JP5880447B2 - Method for transporting microbial cells - Google Patents

Description

  The present invention stably stabilizes the nitrile hydratase activity by filling the sealed microbial cell having nitrile hydratase activity into a sealed container and transporting it to 20% or less of the total volume of the gas phase inside the sealed container. The present invention relates to a method of transporting while holding.

  Enzymes produced by microorganisms are used in many situations as catalysts for chemical conversion reactions. In particular, by using nitrile hydratase, nitrilase, etc., which have nitrile group hydration or hydrolytic ability, it becomes possible to produce amides, carboxylic acids, α-hydroxycarboxylic acids and the like important in the chemical industry at low cost. . Furthermore, by using the above-mentioned enzyme having optical specific hydration or optical specific hydrolyzing ability, it is possible to produce optically active carboxylic acids, amino acids, α-hydroxycarboxylic acids and the like that are important as raw materials for pharmaceuticals and agricultural chemicals. Become.

  In a chemical conversion reaction using a microbial enzyme as a catalyst, it is necessary to stably maintain the enzyme activity of cultured and collected microbial cells until use. That is, the activity of the enzyme must be maintained so that the microbial enzyme's catalytic ability is not lost or deteriorated by contamination or lysis or lysis during storage or transportation. Therefore, in general, by storing in the presence of stabilizers, metabolic inhibitors and high-concentration salts, inactivation, decay, and lysis of microbial enzymes during microbial cell storage are suppressed, and used for chemical conversion reactions. Yes. When the stabilizer is not added, it is stored or transported while maintaining the activity of the enzyme by freezing, refrigeration, or full-time stirring.

  Patent Document 1 (Japanese Patent Laid-Open No. 2004-305066) discloses a method for preserving microbial cells by adding stabilizers such as nitriles, amides, and carboxylic acids, and Patent Document 2 (Japanese Patent Laid-Open No. 2005-295815). Discloses a method for preserving microbial cells by adding a metabolic inhibitor such as an azide compound, and Patent Literature 3 (Japanese Patent Application Laid-Open No. 2001-149065) discloses a culture solution component of microbial cells. A method for preserving microbial cells by adding to a suspension is disclosed. Patent Document 4 (Patent No. 3163224) discloses a storage method by adding high-concentration inorganic salts. Furthermore, Patent Document 5 (Japanese Patent Laid-Open No. 2003-21870) is known as a method of storing by freezing, and Patent Document 6 (Japanese Patent Laid-Open No. 2003-144144) is known as a method of storing by aeration stirring. Are known.

JP 2004-305066 A JP 2005-295815 A Japanese Patent Laid-Open No. 2001-149065 Japanese Patent No. 3163224 JP 2003-21870 A JP 2003-144144 A

  Among the storage methods known so far, the method using an additive such as a stabilizer needs to separate the additive in a later step, which may affect the quality of the product, Furthermore, the manufacturing method becomes complicated. Suspension of microbial cells stored by the method of freezing microbial cells has problems in handling such as complicated freezing and thawing operations, and the enzyme activity is lost with the operation, There is a risk of lowering. In addition, storage by refrigeration is preferable from the viewpoint of preventing proliferation of various bacteria and stabilization of bacterial cells, but requires equipment and electric power to maintain a low temperature, and the burden on cooling cost is large. In the method of storing microbial cells in an aqueous solution of high-concentration inorganic salt, it is necessary to sufficiently wash the microbial cells at the time of use, and there is a demerit that costs are required for the treatment of added inorganic salts and waste water. Although it has been reported that the enzyme activity of the microbial cells is maintained to some extent by aeration stirring, etc., the effect is not uniform depending on the enzyme, and power is required for aeration and agitation, and the cost burden is large. Therefore, in addition to the prior art, a more stable method for preserving bacterial cells having nitrile hydratase activity is desired.

  As a result of intensive studies on conditions for transporting a suspension of microbial cells obtained by culturing and collecting microorganisms at low cost and stably, the gas phase inside the container used for transport is 20%. It has been found that the cells having nitrile hydratase activity can be stably stored and transported when the content is less than or equal to%, and the present invention has been completed.

