JPH0427384A - Production of formic acid-dehydrogenating enzyme - Google Patents

Abstract

PURPOSE:To obtain the subject enzyme with a simple operation in high productivity with making possible of a high-density cultivation of strain by controlling concentration of methanol with successively adding methanol using concentration of dissolved oxygen in a medium as an index. CONSTITUTION:Methanol is successively added using a methanol-utilizing microorganism and concentration of dissolved oxygen in a medium is used as an index to afford the aimed enzyme by a cultivation method. Besides, a methanol- utilizing yeast: candida boidinii is preferably used as said microorganism. Furthermore, as a culturing condition, e.g. an aerobic cultivation in stirring with ventilation at 28 deg.C for 24-72hr is preferable.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention is a method for efficiently producing formate dehydrogenase useful in organic synthesis reactions by culturing formate dehydrogenase-producing microorganisms using methanol as a carbon source. provide a method to do so.

(Prior Art) In recent years, there have been many cases where enzymatic reactions are applied to organic synthesis, particularly to asymmetric synthesis of optically active compounds. Various natural and non-natural L-amino acids are useful as components of nutritional supplements and synthetic intermediates for pharmaceuticals.
Synthesized using the corresponding amino acid dehydrogenase. For example, "Development of new functions of enzymes" (published by Kodansha Scientific, supervised by Sanbe Fukui, edited by Hideaki Yamada), pp. 41-42,
“Asymmetric synthesis bioreactor” (published by Gakkai Publishing Center,
(Supervised by Shu Suzuki, written by Hidekatsu Maeda et al.) pp. 65-67 and 76
-Page 84 introduces a reaction for synthesizing L-leucine using leucine dehydrogenase. Alternatively, a reaction has been proposed in which L-phenylalanine and others are synthesized by phenylalanine dehydrogenase (Yasuhisa Asano, "Development of new microbial enzymes and application to organic synthesis", Journal of the Society of Organic Synthetic Chemistry, Vol. 47, No. 8) 749-759 (1989)
).

The reaction in which these natural and non-natural amino acids are converted from the corresponding keto acids by amino acid dehydrogenase requires nicotine adenine dinucleotide reduced form (hereinafter abbreviated as NADH) as a coenzyme. N.A.D.
H is oxidized (hereinafter abbreviated as "NAD") as a result of the reaction.
is converted to However, since NADH is expensive, when performing this reaction, it is necessary to use NADH for coenzyme regeneration.
A military service-type reaction is required in which an enzyme reaction for recycling D'' into NADH is incorporated into the system.

Formate dehydrogenase is frequently used as an enzyme in the coenzyme regeneration system in the reactions by these NAD''-dependent dehydrogenases. The formic acid produced through metabolism is finally converted into carbon dioxide, and at that time NAD'
Convert to NADH. The great advantage of using this enzyme is! 1) It is in the form of an ammonium formate solution, which can simultaneously supply ammonium ions necessary for the amino acid production reaction. (2) Carbon dioxide, a by-product, becomes a gas,
One of its characteristics is that it is removed from the reaction system and the equilibrium shifts in the direction in which the desired reaction proceeds.

(Problem to be solved by the invention) However, the methanol-assimilating yeasts Candida and Boidini are widely known as microorganisms that possess formate dehydrogenase, but the formate dehydrogenase has a low specific activity, so When applied to the market, it is necessary to obtain a large amount of bacterial cells.

To date, there has been a report on the efficient mass cultivation of Candida and Boidini in an 80-liter culture tank, and an activity of 6,200 units was obtained in the crude extract stage (Shufti et al., Eur. , J.Bioche
m, 62.151-160). However, in order to use the produced enzymes in industrial reactions, it is necessary to further improve productivity.

A major problem in mass culturing methanol-assimilating microorganisms is the difficulty in controlling culture conditions, especially the concentration of methanol supplied as a carbon source.

In other words, formate dehydrogenase is induced when methanol is assimilated, so if other carbon sources are abundant, even if it grows actively, the production of formate dehydrogenase itself will not increase. Therefore, for the production of formate dehydrogenase,
It must be cultured aerobically under conditions such that methanol is the only carbon source.

However, in the case of cultivation using other carbon sources such as glucose and glycerol, the phenomenon that microbial growth is inhibited by adding too much to the medium is common unless the amount is extremely excessive. On the other hand, since methanol is toxic to bacterial cells, if it is given in excess of the amount required for growth, it will significantly inhibit growth.

