How Hydroquinone is Made

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 more efficiently than the 4-hydroxybenzoate 1-hydroxylase found in Candida parapsilosis CBS604 strain. [Solution] The above-mentioned object can be achieved by a method for producing hydroquinone, which includes a step of obtaining hydroquinone by treating 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, which involves an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity that catalyzes the reaction of converting p-hydroxybenzoic acid to hydroquinone. [Background technology]

[0002] Hydroquinone is a dihydric phenol with a structure in which the 1st and 4th positions of the benzene ring are substituted with hydroxy groups (-OH). Hydroquinone is used as a skin-whitening ingredient in cosmetics.

[0003] Hydroquinone has a structure similar to tyrosine and can function as a tyrosine analog. Hydroquinone can inhibit tyrosinase activity by binding to tyrosinase in competition with tyrosine. Since melanin biosynthesis is driven by tyrosinase activity, the inhibition of tyrosinase activity by hydroquinone suppresses melanin biosynthesis. As a result, hydroquinone can exert cosmetic effects on the skin, such as improving pigmentation (e.g., age spots, freckles, and sunburn) and promoting whitening.

[0004] Hydroquinone is mainly produced by chemical synthesis. However, chemical synthesis methods have problems such as the use of substances that are harmful to the human body, such as organic solvents and inorganic metal catalysts, the difficulty of separating the product hydroquinone from raw materials and by-products, and the fact that some raw materials are expensive and not approved as cosmetic ingredients. Therefore, there is a need for a biochemically and biologically safe and easy method for producing hydroquinone.

[0005] One of the hydroquinone-producing enzymes is 4-hydroxybenzoate 1-hydroxylase, which catalyzes the reaction of converting p-hydroxybenzoic acid to hydroquinone. Candida parapsilosis ( White parapsilosis ) 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. Summary of the Invention [Problem to be solved by the invention]

[0007] However, according to the investigations conducted by the present inventors, the 4-hydroxybenzoate 1-hydroxylase possessed by the Candida parapsilosis CBS604 strain has a problem in that it has low catalytic activity and the conversion reaction from p-hydroxybenzoate to hydroquinone is very slow.

[0008] Furthermore, to date, compared to the 4-hydroxybenzoate 1-hydroxylase possessed by Candida parapsilosis CBS604 strain, few enzymes capable of converting p-hydroxybenzoate to hydroquinone with high efficiency, and few microorganisms expressing such enzymes, have been known.

[0009] Therefore, an object of the present invention is to provide a method for producing hydroquinone involving an enzyme that can convert p-hydroxybenzoic acid to hydroquinone more efficiently than the 4-hydroxybenzoic acid 1-hydroxylase contained in Candida parapsilosis CBS604 strain. [Means for solving the problem]

[0010] In an attempt to solve the above problems, the present inventors have conducted extensive research into new hydroquinone-producing enzymes. Spathaspora of passion fruit ) We found that the NRRL Y-27907 strain expresses a protein with 4-hydroxybenzoate 1-hydroxylase activity.

[0011] We then created a transformed microorganism expressing the protein and evaluated its 4-hydroxybenzoate 1-hydroxylase activity. Surprisingly, the 4-hydroxybenzoate 1-hydroxylase activity of the protein was significantly higher than that of the 4-hydroxybenzoate 1-hydroxylase activity of Candida parapsilosis CBS604 strain.

[0012] Based on the above findings, the present inventors have finally succeeded in creating a method for producing hydroquinone involving an enzyme capable of converting p-hydroxybenzoic acid to hydroquinone with high efficiency, thereby solving the problems of the present invention. The present invention has been completed based on the findings and successful examples first obtained by the present inventors.

