CN103055825B - Sarcosine oxidase affinity medium and method for synthesizing and purifying sarcosine oxidase - Google Patents
Sarcosine oxidase affinity medium and method for synthesizing and purifying sarcosine oxidase Download PDFInfo
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Abstract
The invention discloses a sarcosine oxidase affinity medium and a method for synthesizing and purifying sarcosine oxidase, which belong to the technical field of biologic engineering. The sarcosine oxidase affinity medium disclosed by the invention is prepared by connecting a sarcosine oxidase affinity ligand with a chromatography medium; preferably, the sarcosine oxidase affinity ligand is a substrate analog 4-amino pyrrole-2-carboxylic acid, and the chromatography medium is Sepharose or poly-chitosan; the invention further provides a synthesis method of the sarcosine oxidase affinity medium, and a method for purifying the sarcosine oxidase in a large scale by using the sarcosine oxidase affinity medium. By utilizing the sarcosine oxidase affinity medium disclosed by the invention, the sarcosine oxidase can be rapidly separated and purified in a large scale, and a sarcosine oxidase pure product can be obtained under the condition of one step affinity chromatography; and the whole purification step is short in time, the protein and activity recovering rate is high, and thus being applicable to large-scale popularization and application.
Description
Technical field
A method for sarcosine oxidase affinity media and synthetic and large scale purification sarcosine oxidase, belongs to technical field of bioengineering, particularly diagnosis enzyme production technical field.
Background technology
Sarcosine oxidase (sarcosine oxidase, E.C.1.5.3.1) is called for short SOX, is the 3rd enzyme in creatinine katabolism approach, and catalytic substrate methyl amimoacetic acid generates glycine in specific manner.Microorganism is the main source of sarcosine oxidase, it is widely used in clinical diagnosis, measure the content of creatinine in serum by sarcosine oxidase coupling Creatininase, kreatinase, be an important references index that judges renal function, have a good application prospect at field of medicaments.
bacillus(bacillus),
streptomyces sp.(streptomycete),
corynebacterium(corynebacteria),
arthrobacter(arthrobacterium) and
pseudomonasmicroorganisms such as (pseudomonads) can produce sarcosine oxidase.At present, multiple sarcosine oxidase gene is cloned, and proceeds to
e.coligenetic engineering bacteriums such as (Escherichia coli) is expressed.But the purification procedures of sarcosine oxidase has generally included the ammonium sulfate precipitation of multistep, surfactant processing, heat treatment, ion-exchange chromatography, sieve chromatography etc.And too much purification step has caused the purifying time longer, the result that albumen and activity recovery are lower.
For heavy industrialization produce sarcosine oxidase, must reduce the step of separation and purification.Therefore, need to provide a kind of sarcosine oxidase separation method rapidly and efficiently.
Summary of the invention
The object of the invention is to have overcome above-mentioned shortcoming of the prior art, a kind of method of sarcosine oxidase affinity media and synthetic and large scale purification sarcosine oxidase is provided, utilize this sarcosine oxidase affinity media fast separating and purifying sarcosine oxidase on a large scale, can be in the situation that only using a step affinity chromatography, obtain sarcosine oxidase sterling, whole purification step elapsed time is short, and albumen and activity recovery are higher, are suitable for large-scale promotion application.
To achieve these goals, in a first aspect of the present invention, provide a kind of sarcosine oxidase affinity media, be characterized in, described sarcosine oxidase affinity media is formed by connecting by sarcosine oxidase affinity ligand and chromatography media; Sarcosine oxidase affinity ligand is connected as spacerarm by ethylenediamine with described chromatography media.
Preferably, described sarcosine oxidase affinity ligand is 4-amino-pyrroles-2-carboxylic acid, and described chromatography media is Sepharose or chitosan.Be that sarcosine oxidase affinity media is:
A, Sepharose-4-amino-pyrroles-2-carboxylic acid, or B, chitosan-4-amino-pyrroles-2-carboxylic acid.
