JPH0547193B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a new method for immobilizing whole cell enzymes.

Problems to be Solved by the Prior Art and the Invention If whole cell enzymes are used in enzyme reactions instead of purified enzymes, the cost of enzyme purification can be saved and the enzymes are generally more stable than enzymes purified to a protein state. It is extremely advantageous industrially due to its excellent properties. Enzyme immobilization is one of the extremely important steps in the industrial application of enzymes. As conventional methods for immobilizing whole cell enzymes, methods using carriers such as calcium alginate, polyacrylamide, agarose, and collagen have been most commonly used (K. Mosback, Methods of Enzymology, Vol. 44) , Academic Press, New York 1976). Such existing methods require two steps: once cells are cultured in a fermenter, then the cells are collected, and the collected whole cell enzymes are immobilized on a carrier. However, the present invention uses a double capillary membrane bioreactor to simplify these two steps into one step, and furthermore, it is possible to fill the reactor with whole cell enzymes at a high concentration, thereby increasing the production per unit volume of the reactor. It is about new and advanced ways in which sex can be obtained.

The double tubular membrane bioreactor was created by modifying the structure of an existing tubular reactor for aerobic bacterial cell culture, and Robertson and Kim pushed three silicon tubes inside the polypropylene tubular membrane. Liquid nutrients were supplied to the outside of the polypropylene tubular membrane and oxygen was supplied to the inside of the silicone tube, and an aerobic bacterium, Streptomyces oleopheicens, was cultured between these two tubes to conduct research on continuous production of tetracycline. (Robertson and Kim, Biotechno-Bioengineering
27, 1012, 1985). The inventors then inserted a silicon tube on the outside for oxygen supply and three polypropylene tubes for liquid nutrients on the inside, unlike Robertson and Kim's reactor.
During this period, they were the first to succeed in long-term continuous production of lipamycin B by culturing Nocaldea medeterranei (Chiyoung et al., ACS Symposium Series No. 314, 31, 1986).

Means for Solving the Problems The present invention is a method of utilizing the above-mentioned reactor according to the invention of the present inventor for whole-cell enzyme reaction, in which the space between the silicon tube 1 and the polypropylene capillary tube 2 of the double capillary reactor is After inoculating cells into the silicon tube, oxygen is injected into the outside of the silicon tube and liquid nutrients are injected into the inside of the polypropylene tube to culture cells with a specific enzyme activity at a high concentration. To provide a method for immobilizing whole cell enzymes simultaneously with cell growth, by passing temperature-controlled water to the outside and passing a substrate solution to the inside of the polypropylene capillary to carry out continuous enzymatic reactions.

Embodiments The details of the present invention will be explained below with reference to the drawings. Figure 1a is a working diagram of a reactor with one double capillary during cell culture. During cell culture, air 5a passes through the silicone tube 1, and liquid nutrients 3a pass through the polypropylene tube 2 and are transmitted to the cells 6a therebetween, after which the exhausted nutrients 4a are discharged. Figure 1b is a diagram of the operation of a reactor with one double capillary tube during an enzyme reaction after high concentration cell culture, and cells with enzyme activity are immobilized in the reactor at a high concentration due to the cell culture in Figure 1a. be done. During enzyme reaction, substrate solution 3b
is transmitted to the highly concentrated whole cell enzyme 6b, and after the reaction, the product 4b is excreted. At this time, the temperature-adjusted water 5b flows to the silicon outer wall.

FIG. 2 is a cross-sectional view of a double capillary reactor;
Its structure is such that the double capillary tubes described above are arranged in parallel inside a glass tube, and in such a double capillary reactor, silicon tube 1 is connected to glass tube 7.
After putting it inside, fix both ends with silicone bar 8,
An air and temperature-adjusted water inlet 5 and an outlet 5' are provided at the end of the silicone rubber. Thereafter, three polypropylene thin tubes 2 are placed inside each silicon tube 1, and both ends thereof are fixed with silicon rubber 8. A cell inoculation port 6 is installed at the end of the silicon rubber, and a glass tube 7 is inserted into a silicon or tie tube 9. The glass tube is manufactured by connecting the glass tube with an inlet 3 for nutrient and substrate solution and an outlet 3 for the same at both ends of the glass tube. The polypropylene tube used in the reactor production in the present invention is a product of Enca of Germany, and has an inner diameter of 0.033 cm, an outer diameter of 0.063 cm, and an air gap of 0.4 to 0.6 μm. This is a Dow Corning product and the inner diameter
One with an outer diameter of 0.147 cm and an outer diameter of 0.196 cm was used.

Ten double tubes were inserted into a glass tube with an inner diameter of 0.8 cm, and the length that could be in contact with oxygen was 16 cm. That is, materials other than silicon and polypropylene can be used to manufacture the reactor, and the reactor structure can be manufactured in various ways. The number of thin tubes to be inserted into the tube can be variously adjusted according to the specifications of the thin tubes used.

Next, an experimental example of the method of the present invention will be described, but the scope of the present invention is not limited only to this example.

Experimental Example 1 Streptomyces glycius ( _NCTC 7178) having glucose isomerase activity is inoculated between the silicon tube 1 and the polypropylene capillary tube 2 through the cell inoculation port 6, and then cultured.

The medium used was D-xylos (1%), yeast extract (1%), MgSO ₄ (0.1%), CoCl ₂ (0.01%),
KH ₂ PO ₄ (0.3%) and PH was adjusted to 7 with NaOH. The liquid medium is injected through the nutrient inlet 3, and the exhausted nutrients are discharged through the nutrient outlet 3'; the flow rate of the liquid medium at this time is 2 ml/h.
and the air and hot water are connected to the air and temperature-controlled water inlet 5.
The cells were injected through the reactor and discharged through the outlet 5', while air was introduced at a rate of 100 ml/min, and the cells were cultured for 10 days while maintaining the temperature at 30°C to immobilize the cells in the reactor. As a result of supplying a 0.5M glucose substrate solution through the injection port 3 at a flow rate of 2 ml/h to the reactor in which the cells were immobilized as described above, fructose was continuously produced with a productivity of 22.5 g/oh.
This is 12 times higher than the batch method.

Experimental Example 2 An enzymatic conversion experiment of acrylonitrile to acrylamide was conducted in the same manner as in Experimental Example 1 using Brevibacterium isolated from soil. The medium is glucose
15g/, yeast extract 3g/, malt extract 3
g/, K ₂ HPO ₄ 13.4 g/, KH ₂ PO ₄ 6.5 g/
, NaCl 1g/MgSO ₂ 0.2g/, and after culturing in a double capillary reactor for 3 days, the enzymatic reaction was carried out. For the reaction, 5% acrylonitrile was supplied at a rate of 4.5 ml/h, and the reaction temperature was maintained at 4°C.
At this time, the productivity of acrylamide is 106g/
- It was hot.

[Brief explanation of the drawing]

Figure 1a is an operational diagram of a reactor with one double capillary during cell culture, Figure 1b is an operational diagram of a reactor with one double capillary during high concentration cell culture enzyme reaction, and Figure 2 is a diagram of a reactor with a double capillary. FIG. 3 is a cross-sectional view of the structure. 1... Silicon tube, 2... Polypropylene capillary, 3, 5... Inlet, 3', 5'... Outlet, 3a... Liquid nutrients, 3b... Substrate solution, 4
a...Product, 5a...Air, 5b...Temperature-controlled water, 6...Cell inoculation port, 6a...Cell, 6b
... Whole cell enzyme, 7 ... Glass tube, 8 ... Silicone rubber, 9 ... Tycon tube.

Claims (1)

[Claims] 1 After inoculating cells between the silicone tube 1 and the polypropylene tube 2 of the double tube reactor, oxygen is injected into the outside of the silicone tube and liquid nutrients are injected into the inside of the polypropylene tube to have a specific enzyme activity. Cells are cultured at a high concentration and immobilized in a reactor at the same time. Temperature-controlled water is passed outside the silicon tube and a substrate solution is passed through the polypropylene capillary to carry out continuous enzymatic reactions. A method of growing and fixing at the same time.