A creatine amidinohydrolase mutant with higher thermal stability and its application

Abstract

The present invention discloses a creatine amidinohydrolase mutant with higher thermal stability and its application, relating to the field of biotechnology, including mutants V283L, H193Y, E170T, S19L, Y310L, Q151V and / or mutant A180K; their amino acid sequences are shown in SEQ ID NO.2-SEQ ID NO.8 respectively. A method for selecting mutation sites, using deep learning to transform target scoring for the wild type of Creatniase protein of Alcaligenes faecalis; and selecting target sites by structural co-evolution analysis assisted by the crystal structure of the enzyme or homology modeling. Application of the mutant in creatinine detection reagents. The present invention produces a creatine amidinohydrolase mutant with improved thermal stability by site-directed mutagenesis assisted by machine learning.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a creatine amidinohydrolase mutant with higher thermal stability and application thereof. Background Art

[0002] Creatinase (EC 3.5.3.3) is a key enzyme used in enzymatic assays to determine creatinine content. This enzyme is primarily derived from microorganisms such as Pseudomonas, Clostridium, Flavobacterium, Bacillus, and Alcaligenes. However, the yield of creatine amidinohydrolase from primitive bacteria is low, and the inducer is expensive, making it unsuitable for industrial production. In addition, the low substrate affinity and poor thermal stability of creatine amidinohydrolase also limit its application in industrial production.

[0003] Creatine amidinohydrolase is used industrially to measure creatinine levels. It also has important applications in medical diagnostics. Creatinine is the end product of creatine phosphate metabolism in the human body. After being filtered by the kidneys, it is excreted from the blood into the urine. Measuring creatinine levels in serum and urine can assess renal excretion function. Creatinine is the end product of creatine phosphate metabolism in the human body. After being filtered by the kidneys, creatinine in the blood is excreted into the urine. Under normal circumstances, the normal range of human serum creatinine is less than 150. However, when kidney or muscle function is impaired, creatinine levels can rise to 1000. Commonly used methods for creatinine detection include the Jaffe chemical assay and the enzymatic colorimetric assay. Enzymatic assays are gaining increasing attention due to their high sensitivity and selectivity.

[0004] Currently, the properties of creatine amidinohydrolases have been extensively analyzed in strains including Pseudomonas putida, Arthrobacter, and Alcaligenes. The vast majority of creatine amidinohydrolases exhibit an optimal reaction pH range of 7.0-8.0 and remain stable under neutral, weakly alkaline, and weakly acidic conditions. Typically, the optimal reaction temperature for most creatine amidinohydrolases is 30-40°C. When the temperature rises above 45°C, the activity decreases rapidly. Therefore, studying the thermal stability of creatine amidinohydrolases is of great significance.

[0005] Therefore, those skilled in the art are committed to developing a creatine amidinohydrolase with higher thermal stability and activity for use in clinical creatinine detection reagents. Summary of the Invention

[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to develop a creatine amidinohydrolase with higher thermal stability and activity for use in clinical creatinine detection reagents.

[0007] To achieve the above objectives, the present invention provides a creatine amidinohydrolase mutant with higher thermal stability, including a full-length single point mutant V283L, a full-length single point mutant H193Y, a full-length single point mutant E170T, a full-length single point mutant S19L, a full-length single point mutant Y310L, a full-length single point mutant Q151V and / or a full-length single point mutant A180K; the amino acid sequence of the full-length single point mutant V283L is shown in SEQ ID NO.2, the amino acid sequence of the full-length single point mutant H193Y is shown in SEQ ID NO.3, the amino acid sequence of the full-length single point mutant E170T is shown in SEQ ID NO.4, the amino acid sequence of the full-length single point mutant S19L is shown in SEQ ID NO.5, the amino acid sequence of the full-length single point mutant Y310L is shown in SEQ ID NO.6, the amino acid sequence of the full-length single point mutant Q151V is shown in SEQ ID NO. The amino acid sequence of the full-length single-point mutant A180K is shown in SEQ ID NO.7, and the amino acid sequence of the full-length single-point mutant A180K is shown in SEQ ID NO.8.

