A creatine amidine hydrolase mutant for creatinine detection, along with its mutation site selection, purification, and detection method.

Abstract

This invention discloses a creatine amidoside hydrolase mutant for creatinine detection, along with methods for selecting, purifying, and detecting the mutant site. The mutant is a single-point mutation of the wild-type full-length amino acid sequence of the creatine amidoside hydrolase protein (Af-CRE) from Alcaligenes faecalis. This invention utilizes machine learning-assisted site-directed mutagenesis to create a creatine amidoside hydrolase mutant with enhanced activity, obtaining a mutant enzyme with improved activity and certain thermostability. Furthermore, it provides methods for purifying and detecting the mutant, overcoming the shortcomings of existing creatine amidoside hydrolases with poor activity that cannot meet the requirements for reagent applications. This lays the foundation for further expanding the industrial applications of creatine amidoside hydrolases.

Description

Technical Field

[0001] This invention relates to the field of proteins, and more particularly to a creatine amidine hydrolase mutant for creatinine detection, and its purification and detection methods. Background Technology

[0002] Creatine amidine hydrolase (EC 3.5.3.3) is a key enzyme used in enzymatic assays to determine creatinine levels. This enzyme is primarily derived from microorganisms such as *Pseudomonas*, *Clostridium*, *Flavobacterium*, *Bacillus*, and *Alcaligenes*. Creatine amidine hydrolase is used industrially for creatinine determination and also has important applications in medical diagnostics. Creatinine is the final product of phosphocreatine metabolism in the human body. After being filtered by the kidneys, it enters the urine from the blood and is excreted. Detecting creatinine levels in serum and urine can assess kidney function. Normally, serum creatinine levels should be less than 150 μM, but when kidney or muscle function is impaired, creatinine levels can rise to 1000 μM. Currently, commonly used methods for creatinine detection include the Jaffe chemical assay and enzymatic colorimetric methods. Enzymatic detection methods are gaining increasing attention due to their high sensitivity and selectivity. However, the yield of creatine amidine hydrolase from the original bacteria is low, and the inducers are expensive, making them unsuitable for industrial production. Furthermore, the low substrate affinity and poor differential yield of creatine amidine hydrolase also limit its application in industrial production.

[0003] Currently, strains with relatively in-depth analysis of the properties of creatine amidine hydrolases include *Pseudomonas putida*, *Arthrobacter*, and *Alcaligenes*. The optimal reaction pH range for most creatine amidine hydrolases is 7.0-8.0, and they remain stable under neutral, weakly alkaline, and weakly acidic conditions. Typically, the optimal reaction temperature for most creatine amidine hydrolases is 30-40°C; when the temperature exceeds 45°C, their activity decreases rapidly. Therefore, maintaining the activity of creatine amidine hydrolases is crucial for clinical creatinine detection.

[0004] Therefore, those skilled in the art are dedicated to developing a creatine amidine hydrolase mutant with higher activity for creatinine detection. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to obtain a creatine amidine hydrolase mutant with higher activity.

[0006] To achieve the above objectives, the present invention provides a creatine amidine hydrolase mutant for creatinine detection, characterized in that the mutant is a mutant with a single-point mutation in the full-length wild-type amino acid sequence of the Af-CRE protein, wherein the Af-CRE protein is a creatine amidine hydrolase, i.e., Creatniase protein, derived from Alcaligenes faecalis, and the wild-type amino acid sequence of the Af-CRE protein is shown in SEQ ID NO.1.

