JPS62224286A - Method for producing enzyme by gene recombination mold and device therefor - Google Patents

Abstract

PURPOSE:To produce a large amount of the titled enzyme by device control, by cultivating a specific host mold in a medium containing a co-repressor (CRP) while suppressing enzyme formation, removing CRP from the culture solution and carrying out culture in a medium containing no CRP. CONSTITUTION:A host mold (e.g. Escherichia coli C600) transduced with an enzyme gene and an adjusting gene to form an aporepressor is cultivated in a medium containing a need amino acid, glucose and CRP (e.g. tryptophan in 100-200mug/ml concentration in a culture tank 1 at pH about 7 at about 37 deg.C for about 5hr to give a culture solution 12. Then the culture solution is sent through a piping 4, a pump 5 and a flowmeter 8 to a ceramic filter 11 to remove CRP and a prepared filtrate 12 is put in a filtrate tank 15. On the other hand, a mold-containing concentrated solution is circulated through a ball valve 16 and an outlet piping 17 to the culture tank 1. Then a medium containing no CRP is fed from a medium feed tank 26 to the tank 1 to produce an enzyme (e.g. beta-galactosidase).

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an enzyme production method using genetically modified bacteria and an enzyme production apparatus used in the method, and particularly to one used for producing enzymes in large quantities.

[Conventional technology]

In recent years, the production of useful substances using genetically modified bacteria has attracted attention. However, the production of these useful substances remains in the experimental stage in many cases and has not yet reached the stage where mass production is possible.

[Problem that the invention seeks to solve]

The present invention has been made in consideration of the above circumstances, and aims to provide an enzyme production method using genetically modified bacteria that can produce enzymes in large quantities, and an enzyme production device used in this method.

[Means for solving problems]

The enzyme production method using a directly genetically recombinant bacterium of the first invention of the present application involves culturing a host bacterium into which an enzyme gene and a regulatory gene for producing apolipressor have been introduced in a state in which enzyme production is suppressed using a medium containing a corepressor. After that, the corepressor is removed from the culture solution, and the culture is performed using a medium that does not contain the corepressor to produce the enzyme.

Moreover, the enzyme production device of the second invention of the present application generates an enzyme gene and an apolipressor. A culture tank for culturing host bacteria into which a gene has been introduced using a medium containing a corepressor; a cylindrical filter for removing the corepressor by passing the culture solution in the culture tank; The invention is characterized by comprising a circulation system that circulates the medium through a filter, and a medium supply tank that supplies a corepressor-free medium to the culture tank.

[Effect]

In host bacteria into which the enzyme gene and the regulatory gene that produces apolipressor are introduced, apolipressor is produced in the 4A setting. In the method of the present invention, the host fungus is initially cultured using a medium containing a corepressor, so apolipressor produced by the regulatory gene combines with the corepressor to become a repressor, and this repressor acts as a repressor for the enzyme gene. Binds to operator sites and represses transcription.

Therefore, the bacterial cells proliferate while the production of ffj element is suppressed, and the bacterial cell number itself increases. Thereafter, the corepressor is removed from the culture solution and culture is performed using a culture solution that does not contain the corepressor, so that the enzyme is produced by the host bacteria. Since enzymes are produced after increasing the number of bacterial cells in this way, mass production of enzymes becomes possible.

Furthermore, the enzyme production apparatus of the present invention is provided with a culture tank, a cylindrical filter serving as a means for removing corepressors from the culture solution, a circulation system for the culture solution, and a medium supply tank for supplying a medium containing no corepressor. It is something. Since the corepressor is quickly removed by cross-flow filtration in a cylindrical filter, the enzyme production method described in section h can be easily applied, and enzyme production can be controlled and mass-produced.

〔Example〕

Examples in which the present invention is applied to the production of β-galactosidase will be described below with reference to the drawings.

