Method for producing methanol compound from methane gas using cytochrome P450 and use thereof

Abstract

The present invention relates to a novel methane oxidase, a variant thereof, and uses thereof, wherein the enzyme or the variant thereof can convert methane gas into methanol with high efficiency.

Description

Technology Field

[0001] The present invention relates to a method for producing a methanol compound from methane gas using cytochrome P450 and the use thereof. Background Technology

[0003] Methane, the main component of natural gas, shale gas, and biogas, is a primary cause of global warming and a target for reduction, while simultaneously attracting attention as a next-generation carbon resource. Consequently, research on chemical and biological conversion is necessary to utilize methane.

[0004] Although various C1 gas utilization technologies have recently emerged, they are concentrated on the production of basic C1 chemicals or fuels such as methanol and formic acid. However, due to the price of C1 chemicals being only about $300–$500 per ton, it has been difficult to ensure economic viability when producing simple C1 substances by C1 gas refineries.

[0005] Therefore, to ensure economic viability, various novel aldolases have been developed, including enzymes that convert methanol into formaldehyde and those capable of biosynthesizing C4 compounds from formaldehyde or acetaldehyde.

[0006] However, ultimately, there is a need for a methane oxidase capable of converting methane into methanol, and in particular, there is a demand for the discovery of a methane oxidase that can be expressed within the most widely used E. coli. Prior art literature

[0008] Republic of Korea Published Patent Application No. 10-2018-0023735 The problem to be solved

[0009] One aspect of the present invention aims to provide a polypeptide consisting of the amino acid sequence of SEQ ID NO. 1.

[0010] Another aspect of the present invention aims to provide a polypeptide in which any one of the 91st, 115th, 116th, 205th, 266th, 270th, 273rd, 277th, 323rd and 423rd amino acids in the amino acid sequence of SEQ ID NO. 1 is mutated.

[0011] Another aspect of the present invention aims to provide a method for producing methanol comprising the step of reacting methane gas with the polypeptide. means of solving the problem

[0013] One aspect of the present invention provides a polypeptide consisting of the amino acid sequence of SEQ ID NO. 1.

[0014] The amino acid of SEQ ID NO. 1 above is a polypeptide having methane oxidase activity characterized by converting methane gas into methanol, and the specific amino acid sequence and nucleotide sequence are as follows.

