L-lactic dehydrogenase A mutant MALDHAK248A with reduced enzyme activity and application of L-lactic dehydrogenase A mutant MALDHAK248A

Abstract

The invention discloses an L-lactic dehydrogenase A mutant MALDHAK248A with reduced enzyme activity and an application of the L-lactic dehydrogenase A mutant MALDHAK248A. The mutant MALDHAK248A of the L-lactic dehydrogenase A has an amino acid sequence as shown in SEQ ID NO.3. The invention also discloses a preparation method of the mutant MALDHAK248A. The invention provides a mutant MALDHAK248A of L-lactic dehydrogenase A. The amino acid sequence of the mutant MALDHAK248A is as shown in SEQ ID NO.3. The mutant MALDHAK248A can be used for preparing L-lactic dehydrogenase A. The invention further provides a preparation method of the mutant MALDHAK248A. According to the mutant MALDHAK248A, site-specific mutagenesis is carried out on an amino acid sequence as shown in SEQ ID NO.2, Lys at the 248th site is mutated into Ala, so that the activity of the L-lactic dehydrogenase A is reduced, and the enzyme activity of the L-lactic dehydrogenase A is reduced by 31%. The L-lactic dehydrogenase A mutant MALDHAK248A obtained by the invention can be used as a mutant material capable of remarkably reducing the catalytic activity of an enzyme, and provides reference for further research on the functions of the L-lactic dehydrogenase A.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and specifically relates to a mutant MaLDHA of L-lactate dehydrogenase A with reduced enzyme activity K248A and its application. BACKGROUND

[0002] L-lactate dehydrogenase A (LDHA) is an important enzyme belonging to the oxidoreductase class. Its main function is to catalyze the reversible conversion between lactate and pyruvate, accompanied by the reduction of NAD+ to NADH, and vice versa. In plants, LDHA is widely present in various tissues, especially in roots, seedlings, and potato tubers, and its function is closely related to anaerobic metabolism. Under low-oxygen or anaerobic conditions, LDHA helps plant cells maintain the regeneration of NAD+ required in the glycolysis process by converting pyruvate to lactate, thereby continuing to produce energy. In addition, LDHA also plays an important role in other physiological processes of plants. For example, in leaf cells, LDHA may be involved in regulating cell pH and controlling the concentration of reducing equivalents in the cytoplasm. In some plants, LDHA may also be involved in metabolic pathways related to photosynthesis.

[0003] By studying LDHA through site-directed mutagenesis technology, we can gain a deeper understanding of the relationship between its structure and function, and explore potential applications of reducing its activity. For example, by site-directed mutagenesis to change the properties of specific residues in the active site, we can affect the catalytic efficiency and stereoselectivity of the enzyme. This research helps to develop new inhibitors or modulators for regulating plant metabolic processes under specific environmental conditions. Reducing LDHA activity may have important implications for plant stress tolerance research. In some cases, inhibiting LDHA activity can enhance plant tolerance to oxidative stress, as this can reduce lactate accumulation and avoid cell acidification and energy metabolism disorders. In addition, by reducing LDHA activity, we can study the metabolic adaptation mechanisms of plants under different oxygen conditions, providing a theoretical basis for breeding crop varieties with strong stress tolerance. SUMMARY

[0004] The first object of the present application is to provide a mutant MaLDHA of L-lactate dehydrogenase A K248A , whose amino acid sequence is shown in SEQ ID NO. 3.

[0005] The second object of the present application is to provide a gene encoding the above-mentioned mutant MaLDHA K248A .

[0006] The third object of the present application is to provide a recombinant expression plasmid containing a gene encoding the mutant MaLDHA K248A .

[0007] Preferably, the expression plasmid is the expression vector pGEX-4T-3.

[0008] A fourth objective of this invention is to provide a host expression cell containing the above-mentioned recombinant expression plasmid.

[0009] Preferably, the host expression cell is Escherichia coli.

[0010] The fifth objective of this invention is the mutant MaldHA. K248A To obtain NAD through catalysis of NADH + Applications in [the context of the text].

