CN1089111C - New catalase, its gene and composite containing it, and its preparation method - Google Patents
New catalase, its gene and composite containing it, and its preparation method Download PDFInfo
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- CN1089111C CN1089111C CN97120386A CN97120386A CN1089111C CN 1089111 C CN1089111 C CN 1089111C CN 97120386 A CN97120386 A CN 97120386A CN 97120386 A CN97120386 A CN 97120386A CN 1089111 C CN1089111 C CN 1089111C
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Abstract
The present invention provides a new hydrogen peroxidase which is cloned from bacillus thermog lucosidasius with heat resistance. The present invention also provides a method for preparing the high-yield hydrogen peroxidase, which utilizes a genetic engineering technology and hydrogen peroxidase genes cloned from microorganisms to build expression plasmid and conversion cells for obtaining expression products. The present invention also comprises the new recombination plasmid and the conversion cells built in the method. In addition, the present invention also provides a composition for decomposing hydrogen peroxide in residual disinfection physical liquor on contact lenses, which comprises the new hydrogen peroxidase of the present invention.
Description
The present invention relates to a kind of new catalase and application thereof, and the catalatic method of a kind of preparation.Particularly, the invention provides the new catalase of a kind of clone from thermotolerance genus bacillus (Bacillusthermoglucosidasius), comprise this catalatic composition, and a kind ofly utilize engineered method clone catalase gene, make up its expression plasmid, transformant to obtain the catalatic method of high yield.
Hydrogen peroxide (H
2O
2), being commonly called as hydrogen peroxide, it has sterilization, cleaning, bleaching and disinfection efficacy, thereby is usually used in the SYNTHETIC OPTICAL WHITNER of the thimerosal of contact lens and fabric, and is legal foodstuff additive.But hydrogen peroxide is easy to generate highly active free oxygen, the intensive oxygenizement can make protein denaturation, no matter so, all must decompose residual hydrogen peroxide at last, to prevent the injury of hydrogen peroxide to the user with disinfectant with hydrogen peroxide contact lens or bleached woven fabric.
For above-mentioned purpose, industry often utilizes catalase to decompose residual hydrogen peroxide, and this also is known as the method for full blast.For example USP 4,585,488, and USP 5,145,644, and USP 5,362,647, and USP 5,521, and 091 has promptly disclosed in the sterilizing process of contact lens and to add catalase to decompose residual hydrogen peroxide in the thimerosal.In addition, patents such as GB2216149 and JP-A-104781 also once were disclosed in behind the hydrogen peroxide bleaching cloth, must handle to decompose residual hydrogen peroxide through the hydrogen peroxide enzyme, dyeed at last again.
Catalase [oxydo-reductase of hydrogen peroxide (EC11.11.1.6)] can be with bimolecular hydrogen peroxide (H
2O
2) be oxidized to a part oxygen (O
2) and be reduced into two molecular water (H
2O), its catalyzed reaction formula is as follows:
Catalase is present in natural some animal and plant and the microorganism cells, for this type of cell survival in oxygenated environment the enzyme that must possess.Present catalatic preparation is mostly purified by this class cell.Especially the catalase that comes from the beef liver purifying industry catalase of normal use especially.But because of European cows break out chronic viral diseases in nineteen ninety, the mad cow disease that promptly is commonly called as (BSE), and existing this disease that studies show that may infect to human (Dealler and Lorey 1991 Nutr.Health (Bicester) 7:117-134, Dealler and Lacey 1990, Food Microbio 17:253-280), therefore industrial community is inclined to the catalase with the nonmammalian source gradually, and for example microbe-derived catalase replaces beef liver catalase.
The known microorganism that produces peroxidase of fermenting comprises aspergillus niger (Aspergillus niger), some mould (Penicillium notatum) and micrococcus luteus (Micrococcus luteus) etc.For example, USP 3123539 once disclosed from fungies such as aspergillus niger and some moulds and obtained catalase; USP 2,635,069,5,360,732 and WO 93/17721 disclose with aspergillus niger and produce catalase; USP 5521091 then announcement is derived from the catalase of micrococcus luteus and aspergillus niger.The catalase of above-mentioned patent all is a traditional method, via the fermentation specified microorganisms, break thalline, and purifying in addition again.
Yet all extremely low with the hydrogen peroxide production of enzyme that these fermentation modes were obtained, for example according to U.S. Pat P3,123,539 reports in the gained 1000 gram products, only contain 5% catalase with 95 pounds of weight in wet base micrococcus luteus thalline after albumen reclaims; And according to U.S. Pat P, 2,635,069 reports, the catalase of 2.4 units activity is only arranged by the every gram of crude protein extract that aspergillus niger obtained.In order to improve catalatic output, USP 5,360,732 once carried out strain improvement at aspergillus niger, make its every milligram crude protein extract can reach 14.17 units activity, yet calculate as if about 7.5 units activity of the every microgram of reporting according to WO 93/1772 table 1 of the catalatic specific activity of aspergillus niger, this 14.17 units activity is that the catalase by 1.9 micrograms is showed, in other words, by USP 5,360, the 732 crude protein extracts that obtained also only contain and are less than 0.2% catalase.
