JPH02238880A - Regeneration of bioreactor - Google Patents

Abstract

PURPOSE:To efficiently regenerate a bioreactor without calcining a carrier by bringing a bioreactor integrated with an immobilized biocatalyst having reduced activity into contact with an aqueous solution of hypochlorous acid (solt), removing the deactivated catalyst and immobilizing a new catalyst to the carrier. CONSTITUTION:A biocatalyst comprising an enzyme such as pullulanase is dissolved in a buffer solution, brought into contact with a catalytic carrier (e.g. ceramics carrier), allowed to stand at room temperature for 1 hour to prepare an immobilized biocatalyst, which is packed into a column to constitute a bioreactor. A mixed solution of maltose and cyclodextrin is fed to the bioreactor to continuously produce maltosylcyclodextrin, etc. When the catalytic activity is reduced, an aqueous solution of hypochlorous acid (salt) is brought into contact with the catalytic carrier, the deactivated immobilized biocatalyst is removed, a new biocatalyst is brought into contact with the catalytic carrier and immobilized to regenerate the bioreactor.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for regenerating a bioreactor in which a catalyst carrier supports a biocatalyst such as an enzyme, a microbial cell, a plant, an animal cell, or an intracellular organelle. It is. (Prior art) A bioreactor in which a biocatalyst that catalyzes a biochemical reaction is supported on a catalyst carrier can prevent the expensive biocatalyst from being washed away, can increase the density of the biocatalyst, and can reduce the reaction time and make the reactor compact. It is widely used in various fields of biochemical industry because it has many advantages, such as being able to achieve rapid production and continuous production. However, such bioreactors have the unavoidable fate that the activity of the biocatalyst immobilized on the catalyst carrier gradually decreases over time. In such cases, it is desirable to release the deactivated biocatalyst from the catalyst carrier, wash it away, and then reimmobilize a new biocatalyst. However, only when an ion exchange resin is used as a catalyst carrier can a method of injecting a high salt concentration liquid into the reactor to wash away the deactivated biocatalyst be taken, but an inorganic carrier such as ceramics If a bioreactor is used, there is no known method to regenerate the bioreactor with the catalyst carrier still packed inside the reactor.
This is because the inorganic support and the biocatalyst are bound together by a strong bond such as a covalent bond, so they cannot be easily separated. For this reason, in order to regenerate a bioreactor that uses inorganic carriers such as ceramics, the inorganic carrier had to be taken out of the reactor and fired. This was undesirable in terms of bacterial contamination. (Problems to be Solved by the Invention) The present invention solves the conventional problems as described above, and provides a method for removing the carrier from the outside of the reactor when the activity of the bioreactor in which the inorganic carrier is used decreases. This was completed in order to provide a method for regenerating a bioreactor that can be regenerated without any problems. (Means for Solving the Problems) The present invention has been made to solve the above problems, and when the activity of the immobilized biocatalyst in a bioreactor incorporating the immobilized biocatalyst decreases, hypochlorous acid or The feature is that the deactivated immobilized biocatalyst is removed by bringing an aqueous solution of hypochlorite into contact with the catalyst carrier, and then a new immobilized biocatalyst is immobilized on the catalyst carrier. Hypochlorous acid or hypochlorite salts such as sodium hypochlorite used in the present invention are known to have a sterilizing effect due to the oxygen in the generator, but hypochlorous acid itself has the effect of destroying the cell membranes of living cells,
The deactivated immobilized biocatalyst can be removed from the catalyst support. In order to use hypochlorous acid or hypochlorite for the purpose of the present invention, its effective chlorine concentration must be IPP? It is desirable to use it under the conditions that I or more and Pl1 is 9 or less. This is because if the effective chlorine concentration is less than l PPM, the immobilized biocatalyst cannot be effectively removed from the catalyst carrier, and if Pl1 increases, the degree of dissociation of hypochlorite ions decreases, and P}l This is because when the value exceeds 9, the effect is significantly reduced.

As shown in the following example, in the present invention, the deactivated immobilized biocatalyst can be removed from the catalyst support simply by injecting an aqueous solution of hypochlorous acid or hypochlorite into the bioreactor. After that, the inside of the bioreactor can be washed with water, and the new immobilized biocatalyst can be immobilized on the catalyst carrier using a conventional method. Among inorganic carriers such as ceramics, sepiolite is more preferable because it has a large specific surface area and a large amount of enzyme immobilization. (Example) Example 1 5 ml Punorellanase/50 marrow acetate buffer (PH
5.0) A protein concentration of 20 mg/g + 1 was added to 25 g of a ceramic carrier for immobilization (specific surface area 77 M/g) made of sepiolite as a raw material and fired at 900°C, and the
The mixture was allowed to stand for a period of time to prepare immobilized pullulanase. After thoroughly washing this immobilized pullulanase with 50mM acetate buffer, it was prepared with a diameter of 20 am and a length of 150 mm as shown in Figure 1.
■It was filled into the jacket force ram. Next, water at a constant temperature of 80°C is circulated through the column jacket, and after the column reaches a constant temperature, a mixed solution of maltose and cyclodextrin (weight ratio of maltose and cyclodextrin: 4; l
, an aqueous solution with a solid content concentration of 70%) was fed at a flow rate of 20 1/H.