[0007] In a preferred embodiment of the present invention, the single-point mutation mutant is selected from any of the following: full-length single-point mutant L131A as shown in sequence SEQ ID NO.2, full-length single-point mutant W59F as shown in sequence SEQ ID NO.3, full-length single-point mutant N13L as shown in sequence SEQ ID NO.4, full-length single-point mutant S93A as shown in sequence SEQ ID NO.5, full-length single-point mutant M5L as shown in sequence SEQ ID NO.6, full-length single-point mutant F133Y as shown in sequence SEQ ID NO.7, full-length single-point mutant W90Y as shown in sequence SEQ ID NO.8, full-length single-point mutant T27E as shown in sequence SEQ ID NO.9, full-length single-point mutant Y310L as shown in sequence SEQ ID NO.10, full-length single-point mutant F256S as shown in sequence SEQ ID NO.11, and full-length single-point mutant V33L as shown in sequence SEQ ID NO. As shown in SEQ ID NO.12, the full-length single-point mutant A180K is shown in sequence SEQ ID NO.13, the full-length single-point mutant R239D is shown in sequence SEQ ID NO.14, the full-length single-point mutant V241I is shown in sequence SEQ ID NO.15, the full-length single-point mutant G392T is shown in sequence SEQ ID NO.16, the full-length single-point mutant V395Y is shown in sequence SEQ ID NO.17, the full-length single-point mutant N130P is shown in sequence SEQ ID NO.18, the full-length single-point mutant R104Q is shown in sequence SEQ ID NO.19, the full-length single-point mutant W313A is shown in sequence SEQ ID NO.20, the full-length single-point mutant H74Q is shown in sequence SEQ ID NO.21, the full-length single-point mutant W36I is shown in sequence SEQ ID NO.22, and the full-length single-point mutant N31D is shown in sequence SEQ ID NO. As shown in NO.23, the full-length single-point mutant T117A is shown in sequence SEQ ID NO.24, and the full-length single-point mutant I204V is shown in sequence SEQ ID NO.25.

[0008] This invention also provides a method for selecting mutation sites in creatine amidine hydrolase mutant proteins for creatinine detection. The method is characterized by using an existing unsupervised model to score the Af-CRE wild-type amino acid sequence using deep learning, supplemented by crystal structure analysis or homology modeling of the Af-CRE wild-type protein for co-evolutionary analysis to select target sites. In the ranking from high to low scores, the enzyme activity site closest to the target site is selected. The amino acid residues are used as target sites for mutation. This invention also includes the aforementioned method for expressing and purifying creatine amidine hydrolase mutant protein for creatinine detection, characterized in that the method comprises synthesizing a single-point mutant plasmid and transforming it into *E. coli*; inoculating the engineered bacteria from the glycerol tube into 5 ml of LB liquid medium containing 100 μg / mL kanamycin at a ratio of 2%, culturing on a shaker at 37°C and 220 rpm for 12 h; transferring 4 ml of the bacterial culture to a 500 ml shake flask containing 100 μg / mL kanamycin LB liquid medium, culturing at 37°C and 220 rpm for 2 h; when the bacterial OD600 reaches 0.8-1.0, adding 0.1 mM IPTG and transferring to a shaker at 18°C ​​for induced culture for 14-16 h.

[0009] In a preferred embodiment of the present invention, the plasmid is pET28aAf-CRE plasmid.

[0010] In another preferred embodiment of the present invention, the Escherichia coli species is BL21 DE3.

[0011] In another preferred embodiment of the present invention, the method further includes collecting the bacterial cells induced by shaking culture by centrifuging at 4000 rpm for 15-20 min, sonicating them, centrifuging at 10000 rpm and taking the supernatant, performing Ni NTA purification chromatography, dividing the protein into small portions, flash-freezing in liquid nitrogen, and storing at 80°C.

[0012] The present invention also provides a method for detecting the stability of the creatine amidine hydrolase mutant protein used in creatinine detection, characterized in that the method comprises:

[0013] Prepare phosphate buffer and dilute the purified creatine amidine hydrolase mutant proteins to 0.3–0.5 mg / mL. Load the proteins into eight-tube strips and use quantitative real-time PCR to measure the unfolding temperature Tm to characterize thermal stability. Perform three replicates for each experiment.

[0014] In a preferred embodiment of the present invention, the phosphate buffer is 1×PBS with a pH of 8.0.