[0011] The sixth objective of this invention is to provide a method for obtaining the mutant MaldHA. K248A The gene-based method includes the following steps:

[0012] (1) Using the MaldHA gene as a template, PCR was performed using the sequences shown in SEQ ID NO.4 and SEQ ID NO.7 as primers to obtain the first PCR product. The nucleotide sequence of the MaldHA gene is shown in SEQ ID NO.1.

[0013] (2) Using the MaldHA gene as a template, PCR was performed using the sequences shown in SEQ ID NO.5 and SEQ ID NO.6 as primers to obtain the second PCR product;

[0014] (3) Using the first PCR product and the second PCR product as templates, and the sequences shown in SEQ ID NO.4-5 as primers, PCR was performed to obtain the mutant MaldHA containing the restriction enzyme sites. K248A The gene that encodes it.

[0015] Preferably, the PCR program in steps (1)-(3) is as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 20 s, 72℃ extension for 60 s, 35 cycles; 72℃ extension for 5 min.

[0016] This invention provides a mutant of L-lactate dehydrogenase A, MalDHA. K248A The mutant MaldHA K248A The amino acid sequence is shown in SEQ ID NO.3. The mutant MaldHA described in this invention... K248A The amino acid sequence shown in SEQ ID NO.2 was subjected to site-directed mutagenesis, with Lys at position 248 being mutated to Ala, thereby reducing the activity of L-lactate dehydrogenase A by 31%. The L-lactate dehydrogenase A mutant MalDHA obtained in this invention... K248AThis can serve as a mutant material that can significantly reduce enzyme catalytic activity, providing a reference for further research on LDHA. Attached image description:

[0017] Figure 1 The images shown are comparisons of enzyme activity measurements by spectrophotometry in the examples (a and b indicate significant differences between the two groups of enzyme activity measurements, p < 0.05). Detailed implementation method:

[0018] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0019] MaldHA, a mutant of L-lactate dehydrogenase A in this invention K248A The amino acid sequence is shown in SEQ ID NO.3. The mutant MaldHA described in this invention... K248A The mutation involves altering the Lys position at position 248 of the MaldHA amino acid sequence to Ala; the amino acid sequence of MaldHA is shown in SEQ ID NO.2. The mutation method of this invention is not specifically limited, but point mutation is preferred. The MaldHA described in this invention originates from banana, and its gene sequence is shown in SEQ ID NO.1, consisting of 1053 base pairs. The protein encoded by the gene contains 350 amino acids (as shown in SEQ ID NO.2, the protein size is 38.18 kDa), and its corresponding gene number in the banana genome is Ma01_t13550.1.

[0020] The following details the construction of the L-lactate dehydrogenase A mutant, MalDHA, according to the present invention. K248A Methods and activity tests.

[0021] Example 1

[0022] MaldHA, a mutant of L-lactate dehydrogenase A K248A The gene was constructed by PCR, and the primers used for PCR included primer pairs containing restriction enzyme sites and primer pairs containing mutation sites; the sequences of the primer pairs containing restriction enzyme sites are shown in SEQ ID NO.4-5 (restriction enzyme sites are bolded and marked with underline); the sequences of the primer pairs containing mutation sites are shown in SEQ ID NO.6-7 (mutation sites are bolded and marked with underline).

[0023] 1. Sample: The banana variety selected is the Brazilian banana (Musa spp., AAA group cultivar "Brazil").

[0024] 2. Cloning of the MaldHA gene

[0025] Total RNA was extracted from banana fruit using the conventional hot boric acid method. The extracted total RNA was reverse transcribed into cDNA according to the method provided in the PrimeScript™ RT Master Mix (Perfect Real Time, RR036A, TaKara) kit. The reaction system consisted of X μL RNA (<500 ng), 2 μL 5× Mix, and Y μL RNase-free water, mixed to a total volume of 10 μL. The mixture was then placed in a 37°C water bath for 15 min, followed by heat shock in an 85°C metal bath for 5 s, and finally cooled to 4°C.

[0026] Primers containing BamHI restriction sites and primers for mutation sites were designed based on the MaldHA gene sequence in the banana genome (https: / / banana-genome-hub.southgreen.fr / ). The primer sequences are shown in Table 1.

