CN116716281A - Creatine amidinohydrolase mutant with improved heat stability - Google Patents

Creatine amidinohydrolase mutant with improved heat stability Download PDF

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CN116716281A
CN116716281A CN202211447736.4A CN202211447736A CN116716281A CN 116716281 A CN116716281 A CN 116716281A CN 202211447736 A CN202211447736 A CN 202211447736A CN 116716281 A CN116716281 A CN 116716281A
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creatine
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hydrolase
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guanyl
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杨广宇
白雪
罗漫杰
徐灿
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Shanghai Hannover Biotechnology Co ltd
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Abstract

The application discloses a creatine amidino hydrolase mutant with improved thermal stability, and belongs to the technical field of enzyme engineering. The application obtains mutant enzyme with obviously improved thermal stability through consensus design without systematic development bias on creatine amidino hydrolase from alcaligenes. Compared with the half life of the wild type, the highest half life of the optimally combined mutant is improved by 2841 times, which shows that the stability of the mutant is obviously improved compared with the wild type.

Description

Creatine amidinohydrolase mutant with improved heat stability
The application is a divisional application of the following application: filing date: 8 months and 28 days 2020; application number: 202010886832.3; the application discloses a creatine amidinohydrolase mutant with improved heat stability.
Technical Field
The application belongs to the technical field of enzyme engineering, and particularly relates to a creatine amidino hydrolase mutant with improved thermal stability.
Background
Creatine amidinohydrolase is an essential enzyme for the enzymatic detection of creatinine content, which converts creatine into sarcosine and urea, further producing hydrogen peroxide which can be detected chemically. The enzyme mainly originates from microorganisms and has been widely used in industries such as medical diagnosis and organic synthesis.
Creatine amidinohydrolases are used industrially for determining the creatinine content and, in addition, are frequently used in clinical analysis for diagnosing creatinine content in serum and urine and kidney diseases which differ from creatinine content in healthy bodies. Creatinine is the final product of human creatine phosphate metabolism, and after being filtered by kidney, the creatinine enters urine from blood and is discharged out of the body. In general, serum creatinine normally ranges between 35 and 150 μm, but when there is a problem with renal or muscle function, the creatinine content rises to 1000 μm and creatinine content in blood and urine may reflect renal excretion. The most common methods used so far for detecting creatinine content are Jaffe chemical detection methods and enzymatic colorimetry. In contrast, the enzymatic detection method has been attracting attention due to the characteristics of higher sensitivity and selectivity. In the enzymatic detection method, a sample to be detected is continuously converted by creatine hydrolase, creatine guanyl hydrolase and sarcosine oxidase, and finally creatinine is degraded into hydrogen peroxide, and the concentration of the hydrogen peroxide is determined by colorimetric reaction under the catalysis of horseradish peroxidase, so that the purpose of detecting the creatinine content is achieved.
Therefore, in order to better apply creatine guanyl hydrolase to clinical creatinine detection, the creatine guanyl hydrolase mutant with improved thermal stability is obtained by utilizing a consensus design method, 21 amino acid mutation sites are obtained by screening by adopting a consensus method without systematic development bias based on the improvement of a traditional consensus method, and site-directed mutation is carried out on the amino acid mutation sites, so that mutant enzymes with obviously improved thermal stability are obtained, the defect that the existing creatine guanyl hydrolase has poor thermal stability is overcome, the requirement of being applied to reagents cannot be met, and a foundation is laid for widening the industrial application of the creatine guanyl hydrolase.
Disclosure of Invention
