Hydroquinone manufacturing method

Abstract

The object of the present invention is to provide a method for producing hydroquinone involving an enzyme that can convert p-hydroxybenzoic acid to hydroquinone with higher efficiency than the 4-hydroxybenzoic acid 1-hydroxylase possessed by Candida parapsis strain CBS604. [Solution] The above objective can be achieved by a method for producing hydroquinone, which includes the step of obtaining hydroquinone by reacting p-hydroxybenzoic acid with 4-hydroxybenzoic acid 1-hydroxylase derived from Spathaspora passalidarum.

Description

Technical Field

[0001] The present invention relates to a method for producing hydroquinone, in which an enzyme having 4-hydroxybenzoate 1-hydroxylase activity that catalyzes the reaction of converting p-hydroxybenzoic acid to hydroquinone is involved.

Background Art

[0002] Hydroquinone is a diphenol having a structure in which the 1-position and 4-position of the benzene ring are substituted with hydroxy groups (-OH). Hydroquinone is used as a cosmetic raw material as a kind of whitening ingredient.

[0003] Hydroquinone has a structure similar to tyrosine and can function as a tyrosine analog. Thus, hydroquinone can inhibit tyrosinase activity by antagonizing tyrosine and binding to tyrosinase. Since the biosynthesis of melanin is brought about by tyrosinase activity, if tyrosinase activity is inhibited by hydroquinone, the biosynthesis of melanin is suppressed. As a result, hydroquinone can exert a cosmetic improvement effect on the skin, such as improvement of pigmentation such as freckles, age spots, and sunburn, and promotion of whitening.

[0004] Hydroquinone is mainly produced by chemical synthesis. However, the method by chemical synthesis has problems such as using substances harmful to the human body such as organic solvents and inorganic metal catalysts, difficulty in separating the product hydroquinone from raw materials and by-products, and inclusion of expensive substances not recognized as cosmetic ingredients in the raw materials. Therefore, there is a demand for producing hydroquinone biochemically or biologically safely and simply.

[0005] As a kind of hydroquinone-producing enzyme, 4-hydroxybenzoate 1-hydroxylase that catalyzes the reaction of converting p-hydroxybenzoic acid to hydroquinone is known. As a microorganism expressing 4-hydroxybenzoate 1-hydroxylase, Candida parapsilosis ( White parapsilosis The CBS604 strain is known (see, for example, Non-Patent Documents 1 and 2). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] MH Eppinket al, J Bacteriol., 1997 Nov; 179 (21): pages 6680-6687. [Non-Patent Document 2] Willem JH Van Berkel et al, FEMS Microbiology Letters, Volume 121, Issue 2, August 1994, Pages 207-215. [Overview of the project] [Problems that the invention aims to solve]

[0007] However, our inventors have found that the 4-hydroxybenzoic acid 1-hydroxylase present in Candida parapsis strain CBS604 has low catalytic activity, resulting in a very slow conversion reaction from p-hydroxybenzoic acid to hydroquinone.

[0008] Furthermore, to date, there are very few known enzymes that can convert p-hydroxybenzoic acid to hydroquinone with higher efficiency than the 4-hydroxybenzoic acid 1-hydroxylase possessed by Candida parapsis strain CBS604, nor are there any known microorganisms that express such enzymes.

[0009] Therefore, the problem that the present invention aims to solve is to provide a method for producing hydroquinone involving an enzyme that can convert p-hydroxybenzoic acid to hydroquinone with higher efficiency than the 4-hydroxybenzoic acid 1-hydroxylase possessed by Candida parapsis strain CBS604. [Means for solving the problem]

[0010] The inventors of the present invention have diligently investigated new hydroquinone-producing enzymes in an attempt to solve the above problems. As a result, they found that Spasaspora passaridarum ( Spathaspora of passion fruit We discovered that the NRRL Y-27907 strain expresses a protein with 4-hydroxybenzoic acid 1-hydroxylase activity.

[0011] Then, when we created transformed microorganisms expressing the protein and evaluated their 4-hydroxybenzoate 1-hydroxylase activity, we were surprised to find that the 4-hydroxybenzoate 1-hydroxylase activity of the protein was significantly higher than that of the 4-hydroxybenzoate 1-hydroxylase possessed by Candida parapsis strain CBS604.