In a second aspect of the present invention, a kind of synthetic method of above-mentioned sarcosine oxidase affinity media is provided, be characterized in:
Synthesizing of A, Sepharose-4-amino-pyrroles-2-carboxylic acid:
(1) medium activation: the deionized water of 10 times of amounts for 100 g Sepharose CL 4B-be designated as volume V-to wash, drain into wet pie; Be suspended in 50 mL buffer solutions, 40 ℃ of shaking tables shake 2.5 h, then pour in glass frosted funnel, under suction filtration, through the distilled water washing of each V volume, until cleaning solution pH is extremely neutral, then drains into wet pie, obtain the Sepharose CL 4B of activation;
Described buffer solution is the solution of 0.8 M NaOH, 20% methyl-sulfoxide and 10% epoxychloropropane, lower same;
(2) connect spacerarm ethylenediamine: the Sepharose CL 4B chromatography media of activation is suspended in 100 mL deionized waters, adds ethylenediamine according to the molar ratio of 1:2, and gel is 60 ℃ of constant temperature 12 h under agitation; By washed with de-ionized water; Gained amino-Sepharose CL 4 B are suspended in the buffer solution of 50mL again, 40 ℃ of shaking tables shake 2.5 h, then pour in glass frosted funnel, distilled water through each V volume under suction filtration washs, until cleaning solution pH is to neutral, drain into wetter pie, obtain amino-Sepharose CL 4B medium of activation;
(3) connect affinity ligand: take 1g 4-amino-pyrroles-2-carboxylic acid, be dissolved in 80 mL deionized waters, add activation amino-Sepharose CL 4B medium of having drained, at 50-60 ℃ of vibration 24 h, running audit pH in course of reaction, remains between 11-12 solution with 1M NaOH; React complete, fully wash by 500 mL deionized waters, drain, obtain Sepharose-4-amino-pyrroles-2-carboxylic acid;
Synthesizing of B, chitosan-4-amino-pyrroles-2-carboxylic acid
(I) medium activation: the deionized water of 10 times of amounts for 100 g chitosans-be designated as volume V-to wash, drain into wet pie; Be suspended in 50 mL buffer solutions, 40 ℃ of shaking tables shake 2.5 h, then pour in glass frosted funnel, under suction filtration, through the distilled water washing of each V volume, until cleaning solution pH is to neutral, are draining into wet pie, obtain the chitosan medium of activation;
(II) connects spacerarm ethylenediamine: the chitosan dielectric suspension of activation, in 500 mL 0.1M NaOH solution, adds 20 mL ethylenediamine solutions, and gel is 30 ℃ of constant temperature 12 h under agitation; By washed with de-ionized water; Gained NH
2-chitosan is suspended in 50 mL buffer solutions, and 40 ℃ of shaking tables shake 2.5 h, then pours in glass frosted funnel, distilled water through each V volume under suction filtration washs, until cleaning solution pH is to neutral, draining into wet pie, obtain amino-chitosan medium of activation;
(III) connects affinity ligand: the 4-amino-pyrroles-2-carboxylic acid that takes 1 g, be dissolved in 80 mL deionized waters, add activation amino-chitosan medium of having drained, 50-60 ℃ of vibration 24 h, running audit pH in course of reaction, remains between 11-12 solution with 1M NaOH; React complete, fully wash by 500 mL deionized waters, drain, obtain chitosan-4-amino-pyrroles-2-carboxylic acid.
In a third aspect of the present invention, a kind of method of utilizing above-mentioned sarcosine oxidase affinity media large scale purification sarcosine oxidase is provided, be characterized in, the flow of solution that contains sarcosine oxidase is carried out to affine absorption through described sarcosine oxidase affinity media, then adopt cleaning fluid to rinse the sarcosine oxidase affinity media after described absorption, finally with the sarcosine oxidase adsorbing on sarcosine oxidase affinity media described in elution buffer wash-out, thereby obtain the sarcosine oxidase of purifying;
(A) the described solution that contains sarcosine oxidase is to adopt the sarcosine oxidase that gene engineering method obtains to express liquid;
Described sarcosine oxidase express liquid be copy come from rose-red Thermophilic Bacteria (
thermomicrobium roseumdSM 5159) sarcosine oxidase genes of SEQ ID NO:1
,and by Escherichia coli (
escherichia coli) JM109 express obtain;
(B) condition of described affine absorption is pH 7.5 ~ 8.7, and electrical conductivity is 2 ~ 5 mS/cm;
(C) cleaning condition that adopts cleaning fluid to rinse described sarcosine oxidase affinity media is pH value 7.5 ~ 8.7, electrical conductivity 5 ~ 10 mS/cm;
(D) be pH value 8.5 ~ 9.5 with the elution requirement of the sarcosine oxidase adsorbing on sarcosine oxidase affinity media described in elution buffer wash-out, electrical conductivity 40 ~ 80 mS/cm.