That is, the present invention is a method for storing microbial cells having nitrile hydratase activity in a sealable container, wherein a gas phase portion in the container filled with a suspension containing the microbial cells is provided. It is the said method characterized by setting it as 2% or more and 20% or less of the total volume inside a container.
The present invention also relates to a method for transporting microbial cells having nitrile hydratase activity using a sealable container, wherein the gas phase portion in the container filled with a suspension containing the microbial cells. Is 2% to 20% of the total volume inside the container.
In the method of the present invention, the proportion of the gas phase part in the container is preferably 5% or more and 20% or less. The storage or transport temperature is, for example, a temperature of freezing point to 35 ° C. Furthermore, the dispersion medium of the cell suspension is an organic acid aqueous solution, and examples of the organic acid include acrylic acid.

  According to the present invention, by filling the cell suspension so that the gas phase inside the container is 2% or more and 20% or less, nitrile hydratase or the like can be obtained even at room temperature without stirring. It can be stored and transported while maintaining its enzyme activity. Furthermore, according to the present invention, it is possible to greatly reduce the labor and cooling cost required in the conventional storage method, and to provide a method for storing microbial cells that can be industrially satisfied.

The present invention relates to a method for storing microbial cells having nitrile hydratase activity in a sealable container, and a method for transporting the microbial cells using a sealable container. In the method of the present invention, the suspension containing the microbial cells is filled so that the gas phase part in the container is 2% or more and 20% or less of the total volume in the container.
It should be noted that documents cited in the present specification, as well as published gazettes, patent gazettes, and other patent documents are incorporated herein by reference. In addition, the disclosure content of the claims, description, drawings and abstract of Japanese Patent Application No. 2011-005780 (JP2011-005780), which was filed on January 14, 2011 and is the basis for claiming priority of the present application, The entirety is incorporated herein by reference.

In the present invention, the microbial cell having nitrile hydratase activity has the property of producing the target enzyme catalyst and accumulating it in the cell or secreting it outside the cell. These microorganisms include microorganisms isolated from nature and genetically modified microorganisms. Representative examples of such microorganisms include, for example, the genus Rhodococcus having nitrile hydratase activity, the genus Gordona, the genus Pseudomonas, the genus Pseudonocardia, and the genus Geobacillus. The microbial cell to which it belongs is mentioned. Furthermore, the recombinant microorganisms which introduce | transduced the nitrile hydratase gene of these microorganisms are mentioned. Among them, Rhodococcus genus, Gordona genus, and recombinant Escherichia coli and recombinant Rhodococcus bacteria into which nitrile hydratase genes of these microorganisms are introduced are preferable from an industrial viewpoint. For example, specific examples of microorganisms belonging to the genus Rhodococcus include Rhodococcus rhodochrous J-1 described in JP-B-6-55148. This strain was registered under the accession number “FERM BP-1478” on September 18, 1987 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1-1-1 Higashi 1-1-1 Tsukuba City, Ibaraki) The same in the description))).

  Nitrile hydratase refers to an enzyme having the ability to hydrolyze a nitrile compound to produce a corresponding amide compound. Examples of nucleic acids encoding nitrile hydrase and sequences thereof include those described in Patent Document 2. Such nucleic acids can be introduced into microbial cells by conventional molecular biology techniques (see Sambrook, Fritsch and Maniatis, “Molecular Cloning: A Laboratory Manual ”2nd Edition (1989), Cold Spring Harbor Laboratory Press).

  In the present invention, a “sealable container” is a container that can be sealed after being filled with a microbial suspension for transport and / or storage. Any container may be used as long as the contents are not moved inside and outside the container during transportation. For industrial purposes, a drum can, a container, a tank truck, and the like are used. The capacity of the container is not particularly limited, but 200 L is preferable for industrial purposes. The material of the container is not particularly limited, but for industrial purposes, a plastic container such as polyethylene or polypropylene, or a metal container such as iron or stainless steel is preferable. Hereinafter, the container used in the present invention is referred to as a “sealed container”.

  In the present invention, the proportion of the gas phase inside the container is 2% or more and 20% or less. “2% or more and 20% or less” means that the ratio of the volume of the gas phase portion in which the bacterial cell liquid does not exist in the sealed container is 2% or more and 20% or less of the total volume. Refers to that. The gas composition of the gas phase portion is not particularly limited, but air is generally entered for industrial reasons. The gas phase part is usually the upper part in the container.

