CN117448306A - Creatine amidinohydrolase mutant applied to creatinine detection and mutation site selection, purification and detection method thereof - Google Patents

Creatine amidinohydrolase mutant applied to creatinine detection and mutation site selection, purification and detection method thereof Download PDF

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CN117448306A
CN117448306A CN202311414070.7A CN202311414070A CN117448306A CN 117448306 A CN117448306 A CN 117448306A CN 202311414070 A CN202311414070 A CN 202311414070A CN 117448306 A CN117448306 A CN 117448306A
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余元玺
姜帆
康里奇
谈攀
洪亮
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Shanghai Tiandu Technology Co ltd
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Abstract

The invention discloses a creatine amidino hydrolase mutant applied to creatinine detection and a mutation site selection, purification and detection method thereof, wherein the mutant is a mutant which carries out single-point mutation on the whole length sequence of wild amino acid of creatine amidino hydrolase Creatniase protein Af-CRE from alcaligenes Alcaligenes faecalis.

Description

Creatine amidinohydrolase mutant applied to creatinine detection and mutation site selection, purification and detection method thereof
Technical Field
The invention relates to the field of proteins, in particular to a creatine amidino hydrolase mutant applied to creatinine detection and a purification and detection method thereof.
Background
Creatine amidinohydrolase (Creatinase, EC 3.5.3.3) is a key enzyme for measuring creatinine content in enzymatic assays. The enzyme is mainly derived from microorganisms such as Pseudomonas, clostridium, flavobacterium, bacillus, alcaligenes, etc. Creatine amidinohydrolase is used for industrial determination of creatinine content and is also of great importance in medical diagnosis. 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. By detecting creatinine levels in serum and urine, renal excretion can be assessed. Creatinine is the final product of human creatine phosphate metabolism, and after kidney filtration, creatinine in blood enters urine and is discharged out of the body. Normally, the normal range of human serum creatinine should be less than 150 μm, but when there is a problem with renal or muscle function, the creatinine content may rise to 1000 μm. The currently commonly used creatinine detection methods include the Jaffe chemical detection method and the enzymatic colorimetric method. Enzymatic assays are receiving increasing attention due to their high sensitivity and selectivity. However, the yield of creatine amidinohydrolase from the original bacteria is low, and the inducer is expensive, which is not suitable for industrial production. In addition, the low substrate affinity and low differential amount of creatine amidino hydrolase limit the application thereof in industrial production
At present, strains with more intensive analysis of creatine guanyl hydrolase properties include pseudomonas putida, arthrobacter and alcaligenes. Most creatine amidinohydrolases have an optimal reaction pH in the range of 7.0 to 8.0 and remain stable under neutral, weak and weak acid conditions. In general, most creatine guanyl hydrolases have an optimal reaction temperature of 30-40℃and their activity decreases rapidly when the temperature is higher than 45℃and thus maintaining the activity of creatine guanyl hydrolases is important for clinical creatinine detection.
Accordingly, those skilled in the art have been working to develop a creatine amidinohydrolase mutant having higher activity for creatinine detection.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention has been made to solve the technical problem of obtaining a mutant creatine amidinohydrolase having a higher activity.
In order to achieve the above purpose, the invention provides a creatine amidino hydrolase mutant applied to creatinine detection, which is characterized in that the mutant is a mutant with single point mutation on the whole length sequence of wild type amino acid of Af-CRE protein, wherein the Af-CRE protein is creatine amidino hydrolase derived from alcaligenes Alcaligenes faecalis, namely Creatniase protein, and the wild type amino acid sequence of the Af-CRE protein is shown as SEQ ID NO. 1.
In a preferred embodiment of the invention, the mutant of single point mutation is selected from any one of the following: full length single point mutant L131A is shown as SEQ ID NO.2, full length single point mutant W59F is shown as SEQ ID NO.3, full length single point mutant N13L is shown as SEQ ID NO.4, full length single point mutant S93A is shown as SEQ ID NO.5, full length single point mutant M5L is shown as SEQ ID NO.6, full length single point mutant F133Y is shown as SEQ ID NO.7, full length single point mutant W90Y is shown as SEQ ID NO.8, full length single point mutant T27E is shown as SEQ ID NO.9, full length single point mutant Y310L is shown as SEQ ID NO.10, full length single point mutant F256S is shown as SEQ ID NO.11, full length single point mutant V33L is shown as SEQ ID NO.12, full length single point mutant A180K is shown as SEQ ID NO.13 full length single point mutant R239D is shown as sequence SEQ ID NO.14, full length single point mutant V241I is shown as sequence SEQ ID NO.15, full length single point mutant G392T is shown as sequence SEQ ID NO.16, full length single point mutant V395Y is shown as sequence SEQ ID NO.17, full length single point mutant N130P is shown as sequence SEQ ID NO.18, full length single point mutant R104Q is shown as sequence SEQ ID NO.19, full length single point mutant W313A is shown as sequence SEQ ID NO.20, full length single point mutant H74Q is shown as sequence SEQ ID NO.21, full length single point mutant W36I is shown as sequence SEQ ID NO.22, full length single point mutant N31D is shown as sequence SEQ ID NO.23, full length single point mutant T117A is shown as sequence SEQ ID NO.24, and full length single point mutant I204V is shown as sequence SEQ ID NO. 25.
The invention also provides a mutant site selection method of the creatine amidino hydrolase mutant protein applied to creatinine detection, which is characterized in that the modification target point is scored by the existing unsupervised model on the wild type amino acid sequence of the Af-CRE by deep learning, the target site is selected by assistance of the crystal structure of the wild type protein of the Af-CRE or the structural co-evolution analysis of homologous modeling, and the nearest enzyme active site is larger than the nearest enzyme active site in the sorting from high score to low scoreAs target sites for mutation sites. The invention also comprises the method for expressing and purifying the creatine amidino hydrolase mutant protein applied to creatinine detection, which is characterized by comprising the steps of synthesizing single-point mutation plasmids and transforming the single-point mutation plasmids into escherichia coli; inoculating engineering bacteria in glycerol pipe into 5mL LB liquid medium test tube containing 100 mug/mL kanamycin according to the proportion of 2%, culturing for 12h on a shaking table at 37 ℃ and 220rpm, taking 4mL bacterial liquid into 500mL shaking flask containing 100 mug/mL LB liquid medium, culturing for 2h at 37 ℃ and 220rpm, adding IPTG with the concentration of 0.1mM when the bacterial OD600 reaches 0.8-1.0, and transferring to a shaking table at 18 ℃ for induction culture for 14-16h.
In a preferred embodiment of the invention, the plasmid is a pET28aAf-CRE plasmid.
In another preferred embodiment of the present invention, the E.coli species is BL21 DE3.
In another preferred embodiment of the present invention, the method further comprises centrifuging the shaking-induced cultured cells at 4000rpm for 15-20min to collect the cells, performing ultrasonic disruption, centrifuging at 10000rpm at high speed and collecting supernatant, performing Ni NTA purification chromatography, dividing the protein into small parts, quick-freezing with liquid nitrogen, and storing at 80deg.C.
The invention also provides a method for detecting the stability of the creatine amidino hydrolase mutant protein applied to creatinine detection, which is characterized by comprising the following steps:
preparing phosphate buffer solution, diluting the concentration of the purified multiple creatine amidinohydrolase mutant proteins to 0.3-0.5 mg/mL by using the buffer solution, loading the proteins into eight-connecting tubes, and measuring the unfolding temperature Tm by adopting fluorescent quantitative PCR to characterize the thermal stability, wherein each experiment is repeated three times.
In a preferred embodiment of the present invention, the phosphate buffer is 1 XPBS and the pH is 8.0.
The invention also comprises the activity detection method of the creatine amidino hydrolase mutant protein applied to creatinine detection, which is characterized by comprising the following steps: diluting the purified multiple creatine guanylate hydrolase mutant proteins to 1mg/mL by using phosphate buffer solution, and preparing a creatine solution with the concentration of 0.1M; 2g of p-dimethylbenzaldehyde is dissolved in 100mL of dimethyl sulfoxide, and 15mL of concentrated hydrochloric acid is added to prepare a stop solution; 280 mu L of creatine solution is added into an EP tube and is subjected to water bath at 37 ℃ for 5min, then 20 mu L of mutant protein solution is added to start creatine hydrolysis reaction, and after the reaction is carried out for 20min, stop solution is added to stop the reaction; the absorbance of the product was measured at 435nm using an enzyme-labeled instrument.
Technical effects
In order to better apply creatine amidino hydrolase to clinical creatinine detection, the invention prepares the creatine amidino hydrolase mutant with improved activity in a machine learning assisted site-directed mutagenesis mode, obtains mutant enzyme with improved activity and certain thermal stability, provides a corresponding protein purification and activity stability detection method, solves the defect that the prior creatine amidino hydrolase has poor activity and can not meet the requirement of being applied to reagents, and lays a foundation for further widening the industrial application of the creatine amidino hydrolase.
