PRODUCTION METHOD FOR IMMOBILIZED MICROORGANISMS AND PRODUCTION METHOD FOR AMINO ACID USING THE SAME

Abstract

An object of the present invention is to provide a method for producing an immobilized microorganism having high filtration properties, and to provide a method for producing an amino acid using the immobilized microorganism. A method for producing an immobilized microorganism is characterized in that a microorganism is contacted with carboxymethyl cellulose sodium salt and then contacted with polyethylenimine and an alkane dial after the first contact with carboxymethyl cellulose sodium salt.

Claims

1. A method for producing an immobilized microorganism, comprising the steps of: contacting a microorganism with carboxymethyl cellulose sodium salt, and then further contacting the microorganism with polyethylenimine and an alkane dial.

2. The production method according to claim 1, wherein the microorganism is first contacted with the polyethylenimine and then contacted with the alkane dial after the microorganism is contacted with the carboxymethyl cellulose sodium salt.

3. The production method according to claim 1, wherein each contact was made in the presence of a dispersion medium comprising water.

4. The production method according to claim 1, wherein a viscosity of the carboxymethyl cellulose sodium salt measured by the following condition is 50 mPa.Math.s or less, Viscosity measurement condition: a 2% aqueous solution is prepared by precisely weighing 4.4 g of carboxymethyl cellulose sodium salt, adding the weighed carboxymethyl cellulose sodium salt into a 300 mL stoppered conical flask, determining an amount (W) of water by the following formula, and adding the determined amount (W) of water:
Required water amount W(g)=carboxymethyl cellulose sodium salt(g)×(98−water content(%))/2 wherein the water content (%) is a water content in the carboxymethyl cellulose sodium salt and corresponds to a weight loss on drying in the case where the carboxymethyl cellulose sodium salt is dried in a constant temperature dryer of 105±2° C. for 4 hours; the prepared 2% carboxymethyl cellulose sodium salt aqueous solution is left to stand overnight and then stirred using a magnetic stirrer for 5 minutes to obtain a complete solution, the complete solution is added into a covered container having a diameter of 45 mm and a height of 145 mm, the container is immersed in a constant temperature bath of 25±0.2° C. for 30 minutes, the complete solution is slowly stirred using a glass bar after a temperature of the complete solution becomes 25° C., a rotor and a guard of a BM-type viscometer are installed, a scale is read off 3 minutes after the rotor is rotated at a rotation speed of 30 rpm or 60 rpm; and the viscosity value (mPa.Math.s) is calculated by multiplying the following coefficient determined with the Rotor No. and the rotation speed by the read scale, Coefficient in the case of Rotor No. 1 and 60 rpm: 1 Coefficient in the case of Rotor No. 2 and 60 rpm: 5 Coefficient in the case of Rotor No. 3 and 60 rpm: 20 Coefficient in the case of Rotor No. 4 and 60 rpm: 100 Coefficient in the case of Rotor No. 1 and 30 rpm: 2 Coefficient in the case of Rotor No. 2 and 30 rpm: 10 Coefficient in the case of Rotor No. 3 and 30 rpm: 40 Coefficient in the case of Rotor No. 4 and 30 rpm: 200.

5. The production method according to claim 1, wherein the microorganism is recombinant Escherichia coli.

6. The production method according to claim 5, wherein the recombinant Escherichia coli is a transformant having an amino acid dehydrogenase activity.

7. The production method according to claim 5, wherein the recombinant Escherichia coli is a transformant having a leucine dehydrogenase activity and a formate dehydrogenase activity.

8. A method for producing an amino acid, comprising the steps of: producing the immobilized microorganism by the method according to claim 1, and contacting the immobilized microorganism with a keto acid.

9. The method for producing the amino acid according to claim 8, wherein a column is filled with the immobilized microorganism, a solution comprising the keto acid is supplied to an inlet of the column, and a solution comprising the amino acid is discharged from an outlet of the column.

10. The method for producing the amino acid according to claim 8, wherein the keto acid is 3,3-dimethyl-2-oxobutyric acid and the amino acid is tert-leucine.

Description

EXAMPLES

[0124] Hereinafter, the examples are described to demonstrate the present invention more specifically, but the present invention is in no way restricted by the examples, and the examples can be appropriately modified to be carried out within a range which adapts to the contents of this specification. Such a modified example is also included in the range of the present invention.

Production Example 1: Production of Culture Medium for Bacterium

[0125] Production of the plasmid designed to have the ability to express a volume of formate dehydrogenase:

[0126] PCR reaction was performed using the genomic DNA of Thiobacillus sp. KNK65MA (FERM BP-7671) as a template and primers under the PCR condition 1 shown below. Primer 1 is SEQ ID NO: 1 and Primer 2 is SEQ ID NO: 2 in the sequence listing.

