A highly active deoxycytidylate deaminase mutant and its application

Abstract

The present invention discloses a method for preparing a highly active deoxycytidylic acid deaminase mutant, which relates to the field of bioengineering technology and includes the following steps: 1. Synthesis of deoxycytidylic acid deaminase parent and mutant genes; 2. Screening of superior mutants; 3. Purification of deoxycytidylic acid deaminase and mutants; 4. Determination of enzyme activity. The present invention introduces multiple mutation sites through site-directed mutagenesis technology to optimize the catalytic performance of deoxycytidylic acid deaminase, introduces the required mutations at specific positions of the enzyme to achieve precise regulation of the catalytic performance of the enzyme, significantly improves the catalytic activity and reaction rate of the enzyme, and thus improves the efficiency of the entire biocatalytic process. Site-directed mutagenesis technology enhances the thermal stability and pH stability of the enzyme so that it can maintain catalytic activity under a wider range of conditions. Optimizing the amino acid sequence of the enzyme can make it better adapt to specific substrates or reaction conditions, thereby improving the activity of deoxycytidylic acid deaminase in the dCMP deamination reaction.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, in particular to a high-activity deoxycytidylate deaminase mutant and application thereof. Background Art

[0002] At present, the deoxycytidylic acid deaminase with relatively high activity mainly comes from T4 bacteriophage with 220 μmol / mg / min, T2 bacteriophage with 430 μmol / mg / min, and Equus asinus with 730 μmol / mg / min. However, the three have almost no supernatant expression when recombinantly expressed in Escherichia coli BL21 (DE3). The deoxycytidylic acid deaminase derived from Streptococcus mutans has an enzyme activity of 230 μmol / mg / min under 0.1 mM dCTP conditions. The expression amount of the enzyme is acceptable. Therefore, the enzyme from this source is used as the wild enzyme for modification in the present invention.

[0003] The defects of deoxycytidine deaminase in the prior art are:

[0004] 1. Patent document CN110819604B discloses a deoxyribosyltransferase mutant and its application. The deoxyribosyltransferase mutant does not introduce multiple mutation sites to optimize the catalytic performance of deoxycytidylic acid deaminase, and cannot achieve efficient conversion of dCMP, resulting in low activity of deoxycytidylic acid deaminase in the dCMP deamination reaction.

[0005] 2. Patent document CN118480521A discloses a highly active catalase mutant and its application. The catalase mutant does not optimize the amino acid sequence and reaction gradient of the enzyme, cannot precisely regulate the catalytic performance of the enzyme, is not conducive to improving the catalytic activity and reaction rate of the enzyme, and has low efficiency of the entire biocatalytic process.

[0006] 3. Patent document CN116287277B discloses a biomarker of cytidine deaminase activity and its application. This biomarker cannot allow the insertion and cloning of different gene fragments, does not allow the use of different restriction endonucleases to cut the vector and the target gene, and cannot select specific enzymes for cutting as needed to adapt to different gene fragments and experimental requirements.

[0007] 4. Patent document CN106978405B discloses aspartate kinase / homoserine dehydrogenase mutants and their applications. The aspartate dehydrogenase mutants cannot achieve stable expression of the target gene in the host bacteria, and are not convenient for purifying the recombinant protein by affinity chromatography and other methods in subsequent experiments. The purity of the purified enzyme is low. Summary of the invention

[0008] The object of the present invention is to provide a highly active deoxycytidylic acid deaminase mutant and its application to solve the problems raised in the above background technology.

[0009] To achieve the above object, the present invention provides the following technical solution: a highly active deoxycytidylic acid deaminase mutant, comprising the following steps: 1. synthesis of deoxycytidylic acid deaminase parent and mutant genes; 2. screening of advantageous mutants; 3. purification of deoxycytidylic acid deaminase and mutants; 4. determination of enzyme activity;

[0010] The steps for synthesizing the deoxycytidylic acid deaminase parent and mutant genes are as follows:

[0011] Step (1): obtaining the nucleotide sequence of deoxycytidine deaminase from Streptococcus mutans, the sequence number in GenBank is VEF17940.1;

[0012] Step (2): codon optimization of the nucleotide sequence;

[0013] Step (3): synthesizing the optimized nucleotide sequence and cloning it into the pET28a vector to obtain a recombinant plasmid;

[0014] Wherein, the mutant includes at least one of N45S, G46S, G46T, I67N, I67T, T69S, V70N, V70S and V70T.