The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.
Drawings
FIG. 1 is a graph showing the results of an unfolding temperature experiment for different mutants according to a preferred embodiment of the present invention;
FIG. 2 is a graph showing the results of enzyme activity experiments of different mutants according to a preferred embodiment of the present invention.
Detailed Description
The following description of the preferred embodiments of the present invention refers to the accompanying drawings, which make the technical contents thereof more clear and easy to understand. The present invention may be embodied in many different forms of embodiments and the scope of the present invention is not limited to only the embodiments described herein.
Example 1 selection of Single Point Af-CRE mutation sites
The Wild Type (WT) amino acid sequence of Creatniase protein (Af-CRE) of the source alcaligenes Alcaligenes faecalis is shown in SEQ ID NO. 1.
The existing unsupervised model is used for scoring the transformation target point of Af-CRE by deep learning, the target site is selected by assistance of the crystal structure of the enzyme or the structural co-evolution analysis of homologous modeling, and finally the enzyme active site with the mutation point being closest to the mutation point is selected to be larger than the enzyme active siteAs target sites (see table 1).
TABLE 1 target sites and distance to nearest catalytic sites
Example 2 expression and purification of Single Unit mutant proteins
Single point mutation plasmid was synthesized and transformed into pET28aAf-CRE plasmid to E.coli BL21 (DE 3) by Beijing Optimago technologies Co., ltd. The engineering bacteria in the glycerol pipe are inoculated into 5mL LB liquid culture medium test tubes containing 100 mug/mL kanamycin according to the proportion of 2 percent, cultured for 12 hours on a shaking table at 37 ℃ and 220rpm, 4mL bacterial liquid is taken into a 500mL shaking bottle containing 100 mug/mL kanamycin LB liquid culture medium, cultured for 2 hours at 37 ℃ and 220rpm, when the bacterial OD600 reaches 0.8-1.0, the bacterial liquid is added with 0.1mM IPTG and then transferred to an 18 ℃ shaking table for induction culture for 14-16 hours. And centrifuging at 4000rpm for 15-20min to collect thalli, performing ultrasonic crushing, centrifuging at 10000rpm at high speed, collecting supernatant, and performing Ni NTA purification chromatography. Proteins were divided into small portions, snap frozen in liquid nitrogen and stored at 80 ℃.
Example 3 measurement of Heat stability and Activity of Single Unit mutant proteins
Phosphate buffer (1 XPBS, ph 8.0) was prepared and the concentration of the various creatine guanylate hydrolase mutant proteins provided in example 2 was diluted to 0.3-0.5 mg/mL with buffer. Proteins were loaded into eight-tube tubes and the unfolding temperature Tm was measured using fluorescent quantitative PCR to characterize thermal stability, in triplicate for each experiment. As shown in FIG. 1, FIG. 1 shows the stability of proteins at sites of increased Af-CRE activity, and the thermostability is shown in Table 2.
TABLE 2 thermal stability Condition
The results indicate that most of these mutants have no significant decrease in thermostability over the wild-type protein. Wherein the five-point mutant protein simultaneously improves the heat stability and the activity. The points with improved stability or activity can be used as the basis for the subsequent transformation of high-point mutants and can be directly used for creatinine detection, thereby having good application prospect.
Diluting the various creatine amidinohydrolase mutants provided in example 2 to 1mg/ml with phosphate buffer solution to prepare a creatine solution with a concentration of 0.1M; 2g of p-dimethylbenzaldehyde was dissolved in 100mL of dimethyl sulfoxide, and 15mL of concentrated hydrochloric acid was added to prepare a stop solution. 280. Mu.L of creatine solution was added to the EP tube and the mixture was subjected to a water bath at 37℃for 5min, then 20. Mu.L of mutant protein solution was added to start the creatine hydrolysis reaction, and after 20min the reaction was stopped by adding a stop solution. The absorbance of the product was measured at 435nm using an enzyme-labeled instrument.
As can be seen from FIG. 2, the activity of most mutants was not lower than that of the wild type, and the stability was not significantly lowered in most of the mutants with enhanced activity.
The enzyme activity improvement data are shown in table 2, and the single-point mutants with 20 enzyme activities and 6 mutant proteins with higher stability are obtained through screening. FIG. 2 shows the activity improvement at each point, and the results indicate that the activity of the mutant protein is mostly maintained or improved, and the optimal activity reaches 130% of that of the wild type.
TABLE 3 enzyme Activity improvement
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Claims (10)

