Fusion enzyme as well as preparation method and application thereof

Abstract

The invention discloses a fusion enzyme as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. A linker protein Linker is utilized, nucleoside 2 '-deoxyribosyltransferase LlNDT and adenosine deaminase LlADA are used as original enzymes, a fusion enzyme NLA connected with three amino acid sequences is designed, a recombinant plasmid containing a fusion enzyme NLA gene sequence is constructed through an artificial gene synthesis technology, enzyme clear liquid containing the fusion enzyme NLA is obtained through escherichia coli transformation, amplification and inducible expression, and the recombinant plasmid containing the fusion enzyme NLA is obtained. The method is used for synthesizing the nucleoside analogues, two-step enzymatic conversion reaction for synthesizing the nucleoside analogues in the prior art is unified into one-step continuous enzyme reaction, the synthesis process of the nucleoside analogues is simplified, and the production efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a fusion enzyme and a preparation method and application thereof. Background Art

[0002] With the continuous advancement of life science research technology, the mysteries of genes are being continuously deciphered, and nucleic acid drugs based on genes are constantly emerging. Nucleoside analogs (NAs) have the characteristics of antiviral and antitumor activity. They can be incorporated into DNA or RNA to inhibit cell division and viral replication. Therefore, NAs are widely used in clinical treatment, and the global demand for NAs is increasing. In traditional methods, NAs are synthesized using multi-step chemical methods. These methods require protection-deprotection reactions on heterocyclic bases or pentose moieties. Not only are the synthesis steps cumbersome and the yield is low, but also a large amount of organic solvents are used, there are many industrial wastes, it is environmentally unfriendly, and the production cost is high. In this case, novel, economical, safe and environmentally friendly synthesis processes are urgently needed. Enzyme-catalyzed industrial reactions have begun to have an increasing impact on industrial chemical processes due to their uniqueness, such as higher regio- and stereoisomer selectivity, no or fewer intermediates, and more sustainability and environmental friendliness.

[0003] Nucleoside 2′-deoxyribosyltransferase (NDT) is mainly used in the synthesis of nucleoside analogs. NDT has the function of catalyzing the one-step transglycosylation reaction between purine or pyrimidine bases and nucleosides, and can directly transfer the 2′-deoxyribose moiety from the 2′-deoxynucleoside donor to the nucleobase acceptor to achieve controllable conversion between NAs. However, the efficiency of synthesizing 2'-deoxy-2'-fluoroadenosine using NDT in the prior art is low, and a subsequent deamination reaction is required to produce 2'-deoxy-2'-fluoroinosine, which limits its expanded application. Summary of the invention

[0004] The purpose of the present invention is to provide a fusion enzyme and a preparation method and application thereof, so as to realize one-step synthesis of nucleoside analogs and simplify the synthesis process.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The present invention provides a fusion enzyme, the amino acid sequence of the fusion enzyme is shown in SEQ ID NO.1;

[0007] The nucleotide sequence of the fusion enzyme is shown in SEQ ID NO.2.

[0008] The present invention also provides a method for preparing the above fusion enzyme, comprising the following steps:

[0009] S1, connect nucleoside 2′-deoxyribosyltransferase LlNDT and adenosine deaminase LlADA through the linker protein to form the fusion enzyme NLA;

[0010] S2, adding HindIII restriction site and Xho I restriction site to the 5' end and 3' end of the nucleotide sequence of the fusion enzyme NLA by artificial gene synthesis technology, respectively, to obtain the target DNA fragment;

[0011] S3, subclone the target DNA fragment into the pET-28b(+) expression vector to construct the recombinant plasmid pET-28b(+)-NLA;

[0012] S4, transforming the recombinant plasmid pET-28b(+)-NLA into competent Escherichia coli BL21(DE3) cells to obtain recombinant Escherichia coli;

[0013] S5. Streak the recombinant E. coli on solid LB medium and culture at 37°C overnight. Pick out a single clone strain and place it in 1 mL of liquid LB medium. Culture it at 37°C with shaking at 200 rpm for 4 h to obtain a seed solution.

