Cala mutant suitable for medium-high temperature catalytic system and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and discloses a CALA mutant suitable for a medium-high temperature catalytic system and application thereof. The application obtains the CALA mutant by mutating the lysine at the 433th position of the wild type CALA into glycine, the amino acid sequence of the CALA mutant is shown as SEQ ID NO: 2, and the nucleotide sequence is shown as SEQ ID NO: 3. The CALA mutant has higher medium-high temperature catalytic activity and more excellent high temperature stability compared with the wild type, and the catalytic activity of the CALA mutant is better than that of the wild type in the temperature range of 55 DEG C to 85 DEG C, the residual enzyme activity of the CALA mutant is higher than that of the wild type in the range of 30 DEG C to 80 DEG C, and especially when treated at 70 DEG C, the residual enzyme activity of the CALA mutant is about 2.96 times that of the wild type. The CALA mutant suitable for the medium-high temperature catalytic system has good application potential in the fields of oil processing, detergent industry and high temperature enzyme catalytic reaction.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to a CALA mutant suitable for medium and high temperature catalytic systems and its applications. Background Technology

[0002] Lipases (EC 3.1.1.3) are a class of hydrolytic enzymes widely found in microorganisms and plant and animal tissues. They catalyze the hydrolysis of oils and fats, and can also catalyze esterification and transesterification reactions in non-aqueous media. They are widely used in oil processing, detergents, biodiesel production, and chiral synthesis. Lipases are derived from *Candida antarcticis* (…). Candida antarctica CALA, an α / β hydrolase, belongs to the α / β hydrolase family. Its active center is composed of the catalytic triplet Ser184, Asp334, and His366. This enzyme has significant catalytic activity under medium and high temperature conditions, but its thermal stability is insufficient. It is prone to conformational instability and activity decay during long-term continuous catalysis at high temperatures. This makes it difficult to meet the requirements of enzyme preparation stability for industrial-scale continuous operation, thus limiting the application of CALA in medium and high temperature processes.

[0003] Existing technologies have attempted to modify the thermal stability of CALA through site-directed mutagenesis or directed evolution. However, the resulting mutants often exhibit decreased catalytic activity while improving thermal stability, making it difficult to simultaneously achieve both thermal stability and catalytic activity. Furthermore, some studies have employed non-genetic engineering methods such as immobilization and chemical modification to improve CALA stability. For example, immobilizing CALA on mesoporous organosilicon materials or supports like Immobead 150 can improve its thermal stability and reusability to some extent, but immobilized enzymes still experience activity degradation during long-term operation. Therefore, there is an urgent need to provide a CALA mutant that simultaneously possesses high catalytic activity and good thermal stability under medium- and high-temperature conditions to meet the demands of medium- and high-temperature industrial catalysis for enzyme stability and activity. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a CALA mutant suitable for medium- and high-temperature catalytic systems and its applications. This invention mutates lysine at position 433 of the wild-type CALA to glycine to obtain the CALA mutant, the amino acid sequence of which is shown in SEQ ID NO:2, and the nucleotide sequence of which is shown in SEQ ID NO:3. The CALA mutant of this invention exhibits higher medium- and high-temperature catalytic activity and superior high-temperature stability compared to the wild type. The mutant shows superior enzyme activity within the temperature range of 55℃ to 85℃ and higher residual enzyme activity within the temperature range of 30℃ to 80℃, with a particularly significant stability advantage above 50℃. At 70℃, the residual enzyme activity of the wild type decreases to 47.73% ± 3.95%, while the mutant V4 remains as high as 141.13% ± 4.08%, approximately 2.96 times that of the wild type; at 80℃, the residual enzyme activity of the wild type is 42.5% ± 5.28%, while that of the mutant V4 is 84.1% ± 2.79%, approximately 1.98 times that of the wild type. This invention provides a CALA mutant suitable for medium- and high-temperature catalytic systems, which has good application potential in the fields of oil processing, detergent industry and high-temperature enzyme catalytic reactions.

[0005] On the one hand, the present invention provides a CALA mutant suitable for medium- and high-temperature catalytic systems, wherein the medium- and high-temperature range is 55°C to 85°C; The amino acid sequence of the CALA mutant is shown in SEQ ID NO:2.