Shuichi Furuta and Hiroaki Hayashi disclose the rat catalase gene with the genetic engineering technique express recombinant in J.Biochem.107 among the 708-713 (1990).Yet its output is also not high, only has an appointment 16 milligrams/4 liters before purified.
By last sight it, at present the employed catalase of industrial community is all still having great improvement space no matter be aspect source and the preparation method.
Purpose of the present invention promptly is to clone a kind of new catalase.
Further aim of the present invention is to provide a kind of gene engineering method of utilizing to make microbe-derived catalatic method.Not only output is high according to the prepared catalase of the inventive method, and activity is also splendid.
The present invention also provides new recombinant plasmid and transformant constructed in the inventive method.
A further object of the present invention is to provide a kind of composition that is used for decomposing remaining thimerosal hydrogen peroxide on the contact lens, and it comprises new catalase of the present invention.
Fig. 1: the structure of pET20b/kat19 expression plasmid.
Fig. 2: the dna sequence dna of catalase kat19 gene.
Fig. 3: the structure of pET15b/kat19 expression plasmid.
Fig. 4: the structure of pET20b/katTG expression plasmid.
Fig. 5: the dna sequence dna of catalase katTG gene.
Fig. 6: the aminoacid sequence of catalase TG.
Fig. 7: the structure of pET20b/katHP11 expression plasmid.
Fig. 8: the dna sequence dna of catalase HP11 gene.
Fig. 9: each transforms bacterial strain and expresses catalatic 12% policapram gel electrophoresis analysis figure, by the left-to-right pET15b that is followed successively by, pET15b/kat19, pET20b, pET20b/kat19, pET20b/katTG behind pET20b/katHP11 transformed into escherichia coli BL21 (DE3) bacterial strain, expresses catalatic collection of illustrative plates.
Figure 10: the catalatic 12% policapram gel electrophoresis analysis figure of purifying, by left-to-right respectively by pET15b/kat19, pET20b/kat19, pET20b/katTG, the purified catalase of pET20b/katHP11 transformed into escherichia coli BL21 (DE3) cell.
New catalase provided by the present invention is to utilize Bol and Yashin, Gene 109, after 5 ' and 3 ' end dna sequence dna of the bacillus subtilis catalase gene that 31-3 7 (1991) discloses (kat1 9) synthesizes 5 ' and 3 ' suitable primer, clone and get from heat resistance bacillus (Bacillus thermoglucosdasius), the dna sequence dna of its gene as shown in Figure 5, with the similitude of kat1 9 genes be 74.4%. This gene is inserted suitable expression vector, after transformed host cell makes it express again, namely get new catalase of the present invention, its amino acid sequence is shown in Fig. 6. New catalase of the present invention is not by any document illustration.
The present invention also provides a kind of technique for gene engineering that utilizes to prepare catalatic method, and it comprises:
(a) this catalatic genetic insertion of will encoding contains on the expression vector of suitable transcripting promoter with construction recombination plasmid;
(b) this recombinant plasmid transformed is arrived suitable host cell;
(c) express this transformant of cultivation under the condition of catalase gene in being fit to this transformant; And
(d) the expressed hydrogen peroxide zymoprotein of purifying.
Method of the present invention also is applicable to other microbe-derived catalase of clone except that can producing aforesaid new catalase.In detail, the inventive method is that the dna sequence dna with the catalase gene of various microorganisms in the gene pool is that basic design goes out suitable primer, with amplification microbial staining body in catalase gene, construction of expression vector, transformed host cell, and make its expression and make.
" microorganism " that can be used for the inventive method is meant any microorganism that contains catalase gene, be preferably bacterium, for example subtilis (as CCRC 910064), intestinal bacteria (as CCRC 51731), pseudomonas (as CCRC 292607), micrococcus luteus (as CCRC 11034), and the new catalatic thermotolerance genus bacillus (as CCRC 14687) of production the present invention etc., not specificly be limited to what person, those skilled in the art can be according to self being selected for use.
But the mistakeization hydrogenase gene mat art methods that is derived from microorganism is increased, for example polymerase chain reaction (being called for short PCR).Polymerase chain reaction has been that the technology of a kind of specific gene sequence that increases commonly used since the 1980's is (as USP 4,683,195,4,683,202 etc.), implementation step at first is to isolate target nucleic acid sequence in biological sample, its two strands is opened,, utilized polysaccharase in the presence of the deoxynucleoside triphosphate (as dATP, dGTP, dCTP and dTTP etc.) of appropriate amount, to increase again to hybridize according to target gene two terminal sequences institute synthetic primer.Required reaction parameter and reagent are all well-known to those skilled in the art.Above-mentioned polymerase chain reaction can step-by-step operation, yet more common person carries out with the automated installation (temperature cycler) that is purchased.
The catalase gene of amplification gained is to insert in the suitable expression with known method, comprises any expression vector that can be used for bacterium, yeast, mammalian cell and insect cell expression system, for example the plasmid of pBR, pUC, pUB or pET series.Those skilled in the art can or optionally select for use according to its habitual person.