[0015] The present invention also includes the aforementioned method for detecting the activity of creatine amidine hydrolase mutant proteins applied to creatinine detection, characterized in that the method comprises: diluting the purified various creatine amidine hydrolase mutant proteins to 1 mg / mL using phosphate buffer to prepare a 0.1 M creatine solution; dissolving 2 g of p-dimethylbenzaldehyde in 100 mL of dimethyl sulfoxide and adding 15 mL of concentrated hydrochloric acid to prepare a stop solution; adding 280 μL of creatine solution to an EP tube and incubating at 37°C for 5 min, then adding 20 μL of mutant protein solution to start the creatine hydrolysis reaction, and adding the stop solution to terminate the reaction after 20 min; and measuring the absorbance of the product at 435 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0016] Technical effect

[0017] To better apply creatine amidine hydrolase to clinical creatinine detection, this invention creates a creatine amidine hydrolase mutant with enhanced activity through machine learning-assisted site-directed mutagenesis. This yields a mutant enzyme with increased activity and maintained thermal stability. Furthermore, it provides corresponding protein purification and activity stability detection methods, overcoming the shortcomings of existing creatine amidine hydrolases with poor activity that cannot meet reagent requirements. This lays the foundation for further expanding the industrial application of creatine amidine hydrolase.

[0018] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0019] Figure 1 This is a graph showing the experimental results of unfolding temperatures of different mutants according to a preferred embodiment of the present invention;

[0020] Figure 2 This is a graph showing the experimental results of different mutant enzyme activities according to a preferred embodiment of the present invention. Detailed Implementation

[0021] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0022] Example 1: Selection of a single Af-CRE mutation site

[0023] The wild-type (WT) amino acid sequence of the Creatniase protein (Af-CRE) from Alcaligenes faecalis involved in this invention is shown in SEQ ID NO.1.

[0024] The existing unsupervised model was used to score the modification targets of Af-CRE using deep learning. This was supplemented by structural co-evolutionary analysis based on the enzyme's crystal structure or homology modeling to select target sites. Finally, the mutation site was selected based on its proximity to the enzyme's active site. The amino acid residues are used as target sites (see Table 1).

[0025] Table 1 Target sites and distances to the nearest catalytic site

[0026]

[0027] Example 2: Expression and purification of unit point mutant protein

[0028] A single-point mutant plasmid was synthesized by Beijing Qingke Biotechnology Co., Ltd., and the pET28aAf-CRE plasmid was transformed into *E. coli* BL21(DE3). The engineered bacteria from the glycerol tube were inoculated at a ratio of 2% into 5 ml LB broth containing 100 μg / mL kanamycin in a test tube and cultured on a shaker at 37°C and 220 rpm for 12 h. 4 ml of the bacterial culture was then transferred to a 500 ml shake flask containing 100 μg / mL kanamycin in LB broth and cultured at 37°C and 220 rpm for 2 h. When the bacterial OD600 reached 0.8-1.0, 0.1 mM IPTG was added, and the culture was transferred to a shaker at 18°C ​​for 14-16 h of induction culture. The bacterial cells were collected by centrifugation at 4000 rpm for 15-20 min, sonicated, and then centrifuged at 10000 rpm. The supernatant was then purified by NiNTA chromatography. The protein was aliquoted, flash-frozen in liquid nitrogen, and stored at 80°C.

[0029] Example 3: Measurement of the thermal stability and activity of the unit point mutant protein

[0030] Prepare phosphate-buffered saline (1×PBS, pH 8.0) and dilute the various creatine amidine hydrolase mutant proteins provided in Example 2 to a concentration of 0.3–0.5 mg / mL. Load the proteins into eight-tube strips and characterize their thermal stability by measuring the unfolding temperature (Tm) using quantitative real-time PCR. Perform three replicates for each experiment. Figure 1 As shown, Figure 1 The stability of proteins at sites where Af-CRE activity is enhanced is shown in Table 2. The thermal stability is also shown in Table 2.

[0031] Table 2. Thermal stability

[0032]

[0033]

[0034] The results showed that most of these mutants did not exhibit a significant decrease in thermal stability compared to the wild-type protein. Five mutant sites showed improved thermal stability and activity. These sites, which enhance stability or activity, can serve as a basis for further modification of high-site mutants and can be directly used for creatinine detection, demonstrating promising application prospects.

