Preparation of immobilized microorganism (B. SP 3012) by γ-irradiation

Radiat. Phys. Chem. Vol. 20, No. 3, pp. 209-214, 1982 Printed in Great Britain.

0146-5724182]090209-06503.00/0 Pergamon Press Ltd.

PREPARATION OF IMMOBILIZED MICROORGANISM (B. SP 3012) BY y-IRRADIATION LIN TUNG-FU Institute of Technology and Physics, Heilongjiang, Province Academy of Science, Harbin, China

(Received 18 January 1982) Abstract--Gel entrapment using y-ray irradiation provides an efficient method to prepare immobilized fumarase-producing microorganism (B. SP 3012). In the use of such immobilized microorganism for producing L-malic acid from fumaric acid, a high value of the activity of fumarase (13000/.tm/h/g) has been obtained. The enzymatic reaction has been carried out at pH = 6.5 and 37°C. In the case of the column method optimum reaction condition for conversion of fumaric acid to L-malic acid can be easily obtained by adjusting the space velocity of the substrate solution. Due to the sufficient durability of enzymatic activity, the immobilized microorganism can be used repeatedly. INTRODUCTION IMMOBILIZED enzyme and microorganism have been successfully adapted in industry in the production of fermentative products. So far, many studies <'-3> have been reported on the preparation of immobilized enzymes by the gel entrapping method with the use of radiation polymerization, but only a few on microorganism. The process for producing L-malic acid with fumarase-producing Brevibacterium ammoniagene microorganism entrapped by chemical method within a polyacrylamide gel matrix has been used in industry in a production scale, ~4> however, immobilization with irradiation technique yet has to be adequately researched. This article describes a method of immobilizing Brevibacterium SP 3012 microorganism entrapped within polyacrylamide gel matrices by means of y-ray irradiation. The conditions of the enzymatic reaction for the immobilized fumarase-producing microorganism and the effect in a column reactor for the continuous production of L-malic acid are reported. MATERIAL AND METHOD

I Microorganism Brevibacterium SP 3012, i . e . B . SP 3012 (provided by Institute of Industrial Microbiology, Heilongjiang Province A c a d e m y of Science, China). The fumarase activity of the immobilized microorganism treated with an aqueous solution containing cholic acid was 13600/~m/h/g. II Irradiation condition Sources are 6°Co y-ray sources (about 900Ci); Irradiation dose is 50 krad; Dose rate is 7.5 rad/s;

and Irradiation temperature is 15-20°C. Except where otherwise noted, all the irradiation conditions employed in this paper are as described above. III The preparation procedure of the immobilized

microorganism Solution A: 2 g of watery microbial cells of B. SP 3012 (corresponding to 0.4g of dry microbial cells) were suspended in 5 ml of 0.05 M phosphate buffer (pH 6.8) under throughout stirring. Solution B: 1.8g of acrylamide monomers dissolved in 5 ml of 0.05 M phosphate buffer (pH 6.8) and then 28 mg of N,N'-methylene-bisacrylamide added. After their complete dissolution solutions A and B were mixed fully and then the mixture was poured into a polyethylene film pocket of 1 2 × 4 c m . This pocket was then flushed with N2 for about 1 min to remove 02 and sealed in N2 atmosphere. Thus irradiation was carried out by placing it in a water bath at 20°C with both placed in the radiation field. The gel produced was granulated by sieveing. Granules of 2 m m dia., thus obtained were then washed throughout with a physiological saline solution. The fumarase activity of such an immobilized preparation without any treatment was 630/zm/h/g. IV Conditions of treatment to enhance the fumarase activity of the immobilized preparation Cholic acid was used as an activator to enhance the fumarase activity of the immobilized preparation thus obtained. It can cause obvious change in the permeability oi~ the cell wall and can overcome the shielding effect of the cell wall on

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LIN TUNG-FU

the substrate and the product, thereby it can greatly increase the enzyme activity. The treatment procedure was as follows: 7 g of the immobilized microorganism thus obtained (corresponding to 1 g of watery microbial cells) was suspended in 30ml of aqueous IM sodium fumarate solution (pH7.5) containing 0.3% of cholic acid. The mixture was allowed to stand at 37°C for 20 h to enhance the fumarase activity of the immobilized preparation. The immobilized preparation was collected by filtration and washed with a physiological saline solution. After treat-" ment with cholic acid the fumarase activity of the immobilized preparation thus obtained was 13000 p.m/h/g.

V Conditions of enzymatic reaction 30ml of IM sodium fumarate solution was employed as the substrate (pH6.5). 7 g of the immobilized microorganism was taken after treatment with cholic acid. The enzymatic reaction was carried out at 37°C with stirring for 1 h. Then the reaction mixture was filtered to remove the immobilized preparation. The L-malic acid content and the fumaric acid residue content in the filtrate (or upper clean solution) were assayed.