First, by genetic recombination technology, a plasmid p containing the tryptophan promoter trp was created, as shown in Figure 3.
β-galactosidase gene lac Z in oc”r2
, β-galactoside permease gene lac Y, and galactoside transacetylase gene Iac A were introduced into plasmid pMCT98. Promoter P, operator 0, and attenuator At are present at the site trp indicated by the black arrow.

Next, pMC79B and apolipL/-/trpR
The cloned plasmid pRLKl 3 (m4 diagram) was transplanted into Escherichia coli C600 for transformation in the presence of metal ions.

In this transformed E. coli C600, tryptophan, which is a corepressor, is present in plasmid pRLK13.
It combines with the apolypressor produced at the τpR site to produce a repressor, and this repressor
It binds to the trp site operator of MCT98 and suppresses transcription. Conversely, transcription begins in the absence of tryptophan. The table below shows the tryptophan concentration and β-
The relationship with the specific activity of galactosidase is shown. As is clear from the table below, the higher the tryptophan concentration, the lower the specific activity.

1. - Using this E. coli, PIIS, the schematic structure of which is shown in Figure 1, was created.
β-galactosidase was produced using the R production apparatus. First, the enzyme production apparatus shown in FIG. 1 will be explained. In FIG. 1, a culture solution 2 of genetically recombinant E. coli C600 obtained as described above is contained in a culture tank 1. This culture solution 2 is maintained at a constant temperature by constant temperature water sent from a constant temperature bath 3. When performing cross-flow oral filtration, this culture solution 2 is passed through a pipe 7, which is equipped with a pipe 4, a pump 5, and a Pall valve 6. Flowmeter 8 and inlet piping 9
It passes through the ceramic filter 11 provided in the filter case 10.

The oral fluid 12 that has passed through the ceramic filter 11 passes through the oral fluid piping 14 having an electromagnetic valve 13 interposed therebetween, and is stored in the oral fluid tank 15 . On the other hand, the Clii liquid containing bacterial cells passes through the outlet pipe 17 with the Pall valve 16 interposed in the culture tank.
is circulated. Note that a bypass pipe 19 having a pole valve 18 interposed therebetween is connected in parallel with the pump 5 between the pipe 4 and the pipe 7. Moreover, pressure gauges 20 and 21 are provided in the inlet pipe 9 and the outlet pipe 17, respectively. Further, a gas supply pipe 23 for backwashing in which a solenoid valve 22 is interposed is connected to the one-port effluent pipe 14 . Furthermore, the culture tank 1 is provided with a liquid level gauge 24.
New culture solution is supplied from the culture medium supply tank 26 to the culture tank 1 by the pump 25 which is interlocked with the oral fluid.

The ceramic filter 11 shown in FIG. 2 was used. This filter 31 has a total R750mm
Ceramic seals 33 are attached to both ends of a hexagonal prism-shaped alumina filter body 32, and 19 passage holes 34 with a diameter of 4 mm are provided along their longitudinal direction, and the effective opening area is 0. The filter main body 32 has a multilayer structure in which the pore diameter gradually increases around the passage hole 34 (Cerabel Ceramic Filter: a trade name manufactured by Toshiba Ceramics Co., Ltd.). Also, the pore diameter of the alumina in the passage holes of each filter was 0.2 gm.

β-galactosidase was produced using the L enzyme production apparatus in the following manner.

First, using a fed-batch culture medium to which required amino acids, glucose as a carbon source, and tryptophan at a concentration of 100 to 200 μg/ml were added, the culture was carried out at 37° C. and pH 7 for 5 hours.

Next, l containing tryptophan from the medium supply tank 26,
While supplying a fed-batch culture medium with the same composition as above except for The solution was circulated to culture tank 1.

The relationship between the culture time, tryptophan concentration, bacterial cell turbidity, total activity and specific activity of β-galactosidase at this time is shown in FIG. 5. In FIG. 5, the time at which oral passage begins is indicated by a broken line. Furthermore, FIG. 6 shows the relationship between oral fluid volume and tryptophan concentration, and FIG. 7 shows the relationship between culture time and oral wave flux.

