CN110592042A - Transaminase mutants and uses thereof - Google Patents
Transaminase mutants and uses thereof Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12Y206/01—Transaminases (2.6.1)
Abstract
The invention discloses a transaminase mutant and application thereof. Wherein the amino acid sequence of the transaminase mutant consists of SEQ ID NO:1, the mutation at least comprises one of the following mutation site combinations: T7C + S47C, Q78C + a330C, V137C + G313C, a217C + Y252C and L295C + C328C; or an amino acid sequence of the transaminase mutant which has a mutation site in the mutated amino acid sequence and has an amino acid sequence having 80% or more homology with the mutated amino acid sequence. The transaminase mutant provided by the invention realizes the change of protein structure and function, reduces the enzyme dosage, improves the ee value of the product, reduces the difficulty of post-treatment, and is suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a transaminase mutant and application thereof.
Background
Chiral amine compounds are key intermediates for the synthesis of many chiral drugs. There are many important neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs, etc. which are synthesized using chiral amines as intermediates. At present, the synthesis of Chiral amine is mainly realized by a chemical method, such as asymmetric reduction of Schiff base (Chiral amine synthesis: methods, definitions and applications [ M ]. West Sussex, United Kingdom: John Wiley & sons.2010), but the reaction has the defects of harsh reaction conditions, use of toxic transition metal catalyst, low product stereoselectivity and the like.
The transaminase can catalyze the transfer of an amino group from an amino donor to a prochiral acceptor ketone to yield a chiral amine and a byproduct ketone. Because the transaminase has the advantages of high selectivity, high conversion rate, mild reaction conditions and the like, the transaminase is widely applied to the synthesis of chiral amine. A number of important Chiral amines have been prepared by enzymatic (Biocatalytic enzymatic Synthesis of Chiral amines Applied to Chiral amines Manual [ J ] Science,2010,329(5989): 305. about.309.) or by a combination of chemical and enzymatic methods (chemoenzymatic Synthesis of (S) -Rivastigmine using omega-transaminases [ J ] Chemform, 2010,46(30): 0. 5502). However, in the industrial production, most wild transaminases have the defects of low catalytic efficiency, poor stereoselectivity, poor stability and the like, so that the number of transaminases which can be really applied is not large.
The invention patent application with the application publication number of CN108048419A discloses a omega-transaminase mutant F89Y + A417S derived from Chromobacterium violacea, which can catalyze dihydroxy ketal compounds to obtain chiral ammonia products with higher selectivity, and the reaction is as follows:
however, the activity is low, and the amount of enzyme added during the reaction is large.
Disclosure of Invention
The invention aims to provide a transaminase mutant and application thereof to improve the activity of transaminase.
In order to achieve the above object, according to one aspect of the present invention, there is provided a transaminase mutant. The amino acid sequence of the transaminase mutant is represented by SEQ ID NO:1, the mutation at least comprises one of the following mutation site combinations: T7C + S47C, Q78C + a330C, V137C + G313C, a217C + Y252C and L295C + C328C; or an amino acid sequence of the transaminase mutant which has a mutation site in the mutated amino acid sequence and has an amino acid sequence having 80% or more homology with the mutated amino acid sequence.
Further, the mutations include T7C + S47C and at least one of the following mutation sites: q78, a330, V137, G313, a217, Y252, L295, C328, F22, a33, V42, a57, F89, N151, S156, M166, Y168, E171, K249, L283, S292, a299P, a334, F367, H369, V379, Q380, D396, F397, I400, C404, R405, F409, I414, R416, G419, S424, D436, R444, G457.
Further, the mutations include T7C + S47C and at least one of the following mutation sites: Q78C, V137C, G313C, F22P, A33V, A57V/Y, F89A/E/V, N151A/E/F/Q/S/V/W/Y, S156 36156 156A/P/A166A/D/E/F/G/K/S/W/A168A/E/I/M/S/A171A/A249A, L283A, S292A, A299A, A33A, A334A/A367A/A, V379A/D/L/A380A, D396A/P/A397/A/Q/S/A400A, C A/A, F409/C/H/M/Q/T A/A, F409/S A/S/A/S36416/S A/36416, C A/S36416/S A/S/36416, G419W, S424A/C/Q/L/V, D436V/A, R444A/P/Y and G457C/P/W.
Further, the mutation at least comprises one of the following mutation site combinations: T7C + S47C + D436A, T7C + S47C + Q380L + V379L, T7C + S47C + N151W and T7C + S47C + M166F.
Further, the mutation at least comprises one of the following mutation site combinations: t7 + S47 + Q380 + V379, T7 + S47 + Q78 + A330 + Y89, T7 + S47 + V137 + G313 + Y89, T7 + S47 + Q380 + V379 + S156, T7 + S47 + Q78 + A330 + S156, T7 + S47 + K249 + I400 + S156, T7 + S47 + Q380 + V379 + M166, T7 + S47 + H404 + M166, T7 + S47 + H369 + F22 + 166, T7 + S47 + H369 + L283 + M166, T7 + S47 + V3747 + S166 + T7 + V379 + S166 + T7 + V47 + S166 + T7 + S166 + T7 + S166 + S47 + V379 + S166, T7 + S166 + S47 + V379 + K20 + S380 + S166, T7 + S47 + K379 + K20 + K47 + S166, T7 + S166, T47 + S166, T47 + K47 + S166, T7 + S47 + S166 + K47 + S166, T7A 47 + S47 + K47 + S166, T7A 47 + S47 + K37, T7 + S47 + S292 + A299 + M166 + Y168, T7 + S47 + A334 + F367 + M166 + Y168, T7 + S47 + Q380 + V379 + M166 + N151, T7 + S47 + A334 + F367 + M166 + N151, T7 + S47 + Q380 + V379 + M166 + R416, T7 + S47 + Q380 + V379 + R416, T7 + S166 + R416, T7 + S47 + Q380 + V379 + M166 + R166, T7 + S47 + D436 + R444 + M166 + R166 + T7 + T166 + M166 + R416, T7 + M166 + M2 + R416, T7 + M2, T2 + M2, T7 + M2, T2 + M2, T7 + S47 + Q380 + V379 + M166 + R416 + Y168, T7 + S47 + Q380 + V379 + M166 + R416 + N151 + Y168, T7 + S47 + D396 + F397 + M166 + R416 + N151 + Y168, T7 + S47 + Q380 + V379 + M166 + R416 + N151 + S424, T7 + S47 + Q380 + V379 + M166 + R166 + S409, T7 + S47 + Q380 + V379 + V409 + R151 + N409 + S409 + T166 + V409 + F151 + V409 + F151 + V409 + R166 + V151 + V409 + V151 + V409 + F151 + V409 + V151 + F409 + V409 + R166 + V151 + V409 + R166 + V151 + V409 + V151 + V409 + R166 + V151 + V409 + V151 + V409 + R166 + V151 + V409 + V151 + V409 + M2, T7 + V409 + V151 + V409 + R151 + V409 + R166 + V151 + R2, T7C + S47C + Q380L + V379L + M166F + R416D + N151W + S424V + F409C, T7C + S47C + Q380C + V379C + M166C + R416C + N151C + S424C + F409C, T7C + S47C + Q380C + V379C + M166C + R C + N151C + S424C + S C + F409C, T7C + S47C + Q380 + Q C + V379C + M166 + N C + N151C + N36166 + N C + N C + N36409 + N C + F C + F36409 + C + F C + 36409C + F C + F C + F C + 36.
