JPS5951278B2 - Method for producing immobilized cell membrane-bound enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an immobilized cell membrane-bound enzyme that effectively acts as a catalyst in biochemical reactions.

In recent years, with the development of techniques for utilizing enzymes as catalysts in biochemical reactions, various methods have been attempted to immobilize and use microbial cells as enzyme sources.

The method of using the microorganism itself as an enzyme source does not require complicated operations to extract the enzyme from the microorganism, and
Since the stability of the enzyme is relatively high, this method is more convenient than methods using purified enzymes in practical terms.

However, depending on the type of enzyme desired, in many cases the enzyme is tightly bound to the cell membrane, and its activity is not manifested outside the enzyme simply by culturing and harvesting.

In order to express the activity, bacterial cells are brought into contact with an organic solvent, freeze-dried, mineral acid, or a surfactant. In many cases, it is very effective.

On the other hand, when immobilizing bacterial cells, if entrapment fixation using acrylamide gel, which is a commonly used method for immobilization, is adopted, the activity can be expressed before immobilization using the method described above. When using bacterial cells, enzyme activity is often lost during the immobilization process.

For example, when Brevibacterium containing NAD kinase, which is one of the membrane-bound enzymes, is brought into contact with a surfactant to develop its activity and then brought into contact with an acrylamide monomer, it is inhibited as shown in Table 1.

An object of the present invention is to provide a method for producing an immobilized cell membrane-bound enzyme that effectively acts as a catalyst, eliminating the drawbacks of the method for producing immobilized bacterial cells as described above.

As a result of extensive research, the present inventors have found that after binding the desired enzyme to the cell membrane and immobilizing the internal bacterial cells in an acrylamide gel without expressing activity, the immobilized bacterial cells can be transferred to an organic solvent or an interface. It has been found that by contacting with an activator, it is possible to obtain immobilized bacterial cells in which the desired enzyme activity of the bacterial cells is almost completely expressed.

The present invention has been achieved based on the above research results.

The content of the present invention will be explained in detail below. In the present invention, care should first be taken to select bacteria that contain a particularly highly active enzyme bound to the cell membrane.

Examples of enzymes bound to bacterial cell membranes include pyruvate kinase, adenylate kinase, and NAD kinase, which require high-energy phosphoric acid compounds such as ATP as substrates for their reactions; This is an enzyme that does not exhibit any activity once the process has finished.

The present invention is primarily directed to bacteria containing enzymes such as those described above.

Furthermore, when selecting bacteria, it is necessary to select bacteria that have high activity for the desired enzymatic reaction and low activity for other reactions, such as substrate or product decomposition reactions.

For example, if NAD kinase that produces NADP using NAD and ATP as substrates is desired, bacteria with high NAD kinase activity and low NAD, ATP, and NADP degrading enzyme activities are selected.

Similarly, from 2 molecules of ADP, 1 molecule of ATP and 1 molecule of A
When adenylate kinase that produces MP is desired, adenylate kinase activity is high, but APD and A
Select bacteria with low TP degrading activity.

Furthermore, even if a complex enzyme system is desired instead of a single enzyme, the present invention can be applied if a series of target enzymes or a part of a series of enzymes exists in a form bound to the cell membrane. subject to.

The bacteria selected as described above are cultured to collect the bacterial cells, washed if necessary, and then suspended in a buffer solution adjusted to stabilize the enzyme in the bacterial cells.

The bacterial cell suspension thus obtained is mixed with an acrylamide monomer, a crosslinking agent such as N-N'-methylenebisacrylamide, and a suitable polymerization accelerator, and the acrylamide is polymerized to fix the bacterial cells in the gel.

In order to express the desired enzyme activity, the acrylamide gel-immobilized bacterial cells prepared as described above are brought into contact with an organic solvent or a surfactant.

Organic solvents include acetone and l-luene, and surfactants include CTAB (Cetyltnmethyla).
mmonium bronide) etc. are used.

The concentration is preferably 0.5 to 20%, and the contact time is
Preferably 30 minutes to 2 hours.

After the organic solvent or surfactant treatment described above, the immobilized cell membrane-bound enzyme of the present invention can be obtained by washing the immobilized bacterial cells and removing the used organic solvent or surfactant.

