CN101870968A - Triphenylmethane dye decolorization enzyme and application thereof - Google Patents
Triphenylmethane dye decolorization enzyme and application thereof Download PDFInfo
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Abstract
The invention discloses triphenylmethane dye decolorization enzyme, wherein, the amino acid sequence thereof is as shown in SEQ ID No.2. The invention further discloses a gene for expressing the triphenylmethane dye decolorization enzyme, wherein, the nucleotide sequence thereof is as shown in SEQ ID No. 1; and the gene is derived from the triphenylmethane dye decolorization enzyme gene of Pseudomonas putida KT 2440. The invention further discloses a purification method of the triphenylmethane dye decolorization enzyme, and application of triphenylmethane dye decolorization enzyme in removing the triphenylmethane dye. The triphenylmethane dye decolorization enzyme of the invention has efficient decoloring activity and great application prospect.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of triphenylmethane dye decolorization enzyme and application thereof.
Background technology
Triphenhlmethane dye comprises alkalescence, acidity, solvent dye etc., is widely used in fields such as textile printing and dyeing, medicine, biological stain, papermaking.Because its bio-toxicity and carcinogenic, teratogenesis, mutagenic three causing property constitute a threat to human health, caused widely and paid close attention to.Malachite green is a kind of representative in the triphenylmethane dye, it is a kind of industrial dye, is again a kind of mycocide, and is very effective to the control of fish fish molds, protozoal disease etc., therefore, the use of malachite green in culture fishery for a long time is very general.
Biological method is to remove comparatively effective means of dye discoloration at present, has the advantage of nontoxic, noresidue, non-secondary pollution.When the immobilization decolorization enzyme is applied to the decolouring of waste water containing dye and recycle, need not to add substrate, thereby have fine application potential for thalli growth.The now existing relevant report that immobilized azo reductase is applied to contain the treatment of dyeing wastewater of azoic dyestuff.At present, the investigator has found that the decolorization enzyme of multiple degradable triphenylmethane dye reaches and genes involved is studied both at home and abroad.Decolorization enzymes such as the cytochrome P 450 monooxygenases in lignoenzyme system, fungi and the mycobacterium in the fungi, the triphenylmethane dye reductase enzyme TMR in the citric acid bacillus, the triphenylmethane dye decolorization enzyme TpmD in the Aeromonas hydrophila and various decolouring gene have become the research focus.Phanerochaete chrysosporium in the fungi can utilize its lignin peroxidase in the mode of demethylation progressively triphenhlmethane dye Viola crystallina to be decoloured and degraded; Cunninghamella elegans decolours and degraded to malachite green in order to the mode of demethylation by the work of certain unknown enzyme in the cell, and the participation of P450 system is arranged.And the key enzyme of catalysis triphenylmethane dye decolorization (or enzyme system) is up to the present still unclear in the bacterium.
Triphenylmethane dye decolorization enzyme and inquire into the enzymatic property and the mechanism of its decolouring in the further investigation bacterium, can utilize genetic engineering technique that decolorization enzyme is transformed or transfers in the specific carrier, efficiently express, thereby, has huge application potential in this field for the technological design that utilizes the immobilization decolorization enzyme to handle trade effluent provides the science reference.
Summary of the invention
Technical problem to be solved by this invention provides a kind of triphenylmethane dye decolorization enzyme of high vigor.
The technical problem that the present invention also will solve provides the gene of expressing above-mentioned triphenylmethane dye decolorization enzyme.
The technical problem that the present invention also will solve provides the application of above-mentioned triphenylmethane dye decolorization enzyme.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of triphenylmethane dye decolorization enzyme (being dihydropyrimidinase TDE), its aminoacid sequence is shown in SEQ ID No.2, and it has comprised 479 amino acid.
Express the gene of above-mentioned triphenylmethane dye decolorization enzyme, its nucleotide sequence is shown in SEQ ID No.1, and it has comprised the base of 1440bp.
