Immobilization of Enzymes and Microbial Cells Using Carrageenan as Matrix TETSUYA TOSA,* TADASHI SATO, T A K A 0 MORI, KOZO YAMAMOTO, ISAO TAKATA, YUTAKA NISHIDA, and ICHIRO CHIBATA, Department of Biochemisrry, Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co. Ltd., 16-89, Kashima-3-chome, Yodogawa-ku, Osaka, Japan
Summary Conditions for the gelation of k-carrageenan, which is a new polymer for immobilization of enzymes and microbial cells, were investigated in detail. k-Carrageenan was easily induced to gel by contact with metal ions, amines, amino acid derivatives, and water-miscible organic solvents. By using this property of k-carrageenan, the immobilization of enzymes and microbial cells was investigated. Several kinds of enzymes and microbial cells were easily immobilized with high enzyme activities. Immobilized preparations were easily tailor-made to various shapes such as cube, bead, and membrane. The obtained immobilized preparations were stable, and columns packed with them were used for continuous enzyme reaction for a long period. Their operational stabilities were enhanced by hardening with glutaraldehyde and hexamethylenediamine.
INTRODUCTION
At present, the immobilization of enzymes and microbial cells has been the subject of increased interest, and many papers on immobilized enzymes and microbial cells have been published. 132 In 1969, we succeeded in the industrial application of an immobilized enzyme, i.e., immobilized aminoacylase, for continuous production of L-amino acids from acyl-DL-amino acid^.^*^ Since then we also have carried out the industrial application of immobilized microbial cells, and in 1973 we succeeded in the industrial application of these immobilized cells for continuous production of Laspartic acid with Escherichia coli having a high aspartase activit^.^,^ Further, in 1974, we succeeded in the industrial production of L-malic acid using immobilized Brrvibacterium ammoniagenes having a high fumarase activity. 7,8 4
To whom all correspondence should be addressed
Biotechnology and Bioengineering, Vol. XXI, Pp. 1697- 1709 (1979) 0006-3592/79/OO21-1697$01.OO
@ 1979 John Wiley & Sons, Inc.
TOSA ET AL.
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For further improvement of these immobilization systems, we investigated the applicability of many synthetic and natural polymers as a matrix for entrapping enzymes and microbial cells into the gel lattice. As a result, we found that “k-carrageenan,” which is a polysaccharide prepared from seaweeds and is used as a food additive, was the most suitable among the tested polymers for the immobilization of microbial cells.s In this paper, we present the detailed conditions for the immobilization of enzymes and microbial cells using k-carrageenan.
MATERIALS AND METHODS
Carrageenan k-Carrageenan was obtained from Sansyo Co. Ltd. (Osaka, Japan), and the chemical structure is shown in Figure 1 . This polysaccharide is composed of unit structures of p-D-galactose sulfate The molecular weight is around and 3,6-anhydro-a-~-galactose. 100000-800000, and the ester content is 20 to 30% of the unit weight. Other Chemical Compounds Methylenediamine, ethylenediamine, hexamethylenediamine, octamethylenediamine, and p-phenylenediamine were obtained from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan). Hydroxamates of Lhistidine and L-tryptophan, agmatine, and S-2-aminoethyl-~-cysteine were obtained from Sigma Chemical Co. (St. Louis, MO). Poly(L-lysine), &hydroxyl-L-lysine, and DL-histidine hydrazide were obtained from Protein Research Foundation (Osaka, Japan). Other reagents were obtained from Katayama Chemical Industries Co. Ltd. (Osaka, Japan).
0-
H \
OH
H
OH
n = 2502.2000
Fig. 1. Chemical structure of k-carrageenan.
n
ENZYME IMMOBILIZATION USING CARRAGEENAN
1699
Microbial Cells Escherichia coli cells'O and B. ammoniagenes cells' were obtained by methods of previous papers, respectively. Streptomyces phaeochromogenes cells were a gift from Godo Shusei Co. Ltd. (Tokyo, Japan). Enzymes Aminoacyla~e,~ aspartase, and fumarase' were prepared from Aspergillus oryzae, E . coli, and B. ammoniagenes according to previous papers, respectively.