[0006] Specifically, this invention, through experiments, found that mutant V4 exhibits superior catalytic activity at medium to high temperatures compared to the wild type, and its thermal stability is significantly better. The optimal reaction temperature for wild-type CALA is 70°C, at which temperature the enzyme activity is highest, approximately 969.31 ± 9.75 U / mL. The optimal temperature for the CALA mutant (V4) of this invention is also 70°C, at which temperature the enzyme activity is approximately 1014.59 ± 9.70 U / mL, higher than that of wild-type CALA. Furthermore, the residual enzyme activity of mutant V4 was higher than that of the wild type within the temperature range of 30℃ to 80℃, especially in the high-temperature range above 50℃, where the advantage was more obvious. At 70℃ treatment, the residual enzyme activity of the wild type decreased to 47.73%±3.95%, while V4 remained as high as 141.13%±4.08%, approximately 2.96 times that of the wild type. At 80℃ treatment, the residual enzyme activity of the wild type was 42.5%±5.28%, while that of mutant V4 was 84.1%±2.79%, approximately 1.98 times that of the wild type. This indicates that the CALA mutant provided by this invention, suitable for medium- and high-temperature catalytic systems, has higher medium- and high-temperature catalytic activity and superior high-temperature stability compared to the wild type.

[0007] On the other hand, the present invention seeks protection for the gene encoding the above-mentioned CALA mutant, the nucleotide sequence of which is shown in SEQ ID NO:3.

[0008] On the other hand, the present invention seeks protection for a recombinant plasmid carrying the gene encoding the CALA mutant described above.

[0009] On the other hand, the present invention seeks to protect a host cell carrying the gene encoding the CALA mutant or the recombinant plasmid described above.

[0010] On the other hand, the present invention seeks protection for the method of preparing the above-mentioned CALA mutant, which involves mutating the 433rd lysine of wild-type CALA to glycine to obtain the CALA mutant.

[0011] The amino acid sequence of the wild-type CALA is shown in SEQ ID NO:1.

[0012] Furthermore, this invention seeks to protect the application of the aforementioned CALA mutant, or the gene encoding the CALA mutant, or the recombinant plasmid or host cell, in oil processing, detergent preparation, and the preparation of medium- and high-temperature catalytic enzymes.

[0013] Furthermore, the operating temperature of the medium-high temperature catalytic enzyme is 55℃~85℃.

[0014] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention mutates lysine at position 433 of wild-type CALA to glycine to obtain the CALA mutant, the amino acid sequence of which is shown in SEQ ID NO:2, and the nucleotide sequence of which is shown in SEQ ID NO:3. The mutant V4 of this invention exhibits superior catalytic activity and thermal stability compared to the wild type at medium to high temperatures. The optimal reaction temperature for wild-type CALA is 70℃, at which temperature the enzyme activity is highest, approximately 969.31±9.75 U / mL. The optimal temperature for the CALA mutant (V4) of this invention is also 70℃, at which temperature the enzyme activity is approximately 1014.59±9.70 U / mL, higher than that of wild-type CALA. Furthermore, the residual enzyme activity of mutant V4 was higher than that of the wild type within the temperature range of 30℃ to 80℃, especially in the high-temperature range above 50℃. At 70℃, the residual enzyme activity of the wild type decreased to 47.73%±3.95%, while that of mutant V4 remained as high as 141.13%±4.08%, approximately 2.96 times that of the wild type. At 80℃, the residual enzyme activity of the wild type was 42.5%±5.28%, while that of mutant V4 was 84.1%±2.79%, approximately 1.98 times that of the wild type. This indicates that the CALA mutant provided by this invention, suitable for medium- and high-temperature catalytic systems, has higher medium- and high-temperature catalytic activity and better high-temperature stability compared to the wild type. This CALA mutant also exhibits superior substrate selectivity for medium- and long-chain fatty acids C12 and C14. The aforementioned characteristics make CALA mutants promising for applications in oil processing, detergent industry, and high-temperature catalytic reactions. This invention achieves synergistic enhancement of multi-dimensional enzymatic properties through single-point site-directed mutagenesis, providing a high-performance candidate enzyme preparation for high-temperature industrial catalysis. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The image shows the SDS-PAGE results of wild-type CALA and its mutants. M is the protein marker, Con is the blank, WT is wild-type CALA, and V1, V2, V3, and V4 are mutants 1, 2, 3, and 4, respectively.

[0017] Figure 2 This is a graph showing the substrate-specific detection results of wild-type CALA and its mutants. Figure 2 In the figure, A represents the substrate specificity detection results of wild-type CALA; Figure 2 In the figure, B represents the substrate specificity detection results of mutant V1; Figure 2 In the figure, C represents the substrate-specific detection results of mutant V2; Figure 2 In the figure, D represents the substrate-specific detection results of mutant V3; Figure 2 E in the figure represents the substrate-specific detection results of mutant V4.