[0015] Preferably, the step of synthesizing the deoxycytidylic acid deaminase parent and mutant genes further comprises transforming the recombinant plasmid into Escherichia coli BL21 for inducing expression to obtain wet bacteria containing the deoxycytidylic acid deaminase parent and mutants thereof.

[0016] Preferably, the advantageous mutant screening steps are as follows:

[0017] Step (1): obtaining wet bacteria containing the parent deoxycytidylate deaminase and its mutants;

[0018] Step (2): crushing the wet bacterial cells to obtain a crude enzyme solution;

[0019] Step (3): using deoxycytidylic acid as substrate and deoxycytidine triphosphate as co-substrate, using crude enzyme solution to conduct catalytic experiments;

[0020] Step (4): Detect the reaction product by HPLC, and use the relative content of the product as an indicator to screen mutants with improved catalytic performance.

[0021] Preferably, the mutants that improve catalytic performance include single point mutations I67T and T69S.

[0022] Preferably, the deoxycytidine deaminase and mutant purification steps are as follows:

[0023] Step (1): suspending wet cells containing the parent deoxycytidine deaminase and its mutants in a buffer, performing ultrasonic disruption, and centrifuging to obtain the supernatant;

[0024] Step (2): Purify the protein using a Ni affinity column to collect the target protein;

[0025] Step (3): dialyze the target protein in a buffer to obtain purified deoxycytidine deaminase and its mutants.

[0026] Preferably, the buffer comprises 20 mM Tris buffer at pH 8.0, buffer A containing 0.5 M NaCl and 20 mM imidazole for suspending and washing the impurity protein, and buffer B containing 0.5 M NaCl and 400 mM imidazole for eluting the target protein.

[0027] Preferably, the enzyme activity determination steps are as follows:

[0028] Step (1): using an appropriate amount of enzyme solution, 50 mM dCMP, 1 mM dCTP and 0.3 M Tris-HCl buffer to carry out a catalytic reaction at 40° C. and pH 7.0 for 30 minutes;

[0029] Step (2): Detect the reaction product by HPLC and calculate the amount of enzyme required to generate 1 μmol of dUMP per minute, which is defined as one unit of enzyme activity (U);

[0030] Step (3): Determine the protein concentration and calculate the number of activity units per milligram of enzyme protein, i.e., specific enzyme activity (U / mg).

[0031] The invention relates to an application of a highly active deoxycytidylic acid deaminase mutant in the field of biological enzyme catalysis, preferably, the application of the deoxycytidylic acid deaminase in the reaction of catalyzing the conversion of deoxycytidylic acid (dCMP) into deoxyuridylic acid (dUMP), and the effects of different reaction conditions, such as magnesium ion concentration, dCTP concentration, substrate concentration and enzyme amount, on the reaction conversion rate.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The present invention optimizes the catalytic performance of deoxycytidylic acid deaminase by introducing multiple mutation sites through site-directed mutagenesis technology, introduces the required mutations at specific positions of the enzyme to achieve precise regulation of the catalytic performance of the enzyme, and makes the optimization process more efficient and controllable. The introduction of multiple favorable mutations significantly improves the catalytic activity and reaction rate of the enzyme, thereby improving the efficiency of the entire biocatalytic process. The site-directed mutagenesis technology enhances the thermal stability and pH stability of the enzyme so that it can maintain catalytic activity under a wider range of conditions. The optimization of the amino acid sequence of the enzyme can make it better adapt to specific substrates or reaction conditions, thereby broadening the application range of the enzyme. The optimization of the dCTP concentration gradient, the magnesium ion concentration gradient, the reaction temperature gradient and the reaction time gradient realizes the efficient conversion of dCMP, which is particularly important for the enzyme catalytic process in industrial applications, can significantly reduce the production cost and improve the production efficiency, and improves the activity of deoxycytidylic acid deaminase in the dCMP deamination reaction.

[0034] 2. The present invention improves the expression efficiency of deoxycytidine deaminase in Escherichia coli through codon optimization, ensuring the high yield of recombinant protein. The multiple cloning site on the pET28a vector facilitates the insertion and cloning of different gene fragments, allowing the use of different restriction endonucleases to cut the vector and the target gene, thereby providing a variety of options for connecting and cloning gene fragments. Specific enzymes are selected for cutting as needed to adapt to different gene fragments and experimental requirements. The pET28a vector, as an efficient expression vector in Escherichia coli, enables the target gene to be stably expressed in the host bacteria. The specific protein tag contained in the pET28a vector facilitates the purification of the recombinant protein by affinity chromatography and other methods in subsequent experiments, thereby improving the purity of the purified enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a high performance liquid chromatogram of the conversion solution of the present invention;

