JPH07236493A - Conversion of desthiobiotin to biotin - Google Patents

Abstract

PURPOSE:To obtain biotin which is a vitamin participating in the enzymatic reaction for fat metabolism, etc., as a coenzyme by fermentation method in high efficiency by adding flavotoxin to an enzymatic conversion reaction liquid containing desthloblotin and carrying out the reaction of the components. CONSTITUTION:Biotin which is a vitamin participating in the enzymatic reaction for fat metabolism, etc., as a coenzyme is produced in high efficiency by culturing E.coli W3110 (ATCC1498) in a medium, collecting and washing the cells in logarithmic growth stage, extracting the DNA by phenol method, amplifying the flavotoxin gene by PCR method using two synthetic primers positioned at both sides of a translation zone of the flavotoxin gene, linking the gene to a vector to form a recombinant DNA, inserting the DNA into E.coli to obtain a recombinant E.coli, culturing the strain to obtain flavotoxin, adding the flavotoxin to an enzymatic conversion reaction system composed of an enzymatic conversion reaction liquid prepared from E.coli having bioB gene in a biotin operon and subjecting the desthiobiotin to enzymatic conversion reaction.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing biotin.

[0002]

Biotin is an essential vitamin for plants and animals involved in enzymatic reactions such as fat metabolism as a coenzyme of biotin enzyme. Currently, biotin is produced by a chemical synthesis method, but a complicated process is required. Therefore, a biotin production method by fermentation using various biotin-producing bacteria has been developed to replace it.

The microorganisms used in these production methods include α-dehydrobiotin resistant strains (for example, those belonging to the genus Escherichia) (for example,
Japanese Patent Application Laid-Open No. 61-149091), a strain in which feedback inhibition by biotin is released (see, for example, Japanese Patent Application Laid-Open No. 61-202686, see), and a strain in which acetic acid-producing ability is reduced (for example, European Patent Publication No. 1). 031622
No. 9, specification), and a biotin-retaining deficient strain (see, for example, Japanese Patent Publication No. 64-50081).

[0004] In addition to the genus Escherichia, the genus Bacillus (see, for example, JP-A-62-275784), Serratia (S
Microorganisms belonging to the genus erratia (see, for example, JP-A-2-27980) are known, and these patent publications disclose a method for producing biotin using a genetically improved microorganism.

[0005]

Generally, in the method for producing biotin by the fermentation method using a microorganism as described above, since the nutrient source is consumed for the production of bacterial cells, the yield of biotin with respect to sugar is low, and However, it cannot be said that satisfactory productivity is necessarily achieved because the target biotin and the precursor immediately before it, desthiobiotin, tend to accumulate and remain in large amounts. Further, in collecting biotin, it is necessary to collect biotin from a fermentation solution containing various contaminants, which causes a problem of complicated collection and purification steps.

Most of the problems of the biotin production method by these fermentation methods are the biotin production method using a more defined (defined) reaction system as compared with the culture system, that is, a resting microbial cell reaction system or an enzyme reaction system. It can be solved by developing. Regarding the conversion reaction from desthiobiotin to biotin, which is the most important in biotin biosynthesis, for example, a conversion reaction using resting cells of Escherichia coli (Eisenberg, MA et al., Antimicrobial Agents and Che
Motherapy 21, 5, (1982)), a conversion reaction using a cell-free extract of Escherichia coli (for example, JP-A-3-91453).
No. 5, gazette, etc.), but more efficiently,
It is desirable to establish a reaction system that is a simple system consisting only of enzymes, coenzymes, cofactors, etc. involved in this conversion reaction.

It has been conventionally said that this conversion reaction is catalyzed by an enzyme protein encoded by the genotype bioB. However, a recent study of this conversion reaction using a cell-free extract of Escherichia coli and a crude enzyme extract (Biosci. Biotech. Bio
According to chem. (1992) 56, 1780-1785), fructose 1,6 bisphosphate, Fe ²⁺ , NAD are used as reaction promoters.
It has been clarified that PH, S-adenosylmethionine, KCl and the like can act, and it has been shown that proteins other than bioB protein are involved in this conversion reaction, but the conversion reaction mechanism itself has not yet been elucidated. There are many problems to be solved, such as the lack of improvement, and there is much room for improvement in carrying out the conversion of desthiobiotin to biotin with high conversion efficiency using an enzyme reaction system.

[0008]

[Means for Solving the Problems] The inventors of the present invention are required for an enzyme conversion reaction from a cell-free extract prepared from biotin-producing cells in order to establish a more efficient method for converting desthiobiotin to biotin. Various purifications of different enzyme proteins were studied.

As a result, it was found that the conversion activity of desthiobiotin to biotin disappeared by treating the cell-free extract with polyethyleneimine under certain conditions, and further research on recovery of the activity was found. However, it has been known so far that it is only involved in the function as an electron conductor in various metabolic processes and the activation of pyruvate formate lyase (and cobalamin-dependent methionine synthase) in Escherichia coli. The present inventors have completed the present invention by discovering the fact that flavodoxin that does not exist significantly affects this conversion reaction.