[0127] PCR Condition 1

[0128] For the preparation of a reaction solution, 1.25 U, equivalent to 0.25 μL, of Pyrobest DNA polymerase manufactured TAKARA BIO INC., 5 μL of 10×Pyrobest Buffer II manufactured by TAKARA BIO INC., 4 μL from each 2.5 mM dNTP solution, 2 μL from each 20 μM primer solution were added to 100 ng of the DNA template. Distilled water was added to the mixture so that the total volume became 50 μL. The PCR reaction was consisted of three steps, a denaturation step conducted at 96° C. for 30 seconds, an annealing step conducted at 50° C. for 30 seconds, an extension step conducted at 72° C. for 90 seconds, and the PCR sample was cooled down to 4° C. after the cycle was repeated for 25 times.

[0129] The DNA fragment amplified via PCR was digested with restriction endonuclease NdeI and EcoRI and the digested fragment was ligated to the vector plasmid pUCNT with T4 DNAligase. A person with an ordinary skill in the art can produce pUCNT on the basis of WO 94/03613 pamphlet. A plasmid designed to have the ability to express a volume of formate dehydrogenase was obtained.

[0130] Production of the Plasmid Designed to have the Ability to Express a Volume of Leucine Dehydrogenase:

[0131] PCR reaction was performed using the genomic DNA of Bacillus sphaericus NBRC3341 as a template and primers under the above-described PCR condition 1. Primer 3 is SEQ ID NO: 3 and Primer 4 is SEQ ID NO: 4 in the sequence listing.

[0132] The fragment amplified via PCR was digested with restriction endonuclease EcoRI and Sad and the digested fragment was ligated to vector plasmid pUCNT with T4 DNAligase. A person with an ordinary skill in the art can produce pUCNT on the basis of WO 94/03613 pamphlet. The plasmid pUCNT had disrupted recognition site of NdeI by receiving one base replacement. A plasmid designed to have the ability to express a volume of leucine dehydrogenase was obtained.

[0133] The obtained plasmid designed to have the ability to express a volume of leucine dehydrogenase was digested with restriction endonuclease EcoRI and PstI, and the DNA fragment having leucine dehydrogenase gene was recovered by using TaKaRa RECOCHIP manufactured by TAKARA BIO INC.

[0134] The above plasmid designed to have the ability to express a volume of formate dehydrogenase was digested at the recognition sites of EcoRI and PssI located in the downstream of formate dehydrogenase gene, and then DNA fragment was obtained. The DNA fragment was ligated with the above-described DNA fragment having leucine dehydrogenase gene by T4 DNA ligase to obtain a plasmid designed to have the ability to express a volume of both the leucine dehydrogenase and the formate dehydrogenase.

[0135] The competent cell of Escherichia coli HB101 was transformed by mixing with the obtained plasmid, and the transformant having both the leucine dehydrogenase activity and the formate dehydrogenase activity was bred.

[0136] The bred transformant having both the leucine dehydrogenase activity and the formate dehydrogenase activity was inoculated to sterilized culture medium A which contained tryptone 1.6%, yeast extract 1.0%, sodium chloride 0.5% and Ampicillin 0.01% and which prepared by dissolving the components other than Ampicillin in deionized water to obtain a solution having pH of 7.0, sterilizing the solution, and adding Ampicillin thereto, and then the mixture was shaken at 33° C. for 48 hours to aerobically cultivate the transformant.

Example 1: Production of Immobilized Bacterium

[0137] In order to remove 1166 g of supernatant, 1740 g of culture medium of which wet bacteria mass was 35 g and which was obtained in Production example 1 was centrifuged. To the stirred 580 g of residual concentrated culture medium at room temperature, 145 g of 5 mass % aqueous solution of carboxymethylcellulose sodium named Serogen6A and manufactured by DKS. Co. Ltd., was added over 20 minutes. The solution was stirred for another 30 minutes. Then, 66 g of 20% polyethylenimine aqueous solution of which pH was adjusted to 7 using hydrochloric acid was added to the stirred solution at room temperature over 20 minutes. The polyethylenimine aqueous solution called “EPOMIN” p-1000 is manufactured by Nippon Shokubai Co., Ltd. and has 70000 of molecular weight described in a catalog. The mixed solution was stirred for another 30 minutes. To the stirred solution at room temperature, 24 g of 50 mass % glurataldehyde aqueous solution was added for over 20 minutes. The solution was stirred for another 30 minutes. Stirring was stopped, and the solution was allowed to stand for about 5 minutes to generate precipitation. After the supernatant was removed using a pipet, 290 mL of 50 mM Tris-HCl adjusted to pH 7.5 was added thereto, and the mixture was stirred at room temperature for 30 minutes. The above procedure was repeated two more times.