Gelation of Carrageenan k-Carrageenan was dissolved in physiological saline previously warmed at 70 to 80°C at a concentration of 3.4% (w/v), and the solution was kept at 40°C. Gelation of this carrageenan solution was carried out as follows: 1) cooling below 10°C; 2) contact with 0. IM metal salt solutions; 3) contact with 0.5M ammonium chloride dissolved in 0.5M phosphate buffer (pH 7.0); 4) contact with 0.5M diamine hydrochloride dissolved in 0.5M phosphate buffer (pH 7.0), and 5) contact with water-miscible organic solvents.
Measurement of Gel Strength The gel strength of samples was measured by using Rheometer NRM-2002 J type, Fudo Kogyo Co. Ltd. (Tokyo, Japan) with a disk-plate plunger of 10 mm diam, and estimated with load for gel crush when a sample was pressed by the plunger.
Immobilization of Enzymes and Microbial Cells Using k-Carrageenan Cubic type The standard immobilization procedures of enzymes and microbial cells of the cubic type were carried out as follows: 100 mg enzyme or 16 g (wet weight) microbial cells were dissolved or suspended in 32 or 16 ml physiological saline at 25 to SO"C, respectively, and 3.4 g carrageenan are dissolved in 68 ml of the physiological saline at 37 to 60°C. The two were mixed, and the mixture was cooled at around 10°C for 30 min. In order to increase the gel strength, the obtained gel was soaked in a cold 0.3M potassium chloride solution. After this treatment, the resulting stiff gel was formed to a cubic gel of 3 x 3 x 3 mm.
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Bead type
Fifty ml of the mixture of carrageenan and enzyme or microbial cells mentioned above were dropped into a solution containing one of the gel-inducing reagents through a special nozzle having an orifice of I mm in diam at a constant speed. Bead-type gels of 3 mm in diam were obtained by this procedure. Membrane type
Fifty ml of the mixture mentioned above was spread on a plate or sheet (1 x 250 x 200 mm), and soaked in a cold 0.3M potassium chloride solution. In order to estimate enzyme activity, the obtained gel of membrane type was cut to a size of 1 x 10 x 10 mm.
Immobilization of'Enzymes and Microbial Cells Using Polyacrylam ide Gel Enzymes and microbial cells were immobilized by the polyacrylamide gel method as previously r e p ~ r t e d . ~
Hardening Treatment with Glutaraldehyde and Hexamethylenediamine Immobilized preparations (8 to 9 g, wet weight) were suspended in 10 ml O.5M phosphate buffer (pH 7.0) containing 0.3M potassium chloride and 0.02 or 0.12M hexamethylenediamine, and the suspension was gently stirred for 10 min at 5°C. To the suspension 4 ml 0.006 to 0.6M glutaraldehyde were added, and the mixture was gently stirred for 30 min at 5°C. After this treatment, the hardenedimmobilized preparations were sufficiently washed with a cold 0.3M potassium chloride solution.
Estimation of Enzyme ActiviticJs Enzyme activities of a m i n ~ a c y l a s e aspartase,'O ,~ and fumarase' were measured according to previous papers, respectively. Glucose isomerase activity was measured as follows: 3.2 g immobilized cells corresponding to 0.5 g of the cells were incubated with 20 ml 0. IM phosphate buffer (pH 7.0) containing 0. IM glucose and 0.02M MgS04 at 70°C for 30 min with shaking. The enzyme reaction was terminated by filtering off the immobilized cells. The amount of fructose in the filtrate was determined by the cysteine-carbazolesulfate method.I2
ENZYME IMMOBILIZATION USING CARRAGEENAN
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Estimation of HalfLife of a Column Packed with Immobilized Preparation
For determination of the activity decay of a column packed with immobilized preparation the column effluent was collected as a sample at a flow rate giving between 20 and 30% of maximum conversion rate, and the product in the effluent was measured. The apparent half-life was estimated by assuming an exponential decay of enzyme activity versus time.