Therefore, the present invention provides a method for converting desthiobiotin to biotin, which comprises adding flavodoxin to an enzyme reaction conversion solution when converting desthiobiotin into biotin in an enzyme conversion reaction system. The present invention is also a method for converting desthiobiotin into biotin in a culture system or a resting strain system, wherein the cells of the culture system or the resting strain system has a plasmid carrying a flavodoxin gene. A method for converting desthiobiotin to biotin is provided. The present invention also provides a method for producing biotin, which comprises culturing a microorganism having a biotin operon and a flavodoxin gene carried on a plasmid.

[0011]

[Specific Embodiment] The protein added to the enzyme conversion reaction system of desthiobiotin to biotin of the present invention is flavodoxin, and the enzyme conversion reaction system used is bioB gene in the biotin operon (desthiobiotin is converted to biotin. There is an enzyme reaction conversion solution prepared from Escherichia coli cells having a gene encoding an enzyme to be converted.

The enzyme conversion reaction solution referred to in the present invention is a concept including a cell-free extract itself obtained by crushing bacterial cells, and an extract solution purified to various degrees by a commonly used method for separating and purifying enzyme proteins. is there.

Escherichia coli used for the preparation of the cell-free extract is one in which the bioB gene in the biotin operon is contained in the chromosome, the recombinant plasmid, or both, and particularly, the one in which the expression of the bioB gene is enhanced. preferable. For example, DRK3323 [pXBA312] produced by the present inventors as a high biotin-producing strain.
Examples of the strain include a strain (Mikori Kenjo No. 2117) or a recombinant bacterium having an inducible plasmid in which the bioB gene is ligated downstream of an inducible promoter and expression is performed by adding an inducing agent after cell growth.

Such a bioB-inducible plasmid is a commercially available plasmid vector having an inducible promoter (for example, pKK223-3 as a plasmid vector having a taq-type inducible promoter, Pharmacia, etc.) and biotin. A plasmid having an operon, such as the above-mentioned DRK3323 [pX
It can be easily created from pXBA312, which is obtained from the strain BA312] using a plasmid extraction method known per se.

Introduction of the obtained plasmid into the host Escherichia coli is carried out by a conventional calcium treatment method (Mandel, M. et al., Journal of
Molecular Biology 53, 109, (1970), see), and a recombinant bacterium having the plasmid can be selectively obtained by using the drug resistance of the plasmid vector as an index. Thus, Escherichia coli which can be conveniently used in the present invention can be obtained.

[0016] Inducible expression can be performed more effectively by appropriately selecting Escherichia coli used as a host bacterium depending on the type of inducible promoter used. For example, ta
When a q-type promoter is used, for example, E. coli K-12 strain JM105 (Yanisch-Perron, C.
Et al., Gene 33, 103, (1985)), the inducible expression can be performed more effectively. Examples of the recombinant Escherichia coli having the plasmid carrying the bioB gene, the expression of which is regulated by the inducible promoter thus obtained, include JM105 [pTTB15] described in JP-A-3-914535.

The E. coli cells used for preparing the cell-free extract can be obtained by a known culture method for E. coli. As a medium used for culturing, any of a carbon source, a nitrogen source, a synthetic medium containing an inorganic substance, and a natural medium can be used. As a carbon source, glucose,
Carbohydrates such as glycerin, fructose, sucrose, maltose, starch, hydrolyzed starch and molasses can be used.

As the nitrogen source, various inorganic ammonium salts such as ammonia, ammonium chloride, ammonium phosphate and ammonium sulfate, or amino acids, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, defatted soybean meal or its digested product Natural organic nitrogen sources such as can be used. Many of the natural organic nitrogen sources are
It can be a nitrogen source as well as a carbon source. Further, as inorganic substances, potassium dihydrogenphosphate, potassium dihydrogenphosphate, magnesium sulfate, sodium chloride, ferrous sulfate, calcium chloride, zinc chloride, copper sulfate, manganese chloride, cobalt chloride, ammonium molybdate, boric acid, etc. Can be used.

Further, when the produced microorganism is provided with antibacterial drug resistance, contamination of contaminating bacteria can be prevented by adding the corresponding antibacterial agent to the medium. Using a recombinant Escherichia coli having a plasmid carrying a bioB gene whose expression is regulated by an inducible promoter,
In particular, when a taq promoter is used as the inducible promoter, an inducer such as IPTG (isopropyl-β-D-) is used when the bacterial cells reach the logarithmic growth phase after culturing.
Thiogalactopyranoside) is added to induce the expression of the bioB gene, and the cells obtained by continuing the culture for several hours are preferably used for the preparation of the cell-free extract.

For the preparation of the cell-free extract from the cells, a generally used cell disruption method can be applied. That is, ultrasonic crushing, crushing with alumina or glass beads, crushing using a French press or an X press device, and the like can be applied. For example, after collecting the cultured cells with a centrifuge, the cells are washed with physiological saline or the like and resuspended in an appropriate neutral buffer, for example, Tris-HCl buffer, as a stabilizer for the enzyme. For example, the bacterial cell liquid to which dithiothreitol (DTT) or phenylmethylsulfonylfluoride (PMSF) has been added may be treated by the above-mentioned bacterial cell disruption method. After crushing the microbial cells, the microbial cell residue can be removed by a centrifuge and the supernatant can be used as a cell-free extract.

