A highly thermostable adenosine kinase mutant and its application

Abstract

The present invention discloses a highly thermostable adenosine kinase mutant and its application, relating to the field of enzyme engineering technology. The highly thermostable adenosine kinase mutant is obtained by point mutation at position 253 or 82 of the adenosine kinase shown in SEQ ID NO: 1. The present invention uses an enzymatic catalytic method and the adenosine kinase mutant as a biocatalyst for the enzymatic synthesis of 5'-adenylic acid. Adenosine and adenosine triphosphate are reacted to produce adenosine triphosphate, which reduces the generation of byproducts and impurities in the reaction to a certain extent, thereby improving the production efficiency and purity of adenosine triphosphate. Furthermore, the reaction conditions during the enzymatic preparation are mild and will not cause environmental pollution. Furthermore, the highly thermostable adenosine kinase mutant is used in the preparation of adenosine triphosphate, which is beneficial for improving the efficiency and yield of adenosine triphosphate preparation.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, in particular to an adenosine kinase mutant with high thermal stability and application thereof. Background Art

[0002] Adenosine is a compound of adenine, ribose, and phosphate. It is one of the four main mononucleotides that make up the ribonucleic acid in animal cells. Adenosine is widely used in medicine and industry. There are four common production methods. The current process for synthesizing adenosine mainly uses chemical methods. The existing adenosine has three by-products in the preparation process, and their content accounts for a relatively large proportion, which seriously affects the purity and efficiency of the preparation of adenosine.

[0003] The defects of the existing enzymatic method for preparing adenylic acid are:

[0004] 1. Patent document CN107602648B discloses a method for preparing 5'-adenylic acid. The method mainly considers the use of a solvent-free synthesis method to reduce reaction impurities and be environmentally friendly, but does not consider how to improve the yield and conversion efficiency of adenylic acid.

[0005] 2. Patent document CN105603028B discloses a method for the simultaneous enzymatic preparation of glutathione and adenylic acid. The method mainly considers how to simultaneously prepare GSH and AMP, but does not consider how to reduce the interference of other proteins or impurities to more efficiently convert adenosine to adenylic acid.

[0006] 3. Patent document CN103833813B discloses a method for preparing adenosine monophosphate crystals based on isoelectric dissolution coupling. The method mainly focuses on how to prepare adenosine monophosphate crystals and obtain high-purity products in a short period of time, but does not consider how to improve the efficiency of the preparation process and increase the purity of the product.

[0007] 4. The existing adenylate preparation process does not consider how to improve the efficiency of screening, which will help improve the efficiency of adenylate preparation. Summary of the Invention

[0008] The object of the present invention is to provide a highly thermostable adenosine kinase mutant and its application to solve the problems raised in the above background technology.

[0009] To achieve the above objectives, the present invention provides the following technical solutions: a highly thermally stable adenosine kinase mutant and its application.

[0010] In one aspect, the present invention provides a highly thermostable adenosine kinase mutant.

[0011] Specifically, the adenosine mutant is obtained by mutating position 253 or position 82 of the adenosine kinase shown in SEQ ID NO: 1 (nucleotide sequence is 623581 to 624603 of Saccharomyces cerevisiae S288C Chromosome: X; NC_001142.9).

[0012] More specifically, the mutation at position 253 is K253R, and the mutation at position 82 is V82C.

[0013] In another aspect, the present invention provides a gene encoding the above-mentioned adenosine kinase mutant.

[0014] In another aspect, the present invention provides an expression vector comprising the above-mentioned encoding gene.

[0015] Specifically, the vector includes but is not limited to: pET28a(+), pet30a(+), pETduet, pREST or pTriEX.

[0016] In certain specific embodiments of the present invention, the vector is pET28a(+).

[0017] In another aspect, the present invention provides a genetically engineered bacterium comprising the aforementioned adenosine kinase mutant and / or the aforementioned encoding gene and / or the aforementioned expression vector.

[0018] Specifically, the genetically engineered bacteria include but are not limited to Escherichia coli, Salmonella, and Pichia pastoris.

[0019] In certain specific embodiments of the present invention, the engineered bacteria is Escherichia coli BL21 (DE3).