[0012] Based on the above findings, the inventors have finally succeeded in creating a hydroquinone production method involving an enzyme that can efficiently convert p-hydroxybenzoic acid to hydroquinone, thereby solving the problems of the present invention. The present invention is completed based on the first findings and success stories obtained by these inventors.

[0013] Therefore, according to one aspect of the present invention, the following embodiments are provided. [1] A method for producing hydroquinone, p-hydroxybenzoic acid, Spasaspora passaridarum ( Spathaspora of passion fruit The method comprising the step of obtaining hydroquinone by acting on an enzyme having 1-hydroxylase activity of 4-hydroxybenzoic acid derived from ) [2] The enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum is the method according to item [1], having the amino acid sequence of (1) or (2) below. (1) Amino acid sequences having 80% or more sequence identity with the amino acid sequence of Sequence ID No. 2 (2) In the amino acid sequence of Sequence ID No. 2, an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit consisting of 100 amino acids. [3] A hydroquinone production composition for producing hydroquinone from p-hydroxybenzoic acid, The composition comprising, as an active ingredient, an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum. [4] DNA fragments for transforming non-human host organisms, Genes for enzymes possessing 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum, At least one gene different from the said gene and The DNA fragment containing the aforementioned DNA fragment. [5] A transformant capable of producing hydroquinone from p-hydroxybenzoic acid, The transformed organism is obtained by transduction of a gene for an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum as an exogenous gene. [6] The transformant described in item [5] is a transformant selected from the group consisting of microorganisms of the genera Corynebacterium, Escherichia, Rhodococcus, Acinetobacter, Bradyrhizobium, Corynebacterium, Pseudomonas, Rhodopseudomonas, Sinorhizobium, Brevibacterium, Novosphingobium, and Ralstonia. [7] The enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum is a composition, DNA fragment or transformant according to any one of items [3] to [6], having the amino acid sequence of (1) or (2) below. (1) Amino acid sequences having 80% or more sequence identity with the amino acid sequence of Sequence ID No. 2 (2) In the amino acid sequence of SEQ ID NO:2, an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit consisting of 100 amino acids

Advantages of the Invention

[0014] According to the present invention, hydroquinone can be produced from p-hydroxybenzoic acid with high efficiency as compared with the case of using 4-hydroxybenzoate 1-hydroxylase possessed by Candida parapsilosis CBS604 strain. Further, according to the present invention, since a biochemically and / or biologically safe and simple production method can be employed, mass production of hydroquinone on an industrial scale is expected.

Modes for Carrying Out the Invention

[0015] Hereinafter, the details of each aspect of the present invention will be described. However, the present invention is not limited only by the matters in this section, and can take various aspects as long as the object of the present invention is achieved.

[0016] Unless otherwise defined, each term in this specification is used in the meaning usually used by those skilled in the technical fields such as the fields of biochemistry, biotechnology, microbiology, etc., and should not be construed as having an unduly limiting meaning. Further, the assumptions and theories made in this specification are based on the findings and experiences of the present inventors so far, and thus the present invention is not limited only by such assumptions and theories.

[0017] "Comprising" means that elements other than the elements explicitly stated as being included can be added (synonymous with "at least comprising"), and includes "consisting of" and "consisting essentially of". That is, "comprising" can mean including the explicitly stated elements and any one or two or more elements, consisting of the explicitly stated elements, or consisting essentially of the explicitly stated elements. Examples of the elements include restrictive matters such as components, steps, conditions, parameters, etc. "Having" is synonymous with "comprising". The term "and / or" means any one of the plurality of listed related items, or any combination or all combinations of two or more of them. The "~" in a numerical range represents a range that includes the numerical values before and after it, and also includes a range excluding one of the included limit values. For example, "0%~100%" may be 0% or more, 100% or less, or any value between 0% and 100%. "About" means an amount within ±10% of the quantity following the term. For example, "about 100" means 100 ± 10%, that is, 90 to 110. The number of digits of an integer value coincides with the number of significant figures. For example, the significant figure of 1 is 1 digit, and the significant figure of 10 is 2 digits. Also, for a decimal value, the number of digits after the decimal point coincides with the number of significant figures. For example, the significant figure of 0.1 is 1 digit, and the significant figure of 0.10 is 2 digits.

