A highly active, thermostable alkaline protease mutant

Abstract

The present invention relates to the field of enzyme engineering technology and discloses a highly active, thermostable alkaline protease mutant, comprising: a highly active, thermostable alkaline protease mutant obtained by removing amino acid residues from positions 1 to 286 of the amino acid sequence shown in SEQ ID NO: 2, replacing glutamine at position 346 with asparagine, and replacing aspartic acid at position 521 with threonine, wherein the amino acid sequence of the highly active, thermostable alkaline protease mutant is shown in SEQ ID NO: 3 and the gene sequence is shown in SEQ ID NO: 4. Thus, the stability and activity of the serine protease are improved.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, and in particular to a high-activity, high-temperature-resistant alkaline protease mutant. Background Art

[0002] Alkaline proteases, widely used in industrial production, are primarily derived from microorganisms, including bacteria (such as Staphylococcus, Bacillus subtilis, and Bacillus pumilus), actinomycetes (such as Streptomyces griseus), and fungi (such as Aspergillus oryzae and Aspergillus ochraceus). Based on their site of action and chemical properties, alkaline proteases can be divided into serine proteases (such as Novo protease) and metalloproteases (such as Carsberg protease). These enzymes hydrolyze peptide bonds in proteins under alkaline conditions, converting large proteins into smaller peptide chains or amino acids. Alkaline proteases exhibit optimal activity in the pH range of 9 to 11, rapidly losing activity at pH levels below 6 or above 11.

[0003] Among them, serine protease HtrA-like (EC3.4.21) is the most widely used alkaline protease. Its active center is usually serine, and it catalyzes the hydrolysis reaction through the Asp-His-Ser triad, while metal ions (such as Ca 2+ 、Mn 2+ ) can significantly improve its catalytic activity. Due to its mild reaction conditions and wide range of substrates, serine proteases have been widely used in industry.

[0004] Despite their widespread industrial application, these alkaline proteases still face several challenges. First, their unstable enzyme activity easily leads to inactivation under high temperatures, strong acidic or alkaline conditions, necessitating protective measures. Second, their low hydrolysis efficiency in high-osmotic pressure environments limits their application and hinders their widespread adoption in practical production processes. Therefore, improving the stability of serine proteases could enhance their catalytic efficiency in practical production processes, further expanding their potential for industrial application. Summary of the Invention

[0005] This summary is intended to briefly introduce concepts that will be described in detail in the detailed description below. This summary is not intended to identify key features or essential features of the claimed technical solution, nor is it intended to limit the scope of the claimed technical solution.

[0006] The present invention provides a highly active, high-temperature-resistant alkaline protease mutant to solve one or more of the technical problems mentioned in the above background technology section.

[0007] The present invention provides a highly active, thermostable alkaline protease mutant, comprising: a highly active, thermostable alkaline protease mutant, wherein the highly active, thermostable alkaline protease mutant is obtained by removing amino acid residues from positions 1 to 286 of an amino acid sequence as shown in SEQ ID NO: 2, replacing glutamine at position 346 with asparagine, and replacing aspartic acid at position 521 with threonine, wherein the amino acid sequence of the highly active, thermostable alkaline protease mutant is as shown in SEQ ID NO: 3, and the gene sequence is as shown in SEQ ID NO: 4.

[0008] Optionally, the amino acid sequence of the highly active thermostable alkaline protease mutant is:

[0009] MKIAKNSVKSVVTVENDLSNDTTVSDNKNESDNEIGSGVVYKKVGDSIYIFTNAHVVGNNEKQKVTYGNDKSVTGKVIGKDKWSDLAVVKAKVADENIKPMTMGDSNNIKLAEPILVIGNPLGTDFKGSVSQGIVSGLNRHVPVDIDKNDNYDALMKAFQ IDAPVNPGNSGGAVVDRDGRLIGIVSLKIDMHNVEGMAFAIPINDVRKIAKELEHKGKVNYPNTEIKIKNVGDLTDSERNAINLPTKVNHGVLIGEVKENGLGDKSGLKKGDVIVELDGKKIEDNLRYRQVIYSHYDDQKTITAKIYRNGAEKNIKIKLK.

