CN115851681A - Creatine amidino hydrolase mutant with improved activity - Google Patents
Creatine amidino hydrolase mutant with improved activity Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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Abstract
The invention discloses a creatine amidino hydrolase mutant with improved activity, belonging to the technical field of enzyme engineering. The invention utilizes a consensus method without phylogenetic prejudice to analyze the sequence of the creatine amidinohydrolase, single-point mutants with obviously improved activity are obtained by screening and are subjected to site-directed mutation, and the mutant enzymes with improved activity, namely D17V/K351E, T199S/K351E, D17V/T199S/K351E and D17V/T117P/K351E, are obtained, and compared with wild type, the activity of the mutant enzymes is improved by about 2 times.
Description
The present application is a divisional application of the following applications: application date: 8, 28 days in 2020; application No.: 202010888788.X; the invention provides a creatine amidino hydrolase mutant with improved activity.
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a creatine amidino hydrolase mutant with improved activity.
Background
Creatine amidinohydrolase is an essential enzyme for the enzymatic detection of creatinine content, and it converts creatine into sarcosine and urea, further generating hydrogen peroxide which 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 human body, and enters urine from blood after being filtered by kidney, and is discharged out of body. Generally, serum creatinine normally ranges between 35 and 150 μm, but when kidney function or muscle function is compromised, creatinine levels rise to 1000 μm and creatinine levels in blood and urine can reflect renal excretion. The most common methods for measuring creatinine content so far are Jaffe chemical detection and enzymatic colorimetric methods. In contrast, enzymatic assays are increasingly receiving attention due to their high sensitivity and selectivity. In the enzymatic detection method, a sample to be detected is continuously converted by virtue of creatinine hydrolase, creatine amidinohydrolase and sarcosine oxidase, finally creatinine is degraded into hydrogen peroxide, and the concentration of the hydrogen peroxide is determined by virtue of a colorimetric reaction under the catalysis of horseradish peroxidase, so that the aim of detecting the content of the creatinine is fulfilled.
Therefore, in order to better apply the creatine amidino hydrolase to clinical creatinine detection, the invention adopts site-specific mutagenesis to obtain the mutant enzyme with obviously improved activity, solves the problem that the existing creatine amidino hydrolase has poor activity and cannot meet the requirement of being applied to a reagent, and lays a foundation for widening the industrial application of the creatine amidino hydrolase.
Disclosure of Invention
In order to better apply the creatine amidino hydrolase to clinical creatinine detection, the invention adopts site-specific mutagenesis to obtain the mutant enzyme with obviously improved activity, solves the problem that the existing creatine amidino hydrolase has poor activity and cannot meet the requirement of being applied to a reagent, and lays a foundation for widening the industrial application of the creatine amidino hydrolase.
The first purpose of the invention is to provide a creatine amidinohydrolase mutant, which is (a 1) or (a 2) as follows:
(a1) A derived protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.1 and has the same function with the protein shown in SEQ ID NO. 1;
(a2) A derivative protein which is obtained by substituting one or more amino acid residues for one or more positions of the amino acid sequence shown in SEQ ID NO.1 and shows at least 92% homology with the protein shown in SEQ ID NO. 1.
Preferably, the creatine amidino hydrolase mutant, the mutation site of the amino acid sequence shown in SEQ ID NO.1, comprises at least one of: 17 th, 58 th, 117 th, 199 th and 351 th bits.
Further preferably, the creatine amidinohydrolase mutant comprises a single point mutant of any one of the single point mutation sites of D17V, G58D, T117P, T199S and K351E in the amino acid sequence shown in SEQ ID NO. 1.
Further preferably, the creatine amidinohydrolase mutant comprises the amino acid sequence shown in SEQ ID NO.1, D17V/G58D, D17V/T117P, D17V/K351E, D17V/T199S, T199S/K351E, D17V/T199P/T199S, D17V/T117P/K351E.
It is a second object of the present invention to provide a gene encoding a creatine amidinohydrolase mutant.
In one embodiment of the invention, the gene comprises the nucleotide sequence shown in SEQ ID NO. 2.
The third purpose of the invention is to provide a vector containing the gene.
It is a fourth object of the invention to provide cells expressing said mutants.
In one embodiment of the invention, the cell comprises a fungal cell or a bacterial cell.
In one embodiment of the invention, the cell comprises Escherichia coli, yeast or Bacillus subtilis.