[0018] "Foreign gene" means a gene that is not naturally present on the chromosomal (genomic) DNA of the introduced organism, and is also called a heterologous gene. In this specification, genome and chromosome are synonymous terms. "Gene expression" means that through transcription, translation, etc., a protein having an amino acid sequence encoded by a part or all of the nucleotide sequence of a gene (a protein encoded by a gene) is produced so as to have its original structure and activity. "Wild type" means an organism that exists naturally and has not been artificially genetically modified. "Transformant" means an organism that has been artificially genetically modified. "Wild-type gene" means a gene that is originally present on the genomic DNA of a wild type. "Wild-type protein" means a protein encoded by a wild-type gene. A protein having the activity of catalyzing a predetermined reaction is called an "enzyme".

[0019] [Summary of the Invention] One aspect of the present invention is a method for producing hydroquinone. The method for producing hydroquinone according to one embodiment of the present invention comprises subjecting p-hydroxybenzoic acid to Spathaspora of passion fruit The method includes a step of obtaining hydroquinone by acting on an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum. In this specification, the enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum may be referred to as SP-S1H, and the gene encoding the enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum may be referred to as the SP-S1H gene. In addition, either "hydro" or "hydro" is used as the Japanese reading of the English word "hydro".

[0020] Another aspect of the present invention is a hydroquinone production composition for producing hydroquinone from p-hydroxybenzoic acid. One embodiment of the present invention comprises SP-S1H as an active ingredient.

[0021] SP-S1H used in each aspect of the present invention may be an isolated and purified enzyme, or it may be an enzyme-containing material such as a living organism, a biologically processed product, or a biological culture of a transformant transformed to express Spasaspora passaridarum or SP-S1H. Furthermore, SP-S1H may be synthesized using a cell-free protein synthesis system with the SP-S1H gene, which encodes SP-S1H.

[0022] Another aspect of the present invention is a DNA fragment for transforming a non-human host organism. A DNA fragment according to one aspect of the present invention comprises the SP-S1H gene and at least one gene different from said gene.

[0023] Another aspect of the present invention is a transformant capable of producing hydroquinone from p-hydroxybenzoic acid. The transformant according to one embodiment of the present invention is obtained by transduction of the SP-S1H gene as an exogenous gene.

[0024] [Enzyme (SP-S1H) with 1-hydroxylase activity of 4-hydroxybenzoic acid derived from Spasaspora passaridarum] As shown in the following scheme (1), 4-hydroxybenzoic acid 1-hydroxylase has the activity to catalyze the reaction that converts p-hydroxybenzoic acid to hydroquinone (hereinafter also simply referred to as hydroxylase activity). After diligent investigation, it was found that when salicylate hydroxylase derived from Spathaspora passaridarum is treated with 4-hydroxybenzoic acid, the same reaction as in scheme (1) occurs. In other words, it was found that salicylate hydroxylase derived from Spathaspora passaridarum is an enzyme that possesses 4-hydroxybenzoic acid 1-hydroxylase activity. [ka]

[0025] As for 4-hydroxybenzoic acid 1-hydroxylases already known, there are Candida parapsis (as described in Non-Patent Documents 1 and 2) White parapsilosis )CBS604 strain possesses 4-hydroxybenzoic acid 1-hydroxylase. SP-S1H has hydroxylase activity, but its amino acid sequence identity with the 4-hydroxybenzoic acid 1-hydroxylase derived from Candida parapsis CBS604 is 62%. Therefore, SP-S1H may also be an enzyme having an amino acid sequence that has hydroxylase activity and has an amino acid sequence identity of 60% or more, preferably 65% ​​or more, more preferably 70% or more, even more preferably 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence of the wild-type enzyme of SP-S1H (SEQ ID NO: 2).

[0026] There are no particular limitations on the method for determining sequence identity of amino acid sequences, but for example, it can be determined by using a program that aligns the amino acid sequences of two proteins using commonly known methods and calculates the degree of sequence agreement between the two.