[0013] Therefore, according to one aspect of the present invention, the following aspects are provided. [1] A method for producing hydroquinone, comprising: p-Hydroxybenzoic acid was added to Spathaspora passaridarum ( Spathaspora of passion fruit The method according to any one of claims 1 to 4, further comprising the step of reacting the resulting hydroquinone with an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from the above-mentioned benzoin. [2] The method according to item [1], wherein the enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum has the amino acid sequence of (1) or (2) below: (1) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 (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 in the amino acid sequence of SEQ ID NO: 2. [3] A composition for producing hydroquinone from p-hydroxybenzoic acid, comprising: The composition contains, as an active ingredient, an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum. [4] A DNA fragment for transforming a non-human host organism, A gene encoding an enzyme with 4-hydroxybenzoate 1-hydroxylase activity from Spathaspora passaridarum, At least one gene different from the gene The DNA fragment comprising: [5] A transformant capable of producing hydroquinone from p-hydroxybenzoic acid, The transformant is obtained by transducing a gene encoding an enzyme having 4-hydroxybenzoate 1-hydroxylase activity derived from Spathaspora passaridarum as a foreign gene. [6] The transformant according to Item [5], wherein the host organism is selected from the group consisting of microorganisms of the genus Corynebacterium, microorganisms of the genus Escherichia, microorganisms of the genus Rhodococcus, microorganisms of the genus Acinetobacter, microorganisms of the genus Bradyrhizobium, microorganisms of the genus Corynebacterium, microorganisms of the genus Pseudomonas, microorganisms of the genus Rhodopseudomonas, microorganisms of the genus Sinorhizobium, microorganisms of the genus Brevibacterium, microorganisms of the genus Novosphingobium, and microorganisms of the genus Ralstonia. [7] The composition, DNA fragment, or transformant described in any one of items [3] to [6], wherein the enzyme having 4-hydroxybenzoate 1-hydroxylase activity derived from Spathaspora passaridarum has the amino acid sequence (1) or (2) below: (1) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 (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 in the amino acid sequence of SEQ ID NO: 2. [Effects of the Invention]

[0014] According to the present invention, hydroquinone can be produced from p-hydroxybenzoic acid with higher efficiency than when 4-hydroxybenzoate 1-hydroxylase contained in Candida parapsilosis strain CBS 604 is used. Furthermore, since the present invention enables the adoption of a biochemically and biologically safe and simple production method, it is expected that hydroquinone can be produced in large quantities on an industrial scale. DETAILED DESCRIPTION OF THE INVENTION

[0015] Each aspect of the present invention will be described in detail below, but the present invention is not limited to the details of these items and can take various forms as long as the object of the present invention is achieved.

[0016] Unless otherwise specified, each term in this specification is used in the meaning commonly used by those skilled in the art of biochemistry, bioengineering, microbiology, etc., and should not be construed as having an unduly limiting meaning. Furthermore, the speculations and theories made in this specification are based on the inventors' findings and experience to date, and therefore the present invention is not limited solely to such speculations and theories.

[0017] "Comprising" means that elements other than those explicitly stated to be included can be added (same meaning as "comprising at least"), but also encompasses "consisting of" and "consisting essentially of." That is, "comprising" can mean including the explicitly stated elements and any one or more elements, consisting of the explicitly stated elements, or consisting essentially of the explicitly stated elements. Elements include limitations such as ingredients, steps, conditions, parameters, etc. "Having" is synonymous with "including." The term "and / or" means any one or any or all combinations of two or more of the associated listed items. The use of "to" in a numerical range includes both the preceding and following numerical values, and also includes ranges excluding either of the included limits. For example, "0% to 100%" means 0% or more, 100% or less, or 0% or more and 100% or less. "About" means an amount within ±10% of the quantity following the term. For example, "about 100" means 100±10%, i.e., 90 to 110. The number of digits in an integer value matches the number of significant digits. For example, 1 has one significant digit, and 10 has two significant digits. Also, the number of digits after the decimal point in a decimal value matches the number of significant digits. For example, 0.1 has one significant digit, and 0.10 has two significant digits.

[0018] The term "foreign gene" refers to a gene that does not naturally occur in the chromosomal (genomic) DNA of the organism into which it is introduced, and is also called a heterologous gene. Note that, in this specification, genome and chromosome are synonyms. "Gene expression" means the production of 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) with its original structure and activity through transcription, translation, etc. "Wild organism" means a naturally occurring organism that has not been artificially genetically modified. "Transformant" means an organism that has been artificially genetically modified. "Wild-type gene" refers to a gene that is naturally present in the genomic DNA of a wild-type organism. "Wild-type protein" refers to a protein encoded by a wild-type gene. A protein that has the activity of catalyzing a specific reaction is called an "enzyme."

