JPH0449399B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for reducing nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD).

In recent years, it has been found that when enzyme reactions are used to synthesize various substances, substances that are difficult to synthesize using normal chemical means can sometimes be easily obtained. Therefore, there are great expectations for the future of the use of enzymatic reactions as a means of breaking down the barriers of synthesis by chemical means.

There are various types of enzymatic reactions, one of which is redox reaction. When redox reactions are carried out enzymatically, most of them require NAD or NADH (reduced nicotinamide adenine dinucleotide) as a coenzyme. However, NAD and
Since NADH is an extremely expensive substance, it is impractical to use it disposablely, and this has become the biggest problem when performing enzymatic redox reactions.

Using NAD and NADH as coenzymes
NAD is consumed by NADH, and NADH is consumed by NAD. Therefore, NAD from NADH, NADH
If it becomes possible to reproduce the data from the NAD, the above-mentioned problems can be avoided.

From this perspective, research is being conducted on the regeneration of NAD and NADH, and electrochemical methods and enzymatic methods have been proposed. However, electrochemical methods are not preferable because conversion reactions to compounds other than NAD and NADH also occur. Enzymatic methods use various dehydrogenases, but in this case, additional auxiliary raw materials (substrates) are required. and NADH,
This was also not desirable because it required a troublesome process of purification.

The present inventors particularly focused on the regeneration of NADH.
From the above point of view, as a result of intensive research on industrially available methods, we found that allowing specific microorganisms to act on NAD in the presence of high-pressure hydrogen is an excellent method that overcomes the problems seen in conventional methods. The present invention was achieved based on the discovery that a method is possible.

That is, the present invention is a method for reducing NAD, which is characterized in that a microorganism belonging to the genus Clostridium and having the ability to reduce NAD to NADH is allowed to act on NAD under a hydrogen pressure of 10 atmospheres or more.

Hereinafter, the present invention will be explained in detail.

To reduce NAD according to the present invention, for example,
Microorganisms belonging to the genus Clostridium and having the ability to reduce NAD to NADH may be suspended in a buffer containing NAD, and hydrogen may be introduced to cause a reaction under a hydrogen pressure of 10 atmospheres or more. With such a simple operation, NAD is reduced and regenerated into NADH. In this case, no by-products are produced, and only unreacted hydrogen intervenes, and this hydrogen is also removed naturally without the need for special measures, which is extremely advantageous industrially. Normally, it is common knowledge that biological reactions like this are carried out under normal pressure, but this reaction hardly progresses under normal pressure and only progresses under high pressure, which is surprising and overturns conventional wisdom. It was hot.

The microorganisms used in the present invention belong to the genus Clostridium, and examples include Clostridium butylicum, Clostridium acetobutylicum, and Clostridium perifringiens. It may be used after being immobilized on a carrier. In order to carry out the present invention easily, the cultured cells may be used as they are, but using them after immobilization on a carrier has the advantage of having a higher regeneration conversion rate and being able to withstand long-term operation. For immobilization, known methods can be used without any problem. As a carrier,
For example, natural products such as agar, alginic acid, gelatin, and carrageenan, and synthetic products such as polyacrylamide gel are preferably used.

The present invention needs to be carried out under high hydrogen pressure, 10 atmospheres or more, especially 30 atmospheres or more, 50 atmospheres or more,
The pressure is preferably 80 atm or higher, more preferably 100 atm or higher.

There are no particular limitations on the buffer used, and commonly used Tris buffer, phosphate buffer, citrate buffer, tricine buffer, etc. may be used. PH may be set in the range of 6 to 9, preferably 7 to 8. The larger the amount of bacterial cells, the faster the reaction, and the smaller the amount, the slower the reaction; however, this does not mean that the present invention cannot be carried out unless the amount is within a specific range. The NAD concentration does not need to be within a specific range, and reduction can be achieved simply by adding it to the reaction system. The reaction temperature is 20°C to 50°C, preferably 30°C to 40°C. The reaction time varies depending on the conditions, but may be approximately 1 to 10 hours.

As described above, according to the method of the present invention, NADH, which is important as a coenzyme, can be produced and regenerated much more easily than NAD in a shorter time.

Next, the present invention will be specifically explained using examples.

Example 1 Hydrogen-producing microorganism Clostridium butyricum
IFO3858, glucose 1g, peptone 0.4g,
Using 100 ml of a medium (PH7.0) containing 0.4 g of yeast extract, 0.2 g of meat extract, 12.5 g of dipotassium phosphate and 0.05 g of ferrous sulfate, at 37°C under anaerobic conditions.
It was cultured for 9 hours.

6 mg of the bacterial cells obtained in this way was added to 2 ml of
0.8 suspended in 0.1M phosphate buffer (PH7.8)
0.1M phosphate buffer (PH
7.8) and reacted in an autoclave with a hydrogen pressure of 100 atm at 37°C for 5 hours. When the amount of NADH produced was measured from the absorbance at 340 nm, it was 63%.
It was observed that NADH could be obtained from NAD with a conversion rate of .

It was also confirmed that the amount of NADH produced was consistent with the measured value of activity against alcohol dehydrogenase.

For comparison, when the same conditions were used under a hydrogen pressure of 1 atm (normal pressure), almost no NADH was produced.

Example 2 6 mg of Clostridium butylicum cells obtained in Example 1 was added to 1 ml of 0.1M phosphate buffer (PH7.8)
suspended in. This suspension was rapidly mixed with 1 ml of 4% agar solution (using the same phosphate buffer as above) and cooled (0°C) to obtain immobilized bacterial cells.

The immobilized bacterial cells obtained in this way were divided into small pieces (approximately 5
mm ³ ), washed with physiological saline, and added to a phosphate buffer containing NAD in the same manner as in Example 1 to carry out a reaction. Conversion rate is almost 100%
NADH was obtained.

After the above reaction is completed, reuse the same immobilized bacterial cells.
A reduced version of NAD was performed. When the reaction was carried out under the same conditions for 5 hours, the conversion rate was almost 100% and NADH
was gotten. In other words, it was found that the conversion from NAD to NADH can be operated continuously.

Example 3 Cells of the hydrogen-producing microorganism Clostridium acetobutylicum IAM19011 were obtained according to the culture conditions of Example 1. This bacterial cell was immobilized in the same manner as in Example 2, and NAD was reduced under the same conditions.
NADH was obtained with a conversion rate of 76%.

Similarly, hydrogen-producing microorganisms Clostridium acetobutyrical IAM19012 and
When IAM19013 was also used, the same results as above were obtained.

Claims (1)

[Claims] 1. Nicotinamide, characterized in that a microorganism belonging to the genus Clostridium and having the ability to reduce nicotinamide adenine dinucleotide to the reduced form of nicotinamide adenine dinucleotide is made to act on nicotinamide adenine dinucleotide under a hydrogen pressure of 10 atmospheres or more. Reduction method of adenine dinucleotide.