JPS5929236B2 - 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, many attempts have been made to use immobilized microbial cells as enzyme sources.

The method of using the microorganism itself as an enzyme source does not require the extraction of the enzyme through complicated operations such as bacterial cell strength, and the stability of the enzyme is also relatively high. This method is more convenient than methods using purified enzymes.

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 of the cell if it is simply cultured and collected.

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 the blanket immobilization method using acrylamide gel, which is a commonly used method for immobilization, is adopted, bacteria that have expressed activity before immobilization using the method described above can be used. When using a body, enzyme activity is often lost during the immobilization process.

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 intensive research, the present inventors have found that by entrapping the desired enzyme in an acrylamide gel without binding to the cell membrane and expressing the endogenous bacterial activity, the immobilized bacterial cells are freeze-dried. It has been found that by activation, it is possible to obtain immobilized bacterial cells in which the desired four elemental activities of the bacterial cells are 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.

There are various enzymes that are bound to the cell membrane of bacteria, and the type of enzyme that is bound to the cell membrane varies depending on the bacteria, but in general, many enzymes are involved in synthetic reaction systems that require energy.

These are enzymes whose activity does not appear once the culture of the bacterial cells is completed.

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

When selecting bacteria, it is necessary to select bacteria that have high activity against the desired enzymatic reaction, but have low activity against other reactions, such as substrate or product decomposition reactions.

For example, if an NAD kinase that generates NADP using NAD and ATP as substrates is desired, the activity of NADsanase is high and NAD, ATP are generated.

and select bacteria with low NADP-degrading enzyme activity.

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 ADP 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 a pH that is stable for the enzyme in the bacterial cells.

The bacterial cell suspension obtained by mixing the acrylamide monomer with a cross-linking agent such as N-N'-methylenebisacrylamide and a suitable polymerization accelerator is added to polymerize the acrylamide and release the bacterial cells. Fix in gel.

In order to express the desired enzyme activity bias, the acrylamide gel-immobilized bacterial cells prepared as described above are freeze-dried.

During freeze-drying, the acrylamide gel-immobilized bacterial cells are frozen at -20 to -80°C and the vacuum level is approximately 10-4 mmHs.
It is preferable to carry out vacuum drying until the final product temperature reaches 30 to 40° C.

The immobilized cell membrane-bound enzyme produced according to the present invention is easily condensed by suspending it in an appropriate buffer, and is useful for various biochemical reactions, and the reaction may be carried out in a bunch method. This is particularly advantageous when the reaction is carried out continuously in a column.

Furthermore, the immobilized cell membrane #i\h produced by the present invention
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t, /', σ) fri1 type zetogenpa retains its activity for a long period of time and is convenient to handle.

As mentioned above, by immobilizing the enzyme of the present invention in an acrylamide gel while bound to the cell membrane and freeze-drying it, it is possible to provide an immobilized cell membrane-bound enzyme with high activity. It has a lot to contribute to the field of research.

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

[Example 1. ] Proteus vulgaris with NAD kinase activity (
IFO3850) with 2% grease and 0.5% yeast extract.
, C3LI%, pH 7 containing 1 part monobasic potassium phosphate, 1% potassium phosphate, 0.05% magnesium sulfate.
, 5 was aerated and cultured for 8 hours at 30° C. After collection, the cells were washed with water.

The obtained bacterial cells 2402 were suspended in 400-ml water, and then mixed with 802-acrylamide monomer and 82-N-N'-
Methylenebisacrylamide was added and the mixture was cooled to below 10°C, and 200ml each of 5% dimethylamine propionitrile and 5% ammonium persulfate were added thereto, and the mixture was allowed to stand at below 10°C for 1 hour.

After molding the obtained immobilized acrylamide polymer bacterial cells into a gel with a size of 10 to 100 meshes, acrylamide was immobilized using a vacuum freeze-drying machine (VFI) 1200FMS-B manufactured by Inuyasuku. Freeze the bacterial cells at -40°C
The acrylamide-immobilized cell membrane-bound NA was freeze-dried at a vacuum level of 10''-' mmHf until the final product temperature was 30°C.
D-kinase 151 was obtained.

The activity of this product was measured with 5mM NAD and 5mM ATP.

100? ? The dry gel was measured at 20 units/r in 50 mM 9-phosphate buffer (pH 7.5) containing 7M sodium fluoride, 10 iV magnesium chloride, 10 mM sodium azide, 10 mM zinc chloride.

(Lunit is the activity of producing 1 μmole of NADP per hour) On the other hand, no NAD kinase activity was observed in the immobilized bacterial cells that were not subjected to freeze-drying.

Furthermore, when the activity change of the freeze-dried acrylamide-immobilized cell membrane-bound NAD kinase obtained according to the present invention was investigated during storage, no decrease in activity was observed at 4°C or lower for 90 days, and even at 30°C after 90 days. Initial activity 8
It showed 9%.

[Real mouth [1e2li 2. ] Brevibacterium ammoniagenes (IFO12072), which has adenylate kinase activity, was added to 2% glucose, 3% yeast extract, and 0.1% potassium monophosphate.
, potassium phosphate 0.1%, calcium chloride 0.0
Cultured at 30°C for 8 hours in a pH 7.5 medium containing 1%,
Bacterial cells were obtained.

The obtained bacterial cells were immobilized on an acrylamide gel and molded in the same manner as in Example 1, and then lyophilized to obtain acrylamide-immobilized adenylate kinase 15f.

The activity of this substance is 5mM ADP, 2mM magnesium chloride, 100? ? 70mM containing 2M potassium chloride
)! 13.8 unit t/f dry gel measured in J ethanolamine buffer.

On the other hand, no adenylate kinase activity was observed in immobilized bacterial cells that were not subjected to freeze-drying.

Claims (1)

[Claims] 1. Bacterial cells that contain the desired enzyme bound to the cell membrane and do not express enzymatic activity after being cultured and collected are encircled and fixed in an acrylamide gel, and then freeze-dried. 1. A method for producing an immobilized cell membrane-bound enzyme, which is activated by: