosage, by CIP and Mnp with different bioactivity ratios
(1:4,1:2,1:1,2:1,4:1) mixing, is used for lignin degrading.As a result as shown in fig. 7, when the two ratio is the CIP of 4.8U
It is best to the degradation effect of lignin with the compound enzyme system of Mnp (when bioactivity ratio is 2:3) Shi Zucheng of 7.2U, reach
71.5%, illustrate that the compound enzyme system of three kinds of enzymes composition can more efficiently lignin degrading.
The above experiment is comprehensive for the degradation results of lignin as shown in fig. 6, it will be appreciated from fig. 6 that the bacterium that the present invention obtains
Laccase lac1542 can increase substantially degradation when forming compound enzyme system lignin degrading with Mnp, CIP with few enzyme amount
Rate.It can thus be appreciated that lac1542/Mnp+CIP complex enzyme can be sent out in the multiple fields such as papermaking, dye decolored, treatment of Organic Wastewater
The effect of waving.
(4) bacterial laccase and Coprinus cinereus peroxidase complex enzyme tie up to the activity research in decoloration methyl orange
1, the bacterial laccase decoloration methyl orange of embodiment 4 after purification
The detection of decoloration methyl orange, 200 μ L of reaction system, fixation of bacteria paint have been carried out to bacterial laccase lac1542
Enzyme lac1542 0.5-4.0mg (with 2.5mg, 3U is optimal when bioactivity is), containing 100mg/L methyl orange, with NaAC
It (pH3.5) is buffer, the CuSO of 0.02-10mmol/L4(being best with 0.1mmol/L), 0-5mmol/L ABTS (with
1.5mmol/L is best), the content of remaining methyl orange is surveyed in 96 orifice plates under 30 DEG C of reactions 8h, microplate reader 490nm, measures methyl
The percent of decolourization 18.9% of orange.
2, Coprinus cinereus peroxidase decoloration methyl orange
The detection of decoloration methyl orange is carried out to CIP, 200 μ L of reaction system (is most with 15U containing CIP 8-20U
It is good), 100mg/L methyl orange, with NaAC (pH3.5) for buffer, H2O2Concentration is 2-20mmol/L (most preferably 12mmol/L),
The content for surveying remaining methyl orange in 96 orifice plates under 30 DEG C of reactions 8h, microplate reader 490nm, measures the percent of decolourization 15.3% of methyl orange.
3, compound enzyme system decoloration methyl orange
Lac1542 and CIP is formed compound enzyme system methyl orange dye decolorization experiment, through response surface optimization, optimum response item
Part are as follows: methyl orange containing 100mg/L in 200 μ L reaction systems, with NaAC (pH3.5) for buffer, hydrogen peroxide 13mmol/L, laccase
1.45mg (1.47U), CIP 10U, 0.1mM CuSO4, 1.5mM ABTS, 30 DEG C of reaction 8h in 96 orifice plates measure methyl orange
Percent of decolourization 54.8%, the experiment can be scaled up reaction system.
The result of above-mentioned experiment is as shown in figure 8, as can be seen from Figure 8 complex enzyme is tied up in the lower situation of enzyme dosage and taken off
Colour efficiency is much higher than single enzyme.