[0014] S6. Inoculate the seed solution into 100 mL of liquid LB medium at a 1% inoculum volume, culture at 37°C with shaking at 200 rpm until OD600 is 0.6-0.8, add inducer IPTG, the final concentration of inducer IPTG is 0.5 mM, induce at 22°C for 12-16 h, and collect the bacteria by centrifugation after the induction is completed;

[0015] S7. Add the bacteria to PBS buffer for resuspending, ultrasonically disrupt in an ice bath, and then centrifuge. Collect the supernatant to obtain the enzyme supernatant containing the fusion enzyme NLA, and perform SDS-PAGE detection.

[0016] Furthermore, the amino acid sequence of the nucleoside 2′-deoxyribosyltransferase LlNDT is shown in SEQ ID NO.3;

[0017] The nucleotide sequence of the nucleoside 2′-deoxyribosyltransferase L1NDT is shown in SEQ ID NO.4.

[0018] Furthermore, the amino acid sequence of the adenosine deaminase L1ADA is shown in SEQ ID NO.5;

[0019] The nucleotide sequence of the adenosine deaminase L1ADA is shown in SEQ ID NO.6.

[0020] Furthermore, the amino acid sequence of the linker protein Linker is shown in SEQ ID NO.7;

[0021] The nucleotide sequence of the linker protein Linker is shown in SEQ ID NO.8.

[0022] Furthermore, the gene sequence of the target DNA fragment is shown as SEQ ID NO.9.

[0023] Furthermore, the solid LB culture medium and the liquid LB culture medium both contain 50 mg / L of kanamycin sulfate.

[0024] Furthermore, the bacteria and PBS buffer were resuspended by adding 10 mL of PBS disruption buffer to 1 g of wet bacteria.

[0025] The present invention also provides an application of a fusion enzyme, wherein the enzyme serum containing the fusion enzyme NLA prepared by the above-mentioned preparation method is used to synthesize nucleoside analogs.

[0026] Furthermore, the specific steps of synthesizing nucleoside analogs are as follows:

[0027] 1 mM 2'-deoxy-2'-fluorouridine and 1 mM adenine were added to 50 mL of the enzyme supernatant containing the fusion enzyme NLA, the pH was adjusted to 6.0-7.0, and the mixture was placed in a constant temperature shaking incubator to react at 45°C and 200 rpm.

[0028] Beneficial effects of the present invention:

[0029] The present invention utilizes a linker protein, takes nucleoside 2'-deoxyribosyltransferase LlNDT and adenosine deaminase LlADA as original enzymes, and transforms and connects them through genetic engineering technology to form a fusion enzyme for expression, thereby unifying the two-step enzymatic conversion reaction of synthesizing nucleoside analogs into a one-step continuous enzymatic reaction, effectively optimizing the reaction process and improving production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below in conjunction with the accompanying drawings.

[0031] Figure 1 It is a schematic diagram of the reaction process of synthesizing nucleoside analogs of the present invention.

[0032] Figure 2 This is the SDS-PAGE detection diagram of the enzyme supernatant in Example 1 of the present invention;

[0033] Among them, band M is a marker, band 1 is the supernatant of the expression product of the control plasmid pET-28b(+), and band 2 is the supernatant of the expression product of the recombinant plasmid pET-28b(+)-NLA, that is, the enzyme supernatant containing the fusion enzyme NLA.

[0034] Figure 3 It is a map of the recombinant plasmid pET-28b(+)-NLA in the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] In the embodiment, the nucleoside 2′-deoxyribosyltransferase LlNDT is derived from Lactobacillus, the adenosine deaminase LlADA is derived from Lactococcus lactis, and the amino acid sequence is obtained from the biological information website. The amino acid sequence of the nucleoside 2′-deoxyribosyltransferase LlNDT is shown in SEQ ID NO.3, and the nucleotide sequence of the nucleoside 2′-deoxyribosyltransferase LlNDT is deduced according to the codon preference of Escherichia coli as shown in SEQ ID NO.4, and the amino acid sequence of the adenosine deaminase LlADA is shown in SEQ ID NO.5, and the nucleotide sequence of the adenosine deaminase LlADA is deduced according to the codon preference of Escherichia coli as shown in SEQ ID NO.6. The amino acid sequence of the linker protein Linker is shown in SEQ ID NO.7, and the nucleotide sequence of the linker protein Linker is deduced according to the codon preference of Escherichia coli as shown in SEQ ID NO.8.