[0010] Optional, the gene sequence of the highly active high-temperature resistant alkaline protease mutant is:

[0011] atgaaaattgcgaaaaacagcgtgaaaagcgtggtgaccgtggaaaacgatctgagcaacgataccaccgtgagcgataacaaaaacgaaagcgataacgaaattggcagcggcgtggtgtataaaaaagtgggcgatagcatttatatttttaccaacgcgcatgtggtgggcaacaacgaaaaacagaaagtgacctatggcaacgataaaagcgtgaccggcaaagtgattggcaaagataaatggagcgatctggcggtggtgaaagcgaaagtggcggatgaaaacattaaaccgatgaccatgggcgatagcaacaacattaaactggcggaaccgattctggtgattggcaacccgctgggcaccgattttaaaggcagcgtgagccagggcattgtgagcggcctgaaccgccatgtgccggtggatattgataaaaacgataactatgatgcgctgatgaaagcgtttcagattgatgcgccggtgaacccgggcaacagcggcggcgcggtggtggatcgcgatggccgcctgattggcattgtgagcctgaaaattgatatgcataacgtggaaggcatggcgtttgcgattccgattaacgatgtgcgcaaaattgcgaaagaactggaacataaaggcaaagtgaactatccgaacaccgaaattaaaattaaaaacgtgggcgatctgaccgatagcgaacgcaacgcgattaacctgccgaccaaagtgaaccatggcgtgctgattggcgaagtgaaagaaaacggcctgggcgataaaagcggcctgaaaaaaggcgatgtgattgtggaactggatggcaaaaaaattgaagataacctgcgctatcgccaggtgatttatagccattatgatgatcagaaaaccattaccgcgaaaatttatcgcaacggcgcggaaaaaaacattaaaattaaactgaaa。

[0012] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0013] The HtrA protease gene of alkaline protease (as shown in SEQ ID NO.1) was connected to the pET28b plasmid using a one-step cloning method to obtain the expression vector pET28b-HtrA capable of expressing the HtrA protease gene;

[0014] The pET28b-HtrA containing the HtrA protease gene was introduced into E. coli BL21 (DE3) using the heat transformation method to obtain the E. coli BL21 (DE3)-pET28b-HtrA engineered strain;

[0015] Through protein engineering technology, the amino acid residues 1 to 286 of the amino acid sequence of HtrA were removed, and the glutamine (Gln / Q) at position 346 was replaced with asparagine (Asn / N), and the aspartic acid (Asp / D) at position 521 was replaced with threonine (Thr / T), and it was named HtrAP.

[0016] The amino acid sequence corresponding to the mutant HtrAP is SEQ ID NO.3, and the gene sequence is SEQ ID NO.4;

[0017] The mutant HtrAP protease gene (SEQ ID NO. 4) was connected to the pET28b plasmid using a one-step cloning method to obtain an expression vector pET28b-HtrAP capable of expressing the mutant HtrAP protease gene;

[0018] The expression vector pET28b-HtrAP containing the mutant HtrAP protease gene was introduced into E. coli BL21 (DE3) using the thermal transformation method to obtain the E. coli BL21 (DE3)-pET28b-HtrAP engineered strain;

[0019] The present invention provides the gene sequence of HtrA and the gene sequence of mutant HtrAP;

[0020] The present invention provides the amino acid sequence of HtrA and the amino acid sequence of mutant HtrAP;

[0021] The present invention provides an expression vector containing an HtrA gene sequence and an expression vector containing a mutant HtrAP gene sequence;

[0022] The present invention provides an E. coli BL21 (DE3)-pET28b-HtrA engineering strain containing an HtrA gene sequence expression vector and an E. coli BL21 (DE3)-pET28b-HtrAP engineering strain containing a mutant HtrAP gene sequence expression vector;

[0023] The engineered strain containing the HtrA gene sequence expression vector and the engineered strain containing the mutant HtrAP gene sequence expression vector were cultured separately in a fermenter at 37°C until OD600 = 1.3, then cooled to 16°C and induced with IPTG (final concentration of 1.0 mM) for 24 h.

[0024] The engineered strain containing the HtrA gene sequence expression vector and the engineered strain containing the mutant HtrAP gene sequence expression vector were lysed and then purified using Ni2+-NTA affinity chromatography filler;

[0025] The activities of the purified HtrA protease and the purified mutant HtrAP protease were tested at 35°C. The activity test results showed that the activity of the purified HtrA protease at 35°C was 185% of that of the purified mutant HtrAP protease;

[0026] The thermal stability of the purified HtrA protease and the purified mutant HtrAP protease was tested. The results showed that the optimal reaction temperature of the purified HtrA protease was 45°C, while the optimal reaction temperature of the purified mutant HtrAP protease was 65°C, an increase of 20°C.