  Usually, when storing or transporting a liquid in an airtight container, the filling rate of the liquid is freely set according to the situation, but in the present invention, the gas phase part is 20% or less with respect to the container volume. However, if it is less than 2%, the container may be deformed when the contents are expanded or contracted due to a temperature change. Therefore, the gas phase inside the sealed container at the time of transportation can be 2 to 20%, 3 to 20%, 4 to 20%, 5 to 20%, etc., more preferably 3 to 20%. 15%, more preferably 4 to 10%.

  In the present invention, “preservation” means that the bacterial cell suspension is sealed in a sealed container and then allowed to stand. In addition, “preservation” includes leaving it still before it is actually used after transportation. The storage period is the time from the filling of the suspension to the start of transportation (storage 1) and the time from the end of transportation to the start of use at the site (storage 2). For example, in the case of storage 1, it is 1 hour to 180 days, and in the case of storage 2, it is 1 hour to 180 days, but it is not limited to these periods. In the present invention, “transport” means moving the cell suspension using a sealed container. Generally, trucks, forklifts, tank trucks, and ships are loaded with sealed containers and moved.

  Regarding the temperature during storage and transportation, it is more preferable that the temperature is low in order to suppress the decay and decomposition of bacterial cells and enzymes. Specifically, it is carried out at a freezing point to 35 ° C, preferably a freezing point to 30 ° C, more preferably a freezing point to 20 ° C, and even more preferably a freezing point to 10 ° C. Here, “freezing point” means the equilibrium temperature between the solid state and the liquid state of the bacterial cell suspension, and is a temperature that varies depending on the composition of the suspension and the pressure in the storage container.

  In the present invention, after the storage period, the nitrile hydratase activity is 90% or more, preferably 95% or more, more preferably 98% or more, more preferably 99% or more, most preferably before the start of transportation. 100% remains. The residual ratio of nitrile hydratase activity can be measured by any method known in the art. For example, by comparing the reaction rate of acrylamide formation with respect to a substrate (for example, acrylonitrile) before and after storage, etc. It is possible to measure.

  In the present invention, the suspension dispersion medium means a solution used for suspending microbial cells to be stored. The dispersion medium is preferably an organic acid aqueous solution. The concentration of the organic acid in the aqueous organic acid solution is arbitrary, but if it is too low, the enzyme activity is reduced. On the other hand, if it is too high, the operation becomes complicated when removing it in a later step. It is preferable that it is 100 mmol / L.

  In the present invention, the composition of the immersion liquid is not particularly limited as long as it is an organic acid aqueous solution that does not inhibit enzyme activity. Examples of the organic acid include acrylic acid, formic acid, acetic acid, propionic acid, butyric acid, oxalic acid, and carboxylic acid. Among them, acrylic acid is preferable in terms of maintaining the quality of acrylamide.

In the preparation of the suspension of the present invention, the microbial cells having nitrile hydratase activity may be concentrated by any concentration method known in the art, but are preferably concentrated by membrane separation or centrifugation. The When the concentration is performed by membrane separation, it is preferable to use a membrane having a pore size of 0.02 to 0.45 μm. Such a membrane is commercially available, and examples thereof include a hollow fiber membrane module (Kuraray Co., Ltd., pore diameter 0.05 μm, surface area 39000 m ² ). Moreover, when concentrating by centrifugation, it is preferable to use a separation plate type continuous centrifuge.

  EXAMPLES Hereinafter, although the Example demonstrates the implementation method of this invention further in detail, these Examples aim at the illustration of this invention, and do not limit this invention. In the following examples and comparative examples,% display is mass% unless otherwise specified.

Examples 1 and 2 and Comparative Example 1
Culture Rhodococcus rhodochrous J-1 strain (FERM BP-1478) having nitrile hydratase activity is 2% by weight glucose, 1% by weight urea, 0.5% by weight peptone, 0.3% yeast extract. And aerobic culture in a medium (pH 7.0) containing 0.05% by mass of cobalt chloride.

