JP2011072191A - Method for producing target substance by feed culture of microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a target substance by feeding a sugar powder to a culture vessel under a condition of enabling the sterilization without dissolving and caking the sugar at the feed, and using the sugar. <P>SOLUTION: The fermentation processing method includes continuously or intermittently feeding the feed sugar powder containing glucose and sucrose to the culture vessel through a feed pipe to enable high productivity in the method for producing the target substance by culturing microorganisms having target substance-producing abilities in a liquid medium in the culture vessel and collecting the target substance from the medium. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing a target substance using microorganisms, and more particularly, to a method for culturing while feeding sugar powder to an incubator.

Substances such as L-amino acids and nucleic acids are produced by fermentation using microorganisms. In the fermentation method, the yield of the target substance depends on the amount of carbon sources such as glucose and sucrose, but if the sugar concentration in the medium is too high, the growth of microorganisms will be worse, and the productivity of the target substance will be low. In many cases, the culture is performed while feeding a sugar solution according to the consumption of sugar in the medium.
The sugar solution is usually fed through a feed tube for feeding the sugar solution to the fermenter. However, if the sugar concentration is high, the sugar may solidify in the feed tube. Is also about 750 g / L. However, since the amount of the medium in the fermenter increases by feeding the sugar solution, there is a limit to the amount of the sugar solution that can be fed, and the medium components are diluted.

  Moreover, as a carbon source of a medium used for fermentation, a mixture of a plurality of sugars, for example, a mixture of glucose and fructose (Patent Document 1) and a mixture of glucose and sucrose (Patent Document 2) are disclosed.

  However, there is no known report that the physical form or composition of sugar has been studied as a feed sugar.

European Patent Application Publication No. 1225230 JP 2006-204132 A

  In the feed culture, when the sugar solution is fed, the amount of the medium in the incubator such as a fermenter increases, and when the concentration of the sugar solution is increased, the sugar is solidified in the feed tube. It is an object to provide a technique for avoiding the problem.

As a result of intensive studies to solve the above problems, the present inventors have found that the above problems can be solved by feeding powdered sugar to an incubator, and have completed the present invention.
That is, the present invention is as follows: (1) A method for producing a target substance, comprising culturing a microorganism having a target substance-producing ability in a liquid medium in an incubator and recovering the target substance from the medium.
A method of feeding sucrose or a sugar powder containing sucrose and glucose continuously or intermittently to the incubator.
(2) The method according to claim 1, wherein the sugar is a mixture comprising 95% by weight or more of sucrose and 5% by weight or less of glucose.
(3) The method according to claim 1 or 2, wherein the sugar is pasteurized at 140 ° C or higher and 175 ° C or lower.

  According to the present invention, in the feed culture, an increase in the amount of the medium in the incubator can be reduced as compared with the conventional case. In addition, the method of the present invention can overcome the problems of the prior art such as solidification and crystallization of sugar in the feed liquid.

The figure which shows one form of the fermenter used for the method of this invention.

Hereinafter, the present invention will be described in detail.
The present invention is a method for producing a target substance by culturing a microorganism having a target substance-producing ability in a liquid medium in an incubator, and recovering the target substance from the medium, wherein the incubator contains sucrose or sucrose and It is a method characterized by feeding a sugar powder containing glucose continuously or intermittently. Hereinafter, the culture accompanied by the feed sugar feed may be referred to as “fed-batch culture”. In addition, the sugar powder to be fed is sometimes referred to as “feed sugar”.

  In the present invention, the feed sugar includes sucrose, or sucrose and glucose. “Contains” sucrose or sucrose and glucose also means that sucrose or sucrose and glucose alone may be contained, and a saccharide other than sucrose and glucose may be contained in a trace amount. Fructose is mentioned as sugars other than sucrose and glucose. Such sugars other than sucrose and glucose are preferably 50% by weight or less, preferably 10% by weight or less, more preferably 2% by weight or less based on the total amount of feed sugar.

