Carboxypeptidase a mutants with improved thermal stability and activity

Abstract

The application discloses a carboxypeptidase A mutant with improved catalytic activity and thermal stability, and belongs to the field of protein engineering. The carboxypeptidase A mutant D93C / F96C is obtained by introducing a disulfide bond through rational design and site-directed mutagenesis technology. The half-life of the carboxypeptidase A mutant at 65 DEG C is increased by 13.3 min compared with that of the wild type, the half-inactivation temperature is increased by 8.5 DEG C compared with that of the wild type, and the specific activity is also increased by 24.32% compared with that of the wild type. The application breaks the trade-off between stability and activity, realizes the dual improvement of the thermal stability and catalytic activity of the carboxypeptidase A, and is favorable to the important role of the carboxypeptidase A mutant in the industrial application in the fields of food, medicine or feed, etc., such as serving as a food fermenting agent in industrial production, a bitter taste removing agent, and being applied to the removal of ochratoxin A, etc.

Description

Technical Field

[0001] This invention designs a carboxypeptidase A mutant with improved thermal stability and activity, belonging to the field of protein engineering. Background Technology

[0002] Carboxypeptidase A (CPA, EC 3.4.17.1) is a zinc-dependent metallocarboxypeptidase belonging to the M14 family. As a protein hydrolyzing and digestive enzyme, CPA specifically cleaves amino acids one by one from the C-terminus of peptide chains, releasing them. It is commonly used not only for protein sequencing but also for removing bitterness and allergens from various foods. Furthermore, it can be used as a fermentation fortifier in the processing of fermented foods such as soy sauce and cheese. In addition, CPA derived from bovine pancreas was the first enzyme discovered to have OTA-degrading activity, acting on the amide bond of OTA to hydrolyze it into OTα and L-phenylalanine, showing broad application prospects in OTA detoxification in the food, feed processing, and pharmaceutical industries.

[0003] Carboxypeptidase A (CPA), as a mesothermal enzyme, exhibits broad catalytic activity within the 30-50℃ range. However, its activity readily decreases or even disappears completely at high temperatures, severely limiting its application and development in high-temperature industrial processing environments. To address this issue, many researchers have improved its thermal stability by truncating flexible regions in the amino acid sequence of CPA and introducing proline. However, previous modifications to CPA molecules have consistently encountered a problem: increased thermal stability is accompanied by decreased catalytic efficiency. This "stability-activity trade-off"—where an increase in one property often leads to a decrease in another—is common in the modification of biological enzymes, significantly increasing the difficulty of molecular modification. Therefore, employing appropriate strategies to discover CPA mutants that offer both enhanced thermal stability and activity is crucial. Summary of the Invention

[0004] To address the aforementioned problems, this invention employs a rational design and site-directed mutagenesis of bovine pancreatic carboxypeptidase A (PDBID: 1M4L) with an amino acid sequence such as SEQ ID NO:1, using the following scheme:

[0005] The carboxypeptidase A mutant of the present invention is obtained by mutating the amino acid residues at positions 93 and 96 in the amino acid sequence of carboxypeptidase A shown in SEQ ID NO:1.

[0006] Among them, the 93rd aspartic acid is mutated to cysteine;

[0007] The phenylalanine at position 96 is mutated to cysteine;

[0008] The first objective of this invention is to provide a carboxypeptidase A mutant with improved thermal stability and catalytic activity, the amino acid sequence of which is shown in SEQ ID NO:2.

[0009] A second objective of this invention is to protect the gene encoding the above-mentioned mutant, the recombinant vector linking the gene, and the microbial cell carrying the recombinant vector linking the gene.

[0010] A third objective of this invention is to provide a method for screening mutation sites in the aforementioned mutant genes, comprising the following steps:

[0011] Step 1: Analyze the structural characteristics of wild-type carboxypeptidase derived from bovine pancreas using B-factor and molecular dynamics simulations to screen target regions.

[0012] Step 2: For the target region obtained in Step 1, combine the prediction results of Disulfide by design 2.0 and MODIP online server with the conservation analysis of the PSSM matrix to screen target sites.

[0013] The fourth objective of this invention is to provide a recombinant bacterium that expresses the above-mentioned carboxypeptidase A mutant, using plasmid pPIC9K as the expression vector and Pichia pastoris GS115 as the host.