Preparation of microbial cell suspension for storage The cultured microbial cells were collected by centrifugation (12000 rpm, 20 minutes) and then washed with a 0.1% aqueous sodium acrylate solution (pH 7.0). The aqueous solution was added to the washed cells to obtain a cell suspension (10% by mass in terms of dry cells).

Storage of bacterial cell suspension 115 mL (Example 1) and 70 mL (Comparative Example 1) of the bacterial cell suspension prepared by the above method were put in a polyethylene container having a total volume of 120 mL, sealed, and 30 as a transport model. The mixture was shaken at 120 ° C. for 3 weeks. Further, 190 L of the bacterial cell suspension (Example 2) prepared by the above method was placed in a 200 L polyethylene drum, sealed, and allowed to stand at room temperature for 3 months.

Measurement of nitrile hydratase activity The nitrile hydratase activity was determined by using the cell suspension immediately after preparation by the above method and the one contained in these suspensions after shaking or standing for 3 weeks after preparation. It calculated from the acrylamide production | generation reaction rate by a body. The reaction is started by adding an aqueous solution of acrylonitrile as a substrate to the cell suspension. After shaking at 10 ° C. for 10 minutes, the reaction is stopped by filtration separation of the cells and addition of phosphoric acid, and gas chromatography (GC -14B, Shimadzu Corporation). As analysis conditions, a 1 m glass column packed with Porapak PS (Waters) was used, and a FID having a column temperature of 210 ° C. and a detector of 230 ° C. was used. The relative reaction rates with the reaction rate on day 0 as 1 are shown in Table 1 below.

Example 3 and Comparative Example 2
Production of transformants having nitrile hydratase derived from Rhodococcus rhodochrous M8 (1) Preparation of chromosomal DNA from Rhodococcus rhodochrous M8 (hereinafter referred to as M8) M8 (SU1731814) 926).
The M8 strain was cultured with shaking in 100 mL of MYK (0.5% polypeptone, 0.3% bactoeast extract, 0.3% bactomalt extract, 0.2% K2HPO4, 0.2% KH2PO4) medium (pH 7.0) at 30 ° C. for 72 hours. The culture broth was centrifuged, and the collected cells were suspended in 4 mL of Saline-EDTA solution (0.1 M EDTA, 0.15 M NaCl (pH 8.0)). 8 mg of lysozyme was added to the suspension, shaken at 37 ° C. for 1-2 hours, and then frozen at −20 ° C.
Next, 10 mL of Tris-SDS solution (1% SDS, 0.1 M NaCl, 0.1 M Tris-HCl (pH 9.0)) was added to the suspension with gentle shaking. Furthermore, proteinase K (Merck) (final concentration 0.1 mg) was added to the suspension and shaken at 37 ° C. for 1 hour. Next, an equal amount of TE saturated phenol was added and stirred (TE: 10 mM Tris-HCl, 1 mM EDTA (pH 8.0)) and centrifuged. The upper layer was collected, twice the amount of ethanol was added, and the DNA was wound with a glass rod. Thereafter, this was centrifuged sequentially with 90%, 80%, and 70% ethanol to remove phenol.
Next, DNA was dissolved in 3 mL of TE buffer, ribonuclease A solution (100 ° C., heat-treated for 15 minutes) was added to 10 μg / mL, and the mixture was shaken at 37 ° C. for 30 minutes. Further, proteinase K (Merck) was added and shaken at 37 ° C. for 30 minutes. An equal amount of TE-saturated phenol was added thereto, and after centrifugation, the mixture was separated into an upper layer and a lower layer.
The upper layer was further centrifuged with an equal amount of TE saturated phenol, and then separated into an upper layer and a lower layer. This operation was repeated again. Thereafter, the same amount of chloroform (containing 4% isoamyl alcohol) was added to the upper layer and centrifuged to recover the upper layer. Next, twice the amount of ethanol was added to the upper layer, and the DNA was wound with a glass rod and collected to obtain chromosomal DNA.