[0016] Sequence No. 1 MELKSHPAGRCPVAHEAVTGAPTGCPVSARAAAFDPFQDAYMEDPAEFVRWAREQEPVFYAPRLGYWVVTRYQTIKDIFRDPVTFSPSIVLERMSPTSDEALQVLKQYGYAMNRRTLVNEDEPIHMARRRVLMAPFTPEHLAGHVPMVRELVRKAVDRFIDDGRADLVDQLLWEVPFTVALHFLGIDAEDREKMR RFSIAHTVNAFGRPSPEQQVSIAHTVGQFWQFAGEVLEKMRRTPDGPGWMRYSIRQQKLYPDIVTDSYLHSMMMAIIVAAHETTAFASANAIKLLLQHPTAWRDLCEDPALISPAVEECLRYSGSVASWRRRTTRDVVLDGVPVPAESKLLLVVSSANHDGAHFVDPDLFDIRRENSAEHLTFGFGAHQCLGKNI GRMEMQVMIEELSRRLPHMKLAPQTFEYVHNVSFRGPQHLWVEWDPARNPERRDPSILQRRHPVRIGAPHARDKVRPLVVRAAEPVAEGIVRLRLAATDGRPLPQWAPGAHIDIECGETGLSRQYSLCGPLSDTSEWTVAVQLDPASRGGSAWIHRHATPGTVLRARGPRNHFRLDEQARRLIFVAGGIGITPIM AMAERARALGIPYEIHYSVRCRACLAFERELRELHGERLHLYVSEEGRRNDLAGLLATPDPQARIYACGPQRMLEALERLAAGWPEDALRIEHFSGSAPRLDPSCERPFEVELRNSGLRLEVPADRTLLEVLRASNVDVQSDCEEGLCGSCEVGVLEGEVDHRDSVLGRAERQGHRRMMACCSRARSQRLVLDLZ Sequence No. 2 ATGGAGCTGAAGTCCCATCCCGCCGGGCGCTGCCCGGTGGCGCACGAGGCCGTGACCGGTGCGCCCACCGGCTGTCCGGTGAGCGCGCGCGCCGCGGCCTTCGATCCGTTCCAGGACGCGTACATGGAAGACCCGGCCGAGTTCGTGCGCTGGGCACGCGAGCAGGAGCCGGTCTTCTATGCGCCCAGGCTCGGCTACTGGGTGGTGACGCGCTACCAGACGATCAAGGACATCTTCCGCGATCCCGTCACCTTCAGCCCGTCGATCGTGCTCGAGCGCATGTCGCCGACCTCGGACGAGGCGCTGCAGGTGCTGAAGCAGTACGGCTACGCGATGAACCGTACGCTCGTCAACGAGGACGAGCCGATCCACATGGCACGGCGCCGCGTGCTGATGGCGCCGTTCACGCCGGAGCACCTCGCCGGGCATGTCCCGATGGTGCGCGAGCTGGTGCGCAAGGCGGTCGACCGCTTCATCGACGATGGCCGCGCCGACCTGGTGGACCAGCTGCTGTGGGAGGTACCGTTCACCGTCGCGCTGCACTTCCTTGGCATCGACGCGGAAGACCGCGAGAAGATGCGGCGCTTCTCGATCGCGCACACCGTCAACGCGTTCGGGCGGCCCTCGCCCGAGCAGCAGGTGTCGATCGCGCACACCGTCGGGCAGTTCTGGCAGTTCGCTGGCGAGGTGCTCGAGAAGATGCGCCGCACGCCCGACGGGCCGGGCTGGATGCGTTATTCCATCCGGCAGCAGAAGCTCTACCCGGACATCGTCACCGACTCGTACCTGCACTCGATGATGATGGCCATCATCGTCGCGGCGCACGAGACGACGGCGTTCGCCTCGGCCAACGCGATCAAGCTGCTGCTGCAGCATCCGACCGCCTGGCGCGACCTGTGCGAGGACCCGGCGCTGATCTCGCCGGCGGTGGAGGAGTGCCTGCGCTACAGCGGCTCGGTGGCCTCGTGGCGGCGCCGCACCACGCGCGACGTGGTGCTCGACGGCGTGCCGGTGCCGGCGGAGTCCAAGCTGCTGCTGGTCGTCTCGTCGGCCAACCATGACGGCGCGCACTTCGTCGACCCCGACCTGTTCGACATCCGCCGCGAGAACTCGGCCGAGCACCTGACCTTCGGCTTCGGCGCGCACCAGTGCCTGGGCAAGAACATCGGGCGCATGGAGATGCAGGTCATGATCGAGGAGCTGAGCCGCCGGCTGCCGCACATGAAGCTCGCGCCGCAGACCTTCGAGTACGTGCACAACGTGTCGTTCCGCGGCCCGCAGCACCTGTGGGTCGAATGGGACCCGGCGCGCAACCCCGAGCGGCGCGACCCGTCGATCCTGCAGCGCCGGCATCCGGTGCGCATCGGCGCGCCGCACGCGCGCGACAAGGTGCGCCCGCTGGTCGTGCGCGCCGCCGAGCCGGTGGCCGAAGGGATCGTGCGGCTGCGCCTGGCGGCGACGGATGGCCGGCCGCTGCCGCAGTGGGCGCCGGGCGCGCACATCGACATCGAGTGCGGCGAGACCGGGCTGTCGCGCCAGTACTCGCTGTGCGGGCCGCTGTCGGACACCTCCGAGTGGACCGTCGCAGTGCAGCTGGATCCGGCCAGCCGCGGCGGCTCGGCCTGGATCCACCGCCACGCGACGCCGGGCACGGTGCTGCGCGCACGCGGCCCTCGCAACCACTTCCGGCTCGACGAGCAGGCGCGCCGGCTGATCTTCGTCGCAGGCGGCATCGGCATCACGCCGATCATGGCGATGGCCGAGCGTGCCCGGGCGTTGGGGATTCCCTACGAGATCCACTACAGCGTGCGCTGCCGTGCCTGCCTCGCGTTCGAGCGCGAGCTGCGCGAGCTGCACGGTGAACGCCTGCACCTGTACGTCAGCGAGGAAGGGCGGCGCAACGACCTGGCCGGGTTGCTCGCGACGCCGGACCCGCAGGCGCGCATCTACGCCTGCGGGCCGCAGCGCATGCTCGAGGCGCTGGAACGCCTGGCCGCGGGCTGGCCCGAGGACGCGTTGCGCATCGAGCACTTCTCGGGCAGCGCACCACGCCTGGATCCCTCGTGCGAGCGGCCGTTCGAGGTGGAGCTGCGCAACAGCGGGCTCAGGCTCGAGGTGCCCGCCGACCGCACGCTGCTCGAGGTGCTGCGCGCGTCCAACGTCGACGTGCAGAGCGACTGCGAGGAGGGCCTGTGCGGCAGCTGCGAGGTGGGCGTGCTCGAAGGCGAGGTCGACCACCGCGACAGCGTGCTGGGCCGCGCCGAGCGCCAGGGCCACCGGCGCATGATGGCGTGCTGCTCGCGCGCCCGTTCGCAGCGCTTGGTGCTGGACCTC