Beneficial effect of the present invention:
1, the present invention, by adopting substrate analogue 4-amino-pyrroles-2-carboxylic acid as sarcosine oxidase affinity ligand, is connected synthetic sarcosine oxidase affinity media with chromatography media, and rapidly and efficiently purifying sarcosine oxidase, is suitable for large-scale promotion application.
2, the present invention significantly increases the purification efficiency of sarcosine oxidase by optimizing protein adsorption condition, has larger commercial Application potentiality, embodies larger economic benefit.
Accompanying drawing explanation
The structural representation of Fig. 1 sarcosine oxidase affinity ligand of the present invention.
Fig. 2 is take the pH value impact on sarcosine oxidase adsorbance during as sarcosine oxidase affinity media of Sepharose-4-amino-pyrroles-2-carboxylic acid.
Under Fig. 3 alkali condition, be the adsorbance of sarcosine oxidase affinity media Sepharose-4-amino-pyrroles-2-carboxylic acid to sarcosine oxidase.
Fig. 4 is take the elution buffer impact on the sarcosine oxidase rate of recovery during as sarcosine oxidase affinity media of Sepharose-4-amino-pyrroles-2-carboxylic acid.
Fig. 5 is take the pH value impact on sarcosine oxidase adsorbance during as sarcosine oxidase affinity media of chitosan-4-amino-pyrroles-2-carboxylic acid.
Under Fig. 6 alkali condition, be the adsorbance of sarcosine oxidase affinity media chitosan-4-amino-pyrroles-2-carboxylic acid to sarcosine oxidase.
Fig. 7 is take the elution buffer impact on the sarcosine oxidase rate of recovery during as sarcosine oxidase affinity media of chitosan-4-amino-pyrroles-2-carboxylic acid.
The specific embodiment
In order more clearly to understand technology contents of the present invention, describe in detail especially exemplified by following examples.
The preparation of following embodiment 1-2 and comparative example 1-2 sarcosine oxidase solution used: will come from rose-red Thermophilic Bacteria (
thermomicrobium roseum) the sarcosine oxidase gene (GenBank ID:NC_011959.1) of DSM 5159, this gene order is as shown in SEQ ID NO:1, and this gene is synthetic by Shanghai bioengineering Co., Ltd.Escherichia coli (
escherichia colithereby) express in JM109 and give expression to sarcosine oxidase, get the Escherichia coli that 20 mL cultivate
escherichia colijM109 bacterium liquid, adds the broken buffer solutions of 20 mL (pH ~ 8.0, electrical conductivity 2 ~ 5 mS/cm), ultrasonication, power 400 w, ultrasonic 5s interval 1 s, ultrasonic 99 circulations altogether.Get centrifugal 15 min of broken liquid 12000 rpm, get supernatant and use.