According to another aspect of the invention, a DNA molecule is provided. The DNA molecule encodes the above transaminase mutant.
According to still another aspect of the present invention, there is provided a recombinant plasmid. The recombinant plasmid contains the DNA molecule.
Further, the recombinant plasmid is pET-22a (+), pET-22b (+), pET-3a (+), pET-3d (+), pET-11a (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), pET-32a (+), and pET-35b (+), or, pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19.
According to yet another aspect of the present invention, a host cell is provided. The host cell contains the above recombinant plasmid.
Further, host cells include prokaryotic, yeast, or eukaryotic cells; preferably, the prokaryotic cell is an Escherichia coli BL21-DE3 cell or an Escherichia coli Rosetta-DE3 cell.
According to yet another aspect of the present invention, a method of producing a chiral amine is provided. The method comprises the step of carrying out catalytic transamination reaction on ketone compounds and an amino donor by transaminase, wherein the transaminase is any one of the above transaminase mutants.
Further, the ketone compound isThe transamination reaction product isWherein R is1And R2Each independently represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group; r1And R2May be used alone or in combination with each other to form a substituted or unsubstituted ring; preferably, the ketone compound is a dihydroxyketal compound reacted byGeneratingTransamination reaction of (a); preferably, R1And R2Is an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group having 1 to 20 carbon atoms, more preferably an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group having 1 to 10 carbon atoms; preferably, substituted means substituted with a halogen atom, nitrogen atom, sulfur atom, hydroxyl group, nitro group, cyano group, methoxy group, ethoxy group, carboxyl group, carboxymethyl group, carboxyethyl group or methylenedioxy group; preferably, R1Expressed as methyl or halogen-substituted methyl, more preferably,halogen-substituted methyl is CF3，CF2H，CCl3，CCl2H，CBr3Or CBr2H, further preferably CF3Or CF2H; preferably, the ketone compound is
Further, the amino donor is isopropylamine or alanine, preferably isopropylamine.
Further, in a reaction system of the transaminase for carrying out the catalytic transaminase reaction on the ketone compound and the amino donor, the pH value is 7-11, preferably 8-10, and more preferably 9-10; preferably, the temperature of a reaction system for the transaminase to perform the catalytic transaminase reaction on the ketone compound and the amino donor is 25-60 ℃, more preferably 30-55 ℃, and further preferably 40-50 ℃; preferably, the volume concentration of dimethyl sulfoxide in the reaction system for the transaminase to perform the catalytic transamination reaction on the ketone compound and the amino donor is 0-50%, and more preferably 0-20%.
The transaminase mutant of the invention is represented by SEQ ID NO:1, the omega-transaminase mutant F89Y + A417S derived from Chromobacterium violacea is mutated by a site-directed mutagenesis method, so that the amino acid sequence of the mutant is changed, the change of the structure and the function of the protein is realized, the enzyme dosage is reduced, the ee value of the product is improved, the difficulty of post-treatment is reduced, and the mutant is suitable for industrial production.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
The activity of the wild enzyme is lower, and the enzyme consumption is larger. In order to solve the problems, the invention carries out protein modification on wild enzymes by means of directed evolution, improves the activity and stereoselectivity of the enzymes, and develops the transaminase for industrial production.
The inventor carries out evolution on a mutant SEQ ID NO. 1 (application publication number is CN108048419A) of omega-aminotransferase from Chromobacterium violacea by a directed evolution method, thereby improving enzyme activity, reducing the use amount of enzyme and further improving the stereoselectivity of the enzyme.
Specifically, using a ω -transaminase mutant F89Y + a417S (SEQ ID NO:1) derived from Chromobacterium violacea as a template (in the present invention, "F89Y" is taken as an example to indicate "the original amino acid + the site + the mutated amino acid", that is, the 89 th site F is changed to Y), 10 pairs of site-directed mutagenesis primers were first designed to construct 5 pairs of two-site combination mutations (T7C + S47C, Q78C + a330C, V137C + G313C, a217C + Y252C, L295C + C328C) to improve the reactivity of the enzyme under high temperature conditions. A site-directed mutagenesis method is utilized, pET-22b (+) is taken as an expression vector, and a mutant plasmid with a target gene is obtained.
Site-directed mutagenesis: it is intended to introduce a desired change (usually, a change indicating a favorable direction) including addition, deletion, point mutation or the like of a base into a DNA fragment of interest (which may be a genome or a plasmid) by a method such as Polymerase Chain Reaction (PCR). The site-directed mutation can rapidly and efficiently improve the character and the characterization of target protein expressed by DNA, and is a very useful means in gene research work.