  The amount ratio of sucrose and glucose in the feed sugar is not particularly limited as long as each component of the feed sugar does not melt and deliquesce and can be used as a carbon source for the growth of microorganisms or production of a target substance. Since the melting point of sucrose is 186 ° C., the melting point of α-D-glucose is 146 ° C. and the melting point of β-D-glucose is 150 ° C., the quantity ratio of sucrose and glucose is appropriately set according to the sterilization temperature. be able to. Alternatively, the sterilization temperature may be set according to the amount ratio of sucrose and glucose. A suitable amount ratio of sucrose and glucose according to the sterilization temperature can be appropriately set according to the method described in the examples. For example, when the sterilization temperature is 175 ° C., the sucrose is preferably 95% by weight or more, more preferably 99% by weight or more based on the total amount of sucrose and glucose. Moreover, it is preferable that glucose is 5 weight% or less with respect to the total amount of sucrose and glucose, and it is more preferable that it is 1 weight% or less. If the sterilization temperature is lower than this, the amount of glucose may exceed the above range.

  The feed sugar can be prepared by mixing a predetermined amount of sucrose powder and glucose powder. When the feed sugar is composed only of sucrose, the sucrose powder can be used as it is. When the feed sugar contains sugars other than sucrose and glucose, the sugar powder may be mixed with sucrose powder, or sucrose powder and glucose powder. The size (particle size) of the powder is not particularly limited, but is preferably 0.01 mm to 2 mm, more preferably 0.01 mm to 0.5 mm, and still more preferably 0.01 mm to 0.05 mm. In addition, after mixing each solid saccharide | sugar, you may grind | pulverize and may size-regulate as needed.

The feed sugar is sterilized before being introduced into the incubator. The feed sugar may be sterilized after preparation, or the sterilized components may be mixed to prepare the feed sugar. Preferably, each component is mixed and then sterilized.
The method of sterilization is not particularly limited as long as each component of the feed sugar does not melt and deliquesce, and the properties as a carbon source for the growth of microorganisms or the production of target substances are maintained. dry heat), irradiation with infrared rays, irradiation with ultraviolet rays, X-rays or gamma rays.

  In addition, sucrose in feed sugar may decompose | disassemble into glucose and fructose by sterilization etc., and a sugar composition may change. Even if the sugar composition is changed in this manner, such a feed sugar is included in the scope of the present invention as long as the feed sugar can be used as a carbon source for the growth of microorganisms or the production of a target substance.

  Until now, it has not been known in detail how the physical properties change when sugars such as sucrose and glucose, especially solid sugars, are placed at a high temperature. In addition, when changes occur, it has not been known what effect the assimilation by sugar microorganisms has. According to the present invention, it was shown that sugar powder sterilized under suitable conditions is suitable as a carbon source for the growth of microorganisms or the production of useful substances.

  The sterilization temperature of the feed sugar is preferably 140 ° C. or higher and lower than 186 ° C. which is the melting point of sucrose. Further, it is more preferably 175 ° C. or lower. Depending on the proportion of glucose in the feed sugar, a lower temperature is preferred.

  Although the sterilization time may vary depending on the temperature, the purpose can usually be achieved in 15 minutes or longer, or 30 minutes or longer. The sterilization time is preferably 90 minutes or less, but may be longer as long as the object of the present invention can be achieved. Depending on the sterilization method, it may take time to raise the temperature of the feed sugar itself. In such a case, it is preferable to sterilize the feed sugar after the temperature of the feed sugar reaches 140 ° C.

  In the present invention, glucose and sucrose may be purified or may be a crude product. Examples of glucose include tapioca sugar and purified glucose. Examples of sucrose include crude sugar (derived from sugarcane) and purified sucrose.

  In the present invention, the feed sugar may contain other components in addition to sucrose, or sucrose and glucose, and these degradation products that can be contained within an acceptable range, as long as the effects of the present invention are not impaired. Examples of such components include phosphoric acid, potassium phosphate, ammonium sulfate, antifoaming agent, magnesium, required amino acids, and medium components. In addition to the feed sugar of the present invention, other feed liquids containing medium components and the like may be fed as necessary.