RESULTS Gelation of k-Carrageenan
Conditions for gelation of k-carrageenan were tested, and the results are shown in Table I. The compound gelled by cooling at 10°C, which is similar to the behavior of agar. Its gelation occurred by contact with an aqueous solution containing an ammonium ion or metal ions such as alkali metal ions (except for Li+ and Na+). alkaline-earth metal ions, and other bi- or trivalent metal ions. Besides these ions, gelation was caused by contact with amines such as aliphatic or aromatic diamines, amino acids, amino acid derivatives, and with water-miscible organic solvents. In addition, the effect of anions on the gelation of k-carrageenan was investigated by using potassium salts of anions such as CO,'-, SO3'-, SO4'-, S208'-, P20,4-, A1,(S04),'-, Fe(CN)63-, As a result, no marked difference CH3COO-, and -0OC-COO-. was observed in respective anions. Immobilization o f Microbial Cells Using Various Gel-Inducing Reagents
Immobilization of E. coli cells having aspartase activity, and S . phaeochromogenes cells having glucose isomerase activity, into a k carrageenan bead-type gel lattice was investigated by using various gel-inducing reagents. Table. I1 shows the respective activity of immobilized cells prepared by using various gel-inducing reagents, as well as the observed activity yield. In the case of E. cofi, the cells could be immobilized with relatively high retention of activity by a variety of gel-inducing reagents except for ferric chloride, which is an inhibitor of aspartase. On the other hand, in the case
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TABLE I Relation between Condition for Gelation of k-Carrageenan and Gel Strength Condition for gelation
Gel strengtha
+
I ) Cooling at 10°C 2) Contact with ammonium ion
NH,CI 3) Contact with metal ions i) Alkali metal ions LiCI NaCl KCI RbCl CSCl i i ) Alkaline-earth metal ions MgCI, CaCI, SrCI, BaCI, iii) Other bi- or trivalent metal ions AICI, MnCI, FeCI, FeCI, COCI, NiCI, CUCI, ZnCI, Pb (CH,COO), a
+++
++++ +++i
++++ i f
+++
++ ++
+++
++ ++ ++
++ ++ ++ ++ ++
Symbols for gel strength correspond to respective load for gel-crush as follows:
-
(+) 100 to -200 g/cm2; (++) 200 to 500 g/cm*; (+++) 500 to -1000 g/cm2; (+ +) lo00 to 1500 g/cm2; (-) no gelation. Gelation was carried out as described
++
-
in the Material and Methods section.
of S . phaeochromogencs cells, very active preparations were obtained by using potassium chloride or hexamethylenediamine, and the use of an inhibitor of glucose isomerase such as calcium chloride or ferric chloride led to low activity as in the case of E. coli cells. Preparation of Various Shapes of Immobilizd Microbial Cells
Whole cells of E. coli having aspartase activity were immobilized into a k-carrageenan gel lattice in various shapes such as cube, bead, or membrane, and their enzyme activities were compared (Table 111). As shown in Table 111, some differences were observed between the enzyme activities of the different immobilized preparations. This may be due to the difference of the respective surface area of the preparations because the surface area of the preparations varies with shapes of gel as shown in Table 111.
