KR100461906B1 - Process For Preparation of Bacteriorhodopsin Using Halobacterium sp. - Google Patents

Abstract

The present invention relates to a method for producing bacteriododoxin using Halobacterium sp. Specifically, the present invention is a batch culture of the microorganisms of Halobacterium until the end of the logarithmic period, and then a part of the culture medium is used to express the bacteriododocin and the remaining cultures are used to continuously produce the bacteriorodoxin by the high concentration production of the microorganisms of Halobacterium It is about a method. The method for producing bacteriododoxin of the present invention can be effectively used to continuously produce high concentrations of halobacterium sp. Microorganisms and bacteriododoxin within a short time by culturing the culture of the microorganism of the genus Halobacterium and the expression of the bacteriododoxin.

Description

Process for Preparation of Bacteriorhodopsin Using Halobacterium sp.

The present invention relates to a method for producing bacteriododoxin using Halobacterium sp. Specifically, the present invention is batch culture of the microorganisms of the halobacterium until the end of the logarithmic period, and then a part of the culture is used to express the bacteriododocin, and the remaining cultures are used to continuously produce a large amount of the bacteriododocin by the high concentration production of the microorganisms of the Halobacterium. It is about how to.

Bacteriorhodoxin is a chromoprotein that penetrates the cell membranes of Halobacterium sp. Microorganisms and is composed of the protein part bacter ioopsin and the chromophore retinal (Oesterhelt). and Tittor, TIBS 14, 57-61 (1989)). In bacteria, bacterio rhodopsin acts as a photoactivatable proton pump to convert light energy into proton motive force to maintain cell ATP levels. Bacteriododoxin acts to supply energy (ATP) to maintain cellular metabolism by changing the protein structure depending on whether light is irradiated to release or inhale protons. As the reversible photocylce properties of bacteriododocin for intracellular ATP supply have been revealed, studies are actively underway to apply it to optical data storage in biocomputers and bioelectronic devices. (RR Birge, Scientific American March 66-71 (1995); NN Vsevolodov, GR Ivanits, Biophysis 30 (5), 962-967 (1985); US Pat. No. 5,253,198).

A study on the production of bacteriododocin was introduced by Osterhelm in 1974. According to this report, halobacterium halovium was cultured using sodium chloride, magnesium sulfate hydrate, sodium citrate, potassium chloride, and peptone under a light irradiation condition at a temperature of 39 ° C., 4 L / min air supply and agitation at 200 rpm for 90 hours. Incubation produced 20-50 mg / L of bacteriododocin (D. Oesterhelt et al., Methods Enzymol., 31, 667-678 (1974)). At this time, halobacterium halovium was cultured under low dissolved oxygen and light conditions, showing that the growth curve of bacteria and the expression curve of bacteriododocin were not proportional. In other words, the bacteriorhodoxin is expressed from the retardated growth phase of the bacterial growth curve and is maintained until the stationary phase, suggesting that the growth conditions of the halobacterium halovium and the expression conditions of the bacteriorhodoxin are different. .

On the other hand, Cushin cultivated halobacterium halovium in a batch culture method to produce bacteriorhodosin, but the metabolites released during the cultivation process inhibited the growth of halobacterium halovium and limited its mass production (DJ Kushiner, Biotechnol). , 8, 237-245 (1966)).

In addition, Korean Patent Laid-Open Publication No. 1999-16880 discloses a method for producing bacteriododoxin using halobacteria. The present invention is inoculated in the incubator equipped with an inner filter and an outer filter to culture the halo bacteria in aerobic conditions, and then converted to continuous culture to culture the halo bacteria in high concentrations and keep the dissolved oxygen low to express the bacteriododocin When the incubation time was 150 hours or more, 125-200 mg of bacteriododocin was quantified from 1 L of the culture solution. The above-described invention is effective in a small volume of culture, but in the case of a large volume of culture medium or a high concentration of culture solution, a problem may occur that the filter mounted in the incubator is clogged. In addition, since the price of the filter is expensive and the lifetime is limited, it is difficult to apply in a large process for mass production, and the total incubation time is longer than 150 hours, so there is a disadvantage in that productivity is reduced.

Therefore, economic and practical methods for mass production of bacteriododocin, which are still in the spotlight as future bioelectronic devices, have not been developed.

Therefore, the present inventors completed the present invention by developing a process for mass production of bacteriododocin in a short time by performing the step of distinguishing the culture step of the microorganism of the genus Halobacterium and the expression step of the bacteriododoxin while studying the mass production method of the bacteriododocin. .