[0008] Furthermore, the mutant is derived from the wild type of Creatniase protein of Alcaligenes faecalis, and its amino acid sequence is shown in SEQ ID NO.1.

[0009] The present invention also provides a method for selecting a mutation site of creatine amidinohydrolase with higher thermal stability, comprising the following steps:

[0010] Step 1: Using an unsupervised model, the wild-type Creatniase protein of Alcaligenes faecalis from the NCBI database was scored for transformation targets using deep learning to screen mutants.

[0011] Step 2: For the mutants obtained in step 1, target sites are selected by structural co-evolution analysis supplemented by the crystal structure of the enzyme or homology modeling.

[0012] Furthermore, the target score of the mutant in step 1 is greater than or equal to 0.

[0013] Furthermore, the criterion for selecting the target site in step 2 is that the distance between the mutation site and the nearest enzyme active site is greater than

[0014] The present invention also provides a method for expressing and purifying a creatine amidinohydrolase mutant with higher thermal stability, comprising the following steps:

[0015] 1) Synthesize single-point mutation plasmids;

[0016] 2) transforming the single-point mutation plasmid obtained in step 1) into Escherichia coli to obtain an engineered bacterium;

[0017] 3) inoculating the engineered bacteria obtained in step 2) into a liquid culture medium and culturing on a shaker to obtain a bacterial solution; transferring the bacterial solution to a shake flask containing the liquid culture medium and culturing for 2 h, adding IPTG, and then moving the flask to an 18° C. shaker for induction culture; collecting the bacterial cells, disrupting them, and performing high-speed centrifugation to obtain the supernatant for chromatography.

[0018] Furthermore, the single-point mutation plasmid in step 1) includes pET28aAf-CRE plasmid; and the Escherichia coli in step 2) includes Escherichia coli BL21 (DE3).

[0019] Furthermore, step 3) further includes: inoculating the engineered bacteria in the glycerol tube into a 5 mL LB liquid culture medium test tube containing 100 μg / mL kanamycin at a ratio of 2%, culturing on a shaker at 37°C and 220 rpm for 12 hours, taking 4 mL of the bacterial solution into a 500 mL shake flask containing LB liquid culture medium containing 100 μg / mL kanamycin, culturing at 37°C and 220 rpm for 2 hours, and when the OD600 of the bacteria reaches 0.8-1.0, adding IPTG at a concentration of 0.1 mM and moving to an 18°C ​​shaker for induction culture for 14-16 hours; collecting the bacteria by centrifugation at 4000 rpm for 15-20 minutes, ultrasonically disrupting, and then high-speed centrifuging at 10000 rpm and taking the supernatant for Ni NTA purification chromatography; dividing the protein into small portions, quick-freezing with liquid nitrogen, and storing at 80°C.

[0020] The present invention also provides a use of a creatine amidinohydrolase mutant with higher thermal stability in a creatinine detection reagent.

[0021] Furthermore, the creatinine detection reagent can be used within a high temperature range, with the highest temperature in the high temperature range being 58.5°C-59.5°C.

[0022] In preferred embodiment 1 of the present invention, the selection process of the single-point Af-CRE mutation site is described in detail;

[0023] In another preferred embodiment 2 of the present invention, the expression and purification process of the single-site mutant protein is described in detail;

[0024] In another preferred embodiment 3 of the present invention, the process of measuring the thermal stability and activity of single-site mutant proteins is described in detail.