[0027] For calculating the degree of synchrony between two amino acid sequences, for example, the Karlin and Altschul algorithm (Proc.Natl.Acad.Sci.USA 87:2264-2268, 1990; Proc.Natl.Acad.Sci.USA 90:5873-5877, 1993) is known, and a BLAST program using this algorithm has been developed by Altschul et al. (J.Mol.Biol.215:403-410, 1990). Furthermore, Gapped BLAST, a program that determines sequence identity with higher sensitivity than BLAST, is also known (Nucleic Acids Res.25:3389-3402, 1997). Those skilled in the art can use the above programs to search a database for sequences that show high sequence identity with a given sequence. These are available, for example, on the website of the U.S. National Center for Biotechnology Information (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0028] Examples of amino acid sequences with 60% or more sequence identity to the amino acid sequence of the wild-type enzyme include amino acid sequences in which one or more amino acids are deleted, substituted, or added compared to the amino acid sequence of the wild-type enzyme. The range of "several" is determined by the sequence identity of the amino acid sequence. For example, if 100 amino acids in the amino acid sequence are considered as one unit, then each unit may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21-30 amino acids, preferably 1-20, and more preferably 1-10. Furthermore, "amino acid deletion" means the absence or disappearance of an amino acid residue in the sequence, "amino acid substitution" means that an amino acid residue in the sequence is replaced with another amino acid residue, and "amino acid addition" means that a new amino acid residue is inserted into the sequence.

[0029] Specific examples of "deletion, substitution, and addition of amino acids" include cases where an amino acid is replaced by another chemically similar amino acid. For example, this could include substituting one hydrophobic amino acid with another hydrophobic amino acid, or substituting one polar amino acid with another polar amino acid having the same charge. Such chemically similar amino acids are known in the relevant field for each amino acid. Specific examples include nonpolar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged basic amino acids include arginine, histidine, and lysine. Negatively charged acidic amino acids include aspartic acid and glutamic acid.

[0030] SP-S1H may have a tag peptide that facilitates separation and purification. Examples of such tag peptides include His tags, preferably 6×His, which consists of six histidine molecules linked together.

[0031] When attempting to obtain recombinant SP-S1H using genetic engineering technology, it may exist as an intracellular enzyme depending on the host organism used, making enzyme isolation and purification difficult. Therefore, when attempting to obtain recombinant SP-S1H, it is preferable to modify the recombinant SP-S1H to become an extracellular enzyme. However, if recombinant SP-S1H is expressed as an extracellular enzyme in the transformant, extracellular enzyme modification is not necessary.

[0032] Methods for extracellular enzymatic modification of recombinant SP-S1H include, but are not limited to, adding secretory signals such as peptides or proteins that function to cause recombinant SP-S1H to be expressed as an extracellular enzyme to the C-terminal and / or N-terminal side of recombinant SP-S1H.

[0033] A specific embodiment of SP-S1H is an SP-S1H having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 2. Another specific embodiment of SP-S1H is an SP-S1H having an amino acid sequence in which, in the amino acid sequence of SEQ ID NO: 2, 1 to 10 amino acids are deleted, substituted, and / or added per unit consisting of 100 amino acids.

[0034] [Genetic gene (SP-S1H gene) of an enzyme possessing 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum] The SP-S1H gene contains a nucleotide sequence that codes for the amino acid sequence of SP-S1H and expresses SP-S1H. These nucleotide sequences in the SP-S1H gene may be either a coding sequence (CDS) or an open reading frame (ORF), but are preferably ORFs.

[0035] The SP-S1H gene may consist of a nucleotide sequence that codes for SP-S1H, or it may also include transcriptional regulatory sequences such as promoters and terminators, and non-coding sequences such as introns. A gene is not merely sequence information, but a DNA molecule (fragment) made up of linked nucleotides.

[0036] The SP-S1H gene may include a nucleotide sequence optimized for codons, secondary structure, GC content, etc., by utilizing the fact that there are several types of codons corresponding to a single amino acid. Preferably, the codon-modified nucleotide sequence is one which has been modified to be easily expressed in the host organism.

