CN111979217A - Creatine amidino hydrolase with low mie constant and preparation method thereof - Google Patents
Creatine amidino hydrolase with low mie constant and preparation method thereof Download PDFInfo
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Abstract
The application discloses a creatine amidino hydrolase with low Michaelis constant in the first aspect, belonging to the technical field of enzyme engineering; in a second aspect, the application discloses a method for preparing low michaelis constant creatine amidinohydrolase, and in a third aspect, the application also discloses a recombinant plasmid containing related nucleotide sequence; the creatine amidinohydrolase is cloned and expressed in an escherichia coli engineering strain, the problem that the application detection limit is high due to high mie constant of the existing creatine amidinohydrolase is solved, and meanwhile, the creatine amidinohydrolase obtained by the method is high in enzyme activity, and a foundation is laid for widening the application of the creatine amidinohydrolase.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to creatine amidino hydrolase with a low mie constant and a preparation method thereof.
Background
Creatine amidinohydrolase is an essential enzyme for the enzymatic detection of creatinine content, which converts creatine into sarcosine and urea, further generating hydrogen peroxide that can be chemically detected. The enzyme is mainly derived from microorganisms and is widely applied to industries such as medical diagnosis, organic synthesis and the like at present.
Creatine amidinohydrolase is used in industrial determination of creatinine content and, in addition, is often used in clinical analyses for diagnosis of creatinine content in serum and urine and renal diseases different from that in healthy organisms. Creatinine is a final product of creatine phosphate metabolism applied to a human body, and enters urine from blood after being filtered by kidney, and is discharged out of the body. Generally, serum creatinine normally ranges from 35 to 150 μm, but when kidney function or muscle function is problematic, the creatinine level rises to 1000 μm, and the creatinine level in blood and urine can reflect the excretion function of the kidney. The most common methods for measuring creatinine content so far are Jaffe chemical detection and enzymatic colorimetric methods. In contrast, enzymatic assays are gaining increasing attention due to their higher sensitivity and selectivity. In the enzymatic detection method, a sample to be detected is continuously converted by creatinine hydrolase, creatine amidinohydrolase and sarcosine oxidase, finally creatinine is degraded into hydrogen peroxide, and the concentration of the hydrogen peroxide is determined by a colorimetric reaction under the catalysis of horseradish peroxidase, so that the aim of detecting the content of the creatinine is fulfilled.
The Michaelis constant, unit symbol is: km, the Michaelis constant, is used to determine the degree of affinity of the enzyme for the substrate. The small Km value indicates that half of the maximum reaction rate can be achieved with a very low substrate concentration, indicating that the enzyme has a high affinity for the substrate. The Michaelis constant can determine which substrates are natural substrates or optimal substrates for the enzyme (i.e., the substrate with the smallest Km value); the catalytic efficiency of the forward and reverse reactions can be determined, for example, if the forward and reverse directions of a reaction are catalyzed by the same enzyme, the catalytic efficiency of the reaction having a smaller Km value is higher.
Therefore, in order to improve the efficiency of detecting creatinine content and the affinity of creatine amidinohydrolase to reaction substrates, many studies have been conducted, for example, it was disclosed in 1990 by Yasuji KOYAMA that the Km value of Flavobacterium sp expressed in Escherichia coli reaches 40mM (Yasuji KOYAMA et al, agricultural. biol. chem.,54(6):1453-1457)1990), and the higher Km value results in higher detection limit of creatine amidinohydrolase in clinical and industrial applications, which is limited greatly at present.
Therefore, in order to better apply the creatine amidinohydrolase to clinical creatinine detection and industrial application, how to obtain the creatine amidinohydrolase with reduced michaelis constant, further improve the affinity of the creatine amidinohydrolase, and solve the defect of insufficient affinity of the existing creatine amidinohydrolase, so that the detection limit in application is higher, which becomes a prominent technical problem in the field at present.