[0014] The fifth object of the present invention is to provide the application of the above-mentioned mutant in the field of ochratoxin A removal. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below:

[0016] Figure 1 Mean B-factor value of wild-type carboxypeptidase A;

[0017] Figure 2 Root mean square fluctuation (RMSF) of wild-type carboxypeptidase A;

[0018] Figure 3 Predictions of potential disulfide bond formation by wild-type carboxypeptidase A;

[0019] Figure 4 Sequence conservation analysis of wild-type carboxypeptidase A;

[0020] Figure 5 Phenotypic identification of recombinant carboxypeptidase A mutants by PCR;

[0021] Figure 6 Thin-layer chromatography chromatograms of ochratoxin A degradation by wild-type WT and mutant D93C / F96C of carboxypeptidase A;

[0022] Figure 7 Half-life of carboxypeptidase A wild-type WT and mutant D93C / F96C at 65°C;

[0023] Figure 8 Half-inactivation temperature of carboxypeptidase A wild-type WT and mutant D93C / F96C. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the specific implementation methods of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention, which is defined by the claims.

[0025] Unless otherwise specified, all experimental reagents and materials used in the embodiments of this invention are commercially available.

[0026] Unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0027] Example 1: Rational Design of Disulfide Bond Introduction Site for Carboxypeptidase A

[0028] First, the PDB file of the three-dimensional crystal structure of CPA, as shown in SEQ ID NO:1, was uploaded to the HotSpot Wizard 3.0 online server. The amino acid residues with B-Factor values ​​in the first 25% were identified as highly flexible regions. Subsequently, the trajectory of wild-type carboxypeptidase A was simulated using Gromacs 2019.3 molecular dynamics software. Amino acid residues with RMSF values ​​greater than 0.145 nm were identified as highly flexible regions. The results are as follows: Figure 2 As shown. By comparison, the flexible sites common to both methods were identified as potential target regions for disulfide bond introduction.

[0029] To introduce disulfide bonds within the flexible region, the three-dimensional crystal structure PDB file of CPA was uploaded to Disulfide bydesign 2.0 and the MODIP online server. The former screened for bond energies (χ3Angle) at +97° or -87° (±30°) and C α -C β -S λPotential disulfide bonds located around 114.6°±10 were selected, and the latter screened disulfide bond sites in categories A and B. By comparing the disulfide bond potential site prediction results from both software programs, a total of 20 amino acid pairs belonging to both DbD and MODIP category A and B predictions were screened, as shown in the following figures. Figure 3 As shown.

[0030] The conservation of CPA amino acid sequences was analyzed using a position-specific scoring matrix (PSSM). Amino acid residues with scores ≥90 were identified as conserved sites and removed as screening criteria to prevent adverse effects of conserved site mutations on the catalytic activity of CPA.

[0031] The results are as follows Figure 1 As shown, the amino acid residues with B-factor scores in the top 25%, i.e., the regions with high CPA flexibility, are as follows: Ala1-Ser3, Asn5-Thr6, Leu15-Asp16, Glu28-Val33, Gly55-Arg59, Glu88-Asp89, Gly91-Ser95, Ser121-Gln122, Val132-Gys138, Gly152-Gly155, Lys168-Tyr169, Gln211-Ser212, Lys231-Tyr234, Ile247-Gln249, Arg276-Tyr277, and Glu302-Asn307.

[0032] The results are as follows Figure 2 As shown, based on the root mean square deviation (RMSF) calculated using the molecular dynamics simulation software GROMACS2019.3, the flexible sites and regions of CPA can be determined as follows: Ala1-Arg2, Asn5-Thr6, Gly55-Asn58, Gln92-Ser95, Val132-Leu137, Asp148-Gly150, Lys153-Ala154, Lys168-Tyr169, Lys184-Asn188, Lys231-Tyr234, Try248-Gly252, and Asn306-Asn307.

[0033] The results are as follows Figure 3 As shown, in order to obtain more accurate and reliable residue pairs that form potential disulfide bonds, the prediction results of two calculation tools, DbD and MODIP, were compared, and a total of 20 amino acid pairs that belong to both DbD and MODIP A and B grade prediction results were screened out.

[0034] The results are as follows Figure 4 As shown, the CPA sequence contains 151 conserved sites, including the Ser136 site, which is the site to be mutated. Therefore, the Ser136 site is excluded to prevent mutation at this site from causing a decrease in protein activity or even inactivation.

[0035] Example 2 Construction of recombinant strains with carboxypeptidase A mutant

[0036] Based on yeast codon bias, the disulfide bond mutant cpa gene was optimized and inserted between the EcoRI and NotI restriction sites of the expression plasmid pPIC9K, and then transformed into a preserved Escherichia coli DHα strain. The above recombinant plasmid construction process was completed by Shanghai Sangon Biotech Co., Ltd.