As is clear from Fig. 5, the tryptophan concentration in the culture solution decreases rapidly after the start of sips.The decrease in tryptophan concentration immediately after the start of sips is as shown in Fig. 6. It can be seen that tryptophan can be removed quickly.

Moreover, the curve of bacterial cell turbidity in FIG. 5 shows that the bacterial cells are gradually proliferating except immediately after the beginning of oral passage. However, the rate of increase in bacterial cells was greater before the start of oral congestion than after the initiation of oral congestion. This is thought to be due to the fact that before the start of filtration, enzymes are not produced and only bacterial cells proliferate, but after the start of filtration, tryptophan decreases, so the bacteria are producing enzymes and refurbishing. It will be done. This can be seen from the fact that the curves of the total activity and specific activity of β-galactosidase shown in FIG. 5 have very low values before the start of oral laxation, but rapidly increase after the initiation of oral laxation. In this way, enzyme production can be controlled and mass production of enzymes becomes possible.

Moreover, as shown in FIG. 7, although the mouth flux gradually decreases with time, it is understood that the mouth flux can be recovered to some extent by adjusting the backwash interval and backwash pressure.

In Example L, an alumina ceramic filter having 19 passage holes as shown in Fig. 2 was used as a filter for cross-flow filtration, but the filter had uniform pore diameters. If so, it may be made of any material such as metal or synthetic resin, and may have one or more passage holes.

〔Effect of the invention〕

As detailed above, according to the present invention, enzyme production can be controlled, and mass production of enzymes is also possible.
It has a remarkable effect.

[Brief explanation of drawings]

Figure 1 is a schematic configuration diagram of an enzyme production apparatus in an embodiment of the present invention, and Figure 2 shows a filter used in the apparatus.
3 and 4 are respectively explanatory diagrams of the plasmids transplanted into E. coli in the examples of the present invention, and FIG. 5 is the culture time obtained in the examples of the present invention, A characteristic diagram showing the relationship between tryptophan concentration, bacterial cell turbidity, total enzyme activity, and enzyme specific activity: Figure 56 is a characteristic diagram showing the relationship between oral fluid itching obtained in the example of the present invention and tryptophan concentration. Figure 7 shows the 18
FIG. 5 is a characteristic diagram showing the relationship between five hours and oral flux. 1...Culture tank, 2...Culture solution, 3...Thermostat, 4
, 7... Piping, 5... Pump, 6.16.18...
・Pole valve, 8...flow 1 meter, 9...inlet piping,
DESCRIPTION OF SYMBOLS 10... Filter case, 11... Ceramic filter, 12... Mouth filtration liquid, 13.22... Solenoid valve, 14... Mouth filtration liquid piping, 15... Mouth filtration tank, 17・
...Outlet piping, 19...Bypass piping, 20.21.
... Pressure gauge, 23 ... Gas supply piping, 24 ... Liquid level gauge, 25 ... Pump, 26 ... Culture medium supply tank, 31 ...
... Ceramic filter, 32... Filter body, 33... Ceramic seal, 34... Passing hole. Applicant's agent Patent attorney Takehiko Suzue H: HindII+ R: Eco RT 7t3FEJ Figure 4 4 steps fl (hr) F 5 mouths

Claims (2)

[Claims] (1) After culturing a host bacterium into which an enzyme gene and a regulatory gene for producing apolipressor have been introduced in a state in which enzyme production is suppressed using a medium containing a corepressor, the corepressor is removed from the culture solution, and A method for producing an enzyme using a genetically modified bacterium, which is characterized by producing the enzyme by culturing it in a medium that does not contain a corepressor. (2) A culture tank in which a host bacterium into which an enzyme gene and a regulatory gene that produces apolipressor has been introduced is cultured using a medium containing a corepressor, and a culture solution in the culture tank is passed through to remove the corepressor. A cylindrical filter, a circulation system that circulates the culture solution from the culture tank through the filter, and a medium supply tank that supplies a corepressor-free medium to the culture tank. Enzyme production equipment.