In order to better apply creatine guanyl hydrolase to clinical creatinine detection, the creatine guanyl hydrolase mutant with improved thermal stability is obtained by utilizing a consensus design method, 21 amino acid mutation sites are obtained by screening by adopting a consensus method without systematic development bias based on the improvement of a traditional consensus method, and site-directed mutation is carried out on the amino acid mutation sites, so that mutant enzymes with obviously improved thermal stability are obtained, the defect that the existing creatine guanyl hydrolase has poor thermal stability is overcome, the requirement of being applied to reagents cannot be met, and a foundation is laid for widening the industrial application of the creatine guanyl hydrolase.
The first object of the present application is to provide a mutant of creatine guanyl hydrolase having the amino acid sequence shown in the following (a 1) or (a 2):
(a1) A derivative protein with the same function as the protein of SEQ ID NO.1, wherein the amino acid sequence shown in SEQ ID NO.1 is substituted, deleted or added with one or more amino acids;
(a2) A derivative protein which exhibits at least 92% homology with the protein shown in SEQ ID No.1 by substitution of one or more amino acid residues at one or more positions of the amino acid sequence shown in SEQ ID No. 1.
Preferably, the mutant creatine guanyl hydrolase, the mutation site of the amino acid sequence shown in SEQ ID NO.1 comprises at least one of the following: bit 6, bit 17, bit 58, bit 108, bit 117, bit 165, bit 199, bit 251, bit 349, and bit 351.
Further preferred are mutants of creatine guanyl hydrolase, including single point mutants of any single point mutation site of L6P, D17V, G58D, F Y, T117P, Q165I, T199S, T251C, E349V, K351E on the amino acid sequence shown in SEQ ID NO. 1.
Further preferably, the creatine guanyl hydrolase enzyme mutant, the creatine amidinohydrolase mutant comprises L6P/D17V, D V/G58D, D17V/T251C, D17V/K351E, D V/T199S, D V/F108Y, D V/Y109F, D17V/Q165I, D V/E349V, D V/T199S/T251C, D17V/F108Y/T199S, D V/Y109F/T199S, D17V/T199S/K351E, L P/D17V/T199S, L6P/D17V/T199S/K351E, L P/D17V/F108Y/T199S, D V/T199S/L6P/, L6P/D17V/Y109F 199S/T251C, L P/D17V/F108Y/T199S/K E, L P/D17V/Y109F 199S/K351/T351P/T351S/T351F 9P/T351S/T351F/T-L6P/T351F/T-L1 on the amino acid sequence shown in SEQ ID NO. 1.
It is a second object of the present application to provide a gene encoding the creatine guanyl hydrolase mutant.
In one embodiment of the application, the gene comprises the nucleotide sequence of SEQ ID NO. 2.
It is a third object of the present application to provide a vector containing the gene.
It is a fourth object of the present application to provide cells expressing the mutants.
In one embodiment of the application, the cell is a fungal cell or a bacterial cell.
In one embodiment of the application, the cell is E.coli, yeast or Bacillus subtilis.
It is a fifth object of the present application to provide 32 mutants which improve the thermostability of creatine amidino hydrolase, comprising the steps of:
1. searching the NCBI database for the amino acid sequence of SEQ ID NO.1, deleting the repeated identical sequences, and selecting the amino acid sequence with the identity of more than 50% with the amino acid sequence of SEQ ID NO. 1;
2. then carrying out multi-sequence comparison through ClustalX2.1 software, finishing the residual amino acid sequence into fasta files, importing the fasta files into MEGA7.0 software, and constructing a Phylogenetic tree by utilizing NJ algorithm in a Phylogenetic module;
3. according to the branching distance of the phylogenetic tree, weight is introduced, consensus sequence is calculated through a python script, and mutation sites related to thermal stability are selected as L6P, D17V, P20T, V33L, C52N, G58D, W59F, D73T, F108Y, Y109F, T117P, L162A, V340L, Q165I, V362I, T199S, K166A, T251C, C331S, E349V and K351E by combining a homologous modeling structure.
In one embodiment of the application, the mutant is a mutation of the following sites of creatine amidino hydrolase with GenBank accession number BAA 88830.1:
(1) Leucine at position 6 of the amino acid sequence shown in SEQ ID NO.1 is replaced by proline, denoted L6P;
(2) The aspartic acid at position 17 of the amino acid sequence shown in SEQ ID NO.1 is replaced by valine and is marked as D17V;
(3) Glycine at position 58 of the amino acid sequence shown in SEQ ID NO.1 is substituted by aspartic acid, denoted G58D;
(4) Substitution of phenylalanine at position 108 of the amino acid sequence shown in SEQ ID NO.1 with tyrosine, denoted F108Y;
(5) The threonine at position 117 of the amino acid sequence shown in SEQ ID NO.1 is replaced by proline, denoted T117P.
(6) The 165 th glutamine of the amino acid sequence shown in SEQ ID NO.1 is replaced by isoleucine and is denoted as Q165I.
(7) Threonine 199 of the amino acid sequence shown in SEQ ID NO.1 is replaced with serine, denoted as T199S.
(8) Threonine 251 of the amino acid sequence shown in SEQ ID NO.1 is replaced by cysteine, denoted as T251C.