The enzyme conversion reaction system for converting desthiobiotin to biotin in the present invention is the cell-free extract prepared above, or a method for separating and purifying a protein generally used from the cell-free extract, for example, Ammonium sulfate precipitation method,
What was treated by chromatography such as gel filtration or the cell-free extract was treated with polyethyleneimine to form an enzyme conversion reaction solution. As the buffer, a buffer that does not adversely affect the conversion method of the present invention, such as Tris-HCl buffer, is used. The pH of the buffer solution is usually 6.8 to 7.
It is preferably set to 8.

The flavodoxin added to the enzyme conversion reaction system of the present invention may have any amino acid sequence as long as it is a protein having the effect of flavodoxin, but Escherichia coli flavodoxin is preferred. The Escherichia coli flavodoxin can be prepared by, for example, increasing the flavodoxin activity by subjecting it to a method for separating and purifying a protein generally used from a cell-free extract of Escherichia coli having the present protein activity, for example, ammonium sulfate precipitation or chromatography such as gel filtration. The crude product may be used as a crude extract, or as a highly purified protein obtained by a protein purification method using this biotin conversion reaction as an index.

Further, in order to obtain a larger amount of purified flavodoxin, a known flavodoxin gene sequence (J. Bac
teriol. (1991) 173, 1729-1737) and PCR (Po
The flavodoxin gene obtained by the lymerase chain reaction method or the like is ligated to a plasmid and transformed from a cell-free extract of Escherichia coli or, for example, a plasmid vector having a commercially available inducible promoter according to a conventional method (for example, taq). It is efficient to prepare from a cell-free extract of Escherichia coli K-12 strain JM105 strain that has been transformed by ligating to pKK223-3, Pharmacia, etc. as a plasmid vector having a system-inducible promoter. .

The enzyme conversion reaction of the present invention is carried out by adding flavodoxin to the above-mentioned enzyme conversion reaction solution and further adding desthiobiotin, which is a substrate for this conversion reaction. The amount of desthiobiotin added is not limited because the optimum amount varies depending on the constructed enzyme conversion reaction system, but it is preferably used in an amount such that the total amount is dissolved in the system. Desthiobiotin may be D-form or DL-form. The coexistence of L-form does not inhibit the enzyme reaction. The conversion reaction is carried out at a temperature of 30 ° C. to 41 ° C. for 30 minutes to 3 hours, but the reaction may be continued for a longer time as long as the enzyme activity does not disappear.

Fructose 1, is used in the enzyme conversion reaction system.
By adding about 1 mM to 100 mM of 6-bisphosphoric acid, the conversion reaction can be performed more efficiently, and further, as other trace components, Fe ²⁺ and / or KC.
l and / or NAD, NADH, NADP, NA
The conversion efficiency can be further increased by adding at least one of DPH and / or at least one of S-adenosylmethionine and L-methionine to the conversion reaction system.

The optimum addition amount of these components varies depending on the constructed enzyme conversion reaction system, but in general, Fe ²⁺ is 1 mM.
-10 mM, 1 mM or more for KCl, NAD, NAD
For H, NADP, NADPH, 100 μM or more,
L-methionine and S-adenosylmethionine are preferably added so as to have a concentration of 50 μM to 500 μM.

In addition to the above enzyme conversion reaction system, a method of directly utilizing the flavodoxin gene is also effective. For example, a culture system in which the flavodoxin gene and the bioB gene obtained by the above-mentioned method are ligated on, for example, the same plasmid or separate plasmids, transformed E. coli is cultured, and desthiobiotin is converted to biotin in the meantime. After culturing the body, the cells are harvested and suspended in an appropriate buffer to convert desthiobiotin to biotin, and a resting bacterium system is prepared, and a cell-free extract is prepared from the obtained bacterium, and flavodoxin-enhanced enzyme conversion is performed. It is also possible to directly use as a reaction solution for conversion of desthiobiotin to biotin. Similarly, the flavodoxin gene and the biotin operon (bioA, B, F, C, D) are ligated onto, for example, the same or different plasmids, and transformed E. coli is cultured to add desthiobiotin. It is also possible to efficiently carry out de novo synthesis of biotin. The biotin operon used is more preferably a high expression system provided by the present inventors (see, for example, Japanese Patent Application No. 3-235148). Alternatively, a plasmid carrying the prabotoxin gene can be introduced into a microorganism that naturally has the biotin operon, and biotin can be produced using this microorganism.

The biotin converted from desthiobiotin in this way can be recovered from the reaction solution, for example, by
This can be easily achieved by denaturing and precipitating the protein component by heat treatment or the like, removing this by centrifugation, and extracting and purifying biotin from the supernatant by a conventional method. Purified biotin can be obtained by subjecting the supernatant to a treatment with activated carbon and an ion exchange resin, followed by recrystallization from water or alcohol. Compared with, for example, the case where biotin is extracted and purified from the fermentation broth, these steps are significantly easier to accomplish because the types and amounts of the contaminants are smaller.