Synthetic and the application of embodiment 1:Sepharose-4-amino-pyrroles-2-carboxylic acid
Sepharose CL 4 B(100 g), with the deionized water washing of 10 times of volumes, drain into wet pie; Be suspended in 40 ℃ of shaking tables of 50 mL activation buffer solutions (0.8 M NaOH, 20% methyl-sulfoxide, 10% epoxychloropropane) and shake 2.5 h, then pour in glass frosted funnel, under suction filtration, through the distilled water washing of each 10 times of volumes, until cleaning solution pH is extremely neutral, then drain into wet pie.The Sepharose CL 4 B dielectric suspensions of activation, in 100 mL deionized waters, add ethylenediamine according to the molar ratio of 1:2, and gel is stirring (200 rpm) lower 60 ℃ of constant temperature 12 h.Use washed with de-ionized water.NH
2-Sepharose CL 4 B are suspended in (0.8 M NaOH in the buffer solution system of 50mL again, 20% methyl-sulfoxide, 10% epoxychloropropane) 40 ℃ of shaking tables shake 2.5 h, then pour in glass frosted funnel, distilled water through each 10 times of volumes under suction filtration washs, until cleaning solution pH is extremely neutral, then drain into wet pie.Take 1g 4-amino-pyrroles-2-carboxylic acid, be dissolved in 80 mL deionized waters, add activation amino-Sepharose medium of having drained, at 50-60 ℃ of vibration 24 h, running audit pH in course of reaction, remains between 11-12 solution with 1M NaOH.React complete, fully wash by 500 mL deionized waters, drain.
(1) determining of optimal adsorption pH value
Get sarcosine oxidase standard items, being diluted to concentration by deionized water is 600 μ g/mL, is divided into 6 groups, it is 4.7,6.3,7.4 that every group of solution regulates pH value, 8.0,8.7,9.6,10.7, volume 1 mL, adds respectively 0.01 g humid medium, 4 ℃ fully vibrate after 4 h, measure supernatant sarcosine oxidase concentration, the adsorbance of calculation medium, the pH value of selection optimal adsorption.
Initial sarcosine oxidase～0.6 mg/l fully vibrates after 4 h with medium under different pH values, and a large amount of sarcosine oxidases are adsorbed by affinity media, and the content in supernatant reduces, and adsorbance and pH value relation are shown in Fig. 2.Better adsorption conditions is: temperature is 4 ℃, and pH is 7.5-8.7, and electrical conductivity is 2 ~ 5 mS/cm.Optimal adsorption condition is, temperature is 4 ℃, and pH is 8.0, and electrical conductivity is 2 ~ 5 mS/cm.
(2) determining of medium maximal absorptive capacity
On the basis of optimizing in pH value, determine the maximal absorptive capacity of medium.Get sarcosine oxidase standard items, being diluted to concentration by deionized water is 6.311,7.872,9,751,11.869,12.612,13.709mg/mL, regulating pH value is 8.0, adds respectively 0.01 g humid medium, and 4 ℃ fully vibrate after 4 h, measure supernatant sarcosine oxidase concentration, the maximal absorptive capacity of calculation medium.The relation of adsorbance and supernatant SOX concentration, is shown in Fig. 3.Unit of account medium maximal absorptive capacity is 52.7 mg/g media.
(3) determining of optimum washing engaging condition
Cultivating on the basis of adsorption conditions and maximal absorptive capacity, investigating the impact of eluent ionic strength on wash-out result, the results are shown in Figure 4.As seen from the figure, better wash-out is adjusted to pH value 8.5 ~ 9.5, electrical conductivity 40 ~ 80 mS/cm; The suitableeest elution requirement is that pH value is 9.0, electrical conductivity 60 mS/cm.
Synthetic and the application of comparative example 1:Sepharose-3-amino-pyrrolidine
Sepharose CL 4 B(100 g), with the deionized water washing of 10 times of volumes, drain into wet pie; Be suspended in 40 ℃ of shaking tables of 50 ml activation buffer solutions (0.8 M NaOH, 20% methyl-sulfoxide, 10% epoxychloropropane) and shake 2.5 h, then pour in glass frosted funnel, under suction filtration, through the distilled water washing of each 10 times of volumes, until cleaning solution pH is to neutral, draining into wet pie.The Sepharose CL 4 B dielectric suspensions of activation, in 100 ml deionized waters, add ethylenediamine according to the molar ratio of 1:2, and gel is stirring (200 rpm) lower 60 ℃ of constant temperature 12 h.Use washed with de-ionized water.NH
2-Sepharose CL 4 B are suspended in (0.8 M NaOH in the buffer solution system of 50ml again, 20% methyl-sulfoxide, 10% epoxychloropropane) 40 ℃ of shaking tables shake 2.5 h, then pour in glass frosted funnel, distilled water through each 10 times of volumes under suction filtration washs, until cleaning solution pH is extremely neutral, then drain into wet pie.Take 1 g 3-amino-pyrrolidine, be dissolved in 80 ml deionized waters, add activation amino-Sepharose medium of having drained, at 50-60 ℃ of vibration 24 h, running audit pH in course of reaction, remains between 11-12 solution with 1M NaOH.React complete, fully wash by 500 ml deionized waters, drain.