[0019] [Summary of the Invention] One aspect of the present invention is a method for producing hydroquinone. In one aspect of the present invention, p-hydroxybenzoic acid is reacted with Spathaspora passaridarum ( Spathaspora of passion fruit The method includes the step of obtaining hydroquinone by treating the enzyme with 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum. In this specification, the enzyme with 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum is sometimes referred to as SP-S1H, and the gene encoding the enzyme with 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum is sometimes referred to as the SP-S1H gene. The Japanese pronunciation of the English word "hydro" is either "hydro" or "hydro."

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

[0021] The SP-S1H used in each aspect of the present invention may be an isolated and purified enzyme, or an enzyme-containing substance such as a living organism, a processed organism, or a culture of a transformant transformed to express SP-S1H from Spasaspora passaridarum. 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. The DNA fragment of one embodiment of the present invention comprises the SP-S1H gene and at least one gene different from the SP-S1H gene.

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

[0024] [An enzyme with 4-hydroxybenzoate 1-hydroxylase activity from Spathaspora passaridarum (SP-S1H)] As shown in Scheme (1) below, 4-hydroxybenzoate 1-hydroxylase has the activity of catalyzing the reaction of converting p-hydroxybenzoic acid to hydroquinone (hereinafter simply referred to as hydroxylase activity). After extensive investigation, we found that when 4-hydroxybenzoic acid was reacted with salicylate hydroxylase derived from Spathaspora passaridarum, a reaction similar to that shown in Scheme (1) occurred. In other words, salicylate hydroxylase derived from Spathaspora passaridarum was found to be an enzyme with 4-hydroxybenzoate 1-hydroxylase activity. [ka]

[0025] As the already known 4-hydroxybenzoic acid 1-hydroxylase, there is Candida parapsilosis ( White parapsilosis ) 4-hydroxybenzoate 1-hydroxylase possessed by the Candida parapsilosis CBS604 strain. SP-S1H has hydroxylase activity, but shares 62% amino acid sequence identity with 4-hydroxybenzoate 1-hydroxylase derived from Candida parapsilosis CBS604. Therefore, SP-S1H may be an enzyme that has hydroxylase activity and an amino acid sequence that shares 60% or more, preferably 65% ​​or more, more preferably 70% or more, and 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 identity with the amino acid sequence of the wild-type SP-S1H enzyme (SEQ ID NO: 2).

[0026] The method for determining the sequence identity of amino acid sequences is not particularly limited, but for example, it can be determined using a commonly known method, such as aligning the amino acid sequences of two proteins and using a program to calculate the sequence identity between the two sequences.

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

[0028] Examples of amino acid sequences that share 60% or more sequence identity with the amino acid sequence of a wild-type enzyme include amino acid sequences in which one or more amino acids have been deleted, substituted, or added in 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 is defined as one unit, the number of amino acids per unit is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 to 30, preferably 1 to 20, and more preferably 1 to 10. Furthermore, "amino acid deletion" refers to the absence or loss of an amino acid residue in a sequence, "amino acid substitution" refers to the replacement of an amino acid residue in a sequence with another amino acid residue, and "amino acid addition" refers to the addition of a new amino acid residue to the sequence.

[0029] Specific examples of "amino acid deletion, substitution, or addition" include replacement of an amino acid with another chemically similar amino acid. Examples include the replacement of a hydrophobic amino acid with another hydrophobic amino acid, or the replacement of a polar amino acid with another polar amino acid having the same charge. Such chemically similar amino acids are known for each amino acid in the art. 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 a His tag, preferably 6xHis, which is a stretch of six histidines.

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

[0032] Methods for modifying recombinant SP-S1H to produce an extracellular enzyme include, but are not limited to, adding a secretion signal such as a peptide or protein to the C-terminus and / or N-terminus of recombinant SP-S1H, which functions to cause the recombinant SP-S1H to be expressed as an extracellular enzyme.

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

[0034] [The gene encoding an enzyme with 4-hydroxybenzoate 1-hydroxylase activity from Spathaspora passaridarum (SP-S1H gene)] The SP-S1H gene has a nucleotide sequence encoding the amino acid sequence of SP-S1H and expresses SP-S1H. The nucleotide sequence in the SP-S1H gene may be either a coding sequence (CDS) or an open reading frame (ORF), but is preferably an ORF.

[0035] The SP-S1H gene may consist of a nucleotide sequence encoding SP-S1H, and may also include transcriptional regulatory sequences such as promoters and terminators, non-coding sequences such as introns, etc. A gene is not simply sequence information, but is a DNA molecule (fragment) made up of a series of nucleotides.