I703
ENZYME IMMOBILIZATION USING CARRAGEENAN TABLE I. (Continued f r o m previous p a g e . ) Condition for gelation
Gel strengtha
4) Contact with amines
i) Aliphatic diamine Methylenediamine Eth ylenediamine Hexamethylenediamine Octamethylenediamine ii) Aromatic diamine p-Phenylenediamine iii) Amino acids and their derivatives Agmatine Histamine L-Ornithine L-Lysine GHydroxyl-L-iysine L-Lysine hydroxamate L-Histidine hydroxamate DL-Histidine hydrazide L-Tryptophan hydroxamate S-2-Aminoethyl-~-cysteine 5 ) Contact with water-miscible organic solvents Methanol Ethanol Acetone
+++ +++ ++++ ++++ +++ ++++ ++++ ++ ++ +++ +++
+++
++++
++ +++ ++ ++ ++
Itnmobilization of Various Enzymes arid Microbial Cells Immobilization of three kinds of enzymes and four kinds of microbial cells into a k-carrageenan cubic-type gel lattice was investigated using potassium chloride as a gel-inducing reagent, and the enzyme activities were compared with those of the respective native enzymes or intact cells (Table IV). As shown in Table IV, the activities of the obtained immobilized enzymes and microbial cells were high. However, the yield of their activities was around 50%. This may be due to diffusion limitation of substrate and/or product in the gel matrix. Hurdening Treatment for Stabilization of Immobilized Microbial Cells
In order to increase the operational stability of immobilized preparations obtained by using k-carrageenan, those were treated with
46.7 38.2
3.6 44.5 50.0 39.8 57.3
30360 24860
2340 28930 32520 25840 37260
(%)
4280 2480 300 2420 496 I960 I380 4216
glucose isomerase activity (pmol/hr/g cells)
54.1 31.3 3.8 30.6 6.3 24.7 17.4 53.3
(%)
activity yield
Immobilized cells obtained after activation by incubating with a substrate solution at 37°C for 24 hr. On the activation procedure see Ref.
5% absolute
0.3 M
Concentration
activity yield
S. phaeochromogenes
65000 and 7910 pmoVhr/g cells, respectively.
5 . Aspartase activity after sonication of intact cells of E. coli and glucose isomerase activity of intact cells of S . phaeochrornogenes were
a
KCI MgCI, CaCI, Ba-acetate FeCI, NH,CI NH,-fumarate Hexameth ylenediamine Polyeth yleneimine Acetone
Gel-inducing reagent
aspartasea activity (pmol/hr/g cells)
E . coli
TABLE I1 Immobilization of Whole Cells of E . coli and S . phaeochromogcwes into k-Carrageenan Bead-Type Gel Lattice Using Various Gel-Inducing Reagents
1
*r
rn
>
1
4 0 P
ENZYME IMMOBILIZATION USING CARRAGEENAN
I705
TABLE 111 Aspartase Activities of Immobilized E. coli Cells Formed in Various Shapes and Their Activity Yields Surface area of gel (cm*/g cells)
Shape of gel Bead (diameter = 3 mm) Cube (3 x 3 x 3 mm) Membrane (I x 10 x 10 mrn)
124 125 150
Aspartase activitya (pmoVhr/g cells)
Activity yield (%)
30360 32 130 41 120
46.7 49.4 63.3
a After activation by the same condition given in Table 11. Aspartase activity affer sonication of intact cells used was 65000 pmoUhr/g cells.
hardening reagents such as glutaraldehyde and hexamethylenediamine. The results were shown in Table V. The aspartase activity of immobilized E . coli cells was somewhat reduced by this hardening treatment, but the operational stabilities were markedly enhanced. In the case of the glucose isomerase activity of immobilized S. phacochromogcnes cells, there was no noticeable reduction by this hardening treatment, and their stabilities were also markedly improved. TABLE IV Immobilization of Various Enzymes and Microbial Cells Enzyme activitya Immobilization Enzyme and microorganism (enzyme) Arninoacylase Aspartase Fumarase
E . coli (Aspartase) B. ammoniagenes (Fumarase) S . phaeochromogenes
Yield before
after
(%)
20 650 360
10 300 220
50.0 46.2 61.1
30400
46.8
65Wb 967Oe
58Wd
60.0
7910
4280
54.1
(Glucose isomerase) a
Enzyme: prnollhrlmg protein; microbial cells: pmoVhr/g cells. After sonication. In the presence of bile extract. After treatment with bile extract.
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TABLE V Hardening Treatment for Stabilization of Aspartase Activity of Immobilized E . coli Cells and Glucose Isomerase Activity of Immobilized S. phaeochromogenes Cells Hardening treatment/Reagent and final conc E . coli (Aspartase) None HMDA" - GAb (mM) (mM) 85.0 I .7 85.0 17.0 85.0 85.0 85.0 170.0 S . phaeochromogenes (Glucose isomerase) None HMDA - G A (mM) (mM) 20 10 a
Enzyme activity (prnol/hr/g cells)
Half-life at 37°C (day)
30400
50
27500 I9900 21400 I8300
686 443
4280
53
4380
289
7s 168
HMDA: Hexamethylenediamine GA: Glutaraldehyde.