The method for introducing site-directed mutation by utilizing whole plasmid PCR is simple and effective, and is a means which is used more at present. The principle is that a pair of primers (positive and negative) containing mutation sites and a template plasmid are annealed and then are circularly extended by polymerase, wherein the circular extension refers to a cycle that the polymerase extends the primers according to the template, returns to the 5' end of the primers after one circle to terminate, and is repeatedly heated, annealed and extended, and the reaction is different from rolling circle amplification and cannot form a plurality of tandem copies. The extension products of the forward and reverse primers are annealed and then paired to form nicked open-loop plasmids. The template DNA derived from dam + strain can be recognized and cut by DpnI enzyme because of having methylation site, and the plasmid with mutation sequence synthesized in vitro can not be digested because of not having methylation, and the deletion can be naturally repaired after being transformed into Escherichia coli, thus obtaining the clone with mutation plasmid. The mutant plasmid was transformed into E.coli competent cells, plated on a petri dish containing LB solid medium (100. mu.g/mL ampicillin), and cultured overnight at 37 ℃. The single clone grown on the solid medium was activated. After the sequencing was confirmed to be correct, the transaminase expression was induced overnight at 25 ℃ with 0.2mM IPTG. Then obtaining crude enzyme liquid for reaction characteristic detection by a method of centrifuging and ultrasonically breaking cells.
By adopting a computer to carry out butt joint simulation analysis on the three-dimensional structures of the transaminase and the substrate, some amino acids are selected near an enzyme catalysis center and may have a great relation with enzyme activity. Based on the constructed double-point mutation, saturation mutation is carried out on the amino acid sites which may influence the double-point mutation. 31 pairs of saturation mutation primers (F22, A33, V42, A57, F89, N151, S156, M166, Y168, E171, K249, L283, S292, A299, A334, F367, H369, V379& Q380L, D396, F397, I400, C404, R405, F409, I414, R416, G419, S424, D436, R444, G457) were designed to obtain mutants with greatly improved activity.
Among them, saturation mutation is a method for obtaining a mutant in which the amino acid at the target site is substituted with other 19 amino acids in a short time by modifying the coding gene of the target protein. The method is not only a powerful tool for protein directed modification, but also an important means for researching the structure-function relationship of the protein. Saturated mutations tend to yield more desirable evolutionary bodies than single point mutations. These problems that cannot be solved by site-directed mutagenesis are unique to saturation mutagenesis.
On the basis of obtaining the mutant with improved activity by saturation mutation, beneficial amino acid sites can be combined to obtain the mutant with better properties. The construction method of double-point mutation in the combined mutation is the same as that of single-point mutation, and is constructed by adopting a full plasmid PCR method. And simultaneously carrying out multi-point mutation of 2 or more sites by adopting overlap extension PCR amplification to obtain a mutant gene containing the multi-point mutation, carrying out restriction enzyme digestion on two ends of the mutant gene, connecting the two ends of the mutant gene to an expression vector, transforming the expression vector into escherichia coli cells, coating the escherichia coli cells in an LB culture dish containing 100 mu g/mL ampicillin, carrying out overnight culture at 37 ℃ to obtain a combined mutant, and carrying out sequencing and identification.
The overlap extension PCR (gene hybridization by overlap extension PCR, SOEPCR for short) adopts a primer with complementary ends to form overlapped chains on PCR products, so that amplified fragments from different sources are overlapped and spliced together through the extension of the overlapped chains in a subsequent amplification reaction. This technique enables efficient gene recombination in vitro using PCR technology and is often used in the construction of multiple point mutations.
According to an exemplary embodiment of the present invention, a transaminase mutant is provided. The amino acid sequence of the transaminase mutant is represented by SEQ ID NO: 1(MQKQRTTSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDSEGNKIIDGMAGLWCVNVGYGRKDFAEAARRQMEELPFYNTFYKTTHPAVVELSSLLAEVTPAGFDRVFYTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWNGYHGSTIGGASLGGMKYMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGVIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVFVGKRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMVQAFTLVKNKAKRELFPDFGEIGTLCRDIFFRNNLIMRSCGDHIVSAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGLA), wherein the mutation comprises at least one of the following combinations of mutation sites: T7C + S47C, Q78C + a330C, V137C + G313C, a217C + Y252C and L295C + C328C; or an amino acid sequence of the transaminase mutant which has a mutation site in the mutated amino acid sequence and has an amino acid sequence having 80% or more homology with the mutated amino acid sequence.
The transaminase mutant of the invention is represented by SEQ ID NO:1, the omega-transaminase mutant F89Y + A417S derived from Chromobacterium violacea is mutated by a site-directed mutagenesis method, so that the amino acid sequence of the mutant is changed, the change of the structure and the function of the protein is realized, the enzyme dosage is reduced, the ee value of the product is improved, the difficulty of post-treatment is reduced, and the mutant is suitable for industrial production.
The term "homology" as used herein has the meaning generally known in the art and rules, standards for determining homology between different sequences are well known to those skilled in the art. The sequences of the invention defined by different degrees of homology must also simultaneously have an increased transaminase activity. In the above embodiments, it is preferable that the amino acid sequence of the transaminase mutant has the above homology and has or encodes an amino acid sequence having an increased activity. One skilled in the art can obtain such variant sequences under the teachings of the present disclosure.
Preferably, the mutations include T7C + S47C and at least one of the following mutation sites: q78, a330, V137, G313, a217, Y252, L295, C328, F22, a33, V42, a57, F89, N151, S156, M166, Y168, E171, K249, L283, S292, a299P, a334, F367, H369, V379, Q380, D396, F397, I400, C404, R405, F409, I414, R416, G419, S424, D436, R444, G457.
More preferably, the mutation comprises T7C + S47C and at least one of the following mutation sites: Q78C, V137C, G313C, F22P, A33V, A57V/Y, F89A/E/V, N151A/E/F/Q/S/V/W/Y, S156A/P/Q, M166A/D/E/F/G/K/S/W/A168A/E/I/M/S/A171A/A249A, L283A, S292A, A299, A33A, A334/A/36367A/A, V379A/D/L/A380A, D396A/P/A397/Q/S/A400A, C404/A, F409/A/C/H/M/Q/T A, R/S72/S/A, C404/A/F409/V/S/A, C409/V/A, F/V/A/M/A, G419W, S424A/C/Q/L/V, D436V/A, R444A/P/Y and G457C/P/W. Where "/" represents "or".