The present invention provides a method for producing bacteriododocin using microorganisms of the genus Halobacterium. Specifically, the present invention is a halobacterium spp. Culturing step, a halobacterium spp. Batch culture step, the expression step of the bacteriododocin using a part of the culture solution recovered in the batch culture and halo using the remaining cultures recovered in the batch culture It is characterized by consisting of a continuous culture step of microorganisms in the bacterium.

1 is a step-by-step illustration of a method for producing bacteriododocin using a microorganism of the genus Halobacterium according to the present invention.

Figure 2 is a graph showing the growth curve of the halobacterium halobium and the expression curve of bacteriododocin during batch culture according to an embodiment of the present invention.

The present invention provides a method for producing bacteriododocin using halobacterium microorganisms. Specifically, the present invention provides a method of mass-producing bacteriorodopsin in a relatively short time by performing a distinction between the expression stages of the bacteriorodopsin and the culture step of the microorganisms of the genus Halobacterium.

Microorganisms in the genus Halobacterium are basophils and produce ATP using oxygen under aerobic conditions, and ATP by photocirculation of bacteriododocin under anaerobic conditions. The cell membranes of aerobic microorganisms of aerobic growth are red due to many carotenoids, but in anaerobic conditions, a new component, purple, is induced. Bacterio rhodopsin is a newly emerging protein in an anaerobic environment. Therefore, in order to produce bacteriododocin using microorganisms of the genus Halobacterium, the oxygen concentration must be kept low. However, microorganisms in the genus Halobacterium can produce ATP using light in an anaerobic environment, but cannot grow. This means that the expression conditions of bacteriododocin and the culture conditions of the microorganisms of Halobacterium are different, so that the expression stage of the bacteriododoxin and the culture stage of the microorganisms of Halobacterium should be distinguished. In the present invention, the microorganisms of the genus Halobacterium are batch-cultured until the end of the logarithmic period, and then a part of the culture medium is used to express bacteriododocin, and the remaining culture medium is used for the high concentration culture of the microorganisms of the genus Halobacterium, and then the microorganisms of the genus Halobacterium cultured at high concentration. By using a portion of the culture medium of the bacteriododocin again and the remaining cultures for the cultivation of the microorganism of the genus Halobacterium provides a method for continuously producing a large amount of bacteriododocin.

The method for producing bacteriododoxin according to the present invention consists of the following four steps.

First step: seed culture of the halobacterium microorganisms in the seed culture medium and shaken culture until the growth curve of the microorganisms of the halobacterium reaches the end of the logarithmic period;

Second step: inoculate 1-10% (v / v) of the culture medium obtained in the first step into the incubator containing the culture medium until the growth curve of the microorganisms in the halobacterium reaches the end of the log phase Culturing;

Step 3: expressing the bacteriododocin by transferring a part of the culture solution recovered in the second step to a new incubator and culturing until the microorganisms in the halobacterium reach a stationary condition with limited oxygen supply; And

Fourth step: by supplying a new medium to the remaining culture solution recovered in the second step, the growth curve of the microorganisms in the halobacterium is characterized in that it consists of the step of continuous culture followed by adjusting the growth curve.

Preferred method for producing the bacteriododocin of the present invention is as shown in Figure 1 and described in more detail step by step as follows.

First stage

Species Culture Stage of Microorganisms in Halobacterium

Seed culture is for activating microorganisms in the preserved state of Halobacterium to use as an inoculum of the main culture. Inoculation of the bacteria into the culture medium undergoes a lag phase that does not grow for a while and adapts to the new environment of the medium. In order to shorten this induction period, it is necessary to activate the bacteria and then add an optimal inoculation amount. Medium for seed culture in the present invention is sodium chloride 200 ~ 300g / L, magnesium sulfate heptahydrate 10-30g / L, sodium citrate 2-5g / L, potassium chloride 1-3g / L, peptone 5-15g / L and yeast extract Cultures with 0.1-2.0 g / L added can be used. Preferably sodium chloride 220-270 g / L, magnesium sulfate heptahydrate 15-25 g / L, sodium citrate 2-3 g / L, potassium chloride 1.5-2.5 g / L, peptone 8-10 g / L and yeast extract 0.5-1.0 g / Cultures with L added can be used. The pH in the medium is adjusted to 6-8, preferably adjusted to 7.2-7.4. The culture medium for the species is sterilized at about 120 ° C. for about 15 minutes using an autoclave. The microorganisms of the genus Halobacterium are inoculated in sterile medium and shaken at 150-250 rpm at 35-40 ° C, preferably 36-38 ° C. When measured by spectrophotometer (spectrophotometer) the absorbance of the culture solution at 1.0-2.0, preferably 1.2-1.3 at 660nm is stopped and used as an inoculum for batch culture. At this time, the microorganism of the genus Halobacterium may be preferably used Haloacterium halobium (ATCC 29341, 33171, 43214).