Adopt Sepharose-3-amino-pyrrolidine to carry out the determining of optimal adsorption pH value, determining of medium maximal absorptive capacity and determining of optimum washing engaging condition, experimentation is with corresponding experimentation in embodiment 1, and result shows that Sepharose-3-amino-pyrrolidine is substantially without adsorption capacity.
Adopt Sepharose-3-amino-pyrrolidine to carry out the determining of optimal adsorption pH value, determining of medium maximal absorptive capacity and determining of optimum washing engaging condition, experimentation is with corresponding experimentation in embodiment 1, and result shows that Sepharose-3-amino-pyrrolidine is substantially without adsorption capacity.
Embodiment 2: the synthetic and application of chitosan-4-amino-pyrroles-2-carboxylic acid
(100 g) with the deionized water washing of 10 times of volumes, drains into wet pie for chitosan; Be suspended in 40 ℃ of shaking tables of 50 mL activation buffer solutions (0.8 M NaOH, 20% methyl-sulfoxide, 10% epoxychloropropane) and shake 2.5 h, then pour in glass frosted funnel, under suction filtration, through the distilled water washing of each 10 times of volumes, until cleaning solution pH is to neutral, draining into wet pie.The chitosan dielectric suspension of activation, in 500 mL 0.1M NaOH solution, adds 20 mL ethylenediamine solutions, and gel is stirring (200 rpm) lower 30 ℃ of constant temperature 12 h.Drain by washed with de-ionized water, obtain amino-chitosan.Amino-chitosan is suspended in (0.8 M NaOH in 50 ml buffer solution systems, 20% methyl-sulfoxide, 10% epoxychloropropane) 40 ℃ of shaking tables shake 2.5 h, then pour in glass frosted funnel, distilled water through each 10 times of volumes under suction filtration washs, until cleaning solution pH is to neutral, draining into wet pie, obtain amino-chitosan medium of activation.4-amino-pyrroles-2-the carboxylic acid that takes 1 g, is dissolved in 80 ml deionized waters, adds activation amino-chitosan medium of having drained, 50-60 ℃ of vibration 24 h, and running audit pH in course of reaction, remains between 11-12 solution with 1M NaOH.React complete, fully wash by 500 ml deionized waters, drain.
(1) determining of optimal adsorption pH value
Get sarcosine oxidase standard items, being diluted to concentration by deionized water is 600 μ g/mL, is divided into 6 groups, it is 4.7,6.3,7.4 that every group of solution regulates pH value, 7.8,8.7,9.6,10.7, volume 1 mL, adds respectively 0.01 g humid medium, 4 ℃ fully vibrate after 2 h, measure supernatant sarcosine oxidase concentration, the adsorbance of calculation medium, the pH value of selection optimal adsorption.
Initial sarcosine oxidase～0.6 mg/L fully vibrates after 4 h with medium under different pH values, and a large amount of sarcosine oxidases are adsorbed by affinity media, and the content in supernatant reduces, and adsorbance and pH value relation are shown in Fig. 5.Better adsorption conditions is, temperature is 4 ℃, and pH is 7.5-8.7, and electrical conductivity is 2 ~ 5 mS/cm.Optimal adsorption condition is, temperature is 4 ℃, and pH is 8.0, and electrical conductivity is 2 ~ 5 mS/cm.
(2) determining of medium maximal absorptive capacity
On the basis of optimizing in pH value, determine the maximal absorptive capacity of medium.Get sarcosine oxidase standard items, being diluted to concentration by deionized water is 6.311,7.872,9,751,11.869,12.612,13.709mg/mL, regulating pH value is 8.0, adds respectively 0.01 g humid medium, and 4 ℃ fully vibrate after 4 h, measure supernatant sarcosine oxidase concentration, the maximal absorptive capacity of calculation medium.Adsorbance and supernatant protein concentration relation, be shown in Fig. 6.Unit of account medium maximal absorptive capacity is 40.5 mg/g media.