[0027] Purified HtrA protease and purified mutant HtrAP protease were incubated at 45°C for various periods of time, and their relative residual enzyme activities were measured. Purified HtrA protease was completely inactivated within 10 hours, while purified mutant HtrAP protease still retained approximately 75% residual activity after 24 hours.

[0028] An E. coli BL21(DE3)-pET28b-HtrA engineered strain containing an expression vector for the HtrA gene sequence and an E. coli BL21(DE3)-pET28b-HtrAP engineered strain containing an expression vector for the mutant HtrAP gene sequence were immobilized, and their residual enzyme activities were measured when stored at 35°C. The E. coli BL21(DE3)-pET28b-HtrA engineered strain containing the HtrA gene sequence was completely inactivated within 15 days, while the E. coli BL21(DE3)-pET28b-HtrAP engineered strain containing the mutant HtrAP gene sequence still had 50% residual activity after 50 days.

[0029] The present invention has the following beneficial effects: it improves the stability of proteases and can enhance their catalytic efficiency during actual production. Specifically, the alkaline protease gene (SEQ ID NO. 1, NCBI: WP_002499276.1), a serine protease derived from Staphylococcus epidermidis (NIHLM037, Taxonomy ID: 979210), is mutated to remove the large random coil segment at the nitrogen terminus of the protein. Subsequently, amino acid residues on the periphery of the protein that affect protein stability are mutated, resulting in a mutant with significantly improved catalytic activity and thermal stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The above and other features, advantages, and aspects of the various embodiments of the present invention will become more apparent with reference to the following detailed description in conjunction with the accompanying drawings. Throughout the drawings, the same or similar reference numerals represent the same or similar elements. It should be understood that the drawings are schematic and that the elements are not necessarily drawn to scale.

[0031] Figure 1 It is an SDS-PAGE diagram of an E. coli BL21 (DE3)-pET28b-HtrA engineering strain and an E. coli BL21 (DE3)-pET28b-HtrA engineering strain after cytosolic production of a high-activity high-temperature resistant alkaline protease mutant of the present invention;

[0032] Figure 2 It is a bar graph showing the specific enzymatic activity of the purified HtrA protease and the specific enzymatic activity of the purified mutant HtrAP protease of a highly active thermostable alkaline protease mutant of the present invention;

[0033] Figure 3 It is a relative activity curve diagram of the purified HtrA protease and the purified mutant HtrAP protease of a highly active high-temperature-resistant alkaline protease mutant of the present invention;

[0034] Figure 4 The present invention is a high-activity high-temperature resistant alkaline protease mutant purified HtrA protease and purified mutant HtrAP protease treated at 45°C for different time periods, and the residual enzyme activity curves at 35°C are measured;

[0035] Figure 5 The present invention is a graph showing residual enzyme activity of an immobilized engineered bacterium and an immobilized mutant engineered bacterium of a highly active, high-temperature-resistant alkaline protease mutant. DETAILED DESCRIPTION

[0036] The present invention will be described in more detail below with reference to the accompanying drawings. Although certain embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0037] It should also be noted that, for ease of description, only the parts related to the invention are shown in the drawings. In the absence of conflict, the embodiments and features of the embodiments of the present invention may be combined with each other.

[0038] It should be noted that the concepts of "first" and "second" mentioned in the present invention are only used to distinguish different devices, modules or units, and are not used to limit the order or interdependence of the functions performed by these devices, modules or units.

[0039] It should be noted that the modifications of "one" and "multiple" mentioned in the present invention are illustrative rather than restrictive. Those skilled in the art should understand that unless otherwise clearly indicated in the context, it should be understood as "one or more".

[0040] The names of the messages or information exchanged between multiple devices of the present invention are only used for illustrative purposes and are not used to limit the scope of these messages or information.