[0029]

The present invention will be explained more specifically by the following examples, but it is not intended to limit the scope of the present invention to these examples. Example 1 Preparation medium of bacterial cells- A disodium phosphate (12 hydrate) 17.6 g / L monopotassium phosphate 2.4 g / L ammonium sulfate 4.0 g / L yeast extract 10.0 g / L peptone 10.0 g / L Ferrous sulfate (heptahydrate) 0.1 g / L Calcium chloride (dihydrate) 0.05 g /
L manganese chloride (tetrahydrate) 0.05 g /
L Magnesium sulfate (heptahydrate) 0.1 g / L Glucose 5.0 g / L

A recombinant Escherichia coli (JM105 [pTTB15] described in JP-A-3-914535) having a plasmid carrying a bioB gene whose expression is regulated by a high expression inducible promoter was added to a 500 mL plate mouth flask (Shibata). 20 mL of Medium-A in Hario Co., Ltd. was inoculated and shake-cultured at 37 ° C. and 125 strokes / min for 12 to 14 hours to give a preculture. 20 mL of this preculture
Was inoculated into 2 L of Medium-A in a 5 L capacity mini jar fermenter (Maruhishi Bioengineering Co., Ltd.) to start main culture.

The culture conditions were a temperature of 37 ° C., an aeration rate of 2 vvm and a stirring speed of 500 rpm. After 1 to 2 hours from the start of the main culture, IPTG 2 mM was added to induce the taq promoter when the cells grew to a cell concentration (OD ₆₆₀ ) of about 2.0. Cultivation was terminated when the bacterial cell concentration (OD ₆₆₀ ) reached about 12.0 3 to 4 hours after the addition of IPTG, and the obtained culture solution was used as a material for preparing a cell-free extract.

Example 2 Preparation of cell-free extract All the following operations were performed in ice water or at 4 ° C. 2 L of the culture broth prepared in Example 1 was centrifuged at 6000 rpm for 10 minutes to collect the cells, and then the same volume of 0.75% NaCl solution as that of the medium was added to 2 L.
Washed twice, and 200 mL (1/10 volume of culture solution) of 0.1
M Tris-HCl buffer (pH 7.5, 2 mM DTT, 0.2 mM
Resuspended in PMSF).

120 mL of this cell suspension was transferred to a beaker with a capacity of 300 mL, and an ultrasonic disruption device (BRANSON, SONIFER model 450 was used, taking care not to raise the temperature of the suspension above 10 ° C. in an ice bath. ) Was used to ultrasonically disrupt the cells. The obtained cell lysate was transferred to a centrifuge tube and centrifuged at 14000 rpm and 4 ° C for 30 minutes,
The supernatant was used as a cell-free extract.

When the cell lysate had a high viscosity and the cell residue could not be completely removed by one centrifugation, the centrifugation operation was repeated for the supernatant. The protein concentration of the cell-free extract is determined by a protein quantification kit (Pierce, product code 23225).
Was calculated using albumin (BSA). The protein concentration of the prepared cell-free extract is about 15 to 25
It was constant at mg / mL.

Example 3 Polyethyleneimine Treatment The above-mentioned cell-free extract was treated with polyethyleneimine by a conventional method for the purpose of removing nucleic acids. PH at 4 ° C 7.
Tris-HCl buffer adjusted to 5 (0.1
5% polyethyleneimine solution (0.1M Tris-HCl) was added to the cell-free extract at 6% volume at 4 ° C, and the mixture was cooled on ice for 10 minutes and centrifuged at 12,000 rpm to remove the supernatant. The biotin conversion activity was determined.

The reaction solution composition was 5 mM FDP, 5 mM Fe.
Cl ₂ , 1 mM NADPH, 100 μM S-adenosylmethionine, 10 mM KCl, and 50 μM dethiobiotin (DL-DTB) as a substrate were added, and the mixture was added at 35 ° C. for 1 ° C.
The reaction was carried out for 20 minutes. After completion of the reaction, quickly cool with ice,
After heating at 100 ° C for 5 minutes, the mixture was centrifuged, and the amount of biotin in the supernatant was adjusted to Lactobacillus plantarum.
It was measured by a bioassay method using us plantarum ) (ATCC 8014). The results are shown in Table 1.

[0037]

[Table 1]

As shown in Table 1, the pH was 7.5 at 4 ° C.
It was found that the biotin-converting activity disappeared when the treatment with polyethyleneimine was performed in a 0.1 M Tris-HCl buffer adjusted to 1. Further, after treatment with polyethyleneimine (pH 7.5, Tris-HCl buffer 4 ° C.), JM105 / pT was separately added to the cell-free extract (enzyme conversion reaction solution).
0-50% ammonium sulfate precipitation fraction prepared from TB51 strain, 50
The 90% ammonium sulfate precipitation fraction was added at a ratio of 1: 1, and the biotin conversion activity was determined. The results are shown in Table 2.

[0039]

[Table 2]

As shown in Table 2, after the treatment with polyethyleneimine in which the biotin-converting activity disappeared under the above conditions (enzyme conversion reaction solution), separately from the cell-free extract, Biosci. Biotech. Bio
It was revealed that the activity was recovered by adding the 50-90% ammonium sulfate precipitation fraction prepared by the method described in chem. (1992) 56, 1780-1785. bioB protein is 20 ~
Since it is contained in the 50% ammonium sulfate precipitation fraction (Biosci. Biot
ech. Biochem. (1992) 56, 1780-1785), 4 ° C, p.
It was found that the treatment with polyethyleneimine under the condition of H7.5 has a function of precipitating an unidentified protein contained in the 50-90% ammonium sulfate precipitation fraction, which is essential for the biotin conversion reaction, together with the nucleic acid. ′

Example 4 Unidentified protein involved in biotin conversion reaction
Purification of White Matter (1) Ammonium Sulfate Precipitation Fraction of Cell-Free Extract In purifying unidentified proteins involved in biotin conversion reaction, ammonium sulfate precipitation fractionation was attempted for the purpose of crude fractionation of cell-free extract.