  The method for feeding sugar is not particularly limited, and various means for supplying powder can be employed. For example, there is a method in which the feed sugar is put into a tube that communicates the storage tank containing the feed sugar and the incubator, and the feed sugar is pumped to the incubator with a gas such as air. A powder feeder such as a hopper or a screw feeder may be interposed between the tube and the incubator. The sugar feed amount can be adjusted using a sensor, a valve or the like.

  The sterilizer is not particularly limited, and examples thereof include an electric oven and a radiation generator. The position at which the sterilizer is provided in the culture apparatus is not particularly limited, and can be provided, for example, between a feed sugar storage tank for storing the feed sugar and the incubator.

  Microorganism can be cultured in the same manner as normal fermentation except that a predetermined feed sugar powder is fed continuously or intermittently to a culture vessel. As the incubator, a normal incubator such as a fermenter or fermenter can be used. An example of the culture apparatus used in the present invention will be described later.

  The target substance produced in the present invention is not particularly limited as long as it can be produced by microorganisms using glucose and sucrose as a carbon source, and examples thereof include amino acids, nucleic acids, vitamins, antibiotics, growth factors, physiologically active substances, and proteins. Can be mentioned. These target substances may be salts.

  As amino acids, L-glutamic acid, L-glutamine, L-lysine, L-leucine, L-isoleucine, L-valine, L-tryptophan, L-phenylalanine, L-tyrosine, L-threonine, L-methionine, L- Examples include cysteine, L-cystine, L-arginine, L-serine, L-proline, L-aspartic acid, L-asparagine, L-histidine, glycine, and L-alanine. The amino acid may be a free amino acid or a salt such as sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, potassium salt and the like.

  Examples of nucleic acids include inosine, guanosine, xanthosine, adenosine, inosinic acid, guanylic acid, xanthylic acid, adenylic acid and the like. The nucleic acid may be a free nucleic acid or a salt such as sodium salt or potassium salt.

The microorganism used in the present invention is not particularly limited as long as it can produce a target substance using sucrose or sucrose and glucose as a carbon source, and is not limited to Escherichia, Enterobacter, Pantoea Enterobacteria belonging to γ-proteobacteria such as Klebsiella, Raoultella, Serratia, Erwinia, Salmonella, Morganella, etc. In so-called coryneform bacteria belonging to the genus Bacteria, Corynebacterium, Microbacterium, Alicyclobacillus genus, Bacillus genus bacteria, Saccharomyces genus and Candida etc. And yeasts to which it belongs.
Regarding L-amino acid-producing bacteria or nucleic acid-producing bacteria or microorganisms used for breeding thereof, and methods for imparting and enhancing L-amino acid-producing ability and nucleic acid-producing ability, WO2007 / 125954, WO2005 / 095627, US Patent Publication 2004- No. 0166575 and the like.

  The medium should be a medium used for the production of the target substance using ordinary microorganisms, specifically a carbon source, nitrogen source, inorganic salts, and other medium containing organic micronutrients such as amino acids and vitamins as necessary. Can do. Either synthetic or natural media can be used.

  The carbon source contained in the initial culture medium may be sucrose and / or glucose, and may be another carbon source or a mixture thereof. Other carbon sources include glycerol, fructose, maltose, mannose, galactose, starch hydrolysates, sugars such as molasses, organic acids such as acetic acid and citric acid, and alcohols such as ethanol.

  As the nitrogen source, ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, and ammonium acetate, nitrates, and the like can be used.

  Organic micronutrients include amino acids, vitamins, fatty acids, nucleic acids, and peptone, casamino acids, yeast extracts, soybean protein breakdown products, etc. containing these, and auxotrophic mutations that require amino acids for growth. When using a strain, it is preferable to supplement the required nutrients.

  As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like can be used.

  The culture conditions may be set as appropriate according to the microorganism used.