[0041] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0042] Example 1: Construction of expression vector

[0043] The HtrA protease gene was synthesized from the genome of Staphylococcus epidermidis (NIHLM037, Taxonomy ID: 979210) using polymerase chain reaction (PCR). The primers used for PCR synthesis of the HtrA protease gene (SEQ ID NO. 1) were synthesized by Hangzhou Qingke Biotechnology Co., Ltd. The primer sequences for constructing the HtrA protease gene are as follows: F-htrA: aatgggtcgggatccgaattcATGGATAACGATAAAAAACAGGTGAT; R-htrA: ctcgagtgcggccgcaagcttTTTCAGTTTAATTTTAATGTTTTTTTCCG. The resulting PCR product was identified by agarose gel electrophoresis and purified. The purified PCR product was then ligated into the pET28b plasmid treated with EcoRI and HindIII restriction enzymes using the NEBuilder HiFi Assembly Kit to construct the expression vector pET28b-HtrA. The primers used for PCR synthesis of the mutant HtrAP protease gene were synthesized by Hangzhou Qingke Biotechnology Co., Ltd. The primers for constructing the mutant HtrAP protease gene are as follows:

[0044] F-htrAP: aatgggtcgggatccgaattcATGAAAATTGCGAAAAACAGCG;

[0045] R-htrAP:ctcgagtgcggccgcaagcttTTTCAGTTTAATTTTAATGTTTTTTTCCG.

[0046] Then use the following mutation primers:

[0047] F-Q346N: GGGCAACaacGAAAAACAGAAAGTGACCTATGGCA;

[0048] R-Q346N: GTTTTTTCgttGTTGCCCACCACATGCGCGTTG;

[0049] F-D521T:GATCTGaccGATAGCGAACGCAACGCGATTAA;

[0050] R-D521T:TCGCTATCggtCAGATCGCCCACGTTTTTAATT.

[0051] The sequence of the synthesized mutant HtrAP protease gene is shown in SEQ ID NO. 4, and the expression vector finally constructed is pET28b-HtrAP.

[0052] Example 2: Construction of recombinant bacteria

[0053] The expression vector pET28b-HtrA and the expression vector pET28b-HtrAP in Example 1 were extracted and transformed into E. coli BL21 (DE3) using a thermal transformation method (42°C, 30 s). The transformants were spread onto LB solid culture medium containing 50 μg / mL and cultured at 37°C for 16 hours to obtain E. coli BL21 (DE3)-pET28b-HtrA and E. coli BL21 (DE3)-pET28b-HtrAP engineered strains.

[0054] Example 3: Exogenous induced expression

[0055] The method for inducing expression of the target protein HtrA in the E. coli BL21(DE3)-pET28b-HtrA engineered strain is as follows: First, the E. coli BL21(DE3)-pET28b-HtrA engineered strain is cultured in LB medium (37°C) containing 50 μg / mL kanamycin to an OD600 of 1.3. Next, the culture temperature is lowered to 16°C, and isopropyl-β-D-thiogalactopyranoside (IPTG) is added to a final concentration of 1.0 mM to induce expression of the target protein. Induction can be performed at 37°C for 4 to 6 hours or overnight at 16°C to 20°C. This method yields high levels of recombinant protein with good solubility. Finally, the induced engineered cells are harvested and the target protein is extracted by ultrasonication or other methods. The crude protein can be purified using methods such as His-tag affinity chromatography. The expression level and purity of the target protein can be determined by SDS-PAGE analysis. In practice, if necessary, parameters such as IPTG concentration, induction time, and temperature can be further optimized to achieve optimal expression. Throughout the induction expression process, culture conditions must be strictly controlled, and appropriate protein extraction and purification methods must be employed to obtain highly pure and active recombinant target proteins.

[0056] Example 4: Enzyme purification

[0057] Purification of the HtrA protein expressed in the E. coli BL21(DE3)-pET28b-HtrA engineered strain and the HtrAP protein expressed in the E. coli BL21(DE3)-pET28b-HtrAP engineered strain can be achieved by the following steps: First, the cells after induced expression are harvested by centrifugation and resuspended in PBS buffer (pH 7.0). Ultrasonication (5s on, 10s off, 30 cycles) is then used to disrupt the cells and release the target protein. Conditions are optimized to optimize the disruption effect. Next, utilizing the affinity of the N-terminal His-tag of the HtrA protein and the HtrAP protein, preliminary purification is performed using a Ni2+-NTA affinity chromatography column. The disrupted cell lysate is loaded onto the column (PBS buffer containing 20 mM imidazole). After elution (PBS buffer containing 200 mM imidazole), the His-tag-fused HtrA and HtrAP proteins are obtained in relatively pure form. Finally, the eluted HtrA and HtrAP enzyme proteins were desalted using a desalting column (Desalting Column) H to remove high-concentration imidazole, added with 20% glycerol, quickly frozen in liquid nitrogen, and then stored in a refrigerator at -80°C.