The immobilized cell membrane-bound enzyme produced according to the present invention is useful in various biochemical reactions, and although the reaction may be carried out batchwise, it is particularly advantageous to carry out continuous reaction by filling the enzyme into a column.

As described above, the present invention can provide an immobilized cell membrane-bound enzyme with high activity by immobilizing the enzyme in an acrylamide gel while bound to the cell membrane and treating it with an organic solvent or a surfactant. It has much to contribute to fields that utilize reactions.

The present invention will be specifically explained below by way of examples.

Example I Brevibacterium ammoniagenes (IFO12072) having NAD kinase activity was mixed with 2% glucose,
Aerated culture was carried out at 30°C for 8 hours in a pH 7.5 medium containing 3% yeast extract, 0.1% potassium phosphate, 0.1% potassium phosphate, and 0.01% calcium chloride. I got it.

After washing 240 g of the obtained bacterial cells with water, they are suspended in 400 ml of water.

To this, 80 g of acrylamide monomer and 8 g of N-N'-methylenebisacrylamide were added, and after cooling to below 10°C, 5% dimethylaminopropionitrile and 5% dimethylaminopropionitrile were added.
20 ml of ammonium persulfate was added to each, and the mixture was left at 10° C. or lower for 1 hour.

The thus obtained acrylamide-immobilized bacterial cells were crushed and formed into a gel with a size of 10 to 100 meshes, and then treated with toluene by adding 2 μ of 2% toluene and stirring at 30°C for 1 hour. .

After toluene treatment, the bacterial cells were washed with water to remove toluene, and 740 g of acrylamide-immobilized cell membrane-bound NAD kinase was added.
I got it.

Kono also stimulated horn activity with 5mM NAD, 5mMATP, 1
When measured in 50mM'J acid buffer (pH 7.5) containing 00mM7 thorium chloride, 10mM magnesium chloride, 10mM sodium azide, and 10mM zinc chloride, it was found to be 4 units/g gel.

(Lunit is the activity of producing 1 μmole of NADP per hour) On the other hand,)・In the case of immobilized bacterial cells that are not treated with luene, NA
No D kinase activity was observed.

Example 2 Azonile-1... Brevibacterium ammoniagenes (IFO12072') having kinase activity was cultured in the same manner as in Example 1.

After immobilizing this bacterial body on an acrylamide gel and molding it,
, and stirred for 1 hour in a 5% CTAB solution to obtain acrylamide-immobilized cell membrane-bound adenylate kinase.

The activity of this product was measured in 70mM containing 5mM ADP, 2mM magnesium chloride, and 100mM potassium chloride).
4.1u when measured in reethanolamine buffer
nit/g gel.

No adenylate kinase activity was observed in the immobilized bacterial cells that were not treated with CTAB.

Example 3 Proteus vulgaris having NAD kinase activity (
IFO3851) with glucose 2%, C3LI%, yeast extract 0.5%, urea 0.3%, monopotassium phosphate 1
%, potassium phosphate 1%, magnesium sulfate 0.0
Bacterial cells were obtained by aeration culture at 30° C. for 8 hours in a tj' pH 7.5 (7) medium containing 5%.

24 g of the obtained bacterial cells were immobilized with acrylamide in the same manner as in Example 1, and stirred for 1 hour in a 1% acetone solution to obtain 74 g of acrylamide-immobilized cell membrane-bound NAD kinase.

The activity of this product was measured in the same manner as in Example 1 and was found to be 3.2 urut/g gel.

Example 4 Proteus mirabilis having hexokinase activity (
AHU1467) was cultured in the same manner as in Example 3 to obtain bacterial cells.

36 g of the obtained bacterial cells were individualized in the same manner as in Example 1, and then treated with toluene to obtain 111 g of immobilized cell membrane-bound hexokinase having an activity of 0.8 unit/g gel.

Claims (1)

[Claims] 1. Bacterial cells that contain the desired enzyme bound to the cell membrane and do not express enzymatic activity when cultured and collected are immobilized in an acrylamide gel, and then treated with an organic solvent or surfactant. 1. A method for producing an immobilized cell membrane-bound enzyme, which is characterized in that its activity is expressed by bringing it into contact with an enzyme.