The expression vector subsequent transformation appropriate host cell that structure is finished also makes its expression.Can be used for host cell of the present invention and comprise bacterium (as intestinal bacteria or subtilis etc.), yeast (as yeast saccharomyces cerevisiae), mammalian cell (as the mouse fibroblast), or insect cell etc.Wherein be preferably the bacterial cell expression system, special good then is intestinal bacteria.The transformed host cells strain is cultivated under the condition of this external plasmid great expression of facility, at last protein is desired by institute and is separated.Contain 15～50% the active catalase of tool in the crude protein extract that is obtained according to the present invention approximately.Again according to well-established law, give purifying as Histidine affinity chromatography or acetone precipitation after, can obtain the catalase of about 95% purity.
Prepare catalase by the inventive method, not only operate simple and easyly and with low cost, output is high far beyond existing fermentation process gained person also, and activity is also splendid, very be suitable for composition, residue in the composition of hydrogen peroxide on the contact lens in particular for removing as decomposition of hydrogen peroxide.
Therefore, the present invention also provides by what new catalase of the present invention was formed and has been used to decompose the composition that residues in the hydrogen peroxide on the contact lens.
Preparation comprises the method for compositions of catalase product of the present invention, and its composition and using method all are well-known to those skilled in the art, for example can be with reference to the record of all multifiles, and as USP5,521,091,5,362,647,5,145,644,4,585,488 etc.This composition can be made into the form of solution, or is the form of solids composition, as lozenge, capsule etc.
In an embodiment of the present composition, it is to comprise a water-based etc. to open fluid matrix, includes catalase of the present invention.These water-baseds etc. are opened the preferable conventional buffer composition that comprises the amount that can effectively control the pH value of fluid matrix, the buffer composition of the routine of the amount of the pH value of better may command fluid matrix, in the scope of the pH of better may command fluid matrix about in 3 to 10, for example about 6 to 8.
Catalatic amount preferably is enough to decompose all hydrogen peroxide in the hydrogen peroxide matrix of containing that are present in disinfect contact lense, and need not damage contact lens itself and not influence safety and the comfortableness of wearing.Generally speaking, its amount can be preferably in the scope of 20 to 800 catalase activity international unit/milliliters of liquid matrix 10 to 1000.
Another embodiment of the present composition is a solids composition, and it can be forms such as lozenge, capsule, one or more solid particulate.These solids compositions comprise the part (as core body) once coating, and a barrier material or delay release composition.Catalase of the present invention is contained in this in coating layer portion (or core body) promptly.And this barrier material (comprises water miscible ethylene-based polymer, as polyvinylpyrrolidone, polyvinyl alcohol etc., water soluble protein, polyose and derivatived cellulose, as methylcellulose gum etc.) can make the present composition delaying its catalatic disengaging when hydrogen peroxide matrix contacts with containing, make hydrogen peroxide that the sufficient time disinfect contact lense is arranged.Its detailed composition all describes in detail in reference mentioned above.
For making content of the present invention more clear and definite, further describe with following non-limiting examples now.
The structure of embodiment 1 pET20b/kat19 expression plasmid
The extraction of subtilis chromosomal DNA
At first 10 gram Bacto-Tryptoness, 5 gram Bacto-yeast extracts and 10 gram NaCl are dissolved in 1 kilogram the deionized water, adjust potential of hydrogen to pH7.5 with 1N NaOH solution, autoclaving treats that its cooling makes Luria-Bertani (LB) nutrient solution; Be LB/A mp nutrient solution for 2 milliliters if add the penbritin solution of 50 mg/ml.
The LB nutrient solution of 3 milliliters of preparation gained is cultivated subtilis (Bacillus subtilus in 37 degree concussions Celsius, CCRC 910064), after 24 hours centrifugal 15 minutes with 5000rpm, clean thalline once with water, outwell supernatant liquor behind the recentrifuge, add 0.75 ml water and added 0.75 milliliter of phenol extraction 30 minutes, with 12000rpm centrifugal 15 minutes, added 0.75 milliliter of phenol extraction again 30 minutes after taking out upper strata liquid, 12000rpm takes out upper strata liquid again with 0.75 milliliter of chloroform extraction 15 minutes after centrifugal 15 minutes, 12000rpm is centrifugal, adds 2 times of volume ethanol after taking out supernatant liquor, centrifugal 15 minutes (12000rpm), deposit D NA washes once with 75% ethanol, uses TE damping fluid (10mM Tris-HCl again, pH7.5,1mM EDTA) the dissolving chromosomal DNA.
The amplification of subtilis catalase gene (kat-19)
Synthesizing of 5 ' end and 3 ' end primer: according to Bol and Yashin, Gene 109, subtilis (the Bacillus subtilis that 31-37 (1991) is disclosed, YB2003) catalase gene sequence (kat-19, be shown in Fig. 2) 5 ' end and 3 ' end dna sequence dna, synthetic 5 ' end primer: NdeI-kat19 (+), its dna sequence dna is as follows:
T T C A T A T G A G T T C A A A T A A A C T G A C A A C T
This sequence contains NdeI restriction enzyme sequence; And 3 ' end primer: kat19-XhoI (-), its dna sequence dna is as follows:
T T C T C G A G T T A A G A A T C T T T T T T A A T C G G C A A
This sequence contains Xho I restriction enzyme sequence.