(3) determining of optimum washing engaging condition
Cultivating on the basis of adsorption conditions and maximal absorptive capacity, investigating the impact of eluent ionic strength on wash-out result, the results are shown in Figure 7.As seen from the figure, better wash-out is adjusted to pH value 8.5 ~ 9.5, electrical conductivity 40 ~ 80 mS/cm; The suitableeest elution requirement is that pH value is 9.0, electrical conductivity 60 msS/cm.
Comparative example 2: the synthetic and application of chitosan-3-amino-pyrrolidine
(100 g) with the deionized water washing of 10 times of volumes, drains into wet pie for chitosan; Be suspended in 40 ℃ of shaking tables of 50 ml activation buffer solutions (0.8 M NaOH, 20% methyl-sulfoxide, 10% epoxychloropropane) and shake 2.5 h, then pour in glass frosted funnel, under suction filtration, through the distilled water washing of each 10 times of volumes, until cleaning solution pH is to neutral, draining into wet pie.The Sepharose CL 4 B dielectric suspensions of activation, in 100 ml deionized waters, add ethylenediamine according to the molar ratio of 1:2, and gel is stirring (200 rpm) lower 60 ℃ of constant temperature 12 h.Use washed with de-ionized water.NH
2-Sepharose CL 4 B are suspended in (0.8 M NaOH in the buffer solution system of 50ml again, 20% methyl-sulfoxide, 10% epoxychloropropane) 40 ℃ of shaking tables shake 2.5 h, then pour in glass frosted funnel, distilled water through each 10 times of volumes under suction filtration washs, until cleaning solution pH is extremely neutral, then drain into wet pie.Take 1 g 3-amino-pyrrolidine, be dissolved in 80 ml deionized waters, add activation amino-Sepharose medium of having drained, at 50-60 ℃ of vibration 24 h, running audit pH in course of reaction, remains between 11-12 solution with 1M NaOH.React complete, fully wash by 500 ml deionized waters, drain.
Adopt chitosan-3-amino-pyrrolidine to carry out the determining of optimal adsorption pH value, determining of medium maximal absorptive capacity and determining of optimum washing engaging condition, experimentation is with corresponding experimentation in embodiment 2, and result shows that chitosan-3-amino-pyrrolidine is substantially without adsorption capacity.
Adopt chitosan-3-amino-pyrrolidine to carry out the determining of optimal adsorption pH value, determining of medium maximal absorptive capacity and determining of optimum washing engaging condition, experimentation is with corresponding experimentation in embodiment 2, and result shows that chitosan-3-amino-pyrrolidine is substantially without adsorption capacity.
Therefore, the invention provides a kind of method of the separation and purification sarcosine oxidase with commercial Application potentiality, it uses sepharose-4-amino-pyrroles-2-carboxylic acid affinity chromatography one step separation and purification sarcosine oxidase, protein recovery is ~ 30%, activity recovery is ~ 90.8%, and specific activity is ~ 13 U/mg.Use shitosan-4-amino-pyrroles-2-carboxylic acid affinity chromatography one step separation and purification sarcosine oxidase, protein recovery is ~ 30%, and activity recovery is ~ 91%, and specific activity is ~ 14U/mg.
In this description, the present invention is described with reference to its specific embodiment.But, still can make various modifications and conversion obviously and not deviate from the spirit and scope of the present invention.Therefore, description and accompanying drawing are regarded in an illustrative, rather than a restrictive.