It is a fifth object of the present invention to provide a method for increasing creatine amidino hydrolase activity, comprising the steps of:
1. searching the amino acid sequence of SEQ ID NO.1 in an NCBI database, deleting the repeated identical sequence, and selecting the amino acid sequence with the amino acid sequence consistency of more than 50 percent with the amino acid sequence of SEQ ID NO. 1;
2. then, performing multi-sequence comparison through ClustalX2.1 software, arranging the residual amino acid sequences into fasta files, introducing the fasta files into MEGA7.0 software, and constructing a Phylogenetic tree by utilizing an NJ algorithm in a Phylogenetic module of the MEGA7.0 software;
3. introducing weight according to the branch distance of the phylogenetic tree, calculating consensus sequence through a python script, and screening mutation sites related to activity by combining a homologous modeling structure to obtain D17V, G58D, T117P, T199S and K351E.
The amino acids at 6, 17, 20, 52, 58, 73, 108, 166, 351, 33, 59, 109, 162, 117, 165, 199, 251, 349, 362, 340 and 331 of the wild-type creatine amidinohydrolase of the present invention, having GenBank accession BAA88830.1, are mutated.
The technical scheme of the invention has the following advantages:
1. compared with wild-type creatine amidinohydrolase (BAA 88830.1), the single-point mutant and the combined mutant have improved activities at 55 ℃ and 57 ℃, and the optimal activity of the mutant is about 1.6 times that of the wild-type creatine amidinohydrolase (BAA 88830.1). Based on the above, the creatine amidino hydrolase mutant provided by the invention has better activity, and shows excellent catalytic activity when catalyzing creatine to generate sarcosine and urea at higher temperature.
2. The constructed gene engineering bacteria of the creatine amidino hydrolase (BAA 88830.1) can efficiently express creatine amidino hydrolase mutants, and have the advantages of simple culture conditions, short culture period, convenient purification of expression products and the like.
Detailed Description
Mutant naming mode:
"amino acid substituted for the original amino acid position" is used to indicate the mutant. As in G58D, the amino acid at position 58 is replaced by Glu of the parent creatine amidinohydrolase to Asp, the numbering of the positions corresponding to the amino acid sequence of the parent creatine amidinohydrolase.
Example 1: construction of single-site creatine amidinohydrolase (BAA 88830.1) mutant
Wild-type creatine amidino hydrolase plasmid Pany1-CR-AF-WT was deposited in the laboratory, and single-site creatine amidino hydrolase mutants were constructed by the whole plasmid PCR method. The details are as follows: using Pany1-CR-AF-WT as a template, the primers upstream and downstream of each mutation site are shown in Table 1, and are named in the format of "substitution of amino acids by mutation sites", respectively. One round of PCR amplification was performed using the high fidelity DNA Polymerase PrimeSTAR HS DNA Polymerase kit in order to obtain a mutant-containing gene recombinant plasmid. The reaction system is shown in Table 2, and the PCR conditions are as follows: pre-denaturation: 4min at 95 ℃; denaturation: 10s at 98 ℃; annealing: 5s at 55 ℃; extension: 6min at 72 ℃; circulating for 25 times; fully extending: 10min at 72 ℃.
TABLE 2 primer Table
One round of PCR amplification was performed using the high fidelity DNA Polymerase PrimeSTAR HS DNA Polymerase kit in order to obtain a mutant-containing gene recombinant plasmid. The reaction system is shown in Table 2, and the PCR conditions are as follows: pre-denaturation: 4min at 95 ℃; denaturation: 10s at 98 ℃; annealing: 5s at 55 ℃; extension: 6min at 72 ℃; circulating for 25 times; fully extending: 10min at 72 ℃.
TABLE 2 reaction System for the first round of PCR amplification
Example 2: construction of multipoint creatine amidino hydrolase (BAA 88830.1) mutant
To further analyze the effect of different amino acid species at each site on the catalytic properties of the enzyme, the whole plasmid PCR technique was still used to obtain saturated mutant library genes, in reference to the site-directed mutagenesis method, as detailed below: PCR amplification was performed in multiple rounds using the high fidelity DNA Polymerase PrimeSTAR HS DNA Polymerase kit in order to obtain mutant-containing recombinant plasmids. The reaction system, PCR conditions and transformation conditions were the same as those of site-directed mutagenesis.
Example 3: construction of mutant engineering bacteria
The engineering bacteria are constructed by referring to the super competence kit instruction and slightly modifying, and the specific operation is as follows. First, it was confirmed that e.coli BL21 (DE 3) could not grow under Kan resistance; secondly, scribing, separating and activating the E.coli BL21 (DE 3); thirdly, taking a single colony, adding the single colony into an LB culture medium without resistance, and culturing the single colony to OD 600 Preparing competent cells from the solution of the kit between 0.5 and 0.6; fourthly, transforming and smearing the strain on an LB solid medium plate containing Kan resistance, and culturing for 14h; finally, 5 single colonies were picked and usedPCR amplification of the target gene of the bacterial liquid, agarose gel electrophoresis identification of the target band, selection of Suzhou Jinzhi sequencing and engineering bacteria confirmation.