(2) Preparation of nitrile hydratase gene from chromosomal DNA of M8 strain using PCR Non-patent literature (Veiko, VP et al, Cloning, nucleotide sequence of nitrile hydratase gene from Rhodococcus rhodochrous M8, Biotekhnologiia ( Mosc.) 5, 3-5 (1995)), and the base sequence and amino acid sequence of β subunit, α subunit, and activator are shown in SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, and The numbers 5 and 6 are shown. Based on the sequence information, primers described in SEQ ID NOs: 7 and 8 in the sequence listing were synthesized, and PCR was performed using the chromosomal DNA prepared in (1) as a template.

<PCR reaction solution composition>
Template DNA (chromosomal DNA) 200ng
PrimeSTAR Max Premix (Takara Shuzo) 25μl
Primer M8-1 10pmol
Primer M8-2 10pmol
<Primer>
M8-1: GGTCTAGAATGGATGGTATCCACGACACAGGC (SEQ ID NO: 7)
M8-2: cccctgcaggtcagtcgatgatggccatcgattc (SEQ ID NO: 8)
<Reaction conditions>
(98 ℃ 10 seconds, 55 ℃ 5 seconds, 72 ℃ 30 seconds) x 30 cycles
After completion of the PCR, 5 μl of the reaction solution was subjected to 0.7% agarose gel (using Agarose I manufactured by Dojin Chemical Co., Ltd .; agarose concentration 0.7 wt%) to detect a 1.6 kb amplified fragment. The reaction completion solution was purified using Wizard SV Gel and PCR Clean-Up System (Promega Corporation).
The collected PCR product was ligated to a vector (pUC118 / HincII site) using Ligation Kit (Takara Shuzo), and a competent cell of E. coli JM109 was transformed with the reaction solution. Several clones from the obtained transformant colonies were inoculated into 1.5 mL of LB-Amp medium and cultured with shaking at 37 ° C. for 12 hours. After culture, the culture was collected by centrifugation. Plasmid DNA was extracted from the collected cells by using QIAprep Spin Miniprep Kit (Amersham Bioscience). Sequencing kit and auto sequencer CEQ 8000
The base sequence of nitrile hydratase was confirmed using (Beckman Coulter).
Next, the obtained plasmid DNA was cleaved with restriction enzymes XbaI and Sse8387I, and then electrophoresed on a 0.7% agarose gel to recover the nitrile hydratase gene fragment (1.6 kb) and introduced into the XbaI-Sse8387I site of plasmid pSJ042. did.
The obtained plasmid was designated as pSJ-N01A.
PSJ042 was prepared as a plasmid expressing J1 strain nitrile hydratase in Rhodococcus by the method shown in Japanese Patent Application Laid-Open No. 2008-154552, and pSJ023 used for the preparation of pSJ042 is a transformant ATCC12674 / pSJ023 ( FERM BP-6232) was deposited on March 4, 1997 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, 1st Street, Tsukuba, Ibaraki).

(3) Production of competent cells Rhodococcus rhodochrous ATCC 12674 strain (hereinafter referred to as ATCC 12674 strain) was cultured in MYK medium until the early logarithmic growth phase, and the cells were collected using a centrifuge and then in ice-cold sterile water. The cells were washed 3 times and suspended in sterilized water to produce competent cells.

(4) Preparation of Transformant Containing M8 Strain-Derived Nitrile Hydratase The plasmid pSJ-N01A (0.1 μg) obtained was mixed with 20 μl each of a suspension of competent cells of the ATCC12674 strain, and each was ice-cooled. Each liquid mixture was put into a cuvette and subjected to electric pulse treatment at 20 KV / cm and 200 OHMS using a gene introduction apparatus Gene Pulser (BIO RAD). The electric pulse treatment solution was allowed to stand for 10 minutes under ice cooling, and heat shock was performed at 37 ° C. for 10 minutes. Thereafter, 500 μl of MYK medium was added to the cuvette, allowed to stand at 30 ° C. for 5 hours, applied to MYK agar medium containing 50 μg / mL kanamycin, and cultured at 30 ° C. for 3 days.
The plasmid DNA contained in the obtained transformant colony was confirmed, and this recombinant bacterium was named Rhodococcus genus recombinant bacterium (ATCC12674 / pSJ-N01A) having nitrile hydratase derived from M8 strain.

Culturing The obtained recombinant bacteria was aerobic in a medium (pH 7.0) containing 2% by mass of glucose, 1% by mass of urea, 0.5% by mass of peptone, 0.3% of yeast extract and 0.05% by mass of cobalt chloride. Cultured.

