JPH0937764A - Two stage culturing tank type enzyme producing apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a two stage culturing tank type enzyme producing apparatus capable of efficiently mass-producing an enzyme by a microorganism. SOLUTION: This enzyme producing apparatus is equipped with a microbial cell proliferating culture apparatus 1 having a culture tank 10, a heat exchanger 2A forming an inlet 19 of a culture medium at one end and forming an outlet 20 of the culture medium at other end, having indirect heater for heating the culture solution 7 and thermally inducing a microorganism and an enzyme culture apparatus 3 having a culture tank 39. The culture tank 10 of the microbial cell proliferating culture apparatus 1 is connected to an inlet 19 of culture medium of the heat exchanger 2A and the outlet 20 of culture medium of the heat exchanger 2A is connected to the culture tank 39 of the enzyme culture apparatus 3 to carry out microbial cell proliferation and enzymatic culture.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a two-stage fermenter type enzyme production apparatus for efficiently producing an enzyme with a gene recombinant using a fermenter arranged in series with a heat exchanger interposed therebetween.

[0002]

2. Description of the Related Art Conventionally, a multi-stage column tower type gas feeding culture device is disclosed in, for example, Japanese Patent Application Laid-Open No. 49-30581 as a device for culturing microorganisms in multiple stages by arranging culture vessels in series. Further, a multi-stage continuous concentration fermenter in which a plurality of fermenters are connected in series is disclosed in, for example, JP-A-51-91.
It is disclosed in Japanese Patent No. 386. Further, a method for producing high-concentration ethanol by forced feeding in a two-stage serial continuous fermentation by connecting a first-stage fermentation tank and a second-stage fermentation tank in series is disclosed in, for example, JP-A-3-103184. There is. Further, an external heat exchange type fermenter, for example, as a heat exchanger arranged outside the culture tank, is disclosed in, for example, Japanese Patent Publication No.
No. 24512. In addition, a method for culturing a microorganism in which a host bacterium in which a recombinant DNA has been lysogenized is cultured, and then the self-replication of the recombinant DNA is thermally induced, and then further cultured to accumulate the target gene product in the cells, It is disclosed in JP-A-58-28273.

[0003]

However, the present inventor has found that the above-mentioned conventional technique has the following problems. That is, JP-A-49-30
The culture (fermentation) devices disclosed in Japanese Patent Laid-Open No. 581 and Japanese Patent Application Laid-Open No. 3-103184 are merely those in which culture tanks are simply arranged in series, and when the culture solution is transferred from the culture tank to the culture tank, microorganisms Cannot induce heat. In addition, JP-A-5
In the multi-stage continuous concentration and fermentation apparatus disclosed in Japanese Patent Publication No. 1-91386, microorganisms cannot be heat-induced only by controlling the temperature of the culture solution. In addition, JP-A-58-28273
No specific production means is disclosed in the method for culturing a microorganism disclosed in 1. Therefore, the enzyme cannot be efficiently mass-produced by the microorganism.

The object of the present invention is to solve the above-mentioned problems.
It is an object of the present invention to provide a two-stage fermenter-type enzyme production apparatus capable of efficiently producing a large amount of enzymes by microorganisms. The above and other objects and novel features of the present invention will be apparent from the description of the present specification and the accompanying drawings.

[0005]

SUMMARY OF THE INVENTION Among the inventions disclosed in the present application, the outline of a representative one will be briefly described.
It is as follows. (1) The two-stage fermentor-type enzyme production device of the present invention is a bacterial cell growth and culture device having a first fermentor, a culture solution inlet is formed at one end, and a culture solution outlet is formed at the other end. A first culture tank of the microbial cell growth culture apparatus, comprising a heat exchanger that has an indirect heating device that heats a culture solution, heat-induces microorganisms, and an enzyme culture device that has a second culture tank And the culture solution inlet of the heat exchanger are connected, and the culture solution outlet of the heat exchanger and the second culture tank of the enzyme culture device are connected to each other, thereby proliferating bacterial cells and enzymes of the microorganism. It is for culturing.

(2) A two-stage fermentor type enzyme production apparatus of the present invention comprises a bacterial cell growth and culturing apparatus having a first fermentor, a culture solution inlet formed at one end, and a culture solution outlet at the other end. Is formed and has an indirect heating device for heating the culture solution and a gas supply device provided in the vicinity of the culture solution inlet for supplying a gas for activating the microorganism to the culture solution, and heat exchange for heat-inducing the microorganism. And an enzyme culture device having a second culture tank, wherein the first culture tank of the bacterial cell growth culture device and the culture solution inlet of the heat exchanger are connected and the heat The culture solution outlet of the exchanger and the second culture tank of the enzyme culture device are connected to carry out microbial cell growth and enzyme culture.