Sequence table
<210> SEQ ID NO: 1
<211> 1152
<212> DNA
<213> sarcosine oxidase gene
<400> 1
GAATTCATGC GGGAAGAGCG GGCGACAGTC ATCGTGGTCG GTCTGGGCAT CATGGGGTCG 60
GCGACGGCCT GGGCGCTCGC GCGGCGAGGC GTGCGCGTCG TGGGGCTGGA ACAGTATGCG 120
CCCTTTCACG CGCTGGGCTC GTCGCACGGG AAAACGCGGA TCATCCGCGA GGCCTACTTC 180
GAGTCGCCGG AGTACGTGCC GCTGGTCCAG CGCGCTTATG AGCTGTGGGA CGAGCTCGGC 240
GAGCGAACTG GACGGAGGCT TTTGCGGGTG ACCGGTGGTG TGAGTATCGG GCGGCTGGAT 300
AGCCCGTTCA TCGTCGGGGC GCGCGAGAGC GCGCAGCGGC ACGGGCTGGC GCACGAACTG 360
CTGGACGCGC AGGAAGCCCG AAAACGCTTT CCCGTGCTGG CGCTGCCGGA CGACTTCGTC 420
GCGCTCGTCG AAGGGCGAGC GGGGATCCTC TTCGCGGAAG AGTGCTGGCG AGCCTTCTGT 480
GAGGATGCGG TGCGGCATGG GGCGGAGCTG CGCTTCGGTG TGCGTGTGCA CGGGTTTGCG 540
CCGGACGGGG AAGGGATGAC GGTGGAGACC GAGAGCGGTC GACTGCGCGC GGATCGCGTG 600
GTGGTGACTG CCGGACCGTG GTCAACGACA TTGCTGGCTG ATTTGGGATT GCCGCTCGAG 660
GTGCGGCGGG TGCTCGTCGT CCACGTCCAG CCTGACGATC CGACTCGGTT CCGACCAGAG 720
GTGCTGCCGA TTTTCATCAT GGACGTTCCA GAAGGTGAGT ATTATGGTTT CCCCTTCTTG 780
CCGGATCAGG GGGTGAAGTT CGGTCGCCAC GACGATGGCG AGGTGTGCAC GCCGGAATCA 840
GTGCGGAGGA CGGTGACCGA CGATGAGGTG CGCTGGATGA CTGGAGTCCT CCAACGGTAT 900
CTTCCCGGAG CCGCTCGCGA GGTCCTGATG ACGGTGACCT GCCTGTATAC GATGACCCCA 960
GACAGTCATT TCATGATCGA CCGACATCCC GAGTGGCCAC AGGTGGTCTT CGCTGCTGGT 1020
TTTTCCGGAC ACGGGTTCAA GTTCGCGTCG GTGATGGGGG AGGCACTGGC TGATCTCGCA 1080
CTGGAAGGCG CCAGTCGGTT ACCGATCGGC TTTTTGAGCT TCCGCCGATT GGCGAAGACT 1140
CAGTAAGTCG AC 1152
Claims (3)
1. a sarcosine oxidase affinity media, is characterized in that, described sarcosine oxidase affinity media is formed by connecting by sarcosine oxidase affinity ligand and chromatography media; Sarcosine oxidase affinity ligand is connected as spacerarm by ethylenediamine with chromatography media;
Described sarcosine oxidase affinity ligand is 4-amino-pyrroles-2-carboxylic acid, and described chromatography media is Sepharose or chitosan;
Be that sarcosine oxidase affinity media is:
A, Sepharose-4-amino-pyrroles-2-carboxylic acid, or B, chitosan-4-amino-pyrroles-2-carboxylic acid.