  The medium on which the feed culture is performed may be directly inoculated with a microorganism cultured on a solid medium such as an agar medium, but preferably inoculated with a microorganism previously cultured in a liquid medium (seed culture or preculture). . The seed culture or the preculture can be performed by a usual method.

  The feed amount of the feed sugar and the feed timing can be appropriately set according to the sugar consumption in the medium by the microorganism, etc., in accordance with conventional feed culture using a sugar solution. For example, there is a method of measuring the sugar concentration in the medium and feeding when the sugar is depleted or when the sugar concentration becomes a predetermined value or less. Further, feeding may be performed continuously or intermittently.

  A method for collecting the target substance from the culture solution after the completion of the culture may be performed according to a known recovery method according to the type of the target substance. For example, if the target substance is an amino acid, it is collected by a method of concentrating crystallization after removing bacterial cells from the culture solution or ion exchange chromatography. The collection of the target substance from the medium after completion of the culture does not require a special method in the present invention. In addition to the target substance, the target substance collected in the present invention may contain microbial cells, medium components, moisture, and microbial metabolic byproducts.

Below, an example of the culture apparatus for implementing this invention is demonstrated based on FIG. 1, However, This invention is not limited to use of this culture apparatus.
The culture apparatus includes an incubator 1, a feed sugar storage tank 18 for storing the feed sugar fed to the incubator 1, and a feed sugar transfer pipe 17 for supplying the feed sugar from the feed sugar storage tank to the incubator 1. ing. The incubator 1 includes a stirring blade 5 that stirs the culture medium, and a stirring motor 6 that rotates the stirring blade 5. The feed sugar accommodated in the feed sugar storage tank 18 is fed into the feed sugar transfer pipe 17. The amount of feed sugar is adjusted by the first feed amount adjustment valve 19. The feed sugar introduced into the feed sugar transfer pipe 17 is pumped to the metering feeder 13 by the air supplied to the feed sugar transfer pipe 17. The feed sugar is transferred to the sterilization chamber 22 by the conveyor 21 and sterilized by the heater 12 while the feed amount is adjusted by the fixed amount feeder 13. The sterilized feed sugar is flowed down by the hopper 10 to the screw feeder 9 suspended from the discharge port of the hopper 10 and fed to the incubator 1. A level meter 11 is installed in the hopper 10 and is adjusted by the feed sugar addition controller 15 and the second feed amount adjustment valve 14 so that feed sugar exceeding a predetermined amount is not supplied to the hopper 10. The feed sugar fed to the incubator 1 is also adjusted by a third feed amount adjusting valve provided between the screw feeder 9 and the incubator 1. The incubator 1 may be provided with a sugar concentration measurement device (not shown), measure the sugar concentration in the medium, and adjust the feed amount according to the measured value. The culture apparatus may be provided with means for measuring and recording the amount of feed sugar introduced into the incubator 1 over time or cumulatively. The measured value is used as an index of the operation of the quantitative feeder 13 and the feed sugar addition controller 15, for example.

  An air regulating valve 20 is provided upstream of the connecting portion of the feed sugar transfer pipe 17 with the feed sugar storage tank 18. The quantitative feeder 13 communicates with the outside via the bag filter 16, and fine powder of the feed sugar sent to the quantitative feeder 13 is collected by the bug filter 16.

Sterilization of feed sugar in the sterilization chamber can be adjusted by heating temperature and heating time. Moreover, the transfer and sterilization of the feed sugar to the sterilization chamber 22 may be performed continuously or in a batch manner. Furthermore, the sterilization chamber 22 and the heater 12 may be integrated as an electric oven, for example. In addition, the configurations of the sterilization chamber and the heating chamber can be variously changed depending on the sterilization method.

  In order to promote the growth of microorganisms or the production of a target substance, the incubator 1 may be supplied with air or a gas containing oxygen or carbon dioxide from the gas supply pipe 2. The incubator 1 is provided with an exhaust pipe for exhausting gas, and the supplied gas and the gas produced by fermentation or a part thereof are foamed 3, and then released to the outside through the exhaust pipe 4. .

  Hereinafter, the present invention will be described more specifically with reference to examples.