Disclosure of Invention
In order to better apply the creatine amidino hydrolase to clinical creatinine detection, the invention analyzes and compares the prior creatine amidino hydrolase families through bioinformatics, develops a creatine amidino hydrolase with low Michaelis constant (Km), clones the creatine amidino hydrolase and realizes the expression of the creatine amidino hydrolase in an escherichia coli engineering strain, solves the problem of high detection limit in application due to high Michaelis constant of the prior creatine amidino hydrolase, and lays a foundation for widening the application of the creatine amidino hydrolase.
The first aspect of the invention provides creatine amidino hydrolase, and the amino acid sequence of the creatine amidino hydrolase is shown in SEQ ID NO. 1.
Further, the N-terminus of the amino acid sequence of creatine amidinohydrolase contains a histidine Tag (His-Tag).
Further, the nucleotide sequence of the amino acid sequence of the creatine amidino hydrolase is shown as SEQ ID NO. 2.
Further, the nucleotide sequence shown in SEQ ID NO.2 is derived from Alcaligenes.
Further, the creatine amidinohydrolase nucleotide sequence of Alcaligenes exists 60% homology with the creatine amidinohydrolase amino acid sequence from Pseudomonas, Flavobacterium and Arthrobacter.
The second aspect of the present invention provides a preparation method of creatine amidino hydrolase, which specifically comprises the following steps:
1) and (3) excavating creatine amidinohydrolase: obtaining a nucleotide sequence which is shown in SEQ ID NO.2 and codes creatine amidinohydrolase from NCBI database;
2) synthesizing a DNA sequence (with GenBank accession number being BAA88830.1) of creatine amidinohydrolase (afCR) by utilizing the nucleotide sequence in the step 1), constructing the DNA sequence on a pANY1 vector to obtain a plasmid (pANY1-afCR), quickly putting a stored escherichia coli BL21 competent expression strain on ice until the thallus is melted, adding 3-5 mu l of the plasmid (pANY1-afCR) into the competence, standing the mixture on the ice for 25min, putting the mixture into a water bath at 42 ℃ for a heat engine for 1min, and quickly putting the mixture on the ice for 2 min; adding 600 μ l of 2 XYT culture medium, placing in a shaker at 37 deg.C, recovering at 220rpm for 1h to obtain constructed expression strain, uniformly coating the constructed expression strain on 2 XYT culture medium containing kanamycin resistance, and culturing at 37 deg.C overnight; selecting a monoclonal with complete morphology, inoculating the monoclonal into 10ml of 2 XYT culture medium containing kanamycin resistance, placing the monoclonal in a shaking table, and culturing for 12 hours at 37 ℃ and 220 rpm;
3) inoculating the bacterial liquid of escherichia coli BL21 with good growth state after cloning in the step 2), culturing in a 2 XYT culture medium containing kanamycin resistance, and observing the state of the bacterial liquid, wherein when the OD value reaches 0.8-1.0, 0.1 per thousand IPTG is added into the bacterial liquid in an ultraclean workbench for induction culture; transferring the bacteria liquid after induction culture to a centrifugal barrel, balancing and placing in a refrigerated centrifuge, pouring out supernatant after centrifugation, and collecting bacteria for later experiments;
4) adding a proper amount of protein purification Binding-buffer into the thallus collected in the step 3) for resuspension, placing the resuspended bacterial liquid into an ultrasonic cell disruption instrument for ultrasonic disruption to obtain cell disruption liquid, placing the collected cell disruption liquid into a low-temperature refrigerated centrifuge for centrifugation, repeating twice to collect supernatant, collecting the centrifuged supernatant, and filtering the supernatant by using a filter membrane; adding the supernatant collected after low-temperature refrigerated centrifugation into the pretreated Ni column, collecting effluent liquid, and repeatedly passing through the column; washing the residual impure protein in the Ni column by using 10ml Binding buffer, eluting the protein by using 20mM and 400mM solution buffers containing imidazole with different concentrations respectively, and collecting effluent liquid; washing the Ni column with 10ml of Strip buffer, washing with 10ml of deionized water again, adding a Charge buffer regeneration Ni column, and storing at 4 ℃; and (3) filtering and desalting the collected protein liquid by using a concentration tube with the molecular cut-off of 30kDa, concentrating to obtain the creatine amidino hydrolase, and storing at 4 ℃ for subsequent determination of the activity and the stability of the protein.