[0018] Figure 3 This image shows the enzyme activity detection results of wild-type CALA and its mutants on the same substrate. Figure 3 In this context, A represents the different lipases for the substrate pNP. Image of C4 enzyme activity detection results; Figure 3 In this context, B represents the different lipases for the substrate pNP. Image of C6 enzyme activity detection results; Figure 3 In this context, C represents the different lipases for the substrate pNP. Image of C8 enzyme activity detection results; Figure 3 In this context, D represents the different lipases for the substrate pNP. Image of C10 enzyme activity detection results; Figure 3 In this context, E represents the different lipases for the substrate pNP. Image of C12 enzyme activity detection results; Figure 3 In this context, F represents the different lipases for the substrate pNP. Image of C14 enzyme activity detection results; Figure 3 In this context, G represents the different lipases' responses to the substrate pNP. Image of C16 enzyme activity detection results; Figure 3 The H in the figure represents the different lipases for the substrate pNP. Image showing the results of C18 enzyme activity assay.

[0019] Figure 4 The graph shows the enzyme activity detection results of wild-type CALA and its mutants at different reaction temperatures. Figure 4 In the figure, A represents the optimal temperature detection results for wild-type CALA and its mutants; Figure 4 Figure B in the graph shows the results of thermal stability testing of wild-type CALA and its mutants at different temperatures.

[0020] Figure 5 The graph shows the enzyme activity detection results of wild-type CALA and its mutants at different pH values. Figure 5 In the figure, A represents the optimal pH detection results for wild-type CALA and its mutants; Figure 5 Figure B in the graph shows the stability test results of wild-type CALA and its mutants at different pH values. Detailed Implementation

[0021] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially. Unless otherwise specified, the percentages in the following embodiments refer to mass percentages.

[0022] Example 1 This example provides the construction of CALA mutants.

[0023] 1. Obtaining recombinant plasmids of CALA mutants The amino acid sequence of wild-type CALA is shown in SEQ ID NO:1. Three mutants were constructed by deleting the entire sequence from lysine at position 427, lysine at position 433, and phenylalanine at position 435 to the end of the sequence, named V1, V2, and V3, respectively. Lysine at position 433 of wild-type CALA was mutated to glycine, and the resulting amino acid sequence is shown in SEQ ID NO:2. This mutant was named V4. The coding genes of the four mutants were cloned into the pPICZαA vector, recombinant plasmids were extracted, linearized with SacI, and transformed into Pichia pastoris X33 competent cells. The transformation solution was plated on YPD plates containing 100 μg / mL Zeocin and cultured at 30°C until single colonies grew. Single colonies were streaked onto YPD plates containing 200 μg / mL Zeocin for high-resistance selection. Single colonies were then selected for colony PCR verification, and positive clones were sent for sequencing verification.

[0024] 2. Induction and purification of CALA mutants The recombinant strain was inoculated into 50 mL of BMGY medium and cultured at 30°C with shaking at 200 rpm until OD500 was reached. 600 =2~6, collect the bacterial cells by centrifugation, resuspend in BMMY medium, and continue culturing at 30℃ and 230 rpm. Add 1% methanol every 24 h for induction, continuing induction for 3 days. After induction, collect the supernatant by centrifugation to obtain the crude enzyme solution. Perform SDS-PAGE analysis on the crude enzyme solution. Figure 1 The results showed that a specific band appeared at the theoretical molecular weight position of the target protein, indicating that the lipase and its mutants were successfully secreted and expressed.

[0025] The crude enzyme solution was purified by Ni-NTA agarose affinity chromatography. The crude enzyme solution was filtered through a 0.45 μm filter and then loaded onto a Ni-NTA pre-packed column equilibrated with equilibration buffer (20 mM imidazole, PBS, pH 7.4). Impurities were washed with wash buffer containing 40 mM imidazole, and the target protein was eluted with elution buffer containing 300 mM imidazole. The eluent was collected in separate tubes and concentrated using ultrafiltration to obtain the purified lipase.

[0026] Example 2 This embodiment characterizes the enzymatic properties of lipase and its mutants.

[0027] 1. Substrate-specific detection p-Nitrophenyl esters (pNP) with different acyl chain lengths (C4~C18) C4~pNP The substrate specificity of CALA and its mutants was assessed using C18 substrates, and lipase activity was determined spectrophotometrically. 700 µL of Tris-HCl buffer (50 mM, pH 8.5) and 90 µL of 20 mM p-nitrophenyl esters (dissolved in isopropanol) with different acyl chain lengths (C4–C18) were pre-incubated at 40°C for 5 min. Then, 10 µL of lipase enzyme solution was added to initiate the reaction, and the reaction was terminated at 40°C for 10 min with 200 µL of isopropanol. The absorbance was measured at 405 nm using a multi-well microplate reader. Enzyme activity units (U) were defined as the amount of enzyme required to catalyze the release of 1 µmol of p-nitrophenol per minute under the above reaction conditions. The results of substrate specificity assays for lipase and its mutants are shown below. Figure 2 and Figure 3 As shown.