Therefore, in order to efficiently produce formate dehydrogenase, it is necessary to monitor the methanol concentration in the medium and maintain it at a value close to the appropriate concentration at all times. In particular, even if there is a shortage of methanol in the medium, adding an excessive amount must be avoided at all costs.

Therefore, today's fast and accurate methods for tracking the methanol concentration in the culture medium are gas chromatography (GC) or high performance liquid chromatography (HP).
There is an analysis by LC). However, it is not practical from the viewpoint of both cost and labor to constantly operate the GC or HPLC apparatus under constant conditions during the culture lasting from -1 to several days. Furthermore, since it takes at least several tens of minutes per point to subject a sample to analysis and obtain a concentration value, it is difficult to apply the method to automatic control where measurement results are immediately used to set culture conditions.

Further, there is currently no known measurement electrode that can quickly quantify the methanol concentration in a culture solution, and is sterilizable and reusable.

Therefore, there is a need for a method for efficiently propagating methanol-assimilating microorganisms by appropriately maintaining the methanol concentration in the culture medium based on some index.

(Means for Solving the Problems) Therefore, as a result of various studies, the present inventors found that when carrying out aerobic culture using methanol as the only carbon source, as an index for controlling methanol concentration, We have discovered that it is possible to utilize the dissolved oxygen concentration in the medium without directly monitoring the methanol concentration in the medium. The dissolved oxygen concentration in these media can be measured using an oxygen electrode that is normally equipped in a fermentation tank.

In other words, according to the knowledge obtained by the present inventors, while there is sufficient methanol in the culture medium, microorganisms actively assimilate methanol to carbon dioxide, and accordingly actively consume oxygen. The dissolved oxygen concentration in the area is relatively low.

After that, it became clear that when the carbon source, methanol, was completely consumed, oxygen consumption also stopped, and the dissolved oxygen concentration value in the culture medium quickly rose. Then, by adding a sufficient amount of methanol, oxygen consumption starts again and the dissolved oxygen concentration decreases.

The present invention will be explained in detail below.

The culture of the present invention is carried out according to the culture method of each methanol-assimilating microorganism.

For example, the methanol-assimilating yeast Candida boidini species is cultured aerobically at a medium temperature of 25 to 30°C, preferably 28°C, for 24 to 72 hours with aeration and stirring.

The dissolved oxygen concentration is measured using an oxygen electrode that is normally installed in the fermenter, and is normally calibrated before the start of culture. In other words, the reading by an instrument when the electrode is immersed in a saturated aqueous solution of sodium sulfite, which is an oxygen-free solution, is 0%, and the reading when the electrode is immersed after sufficient aeration and agitation is added to the culture medium used for culture. value 100
%, the dissolved oxygen concentration is measured in the following relative value.

Attach the calibrated electrode to the culture tank and start culturing. As the bacterial cells proliferate and the amount of oxygen consumed by the bacterial cells increases, the dissolved oxygen concentration measured by the oxygen electrode decreases even after sufficient aeration and stirring is performed.

However, once the methanol in the culture medium is completely consumed and the culture medium is devoid of methanol, the dissolved oxygen concentration quickly rises to near the value at the start of the culture.

By adding methanol in an amount within the concentration range that does not inhibit growth, the microorganisms will again begin to actively consume methanol, and will continue to use this method until growth is limited by factors other than the carbon source. By repeating the addition of , it is possible to achieve high density growth.

Note that if the methanol concentration exceeds the upper limit of the appropriate concentration, the growth of microorganisms will be significantly inhibited, so it is necessary to determine the upper limit of the appropriate concentration for each microorganism to be used in advance through a preliminary study. . candida,
For Boidini, methanol concentration is important for growth and FDH.
In order to maximize production, it is desirable to set the content to 1 to 1.5%.

The amount of bacterial cell growth during the progress of culture can be determined by measuring the turbidity of an appropriately diluted culture solution using a spectrophotometer at a wavelength of 6000 m.

The methanol concentration in the culture solution can be accurately measured by subjecting the centrifuged supernatant of the culture solution to gas chromatography analysis, if necessary.