The purification process of above-mentioned triphenylmethane dye decolorization enzyme is characterized in that it comprises the steps:
(1) ammonium sulfate precipitation:
(1a) pseudomonas putida (Pseudomonas putida) KT2440 is cultivated centrifugal collection thalline, centrifuging and taking supernatant liquor after the ultrasonication in the LB substratum;
(1b) supernatant liquor that step (1a) is obtained carries out ammonium sulfate precipitation, promptly under continuous agitation condition on the magnetic stirring apparatus, slowly add ammonium sulfate solids to certain saturation degree, continue to stir 30min, centrifugally be collected in the albumen precipitation that 40～50% ammonium sulfate saturation concentration section obtains, to obtain albumen precipitation and heavily be dissolved among pH 8.0, the 20mM Tris-HCl, the ammonium sulfate in the protein solution is removed in dialysis again;
(2) anionresin:
(2a) after DEAE anion-exchange column pH 8.0, the 20mM Tris-HCl balance, the protein solution that obtains after step (1b) dialysis is crossed the DEAE anion-exchange column, wash until the Protein Detection device at A with pH 8.0,20mM Tris-HCl
280The place is 0.05, and flow velocity is 1.0mlmin
-1
(2b) the DEAE anion-exchange column is carried out gradient elution with 0～0.5M NaCl solution, flow velocity is 1.0mlmin
-1, collect and concentrate the protein solution that obtains in 0.23～0.26M NaCl wash-out scope, heavily be dissolved among pH 8.0, the 20mMTris-HCl, the NaCl in the protein solution is removed in dialysis again;
(3) gel permeation chromatography:
(3a) after gel permeation chromatography post pH 8.0, the 20mM Tris-HCl balance, the protein solution that obtains after step (2b) dialysis is crossed the gel permeation chromatography post, flow velocity is 0.5mlmin
-1, the Protein Detection device is at A
280There is the position of albumen absorption peak at the place, is that protein solution is collected by unit with 2ml, is substrate with the malachite green, measures the enzyme of collected each protein solution and lives, and it is strong and weak to live according to enzyme, collects to obtain having that decolouring is active crosses protein solution behind the post;
(3b) the active protein solution of decolouring that has that obtains in the step (3a) is respectively got 15～20 μ l and carried out the SDS-PAGE electrophoresis respectively, behind the coomassie brilliant blue staining, according to the electrophoretic band result, select the active protein solution of decolouring that has of electrophoretogram simple (being that electrophoretic band is more single) correspondence, merge ultrafiltration and concentration.
The application of above-mentioned triphenylmethane dye decolorization enzyme in removing triphenylmethane dye.
Beneficial effect: triphenylmethane dye decolorization enzyme of the present invention has decolouring activity efficiently to triphenylmethane dye, has great application prospect.Triphenylmethane dye has " three cause " effect as a kind of widely used dyestuff, and daily life is constituted big negative impact.Although now decoloring dye waste water is had various physics, chemical process, because all potential hazards of its existence, people more and more pay close attention to adopting the biological method dye wastewater treatment using.In recent years, found that multiple white-rot fungi, mould, yeast etc. have the triphenylmethane dye decolorization activity, both at home and abroad research concentrates on the lignoenzyme system, cytochrome P 450 monooxygenases etc. of fungi to triphenylmethane dye decolorization enzyme.From the eighties in last century, discovery white-rot fungi Phanerochaete chrysosporium Phanerochaetechrysosporium such as Glenn have had some polymeric dyes since the decolored degradation effect, and white-rot fungi becomes the research focus gradually to the decolored degradation of dyestuff.Therefore but, the triphenylmethane dye decolorization enzyme that we found will provide a kind of new approach for dye decolored both at home and abroad to the research of bacterial dye decolorization enzyme also seldom.The dihydropyrimidinase that Pseudomonas putida KT2440 is produced has very strong decolouring activity to triphenylmethane dye-malachite green, for this decolorization enzyme of mass production from now on provides favourable condition.By selecting the suitable carriers plasmid, this gene is expressed in bacterial classification in some environment protection, provide desirable material for making up multi-functional degradation bacteria, improve the performance and the decoloring ability of decolorizing bacterial strain, reduce production and use cost.
Description of drawings
Fig. 1 obtains having the active elution peak of decolouring for separating by the DEAE anion-exchange column in 0.23～0.26M NaCl wash-out scope.
Fig. 2 is for separating by the gel permeation chromatography post, and solution was at A after the Protein Detection device detected post in real time
280The albumen absorption peak at place.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that embodiment only is used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
In following examples, the active detection method of protein decolouring is as follows:
Enzyme activity determination carries out under 45 ℃ of conditions, sets up enzyme constant measuring system alive 800 μ l in 20mM Tris-HCl (pH 8.0) damping fluid, contains TDE enzyme, 20 μ M substrates, 0.1mM NADH that an amount of separation and purification obtains in the system.After adding an amount of enzyme, detecting substrate on the spectrophotometer in initial 1min, the decline of corresponding maximum absorption wave strong point OD value immediately.With the reaction mixture that does not contain decolorization enzyme is contrast.