2nd step

Batch Culture Step of Microorganisms in Halobacterium

Batch culture is a method of culturing a strain in an incubator with no medium supply or removal after the initial filling of the medium. The composition of the medium for the batch culture of the microorganisms of the genus Halobacterium is the same as the composition of the culture medium used in the first step. There is no particular limitation on the size of the incubator as the culture conditions, but preferably 10 L of the incubator is used. At the beginning of the incubation the total working volume is 50-100% (v / v) of the incubator. For example, if a 10 L incubator is used, the culture volume should be 5-10 L. The inoculation amount of the seed culture prepared in the first step is 1-10% (v / v) of the total culture solution, and preferably 1-5% (v / v) is inoculated. The air supply supplies 1.0-1.5 L (1.0-1.5 v / v / m) per liter of culture medium per minute, preferably about 1.0 L (1 v / v / m) per liter of culture medium per minute. Feed at a rate of. Light is continuously irradiated using two 40W daylight fluorescent tubes at a distance of 30 to 60 cm from the surface of the incubator, and no agitation is performed. Incubation temperature is maintained at 35-40 ℃ and preferably maintained at 36-38 ℃. Incubation time stops when the microorganisms in the halobacterium reach the end of the exponential phase. Preferably, the culture is terminated at 1.0-2.0 when the absorbance is measured at about 660 nm using a spectrophotometer. More preferably, the culture is terminated when the absorbance reaches 1.2-1.3. The time until the end of the culture takes 30-40 hours, preferably about 35 hours. When the culture is completed, part of the culture medium is used for the expression of bacteriododocin and the remaining culture medium is used for continuous culture of the microorganisms of the genus Halobacterium. Preferably, 70-90% of the culture is used for the expression of bacteriododocin and 30-10% is used for continuous culture of the genus of Halobacterium. More preferably, 80-85% of the culture is used for the expression of bacteriododocin and 15-20% is used for the continuous culture of the genus of Halobacterium.

3rd step

Expression stage of bacteriododocin

The expression step of bacteriododocin is a step of inducing the expression of bacteriolopsin by transferring a part of the culture medium of the genus of halobacterium obtained in the second step to a new incubator and then irradiating light and keeping the oxygen concentration low. The microorganism of the genus Halobacterium used in the present invention is a basophilic strain, which is a microorganism that survives in extreme conditions with a salt concentration (salt concentration) of 25% or more. Even if contaminated by various bacteria in the culturing step, other microorganisms are difficult to survive in the presence of a high concentration of salt as described above. Therefore, the microbial contamination at the step of transferring the culture medium of the microorganisms of Halobacterium to a new incubator is hardly generated. The method of transferring the culture medium of the halobacterium microorganism obtained in the second step to a new incubator can be performed aseptically or without aseptic manipulation. Preferably, a sterile method is used, and it can be easily carried out by mounting the equipment with appropriate modification according to the manufacturing process, but is not particularly limited. For example, in the process of a large plant unit, by connecting a discharge tube equipped with a switchgear and a sterilizer to the incubator for the batch culture of the microorganisms of the halobacterium in the second stage, when the batch culture is completed, the culture solution is expressed by the bacteriodopsin expression step. Can be transferred to a sterile fresh incubator. In addition, in a small laboratory unit, the flask is connected to a sampling line (sample collection line) of the incubator for batch culture of the microorganisms in Halobacterium, and sterilized together. Can be discharged through

The supply of air under the conditions of expression of bacteriolopsin is supplying the sterilized air at 0.45-0.55L (0.45-0.55v / v / m) per 1L of culture medium, and the light is 40W daylight fluorescent tube 2 Irradiate continuously while incubating at a distance of 30 to 60 cm from the surface of the incubator using dogs and do not stir separately. Incubation temperature is maintained at 35-40 ℃, preferably 36-38 ℃. Incubation time is terminated when the microorganism in the halobacterium reaches the stationary phase and no further cell growth occurs. Preferably, the spectrophotometer is used to terminate the culture when the absorbance of the culture solution is about 1.5-2.5, more preferably 1.8-2.0 at about 660 nm. The time until the end of the culture takes about 24 to 35 hours. At the end of the incubation, the temperature of the culture is cooled to about 20 ° C., and then the cells are precipitated by centrifugation at 5,000-6,000 × g, preferably at about 5,700 × g for 30-60 minutes, preferably about 40 minutes.