[0037] The wild-type SP-S1H gene has the nucleotide sequence of SEQ ID NO: 3. As a specific example of the SP-S1H gene, the wild-type gene is used in the Coryne genus ( Corynebacterium Examples include genes that have a codon-optimized nucleotide sequence of SEQ ID NO: 4 for microorganisms.

[0038] By introducing the SP-S1H gene as an exogenous gene into a host organism, a host organism that was originally unable to express SP-S1H can be transformed to express SP-S1H as a recombinant enzyme. The expression of the SP-S1H gene may be autonomously carried out by the transcriptional regulatory sequence of the gene, or by the transcriptional regulatory mechanism of the host organism, but it is preferably carried out autonomously by the transcriptional regulatory sequence of the gene.

[0039] For the SP-S1H gene to be expressed autonomously within a host organism, it is preferable to include a promoter, ORF, and terminator. The promoter and terminator can be appropriately selected according to the host organism.

[0040] [DNA fragment] A DNA fragment according to one aspect of the present invention includes, in addition to the SP-S1H gene, at least one gene different from the SP-S1H gene. In the DNA fragment, the copy number of the SP-S1H gene may be any number, such as one copy or two or more copies.

[0041] The gene different from the SP-S1H gene can be any gene that codes for a protein different from SP-S1H, and may, for example, be a selection marker gene to facilitate the selection of transformants. Examples of selection marker genes include drug resistance genes such as kanamycin resistance genes and ampicillin resistance genes, but may also be other selection marker genes such as nutritional requirement marker genes. The gene different from the SP-S1H gene may not have a protein-coding nucleotide sequence and may consist of, for example, a transcriptional regulatory sequence or a non-coding sequence.

[0042] The DNA fragment may also be a DNA construct such as a plasmid or vector. A specific example of such a DNA construct is the SP-S1H expression DNA construct described in the examples below.

[0043] [Transformed body] A transformant according to one aspect of the present invention is obtained by transduction of the SP-S1H gene as an exogenous gene. The transformant has the SP-S1H gene introduced as an exogenous gene and expresses the introduced gene.

[0044] Transformed organisms are created by introducing the SP-S1H gene as an exogenous gene into a host organism.

[0045] The host organism is not particularly limited as long as it is an organism that can accept the introduction of the SP-S1H gene as an exogenous gene and express the introduced SP-S1H gene, for example, microorganisms, insect cells and insect bodies, plant cells and plant bodies, animal cells and animal bodies, etc. However, the host organism is not a human. Examples of host organisms include microorganisms of the genera Corynebacterium, Escherichia, Rhodococcus, Acinetobacter, Bradyrhizobium, Pseudomonas, Rhodopseudomonas, Sinorhizobium, Brevibacterium, Sphingovium, Novosphingovium, Ralstonia, Burkholderia, Alkaligenes, and Arthrobacter. In particular, microorganisms such as Pseudomonas, Burkholderia, Alkaligenes, Sphingovium, Rhodococcus, and Arthrobacter, which include strains that degrade p-hydroxybenzoic acid, are preferred. Preferably, the host organism is a Corynebacterium, which has a proven track record as a host organism for genetic modification and does not assimilate the product hydroquinone. In the following, the present invention will be described in detail assuming that the host organism is a microorganism.

[0046] The host microorganism may be a naturally occurring wild-type organism or a transformed microorganism obtained by introducing a mutation into a wild-type organism. For example, if the host microorganism has a gene for an enzyme that has hydroquinone-degrading activity, it is preferable to delete the hydroquinone-degrading enzyme gene in the host microorganism or to inactivate the expression of the hydroquinone-degrading enzyme gene. A transformed microorganism may also be obtained by introducing the SP-S1H gene into a wild-type organism and then deleting or inactivating the hydroquinone-degrading enzyme gene.

[0047] The vectors, culture media, and procedures used to produce transformed microorganisms can be found in the examples described later. For example, methods for transforming host microorganisms by introducing DNA fragments include electroporation, protoplasts, and calcium ion methods, but electroporation is preferred.