[0037] SEQ ID NO.3:

[0038] MPKKTIYFGAGWFTDRQNKAYKEAMEALKENPTIDLENSYVPLDNQYKGIRVDEHPEYLHDKVWATATYNNDLNGIKTNDIMLGVYIPDEEDVGLGMELGYALSQGKYVLLVIPDEDYGKPINLMSWGVSDNVIKMSQLKDFNFNKPRFDFYEGAVY.

[0039] SEQ ID NO.4:

[0040] ATGCCTAAAAAAACCATCTACTTCGGCGCAGGTTGGTTCACTGATCGCCAGAACAAAGCTTATAAGGAAGCGATGGAAGCTCTGAAGGAA AACCCGACCATCGACCTGGAAAACAGCTACGTTCCGCTGGATAACCAGTACAAAGGCATCCGCGTCGATGAACATCCGGAATATCTGCACGACAAAGTTTGGGCTACTGCGACTTATAACAATGACCTGAACGGCATCAAAACCAACGACATTATGCTGGGTGTGTACATCCCAGATGAAGAAGACGTTGGTCTGGGCATGGAACTGGGTTATGCTCTGTCCCAGGGCAAATACGTACTGCTGGTAATTCCGGATGAGGACTACGGTAAACCTATTAACCTGATGTCCTGGGGCGTTTCTGATAACGTTATCAAAATGAGCCAGCTGAAGGACTTCAACTTCAACAAGCCGCGTTTCGATTTCTATGAGGGTGCTGTATAT。

[0041] SEQ ID NO.5:

[0042] MKKKREKLVLKSEIIAQMPKVELHCHLDGSLSLSVIKELAKNAGIHMTMSDEEILEKAQAPENTKNLLEYLQRFDFVLPLLQTYKNLELAAYDVVRQAANDNIKYIEIRFAPSQHLLENLTLEEAVEAVIAGLSRAENDFDIRANALVCGLKQEPIQKLQKLLPLFDKIPDEHLVGFDMAGDELNYPQEKFVDLIHDIKIKGVNVTLHAGECPACEKNILDSIAMGASRIGHGIMTKNLSEAEQKMMIEKQIVLEMAPTSNFQTKAVTELAQYPFKELYDKGIHVTLNTDNRMVSATNLSKEYEKISAWYPDFSLSDFEKVNHYAIDGAFIGQEEKEELHQRFTKEYKKISE

[0043] SEQ ID NO.6:

[0044] ATGAAAAAAAAACGTGAAAAACTGGTACTGAAATCTGAAATTATTGCCCAGATGCCGAAAGTGGAGCTGCATTGTCACCTGGACGGTAGCCTGAGCCTGTCTGTAATCAAGGAACTGGCGAAGAATGCTGGTATCCACATGACCATGAGCGATGAAGAAATCCTGGAAAAAGCCCAGGCGCCTGAGAACACCAAAAACCTGCTGGAATATCTGCAACGCTTTGACTTTGTACTGCCGCTGCTGCAGACGTATAAAAACCTGGAACTGGCTGCTTACGACGTCGTGCGCCAGGCAGCCAACGACAACATCAAATACATTGAAATCCGTTTTGCACCATCCCAACATCTGCTGGAAAACCTGACCCTGGAAGAAGCTGTGGAAGCGGTAATCGCAGGTCTGTCTCGTGCCGAAAACGATTTTGACATCCGTGCGAATGCCCTGGTTTGTGGCCTGAAACAGGAGCCGATTCAGAAACTGCAGAAACTGCTGCCGCTGTTCGATAAAATCCCGGATGAACACCTGGTTGGTTTCGATATGGCCGGTGACGAACTGAACTACCCGCAGGAAAAATTCGTTGATCTGATCCACGACATCAAAATCAAAGGCGTAAACGTTACTCTGCATGCAGGCGAATGTCCGGCATGCGAAAAAAACATTCTGGATTCCATCGCGATGGGTGCGAGCCGTATTGGCCATGGTATTATGACGAAAAACCTGAGCGAAGCCGAGCAGAAGATGATGATTGAAAAACAGATCGTACTGGAAATGGCACCGACCTCTAATTTCCAGACCAAAGCGGTAACTGAGCTGGCGCAGTATCCGTTTAAGGAACTGTACGACAAAGGTATCCACGTGACCCTGAACACCGATAACCGTATGGTTTCCGCTACCAACCTGAGCAAAGAATACGAGAAAATCTCCGCCTGGTACCCAGATTTCTCTCTGAGCGACTTCGAGAAAGTTAACCATTATGCCATCGACGGCGCGTTCATCGGTCAGGAAGAAAAAGAGGAACTGCACCAGCGTTTTACTAAAGAATACAAAAAGATCTCTGAA。