4th step

Continuous culture stage of microorganisms in Halobacterium

The continuous culture step is a culture method that continuously supplies new nutrient media and at the same time removes the culture solution containing the cells and products. The continuous culture step of the microorganisms in the halobacterium is a step of continuous culture of the microorganisms in the halobacterium to the late logarithmic level. That is, by supplying a new medium to a part of the culture solution obtained in the second step to maintain the cell concentration in the early phase of the logarithmic phase, the culture in aerobic conditions to incubate microorganisms in the halobacterium until the end of the logarithmic phase. The microorganism of the genus Halobacterium used in the present invention is a basophilic strain, which is a microorganism that survives in extreme conditions with a salt concentration (salt concentration) of 25% or more. Even if contaminated by various bacteria in the culturing step, other microorganisms are difficult to survive in the presence of a high concentration of salt as described above. Therefore, almost no bacterial contamination occurs at the supply stage of fresh medium and at the discharge stage of the culture medium. Therefore, the method of supplying a new medium and the method of discharging the culture medium completed in the continuous culture step of the microorganisms of Halobacterium can be carried out aseptically or without aseptic operation. Preferably, a sterile method is used, and it can be easily carried out by mounting a device with appropriate modifications according to the manufacturing process, but is not particularly limited. For example, in a large plant unit, the supply of fresh medium may supply the sterilized medium to the incubator via a sterilized transfer line, while in smaller processes, the sterilized fresh medium may be supplied in a sterile environment (ethanol). Sterilization, flame sterilization). Discharge of the culture solution is completed can be carried out in the same manner as described above in the third step. The amount of fresh medium supplied is 400-600% (v / v), preferably about 500% (v / v) of the culture medium in the second stage, so that the absorbance of the culture medium is at the beginning of the log phase of the microorganisms in the Halobacterium. Be sure to Preferably at 660 nm the absorbance is about 0.2-0.3. When incubating the air supply, the sterilized air is supplied at 1.0-1.5L per 1L of the culture medium per minute, preferably about 1.0L per 1L of the culture medium per minute, and the light is provided by two 40W daylight fluorescent tubes. Irradiate continuously during incubation at a point 30 to 60 cm away from the surface of the incubator, using no separate stirring. The incubation time ends when the growth curve of the microorganisms in the halobacterium reaches the end of the logarithmic period. The growth curve of the microorganisms in the halobacterium until the end of the logarithmic period is 20-30 hours, preferably about 24 hours. Preferably, the culture is terminated when the absorbance of the culture liquid is about 1.0-2.0 at about 660 nm. More preferably, the culture is terminated when the absorbance reaches 1.2-1.3. Some of the cultures in which the culture is completed are used for the expression of bacteriododocin, and the new culture is supplied to the remaining cultures to continuously cultivate the microorganisms of the genus Halobacterium. The continuous culture step of the microorganisms of the genus Halobacterium is repeated 20-30 hours, preferably about every 24 hours.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

Example 1

Species Culture of Halobacterium Halobium

Species of halobacterium halovium (ATCC 29341) were cultured using platinum in a colony of halobacterium halovium stored in agar plate state (250 g / L sodium chloride, 20 g / L magnesium sulfate heptahydrate). , 2.5 g / L sodium citrate, 2 g / L potassium chloride, 8 g / L peptone and 0.7 g / L yeast extract, pH7.2) were inoculated at 100 mL and shaken at 38 ° C. and 200 rpm. The culture was terminated when the absorbance reached 1.2 at 660 nm.