Polymerase chain reaction (PCR): get 0.5 microgram subtilis chromosomal DNA, add 10 microlitre 2.5mM dNTPs, 10 microlitres, 10 * PC2 damping fluid (50 μ M Tris-HCl, pH9.1,16mM ammonium sulfate, 3.5mM MgCl
2And 150 mcg/ml BSA), 0.2 mcg/ml, 5 ' the end primer of 10 microlitres, 1.0 0.2 mcg/ml, 3 ' the end primer of microlitre behind the 5U/ microlitre klentaq of 78 microliters of water and 1.0 microlitres (AB peptides company), adds 50 microlitre mineral oil again in the upper strata.The reaction parameter of design temperature circulation instrument (Robocycle, STRATAGENE company) is as follows: 94 degree Celsius 1 minute, 1 circulation; 94 degree Celsius * 30 seconds, 54 degree Celsius * 1 minute, it is a circulation that 72 degree Celsius waited in * 1.5 minutes, 30 circulations of coreaction.Behind the polymerase chain reaction, product with QIA quick Spm PCR purification kit (QI AGEN company) purifying polymerase chain reaction, dash the proposition product with 100 microliters of water, carry out electrophoretic analysis and ethidium bromide staining observation with 0.8% sepharose, the size of determining amplification gained dna fragmentation is as expection, that is about 1.5kb.The structure of pET20b/kat19 expression plasmid (Fig. 1)
Reacted 3 hours down in 37 degree Celsius with NdeI and XhoI Restriction Enzyme, enzyme is cut the PCR product, utilizes the agarose gel electrophoresis DNA isolation, cuts out about 1.5kb fragment, dashes with electrophoresis and proposes dna fragmentation, and is continuous with QI A quick Spm PCR purification kit purify DNA.DNA behind the purifying reacted 16 hours down in 16 degree Celsius with the T4DNA ligase enzyme, be connected on the pET-20b expression vector (Novogen company) that cuts with NdeI and XhoI restriction enzyme, mixture after the ligation is transformed DH5 α bacterial strain, obtaining than pET20b through screening is big plasmid, again via the Restriction Enzyme analysis and utilize Sanger ' s method and Sequence Version 2.0 dna sequencing reagent examinations box (available from United States Biochemical company), (A T T A A T A C GA C T C A C T A T A G G) is primer with the T7 promoter sequence, measure dna sequence dna, determine that the 1.5kb dna fragmentation of being cloned into is the kat-19 gene, and finish the structure of pET20b/kat19 expression plasmid, and being preserved in American type culture collection on November 18th, 1997, preserving number is ATCC209467.The structure (Fig. 3) of embodiment 2 pET15b/kat19 expression plasmids
Utilize NdeI and XhoI restriction enzyme on the pET20b/kat19 expression plasmid, to downcut the kat-19 gene, after agarose gel electrophoresis separates, downcut the dna fragmentation of 1.5kb, dash with electrophoresis and carry and come purify DNA with QIA quick Spin PCR purification kit.Purified DNA spends down in Celsius 16 with the T4DNA ligase enzyme and reacted 16 hours, is connected to the pET-15b that has cut with NdeI and XhoI restriction enzyme and expresses on year grain, is built into the pET15b/kat19 expression plasmid.Utilize the T7 promoter sequence to be primer, measure 5 ' end dna sequence dna, determine to have cloned to finish the pET15b/kat19 expression plasmid.This plasmid is preserved in American type culture collection on November 18th, 1997, and preserving number is ATCC 209469.The structure (Fig. 4) of embodiment 3 pET20b/katTG expression plasmids
Cultivate down thermotolerance genus bacillus (Bacillus thermoglucosdasius, CCRC 14687) in 55 degree Celsius, according to the method for embodiment 1 by cultivating the chromosomal DNA that extracts the thermotolerance subtilis in the thalline.For its catalase gene that increases, at first synthetic its 3 ' end primer (kat TG-XhoI (-)), its dna sequence dna is as follows:
T T C T C G A G T T A A G A A T C T T T T T T A A T C G G C A A
In addition, all the step with embodiment 1 described amplification kat19 gene is identical for all the other materials and methods.Behind polymerase chain reaction, obtain the dna fragmentation of 1.5kb size.After this dna fragmentation given purifying, with the XhoI restriction enzyme in 37 degree Celsius reaction 2 hours down, behind the purifying, preserve down once more in 4 ℃.
Cut the pET20b expression vector with the NdeI restriction enzyme down in 37 degree Celsius, after reacting 2 hours and purifying, mend flat with the klenow enzyme, reacted 2 hours in 37 degree Celsius with the XhoI restriction enzyme behind the purifying once more, with 0.8% sepharose DNA isolation, after cutting-out 3.7kb dna fragmentation dashes proposition with electrophoretic method with it again, continuous with this dna fragmentation of QLA quick Spin PCR purification kit purifying, obtain the pET20b carrier that the NdeI position is put down by mending and the XhoI position is cut open.This carrier and aforesaid 1.5kb fragment are carried out ligation after 16 hours with the T4DNA ligase enzyme down in 16 degree Celsius, transform DH5 α cell.After screening, extract plasmid, obtain the pET20b/katTG expression plasmid, utilize Sanger ' s method and measure the sequence (its dna sequence dna as shown in Figure 5) of the dna fragmentation cloned with Sequence version2.0 sequencing kit, itself and kat19 similarity are 74.4%.Through translating the proteinic aminoacid sequence of gained as shown in Figure 6, vegetalitas catalase (vegetative catalase) similarity of itself and subtilis (Bacillus subtilis) then is 81.0%.