[0045] SEQ ID NO.7:

[0046] EEEEKKKK。

[0047] SEQ ID NO.8:

[0048] GAGGAAGAGGAAAAGAAAAAGAAA。

[0049] Example 1

[0050] Preparation of enzyme supernatant:

[0051] S1. The amino acid sequences of nucleoside 2′-deoxyribosyltransferase LlNDT, adenosine deaminase LlADA and linker protein Linker are connected to form a fusion protein of LlNDT-Linker-LlADA, which is called fusion enzyme NLA. The amino acid sequence of the fusion enzyme NLA is shown in SEQ ID NO.1. The nucleotide sequence of the fusion enzyme NLA is deduced according to the codon preference of Escherichia coli and is shown in SEQ ID NO.2.

[0052] S2. Adding a HindIII restriction site and a Xho I restriction site to the 5' end and 3' end of the nucleotide sequence of the fusion enzyme NLA by artificial gene synthesis technology, respectively, to obtain a target DNA fragment, the gene sequence of which is shown in SEQ ID NO.9;

[0053] S3, subclone the target DNA fragment into the pET-28b(+) expression vector to construct the recombinant plasmid pET-28b(+)-NLA;

[0054] S4, transforming the recombinant plasmid pET-28b(+)-NLA into competent Escherichia coli BL21(DE3) cells to obtain recombinant Escherichia coli;

[0055] S5. Streak the recombinant E. coli on solid LB medium (containing 50 mg / L kanamycin sulfate), culture at 37°C overnight, pick a single clone strain and place it in 1 mL of liquid LB medium (containing 50 mg / L kanamycin sulfate), and culture it at 37°C with shaking at 200 rpm for 4 h to obtain a seed solution;

[0056] S6. Inoculate the seed solution into 100 mL of liquid LB medium (containing 50 mg / L kanamycin sulfate) at a 1% inoculum volume, culture at 37°C with shaking at 200 rpm until OD600 is 0.6-0.8, add inducer IPTG to a final concentration of 0.5 mM, induce at 22°C for 15 h, and collect the cells by centrifugation after the induction is completed;

[0057] S7. Add the bacteria to PBS buffer for resuspending, prepare a ratio of 10 mL PBS disruption buffer to 1 g wet bacteria, and disrupt by ultrasound in an ice bath. Set the ultrasound power to 300 W, ultrasound for 2 seconds, rest for 3 seconds, ultrasound for 10 minutes, and then centrifuge for 20 minutes at a speed of 12000 rpm. Collect the supernatant to obtain the enzyme supernatant containing the fusion enzyme NLA, and perform SDS-PAGE detection. The detection figure is shown in FIG. Figure 2 .