The unidentified protein involved in the biotin conversion reaction was the ammonium sulfate precipitation fraction 5 prepared from the Escherichia coli cell-free extract.
It has been found to exist in 0 to 90% of each (Biosci. Biotech. Biochem. (1992) 56, 178).
0-1785), further detailed ammonium sulfate precipitation fractionation was performed based on this finding. 0-25%, 25-35%, 35-35 from 10 mL of cell-free extract of E. coli JM105 / pTTB51 strain
The protein fractions precipitated with 55%, 55% to 80%, and 80% to 90% ammonium sulfate saturated solution were collected, and 0.1 M Tris-
It was dissolved in HCl (pH 7.5) (1 mL each) and desalted using a PD-10 column (purchased from Pharmacia).

The biotin conversion activity of each ammonium sulfate precipitate fraction as an unidentified protein activity was measured by measuring 25-35% ammonium sulfate precipitate prepared from a cell-free extract of Escherichia coli JM105 / pTTB51 strain for each ammonium sulfate precipitate fraction. The fractions (the fractions containing the bioB protein) were mixed at a ratio of 1: 1 and further added with 5 mM FDP, 5 mM FeCl ₂ , 1 mM NADPH, 100.
μM S-adenosylmethionine, 10 mM KCl, and 50 μM dethiobiotin (DL-DTB) as a substrate were added, and the reaction was carried out at 35 ° C. for 30 minutes.

After completion of the reaction, the mixture was quickly cooled with ice and heated to 100 ° C for 5
After heating for 1 minute, centrifuge and adjust the amount of biotin in the supernatant to Lacto.
bacillus plantarum (ATCC80
It measured by the bioassay method using 14). The results are shown in Table 3.

[0045]

[Table 3]

From the results shown in Table 3, it was revealed that unidentified proteins involved in the biotin conversion reaction were contained in 55-80% ammonium sulfate precipitation fraction (95% of the total recovered activity).
From this, the subsequent purification of the unidentified protein was carried out in Escherichia coli 55-55.
80% ammonium sulfate precipitation fraction was used.

(2) DEAE Ion Exchange Chromatography A 55-80% ammonium sulfate precipitate fraction prepared from a cell-free extract of Escherichia coli JM105 / pTTB51 strain was purified by ion exchange chromatography. Anion exchanger DEA
E-Cellulofine column (26 mm x 280
mm), 20 mM Tris-HCl (pH 7.5),
The protein was eluted with a linear gradient of 0 to 0.8 M KCl at a flow rate of 30 ml / hour. PD-10 column (manufactured by Pharmacia) was used for buffer replacement before sample application.

The results of the DEAE ion exchange chromatography are shown in FIG. 1 (operating conditions, carrier: DEAE Cel
lulofine, column size: 26 mm x 400 mm,
Equilibration buffer: 0.02M Tris-Cl (pH 7.
5), elution: 0 → 0.8 M KCl gradient in the buffer, flow rate: 30 mL / hr). To measure the biotin conversion activity of each fraction, an enzyme conversion reaction solution (polyethyleneimine-treated solution) was added at a ratio of 1: 1, and further 5 mM FDP, 5 mM FeCl ₂ , 1 mM NADPH,
100 μM S-adenosylmethionine, 10 mM KC
l, 50 μM dethiobiotin (DL-DT as substrate)
B) was added and the reaction was carried out at 35 ° C. for 30 minutes.

After the reaction was completed, the reaction mixture was quickly cooled with ice and heated to 100 ° C for 5
After heating for 1 minute, centrifuge and adjust the amount of biotin in the supernatant to Lacto.
bacillus plantarum (ATCC80
It measured by the bioassay method using 14). As is clear from FIG. 1, strong biotin-converting activity was detected in the fraction eluted with 0.4 KCl. This fraction (40m
l) is the ultrafiltration (Collodion Thimble UF Concentration
It was concentrated to 1 ml using System MWCO 10,000; manufactured by Spectrum Co.

(3) Gel filtration The concentrated sample was further purified by gel filtration. Gel filtration was performed using a Sephacryl S200 column (16
mm × 600 mm) was used. 20 containing 0.2 M KCl
After equilibration with mM Tris-HCl (pH 7.5), the same buffer was used to flow out the protein at a flow rate of 1 ml / min.

Measurement of biotin conversion activity of each fraction
Is the enzyme conversion reaction solution (polyethyleneimine treatment solution)
Add 1: 1 ratio and add 5mM FDP, 5mM FDP
eCl ₂1 mM NADPH, 100 μM S-adeno
Sylmethionine, 10 mM KCl, 50 μM as substrate
Dethiobiotin (DL-DTB) of
Reaction was carried out for 30 minutes. After completion of the reaction, quickly cool with ice and
After heating at 00 ° C for 5 minutes, centrifuge and remove the amount of biotinL
actobacillus plantarum(AT
CC8014) for bioassay
It was Collect active peak fractions and use SMART System
(Manufactured by Pharmacia), Fast Desalti
Desalted using a ng PC 3.2 / 10 column.