Above-mentioned PET20b/katTG plasmid is preserved in American type culture collection on November 18th, 1997, and preserving number is ATCC209468.The structure (Fig. 7) of embodiment 4 pET20b/katHP11 expression plasmids
Extract the chromosomal DNA of bacillus coli DH 5 alpha (CCRC 51731) according to the method for embodiment 1.According to J.Bacteriol 173,514-520 discloses synthetic 5 ' the end primer (NdeI HP11 (+)) of dna sequence dna (as Fig. 8) of intestinal bacteria HP11 gene then, and its sequence is as follows:
Contain NdeI restriction enzyme sequence in this primer of T C C C A T A T G T C G C A A C A T A A C G A A A A G A A C.Synthetic in addition 3 ' end primer (HP11-XhoI-(-)), its sequence is as follows:
Contain XhoI restriction enzyme sequence in this primer of T T T C T C G A G G G C A G G A A T T T T G T C A A T C T T A G G.With bacillus coli DH 5 alpha (CCRC 51731) chromosomal DNA is sample, polymeric enzyme reaction condition amplification HP11 gene according to embodiment 1 (wherein changes into 2 minutes except that the extension time with primer, all the other conditions are all identical with embodiment 1), obtain dna fragmentation as the 2.0kb size of expection.
According to the method for embodiment 1, make up the pET20b/katHP11 expression plasmid, through restriction enzyme digestion and dna sequencing, determine that the 2.0kb dna fragmentation of being cloned into is the HP11 gene.This plasmid is preserved in American type culture collection on November 18th, 197, and preserving number is ATCC209470.Embodiment 5 catalatic expression
With pET20b, pET15b, pET20b/kat19, pET20b/katTG and pET20b/katHP11 expression plasmid difference transformed into escherichia coli BL21 (DE3) bacterial strain, when concussion is cultured to O.D.600=2.0 under 37 degree Celsius with 20 milliliters of LB/Amp nutrient solutions then, add 0.1mM isopropylthio-(IPTG) and induce the hydrogen peroxide expression of enzymes, collect thalline after 24 hours.Get 1.5 milliliters of bacterium liquid, centrifugal and outwell supernatant liquor, the 50mM sodium phosphate buffer (pH6.0) that adds 150 microlitres is in thalline, (its prescription is 0.1M dithiothreitol (DTT), 20%SDS, 0.08M Tris-Cl to add 2 times of electrophoresis protein dyestuff of 150 microlitres again, (pH 6.8), 15% glycerine and 0.06% tetrabromophenol sulfonphthalein), mix the back in 95 degree Celsius heating 5 minutes centrifugal 5 minutes down, get upper strata liquid 20 microlitres with 12% policapram gel (SDS-PAGE) electrophoretic analysis bacterial protein with 12000rpm.Analytical results is respectively pET15b as shown in Figure 9 from left to right, pET15b/kat19, RET20b, pET20b/kat19, pET20b/katTG, expressed catalatic situation behind RET20b/HP11 conversion BL2.1 (DE3) bacterial strain.By Fig. 9 result as can be known the amount of catalase account for 15～50% of bacterial protein, that is these host cells that contain the catalase gene expression vector all can be produced a large amount of catalases.The purifying of embodiment 6 subtilis catalases
BL21 (DE3) inoculation that the pET20b/kat19 expression plasmid is transformed in 100 milliliters of LB/Amp nutrient solutions, in 37 degree Celsius down concussion be cultured to O.D.600=2.0.Add 0.1mM IPTG and induced 24 hours, outwell nutrient solution after centrifugal, with 10 milliliters, the 50mM sodium phosphate buffer suspension thalline of pH6.4.Again with the broken thalline of ultrasonic oscillation, with 12000rpm centrifugal 15 minutes, after the precipitation bacterial chip takes out supernatant liquor again, add equivalent acetone down in mycetocyte liquid in four degrees celsius, mixed 30 minutes, outwell supernatant liquor again, 50mM sodium phosphate buffer dissolution precipitation thing with 10 milliliters pH6.4, be statically placed in the four degrees celsius 12 hours, and centrifugal 5 minutes again, took out upper strata liquid and finish purifying with 5000rpm.
In addition, to contain pET15b/kat19, pET20b/katTG, the BL of pET20b/katHP11 expression plasmid (DE3) thalline, be inoculated in 100 milliliters of LB/Amp nutrient solutions, when O.D.600=2.0 is cultivated in concussion under 37 degree Celsius, continue to cultivate 24 hours after adding 0.1mM IPTG, centrifugal, outwell nutrient solution, add 20 milliliters of I MAC-5 (20mM Tris HCl (pH7.9), 0.5M NaCl, 10% glycerine and 5mM imidazoles) behind the suspension thalline, again with the broken thalline of ultrasonic oscillation, centrifugal, the precipitation bacterial chip takes out upper strata liquid, with 2.5 milliliters Histidines in conjunction with affinity chromatography agent (His-Bind resin, Novogen company) purifying catalase, its purification step are fully according to institute of Novogen company proposed steps (pET His T og
TMSystemProtocols, Novogen company).