[0027] Table 1 Cloning Maldha K248A Primers required for gene generation

[0028]

[0029] Using banana cDNA as a template, PCR was first performed using an upstream primer containing the restriction enzyme site (SEQ ID NO.4) and a downstream primer containing the mutation site (SEQ ID NO.7) to obtain product 1. Then, PCR was performed using an upstream primer containing the mutation site (SEQ ID NO.6) and a downstream primer containing the restriction enzyme site (SEQ ID NO.5) to obtain product 2. Finally, using products 1 and 2 as templates, PCR was performed using upstream and downstream primers containing the restriction enzyme sites to obtain the mutant MaldHA containing the restriction enzyme site. K248A product.

[0030] Using banana cDNA as a template, the MaldHA gene was amplified by PCR using an upstream primer (SEQ ID NO.4) and a downstream primer (SEQ ID NO.5) containing restriction enzyme sites.

[0031] The PCR conditions were as follows: pre-denaturation at 98℃ for 3 min, followed by 35 cycles (denaturation at 98℃ for 10 s, annealing at 60℃ for 20 s, extension at 72℃ for 60 s), and final extension at 72℃ for 5 min. After the PCR reaction, the results were detected by 1% agarose gel electrophoresis.

[0032] 3. Construction of MaldHA expression strains

[0033] Maldha K248AThe PCR products and MaldHA gene amplification products were recovered and ligated into the pGEX-4T-3 vector to construct a recombinant expression plasmid. The ligation reaction system used was an in-fusion reaction system (TaKara): Purified RCRfragment, 100 ng; Linearized vector, 200 ng; 5×In-Fusion HD Enzyme Premix, 2 μL, with deionized water added to a final volume of 10 μL. The corresponding negative control consisted of: Linearized vector, 1 μL; 5×In-Fusion HD Enzyme Premix, 2 μL; Deionized water, 7 μL. The pUC19 positive control consisted of: Purified RCRfragment, 2 μL of a 2kb control insert; Linearized vector, 1 μL of a pUC19 control vector; 5×In-Fusion HD Enzyme Premix, 2 μL; Deionized water, 5 μL. The ligation procedure described in this invention is as follows: After gently mixing the above reaction system, incubate at 50°C for 15 minutes, then place on ice to obtain the ligation product. This product can be used for further transformation or stored at -20°C for later use. The ligation product is transformed into *E. coli* DH5α competent cells and cultured in 700 μL LB medium for 1 hour (37°C, 200 rpm). 400 μL of the bacterial culture is spread onto a solid LB agar plate containing 0.05 mg / mL kanamycin and incubated overnight at 37°C. Positive colonies are picked for PCR identification, and the positive bacterial culture is sent to Guangzhou Qingke Sequencing Company for sequencing. The correctly sequenced plasmid is transformed into *E. coli* BL21(DE3) competent cells. Positive bacterial cultures are identified by PCR. The culture is then diluted 1:1 with 50% sterile glycerol and stored at -80°C to obtain MaldHA and MaldHA, respectively. K248A Expression strain.

[0034] 4. Maldha-GST and Maldha K248A -Prokaryotic expression and purification of GST

[0035] (1) Prokaryotic expression

[0036] Maldha and Maldha respectively K248A The expression strain was cultured at 37℃ and 200 rpm. When the bacterial concentration reached OD600 of 0.4-0.6, the bacterial solution was cooled to 15℃ and IPTG protein inducer with a final concentration of 1 mM was added for low-temperature induction (16℃ and 120 rpm). After culturing for 16-18 h, the bacterial cells were collected.

[0037] (2) Protein purification

[0038] First, suspend the bacterial cells in sterile water, centrifuge at 6000g for 10 min at 4°C to remove the culture medium, then add an appropriate volume of protein extraction buffer PBS (to suspend the bacterial cells; add Triton-100 at a ratio of 1:1000 based on the bacterial volume to improve protein extraction efficiency). After fully suspending the bacterial cells, use a low-temperature, ultra-high pressure continuous flow cell disruptor to disrupt the cells for 1 hour (sonication for 30 seconds, pause for 30 seconds). During sonication, keep the container containing the bacterial solution in a full ice bath to prevent the probe from overheating and reducing protein activity. After sonication, add PMSF to a final concentration of 1 mM to inhibit protein degradation. Centrifuge at 9000g for 30 min at 4°C, filter the supernatant through a membrane (0.45 μm), then add the GST (GE Healthcare) purification packing material, which has been equilibrated with protein extraction buffer, to the protein solution, and allow the protein to rotate and bind with the packing material at 4°C for at least 2 hours.