Comparison of Activity Yield and Stability of Iinmobilizod PrcJparations Obtained by Carrageenan and Polyacrylamide Gel Methods
Activity yields and operational stabilities of immobilized enzymes and immobilized microbial cells prepared by using k-carrageenan (cubic-type) were compared with those of immobilized preparations obtained by using polyacrylamide gel (Table VI). As shown in Table VI, the activity yields of immobilized preparations obtained by the carrageenan method were similar to or higher than those of preparations obtained by the polyacrylamide gel method. In addition, the operational stabilities of the preparations obtained by the carrageenan method were enhanced by hardening with glutaraldehyde and hexamethylenediamine, and the hardened preparations were more stable than the corresponding preparations obtained by the polyacrylamide gel method.
29
E . coli
a
12
31
31
60
51
60
34
Hardening with glutaraldehyde and hexamethylenediamine.
53
60
10
75
289a
686a
120 150
I20
60
20 5
30
carrageenan
half-life (day) pol yacrylamide
59a
37
31 60
31
(“c)
temp
51
33a
46 69a
29 12
(Aspartase) S. phasockromogetic,s (Glucose isomerase) B. ammotiiagc,tws (Fumarase) B. Jmum (Fumarase)
50a
carrageenan
50
polyacrylamide
Enzyme activity yield (%)
Aminoacylase Aspartase Glucose isomerase
Enzyme and microorganism (enzyme)
Operational stability
TABLE VI Comparison of Activity Yield and Stability of Immobitized Enzymes and Immobilized Microbial Cells Prepared by Carrageenan and Polyacrylamide Gel Methods
4 0
I .
1708
TOSA ET AL.
DISCUSSION In a previous paper,s we found that k-carrageenan is a suitable polymer for immobilization of S . phaeochromogenes, which is a microorganism displaying glucose isomerase activity. In order to apply this polysaccharide, which is commercially available at low cost, to the immobilization of other microbial cells and enzymes, the detailed conditions for gelation of k-carrageenan were investigated. As a result, it was clarified that the polysaccharide is easily induced to the gel by contact with a solution containing one of a number of gel-inducing reagents such as metal ions, amines, and water-miscible organic solvents as shown in Table I. By using this property of k-carrageenan, the immobilization of enzymes and microbial cells was investigated. Advantages of this method are that the immobilization can be performed under very mild conditions and can be achieved without the use of chemicals that may change the structure of enzyme proteins or microbial cells. Accordingly, as shown in Table IV, the activities and yields of immobilized enzymes and microbial cells obtained by using potassium ion as a gel-inducing reagent were high. Therefore, if a suitable gel-inducing reagent is selected for immobilization of individual enzymes or microbial cells, a preparation of high enzyme activity and operational stability may be obtained. The pore size of this gel matrix is small enough to prevent higher-molecular-weight compounds, such as enzyme proteins, from leaking out from the gel lattice, although the lower-molecularweight substrates and products easily pass through the gel lattice. These immobilized enzymes and immobilized microbial cells were stable, and a column packed with them can be used for continuous enzyme reaction for a long period. If the operational stabilities of the immobilized enzymes and microbial cells are unsatisfactory, preparations of greater stability can be obtained by further treatment with hardening reagents such as glutaraldehyde and hexamethylenediamine. One further advantage of this method is that the various shapes of immobilized enzymes or immobilized microbial cells can be easily tailored for particular application purposes as shown in Table 111. In addition, if immobilized microbial cells, prepared by the method using various gel-inducing reagents, are suspended in physiological saline, the k-carrageenan gel is rapidly dissolved and a stable cell suspension is obtained. This is advantageous for investigating characteristics of microbial cells after immobilization. For example, it
ENZYME IMMOBILIZATION USING CARRAGEENAN
I709
can be easily detected using this k-carrageenan as a matrix whether microbial cells after immobilization are living or not. That is, immobilized cells are thoroughly washed with sterilized potassium chloride solution and are suspended in sterilized physiological saline to dissolve k-carrageenan gel. The resulting cell suspension is suitably diluted with the same saline and is inoculated on an agar plate containing nutrient medium. The number of living cells entrapped in the gel can be counted as colonies. In conclusion, this facile carrageenan method is applicable for immobilization of many kinds of enzymes and microbial cells. Moreover, as this method is capable of making various shapes of immobilized preparations, the design of a suitable reactor for a given application becomes feasible. The authors thank G6do Shusei Co. Ltd., Tokyo, Japan for providing S . phaeochromogrnes cells.