Further, in step 2), the desired E.coli BL21 was stored in a-80 ℃ refrigerator.
Further, in step 3), 5ml of the bacterial solution of E.coli BL21 having a good growth state after cloning was inoculated to 300 ml.
Further, in step 3), the culture conditions in 2 × YT medium were: the culture temperature is 37 ℃, the rotation speed is 220rpm, and the culture period is 3 h.
Further, in step 3), the culture conditions for the induction culture are as follows: the culture temperature is 20 ℃, the rotation speed is 200rpm, and the culture period is 12-16 h.
Further, in the step 4), the precooling temperature of the ultrasonic crusher is 4 ℃, the ultrasonic crushing pressure is 600-.
Further, in the step 4), the centrifugal temperature of the low-temperature refrigerated centrifuge is 4 ℃, the rotating speed is 8000rpm, and the centrifugal time is 20 min.
Further, in step 4), the filter was a 0.22. mu.M filter.
Further, in the step 4), the pretreatment method of the Ni column is: after the Ni column is taken out, the Charge buffer in the column flows dry, and is firstly washed by 10ml of deionized water, and then 10ml of Binding buffer equilibrium column material is added.
The third purpose of the invention is to provide a vector containing the nucleotide sequence shown as SEQ ID NO. 2.
The technical scheme of the invention has the following advantages:
1. compared with the prior art, the creatine amidino hydrolase provided by the invention has the advantages that the Km value is obviously reduced, and the problem of higher detection limit in the detection process can be greatly reduced.
2. The constructed gene engineering bacteria of the creatine amidino hydrolase (BAA88830.1) can efficiently express the creatine amidino hydrolase, and has the advantages of simple culture conditions, short culture period and simple and convenient purification procedure of an expression product.
Drawings
FIG. 1 is a SDS-PAGE electrophoresis of the results of an afCR purification according to a preferred embodiment of the invention;
FIG. 2 is a schematic representation of a phylogenetic tree of members of the creatine amidinohydrolase family;
wherein, 1, supernatant after crushing, 2, precipitation after crushing, 3, supernatant hanging column effluent, 4.20mM imidazole eluting protein effluent, 5.400mM imidazole eluting protein effluent, 6, cleaning Ni column effluent and M.protein Marker.
Detailed Description
The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.
Example 1: and (3) excavating creatine amidinohydrolase:
the creatine amidinohydrolase is used as a key enzyme for detecting the creatinine content by an enzyme method, catalyzes creatine to be hydrolyzed into creatine and urea, and exists in various bacteria such as flavobacterium, pseudomonas, arthrobacter, bacillus and the like. In the present application, the creatine amidinohydrolase family sequence was analyzed using NCBI sequence database, and creatine amidinohydrolase gene derived from Alcaligenes was found and further investigated, which showed about 60% homology with creatine amidinohydrolase amino acid sequences derived from Pseudomonas, Flavobacterium and Arthrobacter, and phylogenetic trees of the creatine amidinohydrolase family members are shown in FIG. 2.
Example 2: cloning of creatine amidinohydrolase
Entrusted with Shanghai to synthesize DNA sequence (GenBank accession number is BAA88830.1) of creatine immomidyl hydrolase (afCR), the sequence is shown as SEQ ID NO.2, constructed on pANY1 vector, taken out Escherichia coli BL21 competent expression strain from-80 deg.C refrigerator, rapidly put on ice until thallus melts, taken out 3-5 μ l of above plasmid (pANY1-afCR) to add into competence, put on ice to stand for 25min, subsequently, put in 42 deg.C water bath to proceed thermal machine for 1min, rapidly put on ice for 2 min. Adding 600 μ l2 XYT medium, placing in a shaker at 37 deg.C, recovering at 220rpm for 1h, and uniformly spreading the constructed expression strain on the 2 XYT medium containing kanamycin resistance, and culturing at 37 deg.C overnight. The growth of colonies on the plates was observed, and morphologically intact single clones were picked, inoculated into 10ml of kanamycin-resistant 2 XYT medium, and cultured in a shaker at 37 ℃ and 220rpm for 12 hours.