[Example 1] Examination of sterilization conditions of sucrose powder (1)
In a test tube, put 1g of crude sugar powder (derived from sugarcane, average powder size 1.5mm, containing 99% by weight of sucrose, hereinafter sometimes referred to as "sucrose"), and using a dry heat sterilizer. Dry heat treatment was performed at 50 ° C, 80 ° C, 110 ° C, or 140 ° C for 30 minutes, 60 minutes, or 90 minutes. These sucrose powders were put into LB medium (50 mL), and cultured with shaking (37 ° C., 115 rpm) for 24 hours, 48 hours, or 72 hours. Each culture solution was appropriately diluted, inoculated into 0.5 mL tryptosa agar medium, and cultured at 37 ° C. After 24 hours, the presence or absence of colonies on the medium was visually determined. The results are shown in Table 1. From this result, it was shown that the sucrose powder can be sterilized by dry heat treatment at 140 ° C. for 30 minutes.

[Example 2] Examination of sterilization conditions of sucrose powder (2)
1 g of sucrose (powder size average 1.5 mm) was thinly laid on an aluminum foil and placed in a dry heat sterilizer at 140 ° C. Thereafter, the sample was taken out at each time of 1 to 30 minutes, dissolved in 50 ml of LB medium in the flask, and cultured at 37 ° C. After 24 hours, 48 hours, and 72 hours, the presence or absence of propagation of various bacteria in the medium was visually evaluated. As a positive control, when culturing was similarly carried out using sucrose not subjected to dry heat sterilization, the number of miscellaneous bacteria was 1 × 10 ⁷ cells / ml. The results are shown in Table 2. The same result was obtained at any culture time. It was shown that sterilization is possible at 140 ° C even by dry heat treatment for 15 minutes.

[Example 3] Examination of sterilization conditions of mixed sugar powder of sucrose and glucose Mixture of sucrose powder (powder size average 0.5 mm) and glucose powder (powder size average 0.5 mm) Was subjected to dry heat treatment at 175 ° C. for 30 minutes, and the presence or absence of melting was visually determined. The results are shown in Table 3. It was shown that when the proportion of glucose was at least 5% by weight or less, it was not melted even by dry heat treatment at 175 ° C.

[Example 4] Cultivation of Escherichia coli using dry heat sterilized sucrose powder L-phenylalanine producing bacteria derived from Escherichia coli K12 strain were inoculated into 300 ml of a medium having the following composition in a 1 L incubator, and 35 ° C. Incubated with shaking (1000 rpm). In the culture J1, sucrose was dissolved in the medium and then autoclaved. In culture J2, sucrose powder (powder size average 1.5 mm) that has been sterilized by dry heat (in a dry heat sterilizer, 140 minutes, 30 minutes) is dissolved in an autoclaved medium, and a predetermined volume (300 ml) is obtained with sterilized water. It was filled up to become. The L-phenylalanine concentration in the culture solution after 21 hours of culture was quantified with an amino acid analyzer. The results are shown in Table 4. The L-phenylalanine concentration was expressed as a relative value where the value at J1 was 1. Moreover, the growth after 21 hours of culture was measured with a turbidimeter. The results are shown in Table 4. Growth was shown as a relative value with a value of J1 being 1.

[Medium composition]
Sucrose 30g / L
MgSO ₄・ 7H ₂ O 1g / L
Soybean hydrolyzate 300mg / L (as total nitrogen content)
H ₃ PO ₄ 0.7g / L
(NH ₄ ) ₂ SO ₄ 1g / L
Vitamin B ₆ 0.7mg / L
Vitamin B ₁₂ 0.01mg / L
FeSO ₄・ 7H ₂ O 10mg / L
MnSO ₄・ 5H ₂ O 8mg / L
KOH 1.7g / L
Defoamer 0.05ml / L
pH6.5

  The growth of Escherichia coli was equivalent in J1 and J2, and as a carbon source for microorganisms, it was shown that sucrose sterilized by dry heat was equivalent to sucrose sterilized by autoclave. Moreover, it was shown that the sucrose pasteurized by dry heat can be used suitably as a carbon source for L-phenylalanine production.