[0036] The SP-S1H gene may contain a nucleotide sequence in which the codons, secondary structure, GC content, etc. have been optimized, taking advantage of the fact that there are several codons corresponding to one amino acid. The codon-modified nucleotide sequence is preferably a nucleotide sequence in which the codons have been modified to facilitate expression in a host organism, for example.

[0037] The wild-type gene of the SP-S1H gene has the nucleotide sequence of SEQ ID NO: 3. In a specific embodiment of the SP-S1H gene, the wild-type gene is Corynebacterium ) A gene having the nucleotide sequence of SEQ ID NO: 4 that has been codon-optimized for a microorganism.

[0038] By introducing the SP-S1H gene into a host organism as a foreign gene, a host organism that is not originally capable of expressing SP-S1H can be transformed so that it expresses SP-S1H as a recombinant enzyme. Expression of the SP-S1H gene may be autonomously mediated by a transcription control sequence contained in the gene or may be mediated by the transcription control mechanism of the host organism, but is preferably autonomously mediated by a transcription control sequence contained in the gene.

[0039] In order to express the SP-S1H gene autonomously in a host organism, the gene preferably contains a promoter, an ORF, and a terminator. The promoter and terminator can be appropriately selected depending on the host organism.

[0040] [DNA fragment] In one embodiment of the present invention, the DNA fragment contains the SP-S1H gene and at least one gene different from the SP-S1H gene. The number of copies of the SP-S1H gene in the DNA fragment may be any number, including one copy or two or more copies.

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

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

[0043] [Transformants] In one embodiment of the present invention, the transformant is obtained by transfection with the SP-S1H gene as a foreign gene. The transformant has the SP-S1H gene introduced as a foreign gene and expresses the introduced gene.

[0044] The transformant is prepared by introducing the SP-S1H gene as a foreign gene into a host organism.

[0045] The host organism is not particularly limited as long as it is an organism into which the SP-S1H gene can be introduced as a foreign gene and into which the introduced SP-S1H gene can be expressed, and may be, for example, a microorganism, an insect cell or insect body, a plant cell or plant body, an animal cell or animal body, etc. However, the host organism is not a human. Examples of host organisms include microorganisms of the genus Corynebacterium, Escherichia, Rhodococcus, Acinetobacter, Bradyrhizobium, Pseudomonas, Rhodopseudomonas, Sinorhizobium, Brevibacterium, Sphingobium, Novosphingobium, Ralstonia, Burkholderia, Alcaligenes, and Arthrobacter. Examples of host organisms include microorganisms of the genus Pseudomonas, Burkholderia, Alcaligenes, Sphingobium, Rhodococcus, and Arthrobacter, which include strains of the genus p-hydroxybenzoic acid decomposing, such as Pseudomonas, Burkholderia, Alcaligenes, Sphingobium, Rhodococcus, and Arthrobacter. However, preferred are microorganisms of the genus Corynebacterium, which have a proven track record as host organisms for genetic recombination and are incapable of assimilating the product hydroquinone. Hereinafter, the present invention will be specifically described assuming that the host organism is a microorganism.

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

[0047] The vectors, media, procedures, etc. used to prepare transformed microorganisms can be found in the Examples section below. For example, methods for transforming a host microorganism by introducing a DNA fragment include electroporation, the protoplast method, and methods using calcium ions, with electroporation being preferred.

[0048] The method for introducing the SP-S1H gene into a host microorganism is not particularly limited, and examples include a method in which a DNA construct such as a plasmid vector incorporating the SP-S1H gene is introduced into the host microorganism so that it replicates autonomously and expresses the gene, and a method in which the SP-S1H gene is incorporated into the genomic DNA of the host microorganism by using homologous recombination, etc. However, since there is a high probability that the gene will be distributed during cell division, the method in which the SP-S1H gene is incorporated into the genomic DNA of the host microorganism is preferred.

[0049] Other molecular biological, biotechnology, and biochemical methods used to prepare transformed microorganisms can be found in references 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 has been introduced as a foreign gene, and the introduced gene is expressed. SP-S1H can be obtained by using the transformed microorganism (1), and the cells, treated cells, and cell cultures of the transformed microorganism (1) can be used as SP-S1H-containing materials. A specific example of the transformed microorganism (1) is the SP-S1H-expressing transformed Coryne microorganism described in the Examples below.

