JPH01101878A - Method for fixing all cell enzymes simultaneously with growth of cell - Google Patents

Abstract

PURPOSE: To perform the subject immobilization at a simplified process and at high productivity by inoculating cells in a space between a silicone tube and polypropylene fine tubes in a double-layered fine tube reactor and subsequently treating the reactor under specific conditions to carry out continuous enzymatic reaction.

CONSTITUTION: Cells are inoculated in a space between a silicone tube 1 and polypropylene fine tubes 2 of a double-layered fine tune reactor. Subsequently, cells having specific enzymatic activity is cultured at a high concentration and simultaneously immobilizing the grown cells in the reactor while an enzyme is placed outside the silicone tube and an liquid nutrient is injected and allowed to flow inside the polypropylene fine tubes. Further, while passing temperature-controlling water outside the silicone tube and a substrate solution inside the polypropylene fine tubes, continuous enzymatic reaction is carried out. Thus, whole cell enzyme is simultaneously cultured and immobilized. This immobilization method enables the simplification from two processes to one process by using a double-layered fine-tube membrane reactor and further enables the filling of whole cell enzyme at a high concentration in a reactor to achieve a high productivity per unit volume of the reactor.

COPYRIGHT: (C)1989,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a new method for immobilizing whole cell ffi cords.

Problems to be Solved by the Prior Art and the Invention If whole class Illusin is used in enzyme reactions instead of purified enzymes, it is possible to save on enzyme production costs and compared to enzymes that are generally purified into protein form. It is extremely advantageous industrially because of its excellent stability. Immobilization of the wl cord is one of the extremely important steps in the industrial application of W9s. As a conventional method for immobilizing all 111Jl enzymes, methods using carriers such as calcium alginate, polyacrylamide, agarose, and :J-Lagen have been most commonly used (K. Mossback, W. G.'s method, Volume 44.

Academic Press, New York, 1976).

Existing methods require two steps: - culturing cells in a fermenter, then collecting the cells, and immobilizing the collected whole cell enzymes on a carrier. However, the present invention uses a double llI tube membrane bioreactor to simplify this 2T to -r, and furthermore, a high llIr! The present invention relates to a new and more advanced method that allows high productivity per unit volume of the reactor to be filled with acetic acid per unit volume of the reactor.

The double tubular membrane bioreactor was created by modifying the structure of an existing tubular reactor for aerobic bacterial culture, and Robertson and Kim pushed three silicone tubes inside the polypropylene tubular membrane. While supplying liquid nutrients to the outside of the polypropylene tube membrane and oxygen to the inside of the silicone tube, we cultivated Streptomyces oleopheicens, an aerobic bacterium, and conducted research on continuous production of tetrazycline. Ta. (Robertson and Kim, Biotechnol. Bioengineering 27, 1 (11
2, 1985). The inventors then inserted a silicon tube for oxygen supply on the outside and three polypropylene tubes for liquid nutrients on the inside, unlike Robertson and Kim's reactor, and inserted Nocaldea Medetera in between. For the first time, the long-term continuous production of lipamycin was successfully achieved by culturing N. neiss (Chuck et al., Symposium Series No. 314.31.1986).

Means for Solving the Problems The present invention is a method of utilizing the reactor according to the inventor's invention described above for a whole-cell enzyme reaction. ) After inoculating the cells between the silicone tube and the enzyme,
Liquid nutrients are injected into the inside of the polypropylene tube, and cells with a specific enzyme activity are cultured in a PI atmosphere, and at the same time, they are fixed in the reactor. Provides a method for immobilizing whole I cell enzymes at the same time as cell growth by passing a substrate solution through them to carry out continuous enzymatic reactions. It is. Figure 1(a) is a working diagram of a reactor with one double capillary during cell culture. It consists of a built-in thin tube (2), and during cell culture, air (5a) passes through the silicone tube (1), and liquid nutrients (3a) pass through the polypropylene tube (2) to remove the darkness. After being transmitted to l1la (6a) in
fj Excrete depleted nutrients (4a). Figure 1(b) is a diagram showing the operation of a reactor with one double tube during an enzyme reaction after culturing high-density a-cells. is immobilized in the reactor. During the enzyme reaction, the substrate solution (3b) is transferred to the highly concentrated whole cell enzyme (6b), and after the reaction, the bioacid (4b) is excreted. At this time, the temperature-controlled water (5b) flows onto the silicon outer wall.