More preferably, the mutation further comprises at least one of the following combinations of mutation sites: T7C + S47C + S292A, T7C + S47C + D436A, T7C + S47C + D396G, T7C + S47C + Q380L, T7C + S47C + a299P, T7C + S47C + F397C, T7C + S47C + S424C, T7C + S47C + V379C, T7C + S47C + M166C, T7C + S47C + M C + C, T7C + S C + C, T7C + S C + N C + 36.
Further preferably, the mutation further comprises at least one of the following combinations of mutation sites: t7 + S47 + Q380 + V379, T7 + S47 + Q78 + A330 + Y89, T7 + S47 + V137 + G313 + Y89, T7 + S47 + Q380 + V379 + S156, T7 + S47 + Q78 + A330 + S156, T7 + S47 + K249 + I400 + S156, T7 + S47 + Q380 + V379 + M166, T7 + S47 + H404 + M166, T7 + S47 + H369 + F22 + 166, T7 + S47 + H369 + L283 + M166, T7 + S47 + V3747 + S166 + T7 + V379 + S166 + T7 + V47 + S166 + T7 + S166 + T7 + S166 + S47 + V379 + S166, T7 + S166 + S47 + V379 + K20 + S380 + S166, T7 + S47 + K379 + K20 + K47 + S166, T7 + S166, T47 + S166, T47 + K47 + S166, T7 + S47 + S166 + K47 + S166, T7A 47 + S47 + K47 + S166, T7A 47 + S47 + K37, T7 + S47 + S292 + A299 + M166 + Y168, T7 + S47 + A334 + F367 + M166 + Y168, T7 + S47 + Q380 + V379 + M166 + N151, T7 + S47 + A334 + F367 + M166 + N151, T7 + S47 + Q380 + V379 + M166 + R416, T7 + S47 + Q380 + V379 + R416, T7 + S166 + R416, T7 + S47 + Q380 + V379 + M166 + R166, T7 + S47 + D436 + R444 + M166 + R166 + T7 + T166 + M166 + R416, T7 + M166 + M2 + R416, T7 + M2, T2 + M2, T7 + M2, T2 + M2, T7 + S47 + Q380 + V379 + M166 + R416 + Y168, T7 + S47 + Q380 + V379 + M166 + R416 + N151 + Y168, T7 + S47 + D396 + F397 + M166 + R416 + N151 + Y168, T7 + S47 + Q380 + V379 + M166 + R416 + N151 + S424, T7 + S47 + Q380 + V379 + M166 + R166 + S409, T7 + S47 + Q380 + V379 + V409 + R151 + N409 + S409 + T166 + V409 + F151 + V409 + F151 + V409 + R166 + V151 + V409 + V151 + V409 + F151 + V409 + V151 + F409 + V409 + R166 + V151 + V409 + R166 + V151 + V409 + V151 + V409 + R166 + V151 + V409 + V151 + V409 + R166 + V151 + V409 + V151 + V409 + M2, T7 + V409 + V151 + V409 + R151 + V409 + R166 + V151 + R2, T7C + S47C + Q380L + V379L + M166F + R416D + N151W + S424V + F409C, T7C + S47C + Q380C + V379C + M166C + R416C + N151C + S424C + F409C, T7C + S47C + Q380C + V379C + M166C + R C + N151C + S424C + S C + F409C, T7C + S47C + Q380 + Q C + V379C + M166 + N C + N151C + N36166 + N C + N C + N36409 + N C + F C + F36409 + C + F C + 36409C + F C + F C + F C + 36.
According to an exemplary embodiment of the present invention, a DNA molecule is provided. The DNA molecule encodes the above transaminase mutant. The transaminase mutant encoded by the DNA molecule has high solubility expression characteristic and high activity characteristic.
The above-described DNA molecules of the invention may also be present in the form of "expression cassettes". An "expression cassette" refers to a nucleic acid molecule, linear or circular, encompassing DNA and RNA sequences capable of directing the expression of a particular nucleotide sequence in an appropriate host cell. Generally, a promoter is included that is operably linked to a nucleotide of interest, optionally operably linked to a termination signal and/or other regulatory elements. The expression cassette may also include sequences required for proper translation of the nucleotide sequence. The coding region typically encodes a protein of interest, but also encodes a functional RNA of interest in the sense or antisense orientation, e.g., an antisense RNA or an untranslated RNA. An expression cassette comprising a polynucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one other component. The expression cassette may also be naturally occurring but obtained with efficient recombinant formation for heterologous expression.
According to an exemplary embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any of the above DNA molecules. The DNA molecule in the recombinant plasmid is placed in a proper position of the recombinant plasmid, so that the DNA molecule can be correctly and smoothly replicated, transcribed or expressed.
Although the term "comprising" is used in the present invention when defining the above DNA molecule, it does not mean that other sequences unrelated to their functions may be arbitrarily added to both ends of the DNA sequence. Those skilled in the art know that in order to satisfy the requirements of recombinant operation, it is necessary to add suitable restriction sites for restriction enzymes at both ends of a DNA sequence, or additionally add initiation codons, termination codons, etc., and thus, if defined by closed expressions, these cases cannot be truly covered.
The term "plasmid" as used in the present invention includes any plasmid, cosmid, phage or Agrobacterium binary nucleic acid molecule, preferably a recombinant expression plasmid, either prokaryotic or eukaryotic, but preferably prokaryotic, selected from the group consisting of pET-22a (+), pET-22b (+), pET-3a (+), pET-3d (+), pET-11a (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23b (+), pET-24a (+), and, pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), pET-32a (+), pET-35b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, QEP 9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pR A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6-p-1, pGEX-6-P-2-pGEX-2 b (+), pET-39b (+), pET-40b (+) pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19. More preferably, the above recombinant plasmid is pET-22b (+).
According to a typical embodiment of the present invention, there is provided a host cell containing any one of the above recombinant plasmids. Host cells suitable for use in the present invention include, but are not limited to, prokaryotic cells, yeast, or eukaryotic cells. Preferably the prokaryotic cell is a eubacterium, such as a gram-negative or gram-positive bacterium. More preferably, the prokaryotic cell is an E.coli BL21 cell or an E.coli DH5 alpha competent cell.