[0048] The method for introducing the SP-S1H gene into a host microorganism is not particularly limited. Examples include introducing a DNA construct, such as a plasmid vector incorporating the SP-S1H gene, into the host microorganism so that it autonomously proliferates and expresses the gene, or incorporating the SP-S1H gene into the host microorganism's genomic DNA by utilizing homologous recombination. However, the method of incorporating the SP-S1H gene into the host microorganism's genomic DNA is preferred because there is a probability that the gene will be distributed during cell division.

[0049] Other molecular biological, biotechnological, and biochemical methods used to produce transformed microorganisms can be found in literature such as Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, and Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons, 1987-1997.

[0050] One specific embodiment of the transformed microorganism is a transformed microorganism (1) in which the host microorganism is Corynebacterium glutamicum, the SP-S1H gene is introduced as an exogenous gene, and the transformed microorganism expresses the introduced gene. SP-S1H can be obtained by using the transformed microorganism (1), and furthermore, the cells, cell treatment products, and cell cultures of the transformed microorganism (1) can be used as SP-S1H-containing products. A specific example of the transformed microorganism (1) is the SP-S1H-expressing transformed Corynebacterium microorganism described in the examples below.

[0051] [Method for producing hydroquinone] A method for producing hydroquinone according to one aspect of the present invention includes the step of obtaining hydroquinone by reacting p-hydroxybenzoic acid with SP-S1H.

[0052] The method for reacting p-hydroxybenzoic acid with SP-S1H is any method in which p-hydroxybenzoic acid and Sp-S1H come into contact and hydroquinone is produced and / or accumulated by SP-S1H. For example, this could involve reacting p-hydroxybenzoic acid with SP-S1H under conditions where the pH is 5 to 9, the temperature is 20°C to 45°C, the time is several minutes to several tens of hours, and the reaction is performed by standing, stirring, or shaking.

[0053] When transformed microorganisms are used as SP-S1H-containing materials, hydroquinone can be produced by culturing the transformed microorganisms under various culture conditions suitable for the transformed microorganisms, using a culture medium containing p-hydroxybenzoic acid and suitable for the growth of the transformed microorganisms. The culture method can be any method suitable for culturing the host microorganism. For example, if the host microorganism is a Corynebacterium, solid culture or liquid culture methods performed under aeration conditions can be used. Alternatively, the recovered transformed microorganisms may be suspended in a liquid medium containing p-hydroxybenzoic acid in the presence of flavin mononucleotide as a coenzyme and NADH as an electron donor, and incubated at a temperature of 20°C to 45°C for several minutes to several tens of hours.

[0054] The method for obtaining hydroquinone from the culture after the completion of cultivation is not particularly limited. For example, the reaction mixture can be subjected to conventional solid-liquid separation treatments such as filtration and centrifugation to separate the solids from the reaction mixture, and hydroquinone can be extracted from the recovered reaction mixture by solid-phase extraction using a column or solvent extraction using a hydroquinone-soluble solvent. The extraction solvent is not particularly limited as long as it is capable of dissolving hydroquinone, and examples include ethyl acetate and diethyl ether. Hydroquinone may also be obtained as a fractionated solution using a chromatograph such as a liquid chromatograph.

[0055] Qualitative or quantitative analysis of hydroquinone may be performed by HPLC as described in the examples below.

[0056] In the method for producing hydroquinone, various steps and operations can be added before, after, or during the above-described steps, as long as the objectives of the present invention can be achieved.

[0057] [Uses of hydroquinone] Generally, hydroquinone is used as a cosmetic ingredient for its expected cosmetic effects on the skin, such as improving pigmentation like age spots, freckles, and sunburn, and promoting skin whitening. Hydroquinone obtained by hydroquinone manufacturing methods can also be used as a cosmetic ingredient as a type of skin whitening agent. Industrial uses include photographic developers, dyes, organic synthesis raw materials, pharmaceutical intermediates, antioxidants, and polymerization inhibitors. Hydroquinone can also be used as a raw material for these industrial materials.