References I . K . Mosbach, Methods in Enzymokigy (Immobilized Enzymes) (Academic, New York, 1976), Vol. 44. 2. 1. Chibata and T. Tosa, Advances in Applied Microbictktgy (Academic, New York, 1977). Vol. 22, p. I . 3. T. Tosa, T. Mori, N. Fuse, and I. Chibata, Enzymologia. 31, 214 (1966). 4. I. Chibata, T. Tosa, T. Sato, T. Mori, and Y. Matuo, Proc. Int. Ferment. Symp., Ferment. Techno/. Today. 4, 383 (1972). 5. I. Chibata, T. Tosa, and T. Sato, Appl. Microbiol.. 27, 878 (1974). 6. T. Tosa, T. Sato, T. Mori, and I. Chibata, Appl. Microbiol.. 27, 886 (1974). 7. K . Yamamoto. T. Tosa, K. Yamashita. and I. Chibata, Eitr. J. Appl. MiCriib i ~ l . 3, . 169 (1976). 8. K. Yamamoto, T. Tosa, K. Yamashita, and I. Chibata, Biotwhno/. Bioeng.. 19, I101 (1977). 9. I. Takata, T. Tosa, and I. Chibata, J. Solid-Phase Biochem.. 2, 225 (1977). 10. T. Tosa, T. Sato, Y. Nishida. and I. Chibata, Biochim. Biophys. Acta. 483, 193 (1977). 1 1 . T. Tosa, T. Sato, K. Yamamoto, Y. Matuo, and I. Chibata, Biochim. Biophys. Acta, 334, I (1974). 12. Z . Dishe and E. Borenfreund. J. Biol. Chem., 92, 583 (1951).
Accepted for Publication November 6, 1978
Summary Conditions for the gelation of k-carrageenan, which is a new polymer for immobilization of enzymes and microbial cells, were investigated in detail. k-Carrageenan was easily induced to gel by contact with metal ions, amines, amino acid derivatives, and water-miscible organic solvents. By using this property of k-carrageenan, the immobilization of enzymes and microbial cells was investigated. Several kinds of enzymes and microbial cells were easily immobilized with high enzyme activities. Immobilized preparations were easily tailor-made to various shapes such as cube, bead, and membrane. The obtained immobilized preparations were stable, and columns packed with them were used for continuous enzyme reaction for a long period. Their operational stabilities were enhanced by hardening with glutaraldehyde and hexamethylenediamine.
INTRODUCTION
At present, the immobilization of enzymes and microbial cells has been the subject of increased interest, and many papers on immobilized enzymes and microbial cells have been published. 132 In 1969, we succeeded in the industrial application of an immobilized enzyme, i.e., immobilized aminoacylase, for continuous production of L-amino acids from acyl-DL-amino acid^.^*^ Since then we also have carried out the industrial application of immobilized microbial cells, and in 1973 we succeeded in the industrial application of these immobilized cells for continuous production of Laspartic acid with Escherichia coli having a high aspartase activit^.^,^ Further, in 1974, we succeeded in the industrial production of L-malic acid using immobilized Brrvibacterium ammoniagenes having a high fumarase activity. 7,8 4
To whom all correspondence should be addressed
Biotechnology and Bioengineering, Vol. XXI, Pp. 1697- 1709 (1979) 0006-3592/79/OO21-1697$01.OO
@ 1979 John Wiley & Sons, Inc.