Example 3: expression of creatine amidino hydrolase
Inoculating 5-8ml of the bacterial liquid with good growth state into 300ml of 2 XYT culture medium containing kanamycin resistance, culturing at 37 ℃ and 220rpm for 3h, observing the state of the bacterial liquid, measuring the OD value of the bacterial liquid, and adding 0.1 per mill IPTG into the bacterial liquid in a super clean bench for induction when the OD value reaches 0.8-1.0, wherein the temperature is 20 ℃, the rpm is 200rpm, and culturing for 12-16 h. Transferring the induced bacterial liquid to a 500ml centrifuge bucket, balancing and placing in a refrigerated centrifuge, centrifuging for 20min at the temperature of 4 ℃ and the rotating speed of 8000rpm, pouring out the supernatant, and collecting the thalli for later experiments.
Example 4: purification of creatine amidino hydrolase
The N end of the target protein contains histidine Tag (His-Tag), and Ni column affinity chromatography is adopted to purify the protein in subsequent protein purification experiments. Adding a proper amount of protein purification Binding-buffer into the collected thalli for resuspension, placing the resuspended bacterial liquid into an ultrasonic cell disruption instrument which is precooled to 4 ℃ for disruption, and disrupting the bacterial liquid under the pressure of 600-; centrifuging the collected cell disruption solution in a low-temperature refrigerated centrifuge at 4 deg.C and 8000rpm for 20min, collecting supernatant twice, and filtering the supernatant with 0.22 μ M filter membrane; pretreating the Ni column, taking out the Ni column, draining the Charge buffer in the column, washing with 10ml of deionized water, and adding 10ml of Binding buffer balance column material; adding the collected supernatant into the pretreated Ni column, collecting effluent liquid, and repeatedly passing through the column; washing the residual impure protein in the Ni column by 10ml Binding buffer, eluting the protein by 20mM of imidazole containing different concentrations and 400mM of Elution buffer respectively, and collecting the effluent liquid. Finally, washing the Ni column by using 10ml of Strip buffer, washing the Ni column by using 10ml of deionized water again, adding a proper amount of Charge buffer to regenerate the Ni column, and storing the Ni column at 4 ℃; and (3) filtering and desalting the collected protein liquid by using a concentration tube with the molecular cut-off of 30kDa, concentrating to a proper concentration, and storing at 4 ℃ for subsequent determination of the activity and the stability of the protein.
Example 5: SDS-PAGE electrophoresis
Collecting precipitate resuspension, supernatant, effluent, and effluent eluted by imidazole of different concentrations, each 15 μ L. Adding 5 μ L of 5 × protein electrophoresis loading buffer, treating in 100 deg.C water bath for 5-10min, and rapidly centrifuging at 12000rpm for 1 min. SDS-PAGE protein gel (12% separation gel and 5% concentrated gel) is prepared, 5 mu L of protein marker and 15 mu L of sample are taken for protein electrophoresis. And (3) placing the protein gel after the electrophoresis into Coomassie brilliant blue staining solution for microwave heating for staining, then oscillating and decolorizing in the decolorizing solution, observing a protein gel image, analyzing and determining an enzyme purification system, wherein the electrophoresis result is shown in figure 1.