(3) In the heat exchanger of the two-stage fermentor type enzyme production apparatus of the present invention, in the means (1) or (2), an indirect heating device is formed in the heat exchange chamber and the heat exchange chamber. The heating medium inlet and the heating medium outlet, and the culture medium pipe communicating with the culture medium inlet and the culture medium outlet of the heat exchanger. (4) In the heat exchanger of the two-stage fermentation tank-type enzyme production device of the present invention, in the means (1) or (2), the indirect heating device has a spiral blade member for stirring the culture solution.

[0008]

[Operation] According to the above-mentioned means, continuous culture is possible because the bacterial cell growth culture apparatus and the enzyme culture apparatus are connected in series via a heat exchanger that heat-induces the microorganisms. The culture is further activated by providing the vessel with a gas supply device.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION

Embodiments Embodiments of the present invention will be described in detail below with reference to the drawings. Here, FIG. 1 is a schematic configuration diagram of a two-stage fermenter-type enzyme production apparatus according to an embodiment of the present invention, FIG. 2 is a sectional view of a heat exchanger according to an embodiment of the present invention, and FIG. The sectional view of the heat exchanger which concerns on the other Example of this is shown. Also, components having the same function are denoted by the same reference symbols throughout the drawings for describing the embodiments, and the repetitive description thereof will be omitted.

In FIG. 1, a two-stage culture tank type enzyme production apparatus comprises a bacterial cell growth culture apparatus 1 for liquid-culturing microorganisms, and a heat exchanger 2A for inducing heat of the microorganisms sent from the bacterial cell growth culture apparatus 1. And an enzyme culture device 3 for culturing a culture solution to produce an enzyme. A pump 4 is connected between the bacterial cell growth culture device 1 and the heat exchanger 2A, and a pump 5 is connected between the heat exchanger 2A and the enzyme culture device 3. Are sequentially transferred to the bacterial cell growth culture device 1, the heat exchanger 2A, and the enzyme culture device 3.

A culture liquid production device 6 for producing a culture liquid 7 is connected to the bacterial cell culture device 1 via a pump 8, and the culture liquid production device 6 via the pumps 9 and 42 has a heat exchanger 2A and an enzyme. The culture medium 7 is connected to the culturing apparatus 3 so that the culture solution 7 is sequentially supplied to the microbial cell growth culturing apparatus 1, the heat exchanger 2A and the enzyme culturing apparatus 3 by the pumps 8, 9, 42.

The cell culture apparatus 1 is a cylindrical culture tank 10
The culture tank 10 has an indirect heating device 11 such as a jacket for controlling the temperature of the culture solution 7, and the culture solution 7
A propeller type stirrer 12 for stirring, a measuring instrument 13 such as a dissolved oxygen meter, a turbidimeter and a pH sensor necessary for setting culture conditions or detecting the amount of cells, and a liquid level controller 14 for the culture solution 7. , A solenoid valve 4 for controlling the temperature of the indirect heating device 11
8 and a pH adjusting device 60 for the culture solution 7 are provided respectively. The pH adjusting device 60 is provided with a solenoid valve 61 for alkali adjustment and a solenoid valve 62 for acid adjustment.

The indirect heating device 11, the agitator 12, the measuring device 13, the liquid level controller 14 and the solenoid valve 48 are connected to a controller 15 such as a personal computer, and the indirect heating device 11, the agitator 12, and the like. Measuring instrument 13, liquid level controller 14
The electromagnetic valve 48 is controlled by the controller 15 to culture the microorganism. Instead of the propeller-type stirrer 12, an ordinary aeration stirrer may be used.

The heat exchanger 2A has a function of thermally inducing a microorganism having an enzyme-producing gene, for example, a lysogen of lysogenic λ phage into host Escherichia coli to express the gene. As shown in FIG. 2, the heat exchanger 2A includes a casing 16 having upper and lower portions formed in a cone shape and a central portion formed in a cylindrical shape.
Has a culture solution outlet 20 at the upper end and a culture solution inlet 19 at the lower end of the lower cone 18.

Disc-shaped partition walls 21 and 22 are provided on the upper and lower portions of the cylindrical portion of the casing 16.
A heat exchange chamber 23 is formed by 22 and the casing 16. In the heat exchange chamber 23, a large number of culture solution tubes 24 extending in the axial direction (vertical direction) of the casing 16, penetrating the upper and lower partition walls 21 and 22 and communicating with the culture solution inlet 19 and the culture solution outlet 20 are formed. ing.