According to an exemplary embodiment of the present invention, a method for producing chiral amines is provided. The method comprises the step of carrying out catalytic transamination reaction on ketone compounds and an amino donor by transaminase, wherein the transaminase is any one of the above transaminase mutants resistant to organic solvents. Because the transaminase mutant has good catalytic activity and specificity, the chiral amine prepared by the transaminase mutant can improve the reaction rate, reduce the enzyme dosage and reduce the difficulty of post-treatment.
Further, the ketone compound isThe transamination reaction product isWherein R is1And R2Each independently represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group; r1And R2May be used alone or in combination with each other to form a substituted or unsubstituted ring; preferably, the ketone compound is dihydroxy ketal compound, and the reaction isGeneratingTransamination reaction of (a);
preferably, R1And R2Is an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group having 1 to 20 carbon atoms, more preferably an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group having 1 to 10 carbon atoms;
preferably, substituted means substituted with a halogen atom, nitrogen atom, sulfur atom, hydroxyl group, nitro group, cyano group, methoxy group, ethoxy group, carboxyl group, carboxymethyl group, carboxyethyl group or methylenedioxy group.
Preferably, R1Expressed as methyl or halogen-substituted methyl, more preferably, halogen-substituted methyl is CF3，CF2H，CCl3，CCl2H，CBr3Or CBr2H, further preferably CF3Or CF2H。
Preferably, the ketone compound is a dihydroxy ketal compound, and the transamination reaction formula is one of the following:
wherein TA is transaminase and PLP is pyridoxal phosphate.
In a typical embodiment of the invention, the amino donor is isopropylamine or alanine, preferably isopropylamine.
In a reaction system for carrying out a catalytic transamination reaction on a ketone compound and an amino donor by using the transaminase of the invention, the pH is 7-11, preferably 8-10, more preferably 9-10, that is, the value of the pH can be selected from 7-11, such as 7, 7.5, 8, 8.6, 9, 10, 10.5, etc. The temperature of the reaction system in which the transaminase catalyzes the transamination reaction of the ketone compound and the amino donor is 25 to 60 ℃, more preferably 30 to 55 ℃, and still more preferably 40 to 50 ℃, that is, the temperature can be selected from 25 to 60 ℃, for example, 30, 31, 32, 35, 37, 38, 39, 40, 42, 45, 48, 50, 51, 52, 55, and the like. The volume concentration of dimethyl sulfoxide in the reaction system of the transaminase for carrying out the catalytic transamination reaction on the ketone compound and the amino donor is 0-50%, for example, 10%, 15%, 18%, 20%, 30%, 35%, 38%, 40%, 42%, 48%, 49%, etc.
It will be apparent to those skilled in the art that many modifications can be made to the present invention without departing from the spirit thereof, and such modifications are intended to be within the scope of the invention. The following experimental methods are all conventional methods unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified.
The following will further illustrate the beneficial effects of the present invention by combining experimental data and examples.
Example 1
10mg of each of substrate 1/substrate 2/substrate 3 was dissolved in 30. mu.L of DMSO to prepare a substrate solution. 10mg of transaminase, 66.6 mu L of isopropylamine hydrochloride solution (6M), 0.1mg of PLP and 0.1M of Tris-Cl buffer (pH9.0) are sequentially added into the reaction system to supplement the total volume to 500 mu L, and finally the prepared substrate solution is added, the pH is adjusted to 9.0, 200rpm is carried out, and the isothermal reaction is carried out for 16h at the temperature of 45 ℃. Adding acetonitrile with 2 times volume into a reaction system, fully and uniformly mixing, standing for 10min, centrifuging at 12000rpm for 10min, taking supernatant, diluting by 10 times, and sending the supernatant to a liquid phase for measuring the conversion rate. ee value detection method: adding acetonitrile with 2 times volume into the reaction system, fully and uniformly mixing, standing for 10min, centrifuging at 12000rpm for 10min, taking the supernatant, adding anhydrous MgSO4Removing water, centrifuging at 12000rpm for 10min, collecting supernatant, and adding N2Blow-drying, adding 1mL of methanol, dissolving, and sending to liquid phase detection. The partial mutant response characteristics are shown in table 1 below:
TABLE 1
The activity is increased by + 5-10 times, the ++ 10-20 times, the +++ 20-50 times, the ++++ 50-100 times, and the ++++ 100 times; indicates ee values of 95% -98%, and > 98%.
Example 2
10mg of substrate 4/substrate 5 were dissolved in 30. mu.L of DMSO, respectively, to prepare substrate solutions. 10mg of transaminase, 66.6 mu L of isopropyl amine hydrochloride solution (6M), 0.1mg of PLP and 0.1M of Tris-Cl buffer (pH9.0) are sequentially added into the reaction system to supplement the total volume to 500 mu L, and finally the prepared substrate solution is added to adjust the pH to 9.0, 200rpm and react for 16h at the constant temperature of 45 ℃. Adding acetonitrile with 2 times volume into a reaction system, fully and uniformly mixing, standing for 10min, centrifuging at 12000rpm for 10min, taking supernatant, diluting by 10 times, and sending the supernatant to a liquid phase for measuring the conversion rate. ee value detection method: adding acetonitrile with 2 times volume into the reaction system, fully and uniformly mixing, standing for 10min, centrifuging at 12000rpm for 10min, taking the supernatant, adding anhydrous MgSO4Removing water, centrifuging at 12000rpm for 10min, collecting supernatant, and adding N2Blow-drying, adding 1mL of methanol, dissolving, and sending to liquid phase detection. The partial mutant response characteristics are shown in table 2 below:
TABLE 2
The activity is increased by + 5-10 times, the ++ 10-20 times, the +++ 20-50 times, the ++++ 50-100 times, and the ++++ 100 times; indicates ee values of 95% -98%, and > 98%.