[0036] Figure 2 The effect of different magnesium ion concentrations on dUMP conversion rate in Example 6 of the present invention;

[0037] Figure 3 The effect of different dCTP concentrations on dUMP conversion rate in Example 7 of the present invention;

[0038] Figure 4 It is the reaction process curve diagram of Example 9 of the present invention. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "front end", "rear end", "two ends", "one end", "the other end" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0041] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "provided with", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection or a movable connection, or a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0042] Example 1, an embodiment provided by the present invention: gene synthesis of deoxycytidine deaminase parent and mutant;

[0043] The deoxycytidine deaminase from Streptococcus mutans is numbered VEF17940.1 in GenBank. The nucleotide sequence was found, codon optimized, and then delivered to Qingke Biotechnology for gene synthesis and cloned into the pET28a vector to obtain a recombinant plasmid.

[0044] Similarly, the mutants N45S, G46S, G46T, I67N, I67T, T69S, V70N, V70S, and V70T of dCD[Sm] were also codon-optimized and then delivered to Qingke Biotechnology for gene synthesis and cloned into the pET28a vector to obtain recombinant plasmids.

[0045] Example 2, based on the above example, transformation and induced expression of deoxycytidine deaminase parent and mutant;

[0046] The constructed plasmid was centrifuged at 10,000 rpm for 10 min, and 50 μL ddH2O was added to dissolve it on a vortex shaker. After dissolution, it was transformed into E. coli BL21 (DE3), and the transformed E. coli was activated, placed at 37°C, 220 rpm, cultured for 1 hour, spread on an LB plate containing 50 μg / mL kanamycin resistance, and inverted and cultured at 37°C overnight;

[0047] A single colony was picked from the plate and inoculated into a TB liquid medium containing kanamycin at a final concentration of 50 μg / mL, and glucose and lactose at a final concentration of 5 g / L were added, and cultured at 37°C and 220 rpm. When OD600 reached 0.8-1.2 (preferably 0.9), the temperature was changed to 28°C, and cultured at 220 rpm for 14 hours, and then centrifuged at 4°C and 8000 rpm / min for 10 minutes to obtain wet cells containing deoxycytidine deaminase and mutants.

[0048] Example 3, this example is based on the above example, deoxycytidine deaminase and mutant library screening;

[0049] The wet cells obtained in Example 2 were centrifuged, resuspended in pure water at a total cell volume of 40 g / L, and crushed on an ice-water mixture for 20 min. The ultrasonic crushing conditions were: 200 W, crushing for 5 s, and pausing for 7 s. After crushing, a crude enzyme solution was obtained.

[0050] The crude enzyme solution of the mutant was used as a catalyst, deoxycytidine acid was used as a substrate, and deoxycytidine triphosphate was used as a co-substrate for catalytic experiments. The reaction system was selected to be 40 ml, the amount of catalyst was 1 g / L based on the total concentration of wet bacteria before crushing, the final concentration of the substrate was 50 mM, the final concentration of deoxycytidine triphosphate was 1 mM, and the final concentration of Tris-HCl buffer was 0.3 mol / L (pH = 7.0). The reaction was static at 40°C for 30 min and 1 h, and samples were taken. 100 μL of the reaction solution was taken, and 6 μL of 3M hydrochloric acid was added to terminate the reaction for 30 min. After termination, 7 μL was taken and added to 1 ml of mobile phase, and passed through a 0.22 μM filter membrane as a HPLC detection sample to screen the superior mutants with the relative content of the product as an indicator. The experimental results are shown in Table 1.

[0051] From the results in Table 1, we can see that the catalytic performance of single point mutations I67T and T69S has been improved to varying degrees. The 1h product UMP conversion rates were 8.22% and 26.30%, respectively, which were 48% and 373% higher than those of the control bacteria. Among them, the T69S point mutation was found to have a significant improvement in its catalytic performance, and the other point mutations were negative mutations.

[0052] Liquid phase detection conditions for dCMP and dUMP: chromatographic column ChromCoreTM 120C18 (5μm 4.6×150mm, NanoChrom, China), mobile phase acetonitrile: 0.1M PBS (pH7.0) volume ratio of 5:95, flow rate 1.0mL / min, detection wavelength 262nm, injection volume 10μL, column temperature 35℃.