[0037] 80 μL of Pichia pastoris GS115 competent cells and 10 μL of linearized recombinant plasmid were mixed thoroughly and electroporated once using an electroporator at 1500 V, 25 μF, and 200 Ω. The bacterial suspension was resuspended in 1 M sterile sorbitol solution and mixed thoroughly. All electroporated material was then transferred to a 1.5 mL centrifuge tube and incubated at 28°C for 30 min. 150 μL of the electroporated material was plated on auxotrophic MD medium and incubated upside down at 28°C for 3–4 days until single colonies appeared. Preliminary screening for HIS was then conducted. + Transformants. HIS were picked up from the MD plate using a sterile toothpick. + Transformants were inoculated onto YPD solid medium containing different concentrations of G418 sulfate (0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 4 mg / mL), totaling 50 HIS transformants. + Transformants were then cultured overnight in a yeast incubator at 28°C, followed by inverted culture for 3–5 days. Genomic DNA was extracted from single colonies and PCR amplification was performed using universal primers 5'AOX1 (5-GACTGGTTCCAATTGACAAGC-3) and 3'AOX1 (5-GCAAATGGCATTCTGACATCC-3) under the following conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, for a total of 35 cycles; 72°C extension for 7 min, followed by incubation at 4°C. The recombinant was identified by 1% agarose gel electrophoresis to determine whether it was a Mut gene capable of utilizing methanol as the sole carbon source. + Phenotype.

[0038] The results are as follows Figure 5As shown, after PCR reaction using the universal AOX1 primers, all recombinants showed two bands. One band was associated with the target gene, approximately 1442 bp in size (the sum of the 950 bp target gene and the 492 bp AOX1 promoter fragment of the recombinant vector), and the other band was the AOX1 gene from the Pichia pastoris genome, approximately 2200 bp in size. The results indicate that the selected HIS... + All recombinants are Mut + The phenotype shows a strong ability to metabolize methanol, allowing it to grow on media where methanol is the sole carbon source, and it can be used for subsequent Pichia pastoris expression experiments.

[0039] Example 3: Expression and purification of carboxypeptidase A mutant protein

[0040] Inoculate 1% of the culture into 25 mL of BMGY liquid medium and culture at 28 °C with shaking at 200 rpm / min until the cell concentration reaches OD500. 600 =2~6. Centrifuge at 4000 rpm for 5 min at room temperature, collect bacterial cells, discard the supernatant, and resuspend the cells in 20 mL of BMMY liquid medium. Subsequently, add methanol to a final concentration of 1% and induce expression at 28℃ and 250 rpm / min for 6 days, supplementing with methanol to a final concentration of 1% every 24 h. After the induction expression is completed, centrifuge the fermentation broth at 4℃ and 10,000 rpm for 5 min, and collect the fermentation supernatant.

[0041] To obtain high-purity enzyme protein, the fermentation supernatant was pretreated with 2M ammonium sulfate, filtered through a 0.22μm filter membrane, and then subjected to hydrophobic chromatography using a HiTrap pre-packed column. TM Purification was performed using Phenyl FF. The target protein was eluted with Tris-HCl buffer (pH 7.4) of different concentrations of ammonium sulfate, and the eluted mutant purified protein was concentrated and removed by ultrafiltration centrifuge tubes and stored at 4°C.

[0042] Example 4: Verification of Ochratoxin A Activity in Carboxypeptidase A Mutant Protein

[0043] Take 300 μL of protein sample and add 2.5 μL of 120 μg / mL OTA solution (final OTA concentration 1 μg / mL). Use an equal volume of 20 mM Tris-HCl buffer (pH 7.4) as a blank control. After the protein and substrate reaction system is thoroughly mixed, incubate at 37 °C in the dark for 72 h. After incubation, 20 μL of 6M HCl was added to acidify the reaction system, followed by 100 μL of chloroform (CHCl3). The mixture was then thoroughly vortexed to facilitate better extraction of OTA and degradation products. The system was then centrifuged at 12,000 rpm for 2 min to ensure complete separation of the organic and inorganic phases. The lower layer (10 μL of CHCl3) was spotted onto a C18 reverse-phase thin-layer plate. An appropriate amount of developing solvent (toluene:ethyl acetate:formic acid = 6:3:1) was added to a sealed container. Development was stopped when the solvent reached 2 / 3 of the plate's height. The developing solvent was recovered, and the C18 reverse-phase thin-layer plate was dried using a blower. The fluorescence intensity was then observed under a 365 nm UV light source. Since OTA emits blue-green fluorescence under 365 nm UV light, the fluorescence intensity compared to the control group can verify whether wild-type and disulfide-mutant recombinant CPA possess OTA degradation activity.

[0044] The results are as follows Figure 6 As shown in the figure, compared to the CK control, the blue-green fluorescence of the carboxypeptidase A mutant D93C / F96C almost disappeared, and a blue-purple fluorescent substance was generated, indicating that the recombinant carboxypeptidase A mutant expressed by Pichia pastoris has OTA degradation activity within a certain reaction time.