[Example 5] L-phenylalanine production culture using dry heat sterilized sucrose powder An L-phenylalanine producing bacterium derived from Escherichia coli K12 strain was cultured in the same manner as in Example 4 (sucrose was autoclaved), The culture solution was inoculated into 250 ml of the main fermentation medium having the following composition in a 1 L incubator so as to be 5.0 V / V%, and cultured with shaking at 36 ° C. (1000 rpm). In the above medium, sucrose and MgSO ₄ · 7H ₂ O are autoclaved separately from the other ingredients, mixed with each ingredient, and finally filled up with sterilized water to a predetermined liquid volume (250 ml). did.

[Main fermentation medium]
Sucrose 40g / L
MgSO ₄・ 7H ₂ O 2g / L
Soybean hydrolyzate 100mg / L (as total nitrogen content)
H ₃ PO ₄ 0.8g / L
(NH ₄ ) ₂ SO ₄ 2g / L
Vitamin B ₆ 0.7mg / L
Vitamin B ₁₂ 0.004mg / L
FeSO ₄・ 7H ₂ O 20mg / L
MnSO ₄・ 5H ₂ O 20mg / L
KOH 0.7g / L
Defoamer 0.02ml / L
pH 7

  During the culture, sucrose was fed at the timing and amount shown in Table 5. The sugar was fed while confirming that there was no residual sugar in the medium. The amount of sucrose listed in Table 5 is a cumulative amount.

In the culture J-1, an autoclave-sterilized sucrose solution (600 g / L) was used as a feed sugar.
In the culture J-2, sucrose powder (powder size average 1.5 mm) sterilized by dry heat at 140 ° C. (30 minutes in a dry heat sterilizer) was used.
In the culture J-3, sucrose powder (powder size average 1.5 mm) sterilized by dry heat at 200 ° C. (in a dry heat sterilizer for 30 minutes) was used. The sucrose sterilized at 200 ° C. was completely dissolved and solidified aseptically at room temperature, and then finely crushed with a hammer (powder size average 1.5 mm).

  After 35 hours of culture, the L-phenylalanine concentration in the medium was quantified. The results are shown in Table 6. L-phenylalanine yield to sugar, productivity (production rate: production amount per unit time), yield (L-phenylalanine concentration in the final medium), and final liquid volume are the values in J-1, respectively. The relative value is 1.

Compared with the conventional method (J-1), the method (J-2) of the present invention has improved productivity and yield. In addition, in the culture (J-3) using sucrose that was over-sterilized, all of yield to sugar, productivity, and yield were low compared to other cultures. The growth of Escherichia coli was similar in J-1 and J-2, but in J3, the growth after sugar feed was inferior to J-1 and J-2.

DESCRIPTION OF SYMBOLS 1 Incubator 2 Gas supply pipe 3 Foam 4 Exhaust pipe 5 Stirring blade 6 Stirring motor 7 Feed amount control valve 8 Feed sugar addition controller 9 Screw feeder 10 Hopper 11 Level meter (powder level meter)
DESCRIPTION OF SYMBOLS 12 Heater 13 Fixed quantity feeder 14 Feed amount control valve 15 Feed sugar addition controller 16 Bag filter 17 Feed sugar transfer pipe 18 Feed sugar storage tank 19 Feed amount control valve 20 Air control valve 21 Conveyor 22 Sterilization room

Claims (3)

A method for producing a target substance, comprising culturing a microorganism having a target substance-producing ability in a liquid medium in an incubator and recovering the target substance from the medium,
A method of feeding sucrose or a sugar powder containing sucrose and glucose continuously or intermittently to the incubator.   The method according to claim 1, wherein the sugar is a mixture comprising 90% by weight or more of sucrose and 10% by weight or less of glucose.   The method according to claim 1 or 2, wherein the sugar is pasteurized at 140 ° C or higher and 175 ° C or lower.

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