TOSA ET AL.
1698
For further improvement of these immobilization systems, we investigated the applicability of many synthetic and natural polymers as a matrix for entrapping enzymes and microbial cells into the gel lattice. As a result, we found that “k-carrageenan,” which is a polysaccharide prepared from seaweeds and is used as a food additive, was the most suitable among the tested polymers for the immobilization of microbial cells.s In this paper, we present the detailed conditions for the immobilization of enzymes and microbial cells using k-carrageenan.
MATERIALS AND METHODS
Carrageenan k-Carrageenan was obtained from Sansyo Co. Ltd. (Osaka, Japan), and the chemical structure is shown in Figure 1 . This polysaccharide is composed of unit structures of p-D-galactose sulfate The molecular weight is around and 3,6-anhydro-a-~-galactose. 100000-800000, and the ester content is 20 to 30% of the unit weight. Other Chemical Compounds Methylenediamine, ethylenediamine, hexamethylenediamine, octamethylenediamine, and p-phenylenediamine were obtained from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan). Hydroxamates of Lhistidine and L-tryptophan, agmatine, and S-2-aminoethyl-~-cysteine were obtained from Sigma Chemical Co. (St. Louis, MO). Poly(L-lysine), &hydroxyl-L-lysine, and DL-histidine hydrazide were obtained from Protein Research Foundation (Osaka, Japan). Other reagents were obtained from Katayama Chemical Industries Co. Ltd. (Osaka, Japan).
0-
H \
OH
H
OH
n = 2502.2000
Fig. 1. Chemical structure of k-carrageenan.
n
ENZYME IMMOBILIZATION USING CARRAGEENAN
1699
Microbial Cells Escherichia coli cells'O and B. ammoniagenes cells' were obtained by methods of previous papers, respectively. Streptomyces phaeochromogenes cells were a gift from Godo Shusei Co. Ltd. (Tokyo, Japan). Enzymes Aminoacyla~e,~ aspartase, and fumarase' were prepared from Aspergillus oryzae, E . coli, and B. ammoniagenes according to previous papers, respectively.
Gelation of Carrageenan k-Carrageenan was dissolved in physiological saline previously warmed at 70 to 80°C at a concentration of 3.4% (w/v), and the solution was kept at 40°C. Gelation of this carrageenan solution was carried out as follows: 1) cooling below 10°C; 2) contact with 0. IM metal salt solutions; 3) contact with 0.5M ammonium chloride dissolved in 0.5M phosphate buffer (pH 7.0); 4) contact with 0.5M diamine hydrochloride dissolved in 0.5M phosphate buffer (pH 7.0), and 5) contact with water-miscible organic solvents.
Measurement of Gel Strength The gel strength of samples was measured by using Rheometer NRM-2002 J type, Fudo Kogyo Co. Ltd. (Tokyo, Japan) with a disk-plate plunger of 10 mm diam, and estimated with load for gel crush when a sample was pressed by the plunger.
Immobilization of Enzymes and Microbial Cells Using k-Carrageenan Cubic type The standard immobilization procedures of enzymes and microbial cells of the cubic type were carried out as follows: 100 mg enzyme or 16 g (wet weight) microbial cells were dissolved or suspended in 32 or 16 ml physiological saline at 25 to SO"C, respectively, and 3.4 g carrageenan are dissolved in 68 ml of the physiological saline at 37 to 60°C. The two were mixed, and the mixture was cooled at around 10°C for 30 min. In order to increase the gel strength, the obtained gel was soaked in a cold 0.3M potassium chloride solution. After this treatment, the resulting stiff gel was formed to a cubic gel of 3 x 3 x 3 mm.
TOSA ET AL.
1700
Bead type
Fifty ml of the mixture of carrageenan and enzyme or microbial cells mentioned above were dropped into a solution containing one of the gel-inducing reagents through a special nozzle having an orifice of I mm in diam at a constant speed. Bead-type gels of 3 mm in diam were obtained by this procedure. Membrane type
Fifty ml of the mixture mentioned above was spread on a plate or sheet (1 x 250 x 200 mm), and soaked in a cold 0.3M potassium chloride solution. In order to estimate enzyme activity, the obtained gel of membrane type was cut to a size of 1 x 10 x 10 mm.