Example 3
10mg of each of substrate 6/substrate 7/substrate 8 was dissolved in 30. mu.L of DMSO to prepare a substrate solution. 10mg of transaminase, 66.6 mu L of isopropylamine hydrochloride solution (6M), 0.1mg of PLP and 0.1M of Tris-Cl buffer (pH9.0) are sequentially added into the reaction system to supplement the total volume to 500 mu L, and finally the prepared substrate solution is added, the pH is adjusted to 9.0, 200rpm is carried out, and the isothermal reaction is carried out for 16h at the temperature of 45 ℃. Is turned to the reverse directionAdding acetonitrile with 2 times volume into the reaction system, fully and uniformly mixing, standing for 10min, centrifuging at 12000rpm for 10min, taking supernatant, diluting by 10 times, and sending the supernatant to a liquid phase for measuring the conversion rate. ee value detection method: adding acetonitrile with 2 times volume into the reaction system, fully and uniformly mixing, standing for 10min, centrifuging at 12000rpm for 10min, taking the supernatant, adding anhydrous MgSO4Removing water, centrifuging at 12000rpm for 10min, collecting supernatant, and adding N2Blow-drying, adding 1mL of methanol, dissolving, and sending to liquid phase detection. The partial mutant response characteristics are shown in table 3 below:
TABLE 3
The activity is increased by + 5-10 times, the ++ 10-20 times, the +++ 20-50 times, the ++++ 50-100 times, and the ++++ 100 times; indicates ee values of 95% -98%, and > 98%.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the transaminase mutant has improved enzyme activity, can reduce the use amount of enzyme when in use, and solves the technical problem that the wild transaminase is not suitable for industrial production.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Kai Lai Ying pharmaceutical group (Tianjin) Ltd
<120> transaminase mutants and uses thereof
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<170> SIPOSequenceListing 1.0
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His His Leu His Pro Phe Thr Asp Thr Ala Ser Leu Asn Gln Ala Gly
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Ala Arg Val Met Thr Arg Gly Glu Gly Val Tyr Leu Trp Asp Ser Glu
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Gly Asn Lys Ile Ile Asp Gly Met Ala Gly Leu Trp Cys Val Asn Val
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Gly Tyr Gly Arg Lys Asp Phe Ala Glu Ala Ala Arg Arg Gln Met Glu
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Glu Leu Pro Phe Tyr Asn Thr Phe Tyr Lys Thr Thr His Pro Ala Val
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Val Glu Leu Ser Ser Leu Leu Ala Glu Val Thr Pro Ala Gly Phe Asp
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Arg Val Phe Tyr Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Met Ile
115 120 125
Arg Met Val Arg Arg Tyr Trp Asp Val Gln Gly Lys Pro Glu Lys Lys
130 135 140
Thr Leu Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr Ile Gly Gly
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Ala Ser Leu Gly Gly Met Lys Tyr Met His Glu Gln Gly Asp Leu Pro
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Ile Pro Gly Met Ala His Ile Glu Gln Pro Trp Trp Tyr Lys His Gly
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Lys Asp Met Thr Pro Asp Glu Phe Gly Val Val Ala Ala Arg Trp Leu
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Glu Glu Lys Ile Leu Glu Ile Gly Ala Asp Lys Val Ala Ala Phe Val
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Gly Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Ala Thr
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Tyr Trp Pro Glu Ile Glu Arg Ile Cys Arg Lys Tyr Asp Val Leu Leu
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Val Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe
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Gly His Gln His Phe Gly Phe Gln Pro Asp Leu Phe Thr Ala Ala Lys
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Gly Leu Ser Ser Gly Tyr Leu Pro Ile Gly Ala Val Phe Val Gly Lys
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Gly Pro Tyr Met Gln Lys Arg Trp Arg Glu Thr Phe Ser Arg Phe Glu
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His Val Asp Asp Val Arg Gly Val Gly Met Val Gln Ala Phe Thr Leu
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Val Lys Asn Lys Ala Lys Arg Glu Leu Phe Pro Asp Phe Gly Glu Ile
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Gly Thr Leu Cys Arg Asp Ile Phe Phe Arg Asn Asn Leu Ile Met Arg
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Ser Cys Gly Asp His Ile Val Ser Ala Pro Pro Leu Val Met Thr Arg
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Ala Glu Val Asp Glu Met Leu Ala Val Ala Glu Arg Cys Leu Glu Glu
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Phe Glu Gln Thr Leu Lys Ala Arg Gly Leu Ala
450 455
Claims (14)
1. A transaminase mutant, characterized in that the amino acid sequence of the transaminase mutant consists of SEQ ID NO:1, the mutation comprises at least one of the following mutation site combinations: T7C + S47C, Q78C + a330C, V137C + G313C, a217C + Y252C and L295C + C328C; or the amino acid sequence of the transaminase mutant has the mutation site in the mutated amino acid sequence and has an amino acid sequence with 80% or more homology with the mutated amino acid sequence.
2. The transaminase mutant according to claim 1, characterized in that the mutations comprise T7C + S47C and at least one of the following mutation sites: q78, a330, V137, G313, a217, Y252, L295, C328, F22, a33, V42, a57, F89, N151, S156, M166, Y168, E171, K249, L283, S292, a299P, a334, F367, H369, V379, Q380, D396, F397, I400, C404, R405, F409, I414, R416, G419, S424, D436, R444, G457.
3. The transaminase mutant according to claim 2, characterized in that the mutations comprise T7C + S47C and at least one of the following mutation sites: Q78C, V137C, G313C, F22P, A33V, A57V/Y, F89A/E/V, N151A/E/F/Q/S/V/W/Y, S156 36156 156A/P/A166A/D/E/F/G/K/S/W/A168A/E/I/M/S/A171A/A249A, L283A, S292A, A299A, A33A, A334A/A367A/A, V379A/D/L/A380A, D396A/P/A397/A/Q/S/A400A, C A/A, F409/C/H/M/Q/T A/A, F409/S A/S/A/S36416/S A/36416, C A/S36416/S A/S/36416, G419W, S424A/C/Q/L/V, D436V/A, R444A/P/Y and G457C/P/W.
4. The transaminase mutant according to claim 2, characterized in that the mutations comprise at least one of the following combinations of mutation sites: T7C + S47C + S292A, T7C + S47C + D436A, T7C + S47C + D396G, T7C + S47C + Q380L, T7C + S47C + a299P, T7C + S47C + F397C, T7C + S47C + S424C, T7C + S47C + V379C, T7C + S47C + M166C, T7C + S47 + S C + C, T7C + S C + N C + 36.