[0058] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples, and can take various forms as long as it can solve the problems of the present invention. [Examples]

[0059] [1. Isolation of hydroquinone-producing enzymes] Hydroquinone-producing enzymes possess enzymatic activity that catalyzes the reaction that converts p-hydroxybenzoic acid (pHBA) to hydroquinone (HQ). Some of these are known as 4-hydroxybenzoate 1-hydroxylase or 4-hydroxybenzoate 1-monooxygenase. The EC number (Enzyme Commission number) for 4-hydroxybenzoate 1-hydroxylase is EC1.14.13.64.

[0060] The inventors searched for a novel hydroquinone-producing enzyme and obtained the SP-S1H protein, which has the amino acid sequence of SEQ ID NO: 2, as a candidate enzyme. The gene encoding SP-S1H is Spasaspora passaridarum ( Spathaspora of passion fruit It was found on the genome of the NRRL Y-27907 strain and consisted of the nucleotide sequence of Sequence ID No. 3. The GenBank accession number version of SP-S1H is XP_007376166.1.

[0061] [2. Construction of the DNA construct for SP-S1H expression] The SP-S1H protein, which has the amino acid sequence of SEQ ID NO: 2, was introduced into a microorganism of the genus Corynebacter ( Corynebacterium glutamic The nucleotide sequence of SEQ ID NO: 4 was obtained by optimizing for the codon of ). pMKsf vector containing the kanamycin resistance gene ( Corynebacterium glutamic A DNA construct for SP-S1H expression was constructed by inserting the nucleotide sequence of Sequence ID No. 4 into the multi-cloning site of the ATCC31808-derived shuttle vector pCG1 (US4617267A).

[0062] [3. Preparation of SP-S1H-expressing transformed microorganisms] Corynebacteria distributed by the National Institute of Technology and Evaluation (NITE) Corynebacterium glutamicATCC13032 (NBRC12168) was transformed by homologous recombination using electroporation and heat shock to obtain CT10 (ΔcglIM-IR-IIR, ΔpobA, ΔpcaHG, ΔgenH, Δpcaben), a transformed Coryne microorganism lacking the cgl gene, pobA gene, pcaHG gene, genH gene, and pcaben gene.

[0063] CT10 was subjected to transduction treatment using electroporation and heat shock with a DNA construct for SP-S1H expression. After transduction treatment, SP-S1H-expressing transformed Coryne microorganisms were selected by culturing CT10 on LB agar medium containing kanamycin at 32°C for 20-24 hours.

[0064] On the other hand, Candida parapsis described in Non-Patent Documents 1 and 2 ( White parapsilosis The amino acid sequence (SEQ ID NO: 1) of 4-hydroxybenzoic acid 1-hydroxylase from strain CBS604 was obtained from GenBank with accession number version XP_036663424.1. This 4-hydroxybenzoic acid 1-hydroxylase was named CP-4HB1H. CP-4HB1H-expressing transformed Corynebacterium microorganisms were selected through codon optimization, DNA construct construction, and transformation, in the same manner as described above. The amino acid sequence identity between SP-S1H and CP-4HB1H was 62%.

[0065] [4. Activity evaluation of SP-S1H] The obtained SP-S1H-expressing transformed Coryne microorganisms and CP-4HB1H-expressing transformed Coryne microorganisms were inoculated into 2 ml of growth medium prepared by mixing CGXIIa medium and LB medium in a 1:1 ratio. Subsequently, the inoculated growth medium was incubated overnight at 32°C with shaking at 250 rpm after adding 25 μg / mL of kanamycin.

[0066] The composition of CGXIIa medium (pH 6.6~6.8) is 83.3 mM urea, 151.4 mM ammonium sulfate, 10 mM dipotassium hydrogen phosphate, 10 mM potassium dihydrogen phosphate, 10% glucose, 250 mg / L magnesium sulfate, 10 mg / L calcium chloride, 200 μg / L biotin, 0.1 mM ferrous sulfate, 10 mg / L manganese sulfate monohydrate, 1 mg / L zinc sulfate heptahydrate, 0.2 mg / L copper sulfate, and 0.02 mg / L nickel chloride hexahydrate.