[0039] First, the supernatant of the settled protein was passed through a nickel column, and the packing material containing the bound protein was also transferred to the protein purification column. The packing material was then washed repeatedly with PBS buffer to remove impurities until no protein was detected by Coomassie Brilliant Blue G250 (Beyotime). After all the supernatant had passed through the nickel column, 1 mL of 10 mM reduced glutathione (prepared with PBS) was added to elute the protein. The eluent was collected in a 1.5 mL centrifuge tube until a high concentration of protein was detected by Coomassie Brilliant Blue G250 (Beyotime). Finally, the purified target protein was stored at -80°C. A portion of the purified protein was compared with the unpurified protein for gel electrophoresis, thus obtaining MaldHA-GST and its mutant MaldHA. K248A -GST protein.

[0040] 5. Protein activity was detected using spectrophotometry. Kits from Suzhou Keming Biotechnology Co., Ltd. were used to determine MaldHA-GST and its mutant MaldHA. K248A - For GST activity, please refer to the Lactate Dehydrogenase (LDH) kit instructions (catalog number: G0804F) for specific operating procedures. Enzyme activity is defined as: one unit of enzyme activity is defined as catalyzing the conversion of 1 nmol of lactate to pyruvate per milligram of protein per minute. The formula for calculating protein activity is as follows:

[0041] LDH(U / mg prot)=[(ΔA-0.0071)÷0.0374]÷(V1×Cpr)÷T=66.9×(ΔA-0.0071)÷Cpr

[0042] The results are as follows Figure 1As shown, from Figure 1 It can be seen that the mutant MaldHA K248A The activity of L-lactate dehydrogenase A was significantly lower than that of MaldHA, resulting in a 31% reduction in its activity.