Example 2

Growth Curves of Halobacterium Halobium and Expression Rates of Bacteriorhodoxin

Halobacterium halovium was batch cultured to determine the growth curve of halobacterium halovium and the expression rate of bacteriododocin. 10 L incubator containing 5 L of this culture medium (250 g / L sodium chloride, 20 g / L magnesium sulfate heptahydrate, 2.5 g / L sodium citrate, 2 g / L potassium chloride, 8 g / L peptone, 0.7 g / L yeast extract, pH 7.3) (New Brunswick Scientific Co., INC., New Jersey, USA) was inoculated with 50 mL (2% (v / v)) of the culture medium of the halobacterium halovium prepared in Example 1. At this time, the culture temperature was maintained at 38 ℃ and air was supplied 1L per 1L of culture solution in 1 minute. The light was continuously irradiated using two 40 W fluorescent lamps (Westinghouse Electric Corp., USA) at a distance of 30 cm from the surface of the incubator, and no agitation was performed. Cultures were sampled at 5 hour intervals to determine the growth rate of bacteria and the expression rate of bacteriododocin. The cell concentration in the culture was measured for absorbance at 660 nm using a spectrophotometer (UV- spectrophotometer, DU-530, Beckman) and the concentration of bacteriodolopsin was measured in 9: 0.5: 5 (4N NH ₄ OH: 4N NaOH). v / v), absorbance was measured at 568 nm, and then exposed to light for 24 hours to separate retinal from bacteriododocin, and then absorbance at 568 nm was quantified from the difference (RS Shand et al., J. Bacteriol. , 173, 4692-4699 (1991)).

As a result, the growth curve of the halobacterium halovium reached a steady state at 35-70 hours through the logarithmic period of 0-35 hours, as shown in Table 1 and FIG. The expression of bacteriodhodopsin did not appear at all until 35 hours and began to express after 35 hours and increased to 70 hours. From this, it was possible to determine the culture time for the expression of the bacteriododocin of the halobacterium halovium and the continuous culture time of the halobacterium halovium.

Cell concentration and bacteriodopsin concentration over time in halobacterium halovium Absorbance / hour 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 Cell A _{660 nm} 0.07 0.09 0.2 0.25 0.4 0.6 0.9 1.2 1.3 1.5 1.6 1.7 1.8 1.8 1.9 Bacteriorhodosin A _568nm 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.06 0.55 0.8 One 1.2 1.35 1.5

Example 3

Batch Culture of Halobacterium Halobium

For continuous mass production of bacteriododocin, the cultured strains were subjected to batch culture, and then a portion of the batch culture was used for the expression of bacteriododocin and the remaining culture was used for the continuous culture of halobacterium halovium.

100 mL of halobacterium halovium cultured in Example 1 was added to the incubator in the culture medium (sodium chloride 225 g / L, magnesium sulfate heptahydrate 25 g / L, sodium citrate 3 g / L, potassium chloride 2.5 g / L, peptone 10 g / L, yeast extract 0.3g / L, pH 7.4) was inoculated in 5L batch culture. At this time, the incubation temperature was maintained at 38 ° C and the sterilized air was supplied 1 L per 1 L of the culture medium, and the light was incubated at 30 cm from the surface of the incubator using two 40 W daylight fluorescent tubes. Irradiation was carried out continuously, and there was no separate stirring.

As a result, the absorbance of the culture solution was 1.2 at 660 nm at 35 hours of culture. This corresponds to the end of the logarithmic phase of halobacterium halovium and the culture was terminated. At the end of the culture, 4.167 L (83% of the total culture) in the culture was transferred to another incubator for the expression of bacteriododocin and the remaining cultures (17% of the total culture) were subjected to continuous culture in the batch batcher used.

Example 4

Expression of bacteriododocin

4.167 L of batch cultured Halobacterium halovium culture (83% (v / v) of the total culture) was collected in a sterile flask connected to the sampling line of the incubator of Example 3 and then transferred to a new sterilized incubator for bacteriododoxin expression. I was. At this time, the sterilized air was supplied at 0.45 L per 1 L of culture medium while maintaining the incubation temperature at 38 ° C., and the light was incubated at 30 cm from the surface of the incubator using two 40 W daylight fluorescent tubes. Irradiated continuously while incubating without incubation. When the absorbance of the culture solution was 2 (cultivation time 35 hours) at 660 nm, the culture was terminated, cooled to 20 ° C, and centrifuged (5,700 xg, 40 minutes) to precipitate the halobacterium halovium. Quantification of the expressed bacteriododocin was performed in the same manner as in Example 2.

As a result, 155 mg of bacteriodopsin was quantified from 1 L of Halobacterium halovium culture.