[0020] In another aspect, the present invention provides use of the adenosine kinase mutant and / or the genetically engineered bacteria of the expression vector in the preparation of adenosine monophosphate.

[0021] In certain specific embodiments of the present invention, the genetically engineered bacteria are cultured and then centrifuged to obtain wet cells.

[0022] In certain specific embodiments of the present invention, the wet bacteria are ultrasonically disrupted to obtain a crude adenosine kinase enzyme solution.

[0023] In certain specific embodiments of the present invention, the adenosine kinase is purified by suspending the wet cells in a buffer solution, disrupting them by ultrasonication, and centrifuging them. The supernatant is collected and the protein is purified using a Ni affinity column.

[0024] Specifically, the genetically engineered bacteria, wet bacteria, crude enzyme solution, and adenosine kinase can all be used for the preparation of adenylic acid.

[0025] In yet another aspect, the present invention provides a method for producing adenosine.

[0026] Specifically, the method uses the adenosine kinase mutant as a catalyst.

[0027] More specifically, the method uses adenosine, disodium adenosine triphosphate, and magnesium chloride hexahydrate as substrates; and adenylate kinase and mutant adenosine kinase as catalysts to construct a reaction system and synthesize adenylate in a one-step reaction.

[0028] In certain specific embodiments of the present invention, the reaction system contains 20-374 mM adenosine, 10-226 mM adenosine disodium triphosphate, and 10-100 mM magnesium chloride hexahydrate; and the reaction system contains 60.7-121.3 U of adenylate kinase and 16.8-33.5 U of mutant adenosine kinase per 100 mL.

[0029] Specifically, the one-step reaction is carried out at 30-50°C.

[0030] In certain embodiments of the present invention, the reaction is carried out at 40°C and 50°C.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention uses an enzymatic catalytic method and utilizes an adenosine kinase mutant as a biocatalyst for the enzymatic synthesis of 5'-adenylic acid. Adenosine and adenosine triphosphate are reacted to produce adenosine triphosphate, which reduces the generation of byproducts and impurities in the reaction to a certain extent, thereby improving the production efficiency and purity of adenosine triphosphate. In addition, the reaction conditions in the enzymatic preparation are mild and will not cause environmental pollution. In addition, the highly thermally stable adenosine kinase mutant is used in the preparation of adenosine triphosphate, which is beneficial to improving the efficiency and yield of adenosine triphosphate preparation.

[0033] 2. The present invention purifies adenosine kinase, thereby reducing interference from other proteins or impurities, and can more effectively convert adenosine into adenylic acid, reducing the occurrence of side reactions, making the final adenylic acid purer. Under the catalysis of highly active purified enzyme, the cost of enzyme use can be reduced, while achieving an efficient catalytic reaction, thereby improving reaction efficiency to a certain extent and reducing costs.

[0034] 3. The present invention can effectively screen out adenosine kinases with poor activity and poor thermal stability by measuring the activity and thermal stability of adenosine kinase, which helps to improve the quality of adenosine kinases that participate in the subsequent adenosine and adenosine triphosphate reaction process, thereby further improving the efficiency of the preparation process and increasing the purity of the product.

[0035] 4. The present invention helps to improve the thermal stability of ArK through ArK molecular dynamics simulation and homologous protein multiple sequence alignment, thereby improving the efficiency of screening and further helping to improve the efficiency of preparing adenylate. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the measurement results of the thermal stability of adenosine kinase at different temperatures of the present invention. DETAILED DESCRIPTION

[0037] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0038] The present invention provides a highly thermostable adenosine kinase mutant, and the preparation method of the adenosine kinase mutant is as follows:

[0039] Step S1: Add an NcoⅠ restriction site before the start codon of the nucleotide sequence of adenosine kinase, then delete the stop codon, and add an XhoⅠ restriction site to the end of the sequence to obtain a new sequence, wherein the nucleotide sequence is the 623581-624603 sequence of Saccharomyces cerevisiae S288CChromosome:X;NC_001142.9, and then perform whole gene synthesis on the obtained new sequence. Then, use the NcoⅠ / XhoⅠ double restriction site to connect the new sequence to the pET28a(+) vector to construct a pET28a(+)-ArK expression vector, followed by centrifugation at a centrifugal speed of 10 rpm for 10 minutes, then add 50 μL of ddH2O thereto, place it on a vortex for dissolution, and then transform the dissolved material into Escherichia coli E.coliB L21 (DE3), and then the transformed Escherichia coli E. coli BL21 (DE3) is activated, wherein the activation temperature is 37 ° C, the stirring speed is 220 rpm, and the time is 1 hour. After activation, it is spread on a 50 μg / mL kanamycin-resistant LB plate and cultured overnight at a culture temperature of 37 ° C. A single colony is picked on the next day and the colony is inoculated into a TB liquid culture medium containing 50 μg / mL kanamycin. Glucose and 20 g / L lactose are added to the culture medium at a final concentration of 5 g / L, and stirred at 220 rpm at 37 ° C until OD600 reaches 0.8-1.2. The TB liquid culture medium is then cooled to 28 ° C, and continued to be stirred at 220 rpm and cultured for 14 hours. After that, it is centrifuged at 8000 rpm for 10 minutes at 4 ° C to obtain wet bacteria containing adenosine kinase;

[0040] Step S2: Use 5 column volumes of buffer A to balance the Ni column until the baseline is stable, then pass the supernatant containing the target protein through the Ni affinity column at a flow rate of 1 mL / min, wherein the concentration of the target protein in the solution is 25-40 mg / mL, then rinse with 6 column volumes of buffer A until the baseline is stable, wherein the rinse flow rate is 1 mL / min, elute with buffer B, and collect the target protein, wherein the elution flow rate is 1 mL / min, and place the obtained target protein in pH 8.0, 20 mM Tris buffer for overnight dialyzation to obtain purified adenosine kinase, and then rinse the Ni column with 5 column volumes of buffer C until the baseline is stable, and rinse with 5 column volumes of buffer C until the baseline is stable. The Ni column was preserved in ultrapure water containing 20% ​​ethanol by volume, and adenosine kinase was finally obtained, wherein buffer A was prepared by mixing NaCl, imidazole and Tris buffer, wherein the concentration of NaCl was 0.5M, the concentration of imidazole was 20mM, the pH was 8.0, and the concentration of Tris buffer was 20mM; buffer B was prepared by mixing NaCl, imidazole and Tris buffer, wherein the concentration of NaCl was 0.5M, the concentration of imidazole was 400mM, the pH was 8.0, and the concentration of Tris buffer was 20mM; buffer C was prepared by mixing NaCl and Tris buffer, wherein the concentration of NaCl was 0.5M, the concentration of Tris buffer was 20mM, and the pH was 8.0;

[0041] Step S3: 13.35 g / L adenosine, 81.5 g / L ATP-Na2, 10 g / L MgCl2·6H2O, and enzyme solution were reacted at 35°C and pH 8.0 for 20 minutes, followed by HPLC analysis. Adenosine kinase was diluted and then placed in environments at 30°C, 35°C, 40°C, 45°C, and 50°C, respectively. Enzyme activity was measured every hour for 24 hours.

[0042] Step S4: Use SWISS-MODEL to predict the three-dimensional structure of ArK, then align the amino acid sequence of ArK with the amino acid sequence of the template protein, and then use GMQE to determine the three-dimensional model and perform modeling. Then use GROMACS to perform molecular dynamics (MD) simulation to generate a trajectory file, and generate RMSD and RMSF by inputting commands. Use the amino acid sequence of ArK as a probe in the NCBI database to query enzyme proteins that are highly similar to the ArK sequence, and select all amino acid sequences with an identity of more than 35% with the ArK sequence. Multiple sequence alignment was performed, and then the homologous proteins were aligned using the BioXM tool. Combining molecular dynamics simulation and homologous protein multiple sequence alignment, the amino acid residue sites were selected to construct a mutation library, and the following 21 single-point mutants were constructed: V6L, V7C, L8F, L8C, V37A, K45H, T69S, A70M, A74Q, V82C, M83G, Y84F, S87C, G117P, G178A, F192L, V200F, T213D, A217G, C226A, K253R;