<110>Nanyang Normal College
<120>a kind of super resistance to Mn2+Bacterial laccase, recombinant vector, recombinant bacterium, enzyme preparation, compound enzyme system and preparation method thereof with answer
With
<160> 4
<170> PatentIn version 3.5
<211> 1542
<212> DNA
<221>bacterial laccase gene sequence
<400> 1
atgacacttg aaaaatttgt ggatgctctc ccaatcccag atacactaaa gccagtacag 60
caatcaaaag aaaaaacata ctacgaagtc accatggagg aatgcaccca tcagctccat 120
cgcgatctcc ctccaacccg cctgtggggc tacaacggct tatttccggg gccgaccatt 180
gaggttaaaa gaaatgaaaa cgtatatgta aaatggatga ataaccttcc ttccacgcat 240
ttccttccga ttgatcacac cattcatcac agtgacagcc agcatgaaga gcccgaggta 300
aagactgttg ttcatttaca cggcggcgtc acgccagatg acagtgacgg gtatcccgag 360
gcttggtttt ccaaagactt tgaacaaaca ggaccttatt tcaaaagaga ggtttatcat 420
tatccaaacc agcagcgcgg ggctatattg tggtatcacg atcacgccat ggcgctcacc 480
aggctaaatg tctatgccgg acttgtcggt gcttatatca ttcatgaccc aaaggaaaaa 540
cgcttaaaac tgccttcaga cgaatacgat gtgccgcttc ttatcacaga ccgcgcgatc 600
aatgaggatg gttctttgtt ttatccgagc gcaccggaaa acccttctcc gtcactgcct 660
aatccttcaa tcgttccggc tttttgcgga gaaaccatac tcgtcaacgg gaaggtatgg 720
ccatacttgg aagtcgagcc aaggaaatac cgattccgtg tcatcaacgc ctccaataca 780
agaacctata atctgtcact cgataatggc ggagagttta ttcaggttgg ttcagatggg 840
gggctcctgc cgcgatctgt taaactgaat tctttcagcc ttgcgcctgc tgaacgttat 900
gatatcatca ttgacttcac agcatatgaa ggagaatcga tcattttggc aaacagcgcg 960
ggctgcggcg gtgatgtcaa tcctgataca gatgcgaata tcatgcaatt cagagtcaca 1020
aaaccattgg cacaaaagga cgaaagcaga aagccgaagt acctcgcctc atacccttcg 1080
gtacagcatg aaagaataca aaacatcaga acgttaaaac tggcaggcac ccaggacgaa 1140
tacggcagac ccgtccttct gcttaataac aaacgctggc acgatcccgt cacagaagca 1200
ccaaaagtcg gcacaactga aatatggtcc attatcaacc caacacgcgg aacacatccg 1260
attcacctgc atctagtctc cttccgtgta ttagaccggc ggccgtttga tatcgcccgt 1320
tatcaagaaa gcggggaatt gtcctatacc ggtccggctg tcccgccgcc gccaagtgaa 1380
aagggctgga aagacaccat tcaagcgcat gcaggtgaag tcctgagaat cgcggcgaca 1440
ttcggtccgt acagcggacg atacgtatgg cattgccata ttctagagca tgaagactat 1500
gacatgatga gaccgatgga tataactgat ccccataact aa 1542
<211> 513
<212> PRT
<221>bacterial laccase amino acid sequence
<400> 2
Met Thr Leu Glu Lys Phe Val Asp Ala Leu Pro Ile Pro Asp Thr
1 5 10 15
Leu Lys Pro Val Gln Gln Ser Lys Glu Lys Thr Tyr Tyr Glu Val
20 25 30
Thr Met Glu Glu Cys Thr His Gln Leu His Arg Asp Leu Pro Pro
35 40 45
Thr Arg Leu Trp Gly Tyr Asn Gly Leu Phe Pro Gly Pro Thr Ile
50 55 60
Glu Val Lys Arg Asn Glu Asn Val Tyr Val Lys Trp Met Asn Asn
65 70 75
Leu Pro Ser Thr His Phe Leu Pro Ile Asp His Thr Ile His His
80 85 90
Ser Asp Ser Gln His Glu Glu Pro Glu Val Lys Thr Val Val His
95 100 105
Leu His Gly Gly Val Thr Pro Asp Asp Ser Asp Gly Tyr Pro Glu
110 115 120
Ala Trp Phe Ser Lys Asp Phe Glu Gln Thr Gly Pro Tyr Phe Lys
125 130 135
Arg Glu Val Tyr His Tyr Pro Asn Gln Gln Arg Gly Ala Ile Leu
140 145 150
Trp Tyr His Asp His Ala Met Ala Leu Thr Arg Leu Asn Val Tyr
155 160 165
Ala Gly Leu Val Gly Ala Tyr Ile Ile His Asp Pro Lys Glu Lys
170 175 180
Arg Leu Lys Leu Pro Ser Asp Glu Tyr Asp Val Pro Leu Leu Ile
185 190 195
Thr Asp Arg Ala Ile Asn Glu Asp Gly Ser Leu Phe Tyr Pro Ser
200 205 210
Ala Pro Glu Asn Pro Ser Pro Ser Leu Pro Asn Pro Ser Ile Val
215 220 225
Pro Ala Phe Cys Gly Glu Thr Ile Leu Val Asn Gly Lys Val Trp
230 235 240
Pro Tyr Leu Glu Val Glu Pro Arg Lys Tyr Arg Phe Arg Val Ile
245 250 255
Asn Ala Ser Asn Thr Arg Thr Tyr Asn Leu Ser Leu Asp Asn Gly
260 265 270