Protein and activity measurement method are as follows: the mensuration of protein mass is to be standard substance with bovine serum albumin (Bovineserum albumin), measures with quantification of protein reagent and method that Bio-RAD company produces.It is in the 50mM sodium phosphate buffer of 25 degree pH7.0 Celsius that catalase activity is measured, and so that hydrogen peroxide is by the zymolytic speed of hydrogen peroxide in the UV240nm measurement solution, the initial concentration of hydrogen peroxide is 20mM in the damping fluid, and distance is 20 seconds then during measurement.Per unit activity (U) is defined as per minute and decomposes 1 micromolar hydrogen peroxide.
Measure purified various catalatic amount and activity according to aforesaid method, and respectively get 5 micrograms with 12% its purity of policapram gel electrophoresis analysis, the result as shown in figure 10, the right side is followed successively by protein standard substance, pET15b/kat19, pET20b/kat19 by a left side, pET20b/katTG, the catalase that the pET20b/katHP11 expression plasmid is expressed, its purity all reaches 95%.The specific activity of these catalases is as follows:
Expression plasmid specific activity (U/ microgram) pET15b/kat19 18-22pET20b/kat19 18-22pET20b/katTG 30-40pET20b/katHP11 10-14
As seen from the above table, the prepared various catalases of the present invention all have excellent activity, and all more commercially available aspergillus niger catalase of its specific activity is that good (according to the catalogue of Merck company, the aspergillus niger catalase is about the 5U/ microgram; And according to the catalogue of SIGMA company, the aspergillus niger catalase then is about the 4-8U/ microgram.) embodiment 7 decomposes the effect of contained hydrogen peroxide in the contact lens disinfection water
General commercially available contact lens disinfecting solutions composition contains 3% (w/v) superoxol, its sterilisation step is contact lens to be soaked to place 10 milliliter of 3% (w/v) aqueous hydrogen peroxide solution sterilization after about 20 minutes earlier, contact lens are contained in the catalatic aqueous solution by taking out and be positioned over 10 milliliters in the aqueous hydrogen peroxide solution, or catalase added in the aqueous hydrogen peroxide solution, take out contact lens after about 10 to 20 minutes and just can wear after the normal saline solution rinse.Effectiveness for hydrogen peroxide in the test catalase decomposition of the present invention contact lens disinfection water, each 50 microgram of the various subtilis catalases that the inventive method is prepared add in 3% (w/v) aqueous hydrogen peroxide solution, and finding only needed 5 minutes just can effectively decompose its hydrogen peroxide to being lower than 0.02% (w/v).It is good that its effect shows more commercially available person.Embodiment 8 solids compositions of the present invention
Prepare a stratiform lozenge, it has a core body, postpones releasing layer coated with one outward.This stratiform lozenge composed as follows:
Core body
89.4 milligrams in sodium-chlor
12.5 milligrams of Sodium phosphate dibasics (anhydrous)
0.87 milligram of phosphoric acid (anhydrous) sodium dihydrogen monohydrate
1.05 milligrams of polyoxyethylene glycol (molecular weight about 3350)
Freeze dried catalase of the present invention 7020 international unit
Coat
8 milligrams of embodiment of Vltra tears, 9 liquid composites of the present invention
Prepare the present composition of two unitary doses (10 milliliters), it is composed as follows:
Sodium-chlor 0.85%
Sodium phosphate dibasic (heptahydrate) 0.402%
SODIUM PHOSPHATE, MONOBASIC monohydrate 0.091%
Stretch disodium ethylene diamine tetraacetate 0.100%
The catalase of the present invention of liquid
(1)260 international unit/milliliters
Pure water is an amount of
(1)The glycerine and 10% ethanol that contain 35-40% (weight ratio) in the liquid hydrogen peroxide enzyme.