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

[0052] The method for reacting p-hydroxybenzoic acid with SP-S1H may be any method that allows contact between p-hydroxybenzoic acid and Sp-S1H and allows hydroquinone to be produced and / or accumulated by SP-S1H, and examples include a method in which p-hydroxybenzoic acid is reacted with SP-S1H under conditions of a pH of 5 to 9, a temperature of 20°C to 45°C, a time of several minutes to several tens of hours, and standing, stirring, or shaking.

[0053] When a transformed microorganism is used as an SP-S1H-containing substance, hydroquinone can be produced by culturing the transformed microorganism under various culture conditions suitable for the transformed microorganism, for example, in a medium containing p-hydroxybenzoic acid and suitable for the growth of the transformed microorganism. Any culture method suitable for the host microorganism may be used. For example, when the host microorganism is a Corynebacterium genus microorganism, solid culture or liquid culture performed under aerobic conditions may be used. Alternatively, the recovered transformed microorganism 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 the culture is not particularly limited. For example, the reaction solution can be subjected to a conventional solid-liquid separation process such as filtration or centrifugation to separate the solid from the reaction solution, and hydroquinone can be extracted from the recovered reaction solution by solid-phase extraction using a column or solvent extraction using a solvent that dissolves hydroquinone. The extraction solvent is not particularly limited as long as it dissolves hydroquinone, and examples include ethyl acetate and diethyl ether. Hydroquinone may also be obtained as a fraction using a chromatograph such as a liquid chromatograph.

[0055] Qualitative or quantitative analysis of hydroquinone may be carried out by HPLC as described in the Examples below.

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

[0057] [Uses of hydroquinone] Hydroquinone is generally used as a cosmetic ingredient for its cosmetic improvement effects on the skin, such as the improvement of pigmentation caused by age spots, freckles, and sunburn, and the promotion of skin whitening. Hydroquinone obtained by the hydroquinone production method can also be used as a cosmetic ingredient as a whitening ingredient. 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 explained in more detail below with reference to examples. However, the present invention is not limited to these examples, and the present invention can take various forms as long as the object of the present invention can be achieved. [Example]

[0059] [1. Isolation of hydroquinone-producing enzyme] Hydroquinone-producing enzymes have the enzyme activity to catalyze the reaction of converting p-hydroxybenzoic acid (pHBA) to hydroquinone (HQ), and some are known as 4-hydroxybenzoate 1-hydroxylases or 4-hydroxybenzoate 1-monooxygenases. The Enzyme Commission number for 4-hydroxybenzoate 1-hydroxylase is EC1.14.13.64.

[0060] The present inventors searched for a novel hydroquinone-producing enzyme and identified the SP-S1H protein having the amino acid sequence of SEQ ID NO: 2 as a candidate enzyme. The gene encoding SP-S1H was cloned from Spathaspora passaridarum ( Spathaspora of passion fruit ) NRRL Y-27907 strain genome and consisted of the nucleotide sequence of SEQ ID NO: 3. The GenBank accession number version of SP-S1H is XP_007376166.1.

[0061] 2. Construction of DNA construct for SP-S1H expression The SP-S1H protein having the amino acid sequence of SEQ ID NO: 2 was isolated from a Corynebacterium spp. Corynebacterium glutamic ) codons to obtain the nucleotide sequence of SEQ ID NO: 4. The pMKsf vector ( Corynebacterium glutamic A DNA construct for expressing SP-S1H was constructed by inserting the nucleotide sequence of SEQ ID NO: 4 into the multicloning site of the ATCC31808-derived shuttle vector pCG1 (modified from US4617267A).

[0062] [3. Construction of SP-S1H-expressing transformed microorganisms] Corynebacterium spp. (Coryne) ( 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 Corynebacterium sp. microorganism lacking the cgl, pobA, pcaHG, genH, and pcaben genes.

[0063] CT10 was subjected to a transduction treatment using a DNA construct for SP-S1H expression, including electroporation and heat shock. After the transduction treatment, CT10 was cultured on LB agar medium containing kanamycin at 32°C for 20 to 24 hours to select transformed Corynebacterium sp. expressing SP-S1H.