[0025] The beneficial technical effects of the present invention are as follows:

[0026] The present invention uses machine learning-assisted site-directed mutagenesis to create mutants of creatine amidinohydrolase with improved thermal stability, resulting in mutant enzymes with improved thermal stability and retained a certain level of activity. Thirty single-point mutant proteins were screened through site-directed mutagenesis, seven of which showed improved stability. Of the seven single-point mutations with improved stability, two also showed improved activity. This method addresses the shortcomings of existing creatine amidinohydrolases, which suffer from poor thermal stability and insufficient high-temperature activity, making them unsuitable for reagent applications, and lays the foundation for expanding the industrial application of creatine amidinohydrolases.

[0027] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a curve chart comparing the thermal stability test results of the mutant protein Q151V and the wild type in a preferred embodiment 3 of the present invention;

[0029] Figure 2 This is a histogram of the thermal stability of a single point mutant (Af-CRE single point mutant protein) according to a preferred embodiment 3 of the present invention;

[0030] Figure 3 This is a bar graph showing the activity data of a single-point mutant protein with improved thermal stability of Af-CRE according to a preferred embodiment 3 of the present invention. DETAILED DESCRIPTION

[0031] The following describes several preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0032] Example 1 Selection of single-point Af-CRE mutation sites

[0033] The wild-type (WT) amino acid sequence of the Creatniase protein (Af-CRE) from Alcaligenes faecalis involved in the present invention is from the NCBI database as shown in SEQ ID NO.1.

[0034] The existing unsupervised model is used to score the target sites of Af-CRE by deep learning, and the target sites are selected by structural co-evolution analysis of the crystal structure or homology modeling of the enzyme. The selection criteria are that the distance between the mutation site and the nearest enzyme active site should be greater than Amino acid residues that meet both criteria were selected as target sites. The distances between the target sites and the nearest catalytic sites are shown in Table 1.

[0035] Table 1 Target site and distance to the nearest catalytic site

[0036]

[0037] Example 2 Expression and purification of single-site mutant proteins

[0038] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize a single-point mutant plasmid and transform the pET28a Af-CRE plasmid into Escherichia coli BL21(DE3). The engineered bacteria from the glycerol tube were inoculated at a 2% ratio into a 5 mL tube of LB liquid medium containing 100 μg / mL kanamycin and incubated at 37°C, 220 rpm, and shaker for 12 hours. Four mL of the bacterial suspension was then transferred to a 500 mL shake flask containing LB liquid medium containing 100 μg / mL kanamycin and incubated at 37°C, 220 rpm, for 2 hours. When the bacterial OD600 reached 0.8-1.0, 0.1 mM IPTG was added and the cells were induced on a shaker at 18°C ​​for 14-16 hours. The cells were harvested by centrifugation at 4000 rpm for 15-20 minutes, disrupted by sonication, and then centrifuged at 10,000 rpm. The supernatant was purified by Ni NTA chromatography. The protein was aliquoted, snap-frozen in liquid nitrogen, and stored at 80°C.

[0039] Example 3 Thermal Stability and Activity Measurement of Single-site Mutant Proteins

[0040] Prepare phosphate buffer (1×PBS, pH 8.0) and dilute the various creatine amidinohydrolase mutant proteins provided in Example 2 to a concentration of 0.3-0.5 mg / mL using the buffer. The proteins were placed in eight tubes and the unfolding temperature (Tm) was measured by fluorescence quantitative PCR to characterize thermal stability. Each experiment was repeated three times. For example, the thermal stability of the mutant protein Q151V was compared with that of the wild type. Figure 1 shown.

[0041] The results showed that the thermal stability of most mutant proteins was maintained, and the T_m of 7 mutants exceeded that of the wild type. The enzyme stability improvement data are shown in Table 2. After screening, 7 single-point mutants with improved enzyme thermal stability were obtained: V283L, H193Y, E170T, Y310L, S19L, Q151V and A180K. The thermal stability bar graph of these 7 single-point mutants with improved enzyme thermal stability (Af-CRE single-point mutant proteins) is shown in Table 2. Figure 2 shown.