After completion of the culture, the bacterial cells were collected by centrifugation, suspended in 10 mM potassium phosphate buffer, and disrupted by ultrasonication, and then the enzyme activity of the water-soluble fraction was measured. The activity of formate dehydrogenase is determined by adding an appropriate amount of the test sample to 100 μmol of sodium formate, 3.0 μmol of NAD, and 2008M potassium phosphate buffer (pH 7.5), and measuring the total amount as 3-. The control was determined from the initial increase rate of absorbance at 340 nm using a spectrophotometer, excluding sodium formate. 1 unit (tJ) of formate dehydrogenase
It is defined as the amount of enzyme that oxidizes 1 μmole of formic acid per minute.

The above-described method of controlling the amount of methanol added using the value measured by the oxygen electrode makes it possible to increase the efficiency of producing formate dehydrogenase with a simple operation, and is useful in the production of this enzyme. This method is not limited to culturing methanol-assimilating yeast, but is also presumed to be effective for other methanol-assimilating microorganisms.

The present invention will be explained below with reference to examples, but the present invention is not limited by these examples.

Example 1 The composition of the medium used for culturing Candida and Boidini yeast is as follows. 0.41% by weight of ammonium chloride, 0.1% by weight of potassium dihydrogen phosphate, 0.1% by weight of dipotassium hydrogen phosphate, 0.05% by weight of magnesium sulfate heptahydrate, 1 volume of vitamin mixture (*) %, and the initial pH was set to 6.8.

(*Vitamin mixture; thiamine, riboflavin, pantothenic acid, nicotinic acid each 1000 tt g/(1, rose-aminobenzoin #200 μg/It, folic acid, biotin each 10 μg/l) A mixed solution with the above composition. 0.1% by weight of yeast extract was added to 4 liters of culture medium, methanol was added to give an initial concentration of 0.8% (w/v), and the mixture was incubated at 28°C in a 101-volume jar fermenter.
Candida and Boydii yeasts were cultured for 4 hours. First, the turbidity of bacterial cells during culture, the dissolved oxygen concentration in the culture solution measured by an oxygen electrode, the cumulative concentration of methanol added to the culture solution midway through the culture, and the change over time in the methanol concentration in the culture solution.
As shown in the figure.

The bacterial cells obtained at the end of the culture had a wet bacterial weight of 256 grams and a total formate dehydrogenase activity of 1830 v.

Comparative Example 1 To 4 liters of the same medium used in Example 1, 0.1% by weight of yeast extract was added, methanol was added to give an initial concentration of 1% (W/V), and then 101 vol. Candida and boidini yeasts were cultured in a jar fermenter at 28°C for 45 hours. Figure 2 shows changes over time in bacterial cell turbidity during culture and methanol concentration in the culture solution.

The bacterial cells obtained at the end of the culture had a wet bacterial weight of 49 grams and a total formate dehydrogenase activity of 400 U.

Comparative Example 2 To 50 d of the same medium used in Example 1, 0.05
Powdered yeast extract was added to each concentration by weight%, 0.1% by weight, and 0.5% by weight, and methyl alcohol was added to each concentration by 0%, 1%, and 2%, and then 200-
In an Erlenmeyer flask, Candida and Boidini yeasts were cultured at 28° C. for 48 hours in a rotating incubator (21 Orpm). The cell turbidity at the end of the culture is shown in Table 1, and the formate dehydrogenase activity per volume of culture solution is shown in Table 2.

Table 1 Cell turbidity after 1148 hours of culture (Ahoo) 2nd
Formate dehydrogenase activity (II/1) after 48 hours of surface culture (effect of the invention) In culturing the enzyme-producing bacteria in the present invention, the methanol concentration in the medium is controlled using the value measured by the oxygen electrode as an index. This enables high-density culture of bacterial cells and increases enzyme productivity. Furthermore, since high enzyme productivity can be achieved with simple operations, this method can also be applied to the cultivation of other methanol-assimilating bacteria.

[Brief explanation of drawings]

FIGS. 1 and 2 are graphs showing the cumulative concentration of methanol added to the culture medium, dissolved oxygen, and bacterial cell turbidity over time in Examples and Comparative Examples of the present invention. Figure 1: Incubation time (?L) Figure 2: Cultivation time (h)

Claims (1)

[Scope of Claims] 1) Production of formate dehydrogenase using a methanol-assimilating microorganism in the production of formate dehydrogenase by a culture method, characterized in that methanol is sequentially added using the dissolved oxygen concentration of the medium as an indicator. Law. 2) The method for producing formate dehydrogenase according to claim 1, wherein the microorganism is the methanol-assimilating yeast Candida boidinii.