[0053] Table 1

[0054]

[0055]

[0056] Example 4, based on the above example, purification of deoxycytidine deaminase and its mutants;

[0057] First, the wet cells of the dCD[Sm] parent and the dominant mutant (in Table 1) obtained in Example 3 were suspended in buffer A (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 20 mM imidazole), ultrasonically disrupted for 15 minutes (ice bath, power 400 W, disruption for 3 seconds, pause for 5 seconds), centrifuged at 4°C and 12,000 rpm for 20 minutes, and the supernatant was collected;

[0058] The protein was purified using a Ni affinity column (1.6 × 10 cm, Bio-Rad, USA) with the following steps: (1) Equilibrate the Ni column with 5 column volumes of binding buffer (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 20 mM imidazole) until the baseline is stable; (2) Load the sample at a flow rate of 1 mL / min with a loading amount of 25–40 mg / mL protein to allow the target protein to adsorb onto the Ni column; (3) Wash the impurities with 6 column volumes of buffer A (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 20 mM imidazole) at a flow rate of 1 mL / min until the baseline is stable; (4) Elute with buffer B (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 400 mM imidazole) at a flow rate of 1 mL / min to collect the target protein; The purified nicotinamide phosphoribosyltransferase was obtained by dialyzing against Tris buffer overnight; (5) the Ni column was rinsed with 5 column volumes of binding buffer (20 mM Tris buffer containing 0.5 M NaCl, pH 8.0) until the baseline was stable, and the Ni column was preserved with 5 column volumes of ultrapure water containing 20% ​​ethanol.

[0059] Example 5, based on the above example, the specific enzyme activity of the parent cytidylate deaminase and its mutants was determined;

[0060] The enzyme activity unit (U) is defined as: the amount of enzyme required to generate 1 μmol of dUMP per minute at 40°C and pH 7.0 is defined as one unit of enzyme activity, U. The specific enzyme activity is defined as the number of activity units per milligram of enzyme protein, U / mg. Standard conditions for enzyme activity detection: 50mM dCMP, 1mM dCTP, appropriate amount of enzyme solution, reaction at 40°C and pH7.0 (0.3M Tris-HCl buffer) for 30 minutes, sample processing and HPLC detection and analysis;

[0061] The protein concentration was determined using a BCA protein assay kit (Shanghai Sangon Biotechnology Development Co., Ltd., Shanghai);

[0062] The specific enzyme activities of the parent cytidylate deaminase and its mutants are shown in Table 2: dCD[Sm]-I67T is 1.42 times the specific enzyme activity of the parent, and dCD[Sm]-T69S is 5.06 times the specific enzyme activity of the parent.

[0063] Table 2

[0064]

[0065] Example 6, an application example provided by the present invention: Application Example 1 of deoxycytidine deaminase;

[0066] According to the description in Example 4, the purified mutant pure enzyme solution was taken, and five experimental groups were set up, and two parallel samples were set up in each experimental group. The reaction base solution was prepared, 100mM dCMP, 1mM dCTP, 300mM Tris, and the concentrations of magnesium chloride hexahydrate were 0mM, 1mM, 10mM, 50mM and 100mM, respectively. The pH was adjusted to 7.5, and 0.5U / mL of deoxycytidine deaminase was added. The reaction was allowed to stand at 40°C for 30min, and the generation of the product dUMP during the reaction was detected by the liquid phase method shown in Example 3;

[0067] The conversion rate of dUMP under different magnesium ion concentrations is as follows Figure 2 As shown: the conversion rate of dUMP is the highest at a magnesium ion concentration of 50 mM, which is 2.67 times the conversion rate without adding magnesium ions. Therefore, the amount of magnesium ions added in the subsequent reaction is 50 mM.

[0068] Example 7, this example is based on the above example, deoxycytidine deaminase application example 2;

[0069] According to the description in Example 6, the purified mutant pure enzyme solution was taken, and five experimental groups were set up, and two parallel samples were set up in each experimental group. The reaction base solution was prepared, 100mM dCMP, 300mM Tris, 50mM magnesium chloride hexahydrate, and the concentrations of dCTP were 1mM, 2mM, 3mM, 4mM and 5mM, respectively. The pH was adjusted to 7.5, and 0.1U / mL of deoxycytidine deaminase was added. The reaction was allowed to stand at 40°C for 30min, and the generation of the product dUMP during the reaction was detected by the liquid phase method shown in Example 3;

[0070] The conversion rate of dUMP under different magnesium ion concentrations is as follows Figure 3 As shown in the figure, the conversion rate of dUMP increases with the increase of dCTP concentration. Although the conversion rate increases slightly when the concentration exceeds 3mM, it is not obvious. In order to save costs, the concentration of dCTP is selected to be 3mM.