(9) The amino acid sequence shown in SEQ ID NO.1 has glutamic acid at position 349 replaced with valine, designated E349V.
(10) The 351 th lysine of the amino acid sequence shown in SEQ ID NO.1 is replaced by glutamic acid, denoted K351E. The technical scheme of the application has the following advantages:
1. the creatine amidino hydrolase mutants provided by the application comprise single-point mutants and combined mutants, and compared with the wild creatine amidino hydrolase (BAA 88830.1), the single-point mutants and the combined mutants have longer half lives at 55 ℃ and 57 ℃; in particular, the combination mutants showed a superposition of the thermal stability of the single point mutations with half-lives approximately 2841 times that of the wild-type creatine amidinohydrolase (BAA 88830.1). Based on the above, the creatine guanyl hydrolase mutant provided by the application has better thermal stability, and the creatine guanyl hydrolase mutant obtained by the construction method provided by the application has excellent thermal stability and catalytic activity when catalyzing creatine to generate sarcosine and urea at a higher temperature.
2. The creatine guanyl hydrolase (BAA 88830.1) gene engineering bacteria constructed by the application can efficiently express the creatine guanyl hydrolase mutant, and has the advantages of simple culture condition, short culture period, convenient purification of expression products and the like.
Detailed Description
Mutant nomenclature:
"amino acid substituted at the original amino acid position" is used to denote a mutant. Like L6P, the substitution of the amino acid at position 6 by Leu of the parent creatine amidino hydrolase with Pro is indicated, the numbering of the positions corresponding to the amino acid sequence of the parent creatine amidino hydrolase.
Example 1: construction of single point creatine amidinohydrolase (BAA 88830.1) mutants
Wild-type creatine guanylate hydrolase plasmid Pany1-CR-AF-WT was deposited from the laboratory and single point creatine guanylate hydrolase mutants were constructed by whole plasmid PCR methods. The details are as follows: using Pany1-CR-AF-WT as a template, the upstream and downstream primers for each mutation site are listed in Table 1, and are named in the format of "mutation site substitution amino acids", respectively. A round of PCR amplification was performed using the high fidelity DNA polymerase PrimeSTAR HS DNA Polymerase kit in order to obtain a recombinant plasmid containing the mutant. The reaction system is shown in Table 2, and PCR conditions: pre-denaturation: 95 ℃ for 4min; denaturation: 98 ℃ for 10s; annealing: 55 ℃ for 5s; extension: 72 ℃ for 6min; cycling 25 times; fully extend: and at 72℃for 10min.
TABLE 1 primer list
A round of PCR amplification was performed using the high fidelity DNA polymerase PrimeSTAR HS DNA Polymerase kit in order to obtain a recombinant plasmid containing the mutant. The reaction system is shown in Table 2, and PCR conditions: pre-denaturation: 95 ℃ for 4min; denaturation: 98 ℃ for 10s; annealing: 55 ℃ for 5s; extension: 72 ℃ for 6min; cycling 25 times; fully extend: and at 72℃for 10min.
TABLE 2 reaction System for first round PCR amplification
Example 2: construction of a mutant of the multipoint creatine amidinohydrolase (BAA 88830.1)
To further analyze the effect of different amino acid species at each site on the catalytic properties of the enzyme, reference was made to site-directed mutagenesis methods, which still employed full plasmid PCR techniques to obtain saturated mutant library genes, as follows: PCR amplification was performed in multiple rounds using the high fidelity DNA polymerase PrimeSTAR HS DNA Polymerase kit in order to obtain a recombinant plasmid containing the mutant. 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 slightly modifying the description of the super competent kit, 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, single colony is taken and added into LB culture medium without resistance to be cultivated to OD 600 Between 0.5 and 0.6, preparing competent cells from the self-contained solution with the kit; fourthly, transforming and coating the strain on a LB solid culture medium plate containing Kan resistance, and culturing for 14 hours; finally, 5 single colonies are selected, target genes are amplified by adopting bacterial liquid PCR, and after agarose gel electrophoresis is adopted to identify target bands, the Suzhou Jin Weizhi sequencing is selected, so that engineering bacteria are confirmed.
Example 4: expression and purification of creatine amidinohydrolase mutant (BAA 88830.1) proteins