[0042] Table 2 Improvement of enzyme thermal stability

[0043]

[0044] The various creatine amidinohydrolase mutants provided in Example 2 were diluted to 1 mg / mL using phosphate buffer. A 0.1 M creatine solution was prepared; 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 in a 37°C water bath for 5 minutes. Then, 20 μL of the mutant protein solution was added to initiate the creatine hydrolysis reaction. After 20 minutes of reaction, the stop solution was added to terminate the reaction. The absorbance of the product was measured at 435 nm using a microplate reader.

[0045] The enzyme activity was expressed as relative enzyme activity, and the data are shown in Table 3.

[0046] Table 3 Enzyme activity

[0047]

[0048]

[0049] The activity data of the single-point mutant protein with improved thermal stability of Af-CRE is shown in the bar graph. Figure 3 As shown in Figure 2 , most mutants with improved thermostability did not show a significant decrease in activity. Specifically, the Y310L and A180K mutants showed improved thermostability and activity. These sites with enhanced stability or activity can serve as a foundation for subsequent engineering of higher-position mutants and can be directly used for creatinine detection, demonstrating promising application prospects.

[0050] The activities of most mutants were no less than those of the wild type, and some mutants showed higher thermal stability than the wild type without a significant decrease in activity.

[0051] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A creatine amidinohydrolase mutant with higher thermal stability, characterized in that: Selected from the full-length single point mutant V283L, the full-length single point mutant E170T, the full-length single point mutant S19L, the full-length single point mutant Y310L or the full-length single point mutant A180K; the amino acid sequence of the full-length single point mutant V283L is shown in SEQ ID NO.2, the amino acid sequence of the full-length single point mutant E170T is shown in SEQ ID NO.4, the amino acid sequence of the full-length single point mutant S19L is shown in SEQ ID NO.5, the amino acid sequence of the full-length single point mutant Y310L is shown in SEQ ID NO.6, and the amino acid sequence of the full-length single point mutant A180K is shown in SEQ ID NO.

8.

2. The creatine amidinohydrolase mutant according to claim 1, wherein The mutant is derived from the wild type of Creatniase protein of Alcaligenes faecalis, and its amino acid sequence is shown in SEQ ID NO.

1.

3. The method for expressing and purifying the creatine amidinohydrolase mutant according to claim 1 or 2, wherein: The following steps are involved: 1) Synthesize single-point mutation plasmids; 2) transforming the single-point mutation plasmid obtained in step 1) into Escherichia coli to obtain an engineered bacterium; 3) inoculating the engineered bacteria obtained in step 2) into a liquid culture medium and culturing on a shaker to obtain a bacterial solution; transferring the bacterial solution to a shake flask containing the liquid culture medium and culturing for 2 h, adding IPTG, and then moving the flask to an 18° C. shaker for induction culture; collecting the bacterial cells, disrupting them, and performing high-speed centrifugation to obtain the supernatant for chromatography.

4. The expression and purification method according to claim 3, wherein The step 3) further comprises: inoculating the engineered bacteria in the glycerol tube into a 5 mL LB liquid culture medium test tube containing 100 μg / mL kanamycin at a ratio of 2%, culturing on a shaker at 37° C. and 220 rpm for 12 hours, taking 4 mL of the bacterial solution into a 500 mL shake flask containing the LB liquid culture medium containing 100 μg / mL kanamycin, culturing at 37° C. and 220 rpm for 2 hours, and when the OD600 of the bacteria reaches 0.8-1.0, adding 0.1 mM IPTG and then moving to an 18° C. shaker for induction culture for 14-16 hours; collecting the bacteria by centrifugation at 4000 rpm for 15-20 minutes, ultrasonically disrupting, and then high-speed centrifuging at 10000 rpm to obtain the supernatant, which is then subjected to Ni NTA purification chromatography; dividing the protein into small portions, quick-freezing with liquid nitrogen, and storing at 80° C.

5. Use of the creatine amidinohydrolase mutant according to claim 1 or 2 in the preparation of a creatinine detection reagent.

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