Example 6: creatine amidino hydrolase activity determination
The activity detection reaction of creatine amidinohydrolase is based on an enzyme coupling catalytic system, wherein creatine is catalyzed in the reaction system to generate sarcosine and urea, the sarcosine can react under the catalysis of Sarcosine Oxidase (SOX), and hydrogen peroxide (H) can be generated at the same time2O2) Hydrogen peroxide can react with toss and 4-AP under the catalysis of horseradish peroxidase to produce purple compounds. Therefore, we evaluated the activity change of creatine-based hydrolase (afCR) by monitoring the amount of change in the UV absorption at a wavelength of 555nm in a single enzymatic reaction system by a UV-2550 UV-visible spectrophotometer (Shimadzu), where the unit activity is defined as the amount of enzyme producing 1. mu.M hydrogen peroxide per minute and the measurement result is 14 u/mg.
Example 7: comparison of Km values for Michaelis constants
The purified enzyme was diluted to 1.0mg/mL in phosphate buffer (0.1M, pH 7.5) and placed in substrate solutions of different concentrations (1mM, 3mM, 5mM, 8mM, 12mM, 16mM, 25mM, 35mM, 50mM) for enzyme activity determination, three replicates were required for each data determination, the data were fitted, and Km was calculated using the formula V ═ Vmax S)/(Km + S). The results of the experiment are shown in table 1.
TABLE 1 results of comparative experiments on Michaelis constants
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Sequence listing
<110> Hannov Biotechnology Limited of Shanghai
<120> creatine amidino hydrolase with low mie constant and preparation method thereof
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Ser Leu Glu Glu Gln Lys Leu Ile Arg Glu Gly Ala Arg Val Cys Asp
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Val Gly Gly Ala Ala Cys Ala Ala Ala Ile Lys Ala Gly Val Pro Glu
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His Glu Val Ala Ile Ala Thr Thr Asn Ala Met Ile Arg Glu Ile Ala
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gcaaagagaa ttggcattga atttgatcat gttaacctgg atttccgccg tcagctggaa 420
gaagcactgc ctggtgttga atttgtggat attagccagc cgagtatgtg gatgcgtacc 480
attaagagtc tggaagaaca gaaactgatt cgcgaaggtg cccgtgtttg cgatgttggt 540
ggtgctgctt gcgcagcagc tattaaggcc ggtgttccgg aacatgaagt tgcaattgcc 600
accaccaatg caatgattcg tgaaattgca aaaagttttc cgtttgtgga actgatggat 660
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Claims (10)
1. The creatine amidinohydrolase is characterized in that the amino acid sequence of the creatine amidinohydrolase is shown in SEQ ID No.1, and the nucleotide sequence for coding the amino acid sequence is shown in SEQ ID No. 2.
2. The creatine amidinohydrolase according to claim 1, wherein the nucleotide sequence set forth in SEQ ID No.2 is derived from alcaligenes sp.
