CN112011528B - Creatine amidino hydrolase mutant with improved thermal stability - Google Patents

Creatine amidino hydrolase mutant with improved thermal stability Download PDF

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CN112011528B
CN112011528B CN202010886832.3A CN202010886832A CN112011528B CN 112011528 B CN112011528 B CN 112011528B CN 202010886832 A CN202010886832 A CN 202010886832A CN 112011528 B CN112011528 B CN 112011528B
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CN112011528A (en
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杨广宇
白雪
罗漫杰
宗迅成
徐灿
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Shanghai Hannover Biotechnology Co ltd
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Priority to CN202010886832.3A priority patent/CN112011528B/en
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Priority to CN202211447736.4A priority patent/CN116716281A/en
Priority to CN202211446981.3A priority patent/CN116410965A/en
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Priority to CN202211451065.9A priority patent/CN115975998A/en
Priority to CN202211447324.0A priority patent/CN115786314A/en
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Abstract

The invention discloses a creatine amidino hydrolase mutant with improved thermal stability, belonging to the technical field of enzyme engineering. The invention obtains the mutant enzyme with obviously improved thermal stability by carrying out consensus design without systematic development prejudice on creatine amidino hydrolase from the alcaligenes. Compared with the wild type half-life period, the maximum half-life period of the optimally combined mutant is increased by 2841 times, which indicates that the stability of the mutant is obviously improved compared with the wild type.