[0058] SEQ ID NO.1:

[0059] MPKKTIYFGAGWFTDRQNKAYKEAMEALKENPTIDLENSYVPLDNQYKGIRVDEHPEYLHDKVWATATYNNDLNGIKTNDIMLGVYIPDEEDVGLGMELGYALSQGKYVLLVIPDEDYGKPINLMSWGVSDNVIKMSQLKDFNFNKPRFDFYEGAVYEEEEKKKKMKKKREKLVLKSEIIAQMPKVELHCHLDGSLSLSVIKELAKNAGIHMTMSDEEILEKAQAPENTKNLLEYLQRFDFVLPLLQTYKNLELAAYDVVRQAANDNIKYIEIRFAPSQHLLENLTLEEAVEAVIAGLSRAENDFDIRANALVCGLKQEPIQKLQKLLPLFDKIPDEHLVGFDMAGDELNYPQEKFVDLIHDIKIKGVNVTLHAGECPACEK NILDSIAMGASRIGHGIMTKNLSEAEQKMMIEKQIVLEMAPTSNFQTKAVTELAQYPFKELYDKGIHVTLNTDNRMVSATNLSKEYEKISAWYPDFSLSDFEKVNHYAIDGAFIGQEEKEELHQRFTKEYKKISE。

[0060] SEQ ID NO.2:

[0061]

[0062] SEQ ID NO.9:

[0063] CCCAAGCTTATGCCTAAAAAAACCATCTACTTCGGCGCAGGTTGGTTCACTGATCGCCAGAACAAAGCTTATAAGGAAGCGATGGAAGCTCTGAAGGAAAACCCGACCATCGACCTGGAAAACAGCTACGTTCCGCTGGATAACCAGTACAAAGGCATCCGCGTCGATGAACATCCGGAATATCTGCACGACAAAGTTTGGGCTACTGCGACTTATAACAATGACCTGAACGGCATCAAAACCAACGACATTATGCTGGGTGTGTACATCCCAGATGAAGAAGACGTTGGTCTGGGCATGGAACTGGGTTATGCTCTGTCCCAGGGCAAATACGTACTGCTGGTAATTCCGGATGAGGACTACGGTAAACCTATTAACCTGATGTCCTGGGGCGTTTCTGATAACGTTATCAAAATGAGCCAGCTGAAGGACTTCAACTTCAACAAGCCGCGTTTCGATTTCTATGAGGGTGCTGTATATGAGGAAGAGGAAAAGAAAAAGAAAATGAAAAAAAAACGTGAAAAACTGGTACTGAAATCTGAAATTATTGCCCAGATGCCGAAAGTGGAGCTGCATTGTCACCTGGACGGTAGCCTGAGCCTGTCTGTAATCAAGGAACTGGCGAAGAATGCTGGTATCCACATGACCATGAGCGATGAAGAAATCCTGGAAAAAGCCCAGGCGCCTGAGAACACCAAAAACCTGCTGGAATATCTGCAACGCTTTGACTTTGTACTGCCGCTGCTGCAGACGTATAAAAACCTGGAACTGGCTGCTTACGAC.

[0064] Example 2

[0065] Synthetic nucleoside analogs:

[0066] 1 mM 2'-deoxy-2'-fluorouridine and 1 mM adenine were added to 50 mL of the enzyme supernatant containing the fusion enzyme NLA prepared in Example 1, the pH was adjusted to 6.0-7.0, and the supernatant was placed in a constant temperature shaking incubator at 45°C and 200 rpm for reaction. The reaction process is shown in the schematic diagram. Figure 1HPLC detection was performed every 12 hours to detect the amount of 2'-fluoro-2'-deoxyinosine produced and the amount of 2'-deoxy-2'-fluorouridine reduced. After 60 hours, the reaction product no longer changed, and the reaction was terminated. The conversion rate of the product 2'-fluoro-2'-deoxyinosine was 91.2%.

[0067] The conditions for HPLC detection are shown in Table 1:

[0068] Table 1

[0069]

[0070] Comparative Example 1

[0071] 2'-Fluoro-2'-deoxyinosine was prepared according to the preparation method provided in Example 7 of Patent No. KR1020190037771A.