Claims (20)
1. catalase gene, it has following nucleotide sequence:
10 20 30 40 50 60ATGAGTTCAA ATAAACTGAC AACTAGCTGG GGAGCACCTG TTGGCGATAA CCAAAACTCG
70 80 90 100 110 120ATAACGGCCG GCAATCCTGG CCCGACATTA ATCCAAGACG TACATCTTAT CGAAAAATTA
130 140 150 160 170 180GCACACTTCA ATAGAGAACG TGTCCCAGAA CGTGTTGTCC ATGCGAAAGG CGCTGGTGCG
190 200 210 220 230 240CACGGCTATT TCGAAGTAAC AAACGATATG TCGAAATACA CAAAAGCGAA AGTGTTTAAC
250 260 270 280 290 300GGTGTTGGCA AACGCACGCC TGTATTCGTC CGCTTCTCTA CTGTCGCCGG TGAATTGGGA
310 320 330 340 350 360TCTGCGGATA CAGTCCGCGA CCCGCGCGGT TTTGCCGTCA AATTTTATAC CGAAGAAGGA
370 380 390 400 410 420AACTATGACA TCGTTGGCAA CAACACACCG ATTTTCTTCA TTCGTGATGC GATTAAATTC
430 440 450 460 470 480TCGGATTTTA TCCATACACA AAAACGCGAC CCGCGCACCC ATTTGATTTA TCCGACAGCA
490 500 510 520 530 540ATGTGGGATT TCTTGTCTTT ATCTCCGGAA TCTTTGCACC AAGTCACTTA TTTATTCGGG
550 560 570 580 590 600GATCGCGGCA TCCCATTGAC ATACCGCCAT ATGAACGGAT ACGGAAGCCA TACATTCAAA
610 620 630 640 650 660TGGGTGAATG AAAAAGGCGA AGCGGTATGG GTAAAATACC ACTTTAAAAC AAACCAAGGC
670 680 690 700 710 720GTGAAAAACA TGGATCCGGA ACTAGCGGTT AAAATCGCCG GAGAAAATCC GGATTACCAT
730 740 750 760 770 780ACGGAAGATT TATATAACGC CATCGAAAAA GGCGACTATC CATCTTGGAC ATTATATGTG
790 800 810 820 830 840CAAATTATGC CGTTAGAAGA CGCAAAAACA TACCGTTTCA ATCCATTTGA TGTCACAAAA
850 860 870 880 890 900GTTTGGTCAC ATAAAGATTA TCCGTTAATT GAAGTCGGCC GTATGGTATT AAACCGCAAT
910 920 930 940 950 960CCAGAAAATT ATTTTGCCGA AGTCGAACAA GCGACATTCT CTCCTGGAAA CCTTGTTCCT
970 980 990 1000 1010 1020GGCGTTGAAC CATCGCCGGA TAAAATCTTG CAAGCCCGTT TGTTCGCTTA TGCGGATGCG
1030 1040 1050 1060 1070 1080CACCGTTACC GCGTCGGCGT GAACCATAAC TTGCTTCCGA TCAACCGCCC GCGCGTGGAA
1090 1100 1110 1120 1130 1140GTAAACAATT ATCAACGTGA CGGCTTCATG CGCTTTGACA ATAATGGCGG CGGTTCGGTC
1150 1160 1170 1180 1190 1200AACTACGAAC CAAACAGCTT CGGCGGACCG ACAGAAGTGC CAGAACATAA AACGACCCCA
1210 1220 1230 1240 1250 1260TTCCCGGTAT CCGGCGTGGC AGAAAGCGTG CCATATGACG ACGATGATCA TTATACGCAA
1270 1280 1290 1300 1310 1320GCAGGCGACT TATACCGTCT CATGAGCGAA GAAGAAAAAG CGCGCCTTGT GAAAAACATT
1330 1340 1350 1360 1370 1380GTCGAATCAT TGAAACAAGT AACAAAAGAA GAAATTAAAC TTCGCCAAAT CCGCCACTTC
1390 1400 1410 1420 1430 1440TACAAAGCAG ACCCTGACTA CGGCCGCCGC GTTGCCGAAG GTCTTGGATT GCCGATTAAA
1,450 1,460 1,470 1,480 1490 1500AAAGATTCT and degenerate sequences thereof.
2. catalase, it has following aminoacid sequence
10 20 30 40 50 60MSSNKLTTSW GAPVGDNQNS ITAGNPGPTL IQDVHLIEKL AHFNRERVPE RVVHAKGAGA
70 80 90 100 110 120HGYFEVTNDM SKYTKAKVFN GVGKRTPVFV RFSTVAGELG SADTVRDPRG FAVKFYTEEG
130 140 150 160 170 180NYDIVGNNTP IFFIRDAIKF SDFIHTQKPD PRTHLIYPTA MWDFLSLSPE SLHQVTYLFG
190 200 210 220 230 240DRGIPLTYRH MNGYGSHTFK WVNEKGFAVW VKYHFKTNQG VKNMDPFLAV KLAGENPDYH
250 260 270 280 290 300TEDLYNAIEK GDYPSWTLYV QIMPLEDAKP YPFNPFDVTK VWSHKDYPLI EVGRMVLNPN
310 320 330 340 350 360PENYFAEVEQ ATFSPGNLVP GVEPSPDKIL QARLFAYADA HRYRVGVNHN LLPINRPRVE
370 380 390 400 410 420VNNYQRDGFM RFDNNGGGSV NYFPNSFGGP TEVPEHKTTP FPVSGVAESV PYDDDCHYTQ
430 440 450 460 470 480AGDLYRLMSE EEKARLVKNI VESLKQVTKE ETKLRQIRHF YKADPDYGRR VAEGLGLPIK
490 500 510 520 530 540KDS....... .......... .......... .......... .......... ..........。
3. one kind is utilized genetic engineering technique to prepare microbe-derived catalatic method, and it comprises:
(a) catalase gene according to claim 1 is embedded in the expression vector that contains suitable transcripting promoter with construction recombination plasmid;
(b) with this recombinant plasmid transformed in appropriate host cell;
(c) express this transformant of cultivation under the condition of catalase gene in being fit to this transformant; And
(d) the expressed hydrogen peroxide zymoprotein of purifying.
4. method according to claim 3, wherein this host cell is selected from bacterium, yeast, mammalian cell and insect cell.
5. method according to claim 4, wherein this host cell is a bacterium.