[0067] The composition of LB medium (pH 7.0) is 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0068] The obtained culture solution was subjected to centrifugation (13,000 rpm, 2 minutes), and the cell was recovered by removing the supernatant. The obtained wet cell was added to CGXIIa medium containing 10 mM pHBA and thoroughly suspended, and subjected to the HQ conversion reaction at 37°C overnight.

[0069] The solution after the HQ conversion reaction was subjected to centrifugation (13,000 rpm, 2 minutes), and the supernatant was collected as the reaction solution. The HQ concentration of the obtained reaction solution was measured by HPLC analysis.

[0070] HPLC analysis was performed using a degasser "DGU-20A", liquid chromatograph "LC-20AR", autosampler "SIL-20AC", column oven "CTO-20AC", and detector "SPD-M20A" (all manufactured by Shimadzu Corporation). An Inertsil ODS-3 5μm column (diameter 4.6 mm, length 250 mm, particle size 5 μm, manufactured by GL Science) was used and maintained at 30°C. A gradient elution mode was used (solvent A: 0.1% (v / v) formic acid, solvent B: 100% (v / v) acetonitrile). After equilibration with solvent A, the proportion of solvent B was maintained at 30% for 2 minutes from the start of the analysis, then increased to 50% over 2 minutes, and then decreased to 30% over 1 minute. The mobile phase flow rate was set to 1.0 mL / min, the measurement wavelength for HQ was 290 nm, and the measurement wavelength for pHBA was 255 nm.

[0071] [5. Evaluation Results] When the HQ concentration was measured, the HQ concentration in the reaction solution obtained using CP-4HB1H-expressing transformed Coryne microorganisms was 0.42 g / L, while the HQ concentration in the reaction solution obtained using SP-S1H-expressing transformed Coryne microorganisms was 0.90 g / L.

[0072] Therefore, if the HQ-converting enzyme activity of CP-4HB1H is set to 100%, the HQ-converting enzyme activity of SP-S1H was 215%. In this way, we were able to obtain a hydroquinone-producing enzyme with very high enzymatic activity that catalyzes the reaction that converts pHBA to HQ.

[0073] [6. Sequence Listing] The sequences listed in the sequence listing are shown in Table 1 below.

[0074] [Table 1] [Industrial applicability]

[0075] By utilizing the transformed microorganism and production method according to one aspect of the present invention, beneficial hydroquinones with physiological activity can be produced on an industrial scale from p-hydroxybenzoic acid through a biochemically or biologically safe and simple production method.

Claims

1. A method for producing hydroquinone, The method comprising the step of obtaining hydroquinone by reacting p-hydroxybenzoic acid with an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passalidarum.

2. The method according to claim 1, wherein the enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum has the following amino acid sequence (1) or (2). (1) Amino acid sequences having 80% or more sequence identity with the amino acid sequence of Sequence ID No. 2 (2) In the amino acid sequence of Sequence ID No. 2, an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added to a unit consisting of 100 amino acids.

3. A hydroquinone production composition for producing hydroquinone from p-hydroxybenzoic acid, The composition comprising, as an active ingredient, an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum.

4. DNA fragments for transforming non-human host organisms, The gene for the enzyme possessing 4-hydroxybenzoic acid 1-hydroxylase activity, derived from Spathaspora passaridarum, At least one gene different from the said gene and The DNA fragment containing the aforementioned DNA fragment.

5. A transformant capable of producing hydroquinone from p-hydroxybenzoic acid, The transformed organism is obtained by transduction of a gene for an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum as an exogenous gene.

6. The transformant according to claim 5, wherein the host organism is selected from the group consisting of microorganisms of the genera Corynebacterium, Escherichia, Rhodococcus, Acinetobacter, Bradyrhizobium, Corynebacterium, Pseudomonas, Rhodopseudomonas, Sinorhizobium, Brevibacterium, Novosphingovium, and Ralstonia.

7. The enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spasaspora passaridarum has the amino acid sequence of (1) or (2) below, as described in any one of claims 3 to 6, the composition, DNA fragment or transformant. (1) Amino acid sequences having 80% or more sequence identity with the amino acid sequence of Sequence ID No. 2 (2) In the amino acid sequence of Sequence ID No. 2, an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added to a unit consisting of 100 amino acids.

Images