With the needed enzyme amount of per minute catalysis 1.0 μ mol substrate dyestuffs is 1 enzyme unit (U) alive.
Embodiment 1: proteic purifying.
1, ammonium sulfate precipitation:
(1a) under 30 ℃ of conditions, pseudomonas putida (Pseudomonas putida) KT2440 cultivated 36 hours in the LB substratum, the centrifugal 5min of 15000 * g collects thalline, thalline is centrifugal again after washing once with 20mM Tris-HCl (pH 8.0), then thalline is resuspended in 20mM Tris-HCl (pH 8.0), after the ultrasonication, 20000 * g high speed centrifugation 30min gets supernatant liquor on ice.
(1b) supernatant liquor mild stirring on magnetic stirring apparatus that step (1a) is obtained, add solid ammonium sulfate simultaneously to different saturation concentration section precipitating proteins (this process control is finished) in 5～10min, stir 30min after adding to each respective concentration, 20000 * g high speed centrifugation 30min, the albumen precipitation of centrifugal back gained heavily is dissolved among an amount of 20mMTris-HCl (pH 8.0), measure the decolouring activity of the corresponding protein precipitation of different ammonium sulfate concentrations sections simultaneously, get have the active ammonium sulfate concentrations section of the higher decolouring protein of (40～50% saturated ammonium sulphate concentration section), the protein precipitation that this concentration section is obtained after centrifugal heavily is dissolved among an amount of 20mM Tris-HCl (pH 8.0), and the ammonium sulfate of protein solution middle and high concentration is removed in dialysis again.
2, anionresin:
(2a) DEAE anion-exchange column (DEAE Sefinose FF; sales company of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; 2.5cm * 20cm) with after 20mM Tris-HCl (pH 8.0) balance; the protein solution that obtains after step (1b) dialysis is crossed the DEAE anion-exchange column; according to the different initial gross separation protein of different proteins charge property; after protein soln advances post; wash until Protein Detection instrument (HD-4 computer nucleic acid-protein detector, Shanghai Hu Xi analytical instrument factory) at A with 20mM Tris-HCl (pH 8.0)
280The place is 0.05,, the control flow velocity is 1.0mlmin
-1
(2b) the DEAE anion-exchange column is carried out gradient elution with 0～0.5M NaCl solution, the control flow velocity is 1.0mlmin
-1The corresponding enzyme of each elution peak of The real time measure Protein Detection instrument 280nm place is lived, in 0.23～0.26M NaCl wash-out scope, obtain having decolouring active elution peak (Fig. 1), collect the protein sample that concentrates in this elution peak, heavily be dissolved among an amount of 20mM Tris-HCl (pH 8.0), protein solution middle and high concentration NaCl is removed in dialysis again.
3, gel permeation chromatography:
(3a) gel permeation chromatography post (Sephacryl S-200HR, GE Healthcare, 1.5cm * 100cm) with after 20mM Tris-HCl (pH 8.0) balance, the protein solution that obtains after step (2b) dialysis is crossed the gel permeation chromatography post, according to the further separation and purification protein of protein molecular weight size, the control flow velocity is 0.5mlmin
-1, detected post in real time with the Protein Detection device after solution at A
280The absorbing state at place at the position that shows the albumen absorption peak (Fig. 2), is that protein solution is collected by unit with 2ml, measures the enzyme of each collected protein solution respectively and lives, and obtains having that decolouring is active crosses protein solution behind the post.
(3b) the high enzyme protein sample alive that has that obtains in the step (3a) is respectively got 15～20 μ l respectively through the SDS-PAGE electrophoresis, after coomassie brilliant blue staining and corresponding decolouring processing, according to the electrophoretic band result, select the active protein solution of decolouring that has of protein solution purer (being that electrophoretic band is single) to merge, ultrafiltration and concentration ,-20 ℃ frozen.
Embodiment 2: determined amino acid sequence.
The single protein band that embodiment 1 is finally prepared carries out the N-terminal order-checking through the Edman chemical degradation method, obtains the aminoacid sequence of this decolorization enzyme, shown in SEQ ID No.2.