2. the synthetic method of sarcosine oxidase affinity media described in claim 1, is characterized in that:
Synthesizing of A, Sepharose-4-amino-pyrroles-2-carboxylic acid:
(1) medium activation: the deionized water of 10 times of amounts for 100 g Sepharose CL 4B-be designated as volume V-to wash, drain into wet pie; Be suspended in 50 mL buffer solutions, 40 ℃ of shaking tables shake 2.5 h, then pour in glass frosted funnel, under suction filtration, through the distilled water washing of each V volume, until cleaning solution pH is extremely neutral, then drains into wet pie, obtain the Sepharose CL 4B of activation;
Described buffer solution is the solution of 0.8 M NaOH, 20% methyl-sulfoxide and 10% epoxychloropropane, lower same;
(2) connect spacerarm ethylenediamine: the Sepharose CL 4B chromatography media of activation is suspended in 100 mL deionized waters, adds ethylenediamine according to the molar ratio of 1:2, and gel is 60 ℃ of constant temperature 12 h under agitation; By washed with de-ionized water; Gained amino-Sepharose CL 4 B are suspended in the buffer solution of 50mL again, 40 ℃ of shaking tables shake 2.5 h, then pour in glass frosted funnel, distilled water through each V volume under suction filtration washs, until cleaning solution pH is to neutral, drain into wetter pie, obtain amino-Sepharose CL 4B medium of activation;
(3) connect affinity ligand: take 1g 4-amino-pyrroles-2-carboxylic acid, be dissolved in 80 mL deionized waters, add activation amino-Sepharose CL 4B medium of having drained, at 50-60 ℃ of vibration 24 h, running audit pH in course of reaction, remains between 11-12 solution with 1M NaOH; React complete, fully wash by 500 mL deionized waters, drain, obtain Sepharose-4-amino-pyrroles-2-carboxylic acid;
Synthesizing of B, chitosan-4-amino-pyrroles-2-carboxylic acid
(I) medium activation: the deionized water of 10 times of amounts for 100 g chitosans-be designated as volume V-to wash, drain into wet pie; Be suspended in 50 mL buffer solutions, 40 ℃ of shaking tables shake 2.5 h, then pour in glass frosted funnel, under suction filtration, through the distilled water washing of each V volume, until cleaning solution pH is extremely neutral, then drains into wet pie, obtain the chitosan medium of activation;
(II) connects spacerarm ethylenediamine: the chitosan dielectric suspension of activation, in 500 mL 0.1M NaOH solution, adds 20 mL ethylenediamine solutions, and gel is 30 ℃ of constant temperature 12 h under agitation; By washed with de-ionized water; Gained NH
2-chitosan is suspended in 50 mL buffer solutions, and 40 ℃ of shaking tables shake 2.5 h, then pours in glass frosted funnel, distilled water through each V volume under suction filtration washs, until cleaning solution pH is extremely neutral, then drains into wet pie, obtain amino-chitosan medium of activation;
(III) connects affinity ligand: the 4-amino-pyrroles-2-carboxylic acid that takes 1 g, be dissolved in 80 mL deionized waters, add activation amino-chitosan medium of having drained, 50-60 ℃ of vibration 24 h, running audit pH in course of reaction, remains between 11-12 solution with 1M NaOH; React complete, fully wash by 500 mL deionized waters, drain, obtain chitosan-4-amino-pyrroles-2-carboxylic acid.
3. one kind is utilized the method for the sarcosine oxidase affinity media large scale purification sarcosine oxidase described in claim 1, it is characterized in that, the flow of solution that contains sarcosine oxidase is carried out to affine absorption through described sarcosine oxidase affinity media, then adopt cleaning fluid to rinse the sarcosine oxidase affinity media after described absorption, finally with the sarcosine oxidase adsorbing on sarcosine oxidase affinity media described in elution buffer wash-out, thereby obtain the sarcosine oxidase of purifying;
(A) the described solution that contains sarcosine oxidase is to adopt the sarcosine oxidase that gene engineering method obtains to express liquid;
Described sarcosine oxidase express liquid be copy come from rose-red Thermophilic Bacteria (
thermomicrobium roseum) the sarcosine oxidase genes of SEQ ID NO:1 of DSM 5159
,and by Escherichia coli (
escherichia coli) JM109 express obtain;
(B) condition of described affine absorption is pH 7.5 ~ 8.7, and electrical conductivity is 2 ~ 5 mS/cm;
(C) cleaning condition that adopts cleaning fluid to rinse described sarcosine oxidase affinity media is pH value 7.5 ~ 8.7, electrical conductivity 5 ~ 10 mS/cm;
(D) be pH value 8.5 ~ 9.5 with the elution requirement of the sarcosine oxidase adsorbing on sarcosine oxidase affinity media described in elution buffer wash-out, electrical conductivity 40 ~ 80 mS/cm.
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| CN201310022527.XA CN103055825B (en) | 2013-01-21 | 2013-01-21 | Sarcosine oxidase affinity medium and method for synthesizing and purifying sarcosine oxidase |
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