Figure 2 is a cross-sectional view of a double capillary reactor, and its structure is such that the double capillary tubes described above are arranged in parallel inside a glass tube. After putting the silicone tube (1) inside the glass tube (7), fix both ends with silicone rubber (8), and one end of the silicone rubber has an air and temperature-controlled water inlet (5) and its outlet (
5') is provided. Each silicone tube (1
), put three polypropylene thin tubes (2) into it, fix their ends with silicone rubber (8), install a cell inoculation boat 6) at one end of the silicone rubber, and insert the glass tube (
7) are connected with a silicone or tie-con tube (9),
Nutrient and substrate solution injection ports (3
) and its discharge port (3). The polypropylene capillary tube used in the reactor production in the present invention is a product of Enca, Germany, and has an inner diameter of 0.033IJI and an outer diameter of 0.
．． 063 preparation.

A silicone tube with an air gap size of 0.4 to 0.6 μm was used, and a silicone tube manufactured by Da Corning of the United States with an inner diameter of 0.147α and an outer diameter of 0.196 w was used.

A 10WA II III tube was inserted into a glass tube with an inner diameter of 0.8a11, and the length that could come into contact with oxygen was 16 (J).

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 experimental example.

Experimental example 1゜Glucose isomerization! Streptomyces glyceus (NcTC7178), which has the activity of 1, is inoculated between the silicone tube (1) and the polypropylene tubule (2) through the mr11a seed boat (6), and then cultured.

The media used were D-Kijiros (1%) and yeast extract (1%).
%), MQ80< (0,1%), Co(,22(0,
01%), KH2P○, (0,3%), and PH1t
Adjusted to 7 with NaOH. The liquid medium is supplied through the nutrient inlet (3).
), and the consumed nutrients are injected through the nutrient outlet (3).
' ), the flow rate of the liquid medium at this time is 2 d/h, and while air and hot water are injected through the air and temperature-controlled water inlet (5), it is discharged to the outlet C1 (5'). Air is introduced at a speed of 100d/Win,
The cells were cultured for 10 days while maintaining the temperature at 30°C, and the cells were immobilized in the reactor. As a result of feeding fructose at a flow rate of 2 d/h through 0.5 M llI'@sugar substrate glycobase solution (3) to the reactor in which ma was immobilized as described above, fructose was continuously produced, and the productivity was It was 22.5 SF/l oh.

This is 12 times higher than the batch method.

Experiment IA2 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 was @sucrose159/f1. M mother extract 39/i,? Ruto extract 3g/2 HPO4
13,49/2゜KH2PO46,5g/e,
NaC21'J/Il.

The liquid medium had a composition of MgSO20,2'j/e, and was cultured in a double capillary reactor for 3 days, followed by an enzyme reaction.

For the reaction, 5% acrylonitrile was supplied at a rate of 4.5 d/h, and the reaction temperature was maintained at 4°C. At this time, the productivity of acrylamide was 106 g/4-h.

[Brief explanation of the drawing]

Figure 1(a) is a diagram of the operation of a reactor with one double tube during cell culture, and Figure 1(b) is a diagram of the operation of a reactor with one double tube during fermentation reaction with high concentration cell culture. FIG. 2 is a cross-sectional view of a double capillary reactor structure. DESCRIPTION OF SYMBOLS 1... Silicone tube, 2... Polypropylene capillary, 3, 5... Inlet, 3', 5'... Outlet, 3a... Liquid nutrient, 3b... Substrate solution, 4a.
...Product, 5a...Air, 5b...Temperature-controlled water,
6... Cell inoculation boat, 6a... Cell, 6b...
All lllll11 enzymes, 7... Gans tube, 8... silicone rubber, 9... Ticon tube. Patent Applicant Korea Adventist Institute of Science and Technology Figure 1 (CI) b (b)

Claims (1)

[Claims] After inoculating cells between the silicone tube (1) and 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 release a specific enzyme. Active cells are cultured at a high concentration and immobilized in a reactor at the same time, and temperature-controlled water is passed through the outside of the silicone tube and a substrate solution is passed through the inside of the polypropylene tube to carry out continuous enzymatic reactions. A method to immobilize enzymes simultaneously with cell growth.