6. method according to claim 5, wherein this host cell is intestinal bacteria.
7. method according to claim 3 wherein is with Histidine affinity chromatography or acetone precipitation expressed hydrogen peroxide zymoprotein to be given purifying.
8. catalatic recombinant plasmid of expressing the thermotolerance genus bacillus, this recombinant plasmid are to utilize genetic engineering technique that catalase gene according to claim 1 is embedded in the expression vector.
9. recombinant plasmid according to claim 8, wherein expression vector is the expression vector that is selected from bacterium, yeast, mammalian cell and insect cell expression system.
10. recombinant plasmid according to claim 9, wherein expression vector is the expression vector of bacterial expression system.
11. recombinant plasmid according to claim 10, wherein expression vector is the expression vector of escherichia expression system.
12. recombinant plasmid according to claim 8, it is pET20b/katTG (ATCC209468).
13. express catalatic transformant according to claim 2 for one kind, it is each a recombinant plasmid institute transformed host cells in according to Claim 8-12.
14. according to the transformant of claim 13, wherein this host cell is selected from bacterium, yeast, mammalian cell and insect cell.
15. according to the transformant of claim 14, wherein this host cell is a bacterium.
16. according to the transformant of claim 15, wherein this host cell is intestinal bacteria.
17. according to the transformant of claim 16, wherein these intestinal bacteria are e. coli bl21 (DE3).
18. according to the transformant of claim 17, wherein this intestinal bacteria 35BL21 (DE3) transforms through pET20b/katTG (ATCC209468) recombinant plasmid.
19. a composition that is used for decomposing remaining thimerosal hydrogen peroxide on the contact lens, it comprises the catalase according to claim 2 of significant quantity.
20. according to the composition of claim 19, it is to be solution or solid state.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97120386A CN1089111C (en) | 1997-12-11 | 1997-12-11 | New catalase, its gene and composite containing it, and its preparation method |
| US09/027,166 US6022721A (en) | 1997-01-03 | 1998-02-20 | Catalase, the gene thereof and composition comprising the same, and process for preparing catalase using genetic engineering technology |
| CA 2228394 CA2228394C (en) | 1997-12-11 | 1998-02-25 | Novel catalase, the gene thereof and composition comprising the same, and process for preparing catalase using genetic engineering technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97120386A CN1089111C (en) | 1997-12-11 | 1997-12-11 | New catalase, its gene and composite containing it, and its preparation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1219588A CN1219588A (en) | 1999-06-16 |
| CN1089111C true CN1089111C (en) | 2002-08-14 |
Family
ID=5175932
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN97120386A Expired - Fee Related CN1089111C (en) | 1997-01-03 | 1997-12-11 | New catalase, its gene and composite containing it, and its preparation method |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1089111C (en) |
| CA (1) | CA2228394C (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1300842A (en) * | 1999-12-21 | 2001-06-27 | 复旦大学 | Polypeptide-hydrogen peroxidase 10 and polynucleotide for coding this polypeptide |
| CN104212820B (en) * | 2014-09-15 | 2016-09-21 | 青岛蔚蓝生物集团有限公司 | A kind of enzyme with catalase activity and encoding gene thereof |
| CN104312990A (en) * | 2014-09-19 | 2015-01-28 | 中国科学院南海海洋研究所 | Catalase, coding gene and application thereof |
| CN111363754A (en) * | 2020-04-07 | 2020-07-03 | 上海海洋大学 | Recombinant low-temperature catalase and recombinant vector and engineering bacteria thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6317693A (en) * | 1986-07-09 | 1988-01-25 | Mitsui Toatsu Chem Inc | Human catalase gene, chimera dna containing said gene and production of catalase using said chimera dna |
| JPS6486879A (en) * | 1987-09-30 | 1989-03-31 | Mitsubishi Chem Ind | Dna fragment |
| WO1993018166A2 (en) * | 1992-03-04 | 1993-09-16 | Genencor International, Inc. | PRODUCTION OF $i(ASPERGILLUS NIGER CATALASE-R) |
| WO1996034962A1 (en) * | 1995-05-05 | 1996-11-07 | Novo Nordisk Biotech, Inc. | Scytalidium catalase gene |
-
1997
- 1997-12-11 CN CN97120386A patent/CN1089111C/en not_active Expired - Fee Related
-
1998
- 1998-02-25 CA CA 2228394 patent/CA2228394C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6317693A (en) * | 1986-07-09 | 1988-01-25 | Mitsui Toatsu Chem Inc | Human catalase gene, chimera dna containing said gene and production of catalase using said chimera dna |
| JPS6486879A (en) * | 1987-09-30 | 1989-03-31 | Mitsubishi Chem Ind | Dna fragment |
| WO1993018166A2 (en) * | 1992-03-04 | 1993-09-16 | Genencor International, Inc. | PRODUCTION OF $i(ASPERGILLUS NIGER CATALASE-R) |
| WO1996034962A1 (en) * | 1995-05-05 | 1996-11-07 | Novo Nordisk Biotech, Inc. | Scytalidium catalase gene |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2228394A1 (en) | 1999-06-11 |
| CA2228394C (en) | 2002-08-20 |
| CN1219588A (en) | 1999-06-16 |
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