(4) Micropurification by SMART System The protein purified by gel filtration was further purified by SMART.
Micropurification was performed using System (manufactured by Pharmacia). Anion Exchanger MonoQ PC1.6 / 5
Using a column, 20 mM Tris-HCl (pH 7.
5), the protein was eluted with a linear gradient of 0 to 0.8 MKCl.

Measurement of biotin conversion activity of each fraction
Is the enzyme conversion reaction solution (polyethyleneimine treatment solution)
Add 1: 1 ratio and add 5mM FDP, 5mM FDP
eCl ₂1 mM NADPH, 100 μM S-adeno
Sylmethionine, 10 mM KCl, 50 μM as substrate
Dethiobiotin (DL-DTB) of
Reaction was carried out for 30 minutes. After completion of the reaction, quickly cool with ice,
After heating at 100 ° C for 5 minutes, centrifuge and the amount of biotin in the supernatant
ToLactobacillus plantarum
Measured by bioassay method using (ATCC8014)
Decided SDS-PAGE binding of active fractions
The results are shown in FIG. It was purified to almost a single band.

Example 5 N-terminal amino acid structural analysis The primary structural analysis of the N-terminal amino acid of an unidentified protein involved in the purified biotin conversion reaction was performed by Edman degradation, which is an automatic analyzer Protein Sequencer 471A (App
Lide Biosystems) was used. About 30 picomoles of protein was used. The sequence results are shown below based on the three letter symbols of amino acids. In addition, 18 amino acids (only one amino acid is unknown) were determined from the N-terminal.

[0055]

[Chemical 1]

Example 6 Software, GE based on the amino acid sequence determined by homology search
NETYX-CD (manufactured by SDC) is used, and SWISS-PROT Rel. 24 (Euro
Homology search was performed using pean Molecular Biology Laboratory). The results are shown below based on the one-letter symbols of amino acids.

[0057]

[Chemical 2] It was revealed that the unidentified protein involved in the purified biotin conversion reaction was Escherichia coli flavodoxin itself.

Example 7 Cloning of Escherichia coli flavodoxin gene
Ningu (1) Preparation of chromosomal DNA Escherichia coli W3110 (ATCC1498) the strain L- medium (peptone 10 g / l, yeast extract 5 g / l, glucose 1 g / l, chloride Natorumu 5 g / l,) in 37 ° C. 3 hours with shaking at After culturing, the bacterial cells in the logarithmic growth phase are collected and washed, and then the phenol method [Biochim. Biophys. Acta, 72, 619 (1963)]
A chromosomal DNA was obtained by extraction and purification by the usual DNA extraction method described in 1.

(2) Amplification of flavodoxin gene by PCR method Two synthetic primers flanking the translation region of flavodoxin gene (see J. Bacteriol. (1991) 173, 1729-1737) using the Escherichia coli chromosomal DNA as a template (see 5'-GCA
ATATGAGCCGTAGCGACTG-3 ′, 5′-GCCACTTGAACACCGGGAC
ATTG-3 ') (Takara custom synthesis)
Using neAmpR PCR Reagent Kit (purchased from Takara), DNA denaturation was performed at 92 ° C. for 1 minute, primer annealing was performed at 60 ° C. for 2 minutes, and primer extension reaction was performed at 72 ° C. for 3 minutes to perform 30 cycles of PCR reaction.

(3) Cloning of flavodoxin gene After adding 5'-P to the PCR product with T4 DNA polynucleotide kinase (purchased from Takara), Klenow fragment (purchased from Takara)
The ends were made blunt by treatment and the polylinker cloning site (EcoRI-SacI-Kpn) of the cloning vector pUC19 (purchased from Takara) was used.
I-SmaI-XbaI-SalI-PstI-Sph
I-HindIII) was inserted into the SmaI site, and pAFL4 in which the flavodoxin gene was inserted was constructed to obtain a recombinant Escherichia coli JM105 [pAFL4] strain.

Example 8 A flavodoxin purified recombinant Escherichia coli JM105 [pAFL4] strain was cultured in the same manner as in Example 1, and a cell-free extract was prepared in the same manner as in Example 2, and then, (1, 2, Flavodoxin was purified by the same method as 3). The protein concentration of the purified flavodoxin-containing solution was 1.9 mg / ml.

Example 9 Biotin conversion reaction 0 to 20% by volume of flavodoxin (1.9 mg / ml) was added to an enzyme conversion reaction system (polyethyleneimine-treated solution), and 5 mM FDP, 5 mM FeCl ₂ , 1 mM NADP was further added.
H, 100 μM S-adenosylmethionine, 10 mM KCl, 50 μM dethiobiotin (DL-
DTB) to make the total volume 100 μL,
Reaction was carried out for 30 minutes. After completion of the reaction, quickly cool with ice,
After heating at 100 ° C for 5 minutes, centrifuge and remove the amount of biotin in the supernatant.
Lactobacillus plantarum (A
It was measured by a bioassay method using TCC8014). The results are shown in Table 4.

[0063]

[Table 4]

As is clear from Table 4, addition of flavodoxin to the enzyme conversion reaction solution markedly increased the biotin conversion activity.