Preparation of microbial cell suspension for storage The cultured microbial cells were collected by centrifugation (12000 rpm, 20 minutes) and then washed with a 0.1% aqueous sodium acrylate solution (pH 7.0). The aqueous solution was added to the washed cells to obtain a cell suspension (10% by mass in terms of dry cells).

Storage of the cell suspension 100 mL (Example 3) and 80 mL (Comparative Example 2) of the cell suspension prepared by the above method were put in a polyethylene container having a total volume of 120 mL and sealed, and at 30 ° C. and 120 rpm. Shake for 3 weeks.

Measurement of nitrile hydratase activity The nitrile hydratase activity was measured in the same manner as in Example 1 using the cell suspension immediately after preparation by the above method and the suspension after 7 days and 21 days after preparation. did. The relative reaction rates with the reaction rate on day 0 as 1 are shown in Table 2 below.

Example 4, Comparative Example 3
Culture Pseudomonas marginalis DSM16275 (Pseudomonas marginalis DSM16275) having nitrile hydratase activity 0.2% by mass of glycerol, 0.05% by mass of citric acid, 0.15% of yeast extract, 0.3% by mass of potassium dihydrogen phosphate , A medium containing 0.7% by mass of dipotassium hydrogen phosphate, 0.0004% by mass of iron sulfate, 0.01% by mass of manganese sulfate, 1.25% by mass of methylurea, 0.001% by mass of cobalt hydrochloride (pH 7.0) ) Was aerobically cultured.

Preparation of microbial cell suspension for storage The cultured microbial cells were collected by centrifugation (12000 rpm, 20 minutes) and then washed with a 0.1% aqueous sodium acrylate solution (pH 7.0). The aqueous solution was added to the washed cells to obtain a cell suspension (10% by mass in terms of dry cells).

  100 mL (Example 4) and 80 mL (Comparative Example 3) of the cell suspension prepared by the above method were put in a polyethylene container having a total volume of 120 mL, sealed, and shaken at 30 ° C. and 120 rpm for 1 week.

Measurement of nitrile hydratase activity The nitrile hydratase activity was measured in the same manner as in Example 1 using the cell suspension immediately after preparation by the above method and the suspension after 7 days of preparation. The relative reaction rates with the reaction rate on day 0 as 1 are shown in Table 3 below.

  From the results of Examples 1, 2, 3 and 4 above, when a microbial cell having nitrile hydratase activity was transported using the transport method of the present invention, the microbial cell exhibited nitrile hydratase activity before storage. It is shown that it is stored in a state maintained at the same level.

  The present invention provides a method for preserving microbial cells that is industrially satisfactory. According to the present invention, a large amount of cells can be stored and transported at room temperature even without agitation while maintaining an enzyme activity such as nitrile hydratase. The method of the present invention is extremely useful because the cooling cost can be greatly reduced.

Rhodococcus rhodochrous J-1: Accession number “FERM BP-1478” on September 18, 1987, Patent Biodeposition Center, National Institute of Advanced Industrial Science and Technology (1st East, Tsukuba City, Ibaraki Prefecture) -1-1 Deposited at the center 6).
Pseudomonas marginalis DSM16275 (Pseudomonas marginalis DSM16275): Deposited to Deutsche Zammlung von Microorganismen und Zerkl Turren Gaembecher (DSMZ) on March 4, 2004 as the deposit number “DSM16275”.

Sequence number 7: Synthetic DNA
Sequence number 8: Synthetic DNA

Claims (3)

A method for storing microbial cells having nitrile hydratase activity in a sealable container,
The gas phase portion in the container filled with the suspension containing the microbial cells and acrylic acid is 2% to 20% of the total volume inside the container, and is stored at a temperature of freezing point to 35 ° C. Said method. A method of transporting microbial cells having nitrile hydratase activity using a sealable container,
The gas phase part in the container filled with the suspension containing the microbial cells and acrylic acid is 2% to 20% of the total volume inside the container, and is transported at a temperature of freezing point to 35 ° C. Said method. The method according to claim 1 or 2, wherein a ratio of a gas phase portion in the container is 5% or more and 20% or less.