In addition, the heating medium inlet 26 for introducing a heating medium such as steam or hot water to heat, ie, heat, the culture medium 7 flowing through the culture medium pipe 24 and the heating medium are discharged into the heat exchange chamber 23. The heating medium outlet 27 is formed in communication with each other, and the heating medium inlet 26, the heating medium outlet 27, and the heat exchange chamber 23 form an indirect heating device 28a.

In the lower cone 18, there is provided an oxygen supply device 29 for supplying oxygen to the culture solution 7 to further activate the microorganisms. The oxygen supply device 29 is provided on the upper surface of the holding member 33 and the holding member 33 that are formed in a so-called streamline shape with a diameter reduced toward the upstream in order to smooth the flow of oxygen, and a large number of small holes 31 are bored. It is composed of a mesh-shaped air bubble refining plate 32. Further, a gas introduction port 29a opened to the outside is formed on the upstream side of the holding member 33. The bubble-refining plate 32 has a function of reducing bubbles such as oxygen to be easily absorbed in the culture solution 7.

In the casing 16 in the vicinity of the culture solution inlet 19, the nutrient solution-rich culture solution 7 produced by the culture solution producing apparatus 6 is added to the culture solution 7 which has been absorbed by microorganisms and has become nutrient-deficient.
A culture solution supply port 37 for additionally supplying the culture solution is formed. Further, 43 is an air vent valve for discharging the gas supplied from the oxygen supply device 29, which is not absorbed by the culture solution 7, and 49 is an electromagnetic valve for controlling the amount of the heating medium.

The enzyme culturing device 3 has substantially the same construction as the microbial cell growth culturing device 1, and includes a culture tank 39 having an indirect heating device 38, an agitator 40, a dissolved oxygen meter, a turbidimeter, and a p-meter.
It is equipped with a measuring device 47 such as an H sensor or a spectrophotometer, which is necessary for setting culture conditions or detecting the produced enzyme, and a liquid level controller 46. These are controlled in the same manner as the bacterial cell growth culture device 1. While being controlled by machine 15,
The pump 42 allows the nutrient solution-rich culture solution 7 to be introduced into the culture tank 39 as needed. Further, the culture tank 39 is connected via a pump 44 to an enzyme separation device 45 using, for example, a centrifuge. further,
Reference numeral 50 is an electromagnetic valve for controlling the temperature of the indirect heating device 38, and 65 is a pH adjusting device for the culture solution 7 having an electromagnetic valve 63 for alkali adjustment and a solenoid valve 64 for acid adjustment.

Next, the operation of the two-stage culture tank type enzyme production apparatus having the above construction will be described. First, the case of the continuous type will be described. First, the culture medium 7 is produced by the culture medium producing apparatus 6, and the culture medium 7 may be a normal one, and examples thereof include glycerol as a carbon source, ammonia as an nitrogen source, and an inorganic salt. , Magnesium sulfate,
It is possible to use iron sulfate, zinc sulfate, etc., to which vitamins, nucleic acids, etc. are appropriately added, if necessary.

The culture solution 7 produced in this manner is supplied to the culture tank 10 of the bacterial cell culture apparatus 1 by a pump 8.
In the bacterial cell growth culture device 1, bacteria are added to the culture solution 7, the bacteria are cultured under normal culture conditions, and the bacterial cells are grown. Next, the growth of the bacterial cells is detected by, for example, a turbidimeter, and a part of the culture solution 7 is sent to the heat exchanger 2A by the pump 4. At this time, the culture solution 7 is replenished from the culture solution manufacturing apparatus 6 by the amount discharged. The replenishment amount of the culture solution 7 is controlled based on the liquid level controller 14 of the bacterial cell culture apparatus 1 or the concentration of the culture solution 7.

The culture solution 7 sent to the heat exchanger 2A is heated and the enzyme-producing gene is expressed. Then, the culture solution 7 containing the bacterial cells is sent to the enzyme culture device 3 and is cultured there under normal conditions. The production of the enzyme is confirmed by a spectrophotometer or the like, and then the pump 44 sends the enzyme to the enzyme separation device 45 to collect the enzyme. Next, in the case of batch type, first,
The culture solution 7 is supplied to the culture tank 10 of the bacterial cell culture apparatus 1,
The cells are then cultured. After detecting the growth of bacterial cells with a turbidimeter, the whole amount is sent to the heat exchanger 2A by the pump 4. After being heated by the heat exchanger 2A to express the enzyme-producing gene, the enzyme is further pumped to the culture tank 39 of the enzyme culture apparatus 3. Here, it is further cultured to produce the enzyme. After confirming the production of the enzyme by the spectrophotometer, the whole amount of the culture solution 7 is sent to the enzyme separation device 45 by the pump 44 to collect the enzyme.