Embodiment 3: enzyme constant measuring alive.
The maximum absorption wavelength of Victoria Green WPB, brilliant green, Viola crystallina and magenta is respectively 617nm, 630nm, 590nm and 545nm.
For measuring the kinetic constant of this decolorization enzyme, the final concentration of each substrate dyestuff (S) is made as 5.0～20 μ M, by calculating the initial velocity (V) that enzyme is lived and reacted under each substrate final concentration condition, try to achieve TDE enzyme constant (as shown in table 1) alive by the most frequently used Lineweaver-Burk double-reciprocal plot method.
Table 1
From the K of resulting this triphenylmethane dye decolorization enzyme to above four kinds of dyestuffs (malachite green, brilliant green, Viola crystallina, magenta)
mNumerical value can be inferred, this decolorization enzyme all has decolouring active to these four kinds of triphenylmethane dyes, and wherein the decolouring activity to malachite green is the strongest, is followed successively by brilliant green, magenta, Viola crystallina thereafter.
Nucleotide or aminoacid sequence
SEQUENCE?LISTING
＜110〉Nanjing University
＜120〉triphenylmethane dye decolorization enzyme and application thereof
<130>nju100424
<160>2
<170>PatentIn?version?3.3
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Met?Ser?Leu?Leu?Ile?Arg?Gly?Ala?Thr?Val?Val?Thr?His?Glu?Glu?Ser
1 5 10 15
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Tyr?Pro?Ala?Asp?Val?Leu?Cys?Ala?Asp?Gly?Leu?Ile?Arg?Ala?Ile?Gly
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caa?aac?ctc?gaa?ccg?ccc?acc?gac?tgc?gag?atc?ctc?gac?ggc?agc?ggc 144
Gln?Asn?Leu?Glu?Pro?Pro?Thr?Asp?Cys?Glu?Ile?Leu?Asp?Gly?Ser?Gly
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Gln?Tyr?Leu?Met?Pro?Gly?Gly?Ile?Asp?Pro?His?Thr?His?Met?Gln?Leu
50 55 60
ccg?ttc?atg?ggc?acg?gtg?gcc?agc?gag?gac?ttc?ttc?agc?ggc?acc?gct 240
Pro?Phe?Met?Gly?Thr?Val?Ala?Ser?Glu?Asp?Phe?Phe?Ser?Gly?Thr?Ala
65 70 75 80
gcg?ggc?ctg?gct?ggc?ggc?acc?acc?tcg?atc?atc?gac?ttc?gtc?att?ccc 288
Ala?Gly?Leu?Ala?Gly?Gly?Thr?Thr?Ser?Ile?Ile?Asp?Phe?Val?Ile?Pro
85 90 95
aac?ccg?cag?cag?tcg?ttg?ctg?gag?gcc?ttc?cac?acc?tgg?cgt?ggc?tgg 336
Asn?Pro?Gln?Gln?Ser?Leu?Leu?Glu?Ala?Phe?His?Thr?Trp?Arg?Gly?Trp
100 105 110
gcg?cag?aaa?agc?gcc?agc?gac?tat?ggc?ttc?cac?gtc?gcc?att?acc?tgg 384
Ala?Gln?Lys?Ser?Ala?Ser?Asp?Tyr?Gly?Phe?His?Val?Ala?Ile?Thr?Trp
115 120 125
tgg?agc?gag?cag?gtg?gcc?gaa?gag?atg?ggc?gag?ctg?gtg?gcc?aag?cac 432
Trp?Ser?Glu?Gln?Val?Ala?Glu?Glu?Met?Gly?Glu?Leu?Val?Ala?Lys?His
130 135 140
ggg?gtg?aac?agc?ttc?aag?cat?ttc?atg?gcc?tac?aag?aat?gcg?atc?atg 480
Gly?Val?Asn?Ser?Phe?Lys?His?Phe?Met?Ala?Tyr?Lys?Asn?Ala?Ile?Met