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Except where otherwise noted, all the enzymatic reactions were carried out according to the conditions described above. VI Analytical method The L-malic acid content in the filtrate was assayed in accordance with the method described by Goodban et al.~5~ A 721 model spectrophotometer (made in China) was used (wave length 390/zm). The fumaric acid residue content was assayed by using the potassium permanganate oxidationreduction method. R E S U L T S AND D I S C U S S I O N I The radiation polymerization mechanism of acrylamide The radiation polymerization of the acrylamide results from a considerable amount of free radicals (such as H, OH, H:O2, etc.) produced by y-rays in the aqueous solution of monomener, rather than from the direct effect of y-rays. These radicals might induce the radiation polymerization of the acrylamide. "'6~ The reaction mechanism may be shown as following: (1) Chain propagation

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other conditions were the same as described in the Material and Method Section in this paper. III Effect of y-rays irradiation on the fumarase

II Optimum irradiation dose and dose rate When acrylamide solution of pH--<7 without addition of any crosslinking agent was irradiated, network polymers of higher crosslinking degree can be formed (') only under a dose more than I Mrad at a rate of 25-60krad/h. In order to prevent depolymerization due to oxygen, the radiation polymerization must be carried out in a N2 atmosphere. However, when 0.02% of N,N'-methylene-bisacrylamide was added, the doses required to polymerize acrylamide into more suitable gels would obviously decrease. Gels formed at various dose rates and doses were presented in Table 1. It can be seen that even under higher doses, the quality of gel formed remains poor if the dose rate is low. Due to insufficient crosslinking of acrylamide, the gel would not be suitable for use because of the considerable swelling in aqueous solution. Except for the dose and the dose rate,

activity of the immobilized microorganism The solution of the microbial cells of microorganism and monomers was irradiated at a rate of 7.5 rad/s and immobilized at various doses, the curve for the fumarase activity of the immobilized preparation is shown in Fig. 1. Only when irradiation doses exceeds I Mrad may it appear that the enzyme activity is slowly decreasing. This implies that the immobilized fumarase-producing microorganism possess higher resistance to radiation. In the case of this test the dose selected were considerable below 1 Mrad, so as to ensure no reduction of enzyme activity.

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IV Optimum concentration of the acrylamide and

quantity of N,N'-methylene-bis-acrylamide added The dependence of the fumarase activity of the immobilized preparation obtained by y-irradiation on the acrylamide content in gel was shown in Fig. 2. Here, the optimum content of the acrylamide was 0.9g per 1 g of microbial cells, which corresponds to 12.85% of total amount of the gel. When the acrylamide content was more than 0.9 g per 1 g of microbial cells, the fumarase activity of the immobilized preparation would decrease as the content of acrylamide increases. This mainly results from that the thickening of the entrapping layers directly influencing the velocity of diffusion of the reaction mixture during the course of the enzymatic reaction. However, if the acrylamide content was less than 0.9g per 1 g of microbial cells, owing to the softness, viscosity and poor strength of the gel its use became disadvantageous. The content of N,N'-methylene-bis-acrylamide was only 0.2% of the total amount of the gel in this test. The gel would become brittle if the content of N,N'-methylene-bis-acrylamide were excessive. However, in the chemical polymerization gel system of acrylamide, the content of N,N'methylene-bis-acrylamide was about 2-5% of the total amount of the gel, (4'9) this amount was con-

siderably more than in the case of radiation polymerization.

V Optimum pH value of the immobilized fumarase-producing microorganism prepared by the gel entrapping method using ~/-rays irradiation The optimum pH value of the immobilized fumarase-producing microorganism (B. SP 3012) was in the range of 6.0-7.5. It seemed that the optimum pH value for the immobilized fumaraseproducing microorganism prepared by the radiation polymerization method was higher than that by the chemical polymerization method. However, when pH > 7.5, the enzyme activity was decreased sharply (Fig. 3). VI Temperature curve for the immobilized fumarase-producing microorganism (B. SP 3012) prepared by the means of y-rays irradiation The optimum temperature for the immobilized fumarase-producing microorganism (B. SP 3012) was 55°C. This optimum temperature was higher than that for the native microorganism. However, the optimum temperature for the immobilized micoorganism prepared by means of y-irradiation polymerization was lower than that by chemical polymerization (its optimum temperature was 65°C) (Fig. 4).