[0043] Step S5: Amplify the target DNA fragment by PCR amplification technology, catalyze with KOD high-fidelity enzyme polymerase at 95°C for 2 minutes, and then continue to complete 30 cycles, each cycle includes maintaining at 55°C for 20 seconds, then maintaining at 71°C for 100 seconds, and maintaining at 72°C for 10 minutes after the cycle is completed. Then, perform agarose gel electrophoresis to separate the target DNA fragment, and then cut out the gel portion containing the target DNA fragment by gel recovery, and recover the PCR product. Then, add DpnI enzyme to the PCR product recovered by gel, incubate at 37°C for 2 hours, degrade the initial template, and finally leave a pure PCR product. The PCR product obtained in step S52 is digested by restriction endonuclease. The digested DNA fragments were transformed with Escherichia coli BL21 (DE3) strains, and the transformed cells were plated on LB agar plates containing 50 μg / mL kanamycin. The LB agar plates were placed in a 37°C incubator for overnight culture. The next day, positive clones containing the correct target DNA fragments were obtained by antibiotic screening and sequencing verification, i.e., recombinant bacteria containing ArK enzyme mutants. The obtained recombinant bacteria were treated according to the method in step S1 to obtain a crude enzyme solution of the ArK enzyme mutant, and the mutant was subjected to a thermal stability test according to the method in step S3, wherein the test temperature was 50°C. The crude mutant enzyme solution was then purified according to the purification method in step S2 to finally obtain a pure mutant enzyme solution, and the obtained highly thermally stable adenosine kinase mutant was used for adenylate preparation.

[0044] Example 1:

[0045] The highly thermostable adenosine kinase mutant K253R was used in the preparation of adenosine. NaOH solution was first added to dissolve adenosine, and then adenosine triphosphate was added to make the molar ratio of adenosine to adenosine triphosphate 2:1. The concentration of adenosine was 187 mM, and the amount added was 50 g. The concentration of adenosine triphosphate was 93.5 mM, and the amount added was 54.5 g. Then, 4.07 g of magnesium chloride hexahydrate with a concentration of 20 mM and a pH of 7.0 was added. The resulting reaction system was 500 ml, and the adenosine kinase mutant K253R was added to the reaction system. The reaction mixture contained 16,800 U of ATP and 60,700 U of adenylate kinase. Adenosine and adenosine triphosphate were reacted to generate adenylic acid and adenosine diphosphate under the action of adenosine kinase. Adenosine diphosphate was then reacted to generate adenylic acid and adenosine triphosphate under the action of adenylate kinase. The reaction was allowed to proceed at 50°C for 1 hour. The pH was then adjusted to 7.0 with NaOH solution. Samples were taken during the reaction for HPLC analysis. The final reaction data were AMP: 92.234%, Ar: 6.653%, ADP: 1.054%, and ATP: 0.022%.

[0046] After the reaction liquid is heated to a temperature of 1 000 ℃, the protein and other insoluble substances are removed by filtration. The A259nm is measured to be 4423. The solution is then preheated to 60°C, stirred and adjusted to pH 3.0 with HCl solution. The heating is turned off. After crystals precipitate, HCl solution is gradually added to adjust the pH to 2.0. After stabilizing and cooling to room temperature, the crystallization mother liquor is filtered and the A259nm is measured to be 587. The yield is low and the purity of the wet powder is 99.256%.

[0047] Example 2:

[0048] The highly thermostable adenosine kinase mutant K253R was used to prepare adenosine. NaOH solution was first added to dissolve adenosine, and then adenosine triphosphate was added to make the molar ratio of adenosine to adenosine triphosphate 2:1. The concentration of adenosine was 374 mM, and the amount added was 100 g. The concentration of adenosine triphosphate was 187 mM, and the amount added was 109 g. Then, 4.07 g of magnesium chloride hexahydrate with a concentration of 20 mM and a pH of 7.0 was added. The resulting reaction system was 1000 ml, and the adenosine kinase mutant was added to the reaction system. 33,500 U, adenylate kinase 121,300 U, adenosine and adenosine triphosphate are reacted with adenosine kinase to generate adenylic acid and adenosine diphosphate, and adenosine diphosphate is reacted with adenylic acid and adenosine triphosphate. The reaction is allowed to stand at 50°C for 1 hour, and then the pH is adjusted to 7.0 with NaOH solution. Samples are taken during the reaction for HPLC analysis. The final reaction data are AMP: 91.525%, Ar: 6.967%, ADP: 1.387%, and ATP: 0.022%;