[0017] In the present invention, polypeptide is used in the conventional sense, that is, meaning an arrangement of amino acids. Although polypeptide is not limited to a specific length, in the context of the present invention it generally refers to a full-length protein fragment and may include post-translational modifications, such as glycosylation, acetylation, phosphorylation, etc., and other modifications known in the art (naturally occurring modifications and unnaturally occurring modifications). The polypeptides and proteins of the present invention may be prepared using any various known recombinant and / or synthetic techniques. The present invention includes, without limitation, any protein having the function intended by the present invention, as an amino acid sequence having homology of at least 55%, preferably at least 75%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% with respect to the sequence as well as the amino acid sequence having such homology, and it is obvious that the present invention is also included in the scope of the present invention even if some sequences have deleted, modified, substituted, or added amino acid sequences, provided that the amino acid sequences having such homology have biological activity substantially identical to or corresponding to that of SEQ ID NO. 1.

[0018] In the present invention, homology refers to the degree of correspondence with a given amino acid sequence or base sequence and may be expressed as a percentage. In this specification, a homologous sequence having the same or similar activity as a given amino acid sequence or base sequence is indicated as "% homology." For example, this can be verified by using standard software that calculates parameters such as score, identity, and similarity, specifically BLAST 2.0, or by comparing sequences by Southern hybridization experiments under defined strict conditions, and the defined appropriate hybridization conditions are within the scope of the art (e.g., see Sambrook et al., 1989, infra) and can be determined by methods well known to those skilled in the art.

[0020] The above polypeptide is specifically derived from Escherichia coli and may be a cytochrome p450 variant. The protein of SEQ ID NO. 1 is specifically derived from a microorganism and corresponds to cytochrome p450, and the present invention provides a cytochrome p450 wild-type enzyme and a variant thereof (an enzyme in which the 91st, 115th, 117th, 205th, 266th, 270th, 273rd, 277th, 323rd, and 423rd amino acids are substituted with alanine).

[0021] In the present invention, the cytochrome p450 is an enzyme involved in drug metabolism, such as breaking down substrates like drugs absorbed from outside the cell. It is evenly distributed throughout somatic cells and is mainly found in the endoplasmic reticulum and mitochondria. This cytochrome P450 is not a single form but a complex form composed of several isoenzymes having different molecular structures, and its types include CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A, etc. The activity of these CYP isoenzymes varies among individuals.

[0022] In the present invention, the polypeptide may have high activity at 40 to 60 ℃, 45 to 60 ℃, 50 to 60 ℃, 55 to 60 ℃, 40 to 55 ℃, 45 to 55 ℃, 50 to 55 ℃, 40 to 50 ℃, or 45 to 50 ℃, and more specifically, may have optimal activity at 40, 45, 50, 55, or 60 ℃. The activity of the polypeptide is the relative activity (%) of the enzyme, and high activity or optimal activity may mean an activity of 95% to 100%.