As described above, in this embodiment, since the bacterial cell culture apparatus 1 and the enzyme culture apparatus 3 are connected in series via the heat exchanger 2A for thermally inducing the microorganisms, continuous culture becomes possible. By providing the oxygen supply device 29 in the heat exchanger 2A, the culture is further activated. As described above, the invention made by the present inventor has been specifically described based on the embodiment. However, the present invention is not limited to the embodiment, and can be variously modified without departing from the gist thereof. It goes without saying that.

The heat exchanger 2B shown in FIG.
Is omitted, and a jacket 35 is provided on the outer periphery of the casing 16. The jacket 35 is an indirect heating device 28b. If a large number of spiral blade members 36, that is, fixed agitators are provided on the inner peripheral wall 51 of the casing 16, the culture solution 7 is agitated while being transferred, and heat exchange is efficiently performed. Therefore, the same effect can be expected by using the heat exchanger 2B instead of the heat exchanger 2A.

[0025]

The effects obtained by the typical ones of the inventions disclosed in this application will be briefly described as follows.
It is as follows. According to the present invention, since the bacterial cell growth culture device and the enzyme culture device are connected in series via a heat exchanger for thermally inducing microorganisms, continuous culture becomes possible, and a gas supply device for the heat exchanger is provided. By providing, the culture is further activated. Therefore, the enzyme can be efficiently mass-produced by the microorganism.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of a two-stage fermenter-type enzyme production apparatus that is an embodiment of the present invention.

FIG. 2 is a cross-sectional view of a heat exchanger that is an embodiment of the present invention.

FIG. 3 is a sectional view of a heat exchanger according to another embodiment of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Bacterial cell growth culture apparatus 2A, 2B ... Heat exchanger 3 ... Enzyme culture apparatus 6 ... Culture solution manufacturing apparatus 7 ... Culture solution 10,39 ... Culture tank 12,40 ... Stirrer 13, 47 ... Measuring machine 14, 46 ... Liquid level controller 15 ... Controller 19 ... Culture solution inlet 20 ... Culture solution outlet 24 ... Culture solution pipe 11, 28a, 28b, 38 ... Indirect heating device 29 ... Oxygen supply device 45 ... Enzyme separation device

Front page continuation (72) Inventor Eiichi Nakano 339 Noda, Noda City, Chiba Prefecture Kikkoman Corporation

Claims (4)

[Claims] 1. A cell growth and culturing apparatus having a first culture tank, and an indirect heating device for heating the culture solution, the culture solution inlet being formed at one end and the culture solution outlet being formed at the other end. And a heat exchanger for thermally inducing a microorganism, and an enzyme culture device having a second culture tank, the first culture tank of the bacterial cell growth culture device and the culture solution inlet of the heat exchanger. Is connected and the culture solution outlet of the heat exchanger and the second culture tank of the enzyme culture device are connected to perform microbial cell growth and enzyme culture. Tank-type enzyme production equipment. 2. An indirect heating device for heating a culture solution, comprising: a bacterial cell growth culture apparatus having a first culture tank; a culture solution inlet formed at one end and a culture solution outlet formed at the other end; It has a gas supply device that is provided near the culture solution inlet and supplies a gas for activating the microorganisms to the culture solution,
A heat exchanger for inducing heat of microorganisms; and an enzyme culture device having a second culture tank, wherein the first culture tank of the bacterial cell growth culture device and the culture solution inlet of the heat exchanger are A two-stage culture tank, which is connected to the culture solution outlet of the heat exchanger and the second culture tank of the enzyme culture device to perform bacterial cell growth and enzyme culture of microorganisms. Type enzyme production equipment. 3. The indirect heating device communicates with a heat exchange chamber, a heating medium inlet and a heating medium outlet formed in the heat exchange chamber, and the culture solution inlet and the culture solution outlet of the heat exchanger. The two-stage fermentor type enzyme production device according to claim 1 or 2, which comprises a culture solution tube. 4. The two-stage fermentor type enzyme production device according to claim 1, wherein the indirect heating device has a spiral blade member for stirring the culture solution.