Example 4: expression and purification of creatine amidino hydrolase mutant (BAA 88830.1) protein
Inoculating the engineering bacteria in the glycerin pipe into a 4mL 2YT liquid culture medium test tube containing 100 mug/mL kanamycin (Kan +) according to the volume ratio of 1%, and culturing for 11h at 37 ℃ and 220 rpm; then transferring the 4mL of the bacterial liquid to a 1L shake flask containing a 2YT liquid culture medium containing 50 ug/mL kanamycin (Kan +), and culturing at 37 ℃ and 220rpm for about 3h to make OD600 reach about 0.8; then 0.1mM IPTG inducer was added, and the mixture was subjected to induction culture at 25 ℃ and 200rpm for 11-17 hours, in this example for 14 hours. And (3) centrifuging the escherichia coli thallus suspension obtained after induction expression, and performing one-step Ni-NTA affinity chromatography treatment to obtain the creatine amidino hydrolase protein with the purity of more than 95%.
Example 5: enzyme activity determination of creatine amidino hydrolase mutant
The activity of the optimized wild-type creatine amidino hydrolase (BAA 88830.1) and various creatine amidino hydrolase mutants provided by the embodiment 3 is tested, and the method for measuring the activity of the creatine amidino hydrolase specifically comprises the following steps:
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 time 2 O 2 ) Hydrogen peroxide can react with toss (N-ethyl-N- (2-hydroxy-3-sulfopropyl) m-toluidine sodium salt) and 4-AP (4-aminoantipyrine ) under the catalysis of horseradish peroxidase to produce purple compounds. Therefore, we assessed the change in activity of creatine amidinohydrolase by monitoring the amount of change in UV absorption of a single enzymatic reaction system at a wavelength of 555nm by a UV-2550 UV-visible spectrophotometer (Shimadzu), where unit activity is defined as the amount of enzyme producing 1. Mu.M hydrogen peroxide per minute.
The enzyme reaction system is as follows: 0.5mM TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) M-toluidine sodium salt), 0.45mM 4-AP (4-aminoantipyrine ), 900U/L horseradish peroxidase, 0.1M potassium phosphate buffer (pH 7.5).
1) The activity of creatine amidinohydrolase is measured by an enzyme multi-stage coupling method under the catalytic action of sarcosine oxidase and horseradish peroxidase, and a to-be-detected sample enzyme concentration is diluted to 1mg/ml by using a phosphate buffer solution (0.1M, pH 7.5). The substrate solution was prepared from 500. Mu.M creatine, 0.45mM 4-AA (4-aminoantipyrine), 0.5mM TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline) and phosphate buffer (0.1M, pH 7.5), and incubated at 37 ℃. The activity of the enzyme was measured by taking 950. Mu.L of the substrate solution and adding 50. Mu.L of the enzyme of the sample to be tested thereto, and the change in the absorption of ultraviolet light at 555nm in the enzyme reaction system was monitored by a UV2550 spectrophotometer (Shimadzu), the unit activity being defined as the amount of the enzyme which generates 1. Mu.M hydrogen peroxide per minute.
The creatine amidino hydrolase mutants provided by the invention comprise single-site mutants and combined mutants (shown in table 3), and the activity of wild-type creatine amidino hydrolase (BAA 88830.1) and creatine amidino hydrolase mutants at 57 ℃ is determined, so that compared with the optimized wild-type creatine amidino hydrolase (BAA 88830.1), the activity of four creatine amidino hydrolase mutants at 57 ℃ is obviously improved, and the four creatine amidino hydrolase mutants are respectively: K351E/L6P/T199S/T251C, K351E/L6P/F108Y/Y109F, Q165I/L6P/F108Y/Y109F/E349V, and K351E/L6P/F108Y/Y109F/Q165I, as shown in Table 3.
TABLE 3 wild-type creatine amidino hydrolase and its mutant activity
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (6)
1. The creatine amidinohydrolase mutant is any one of combined mutants of Q165I/L6P/F108Y/Y109F/E349V and D17V/L6P/T199S of an amino acid sequence shown in GenBank accession number BAA 88830.1.
2. A gene encoding the creatine amidinohydrolase mutant according to claim 1.
3. A recombinant plasmid comprising the gene of claim 2.
4. An immobilized or engineered bacterium comprising the creatine amidinohydrolase mutant of any one of claims 1.
5. The engineered bacterium of claim 4, wherein said engineered bacterium comprises a fungal cell, a bacterial cell.
6. The engineered bacterium of claim 5, wherein said engineered bacterium comprises Escherichia coli, yeast or Bacillus subtilis.
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