3. A recombinant plasmid comprising the nucleotide sequence of claim 1.
4. A process for the preparation of the creatine amidinohydrolase according to claim 1, comprising the steps of:
1) mining creatine amidinohydrolase, and obtaining the nucleotide sequence which is shown in SEQ ID NO.2 and codes the creatine amidinohydrolase from NCBI database;
2) cloning creatine amidinohydrolase, synthesizing DNA sequence (BAA88830.1 with GenBank accession number) of creatine amidinohydrolase (afCR) by using the nucleotide sequence in the step 1), constructing the DNA sequence on pANY1 vector to obtain plasmid (pANY1-afCR), rapidly putting the stored escherichia coli BL21 competent expression strain on ice until the thallus is melted, taking 3-5 μ l of the plasmid (pANY1-afCR) to add into competence, standing on ice for 25min, then putting the strain in 42 ℃ water bath for thermomechanical 1min, and rapidly putting on ice for 2 min; adding 600 μ l of 2 XYT culture medium, placing in a shaker at 37 deg.C, recovering at 220rpm for 1h to obtain constructed expression strain, uniformly coating the constructed expression strain on 2 XYT culture medium containing kanamycin resistance, and culturing at 37 deg.C overnight; selecting a monoclonal with complete morphology, inoculating the monoclonal into 10ml of 2 XYT culture medium containing kanamycin resistance, placing the monoclonal in a shaking table, and culturing for 12 hours at 37 ℃ and 220 rpm;
3) inoculating the bacterial liquid of escherichia coli BL21 with good growth state after cloning in the step 2), culturing in a 2 XYT culture medium containing kanamycin resistance, and observing the state of the bacterial liquid, wherein when the OD value reaches 0.8-1.0, 0.1 per thousand IPTG is added into the bacterial liquid in an ultraclean workbench for induction culture; transferring the bacteria liquid after induction culture to a centrifugal barrel, balancing and placing in a refrigerated centrifuge, pouring out supernatant after centrifugation, and collecting bacteria for later experiments;
4) adding a proper amount of protein purification Binding-buffer into the thalli collected in the step 3) for resuspension, placing the bacterial liquid after the resuspension into an ultrasonic cell disruption instrument for ultrasonic disruption to obtain cell disruption liquid, placing the collected cell disruption liquid into a low-temperature freezing centrifuge for centrifugation, repeating twice to collect supernatant, collecting the centrifuged supernatant, and filtering the supernatant by using a filter membrane; adding the supernatant collected after low-temperature refrigerated centrifugation into the pretreated Ni column, collecting effluent liquid, and repeatedly passing through the column; washing the residual impure protein in the Ni column by using 10ml Binding buffer, eluting the protein by using 20mM and 400mM solution buffers containing imidazole with different concentrations respectively, and collecting effluent liquid; washing the Ni column with 10ml of Strip buffer, washing with 10ml of deionized water again, adding a Charge buffer regeneration Ni column, and storing at 4 ℃; and (3) filtering and desalting the collected protein liquid by using a concentration tube with the molecular cut-off of 30kDa, concentrating to obtain the creatine amidinohydrolase, and storing at 4 ℃.
5. The method according to claim 4, wherein in step 2), the Escherichia coli BL21 is stored in a refrigerator at-80 ℃.
6. The method according to claim 4, wherein the E.coli BL21 bacterial suspension having a good growth state after cloning in step 3) is inoculated from 5ml to 300 ml.
7. The method according to claim 4, wherein the culture conditions in the 2 XYT medium in step 3) are: the culture temperature is 37 ℃, the rotation speed is 220rpm, and the culture period is 3 h.
8. The preparation method according to claim 4, wherein in the step 4), the pre-cooling temperature of the ultrasonication device is 4 ℃, the ultrasonication pressure is 600 kpa, and the ultrasonication time is 2-3 min.
9. The method according to claim 4, wherein in the step 4), the low-temperature refrigerated centrifuge has a centrifugation temperature of 4 ℃, a rotation speed of 8000rpm, and a centrifugation time of 20 min.
10. The method according to claim 4, wherein in the step 4), the filter is a 0.22 μm filter.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000031245A1 (en) * | 1998-11-25 | 2000-06-02 | Kikkoman Corporation | Creatine amidinohydrolase and process for producing the same |
| US20030119084A1 (en) * | 2001-09-20 | 2003-06-26 | Zhixin Shao | Variants of Erwinia-type creatinase |
| CN107257855A (en) * | 2015-02-27 | 2017-10-17 | 雷迪奥米特医学公司 | Modified kreatinase |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000031245A1 (en) * | 1998-11-25 | 2000-06-02 | Kikkoman Corporation | Creatine amidinohydrolase and process for producing the same |
| US20030119084A1 (en) * | 2001-09-20 | 2003-06-26 | Zhixin Shao | Variants of Erwinia-type creatinase |
| US6958231B2 (en) * | 2001-09-20 | 2005-10-25 | Roche Diagnostics Operations, Inc. | Variants of Erwinia-type creatinase |
| CN107257855A (en) * | 2015-02-27 | 2017-10-17 | 雷迪奥米特医学公司 | Modified kreatinase |
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