[0035] The various creatine amidine hydrolase mutants provided in Example 2 were diluted to 1 mg / mL using phosphate buffer to prepare a 0.1 M creatine solution. 2 g of p-dimethylbenzaldehyde was dissolved in 100 mL of dimethyl sulfoxide, and 15 mL of concentrated hydrochloric acid was added to prepare a stop solution. 280 μL of creatine solution was added to an EP tube and incubated at 37°C for 5 min. Then, 20 μL of the mutant protein solution was added to initiate the creatine hydrolysis reaction. The reaction was terminated by adding the stop solution after 20 min. The absorbance of the product was measured at 435 nm using a microplate reader.

[0036] Depend on Figure 2 It can be seen that the activity of most mutants is no less than that of wild type, and most of the mutants with increased activity do not show a significant decrease in stability.

[0037] The enzyme activity enhancement data are shown in Table 2. After screening, 20 single-point mutants with enhanced enzyme activity were obtained, and 6 mutant proteins also had high stability. Figure 2 The activity enhancement at each site was shown, and the results indicated that the activity of most mutant proteins was maintained or enhanced, with the optimal activity reaching 130% of that of the wild type.

[0038] Table 3. Enzyme Activity Increase

[0039]

[0040]

[0041] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A creatine amidine hydrolase mutant for creatinine detection, characterized in that, The mutant is a single-point mutation in the full-length wild-type amino acid sequence of the Af-CRE protein. The Af-CRE protein is a creatine amidine hydrolase protein derived from Alcaligenes faecalis. The wild-type amino acid sequence of the Af-CRE protein is shown in SEQ ID NO. 1, and the full-length single-point mutant V395Y is shown in SEQ ID NO.

17.

2. The method for expression and purification of creatine amidine hydrolase mutant protein for creatinine detection as described in claim 1, characterized in that, The method includes synthesizing a single-point mutant plasmid and transforming it into Escherichia coli; inoculating the engineered bacteria from the glycerol tube into 5 ml of LB liquid medium containing 100 μg / mL kanamycin at a ratio of 2%, and culturing it on a shaker at 37°C and 220 rpm for 12 h; taking 4 ml of the bacterial culture into a 500 mL shake flask containing 100 μg / mL kanamycin, and culturing it at 37°C and 220 rpm for 2 h; when the bacterial OD600 reaches 0.8-1.0, adding 0.1 mM IPTG and transferring it to a shaker at 18°C ​​for induction culture for 14-16 h.

3. The method for expression and purification of creatine amidine hydrolase mutant protein for creatinine detection as described in claim 2, characterized in that, The plasmid is pET28a Af-CRE plasmid, and the Escherichia coli species is BL21 DE3.

4. The method for expression and purification of creatine amidine hydrolase mutant protein for creatinine detection as described in claim 2, characterized in that, The method further includes collecting the bacterial cells by centrifuging them at 4000 rpm for 15-20 min in a shaker, sonicating them, centrifuging them at 10000 rpm and taking the supernatant, performing Ni NTA purification chromatography, dividing the protein into small portions, flash-freezing them in liquid nitrogen, and storing them at 80°C.

5. A method for detecting the stability of the creatine amidine hydrolase mutant protein of claim 1 used for creatinine detection, characterized in that, The method includes: Prepare phosphate buffer and dilute the purified creatine amidine hydrolase mutant proteins to 0.3-0.5 mg / mL. Load the proteins into eight-tube strips and use quantitative real-time PCR to measure the unfolding temperature Tm to characterize thermal stability. Perform three replicates for each experiment.

6. The stability testing method as described in claim 5, characterized in that, The phosphate buffer solution is 1×PBS with a pH of 8.

0.

7. A method for detecting the activity of the creatine amidine hydrolase mutant protein of claim 1 for the detection of creatinine, characterized in that, The method includes: diluting the purified creatine amidine hydrolase mutant proteins to 1 mg / mL using phosphate buffer to prepare a 0.1 M creatine solution; dissolving 2 g of p-dimethylbenzaldehyde in 100 mL of dimethyl sulfoxide and adding 15 mL of concentrated hydrochloric acid to prepare a stop solution; adding 280 μL of creatine solution to an EP tube and incubating at 37°C for 5 min, then adding 20 μL of mutant protein solution to start the creatine hydrolysis reaction, and adding the stop solution to terminate the reaction after 20 min; and measuring the absorbance of the product at 435 nm using an ELISA reader.

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