[0023] In the present invention, the polypeptide may have high activity at pH 5 to 8, pH 6 to 8, pH 7 to 8, pH 5 to 7, or pH 6 to 7, and more specifically, may have optimal activity at pH 5, 6, 7, or 8. The activity of the polypeptide is the relative activity (%) of the enzyme, and high activity or optimal activity may mean an activity of 95% to 100%.

[0025] One aspect of the present invention provides a polypeptide in which any one of the 91st, 115th, 117th, 205th, 266th, 270th, 273rd, 277th, 323rd, and / or 423rd amino acids in the amino acid sequence of SEQ ID NO. 1 is mutated.

[0026] The method of the above mutation may be carried out by a known method, and in one embodiment of the present invention, the mutation may occur in any one of L91, T115, V117, F205, M266, I270, A273, T277, W323A, and F423, or more specifically, the mutation may be any one of L91A, T115A, V117A, F205A, M266A, I270A, A273Q, T277A, W323A, and F423A.

[0028] One aspect of the present invention provides a method for producing methanol comprising the step of reacting methane gas with the polypeptide.

[0029] The above reaction step is a step of reacting methane gas with the polypeptide, and the methane gas may be reacted directly with the polypeptide, or the reaction may be carried out by reacting it with a medium containing microorganisms that express the polypeptide.

[0030] The above polypeptide may be the polypeptide of SEQ ID NO. 1 or a mutated polypeptide.

[0031] In one embodiment of the present invention, the polypeptide may be 100 to 1000 pmol.

[0032] In one embodiment of the present invention, the reaction step may be performed at 40 to 60°C, 45 to 60°C, 50 to 60°C, 55 to 60°C, 40 to 55°C, 45 to 55°C, 50 to 55°C, 40 to 50°C, or 45 to 50°C, and more specifically, may be performed at 40, 45, 50, 55, or 60°C.

[0033] In one embodiment of the present invention, the reaction step may be carried out at a pH of 5 to 8, a pH of 6 to 8, a pH of 7 to 8, a pH of 5 to 7, or a pH of 6 to 7, and more specifically, at a pH of 5, 6, 7, or 8. If the reaction is carried out outside the above temperature and pH ranges, the efficiency of the reaction may decrease.

[0034] In one embodiment of the present invention, the reaction step may be performed for 10 to 1500 minutes. However, although the upper limit of the reaction step is described as 1500 minutes for clarity of the present invention, it may be performed for 1500 minutes or more considering the continuity of the process. More specifically, it may be performed for 10 to 30 minutes, 15 to 25 minutes, 19 to 21 minutes, or 20 minutes. Effects of the invention

[0036] The polypeptide or polypeptide variant of the present invention is a novel methane oxidase capable of converting methane gas into methanol with high efficiency, and has the advantage of enabling the construction of a high-efficiency microbial strain and process that can directly produce a final product from methane by utilizing it. Brief explanation of the drawing

[0038] Figures 1 to 3 show the results of verifying whether the polypeptide of the present invention can convert methanol from methane gas. Figure 4 is the result of confirming the methanol conversion level according to the concentration of the polypeptide of the present invention. Figure 5 is the result of confirming the methanol conversion level of the polypeptide of the present invention at different temperatures. Figure 6 is the result of confirming the methanol conversion level of the polypeptide of the present invention according to pH. Figure 7 is the result of confirming the methanol conversion level of the polypeptide of the present invention according to reaction time. Figure 8 shows the results of confirming the substrate affinity of methane oxidase for methane. Figure 9 is the result of confirming the methanol conversion level of the polypeptide mutation of the present invention. Specific details for implementing the invention

[0039] One or more specific examples are described in more detail below through embodiments. However, these embodiments are intended to illustrate one or more specific examples and the scope of the present invention is not limited to these embodiments.