145 150 155 160
gcc?gcg?gac?gat?acc?ctg?gtg?gcc?agc?ttc?gag?cgt?tgc?ctg?caa?ctg 528
Ala?Ala?Asp?Asp?Thr?Leu?Val?Ala?Ser?Phe?Glu?Arg?Cys?Leu?Gln?Leu
165 170 175
ggc?gcg?gtg?ccc?acc?gtg?cat?gcc?gaa?aac?ggc?gag?ctg?gtg?tac?cac 576
Gly?Ala?Val?Pro?Thr?Val?His?Ala?Glu?Asn?Gly?Glu?Leu?Val?Tyr?His
180 185 190
ctg?cag?aaa?aag?ctg?ctc?gcc?cag?ggc?atg?acc?ggg?ccg?gaa?gcc?cac 624
Leu?Gln?Lys?Lys?Leu?Leu?Ala?Gln?Gly?Met?Thr?Gly?Pro?Glu?Ala?His
195 200 205
ccg?ctg?tcg?cgc?ccc?tcg?cag?gtt?gaa?ggc?gaa?gcg?gcc?agc?cgc?gcc 672
Pro?Leu?Ser?Arg?Pro?Ser?Gln?Val?Glu?Gly?Glu?Ala?Ala?Ser?Arg?Ala
210 215 220
atc?cgc?att?gct?gaa?acc?ctc?ggc?acg?ccg?ttg?tac?ctg?gtg?cac?att 720
Ile?Arg?Ile?Ala?Glu?Thr?Leu?Gly?Thr?Pro?Leu?Tyr?Leu?Val?His?Ile
225 230 235 240
tcc?agc?cgc?gag?gca?ctg?gat?gaa?atc?gcc?tat?gcc?cga?ggc?aag?ggc 768
Ser?Ser?Arg?Glu?Ala?Leu?Asp?Glu?Ile?Ala?Tyr?Ala?Arg?Gly?Lys?Gly
245 250 255
cag?ccg?gtg?tat?ggc?gag?gtg?ctg?ccc?gga?cat?ctg?ctg?ctg?gac?gac 816
Gln?Pro?Val?Tyr?Gly?Glu?Val?Leu?Pro?Gly?His?Leu?Leu?Leu?Asp?Asp
260 265 270
agt?gtc?tac?cgt?gac?ccg?gac?tgg?acc?acc?gcc?gcc?ggt?tat?gtg?atg 864
Ser?Val?Tyr?Arg?Asp?Pro?Asp?Trp?Thr?Thr?Ala?Ala?Gly?Tyr?Val?Met
275 280 285
agc?ccg?ccg?ttc?cgc?cca?cgc?gaa?cac?cag?gag?gcg?ctg?tgg?cgc?ggc 912
Ser?Pro?Pro?Phe?Arg?Pro?Arg?Glu?His?Gln?Glu?Ala?Leu?Trp?Arg?Gly
290 295 300
ttg?cag?tcg?ggc?aac?ctg?cac?acc?acc?gcc?acc?gac?cac?tgc?tgc?ttc 960
Leu?Gln?Ser?Gly?Asn?Leu?His?Thr?Thr?Ala?Thr?Asp?His?Cys?Cys?Phe
305 310 315 320
tgc?gcc?gag?cag?aaa?gcc?atg?ggc?cgc?gac?gac?ttc?agc?cgc?atc?ccc 1008
Cys?Ala?Glu?Gln?Lys?Ala?Met?Gly?Arg?Asp?Asp?Phe?Ser?Arg?Ile?Pro
325 330 335
aac?ggc?acc?gcc?ggc?atc?gaa?gac?cgc?atg?gca?gta?ctg?tgg?gat?gcc 1056
Asn?Gly?Thr?Ala?Gly?Ile?Glu?Asp?Arg?Met?Ala?Val?Leu?Trp?Asp?Ala
340 345 350
ggg?gtc?aac?agc?ggg?cgt?ttg?tcg?atg?cac?gaa?ttc?gtc?gcg?ctg?acc 1104
Gly?Val?Asn?Ser?Gly?Arg?Leu?Ser?Met?His?Glu?Phe?Val?Ala?Leu?Thr
355 360 365
tct?acc?aac?acg?gcg?aaa?atc?ttc?aac?ctg?ttc?ccg?cgc?aag?ggc?gct 1152
Ser?Thr?Asn?Thr?Ala?Lys?Ile?Phe?Asn?Leu?Phe?Pro?Arg?Lys?Gly?Ala
370 375 380
atc?cgc?gtg?ggt?gcc?gat?gcc?gac?ctg?gtg?ctg?tgg?gac?ccg?cag?ggc 1200
Ile?Arg?Val?Gly?Ala?Asp?Ala?Asp?Leu?Val?Leu?Trp?Asp?Pro?Gln?Gly
385 390 395 400
acc?cgc?acc?atc?tcc?gcc?aaa?acc?cac?cac?cag?cag?gtg?gac?ttc?aac 1248
Thr?Arg?Thr?Ile?Ser?Ala?Lys?Thr?His?His?Gln?Gln?Val?Asp?Phe?Asn
405 410 415
att?ttc?gaa?ggc?cgc?acc?gtg?cgc?ggc?atc?cca?agc?cac?acc?atc?agc 1296
Ile?Phe?Glu?Gly?Arg?Thr?Val?Arg?Gly?Ile?Pro?Ser?His?Thr?Ile?Ser
420 425 430
cag?ggc?aag?gtg?ctc?tgg?gcc?gat?ggc?gac?ctg?cgc?gcc?gag?ccg?ggc 1344
Gln?Gly?Lys?Val?Leu?Trp?Ala?Asp?Gly?Asp?Leu?Arg?Ala?Glu?Pro?Gly
435 440 445