Description

Creatine amidino hydrolase mutant with improved thermal stability
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a creatine amidinohydrolase mutant with improved thermal stability.
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 analysis for diagnosis of creatinine content in serum and urine and kidney diseases different from creatinine content 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 reflect renal excretion. The most common methods for measuring creatinine content so far are Jaffe chemical assay and enzymatic colorimetric assay. In contrast, enzymatic assays are gaining 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 obtains creatine amidino hydrolase mutants with improved thermal stability by using a consensus design method, and the invention screens 21 amino acid mutation sites by using a consensus method without systematic developmental bias improved based on the traditional consensus method and performs site-specific mutation on the amino acid mutation sites to obtain the mutant enzymes with obviously improved thermal stability, thereby solving the problem that the existing creatine amidino hydrolase has poor thermal stability and cannot meet the requirements of being applied to reagents and laying 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 obtains creatine amidino hydrolase mutants with improved thermal stability by using a consensus design method, screens 21 amino acid mutation sites by using a consensus method which is improved based on a traditional consensus method and has no systematic developmental bias, performs site-specific mutation on the amino acid mutation sites, obtains the mutant enzymes with obviously improved thermal stability, solves the problem that the existing creatine amidino hydrolase has poor thermal stability and cannot meet the requirements of being applied to reagents, 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 amidino hydrolase mutant, the amino acid sequence of which is shown as (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: 6 th, 17 th, 58 th, 108 th, 117 th, 165 th, 199 th, 251 th, 349 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 L6P, D17V, G58D, F108Y, T117P, Q165I, T199S, T251C, E349V and K351E in the amino acid sequence shown in SEQ ID NO. 1.
Further preferably, the creatine amidinohydrolase mutants comprise the combination of L6P/D17V, D17V/G58D, D17V/T251C, D17V/K351E, D17V/T199S, D17V/F108Y, D17V/Y109F, D17V/Q165I, D17V/E349V, D17V/T199S/T251C, D17V/F108Y/T199S, D17V/Y109F/T199S, D17V/T199S/K351E, L6P/D17V/T199S/K351E, L6P/D17V/T199S, D17V/T199S, L17V/T199S/L6P/T199S/T351K 351E, L6P/D17V/T199S 108K 199S, T17V/T199S 108K 199S/T199S, T17V/T199S K108K 199S, T199S/T199S and the like on the amino acid sequence shown in SEQ ID NO. 1.
It is a second object of the present invention to provide a gene encoding the creatine amidinohydrolase mutant.
In one embodiment of the invention, the gene comprises the nucleotide sequence of 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 is a fungal cell or a bacterial cell.
In one embodiment of the invention, the cell is Escherichia coli, yeast or Bacillus subtilis.
The fifth objective of the invention is to provide 32 mutants capable of improving the thermal stability of creatine amidinohydrolase, which comprises the following steps:
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, importing the fasta files into MEGA7.0 software, and constructing a Phylogenetic tree by using an NJ algorithm in a Phylogenetic module of the MEGA7.0 software;
3. introducing weight according to the branch distance of a phylogenetic tree, calculating consensus sequence through a python script, and screening mutation sites related to thermal stability by combining a homologous modeling structure into L6P, D17V, P20T, V33L, C52N, G58D, W59F, D73T, F108Y, Y109F, T117P, L162A, V340L, Q165I, V362I, T199S, K166A, T251C, C331S, E349V and K351E.
In one embodiment of the invention, the mutant is a creatine amidinohydrolase with GenBank accession number BAA88830.1, mutated at the following sites:
(1) The leucine at the 6 th site of the amino acid sequence shown in SEQ ID NO.1 is replaced by proline, and is marked as L6P;
(2) The aspartic acid at the 17 th site of the amino acid sequence shown in SEQ ID NO.1 is replaced by valine, which is marked as D17V;
(3) The 58 th glycine of the amino acid sequence shown in SEQ ID NO.1 is replaced by aspartic acid and is marked as G58D;
(4) The 108 th phenylalanine of the amino acid sequence shown in SEQ ID NO.1 is substituted by tyrosine and is marked as F108Y;
(5) Threonine 117 of the amino acid sequence shown in SEQ ID NO.1 was substituted with proline and designated as T117P.
(6) The glutamine at position 165 of the amino acid sequence shown in SEQ ID NO.1 is substituted with isoleucine and is designated as Q165I.
(7) The amino acid sequence shown in SEQ ID No.1 has the amino acid sequence with threonine at position 199 substituted by serine, which is denoted as T199S.
(8) The 251 st threonine of the amino acid sequence shown in SEQ ID NO.1 is substituted by cysteine and is denoted as T251C.
(9) The glutamic acid at position 349 of the amino acid sequence shown in SEQ ID NO.1 is substituted by valine and is marked as E349V.
(10) The 351 th lysine of the amino acid sequence shown in SEQ ID NO.1 is replaced by glutamic acid and is marked as K351E. The technical scheme of the invention has the following advantages:
1. the creatine amidino hydrolase mutant provided by the invention comprises a single-point mutant and a combined mutant, and compared with wild creatine amidino hydrolase (BAA 88830.1), the single-point mutant and the combined mutant have longer half lives at 55 ℃ and 57 ℃; especially the combination mutant, shows the additive effect of single-point mutant thermal stability, and the half-life is about 2841 times of that of wild-type creatine amidinohydrolase (BAA 88830.1). Based on the above, the creatine amidinohydrolase mutant provided by the invention has better thermal stability, and the creatine amidinohydrolase mutant obtained by the construction method provided by the invention has excellent thermal stability and catalytic activity when catalyzing creatine to generate sarcosine and urea at higher temperature.
2. The constructed gene engineering bacteria of the creatine amidinohydrolase (BAA 88830.1) can efficiently express creatine amidinohydrolase mutants, and have the advantages of simple culture conditions, short culture period, convenient purification of expressed products and the like.
Detailed Description
Mutant naming mode:
the mutant is represented by "amino acid substituted at original amino acid position". E.g., L6P, indicates that the amino acid at position 6 is replaced by Leu to Pro of the parent creatine amidinohydrolase, 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 1 primer Table
Figure GDA0003807995950000041
One round of PCR amplification was performed using high fidelity DNA Polymerase PrimeSTAR HS DNA Polymerase kit to obtain a recombinant plasmid containing the mutant. 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
Figure GDA0003807995950000051
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, with reference to the site-directed mutagenesis method, as follows: 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 are picked, the target genes of the single colonies are amplified by adopting a bacterial liquid PCR, and after a target band is identified by agarose gel electrophoresis, the single colonies are selectedAnd (5) sending the sequence to the Jinwei Zhi of Suzhou to confirm the engineering bacteria.
Example 4: expression and purification of creatine amidine hydrolase mutant (BAA 88830.1) protein
Inoculating the engineering bacteria in the glycerin pipe to 100 mug/mL kanamycin (Kan) according to the volume ratio of 1 percent + ) Culturing in a 4mL 2YT liquid culture medium test tube at 37 ℃ and 220rpm for 11h; then, the 4mL of the bacterial suspension was transferred to a cell line containing 50. Mu.g/mL kanamycin (Kan) + ) 2YT liquid medium 1L flask, at 37 degrees C, 220rpm under about 3h culture, to make OD600 to 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: characterization of Properties of creatine amidinohydrolase mutant
The optimized wild-type creatine amidino hydrolase (BAA 88830.1) and various creatine amidino hydrolase mutants provided by the embodiment 3 are subjected to a thermal stability test, and the creatine amidino hydrolase activity determination method 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 be reacted 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 a purple compound. 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.
2) The concentration of the purified enzyme was diluted to 1.0mg/mL in a phosphate buffer (0.1M, pH 7.5) and incubated at 55 ℃ and 57 ℃ for various periods of 0min,5min,10min,20 min,30min to carry out an enzyme inactivation preliminary experiment, estimating the half-life t of wild-type creatine amidinohydrolase (BAA 88830.1) 1/2 . Half-life calculation formula: t is t 1/2 K is the slope of a line plotting the natural log value of the remaining relative activity of the enzyme versus the time of heat treatment. The experimental results show that the thermal stability of the single-point mutant and the combined mutant is obviously improved, and the results are shown in table 3:
as shown in table 3, the creatine amidinohydrolase mutants provided by the present invention include single-site mutants and combinatorial mutants, and it was found that by determining the half-lives of wild-type creatine amidinohydrolase (BAA 88830.1) and creatine amidinohydrolase mutants at 57 ℃, the thermal stabilities at 57 ℃ of the eight creatine amidinohydrolase mutants are significantly improved compared with the optimized wild-type creatine amidinohydrolase (BAA 88830.1), and the eight creatine amidinohydrolase mutants are: T117P/T199S/T251C, F108Y/T117P/T199S, T199S/D17V/K351E, L6P/T117P/T199S, L6P/T117P/F108Y/T199S, L6P/D17V/T199S/T251C, L6P/T117P/F108Y/T199S/T251C and L6P/T117P/T199S/T251C/K351E, with the specific results shown in Table 3.
TABLE 3 characterization of enzymatic Properties of wild-type creatine amidino hydrolase and its mutants
Figure GDA0003807995950000071
Figure GDA0003807995950000081
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.
Sequence listing
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<213> Artificial Sequence (Artificial Sequence)
<400> 5
gtgaaaaagt ttacagtccg tttagtgatg ccgaaatg 38
<210> 6
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggactgtaaa ctttttcacc attatgccat ttcatcac 38
<210> 7
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tactacagtg attggctgta ttgttatttt ggtcg 35
<210> 8
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atacagccaa tcactgtagt aattaataca atgatagc 38
<210> 9
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cgtgataatt attatcgtgc agtgcgccag ctgaccac 38
<210> 10
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ctgcacgata ataattatca cgacgccaat cggtatag 38
<210> 11
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gataattttt ttcgtgcagt gcgccagc 28
<210> 12
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ctattaaaaa aagcacgtca cgcggtcg 28
<210> 13
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gaagaaatta aactgattcg cgaaggtgcc 30
<210> 14
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cttctttaat ttgactaagc gcttccacgg 30
<210> 15
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gcctctacca atgcaatgat tcgtgaaatt g 31
<210> 16
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cggagatggt tacgttacta agcatttaac 30
<210> 17
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gaattgcttt ccgatgattt ttggc 25
<210> 18
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cttaacgaaa ggctactaaa aaccg 25
<210> 19
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gataccgttc tgaaaccggg tatggttg 28
<210> 20
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ctatggcaag actttggccc cataccaac 29
<210> 21
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gataccgaac tggaaccggg tatggttgtt agtatg 36
<210> 22
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
catacccggt tccagttcgg tatcaatatc ttcgcg 36