[0049] After the reaction liquid heat is terminated, the protein and other insoluble matter are removed by suction filtration. Then, the solution is preheated to 60°C, and the pH is adjusted to 3.0 with HCl solution while stirring. The heating is turned off. After crystals precipitate, HCl solution is gradually added to adjust the pH to 2.0. After stabilizing and cooling to room temperature, the crude adenylate is obtained by filtration. The purity is 99.063%, the wet weight is 282 g, and the finished adenylate is obtained after purification. The net weight is 185.77 g, the purity is 99.646%, and the content is 99.5%.

[0050] Example 3:

[0051] The highly thermostable adenosine kinase mutant K253R was used to prepare adenosine. Adenosine and adenosine triphosphate were mixed at a molar ratio of 2:1.1. The concentration of adenosine was 374 mM and the amount added was 100 g. The concentration of adenosine triphosphate was 206 mM and the amount added was 120 g. Then, 4.07 g of magnesium chloride hexahydrate at a concentration of 20 mM and a pH of 7.0 was added to the resulting reaction system of 1000 ml. 33,500 U of the adenosine kinase mutant and 12. 130,000 U, adenosine and adenosine triphosphate are reacted with adenosine kinase to generate adenylate and adenosine diphosphate, and adenosine diphosphate is reacted with adenylate kinase to generate adenylate and adenosine triphosphate. The reaction is stirred at 50°C. The pH is automatically controlled at 7.0 for 4 hours before the reaction and at 7.5 after 4 hours using an automatic pH adjustment system. Samples are taken during the reaction for HPLC analysis. The final data after 18 hours of reaction are AMP: 93.967%, Ar: 2.579%, ADP: 3.240%, and ATP: 0.098%.

[0052] After the reaction liquid heat is terminated, the protein and other insoluble matter are removed by suction filtration. Then, the solution is preheated to 60°C, and the pH is adjusted to 3.0 with HCl solution while stirring. The heating is turned off. After crystals precipitate, HCl solution is gradually added to adjust the pH to 2.0. After stabilizing and cooling to room temperature, the crude adenylate is obtained by filtration. After drying, the finished adenylate is obtained, with a net weight of 165.62 g, a purity of 99.683%, and an assay of 99.3%.

[0053] Example 4:

[0054] The highly thermostable adenosine kinase mutant K253R was used to prepare adenosine. Adenosine and adenosine triphosphate were mixed at a molar ratio of 2:1.2. The concentration of adenosine was 374 mM and the amount added was 100 g. The concentration of adenosine triphosphate was 225 mM and the amount added was 131 g. Then, 4.07 g of magnesium chloride hexahydrate at a concentration of 20 mM and a pH of 7.0 was added to the resulting reaction system of 1000 ml. 33,500 U of the adenosine kinase mutant and 12. 130,000 U, adenosine and adenosine triphosphate are reacted with adenosine kinase to generate adenylate and adenosine diphosphate, and adenosine diphosphate is reacted with adenylate kinase to generate adenylate and adenosine triphosphate. The reaction is stirred at 50°C. The pH is automatically controlled at 7.0 for 4 hours before the reaction and at 7.5 after 4 hours using an automatic pH adjustment system. Samples are taken during the reaction for HPLC analysis. After 18 hours of reaction, the final data are AMP: 90.266%, Ar: 0.981%, ADP: 8.323%, and ATP: 0.325%.

[0055] After the reaction liquid heat is terminated, the protein and other insoluble matter are removed by suction filtration. Then, the solution is preheated to 60°C, and the pH is adjusted to 3.0 with HCl solution while stirring. The heating is turned off. After crystals precipitate, HCl solution is gradually added to adjust the pH to 2.0. After stabilizing and cooling to room temperature, the wet product of adenylate is obtained by filtration. After drying, the finished product of adenylate is obtained, with a net weight of 170.53 g, a purity of 99.541%, and an assay of 99.2%.