[0041] Example 1: Selection of novel polypeptides (methane oxidase, cytochrome p450)

[0042] In the case of cytochrome p450 (CYP450, p450), when it generally binds to carbon monoxide (CO), the maximum absorption wavelength of the enzyme at a wavelength around 450 nm can be confirmed using a spectrophotometer. In this invention, utilizing the characteristics of cytochrome p450 and methane gas and a novel methane oxidase, the inventors confirmed whether the enzyme (STCYP) identified by the inventors using a spectrophotometer exhibits activity at around 450 nm (Fig. 1). As a result, it was confirmed that the spectrum peak shifted when only the novel methane oxidase was present and when methane gas was injected. This indicates that the novel methane oxidase can bind to methane gas.

[0044] Example 2: Confirmation of activity of selected polypeptides

[0045] The methanol production activity from methane gas of the polypeptide (methane oxidase, cytochrome p450) selected in Example 1 was confirmed.

[0046] Specifically, after adjusting the pH to 7.4 with 100 mM Kpi, 30 μL of 10 mM NADPH and 10–100 μL of polypeptide (methanooxidase, cytochrome p450) CYP116B ST8 were mixed. Then, 5 mL of CH4 was mixed with 5 mL of air and supplied.

[0047] Subsequently, it was incubated at 37°C for 30 minutes, and 5 mL of CH4 was added. It was then incubated overnight at 37°C. Then, 100 μL of 2M HCl was added, centrifuged at 13,000 rpm for 10 minutes, filtered, and analyzed by GCMS.

[0048] As a result, as shown in Figure 2, it was confirmed that the activity was superior when CYP116B ST8 and methane were mixed compared to when only methane or CYP116B ST8 was added, and it was confirmed that the activity increased in a concentration-dependent manner when mixed with CYP116B ST8.

[0049] The graph on the right in Figure 2 shows the results of analyzing methanol at different concentrations using standard methanol to identify methanol that can be produced from methane oxidase, while the graph on the left confirms that the methanol peak increases at the peak where methane oxidase and methane gas were injected. As confirmed in Figure 2, it can be seen that the activity is superior when CYP116B ST8 and methane are mixed compared to when only methane or CYP116B ST8 is added, and it was confirmed that the activity increases in a concentration-dependent manner of CYP116B ST8 when mixed.

[0051] Meanwhile, the activity toward methane by CYP116B ST was analyzed. Specifically, the above CYP116B ST was 37 o Incubated at C for 30 minutes, 5 mL of CH4 was added, incubated at 37°C for 3 hours, 100 μL of 2M HCl was added, centrifuged at 13,000 rpm for 10 minutes, filtered, and analyzed by GCMS.

[0052] As a result, as confirmed in Figure 3, a methanol peak was detected in CYP116B ST8 + NADPH + CH4, and MS confirmed that it was consistent with methanol fragmentation.

[0054] Example 3: Confirmation of Optimal Conditions for Selected Methane Oxidation Enzymes

[0055] 3-1. Confirmation of Optimal Enzyme Concentration

[0056] To determine the optimal methane oxidase concentration, 10–500 pmol of CYP116B ST8 and 5 mL of CH4 were added to a medium adjusted to pH 7.4 with 100 mM Kpi (potassium phosphate buffer) and supplemented with 30 μL of 10 mM NADPH. Subsequently, 37 o After incubating at C for 30 minutes, an additional 5 mL of CH4 was added. Subsequently, 37 o Incubated at C for 1 hour or overnight.

[0057] Afterwards, 100 μL of 2M HCl was added, centrifuged at 13,000 rpm for 10 minutes, filtered, and analyzed by GCMS.

[0058] As a result, as confirmed in Figure 4, it was observed that when cultured for 1 hour, the methanol production increased in a concentration-dependent manner from a concentration of 100 pmol or higher up to 500 pmol. These results also confirmed a concentration-dependent increase in methanol production when cultured overnight. In particular, when comparing the results of ST8 at 25 pmol and 50 pmol left for 1 hour with those left for 12 hours or more, it was confirmed that the amount of methanol produced also increased as the reaction time increased.