Claims (4)

1. A creatine amidinohydrolase mutant, wherein the creatine amidinohydrolase mutant is a single point mutant of D17V single point mutation site in the amino acid sequence of GenBank accession number BAA88830.1 or any combination of mutations of D17V/G58D, T199S/D17V/K351E, L6P/D17V/T199S/T251C, L6P/D17V/T199S/K351E, T199S/D17V/K351E/G58D/T251C, L6P/T117P/T199S/K351E/D17V, T199S/D17V/K351E/G58D/T251C/F108Y, L6P/T117P/F108Y/G58D/T251C/D17V/K351E, D17V/K E/G58D/T251C/F108Y, T6P/T117P/F108Y/G58D/T251C/D17V/K351E, D17V/K351E/G58D/T351D/K351E/T251C/T108Y, T199V/K108E/K108D/K108E/T/K108C/K108Y, T48E/K108K and T199S/K108E 108K.
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 according to any one of claims 1.
CN202010886832.3A 2020-08-28 2020-08-28 Creatine amidino hydrolase mutant with improved thermal stability Active CN112011528B (en)

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CN202211451065.9A CN115975998A (en) 2020-08-28 2020-08-28 Creatine amidinohydrolase mutant with improved thermal stability
CN202211447736.4A CN116716281A (en) 2020-08-28 2020-08-28 Creatine amidinohydrolase mutant with improved heat stability
CN202211447041.6A CN115851682A (en) 2020-08-28 2020-08-28 Creatine amidino hydrolase mutant with improved thermal stability
CN202211446983.2A CN115806961A (en) 2020-08-28 2020-08-28 Creatine amidinohydrolase mutant with improved thermal stability
CN202211446967.3A CN116716280A (en) 2020-08-28 2020-08-28 Creatine amidinohydrolase mutant with improved heat stability
CN202211447324.0A CN115786314A (en) 2020-08-28 2020-08-28 Creatine amidino hydrolase mutant with improved thermal stability
CN202211446969.2A CN115896077A (en) 2020-08-28 2020-08-28 Creatine amidino hydrolase mutant with improved thermal stability
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CA2404293C (en) * 2001-09-20 2007-05-15 F. Hoffmann-La Roche Ag Variants of an erwinia-type creatinase
EP1298213A1 (en) * 2001-09-20 2003-04-02 Roche Diagnostics GmbH Variants of an Erwinia-type creatinase
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