[0058] Example 5: Enzyme activity assay

[0059] The specific enzymatic activity of the purified HtrA protease and the purified mutant HtrAP protease is expressed as enzyme activity per microgram of protease (U / μg). The definition of the enzyme activity unit (U) is the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine (Tyr) per minute. The determination method is to first use β-casein as a substrate to determine the rate at which the protease catalyzes the hydrolysis of the substrate at 35°C and pH 7.0 to obtain the enzyme activity value. Then, a protein concentration kit is used to determine the total protein content of the purified enzyme solution. Finally, the measured enzyme activity is divided by the total protein content to obtain the specific activity of the protease. It was determined that the specific enzymatic activity of the purified HtrA protease was 12568±4217.9U / μg, and the specific enzymatic activity of the purified mutant HtrAP protease was 23250.8±1590.8U / μg (e.g. Figure 2 shown).

[0060] Example 6: Thermal stability determination

[0061] The relative activities of the purified HtrA protease and the purified mutant HtrAP protease were measured in PBS buffer. The highest enzyme activity of the purified HtrA protease and the purified mutant HtrAP protease was set as 100%, and the relative enzyme activity was calculated. The relative enzyme activity curve with temperature as the variable was drawn (e.g. Figure 3The optimal temperature range for purified HtrA protease and purified mutant HtrAP protease was 10-80°C. The results showed that the optimal temperature for purified HtrA protease was 45°C, while the optimal temperature for purified mutant HtrAP protease was 65°C.

[0062] After the purified HtrA protease and the purified mutant HtrAP protease were treated at 45°C for different time periods, their residual enzyme activities at 35°C, i.e., thermal stability (e.g. Figure 4 The horizontal axis represents temperature (°C), and the vertical axis represents relative enzyme activity (%). The purified HtrA protease was completely inactivated within 10 hours, while the purified mutant HtrAP protease still had approximately 75% residual enzyme activity after 24 hours.

[0063] Example 7: Immobilization of whole cells containing recombinant enzyme and detection of residual enzyme activity

[0064] First, the E. coli BL21(DE3)-pET28b-HtrA engineered strain containing the HtrA gene expression vector and the E. coli BL21(DE3)-pET28b-HtrAP engineered strain containing the mutant HtrAP gene expression vector were harvested by low-speed centrifugation. The culture medium was then washed repeatedly with saline or buffer to remove impurities. Due to the specificity of the protease substrate, the cell wall was treated by three freeze-thaw cycles to increase permeability. Next, a suitable immobilization support, namely, polymer microspheres composed of polyacrylonitrile, was selected. The immobilization process then began. The pretreated engineered bacteria were mixed with the selected immobilization support, and an appropriate amount of glutaraldehyde crosslinking agent was added. The reaction was allowed to proceed at a mild temperature of approximately 4°C for 12 hours to firmly bind the bacteria to the support surface. Finally, the immobilized engineered bacteria preparation was obtained after two washes with saline and drying.

[0065] In order to evaluate the enzyme activity of the immobilized engineered bacteria, it is necessary to first determine its initial enzyme activity as a benchmark for subsequent comparison. The same enzyme activity determination method as before can be used for determination using the corresponding substrate and detection conditions. Next, a preservation experiment is carried out. The prepared immobilized engineered bacteria preparation is placed in a beaker and placed in a constant temperature incubator, maintained at a constant temperature of 35°C for 50 days. Every five days, the residual enzyme activity of the immobilized bacterial cells during the preservation process is determined using the same method as the initial determination. The enzyme activity data at different time points are compared, and the percentage of the residual enzyme activity to the initial value is calculated to analyze the enzyme activity stability of the immobilized engineered bacteria after 50 days of storage at 35°C. The immobilized engineered bacteria corresponding to E. coli BL21 (DE3) -pET28b-HtrA are completely inactivated within 15 days, and the immobilized mutant engineered bacteria corresponding to E. coli BL21 (DE3) -pET28b-HtrAP can still maintain about 50% residual enzyme activity within 50 days (such as Figure 5 shown).