Example 10 To 20 μl of flavodoxin (1.9 mg / mL) was added to 50 μl of cell-free extract of biotin conversion reaction (protein concentration 17.8 mg / mL), and further 5 mM FDP, 5 mM FeCl ₂ , 1 mM N
ADPH, 100 μM S-adenosylmethionine, 1
0 mM KCl and 50 μM dethiobiotin (DL-DTB) as a substrate were added to make the total amount 100 μL,
The reaction was carried out at 35 ° C for 120 minutes. After completion of the reaction, the reaction mixture was quickly ice-cooled, heated at 100 ° C. for 5 minutes, and then centrifuged, and the amount of biotin in the supernatant was adjusted to Lactobacillus planta.
It was measured by a bioassay method using rum (ATCC8014). The results are shown in Table 5.

[0066]

[Table 5] As is clear from Table 5, addition of flavodoxin to the enzyme conversion reaction solution markedly increased the biotin conversion activity.

Example 11 Construction of recombinant plasmid (I) Escherichia coli DRK3323 [p provided by the present inventors
XBA312] strain (Mikoken Kenjoyori No. 2117) from a conventional alkali extraction method [Nucleic acidRese
arch, 7, 1513 (1979)].
Obtained XBA312.

This plasmid was completely digested with the restriction enzyme XbaI, subjected to 1% agarose gel electrophoresis using low temperature gel agarose, and stained with ethidium bromide to give about 6 kbp.
The DNA fragment containing the biotin operon of
After heating for 5 minutes at ℃, TE buffer (10 mM Tris-hydrochloric acid buffer pH 8.0, 0.1 mM EDTA) saturated phenol was added in approximately the same amount, mixed well, and an aqueous phase was obtained by centrifugation.
DNA was recovered from this aqueous phase by ethanol precipitation.

On the other hand, Escherichia coli JM105 obtained in Example 7
The plasmid pAFL4 obtained from the [pAFL4] strain by a usual alkali extraction method, and the control plasmid p
After MW119 was completely digested with the restriction enzyme XbaI, each was ligated with the above-mentioned DNA fragment containing the biotin operon using a DNA ligation kit (Takara).

For screening of recombinant Escherichia coli, the above ligation reaction solutions were used as described in JP-A-62-155.
The BR-4 strain, which is a biotin-requiring strain obtained by mutating the Escherichia coli JA221 strain described in JP-A-0881, was added to the conventional calcium method [J. Mol. Biol. , 53, 109
(1970)] and ampicillin 50μ
Biotin-free minimal agar plate containing g / ml (glucose 0.5%, ammonium sulfate 0.4%, potassium dihydrogen phosphate 0.2%, potassium dihydrogen phosphate 0.01%, vitamin-free It was carried out by obtaining colonies grown on casamino acid 0.4%, tryptophan 0.002%, agar 1.5%).

After culturing the obtained strain, a plasmid pFLB401 having a biotin operon and a flavodoxin gene and a plasmid pBMW400 having only a biotin operon were obtained by an alkaline extraction method.

Example 12 Construction of Recombinant Plasmid (II) Escherichia coli DRAT9 strain provided by the present inventors as a high expression system biotin operon having a point mutation in the operator region of the biotin operon (Microtech Lab. No. 1237
No. 8) obtained by the method described in Japanese Patent Application No. 3-235148, the plasmid pAMP912 having a high expression system biotin operon was digested with the restriction enzyme NcoI (a cleavage site present in the start codon of bioB gene), EcoT221 (b).
(Cleavage site present in ioA gene) After complete digestion, 1% agarose gel electrophoresis using low temperature gel agarose was performed, and a DNA fragment containing a control region of biotin operon of about 1 kbp was excised after staining with ethidium bromide.
After heating at 0 ° C. for 5 minutes, TE buffer saturated phenol was added in approximately the same amount, mixed well, and an aqueous phase was obtained by centrifugation.

DNA was precipitated from this aqueous phase by ethanol precipitation.
Was recovered. On the other hand, the plasmid pF obtained in Example 11
Restriction enzyme N for LB401 and pBMW400, respectively
After complete digestion with coI and EcoT221, about 10 kbp and 9.2 kbp DNA fragments from which the control region of the biotin operon was deleted were recovered by the same method as described above. Each of these DNA fragments was ligated with a 1 kbp DNA fragment containing the control region of the biotin operon of the high expression system using a DNA ligation kit (Takara).

Screening of recombinant E. coli was done in Example 11
The ligation reaction solution was transformed into the BR-4 strain which is a biotin-requiring strain by the same method as described in (1) above, and colonies grown on the minimal agar plate containing no biotin and supplemented with 50 μg / ml of ampicillin were obtained. Went by. After culturing the obtained strain, a plasmid pFLM922 having a high expression type biotin operon and a flavodoxin gene and a plasmid pMMW900 having only a high expression type biotin operon were obtained by an alkaline extraction method.

Example 13 Plasmid pFLB40 constructed in Biotin conversion method examples 11 and 12
1, pBMW400, pFLM922, and pMMW9
00 was transformed into E. coli W3110 (ATCC1498) to obtain W3110 [pFLB401] strain, W3110.
[PBMW400] strain, W3110 [pFLM922]
Strain and W3110 [pMMW900] strain were acquired.