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FIG. 4. Dependence of the fumarase activity of the immobilized microorganism prepared by the means of 3'irradiation on temperature. Except for the temperature, other conditions were that as described in the Material and Method Section in this paper. VII Heat stability of the immobilized fumaraseproducing microorganism (B. SP 3012) prepared by the means of y-irradiation The curve for the heat stability of the immobilized fumarase-producing microorganism (B. SP 3012) prepared by the means of y-irradiation was shown in Fig. 5. The procedure used was as

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following: Several examples each containing 7 g of gel granules of the immobilized microorganism were treated with cholic acid were suspended in a physiological saline solution and heated at each given temperature with stirring for 1 h. After rapid cooling, the immobilized microorganism were filtered and collected. Then the enzymatic reactions were carried out at given various temperature. As the immobilized microorganisms were heated over 50°C, the enzyme activity of the immobilized microogranism decreased sharply. At this point, the feature of the immobilized microorganism prepared by means of y-irradiation was very similar to that prepared by means of chemical polymerization. Compared with the native microorganism, the decrease in heat stability for the immobilized fumarase-producing microorganism (B. SP 3012) was an unexpected phenomenon. VIII Condition for the unexpected production of

L-malice acid employing the immobilized [umarase-producing microorganism in the column reactor 105 g Of gel granules of the immobilized microorganism were washed with a physiological saline solution many times. The granules treated with cholic acid were charged into a 3 0 × 3 cm glass column. The enzymatic reaction of the substrate was carried out continuously at constant temperature (37°C) (descent method). (1) Determination of the maximum space volume. The balance point of the conversion of fumaric acid to L-malic acid for the enzymatic reaction of the immobilized fumarase-producing microorganism was attained as the percentage conversion was about 82%. ~8) When the space velocity of reaction column (i.e. SV) < 1, all of the percentage conversion was about 82%. When SV -> I, the percentage conversion began to decrease. As SV = 4, the percentage conversion was 50% only (Fig. 6). (2) Stability of the reaction column. In the practical operation, if SV = 0.25 and flow rate per hour was 52.5 ml, the percentage conversion of the sodium fumarate to the sodium L-malate for the enzymatic reaction of the immobilized fumaraseproducing microorganism remained at 70% after 25 days. The decrease tendency of the percentage conversion is shown in Table 2. (3) The effluent was acidified with concentrated hydrochloric acid, and then filtered to remove the precipitate of fumaric acid. The excess calcium carbonate was added to the filtrate. The precipitates were collected by filtration, washed with water and then dried to give the crude product.

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LIN TUNG-FU

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TABLE 2. DECREASE IN PERCENTAGE CONVERSION OF SODIUM FUMARATE TO SODIUM L-MALATE

Operation days The percentage conversion of the sodium fumarate to the sodium Lmalate (%) (37°C, SV = 0.25) 5 10 15 25

82 80 80 70

CONCLUSIONS (1) The fumarase activity of the immobilized microorganism prepared by the gel entrapping method using y-irradiation, treated with the cholic

acid solution, was 13,000/xm/h/g of microbial cells. This value was higher than that prepared by the means of chemical immobilizing techniques. (2) Owing to the fast rate of immobilization and the fast diffusion of heat from thin entrapped matrix, the enzyme activity loss induced by polymerization heat for the immobilized microorganism prepared by the gel entrapping method using y-irradiation can be diminished. (3) The method of polymerization of acrylamide induced by y-irradiation was used, in which microorganism was crosslinked in a network matrix structure and without any reagent as is required in chemical immobilizing techniques. The amount of crosslinking agent was only 0.2%, so that the factor that caused the lowering of the enzyme activity was also decreased. (4) Because of its advantages, simple and convenient, the technique product pure, low cost, the good immobilizing effect, etc. the technique of producing immobilized microorganism preparation by the gel entrapping method using y-irradiation is more advantageous and of benefit in industrial production. REFERENCES 1. H. MAEDA and H. SUZUKI, Biochem. Biophys. Acta. 1973, 315, 18. 2. I. KAETSU.,A. ITO and K. HAVASHI,J. PolymerSci. A-l, 1973, 11, 1811. 3. K. KAWASHIMAand K. UMEDA,Biotech. Bioeng. 1974, 16, 609. 4. I. CHIBATA, T. TOSA and T. SATO, U,S. Pat. (1975), 3, 922, 195. 5. A. E. GOODI3ANand B. STARK, Anal. Chem. 1967, 29, 2, 283. 6. T. WADA,J.Polym. Sci. Polymer Chem. Ed. 1975,13, 10. 7. An Introduction to Experimental Technique of Biochemistry The Department of Biologies. Zhongshan University, China (in Chinese), p. 241, 1979. 8. K. YAMAMOTO,Biotech. Bioeng. 1977, 19, 1115. 9. I. CHIBATA, Yuki Gosei Kagaku Kaishi 1974, 32, 2, 286.