[0072] Combining Comparative Example 1 and Example 2, it can be seen that in Example 2, the enzyme supernatant containing the fusion enzyme NLA of Example 1 is used to prepare 2'-fluoro-2'-deoxyinosine, and 2'-fluoro-2'-deoxyinosine can be converted and synthesized in one step. Compared with Comparative Example 1, the synthesis time is short, the synthesis steps are simplified, and the conversion rate is higher.

[0073] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0074] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A fusion enzyme, characterized in that The amino acid sequence of the fusion enzyme is shown in SEQ ID NO.1; The nucleotide sequence of the fusion enzyme is shown in SEQ ID NO.

2.

2. A method for preparing the fusion enzyme according to claim 1, characterized in that: The following steps are involved: S1, connect nucleoside 2′-deoxyribosyltransferase LlNDT and adenosine deaminase LlADA through the linker protein to form the fusion enzyme NLA; S2, adding Hind III restriction site and Xho I restriction site to the 5' end and 3' end of the nucleotide sequence of the fusion enzyme NLA by artificial gene synthesis technology, respectively, to obtain the target DNA fragment; S3, subclone the target DNA fragment into the pET-28b(+) expression vector to construct the recombinant plasmid pET-28b(+)-NLA; S4, transforming the recombinant plasmid pET-28b(+)-NLA into competent Escherichia coli BL21(DE3) cells to obtain recombinant Escherichia coli; S5. Streak the recombinant E. coli on solid LB medium and culture at 37°C overnight. Pick out a single clone strain and place it in 1 mL of liquid LB medium. Culture it at 37°C with shaking at 200 rpm for 4 h to obtain a seed solution. S6. Inoculate the seed solution into 100 mL of liquid LB medium at a 1% inoculum volume, culture at 37°C with shaking at 200 rpm until OD600 is 0.6-0.8, add inducer IPTG, the final concentration of inducer IPTG is 0.5 mM, induce at 22°C for 12-16 h, and collect the bacteria by centrifugation after the induction is completed; S7. Add the bacteria to PBS buffer for resuspending, ultrasonically disrupt in an ice bath, and then centrifuge. Collect the supernatant to obtain the enzyme supernatant containing the fusion enzyme NLA, and perform SDS-PAGE detection.

3. The method for preparing a fusion enzyme according to claim 2, characterized in that: The amino acid sequence of the nucleoside 2′-deoxyribosyltransferase L1NDT is shown in SEQ ID NO.3; The nucleotide sequence of the nucleoside 2′-deoxyribosyltransferase L1NDT is shown in SEQ ID NO.

4.

4. The method for preparing a fusion enzyme according to claim 2, characterized in that: The amino acid sequence of the adenosine deaminase L1ADA is shown in SEQ ID NO.5; The nucleotide sequence of the adenosine deaminase L1ADA is shown in SEQ ID NO.

6.

5. The method for preparing a fusion enzyme according to claim 2, characterized in that: The amino acid sequence of the linker protein Linker is shown in SEQ ID NO.7; The nucleotide sequence of the linker protein Linker is shown in SEQ ID NO.

8.

6. The method for preparing a fusion enzyme according to claim 2, characterized in that: The gene sequence of the target DNA fragment is shown in SEQ ID NO.

9.

7. The method for preparing a fusion enzyme according to claim 2, characterized in that: The solid LB culture medium and the liquid LB culture medium both contain 50 mg / L of kanamycin sulfate.

8. The method for preparing a fusion enzyme according to claim 2, characterized in that: The bacteria and PBS buffer were resuspended by adding 10 mL of PBS disruption buffer to 1 g of wet bacteria.

9. An application of a fusion enzyme, characterized in that: Nucleoside analogs are synthesized using the enzyme serum containing the fusion enzyme NLA prepared by the preparation method according to any one of claims 2 to 7.

10. The use of a fusion enzyme according to claim 9, characterized in that: The specific steps of synthesizing nucleoside analogs are as follows: 1 mM 2'-deoxy-2'-fluorouridine and 1 mM adenine were added to 50 mL of the enzyme supernatant containing the fusion enzyme NLA, the pH was adjusted to 6.0-7.0, and the mixture was placed in a constant temperature shaking incubator to react at 45°C and 200 rpm.

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