These strains were mixed with 20 ml of Medium-A (ampicillin 5) in a 500 ml Sakaguchi flask (Shibata Hario Co.).
0 μg / ml, dl-desthiobiotin 10 mg / l added)
The cells were inoculated with the strain and incubated at 37 ° C. for 125 hours at 125 strokes / min to carry out a conversion reaction. After the reaction was completed, the bacterial cells were removed by centrifugation and the amount of biotin produced in the supernatant was adjusted to Lactobacil
bioassay using lus plantarum
It was measured by the method. The results are shown in Table 6.

[0077]

[Table 6]

Example 14 Biotin conversion method W3110 [pFLB401] strain obtained in Example 13, W
3110 [pBMW400] strain, W3110 [pFLM]
922] strain and W3110 [pMMW900] strain
20 ml Desthiobiotin-free medium-A (ampicillin 50 μm) in a 00 ml Sakaguchi flask (Shibata Hario)
g / ml added), 37 ° C, 125 strokes /
The conversion reaction was carried out by culturing with shaking for 24 hours. After the reaction was completed, the bacterial cells were removed by centrifugation and the amount of biotin produced in the supernatant was adjusted to Lact.
bi using obacillus plantarum
It was measured by the oassay method. The results are shown in Table 7.

[0079]

[Table 7]

[0080]Example 15．Biotin conversion method W3110 [pFLM922] strain obtained in Example 13, and
And W3110 [pMMW900] strain 500ml capacity Sakaguchi
20 ml of Medium-A (Ampi) in a flask (Shibata Hario)
Sillin (50 μg / ml added) at 37 ° C for 125 seconds
The cells were shake-cultured for 6 hours at Trox / min. Then 600
After centrifuging at 0 rpm for 10 minutes to collect the cells,
Wash twice with 0.75% NaCl solution and wash with 2 ml of 0.1
M Tris-HCl buffer (pH 7.5, 100 μg / ml black
Resuspended in lamphenicol) to give resting cells.
To 1 ml of resting cells, 5 mM glucose, 1 mM L-system
In, 5 mM FeCl ₂, 50 μM dl- as substrate
Add Desthiobiotin and use a test tube of φ20mm
30 minutes at 150 strokes / min in a 37 ° C constant temperature bath
The biotin conversion reaction was carried out by shaking. After the reaction is over
Quickly cool with ice, heat at 100 ° C for 5 minutes, then centrifuge
The amount of clean biotinLactobacillus pla
ntarumBioas using (ATCC8014)
It was measured by the say method. The results are shown in Table 8.

[0081]

[Table 8]

[0082]

The present invention provides an efficient method for producing biotin, particularly an efficient method for converting desthiobiotin to biotin. The conversion efficiency can be increased by adding flavodoxin or the flavotoxin gene to the enzyme conversion reaction solution.

[Brief description of drawings]

FIG. 1 is a diagram showing the elution behavior of a DEAE ion exchange chromatograph of a 55-80% ammonium sulfate precipitation fraction derived from the Escherichia coli cell-free extract described in Example 4 (2).

FIG. 2 SDS-PE of purified protein described in Example 4 (4)
Shows GE. Lane 1 is a molecular weight marker, and lane 2 is a sketch diagram showing the purified protein.

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Claims (18)

[Claims] 1. A method for converting desthiobiotin to biotin, which comprises adding flavodoxin to an enzyme conversion reaction solution when converting desthiobiotin to biotin in an enzyme conversion reaction system. 2. The method according to claim 1, wherein the enzyme conversion reaction system comprises an enzyme conversion reaction solution prepared from Escherichia coli having the bioB gene in the biotin operon. 3. The method according to claim 2, wherein the bioB gene is carried by a plasmid. 4. The method according to claim 3, wherein the bioB gene is a highly expressed bioB gene. 5. The method according to claim 1, wherein the flavodoxin is Escherichia coli flavodoxin. 6. The flavodoxin is provided by expression of the flavodoxin gene in a plasmid.
Or the method according to 5. 7. The bioB gene and the flavodoxin gene are carried by the same plasmid,
The method according to claim 3, 4 or 6. 8. A method for converting desthiobiotin into biotin in a culture system or a resting cell system, wherein the cells of the culture system or the resting cell system have a plasmid carrying a flavodoxin gene. How to convert desthiobiotin to biotin. 9. The method according to claim 8, wherein the bacterial cell is an Escherichia coli bacterial cell. 10. The method according to claim 8 or 9, wherein the flavodoxin gene is an Escherichia coli flavodoxin gene. 11. The method according to claim 8, wherein the bacterial cell has a plasmid carrying at least the bioB gene in the biotin operon. 12. The bioB gene highly expressing the bioB gene
The method according to claim 11, which is a gene. 13. The flavodoxin gene and the b
The method according to any one of claims 8 to 12, wherein the ioB gene is carried by the same plasmid. 14. A method for producing biotin, which comprises culturing a microorganism having a biotin operon and a flavodoxin gene carried on a plasmid. 15. The method according to claim 14, wherein the biotin operon is carried on a plasmid. 16. The method according to claim 1, wherein the microorganism is Escherichia coli.
The method according to 4. 17. The method according to claim 14 or 15, wherein the biotin operon and flavodoxin gene are carried on the same plasmid. 18. The biotin operon and flavodoxin gene are derived from Escherichia coli.
The method according to any one of items 1 to 17.