gcg?ggg?cgc?tat?gtg?gaa?cgg?ccg?gcg?tac?ccg?gcg?gtg?tat?gag?gtg 1392
Ala?Gly?Arg?Tyr?Val?Glu?Arg?Pro?Ala?Tyr?Pro?Ala?Val?Tyr?Glu?Val
450 455 460
ctg?ggg?cgc?cgg?gcc?gag?cat?cag?cgg?ccg?atg?cct?gtt?ccg?cgc?tga 1440
Leu?Gly?Arg?Arg?Ala?Glu?His?Gln?Arg?Pro?Met?Pro?Val?Pro?Arg
465 470 475
<210>2
<211>479
<212>PRT
＜213〉pseudomonas putida (Pseudomonas putida) KT2440
<400>2
Met?Ser?Leu?Leu?Ile?Arg?Gly?Ala?Thr?Val?Val?Thr?His?Glu?Glu?Ser
1 5 10 15
Tyr?Pro?Ala?Asp?Val?Leu?Cys?Ala?Asp?Gly?Leu?Ile?Arg?Ala?Ile?Gly
20 25 30
Gln?Asn?Leu?Glu?Pro?Pro?Thr?Asp?Cys?Glu?Ile?Leu?Asp?Gly?Ser?Gly
35 40 45
Gln?Tyr?Leu?Met?Pro?Gly?GlyI?le?Asp?Pro?His?Thr?His?Met?Gln?Leu
50 55 60
Pro?Phe?Met?Gly?Thr?Val?Ala?Ser?Glu?Asp?Phe?Phe?Ser?Gly?Thr?Ala
65 70 75 80
Ala?Gly?Leu?Ala?Gly?Gly?Thr?Thr?Ser?Ile?Ile?Asp?Phe?Val?Ile?Pro
85 90 95
Asn?Pro?Gln?Gln?Ser?Leu?Leu?Glu?Ala?Phe?His?Thr?Trp?Arg?Gly?Trp
100 105 110
Ala?Gln?Lys?Ser?Ala?Ser?Asp?Tyr?Gly?Phe?His?Val?Ala?Ile?Thr?Trp
115 120 125
Trp?Ser?Glu?Gln?Val?Ala?Glu?Glu?Met?Gly?Glu?Leu?Val?Ala?Lys?His
130 135 140
Gly?Val?Asn?Ser?Phe?Lys?His?Phe?Met?Ala?Tyr?Lys?Asn?Ala?Ile?Met
145 150 155 160
Ala?Ala?Asp?Asp?Thr?Leu?Val?Ala?Ser?Phe?Glu?Arg?Cys?Leu?Gln?Leu
165 170 175
Gly?Ala?Val?Pro?Thr?Val?His?Ala?Glu?Asn?Gly?Glu?Leu?Val?Tyr?His
180 185 190
Leu?Gln?Lys?Lys?Leu?Leu?Ala?Gln?Gly?Met?Thr?Gly?Pro?Glu?Ala?His
195 200 205
Pro?Leu?Ser?Arg?Pro?Ser?Gln?Val?Glu?Gly?Glu?Ala?Ala?Ser?Arg?Ala
2102 15 220
Ile?Arg?Ile?Ala?Glu?Thr?Leu?Gly?Thr?Pro?Leu?Tyr?Leu?Val?His?Ile
225 230 235 240
Ser?Ser?Arg?Glu?Ala?Leu?Asp?Glu?Ile?Ala?Tyr?Ala?Arg?Gly?Lys?Gly
245 250 255
Gln?Pro?Val?Tyr?Gly?Glu?Val?Leu?Pro?Gly?His?Leu?Leu?Leu?Asp?Asp
260 265 270
Ser?Val?Tyr?Arg?Asp?Pro?Asp?Trp?Thr?Thr?Ala?Ala?Gly?Tyr?Val?Met
275 280 285
Ser?Pro?Pro?Phe?Arg?Pro?Arg?Glu?His?Gln?Glu?Ala?Leu?Trp?Arg?Gly
290 295 300
Leu?Gln?Ser?Gly?Asn?Leu?His?Thr?Thr?Ala?Thr?Asp?His?Cys?Cys?Phe
305 310 315 320
Cys?Ala?Glu?Gln?Lys?Ala?Met?Gly?Arg?Asp?Asp?Phe?Ser?Arg?Ile?Pro
325 330 335
Asn?Gly?Thr?Ala?Gly?Ile?Glu?Asp?Arg?Met?Ala?Val?Leu?Trp?Asp?Ala
340 345 350
Gly?Val?Asn?Ser?Gly?Arg?Leu?Ser?Met?His?Glu?Phe?Val?Ala?Leu?Thr
355 360 365
Ser?Thr?Asn?Thr?Ala?Lys?Ile?Phe?Asn?Leu?Phe?Pro?Arg?Lys?Gly?Ala
370 375 380
Ile?Arg?Val?Gly?Ala?Asp?Ala?Asp?Leu?Val?Leu?Trp?Asp?Pro?Gln?Gly
385 390 395 400
Thr?Arg?Thr?Ile?Ser?Ala?Lys?Thr?His?His?Gln?Gln?Val?Asp?Phe?Asn
405 410 415
Ile?Phe?Glu?Gly?Arg?Thr?Val?Arg?Gly?Ile?Pro?Ser?His?Thr?Ile?Ser
420 425 430
Gln?Gly?Lys?Val?Leu?Trp?Ala?Asp?Gly?Asp?Leu?Arg?Ala?Glu?Pro?Gly
435 440 445
Ala?Gly?Arg?Tyr?Val?Glu?Arg?Pro?Ala?Tyr?Pro?Ala?Val?Tyr?Glu?Val
450 455 460
Leu?Gly?Arg?Arg?Ala?Glu?His?Gln?Arg?Pro?Met?Pro?Val?Pro?Arg
465 470 475
Claims (4)