1. The creatine amidino hydrolase mutant is characterized by being a mutant with single point mutation on the full length sequence of wild type amino acid of Af-CRE protein, wherein the Af-CRE protein is creatine amidino hydrolase derived from alcaligenes Alcaligenes faecalis, namely Creatniase protein, and the wild type amino acid sequence of the Af-CRE protein is shown as SEQ ID No. 1.
2. A creatine amidinohydrolase mutant according to claim 1 for use in creatinine detection, wherein said mutant of single point mutation is selected from any one of the following: full length single point mutant L131A is shown as SEQ ID NO.2, full length single point mutant W59F is shown as SEQ ID NO.3, full length single point mutant N13L is shown as SEQ ID NO.4, full length single point mutant S93A is shown as SEQ ID NO.5, full length single point mutant M5L is shown as SEQ ID NO.6, full length single point mutant F133Y is shown as SEQ ID NO.7, full length single point mutant W90Y is shown as SEQ ID NO.8, full length single point mutant T27E is shown as SEQ ID NO.9, full length single point mutant Y310L is shown as SEQ ID NO.10, full length single point mutant F256S is shown as SEQ ID NO.11, full length single point mutant V33L is shown as SEQ ID NO.12, full length single point mutant A180K is shown as SEQ ID NO.13 full length single point mutant R239D is shown as sequence SEQ ID NO.14, full length single point mutant V241I is shown as sequence SEQ ID NO.15, full length single point mutant G392T is shown as sequence SEQ ID NO.16, full length single point mutant V395Y is shown as sequence SEQ ID NO.17, full length single point mutant N130P is shown as sequence SEQ ID NO.18, full length single point mutant R104Q is shown as sequence SEQ ID NO.19, full length single point mutant W313A is shown as sequence SEQ ID NO.20, full length single point mutant H74Q is shown as sequence SEQ ID NO.21, full length single point mutant W36I is shown as sequence SEQ ID NO.22, full length single point mutant N31D is shown as sequence SEQ ID NO.23, full length single point mutant T117A is shown as sequence SEQ ID NO.24, and full length single point mutant I204V is shown as sequence SEQ ID NO. 25.
3. The method for selecting mutant site of creatine guanyl hydrolase according to claim 1, wherein the wild-type amino acid sequence of Af-CRE is selected by the existing unsupervised modelTarget site selection by deep learning-based target scoring, assisted by analysis of crystal structure or homology modeling of Af-CRE wild-type protein structure co-evolution, and in ranking the scores from high to low, the nearest enzyme activity site is greater thanAs target sites for mutation sites.
4. The method for expressing and purifying a creatine amidinohydrolase mutant protein for creatinine assay according to claim 1, wherein the method comprises synthesizing single point mutation plasmid and transforming into colibacillus; inoculating engineering bacteria in glycerol pipe into 5mL LB liquid medium test tube containing 100 mug/mL kanamycin according to the proportion of 2%, culturing for 12h on a shaking table at 37 ℃ and 220rpm, taking 4mL bacterial liquid into 500mL shaking flask containing 100 mug/mL LB liquid medium, culturing for 2h at 37 ℃ and 220rpm, adding IPTG with the concentration of 0.1mM when the bacterial OD600 reaches 0.8-1.0, and transferring to a shaking table at 18 ℃ for induction culture for 14-16h.
5. The method for expressing and purifying a mutant protein of creatine guanylate hydrolase for creatinine assay according to claim 4 wherein the plasmid is pET28aAf-CRE plasmid.
6. The method for expressing and purifying a mutant protein of creatine guanyl hydrolase for creatinine assay according to claim 4, wherein the E.coli is BL21 DE3.
7. The method for expressing and purifying a mutant protein of creatine amidinohydrolase for creatinine assay according to claim 4, further comprising centrifuging the cultured cells induced by a shaker at 4000rpm for 15-20min to collect the cells, sonicating, centrifuging at 10000rpm at high speed and collecting supernatant, subjecting to Ni NTA purification chromatography, dividing the protein into small portions, quick freezing with liquid nitrogen, and storing at 80 ℃.
8. A method for detecting the stability of the creatine amidinohydrolase mutant protein for creatinine assay of claim 7, comprising:
preparing phosphate buffer solution, diluting the concentration of the purified multiple creatine amidinohydrolase mutant proteins to 0.3-0.5 mg/mL by using the buffer solution, loading the proteins into eight-connecting tubes, and measuring the unfolding temperature Tm by adopting fluorescent quantitative PCR to characterize the thermal stability, wherein each experiment is repeated three times.
9. The method for detecting stability according to claim 8, wherein the phosphate buffer is 1 XPBS and the pH is 8.0.
10. A method for detecting the activity of the creatine amidinohydrolase mutant protein for creatinine assay of claim 7, comprising: diluting the purified multiple creatine guanylate hydrolase mutant proteins to 1mg/mL by using phosphate buffer solution, and preparing a creatine solution with the concentration of 0.1M; 2g of p-dimethylbenzaldehyde is dissolved in 100mL of dimethyl sulfoxide, and 15mL of concentrated hydrochloric acid is added to prepare a stop solution; 280 mu L of creatine solution is added into an EP tube and is subjected to water bath at 37 ℃ for 5min, then 20 mu L of mutant protein solution is added to start creatine hydrolysis reaction, and after the reaction is carried out for 20min, stop solution is added to stop the reaction; the absorbance of the product was measured at 435nm using an enzyme-labeled instrument.
CN202311414070.7A 2023-10-27 2023-10-27 Creatine amidinohydrolase mutant applied to creatinine detection and mutation site selection, purification and detection method thereof Pending CN117448306A (en)

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