[0071] Example 8, this example is based on the above example, deoxycytidine deaminase application example 2;

[0072] According to the description of Example 7, the purified mutant pure enzyme solution was taken to perform a low concentration dCMP deamination reaction, and two parallel samples were set up, each sample was 100 mL, the amount of dCMP added was 7.68 g, the amount of magnesium chloride hexahydrate added was 1.02 g, the amount of dCTP added was 0.14 g, the pH was adjusted to 7.5, and 1000 U of deoxycytidine deaminase was added. The reaction was allowed to stand at 40°C;

[0073] The formation of the product dUMP during the reaction was detected by the liquid phase method shown in Example 3. The conversion rate of dUMP in the reaction was above 99%.

[0074] Example 9, this example is based on the above example, application example 2 of deoxycytidine deaminase;

[0075] According to the description in Example 8, the purified mutant pure enzyme solution was taken, the dCMP concentration was increased for deamination reaction, two parallel samples were set, each sample was 1 L, the amount of dCMP added was 261.12 g, the amount of magnesium chloride hexahydrate added was 10.20 g, the amount of dCTP added was 1.40 g, the pH was adjusted to 7.5, 10000 U of deoxycytidine deaminase was added, and the reaction was allowed to stand at 40°C;

[0076] The formation of the product dUMP during the reaction was detected by the liquid phase method shown in Example 3. The conversion rate of dUMP in the reaction was as follows: Figure 4 As shown: after 3 hours of reaction, the conversion rate of dUMP is 73%, and after 19.5 hours of reaction, the conversion rate of dUMP is 99.30%. At this time, the output of dUMP disodium is 280 g / L, while a small amount of dCMP remains in the reaction system, which requires a long time of reaction to be reduced slightly.

[0077] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A highly active deoxycytidylate deaminase mutant, characterized in that: The sequence of the deoxycytidylate deaminase is numbered VEF17940.1 in GenBank; the mutant is a single point mutation I67T or T69S, and the sequence numbered VEF17940.1 in GenBank is shown as SEQ ID NO.

1.

2. A highly active deoxycytidylate deaminase mutant according to claim 1, characterized in that: The method comprises the following steps:

1. synthesis of deoxycytidylic acid deaminase parent and mutant genes; 2. screening of advantageous mutants; 3. purification of deoxycytidylic acid deaminase and mutants; 4. determination of enzyme activity; The steps for synthesizing the deoxycytidylic acid deaminase parent and mutant genes are as follows: Step (1): obtaining the nucleotide sequence of deoxycytidine deaminase from Streptococcus mutans; Step (2): codon optimization of the nucleotide sequence; Step (3): The optimized nucleotide sequence is synthesized and cloned into the pET28a vector to obtain a recombinant plasmid.

3. A highly active deoxycytidylate deaminase mutant according to claim 2, characterized in that: The step of synthesizing the deoxycytidylic acid deaminase parent and mutant genes further comprises transforming the recombinant plasmid into Escherichia coli BL21 for inducing expression to obtain wet bacteria containing the deoxycytidylic acid deaminase parent and mutants thereof.

4. A highly active deoxycytidylate deaminase mutant according to claim 2, characterized in that: The advantageous mutant screening steps are as follows: Step (1): obtaining wet bacteria containing the parent deoxycytidylate deaminase and its mutants; Step (2): crushing the wet bacterial cells to obtain a crude enzyme solution; Step (3): using deoxycytidylic acid as substrate and deoxycytidine triphosphate as co-substrate, using crude enzyme solution to conduct catalytic experiments; Step (4): Detect the reaction product by HPLC, and use the relative content of the product as an indicator to screen mutants with improved catalytic performance.

5. A highly active deoxycytidylate deaminase mutant according to claim 2, characterized in that: The enzyme activity determination steps are as follows: Step (1): using an appropriate amount of enzyme solution, 50 mM dCMP, 1 mM dCTP and 0.3 M Tris-HCl buffer to carry out a catalytic reaction at 40°C and pH 7.0 for 30 minutes; Step (2): Detect the reaction product by HPLC and calculate the amount of enzyme required to generate 1 µmol of dUMP per minute, which is defined as one unit of enzyme activity (U); Step (3): Determine the protein concentration and calculate the number of activity units per milligram of enzyme protein, i.e., specific enzyme activity (U / mg).

6. The use of a highly active deoxycytidylate deaminase mutant according to claim 1 in the field of biological enzyme catalysis, characterized in that: Application of deoxycytidylic acid deaminase mutants in catalyzing the conversion of deoxycytidylic acid (dCMP) to deoxyuridylic acid (dUMP).

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