[0066] In these examples, improving the stability of the protease can enhance its catalytic efficiency during actual production. Specifically, the alkaline protease gene (SEQ ID NO. 1, NCBI: WP_002499276.1), a serine protease derived from Staphylococcus epidermidis (NIHLM037, Taxonomy ID: 979210), was mutated to remove the large random coil segment at the nitrogen terminus of the protein. Subsequently, amino acid residues on the periphery of the protein that affect protein stability were mutated, resulting in a mutant with significantly enhanced catalytic activity and thermal stability.

[0067] The above descriptions are merely some preferred embodiments of the present invention and illustrate the underlying technical principles. Those skilled in the art should understand that the scope of the present invention is not limited to technical solutions formed by specific combinations of the aforementioned technical features. It also encompasses other technical solutions formed by any combination of the aforementioned technical features or their equivalents, without departing from the aforementioned inventive concept. For example, a technical solution formed by replacing the aforementioned features with (but not limited to) technical features with similar functions disclosed in this invention.

Claims

1. A highly active, high-temperature-resistant alkaline protease mutant, characterized in that: include: A highly active, thermostable alkaline protease mutant is obtained by removing the amino acid residues from positions 1 to 286 of the amino acid sequence shown in SEQ ID NO: 2, replacing the glutamine at position 346 with asparagine, and replacing the aspartic acid at position 521 with threonine, wherein the amino acid sequence of the highly active, thermostable alkaline protease mutant is shown in SEQ ID NO: 3 and the gene sequence is shown in SEQ ID NO:

4.

2. A highly active, high-temperature-resistant alkaline protease mutant according to claim 1, characterized in that: The amino acid sequence of the highly active high-temperature resistant alkaline protease mutant is: MKIAKNSVKSVVTVENDLSNDTTVSDNKNESDNEIGSGVVYKKVGDSIYIFTNAHVVGNNEKQKVTYGNDKSVTGKVIGKDKWSDLAVVKAKVADENIKPMTMGDSNNIKLAEPILVIGNPLGTDFKGSVSQGIVSGLNRHVPVDIDKNDNYDALMKAFQ IDAPVNPGNSGGAVVDRDGRLIGIVSLKIDMHNVEGMAFAIPINDVRKIAKELEHKGKVNYPNTEIKIKNVGDLTDSERNAINLPTKVNHGVLIGEVKENGLGDKSGLKKGDVIVELDGKKIEDNLRYRQVIYSHYDDQKTITAKIYRNGAEKNIKIKLK.

3. A highly active, high-temperature-resistant alkaline protease mutant according to claim 2, characterized in that: The gene sequence of the highly active high-temperature resistant alkaline protease mutant is: atgaaaattgcgaaaaacagcgtgaaaagcgtggtgaccgtggaaaacgatctgagcaacgataccaccgtgagcgataacaaaaacgaaagcgataacgaaattggcagcggcgtggtgtataaaaaagtgggcgatagcatttatatttttaccaacgcgcatgtggtgggcaacaacgaaaaacagaaagtgacctatggcaacgataaaagcgtgaccggcaaagtgattggcaaagataaatggagcgatctggcggtggtgaaagcgaaagtggcggatgaaaacattaaaccgatgaccatgggcgatagcaacaacattaaactggcggaaccgattctggtgattggcaacccgctgggcaccgattttaaaggcagcgtgagccagggcattgtgagcggcctgaaccgccatgtgccggtggatattgataaaaacgataactatgatgcgctgatgaaagcgtttcagattgatgcgccggtgaacccgggcaacagcggcggcgcggtggtggatcgcgatggccgcctgattggcattgtgagcctgaaaattgatatgcataacgtggaaggcatggcgtttgcgattccgattaacgatgtgcgcaaaattgcgaaagaactggaacataaaggcaaagtgaactatccgaacaccgaaattaaaattaaaaacgtgggcgatctgaccgatagcgaacgcaacgcgattaacctgccgaccaaagtgaaccatggcgtgctgattggcgaagtgaaagaaaacggcctgggcgataaaagcggcctgaaaaaaggcgatgtgattgtggaactggatggcaaaaaaattgaagataacctgcgctatcgccaggtgatttatagccattatgatgatcagaaaaccattaccgcgaaaatttatcgcaacggcgcggaaaaaaacattaaaattaaactgaaa。

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