[0064] On the other hand, Candida parapsilosis ( White parapsilosis The amino acid sequence of 4-hydroxybenzoate 1-hydroxylase from the CBS604 strain (SEQ ID NO: 1) was obtained from GenBank under the accession number version XP_036663424.1. This 4-hydroxybenzoate 1-hydroxylase was designated CP-4HB1H. After codon optimization, DNA construct construction, and transformation, a Corynebacterium sp. microorganism expressing CP-4HB1H was selected as described above. The amino acid sequence identity between SP-S1H and CP-4HB1H was 62%.

[0065] [4. Evaluation of SP-S1H activity] The resulting SP-S1H-expressing Corynebacterium spp. and CP-4HB1H-expressing Corynebacterium spp. were each inoculated into 2 ml of a growth medium prepared by mixing CGXIIa medium and LB medium at a 1:1 ratio. 25 μg / mL of kanamycin was then added to the inoculated growth medium, which was then cultured overnight at 32°C with shaking at 250 rpm.

[0066] CGXIIa medium (pH 6.6-6.8) contains 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 iron 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 resulting culture medium was centrifuged (13,000 rpm, 2 minutes) and the supernatant was removed to recover the bacterial cells. The resulting wet bacterial cells were added to CGXIIa medium containing 10 mM pHBA, thoroughly suspended, and subjected to an HQ conversion reaction at 37°C overnight.

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

[0070] HPLC analysis was performed using a degasser (DGU-20A), a liquid chromatograph (LC-20AR), an autosampler (SIL-20AC), a column oven (CTO-20AC), and a detector (SPD-M20A) (all manufactured by Shimadzu Corporation). The column used was an Inertsil ODS-3 5 μm column (4.6 mm diameter, 250 mm length, 5 μm particle size, manufactured by GL Science) and was maintained at 30 °C. A gradient elution mode (solvent A: 0.1% (v / v) formic acid, solvent B: 100% (v / v) acetonitrile) was used. After equilibration with solvent A, the solvent B ratio was held at 30% for 2 min, then increased to 50% over 2 min, and then decreased to 30% over 1 min. The flow rate of the mobile phase was 1.0 mL / min, and 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 the transformed Corynebacterium sp. microorganism expressing CP-4HB1H was 0.42 g / L, while the HQ concentration in the reaction solution obtained using the transformed Corynebacterium sp. microorganism expressing SP-S1H was 0.90 g / L.

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

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

[0074] [Table 1] [Industrial Applicability]

[0075] By utilizing the transformed microorganism and production method according to one embodiment of the present invention, it is possible to produce physiologically active and valuable hydroquinone from p-hydroxybenzoic acid on an industrial scale through a biochemically and biologically safe and simple production method.

Claims

1. A method for producing hydroquinone, comprising: The method includes a step of obtaining hydroquinone by treating p-hydroxybenzoic acid with an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passalidarum; and The method as described above, wherein the enzyme having 4-hydroxybenzoate 1-hydroxylase activity derived from Spathaspora passaridarum has the following amino acid sequence (1) or (2): (1) an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 (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 in the amino acid sequence of SEQ ID NO:

2.

2. A composition for producing hydroquinone from p-hydroxybenzoic acid, comprising: The present invention comprises an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum as an active ingredient, and The composition as described above, wherein the enzyme having 4-hydroxybenzoate 1-hydroxylase activity derived from Spathaspora passaridarum has the following amino acid sequence (1) or (2): (1) an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 (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 in the amino acid sequence of SEQ ID NO:

2.

3. A transformant capable of producing hydroquinone from p-hydroxybenzoic acid, The gene encoding an enzyme having 4-hydroxybenzoic acid 1-hydroxylase activity derived from Spathaspora passaridarum has been transduced as a foreign gene, and The transformant as described above, wherein the enzyme having 4-hydroxybenzoate 1-hydroxylase activity derived from Spathaspora passaridarum has the following amino acid sequence (1) or (2): (1) an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 (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 in the amino acid sequence of SEQ ID NO:

2.

4. The transformant according to claim 3, wherein the host organism is selected from the group consisting of microorganisms of the genus Corynebacterium, Escherichia, Rhodococcus, Acinetobacter, Bradyrhizobium, Corynebacterium, Pseudomonas, Rhodopseudomonas, Sinorhizobium, Brevibacterium, Novosphingobium, and Ralstonia.

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