[0045] Example 5: Determination of the activity and thermal stability of carboxypeptidase A mutant protein

[0046] A reaction system was prepared by mixing 108 μL of substrate Z-Phe-Leu solution (final concentration 1 mM) and 12 μL of recombinant CPA (final concentration 10 mg / mL), and incubated at 37 °C with shaking for 15 min. Subsequently, 240 μL of cadmium-ninhydrin reagent was added to the reaction system, mixed thoroughly, and heated in a water bath at 84 °C for 5 min. After cooling to room temperature, the mixture was diluted 6-fold with 20 mM Tris-HCl buffer, and the absorbance value A at 507 nm was measured. 507CPA enzyme activity is defined as the number of micromoles of leucine produced by wild-type and mutant CPA from the substrate Z-Phe-Leu under conditions of pH 7.4 and 37°C, expressed in U. The enzyme activity calculation formula is: W = 1000XYf / T. Where W is the CPA enzyme activity (μmol / min), X is the L-leucine concentration (μmol / L), Y is the total volume of the reaction system between CPA and substrate Z-Phe-Leu (L), T is the total reaction time between CPA and substrate Z-Phe-Leu (min), and f is the dilution factor of the reaction volume. Specific enzyme activity is the enzyme activity per milligram of carboxypeptidase A. Specific enzyme activity is expressed as enzyme activity / mg (U / mg).

[0047] An appropriate amount of recombinant CPA was incubated at 65℃ for 10 min, 20 min, 30 min, 40 min, and 50 min, respectively, and then immediately placed on ice for 10 min. 12 μL of the treated recombinant CPA was added to 108 μL of Z-Phe-Leu substrate solution (final concentration 1 mM) to form a reaction system, and enzyme activity was measured according to the enzyme activity method described above. The enzyme activity of the untreated sample was set as 100%, and each group was divided into three replicates. The ordinate represents the natural logarithm of the remaining activity of wild-type and mutant CPA, and the abscissa represents time t. A linear fit was performed, and the slope of the line represents the inactivation rate constant k of CPA. d The formula for calculating half-life is: t 1 / 2 =ln2 / K d .

[0048] Take appropriate amounts of recombinant CPA and heat-treat them at 40℃, 50℃, 60℃, and 70℃ for 15 min each, then immediately place the samples on ice for 10 min. Add 12 μL of the treated recombinant CPA to 108 μL of substrate Z-Phe-Leu solution (final concentration 1 mM) to form the reaction system. Determine the enzyme activity according to the method in 4.3.1.1. Set the enzyme activity of the untreated samples as 100%, and set three replicates for each group. The temperature at which the remaining activity decreases to 50% of the initial activity is defined as T. 50 15 .

[0049] The results are shown in Table 1. It can be seen that the catalytic activity of the carboxypeptidase A mutant on the substrate Z-Phe-Leu is 24.32% higher than that of WT.

[0050] Table 1. Specific enzyme activities of carboxypeptidase A wild-type WT and mutant D93C / F96C

[0051]

[0052] The results are as follows Figure 7 As shown, the half-life t of the carboxypeptidase A mutant D93C / F96C at 65℃ 1 / 2The effective time was 50.8 min, an increase of 13.3 min compared to the WT. The results indicate that the introduction of disulfide bond mutations enhances the heat resistance of CPA, allowing its activity to be maintained for a longer period at higher temperatures.

[0053] The results are as follows Figure 8 As shown, WT's T 50 15 The T value of mutant D93C / F96C was 58.0℃. 50 15 The temperature was 66.5℃, an increase of 8.5℃ compared to the WT. These results indicate that the introduction of disulfide bond mutations enhances the thermostability of carboxypeptidase A, enabling it to maintain higher activity at higher temperatures.

[0054] Finally, the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail above, modifications can still be made to the technical solutions in the above embodiments or equivalent substitutions can be made to some of the technologies. Therefore, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A carboxypeptidase A mutant, characterized in that, The mutant uses bovine pancreatic carboxypeptidase A, whose amino acid sequence is shown in SEQ ID NO:1, as the wild type. The aspartic acid at position 93 and the phenylalanine at position 96 of the wild type carboxypeptidase A are both mutated to cysteine. The amino acid sequence of the mutant carboxypeptidase A is shown in SEQ ID NO:

2.

2. The gene encoding the carboxypeptidase A mutant as described in claim 1.

3. A recombinant expression vector carrying the gene as described in claim 2.

4. A recombinant strain carrying the gene as described in claim 2 or carrying the recombinant expression vector as described in claim 3.

5. The recombinant strain according to claim 4, characterized in that, The recombinant strain is a bacterium or a fungus.

6. The application of the carboxypeptidase A mutant as described in claim 1 in the field of ochratoxin A removal.

7. The application according to claim 6, characterized in that, The crude enzyme solution or purified protein of the carboxypeptidase A mutant is added to the ochratoxin A degradation system to achieve the degradation of ochratoxin A.

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