[0059] From these results, it was confirmed that the polypeptide CYP116B ST8 of the present invention must react with an enzyme of 100 pmol or more for a reaction time of less than 1 hour to produce methanol. Accordingly, it is preferable to use 100 pmol or more of polypeptide CYP116B ST8 to produce effective methanol.

[0061] 3-2. Verify Optimal Reaction Temperature

[0062] The optimal reaction temperature of the polypeptide CYP116B ST8 of the present invention was determined.

[0063] Specifically, 200 pmol of CYP116B ST8 was added to a medium adjusted to pH 7.4 with 100 mM Kpi and 30 μL of 10 mM NADPH, followed by the addition of 5 mL of CH4. Afterward, the mixture was incubated at 30 to 60°C for 30 minutes, after which an additional 5 mL of CH4 was added. Subsequently, the mixture was left at 30 to 60°C for 1 hour or at least 12 hours.

[0064] Then, 100 μL of 2M HCl was added, centrifuged at 13,000 rpm for 10 minutes, filtered, and analyzed by GCMS (1 μL dispensing).

[0065] As a result, as confirmed in Figure 5, the reactivity began to increase from 45°C, showed high activity up to 55°C, and then showed a level of activity similar to that at 30°C or 40°C at 60°C. This is believed to be due to the polypeptide being denatured by high heat, and it was confirmed that the polypeptide of the present invention exhibits optimal reactivity at 40 to 60°C and peak activity at 50°C.

[0067] 3-3. Checking the Optimal pH Range

[0068] The optimal reaction pH of the polypeptide CYP116B ST8 of the present invention was determined.

[0069] Specifically, 200 pmol of CYP116B ST8 was added to a medium adjusted to pH 5.5 to 8.0 with 100 mM Kpi and 30 μL of 10 mM NADPH, followed by the addition of 5 mL of CH4. Afterward, the mixture was incubated at 50°C for 30 minutes, after which an additional 10 mL of CH4 was added. Subsequently, the mixture was left at 50°C for 1 hour or at least 12 hours.

[0070] Afterward, the sample was centrifuged at 13,000 rpm for 10 minutes, filtered, and analyzed by GCMS (4 μL aliquot).

[0071] As a result, as confirmed in Figure 6, reactivity appeared from the pH 5.5 range and showed high reactivity up to pH 7.0.

[0073] 3-4. Verify Optimal Response Time

[0074] The optimal reaction time of the polypeptide CYP116B ST8 of the present invention was determined.

[0075] To determine the optimal reaction time, the solution was adjusted with 100 mM potassium phosphate buffer (Kpi buffer, pH 7.0), 30 μL of 10 mM NADPH and 200 pmol of CYP116B ST8 were added, and 5 mL of CH4 was added. Afterward, the solution was incubated at 50 ℃ for 30 minutes, followed by the addition of 10 mL of CH4. Subsequently, the solution was incubated at 50 ℃ for 5 minutes to 24 hours at hourly intervals.

[0076] Afterward, 100 μL of 2M HCl was added, centrifuged at 13,000 rpm for 10 minutes, filtered, and analyzed by GCMS (4 μL dispensing).

[0077] As a result, as shown in Figure 7, methanol production capacity was confirmed even after a reaction of 5 minutes, and methanol production capacity was also confirmed at 1500 minutes, which is over 24 hours.

[0079] Example 4: Confirmation of substrate affinity of methane oxidase for methane

[0080] The substrate affinity for methane was confirmed using the polypeptide discovered in Example 1.

[0081] Specifically, the solution was adjusted to 100 mM potassium phosphate buffer (Kpi buffer, pH 7.0), 30 μL of 10 mM NADPH and 200 pmol of CYP116B ST8 were added, and CH4 was added at various concentrations. Afterward, the mixture was left at 50°C for 30 minutes, followed by the addition of 10 mL of CH4. The mixture was then left at 50°C for 1 hour.