[0138] The obtained mixed liquid was filtrated with a filter paper 5 A manufactured by Kiriyama Glass Works Co. having an area of 15.2 cm.sup.2 in a condition of a filtration pressure of 1.0 kgf/m.sup.2 and a cake thickness of 3 cm, the filtration property was excellent as the filtration rate resistance coefficient was 1.5×10.sup.9 m/kg. In this way, 63 g of immobilized bacterium was obtained.

[0139] A pressure proof glass column (Omnifit) having an internal diameter of 10 mm was filled with about 9 g of the obtained immobilized bacterium, put into a column oven controlled at 30° C., standing and fixing the column upright, and then distilled water was supplied into the column using a syringe. It could be confirmed that a column with a low pressure loss can be produced, since distilled water could run through the column without applying a high pressure on the syringe.

Example 2: Evaluation of Immobilized Vacterium's Activity Yield

[0140] To 1 mL of a reaction mixture of which pH was adjusted to 7.3 as a 50 mM potassium phosphate buffer solution containing 200 mg of trimethyl pyruvate, 1.45 mg of NAD.sup.+, 20 mg of zinc sulfate heptahydrate, 150 mg of ammonium formate and 60 mg of ammonium sulfate, 200 mg of the obtained immobilized bacterium or 9.2 mL of the obtained concentrated culture medium ultrasonically pulverized was added. The mixture was stirred at 30° C. for 1 hour. The yield and optical purity of the remained trimethyl pyrate and produced L-tert-Leucine were analyzed with high-performance liquid chromatography, HPLC, as a result, the activity yield obtained by dividing total activity of immobilized bacterium by total activity of culture medium used for immobilization of the immobilized bacterium was 0.4.

[0141] HPCL analysis condition for trimethyl pyruvate

[0142] Column: COSMOSIL 5C18-AR, 4.6 mm×250 mm in size, manufactured by NACALAI TESQUE, INC.

[0143] Mobile phase: 10 mM potassium phosphate buffer solution adjusted to pH 2.0/acetonitrile=95/5 (V/V)

[0144] Flow rate: 1 mL/min

[0145] Column temperature: 40° C.

[0146] Detection: 210 nm

[0147] HPCL analysis condition for L-tert-Leucine

[0148] Column: SUMICHIRAL OA-5000, 4.6 mm×250 mm in size, manufactured by Sumika Chemical Analysis Service, Ltd.

[0149] Mobile phase: 2 mM copper sulfate aqueous solution/methanol=95/5 (V/V)

[0150] Flow rate: 1 mL/min

[0151] Column temperature: 35° C.

[0152] Detection: 254 nm

Comparative Example 1: Production of Immobilized Bacterium

[0153] In order to remove 1166 g of supernatant, 1740 g of culture medium of which wet bacteria mass was 35 g and which was obtained in Production example 1 was centrifuged. To the stirred 580 g of residual concentrated culture medium at room temperature, 66 g of 20 mass % polyethylenimine called “EPOMIN” and manufactured by Nippon Shokubai Co., Ltd. aqueous solution of which pH was adjusted to 7 by a hydrochloric acid was added over 20 minutes. The solution was stirred for another 30 minutes. To the stirred solution at room temperature, 24 g of 50 mass % glurataldehyde aqueous solution was added for over 20 minutes. The solution was stirred for another 30 minutes. Stirring was stopped, and the solution was allowed to stand for about 5 minutes to generate precipitation. After the supernatant was removed using a pipet, 290 ml of 50 mM Tris-HCl adjusted to pH 7.5 was added thereto, and the mixture was stirred at room temperature for 30 minutes. The above procedure was repeated two more times.

[0154] The obtained mixed liquid was filtered with a filter paper 5A manufactured by Kiriyama Glass Works Co. having an area of 15.2 cm.sup.2 under reduced pressure of 15 mmHg; as a result it was confirmed that efficient production of immobilized bacterium was difficult.

[0155] Instead of filtrating the mixed liquid, immobilized bacterium was obtained by using a large amount of paper towel to absorb liquid component of the mixed liquid. A pressure proof glass column (Omnifit) having an internal diameter of 10 mm was filled with about 9 g of the obtained immobilized bacterium, put in a column oven controlled at 30° C., standing and fixing the column upright, and then, distilled water was supplied into the column using a syringe. It was confirmed that the pressure loss became huge when a column was packed with the immobilized microorganism, since higher pressure was needed to allow distilled water to pass through the column.