[0043] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0044] SEQ ID NO.1

[0045] ATGAAGAAGGTGTCGTCTCTGACCGAGCTCGGCTTCGCTGATGATGTCCACCGGGCTCTG

[0046] TTCCGCCCGATCCAACAGGCGGCACCGCCCTCCCCGACGAAGCGCCACACGAAGATCTCC

[0047] GTGATCGGCGCCGGGAACGTCGGCATGGCCATCGCGCAGACGATCCTGACGCAGGACCTG

[0048] ACGGACGAGCTGGCGCTGGTGGACGCCAAGCCCGACAAGCTCCGGGGGGAGATGCTGGAT

[0049] CTGCAGCACGCCGCCGCCTTCCTCCCCCGCACCCGGATCCTGGCGTCGCCGGACTACGCG

[0050] GTGACGGTCGACTCCGACCTCTGCATCATCACGGCGGGCGCCCGGCAGATCCCCGGGGAG

[0051] ACGCGCCTCAACCTCCTCCAGCGGAACCTGTCCCTGTTCAAGGAGATCGTGCCGCCGCTC

[0052] GCCCGGTACTCCCCGGGGGCGCTGCTGCTGGTGGTGTCGAATCCGGTGGACGTGTTGACG

[0053] TACATCGCGTGGAAGCTGTCCGGCTTCCCTCCCAATCGGGTGATCGGATCCGGCACCAAT

[0054] CTCGATTCCTCGCGCTTCAGGTTCCTGCTCGCCGACCACCTCGAGGTCAACGCTCAAGAT

[0055] GTCCAGGCATACATGGTGGGGGAGCACGGAGACAGCTCTGTGGCGCTGTGGTCGAGCATA

[0056] AGCGTGGGGGGAGTGCCAATACTGAGCCAATTCACCAAGGATGTGGCGGCAATCGAGCAA

[0057] GGAGTGCTGGAGCGGATCAGGAAGGCGGTGGTGGACAGCGCGTACGAGGTCATCCGCCTC

[0058] AAAGGCTACACGTCCTGGGCCATCGGCTACTCGGTGGCGAGCCTCGCCCGATCCCTTCTC

[0059] CGCGACCAACACCGAATCCACCCGGTTTCTCTGCTCGCCAAGGGATTCTACGGCATTCCC

[0060] GATGACCGCGAGGTGTTCCTTAGCCTTCCGGCGCAGCTCGGCCGCAGCGGCATTCTCAGC

[0061] GTAGCCAACATCCAACTCACCGACGAGGAGGCAGGCCGCCTTCAGCGATCCGCCGAGGCT

[0062] CTCTGGGACCTGCAACAAAAGCTTGACCTCTGA

[0063] SEQ ID NO.2

[0064] MKKVSSLTELGFADDVHRALFRPIQQAAPPSPTKRHTKISVIGAGNVGMAIAQTILTQDL

[0065] TDELALVDAKPDKLRGEMLDLQHAAAFLPRTRILASPDYAVTVDSDLCIITAGARQIPGE

[0066] TRLNLLQRNLSLFKEIVPPLARYSPGALLLVVSNPVDVLTYIAWKLSGFPPNRVIGSGTN

[0067] LDSSRFRFLLADHLEVNAQDVQAYMVGEHGDSSVALWSSISVGGVPILSQFTKDVAAIEQ

[0068] GVLERIRKAVVDSAYEVIRLKGYTSWAIGYSVASLARSLLRDQHRIHPVSLLAKGFYGIP

[0069] DDREVFLSLPAQLGRSGILSVANIQLTDEEAGRLQRSAEALWDLQQKLDLSEQ ID NO.3

[0070] MKKVSSLTELGFADDVHRALFRPIQQAAPPSPTKRHTKISVIGAGNVGMAIAQTILTQDL

[0071] TDELALVDAKPDKLRGEMLDLQHAAAFLPRTRILASPDYAVTVDSDLCIITAGARQIPGE

[0072] TRLNLLQRNLSLFKEIVPPLARYSPGALLLVVSNPVDVLTYIAWKLSGFPPNRVIGSGTN

[0073] LDSSRFRFLLADHLEVNAQDVQAYMVGEHGDSSVALWSSISVGGVPILSQFTKDVAAIEQ

[0074] GVLERIRAAVVDSAYEVIRLKGYTSWAIGYSVASLARSLLRDQHRIHPVSLLAKGFYGIP

[0075] DDREVFLSLPAQLGRSGILSVANIQLTDEEAGRLQRSAEALWDLQQKLDL

Claims

1. Mutant MaLDHA of L-lactate dehydrogenase A K248A characterized in that The amino acid sequence is shown as SEQ ID NO.

3.

2. A gene encoding the mutant MaLDHA of claim 1. K248A 2. The method of claim 1, wherein the mutant MaLDHA is encoded by a gene having the nucleotide sequence of SEQ ID NO:

2.

3. A mutant containing the coding variant MaldHA as described in claim 2 K248A Recombinant expression plasmids of the gene.

4. The recombinant expression plasmid of claim 3, wherein, The expression plasmid is an expression vector pGEX-4T-3.

5. A host expression cell containing the recombinant expression plasmid of claim 3 or 4.

6. The expression cell of claim 5, wherein, The host expression cell is Escherichia coli.

7. The mutant MaLDHA of claim 1 K248A Use in reducing cytoplasmic acidification under hypoxic conditions.

8. A method of obtaining the gene of the mutant MaLDHA of claim 2, characterized in that, K248A The method comprises the following steps: ​ (1) performing PCR with MaLDHA gene as a template and the sequences shown as SEQ ID NO. 4 and SEQ ID NO. 7 as primers to obtain a first PCR product, wherein the nucleotide sequence of the MaLDHA gene is shown as SEQ ID NO. 1; (2) performing PCR with MaLDHA gene as a template and the sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6 as primers to obtain a second PCR product; (3) using the first PCR product and the second PCR product as templates, and using the sequences shown in SEQ ID NO. 4-5 as primers to perform PCR to obtain a mutant MaLDHA containing a restriction site K248A encoding gene.

9. The method of claim 8, wherein, The PCR procedure in steps (1)-(3) is as follows: 98 ℃ pre-denaturation for 3 min; 98 ℃ denaturation for 10 s, 60 ℃ annealing for 20 s, 72 ℃ extension for 60 s, 35 cycles; 72 ℃ extension for 5 min.

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