[0082] Subsequently, 100 μL of 2 M HCl was added, and after centrifugation at 13,000 rpm for 10 minutes, the mixture was filtered and analyzed by GCMS. (4 μL aliquot)

[0083] As a result, as confirmed in Fig. 8, methane oxidase's K m The value is 0.274 mM, k cat The value is 6.006 min -1 was, and the catalytic efficiency was 21.92 min -1 mM -1 It was confirmed that it is.

[0086] Example 5: Confirmation of activity of methane oxidase variant

[0087] A variant exhibiting higher methanol conversion ability was identified by adding additional mutations to the polypeptide discovered in Example 1. By performing PCR using the primer pairs listed in the table below and the QuikChange II Site-Directed Mutagenesis Kit (Agilent), a recombinant expression vector was constructed with PCR products containing the nucleotide sequences of L91A, T115A, V117A, F205A, M266A, I270A, A273Q, T277A, W323A, and F423A inserted.

[0088] amplification target Primer pair Sequence (5′ to 3′) L91A Forward Primer cccgtcgatcgtggccgagcgcatgtcg L91A Reverse primer cgacatgcgctcggccacgatcgacggg T115A Forward Primer cggctacgcgatgaaccgtgcgctcgtcaa T115A Reverse primer ttgacgagcgcacggttcatcgcgtagccg V117A Forward Primer gaaccgtacgctcgccaacgaggacgagc V117A Reverse primer gctcgtcctcgttggcgagcgtacggttc F205A Forward Primer caccgtcaacgcggccgggcggccctcg F205A Reverse primer cgagggccgcccggccgcgttgacggtg M266A Forward Primer actcgtacctgcactcggcgatgatggccatcatcg M266A Reverse primer cgatgatggccatcatcgccgagtgcaggtacgagt I270A Forward Primer cgatgatgatggccgccatcgtcgcggcgc I270A Reverse primer gcgccgcgacgatggcggccatcatcatcg A273Q Forward Primer ggccatcatcgtccaggcgcacgagacg A273Q Reverse primer cgtctcgtgcgcctggacgatgatggcc T277A Forward Primer cggcgcacgaggcgacggcgttc T277A Reverse primer gaacgccgtcgcctcgtgcgccg W323A Forward Primer cggtggcctcggcgcggcgccgca W323A Reverse primer tgcggcgccgcgccgaggccaccg F422A Forward Primer gcacaacgtgtcggcccgcggcccgcag F422A Reverse primer ctgcgggccgcgggccgacacgttgtgc

[0089] The activity of the manufactured mutant was confirmed.

[0090] Specifically, the solution was adjusted with 100 mM potassium phosphate buffer (Kpi buffer, pH 7.0), 30 μL of 10 mM NADPH and 200 pmol of CYP116B ST8 were added, and 5 mL of CH4 was added. After incubating at 50 ℃ for 30 minutes, an additional 10 mL of CH4 was added. Subsequently, the solution was incubated at 30 ℃ for 5 minutes to 24 hours at hourly intervals. Afterward, 100 μL of 2M HCl was added, followed by centrifugation at 13,000 rpm for 10 minutes, filtration, and analysis by GCMS (4 μL aliquot).

[0091] As a result, as confirmed in Figure 9, it was confirmed that variants with the mutations L91A, T115A, V117A, F205A, M266A, I270A, A273Q, T277A, W323A, and F423A had superior activity compared to the polypeptide (wild type, WT) discovered in Example 1. In particular, A273Q and F423A showed nearly twice as much superior enzyme activity compared to the wild type.

[0093] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 Polypeptide containing any one of the mutations L91A, V117A, F205A, M266A, I270A, A273Q, T277A, W323A, F423A in the amino acid sequence of SEQ ID NO.

1. Claim 6 delete Claim 7 A method for producing methanol comprising the step of reacting methane gas with the polypeptide of claim 5. Claim 8 A method of preparation according to claim 7, wherein the polypeptide is 100 to 1000 pmol. Claim 9 A manufacturing method according to claim 7, wherein the reaction step is carried out at 40 to 60 ℃. Claim 10 A manufacturing method according to claim 7, wherein the reaction step is carried out at a pH of 5 to 8. Claim 11 A manufacturing method according to claim 7, wherein the reaction step is performed for 10 to 1500 minutes.

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