Comparative Example 2: Synthesis of L-tert-Leucine by Batch Method

[0156] In a glass reaction vessel, 102.1 mg of immobilized bacterium obtained in Comparative example 1 was mixed with each 10 ml of Solution A and Solution B. After the mixture was stirred for 16 hours at room temperature, a sample of the reaction mixture was analyzed with HPLC; as a result, mole conversion ratio from trimethyl pyruvate to L-tert-Leucine was 20.3%.

[0157] Preparation Method of Solution A

[0158] To a trimethyl pyruvate solution of 5.80 g (66 wt %), 6 N NaOH aqueous solution and 50 mM potassium phosphate buffer solution were added, and pH of the solution was adjusted to 7. Then, the volume of the solution was increased to 20 mL with 50 mM potassium phosphate.

[0159] Preparation Method of Solution B

[0160] After NAD.sup.+ of 2.9 mg, zinc sulfate heptahydrate of 4.0 mg, ammonium formate of 3.0 g, ammonium sulfate of 1.2 g and 1 ml of 1 M potassium phosphate buffer solution adjusted to pH 7 were mixed, the volume of the solution was increased to 20 mL with distilled water.

Example 3: Synthesis of L-tert-Leucine by Flow Method 1

[0161] A pressure proof glass column (Omnifit) having an internal diameter of 10 mm was filled with 2.98 g of the immobilized bacterium obtained in Example 1, put in a column oven controlled at 30° C., standing and fixing the column upright, and then distilled water was pumped into the column at a flow rate of 0.05 ml/min with a syringe pump manufactured by YMC.CO., LTD. Subsequently, a raw material solution prepared by mixing 19 ml each of Solution B and Solution C was pumped into the column at a flow rate of 0.03 ml/min, SV of 0.45 hr.sup.−1, with a syringe pump manufactured by YMC.CO., LTD. for 68 hours in total to obtain a reaction mixture containing the L-tert-leucine from the column outlet. HPLC recovery rate was 99%. Mole conversion ratios of the obtained reaction mixture at times of 23, 46 and 68 hours were 97%, 99% and 97% respectively.

[0162] Preparation Method of Solution C

[0163] Distilled water of 2.9 g was added to trimethyl pyruvate aqueous solution of 2.90 g (66 wt %). Then, the pH of the solution was adjusted to 7 with 6 N NaOH aqueous solution and 50 mM potassium phosphate buffer solution, and finally the volume of the solution was increased to 20 mL with 50 mM potassium phosphate buffer solution.

Example 4: Synthesis of L-tert-Leucine by Flow Method 2

[0164] A pressure proof glass column (Omnifit) having an internal diameter of 10 mm was filled with 8.94 g of the immobilized bacterium obtained in Example 1, put in a column oven controlled at 30° C., standing and fixing the column upright, and then distilled water was pumped into the column at a flow rate of 0.1 ml/min with a plunger pump manufactured by FLOM corporation. A raw material solution prepared by mixing 140 ml each of Solution D and Solution E was pumped into the column with a plunger pump manufactured by FLOM Corp. at a flow rate of 0.09 ml/min, SV of 0.45 hr.sup.−1, for 56 hours totally, and the reaction mixture remaining in the column was extruded by pumping distilled water into the column at the same flow rate. As a result, the reaction mixture containing 11.9 g of the L-tert-leucine was obtained, and mole conversion rate was 99% and HPLC recovery rate was 87%. Mole conversion rates of the obtained reaction mixture at times of 20, 26, and 44 hours were all 99%. Moreover, the height of the immobilized bacterium filled in the column didn't change from 5.1 cm measured at the start of the reaction, it was considered that the volume of the immobilized bacterium didn't change before and after the reaction and no elution of the bacterium from the immobilized bacterium occurred and the carrier for the immobilization was not dissolved.

[0165] Preparation Method of Solution D

[0166] To trimethyl pyruvate solution of 20.3 g (70 wt %), distilled water of 20.3 g was added. The solution was adjusted to pH 7 with 6 N NaOH aqueous solution and 50 mM potassium phosphate buffer solution. To the solution, 50 mM potassium phosphate buffer solution was added so that the total volume became 140 mL.

[0167] Preparation Method of Solution E

[0168] After NAD.sup.+ of 20.3 mg, zinc sulfate heptahydrate of 28.0 mg, ammonium formate of 21.0 g, ammonium sulfate of 8.4 g and 7 ml of 1 M potassium phosphate buffer solution adjusted to pH 7 were mixed, distilled water was added thereto so that the total volume became 140 mL.

INDUSTRIAL APPLICABILITY

[0169] An immobilized catalyst having an excellent property can be produced by the present invention in a simple way, and the present invention can be advantageously utilized for various synthesis reaction, preferably an enantioselective synthesis reaction.