[0056] Embodiment 5:

[0057] The highly thermostable adenosine kinase mutant K253R was used in the preparation of adenosine triphosphate. Adenosine was mixed with adenosine triphosphate at a molar ratio of 2:1.1. The concentration of adenosine was 374 mM and the amount added was 100 g. The concentration of adenosine triphosphate was 206 mM and the amount added was 120 g. Then, 4.07 g of magnesium chloride hexahydrate with a concentration of 20 mM and a pH of 7.0 was added. The resulting reaction system was 1000 ml. 16,800 U of adenosine kinase mutant and 6.0 U of adenylate kinase were added to the reaction system. 70,000 U, adenosine and adenosine triphosphate generate adenylate and adenosine diphosphate under the action of adenosine kinase, and adenosine diphosphate generates adenylate and adenosine triphosphate under the action of adenylate kinase. The reaction is stirred at 40°C. The pH is automatically controlled at 7.0 for 22 hours before the reaction and at 7.5 after 22 hours using an automatic pH adjustment system. Samples are taken during the reaction for HPLC analysis. The final data after 36 hours of reaction are AMP: 93.551%, Ar: 1.755%, ADP: 4.506%, and ATP: 0.101%.

[0058] After the reaction liquid heat is terminated, the protein and other insoluble matter are removed by suction filtration. Then, the solution is preheated to 60°C, and the pH is adjusted to 3.0 with HCl solution while stirring. The heating is turned off. After crystals precipitate, HCl solution is gradually added to adjust the pH to 2.0. After stabilizing and cooling to room temperature, the wet product of adenylate is obtained by filtration. After drying, the finished adenylate product is obtained, with a net weight of 167.34 g, a purity of 99.663%, and an assay of 99.4%.

[0059] The results of the thermal stability test of adenosine kinase mutants at 50°C are as follows:

[0060]

[0061]

[0062] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

Claims

1. A highly thermostable adenosine kinase mutant, characterized in that: The highly thermostable adenosine kinase mutant is obtained by performing a point mutation at position 253 or 82 of the adenosine kinase obtained by translating the nucleotide sequence shown in SEQ ID NO: 1; The mutation position at position 253: K253R; The mutation position at site 82 is: V82C.

2. A gene encoding the adenosine kinase mutant according to claim 1.

3. An expression vector comprising the coding gene according to claim 2.

4. The expression vector according to claim 3, characterized in that: The expression vector was constructed based on pET28a(+).

5. A genetically engineered bacterium comprising the adenosine kinase mutant according to claim 1, the encoding gene according to claim 2, or the expression vector according to any one of claims 3 to 4.

6. The genetically engineered bacterium according to claim 5, characterized in that: The genetically engineered bacteria is Escherichia coli BL21 (DE3).

7. Use of the adenosine kinase mutant according to claim 1, the encoding gene according to claim 2, the expression vector according to any one of claims 3 to 4, or the genetically engineered bacterium according to claim 6 in the production of adenosine monophosphate.

8. A method for preparing adenylic acid, characterized in that: The method uses the adenosine kinase mutant according to claim 1 or the genetically engineered bacteria according to any one of claims 5 to 6 as a catalyst.

9. The method according to claim 8, characterized in that: The method uses adenosine, disodium adenosine triphosphate and magnesium chloride hexahydrate as substrates; uses adenylate kinase and adenosine kinase mutants as catalysts, constructs a reaction system, and synthesizes adenylate in a one-step reaction.

10. The method according to claim 9, characterized in that: The reaction system contains 20-374 mM adenosine, 10-226 mM adenosine disodium triphosphate and 10-100 mM magnesium chloride hexahydrate.

11. The method according to claim 9, wherein: The reaction system contains 60.7-121.3 U of adenylate kinase and 16.8-33.5 U of adenosine kinase mutant per 100 mL.

12. The method according to claim 9, wherein: The one-step reaction is carried out at 30-50°C.

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