5. The transaminase mutant according to claim 1, characterized in that the mutations comprise at least one of the following combinations of mutation sites: t7 + S47 + Q380 + V379, T7 + S47 + Q78 + A330 + Y89, T7 + S47 + V137 + G313 + Y89, T7 + S47 + Q380 + V379 + S156, T7 + S47 + Q78 + A330 + S156, T7 + S47 + K249 + I400 + S156, T7 + S47 + Q380 + V379 + M166, T7 + S47 + H404 + M166, T7 + S47 + H369 + F22 + 166, T7 + S47 + H369 + L283 + M166, T7 + S47 + V3747 + S166 + T7 + V379 + S166 + T7 + V47 + S166 + T7 + S166 + T7 + S166 + S47 + V379 + S166, T7 + S166 + S47 + V379 + K20 + S380 + S166, T7 + S47 + K379 + K20 + K47 + S166, T7 + S166, T47 + S166, T47 + K47 + S166, T7 + S47 + S166 + K47 + S166, T7A 47 + S47 + K47 + S166, T7A 47 + S47 + K37, T7 + S47 + S292 + A299 + M166 + Y168, T7 + S47 + A334 + F367 + M166 + Y168, T7 + S47 + Q380 + V379 + M166 + N151, T7 + S47 + A334 + F367 + M166 + N151, T7 + S47 + Q380 + V379 + M166 + R416, T7 + S47 + Q380 + V379 + R416, T7 + S166 + R416, T7 + S47 + Q380 + V379 + M166 + R166, T7 + S47 + D436 + R444 + M166 + R166 + T7 + T166 + M166 + R416, T7 + M166 + M2 + R416, T7 + M2, T2 + M2, T7 + M2, T2 + M2, T7 + S47 + Q380 + V379 + M166 + R416 + Y168, T7 + S47 + Q380 + V379 + M166 + R416 + N151 + Y168, T7 + S47 + D396 + F397 + M166 + R416 + N151 + Y168, T7 + S47 + Q380 + V379 + M166 + R416 + N151 + S424, T7 + S47 + Q380 + V379 + M166 + R166 + S409, T7 + S47 + Q380 + V379 + V409 + R151 + N409 + S409 + T166 + V409 + F151 + V409 + F151 + V409 + R166 + V151 + V409 + V151 + V409 + F151 + V409 + V151 + F409 + V409 + R166 + V151 + V409 + R166 + V151 + V409 + V151 + V409 + R166 + V151 + V409 + V151 + V409 + R166 + V151 + V409 + V151 + V409 + M2, T7 + V409 + V151 + V409 + R151 + V409 + R166 + V151 + R2, T7C + S47C + Q380L + V379L + M166F + R416D + N151W + S424V + F409C, T7C + S47C + Q380C + V379C + M166C + R416C + N151C + S424C + F409C, T7C + S47C + Q380C + V379C + M166C + R C + N151C + S424C + S C + F409C, T7C + S47C + Q380 + Q C + V379C + M166 + N C + N151C + N36166 + N C + N C + N36409 + N C + F C + F36409 + C + F C + 36409C + F C + F C + F C + 36.
6. A DNA molecule encoding the transaminase mutant of any one of claims 1 to 5.
7. A recombinant plasmid comprising the DNA molecule of claim 6.
8. The recombinant plasmid of claim 7, wherein the recombinant plasmid is pET-22a (+), pET-22b (+), pET-3a (+), pET-3d (+), pET-11a (+), pET-12a (+), pET-14b (+), pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), or, pET-32a (+), pET-35b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc 53999, pTwin1, pZZ 685 18, pK 232-18, pK-18-19 or pK-19.
9. A host cell comprising the recombinant plasmid of claim 7 or 8.
10. The host cell of claim 9, wherein the host cell comprises a prokaryotic cell, a yeast, or a eukaryotic cell; preferably, the prokaryotic cell is an Escherichia coli BL21-DE3 cell or an Escherichia coli Rosetta-DE3 cell.
11. A process for producing chiral amines comprising the step of subjecting a ketone compound and an amino donor to a catalyzed transamination reaction by a transaminase, wherein the transaminase is the transaminase mutant of any one of claims 1 to 5.
12. The method of claim 11, wherein the ketone compound isThe transamination reaction product isWherein R is1And R2Each independently represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group; r1And R2May be used alone or in combination with each other to form a substituted or unsubstituted ring;
preferably, the ketone compound is dihydroxy ketal compound, and the reaction isGeneratingTransamination reaction of (a);
preferably, R1And R2Is an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group having 1 to 20 carbon atoms, more preferably an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group having 1 to 10 carbon atoms;
preferably, the substitution means substitution by a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group;
preferably, R1Expressed as methyl or halogen-substituted methyl, more preferably, the halogen-substituted methyl is CF3，CF2H，CCl3，CCl2H，CBr3Or CBr2H, further preferably CF3Or CF2H；