Inoculating engineering bacteria in glycerol pipe to kanamycin containing 100 μg/mL (Kan) + ) In a 4mL 2YT liquid medium tube at 3Culturing at 7deg.C and 220rpm for 11 hr; the 4mL of the bacterial suspension was then transferred to a strain containing 50. Mu.g/mL kanamycin (Kan + ) In a 1L shake flask of the 2YT liquid medium, culturing at 37 ℃ and 220rpm for about 3 hours to reach an OD600 of about 0.8; then, 0.1mM IPTG inducer was added, and the culture was induced at 25℃and 200rpm for 11-17 hours, in this example, 14 hours. And centrifuging the escherichia coli bacterial suspension obtained after induced expression, and carrying out one-step Ni-NTA affinity chromatography treatment to obtain the creatine amidino hydrolase protein with the purity of more than 95%.
Example 5: characterization of creatine amidinohydrolase mutants
The optimized wild-type creatine guanyl hydrolase (BAA 88830.1) and the various creatine guanyl hydrolase mutants provided in example 3 were subjected to a thermal stability test, and the creatine guanyl hydrolase activity assay method specifically comprises:
the activation-detection reaction of creatine amidinohydrolase is based on an enzyme-coupled catalytic system which catalyzes creatine to produce sarcosine and urea in the reaction system, and the sarcosine can react under the catalysis of Sarcosine Oxidase (SOX) and can simultaneously produce hydrogen peroxide (H) 2 O 2 ) Hydrogen peroxide can be reacted with tos (N-ethyl-N- (2-hydroxy-3-sulfopropyl) m-toluidine sodium salt) and 4-AP (4-aminoantipyrine ) under the catalysis of horseradish peroxidase to produce a purple compound. Thus, we evaluate the change in creatine amidinohydrolase activity, where unit activity is defined as the amount of enzyme that produces 1. Mu.M hydrogen peroxide per minute, by monitoring the change in UV absorbance at 555nm wavelength of a single enzymatic reaction system by a UV-2550 UV-visible spectrophotometer (Shimadzu).
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) Under the catalysis of sarcosine oxidase and horseradish peroxidase, the activity of creatine guanyl hydrolase is measured by an enzyme multistage coupling method, and the enzyme concentration of a sample to be detected is diluted to 1mg/ml by using 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 determined by taking 950. Mu.L of the substrate solution and adding 50. Mu.L of the sample enzyme 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), and the unit activity was defined as the amount of enzyme producing 1. Mu.M hydrogen peroxide per minute.
2) The concentration of the purified enzyme was diluted to 1.0mg/mL in phosphate buffer (0.1M, pH 7.5) and incubated at 55deg.C and 57deg.C for different times of 0min,5min,10min,15min,20min,30min, respectively, for pre-experiments of enzyme inactivation, estimating half-life t of wild-type creatine guanyl hydrolase (BAA 88830.1) 1/2 . Half-life calculation formula: t is t 1/2 = -ln (2)/k, k is the slope of the line of the natural log of the residual relative activity of the enzyme plotted against the heat treatment time. Experimental results show that the thermal stability of the single-point mutant and the combined mutant is obviously improved, as shown in Table 3:
as can be seen from Table 3, the creatine amidino hydrolase mutants provided by the application comprise single-point mutants and combined mutants, and the half lives of the wild-type creatine amidino hydrolase (BAA 88830.1) and the creatine amidino hydrolase mutants at 57 ℃ are detected, compared with the optimized wild-type creatine amidino hydrolase (BAA 88830.1), the thermal stability of the eight creatine amidino hydrolase mutants at 57 ℃ is obviously improved, and the eight creatine amidino hydrolase mutants are respectively: the specific results of T117P/T199S/T251C, F Y/T117P/T199S, T199S/D17V/K351E, L P/T117P/T199S, L P/T117P/F108Y/T199S, L P/D17V/T199S/T251C, L P/T117P/F108Y/T199S/T251C and L6P/T117P/T199S/T251C/K351E are shown in Table 3.
TABLE 3 characterization of the enzymatic Properties of wild-type creatine amidinohydrolases and their mutants
While the application has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the application as defined in the appended claims.

Claims (6)

1. The creatine guanyl hydrolase mutant is characterized by being an F108Y single-point mutant with the amino acid sequence shown in GenBank accession number BAA 88830.1.
2. A gene encoding the creatine guanyl hydrolase enzyme mutant of claim 1.
3. A recombinant plasmid comprising the gene of claim 2.
4. An immobilized enzyme or engineered bacterium comprising the creatine amidinohydrolase mutant of any one of claim 1.
5. The engineering bacterium according to claim 4, wherein the engineering bacterium comprises a fungal cell and a bacterial cell.
6. The engineering bacterium according to claim 5, wherein the engineering bacterium comprises escherichia coli, yeast or bacillus subtilis.
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