Example 5

Continuous culture of halobacterium halovium

In Example 3, except for the culture medium used for the expression of bacteriododocin after the batch culture, fresh medium was supplied to 0.833 L of the remaining culture solution (17% of the total culture solution), and the continuous culture of the halobacterium halovium was performed. The fresh medium was sterilized by spraying 70% ethanol around the incubator and sterilized with the sterilized medium under flame sterilization around the inlet of the incubator. The fresh medium was fed 4.167 L, which is 500% (v / v) of 0.833 L of the culture. The incubation temperature was maintained at 38 ° C. The sterilized air was supplied 1 L per 1 L of culture medium per minute, and the light was continuously incubated at 30 cm from the surface of the incubator using two 40 W daylight fluorescent tubes. Irradiation was not carried out and no agitation was performed and culture was terminated after 24 hours. When the culture was terminated, 83% of the total culture solution was supplied to a new incubator for the expression of bacteriododocin, and the remaining cultures were subjected to continuous culture of halobacterium halovium in the same manner as described above.

As a result, the absorbance of the culture diluted by the supply of fresh medium was 0.24 at 660 nm, which corresponded to the beginning of log phase in the growth curve of Halobacterium halovium. After 24 hours of incubation, the absorbance of the culture was 1.2 at 660 nm, corresponding to the end of logarithmic period.

The present invention provides a method for producing bacteriododocin using microorganisms of the genus Halobacterium. The method for producing bacteriododocin of the present invention is the batch culture of the microorganisms of Halobacterium until the end of the logarithmic period, and then a part of the culture medium is used to express the bacteriorodoxin and the remaining culture medium is used for the high concentration production of the microorganisms of the halobacterium in a short time continuously. It can be useful for mass production.

Claims (12)

In the method for producing a bacteriododocin by culturing the microorganism of the genus Halobacterium, i) seeding the halobacterium microorganisms in the cultivation medium and shaking culture to seed the halobacterium microorganisms until the absorbance of the culture solution is 1.0-1.2 at 660 nm; ii) inoculating 1-10% (v / v) of the culture medium obtained in step i) in the incubator containing the culture medium and culturing the batch until the absorbance of the culture solution is 1.0-1.2 at 660 nm; iii) transferring a part of the culture solution recovered in step ii) to a new incubator to express the bacteriododocin until the absorbance of the culture solution is 1.8-2.0 at 660 nm under limited oxygen supply; iv) supplying a fresh medium to the remaining culture solution recovered in step ii) to adjust the growth curve of the microorganisms in the halobacterium to the beginning of the log phase, and then continuously cultivating it; v) a part of the halobacterium microbial culture medium continuously cultured in step iv) is transferred to a new incubator for use in the expression of bacteriododocin, and the remaining culture medium is used for continuous culture of the microorganisms in halobacterium. Method for preparing bacteriododocin. The culture medium according to claim 1, wherein the culture medium or the main culture medium is 200-300 g / L sodium chloride, 10-30 g / L magnesium sulfate heptahydrate, 2-5 g / L sodium citrate, 1-3 g / L potassium chloride, 5-15 g peptone. / L and yeast extract 0.1-2.0g / L and the method of producing bacteriododocin characterized in that the pH is 6-8. The method of claim 1, wherein the species is cultured at 35-40 ℃ 150-250rpm with agitation method for producing bacteriodopsin. According to claim 1, batch culture and continuous culture is supplied with 1.0-1.5L (1.0-1.5v / v / m) per 1L culture medium per minute air sterile at 35-40 ℃, irradiated with light Method for producing bacteriododocin, characterized in that the culture in a bath. delete The method according to claim 1, wherein 70-90% of the culture medium of the batch of Halobacterium sp. Microorganism is used for the expression of bacteriorhodoxin and 30-10% of the culture medium of the bacterio rhodopsin is used. Manufacturing method. The method of claim 1, wherein the expression of bacteriododoxin is supplied at 0.45-0.55L (0.45-0.55v / v / m) per liter of culture medium at 1-40 minutes with the sterilized air at 35-40 ° C. Method for producing bacteriododocin, characterized in that the culture. 8. The method of claim 4 or 7, wherein the irradiation of light is continuously irradiated during incubation of two 40W fluorescent lamps 30 to 60cm from the surface of the incubator. delete The method of claim 1, wherein the supply of fresh medium in continuous culture is 400-600% (v / v) of the culture medium for the production of bacteriodopsin. The method of claim 1, wherein the microorganism of the genus Halobacterium is selected from Halobacterium halovium ATCC 29341, ATCC 33171 or ATCC 43214. delete