Preferably, the ketone compound is
13. The process according to claim 11, wherein the amino donor is isopropylamine or alanine, preferably isopropylamine.
14. The method of claim 11, wherein the pH of the reaction system for the transaminase-catalyzed transamination of a ketone compound and an amino donor is 7 to 11, preferably 8 to 10, and more preferably 9 to 10;
preferably, the temperature of a reaction system for the transaminase to perform the catalytic transaminase reaction on the ketone compound and the amino donor is 25-60 ℃, more preferably 30-55 ℃, and further preferably 40-50 ℃;
preferably, the volume concentration of dimethyl sulfoxide in the reaction system for the transaminase to perform the catalytic transamination reaction on the ketone compound and the amino donor is 0-50%, and more preferably 0-20%.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110950788A (en) * | 2020-02-26 | 2020-04-03 | 凯莱英医药集团（天津）股份有限公司 | Synthetic method of fluorine-containing chiral amine compound |
| CN110982801A (en) * | 2019-12-27 | 2020-04-10 | 中国科学院苏州生物医学工程技术研究所 | Transaminase mutant and construction method and application thereof |
| CN112458123A (en) * | 2021-02-04 | 2021-03-09 | 凯莱英医药集团（天津）股份有限公司 | Method for synthesizing chiral amine |
| WO2021168987A1 (en) * | 2020-02-26 | 2021-09-02 | 凯莱英医药集团（天津）股份有限公司 | Method for synthesizing chiral diamine compound |
| CN113801858A (en) * | 2021-11-18 | 2021-12-17 | 广东金骏康生物技术有限公司 | Dehydrogenase mutant L283V/L286V and preparation method and application thereof |
| CN114277011A (en) * | 2021-12-29 | 2022-04-05 | 凯莱英医药集团（天津）股份有限公司 | Transaminase mutants and uses thereof |
| WO2022166103A1 (en) * | 2021-02-04 | 2022-08-11 | 凯莱英医药集团（天津）股份有限公司 | Transaminase mutant and application thereof |
| CN115677694A (en) * | 2022-11-30 | 2023-02-03 | 南通常佑药业科技有限公司 | Synthetic method of rimazepam |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3085776A1 (en) * | 2015-04-08 | 2016-10-26 | Industry-Academic Cooperation Foundation, Yonsei University | Omega-transaminase mutants with activity improvements toward ketones and methods for producing optically pure amines |
| CN108048419A (en) * | 2017-11-15 | 2018-05-18 | 凯莱英生命科学技术(天津)有限公司 | Transaminase mutant and its application |
| CN108384767A (en) * | 2018-02-05 | 2018-08-10 | 凯莱英生命科学技术(天津)有限公司 | Transaminase mutant and its application |
-
2019
- 2019-10-28 CN CN201911033481.5A patent/CN110592042B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3085776A1 (en) * | 2015-04-08 | 2016-10-26 | Industry-Academic Cooperation Foundation, Yonsei University | Omega-transaminase mutants with activity improvements toward ketones and methods for producing optically pure amines |
| CN108048419A (en) * | 2017-11-15 | 2018-05-18 | 凯莱英生命科学技术(天津)有限公司 | Transaminase mutant and its application |
| CN108384767A (en) * | 2018-02-05 | 2018-08-10 | 凯莱英生命科学技术(天津)有限公司 | Transaminase mutant and its application |
Non-Patent Citations (2)
| Title |
|---|
| DESZCZ, DAWID等: "Single active-site mutants are sufficient to enhance serine:pyruvate -transaminase activity in an -transaminase", 《FEBS JOURNAL》 * |
| 高新星等: "ω-转氨酶分子改造研究进展", 《生物工程学报》 * |
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110982801A (en) * | 2019-12-27 | 2020-04-10 | 中国科学院苏州生物医学工程技术研究所 | Transaminase mutant and construction method and application thereof |
| EP4112733A4 (en) * | 2020-02-26 | 2023-09-06 | Asymchem Laboratories (Tianjin) Co., Ltd. | Method for synthesizing chiral diamine compound |
| CN110950788B (en) * | 2020-02-26 | 2020-06-16 | 凯莱英医药集团（天津）股份有限公司 | Synthetic method of fluorine-containing chiral amine compound |
| JP7431999B2 (en) | 2020-02-26 | 2024-02-15 | アシムケム ラボラトリーズ （ターンジン） カンパニー リミテッド | Method for synthesizing fluorine-containing chiral amine compounds |
| EP4116289A4 (en) * | 2020-02-26 | 2023-09-27 | Asymchem Laboratories (Tianjin) Co., Ltd. | Synthesis method for fluorine-containing chiral amine compound |
| CN110950788A (en) * | 2020-02-26 | 2020-04-03 | 凯莱英医药集团（天津）股份有限公司 | Synthetic method of fluorine-containing chiral amine compound |
| WO2021168988A1 (en) * | 2020-02-26 | 2021-09-02 | 凯莱英医药集团（天津）股份有限公司 | Synthesis method for fluorine-containing chiral amine compound |
| WO2021168987A1 (en) * | 2020-02-26 | 2021-09-02 | 凯莱英医药集团（天津）股份有限公司 | Method for synthesizing chiral diamine compound |
| CN112980899B (en) * | 2021-02-04 | 2021-08-20 | 凯莱英医药集团（天津）股份有限公司 | Method for synthesizing chiral amine |
| WO2022166104A1 (en) * | 2021-02-04 | 2022-08-11 | 凯莱英医药集团（天津）股份有限公司 | Method for synthesizing chiral amine compound |
| WO2022166103A1 (en) * | 2021-02-04 | 2022-08-11 | 凯莱英医药集团（天津）股份有限公司 | Transaminase mutant and application thereof |
| CN112980899A (en) * | 2021-02-04 | 2021-06-18 | 凯莱英医药集团（天津）股份有限公司 | Method for synthesizing chiral amine |
| CN112458123A (en) * | 2021-02-04 | 2021-03-09 | 凯莱英医药集团（天津）股份有限公司 | Method for synthesizing chiral amine |
| CN113801858B (en) * | 2021-11-18 | 2022-02-22 | 广东金骏康生物技术有限公司 | Dehydrogenase mutant L283V/L286V and preparation method and application thereof |
| CN113801858A (en) * | 2021-11-18 | 2021-12-17 | 广东金骏康生物技术有限公司 | Dehydrogenase mutant L283V/L286V and preparation method and application thereof |
| US11873515B2 (en) | 2021-11-18 | 2024-01-16 | Golden Health (Guangdong) Biotechnology Co., Ltd. | Dehydrogenase mutant L283V/L286V, and preparation method and use thereof |
| CN114277011A (en) * | 2021-12-29 | 2022-04-05 | 凯莱英医药集团（天津）股份有限公司 | Transaminase mutants and uses thereof |
| WO2023123589A1 (en) * | 2021-12-29 | 2023-07-06 | 凯莱英医药集团（天津）股份有限公司 | Transaminase mutant and use thereof |
| CN114277011B (en) * | 2021-12-29 | 2024-02-06 | 凯莱英医药集团（天津）股份有限公司 | Transaminase mutants and uses thereof |
| CN115677694A (en) * | 2022-11-30 | 2023-02-03 | 南通常佑药业科技有限公司 | Synthetic method of rimazepam |
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