1. triphenylmethane dye decolorization enzyme, its aminoacid sequence is shown in SEQ ID No.2.
2. express the gene of the described triphenylmethane dye decolorization enzyme of claim 1, its nucleotide sequence is shown in SEQ IDNo.1.
3. the purification process of the described triphenylmethane dye decolorization enzyme of claim 1 is characterized in that it comprises the steps:
(1) ammonium sulfate precipitation:
(1a) pseudomonas putida (Pseudomonas putida) KT2440 is cultivated centrifugal collection thalline, centrifuging and taking supernatant liquor after the ultrasonication in the LB substratum;
(1b) in the supernatant liquor that step (1a) obtains, add ammonium sulfate solids, on magnetic stirring apparatus, constantly stir simultaneously, centrifugal then, collect the albumen precipitation of 40～50% saturated ammonium sulphate concentration sections, the albumen precipitation that obtains heavily is dissolved among pH 8.0, the 20mM Tris-HCl, and the ammonium sulfate in the protein solution is removed in dialysis again;
(2) anionresin:
(2a) after DEAE anion-exchange column pH 8.0, the 20mM Tris-HCl balance, the protein solution that obtains after step (1b) dialysis is crossed the DEAE anion-exchange column, wash until the Protein Detection instrument at A with pH 8.0,20mM Tris-HCl
280The place is 0.05, and flow velocity is 1.0mlmin
-1
(2b) the DEAE anion-exchange column is carried out gradient elution with 0～0.5M NaCl solution, flow velocity is 1.0mlmin
-1, collect and be concentrated in the protein solution that obtains in 0.23～0.26M NaCl wash-out scope, heavily be dissolved among pH 8.0, the 20mM Tris-HCl, the NaCl in the protein solution is removed in dialysis again;
(3) gel permeation chromatography:
(3a) after gel permeation chromatography post pH 8.0, the 20mM Tris-HCl balance, the protein solution that obtains after step (2b) dialysis is crossed the gel permeation chromatography post, flow velocity is 0.5mlmin
-1, the Protein Detection instrument is at A
280There is the position of albumen absorption peak at the place, is that protein solution is collected by unit with 2mL, is substrate with the malachite green, measures the enzyme of collected each protein solution respectively and lives, and obtains having that decolouring is active crosses protein solution behind the post;
(3b) the active protein solution of decolouring that has that obtains in the step (3a) is respectively got 15～20 μ l and carried out electrophoresis respectively,, select the single corresponding active protein solution of decolouring that has of electrophoretic band, merge ultrafiltration and concentration according to the electrophoretic band result.