Gly Glu Phe Ile Gln Val Gly Ser Asp Gly Gly Leu Leu Pro Arg
275 280 285
Ser Val Lys Leu Asn Ser Phe Ser Leu Ala Pro Ala Glu Arg Tyr
290 295 300
Asp Ile Ile Ile Asp Phe Thr Ala Tyr Glu Gly Glu Ser Ile Ile
305 310 315
Leu Ala Asn Ser Ala Gly Cys Gly Gly Asp Val Asn Pro Asp Thr
320 325 330
Asp Ala Asn Ile Met Gln Phe Arg Val Thr Lys Pro Leu Ala Gln
335 340 345
Lys Asp Glu Ser Arg Lys Pro Lys Tyr Leu Ala Ser Tyr Pro Ser
350 355 360
Val Gln His Glu Arg Ile Gln Asn Ile Arg Thr Leu Lys Leu Ala
365 370 375
Gly Thr Gln Asp Glu Tyr Gly Arg Pro Val Leu Leu Leu Asn Asn
380 385 390
Lys Arg Trp His Asp Pro Val Thr Glu Ala Pro Lys Val Gly Thr
395 400 405
Thr Glu Ile Trp Ser Ile Ile Asn Pro Thr Arg Gly Thr His Pro
410 415 420
Ile His Leu His Leu Val Ser Phe Arg Val Leu Asp Arg Arg Pro
425 430 435
Phe Asp Ile Ala Arg Tyr Gln Glu Ser Gly Glu Leu Ser Tyr Thr
440 445 450
Gly Pro Ala Val Pro Pro Pro Pro Ser Glu Lys Gly Trp Lys Asp
455 460 465
Thr Ile Gln Ala His Ala Gly Glu Val Leu Arg Ile Ala Ala Thr
470 475 480
Phe Gly Pro Tyr Ser Gly Arg Tyr Val Trp His Cys His Ile Leu
485 490 495
Glu His Glu Asp Tyr Asp Met Met Arg Pro Met Asp Ile Thr Asp
500 505 510
Pro His Asn
513
<211> 29
<212> DNA
<213>sequence
<221>primer LacF
<400> 3
gcgaattcat gacacttgaa aaatttgtg 29
<211> 30
<212> DNA
<213>sequence
<221>primer LacR
<400> 4
cccaagcttg ttatggggat cagttatatc 30
Claims (10)
1. a kind of super resistance to Mn2+Bacterial laccase, it is characterised in that: the amino acid sequence of the bacterial laccase such as SEQ ID NO.2
It is shown.
2. bacterial laccase according to claim 1, it is characterised in that: the gene for encoding the bacterial laccase is Lac1542
Gene, nucleotide sequence is as shown in SEQ ID NO.1.
3. a kind of recombinant vector comprising encoding the genetic fragment of bacterial laccase as described in claim 1.
4. a kind of preparation method of recombinant vector as claimed in claim 3, it is characterised in that: include: that will encode the bacterium paint
The genetic fragment of enzyme is inserted into the carrier that sets out, after conversion screen to get.
5. a kind of recombinant bacterium comprising recombinant vector as claimed in claim 3.
6. a kind of preparation method of recombinant bacterium as claimed in claim 5, it is characterised in that: include: to convert the recombinant vector
Enter in host cell, culture, screen to get.
7. a kind of method for preparing bacterial laccase using recombinant bacterium as claimed in claim 5, it is characterised in that: including walking as follows
It is rapid: cultivate the recombinant bacterium, inducing expression bacterial laccase, purification of bacterial laccase to get.
8. a kind of enzyme preparation comprising bacterial laccase as described in claim 1.
9. a kind of compound enzyme system comprising bacterial laccase as described in claim 1, it is characterised in that: further include manganese peroxidase
One or both of with Coprinus cinereus peroxidase.
10. bacterial laccase as described in claim 1, enzyme preparation according to any one of claims 8 or complex enzyme as claimed in claim 9
Application in terms of tying up to lignin degrading or methyl orange of decolourizing.
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| CN113430182B (en) * | 2021-08-09 | 2023-01-13 | 云南师范大学 | Bacterial laccase from Astrospiraceae of elephant intestinal tract and gene thereof |
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| CN106947748A (en) | 2017-07-14 |
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