[0028] Depend on Figure 2 and Figure 3 It can be seen that WT is related to pNP C16 showed the highest activity; in comparison, all mutants were at pNP level. The highest catalytic activity is achieved at C14, the optimal substrate chain length is changed to C14, and the activity of mutant V4 is significantly higher than that of other mutants.

[0029] 2. Optimal reaction temperature detection The optimal reaction temperature for wild-type CALA and its mutants was determined within the temperature range of 30℃ to 90℃. The enzyme activity detection method was the same as above, using p-nitrophenol myristate (pNP-C14) as the substrate. The detection results are as follows. Figure 4As shown in A in the figure. Wild-type CALA and its mutants were incubated at 30℃~80℃ for 1 h, and then the residual activity was measured at their optimal reaction temperature. The enzyme activity at 40℃ without incubation was defined as 100%, and the residual enzyme activity at other temperatures was calculated. The test results are shown in Figure A. Figure 4 As shown in B in the diagram.

[0030] Depend on Figure 4 As shown in Figure A, the temperature-activity curves of all lipases exhibit a typical single-peak distribution. The optimal reaction temperature for wild-type CALA protein is 70℃, at which point the enzyme activity reaches its peak, approximately 969.31±9.75 U / mL. The optimal temperature for mutant V4 is also 70℃, at which point the enzyme activity is approximately 1014.59±9.70 U / mL, higher than that of WT. Throughout the entire testing temperature range of 30℃ to 80℃, the residual enzyme activity of mutant V4 is higher than that of wild-type. Especially in the high-temperature range above 50℃, the residual enzyme activity of wild-type decreases significantly with increasing temperature, while mutant V4 maintains a high level of residual enzyme activity. At 70℃, the residual enzyme activity of the wild-type decreased to 47.73%±3.95%, while that of the mutant V4 remained as high as 141.13%±4.08%, approximately 2.96 times that of the wild-type. At 80℃, the residual activity of the wild-type was 42.5%±5.28%, while that of the mutant V4 was 84.1%±2.79%, approximately 1.98 times that of the wild-type. These results indicate that the mutant V4 exhibits significantly better enzyme activity retention after high-temperature treatment than the wild-type, demonstrating a wider temperature tolerance range and superior high-temperature stability.

[0031] 3. Optimal pH detection The optimal pH for determining the enzyme activity of wild-type CALA and its mutants at 40℃ and within the pH range of 4.0–10.0 was determined using the same method as above, with p-nitrophenol myristate (pNP-C14) as the substrate. The results are as follows. Figure 5 As shown in A in Figure 5. pH stability was assessed by measuring residual activity after mixing the lipase with equal volumes of buffer solutions of different pH values ​​and incubating at 40°C for 1 h, with results shown in B in Figure 5.

[0032] Depend on Figure 5 It is known that the optimal pH for both wild-type CALA and its mutants is 8.5, and they are most stable at pH 7.5, indicating that the mutation described in this invention does not change the optimal pH and pH stability characteristics of the enzyme.

[0033] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A CALA mutant suitable for medium- and high-temperature catalytic systems, characterized in that, The temperature range of the medium and high temperature is 55℃~85℃; The amino acid sequence of the CALA mutant is shown in SEQ ID NO:

2.

2. The gene encoding the CALA mutant of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

3.

3. A recombinant plasmid, characterized in that, Carrying the gene as described in claim 2.

4. A host cell, characterized in that, Carrying the gene as described in claim 2 or the recombinant plasmid as described in claim 3.

5. The method for preparing the CALA mutant according to claim 1, characterized in that, The CALA mutant was obtained by mutating lysine at position 433 of the wild-type CALA to glycine.

6. The preparation method according to claim 5, characterized in that, The amino acid sequence of the wild-type CALA is shown in SEQ ID NO:

1.

7. The application of the CALA mutant of claim 1, the gene of claim 2, the recombinant plasmid of claim 3, or the host cell of claim 4 in oil processing, detergent preparation, and preparation of medium- and high-temperature catalytic enzymes.

8. The application according to claim 7, characterized in that, The operating temperature of the medium-high temperature catalytic enzyme is 55℃~85℃.

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