4. the application of the described triphenylmethane dye decolorization enzyme of claim 1 in removing triphenylmethane dye.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010175228.6A CN101870968A (en) | 2010-05-18 | 2010-05-18 | Triphenylmethane dye decolorization enzyme and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010175228.6A CN101870968A (en) | 2010-05-18 | 2010-05-18 | Triphenylmethane dye decolorization enzyme and application thereof |
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| Publication Number | Publication Date |
|---|---|
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105621630A (en) * | 2016-01-18 | 2016-06-01 | 黑龙江大学 | Beta-mannase for decoloration and application of beta-mannase |
| CN109112115A (en) * | 2017-06-23 | 2019-01-01 | 武汉轻工大学 | A kind of triphenylmethane dye decolorization enzyme and encoding gene from macro genome |
| CN110172414A (en) * | 2019-04-18 | 2019-08-27 | 宋金龙 | Wo Shi pseudomonad composition and its processing the material containing dyestuff application |
| CN110563157A (en) * | 2019-09-20 | 2019-12-13 | 东北师范大学 | Application of citrobacter in removal of dye wastewater |
| CN110668582A (en) * | 2019-10-29 | 2020-01-10 | 盘林(厦门)生物科技有限责任公司 | Biological compound water treatment agent and preparation method and application thereof |
| CN114686465A (en) * | 2021-11-21 | 2022-07-01 | 宁波酶赛生物工程有限公司 | Method for synthesizing hydrolase and (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid |
-
2010
- 2010-05-18 CN CN201010175228.6A patent/CN101870968A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 《NCBI》 20091015 D-hydantoinase [Pseudomonas putida] AAQ90019 , 2 * |
| 《NCBI》 20091015 Pseudomonas putida strain YZ-II6 D-hydantoinase gene AY387829 , 2 * |
| 《高校化学工程学报》 20050630 李家璜等 Burkholderia cepecia.njut1中的海因酶的纯化及性质 第19卷, 第3期 2 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105621630A (en) * | 2016-01-18 | 2016-06-01 | 黑龙江大学 | Beta-mannase for decoloration and application of beta-mannase |
| CN109112115A (en) * | 2017-06-23 | 2019-01-01 | 武汉轻工大学 | A kind of triphenylmethane dye decolorization enzyme and encoding gene from macro genome |
| CN109112115B (en) * | 2017-06-23 | 2021-08-13 | 武汉轻工大学 | Triphenylmethane dye decolorizing enzyme derived from metagenome and encoding gene |
| CN110172414A (en) * | 2019-04-18 | 2019-08-27 | 宋金龙 | Wo Shi pseudomonad composition and its processing the material containing dyestuff application |
| CN110172414B (en) * | 2019-04-18 | 2020-12-18 | 宋金龙 | Revolve Pseudomonas stutzeri and application thereof in treatment of dye-containing sewage and soil |
| CN110563157A (en) * | 2019-09-20 | 2019-12-13 | 东北师范大学 | Application of citrobacter in removal of dye wastewater |
| CN110668582A (en) * | 2019-10-29 | 2020-01-10 | 盘林(厦门)生物科技有限责任公司 | Biological compound water treatment agent and preparation method and application thereof |
| CN114686465A (en) * | 2021-11-21 | 2022-07-01 | 宁波酶赛生物工程有限公司 | Method for synthesizing hydrolase and (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid |
| CN114686465B (en) * | 2021-11-21 | 2024-03-22 | 宁波酶赛生物工程有限公司 | Synthesis method of hydrolase and (R) - (-) -3- (carbamoylmethyl) -5-methylhexanoic acid |
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