Next, the amount of maltosylcyclodextrin produced at the outlet of this reaction ram was measured by HPLC, and the branching rate was 58%. After that, we continued to measure the branching rate, and the branching rate decreased to 20% on the 30th day. Therefore, after washing the reaction column with distilled water, it was repacked into five columns of 5 g each. Then 1 for each of these columns,
Sodium hypochlorite (PH 7.0) with an effective chlorine concentration of 100, 1,000, and 5000 PPM was pumped at a rate of 41/{1r, and the washing liquid at the outlet was collected in fraction collectors, and the protein The amount was measured.

The results are shown in Figure 2. The total amount of protein eluted with sodium hypochlorite at IOPPM or higher reached 90% of the initially immobilized protein amount. In addition, in order to investigate the presence or absence of protein remaining on the carrier, the amount of Kjeldahl nitrogen on the ceramic carrier was measured using the Kjeldahl decomposition method, and it was found to be NO. In addition, when the protein amount was similarly measured on a column washed with IPP- sodium hypochlorite, 30% of the initially immobilized protein amount was detected. For the four columns that were washed with sodium hypochlorite of IOPPM or higher, after thoroughly washing them with water,
Immobilization of pullulanase was attempted using the same procedure as the first time.

The resulting branching rate was 58.2%, confirming that the column had been regenerated to its original state. Example 2 Ceramic carrier for enzyme immobilization (specific surface area 77 M/g) 2 made of sepiolite as the main raw material and fired at 900'C
20g/ml of 51 invertase solution/50mM acetate buffer (PH 4.0) was added to 6g and left to stand at room temperature for 1 hour to prepare immobilized invertase. After thoroughly washing this invertase with 50-monoacetic acid buffer, 200mg of the immobilized enzyme was added to 2% sucrose/50mM acetate buffer-50H at 37°C, and the amount of glucose produced was measured using Wako Pharmaceutical's Glucose B test. It was measured with At this time, the activity per 1 g of immobilized invertase was 600 U when calculated as the amount of enzyme that produces 1 μmol of glucose per minute + 1 U. This immobilized invertase has a diameter of 10go and a length of 100mm.
Fill each column with 5 sg, Pllll,
Aqueous sodium hypochlorite solutions with an effective chlorine concentration of 0.1% prepared in Examples 9, 7, 5, and 3 were fed into the column at a flow rate of 4 I/Hr. Next, lal portions of the eluate were collected from each column outlet and the protein concentration was measured, resulting in an elution profile as shown in Figure 3. This result shows that when the pH of the sodium hypochlorite aqueous solution exceeds 9, the amount of immobilized inherutase eluted decreases. (Effects of the Invention) As explained above, the present invention regenerates a bioreactor using an inorganic carrier such as ceramics by bringing an aqueous solution of hypochlorous acid or hypochlorite into contact with a catalyst carrier. Since the catalyst carrier can be regenerated while remaining inside the reactor, there is no need to worry about contamination by bacteria, and complete regeneration is possible.

Therefore, the present invention will greatly contribute to the development of industry as a method for regenerating pyroreactors that solves the problems of the conventional method.

[Brief explanation of drawings]

FIG. 1 is a front view showing the continuous synthesis apparatus for immobilized pullulanase in the first example, FIG. 2 is a graph showing the difference in enzyme elution pattern depending on the available chlorine concentration in the first example, and FIG. 2 is a graph showing differences in enzyme elution patterns due to differences in eluent pH in Example 2. Figure 1

Claims (1)

[Scope of Claims] 1. When the activity of the immobilized biocatalyst in the bioreactor incorporating the immobilized biocatalyst decreases, an aqueous solution of hypochlorous acid or hypochlorite is brought into contact with the catalyst carrier to be depleted. A method for regenerating a bioreactor, comprising removing an active immobilized biocatalyst and then immobilizing a new immobilized biocatalyst on a catalyst carrier. 2. The method for regenerating a bioreactor according to claim 1, wherein an aqueous solution of hypochlorous acid or hypochlorite having an effective chlorine concentration of 1 PPM or more is brought into contact with the catalyst carrier under a pH of 9 or less.