Immobilization of'Enzymes and Microbial Cells Using Polyacrylam ide Gel Enzymes and microbial cells were immobilized by the polyacrylamide gel method as previously r e p ~ r t e d . ~
Hardening Treatment with Glutaraldehyde and Hexamethylenediamine Immobilized preparations (8 to 9 g, wet weight) were suspended in 10 ml O.5M phosphate buffer (pH 7.0) containing 0.3M potassium chloride and 0.02 or 0.12M hexamethylenediamine, and the suspension was gently stirred for 10 min at 5°C. To the suspension 4 ml 0.006 to 0.6M glutaraldehyde were added, and the mixture was gently stirred for 30 min at 5°C. After this treatment, the hardenedimmobilized preparations were sufficiently washed with a cold 0.3M potassium chloride solution.
Estimation of Enzyme ActiviticJs Enzyme activities of a m i n ~ a c y l a s e aspartase,'O ,~ and fumarase' were measured according to previous papers, respectively. Glucose isomerase activity was measured as follows: 3.2 g immobilized cells corresponding to 0.5 g of the cells were incubated with 20 ml 0. IM phosphate buffer (pH 7.0) containing 0. IM glucose and 0.02M MgS04 at 70°C for 30 min with shaking. The enzyme reaction was terminated by filtering off the immobilized cells. The amount of fructose in the filtrate was determined by the cysteine-carbazolesulfate method.I2
ENZYME IMMOBILIZATION USING CARRAGEENAN
1701
Estimation of HalfLife of a Column Packed with Immobilized Preparation
For determination of the activity decay of a column packed with immobilized preparation the column effluent was collected as a sample at a flow rate giving between 20 and 30% of maximum conversion rate, and the product in the effluent was measured. The apparent half-life was estimated by assuming an exponential decay of enzyme activity versus time.
RESULTS Gelation of k-Carrageenan
Conditions for gelation of k-carrageenan were tested, and the results are shown in Table I. The compound gelled by cooling at 10°C, which is similar to the behavior of agar. Its gelation occurred by contact with an aqueous solution containing an ammonium ion or metal ions such as alkali metal ions (except for Li+ and Na+). alkaline-earth metal ions, and other bi- or trivalent metal ions. Besides these ions, gelation was caused by contact with amines such as aliphatic or aromatic diamines, amino acids, amino acid derivatives, and with water-miscible organic solvents. In addition, the effect of anions on the gelation of k-carrageenan was investigated by using potassium salts of anions such as CO,'-, SO3'-, SO4'-, S208'-, P20,4-, A1,(S04),'-, Fe(CN)63-, As a result, no marked difference CH3COO-, and -0OC-COO-. was observed in respective anions. Immobilization o f Microbial Cells Using Various Gel-Inducing Reagents
Immobilization of E. coli cells having aspartase activity, and S . phaeochromogenes cells having glucose isomerase activity, into a k carrageenan bead-type gel lattice was investigated by using various gel-inducing reagents. Table. I1 shows the respective activity of immobilized cells prepared by using various gel-inducing reagents, as well as the observed activity yield. In the case of E. cofi, the cells could be immobilized with relatively high retention of activity by a variety of gel-inducing reagents except for ferric chloride, which is an inhibitor of aspartase. On the other hand, in the case
TOSA ET AL.
1702
TABLE I Relation between Condition for Gelation of k-Carrageenan and Gel Strength Condition for gelation
Gel strengtha
+
I ) Cooling at 10°C 2) Contact with ammonium ion
NH,CI 3) Contact with metal ions i) Alkali metal ions LiCI NaCl KCI RbCl CSCl i i ) Alkaline-earth metal ions MgCI, CaCI, SrCI, BaCI, iii) Other bi- or trivalent metal ions AICI, MnCI, FeCI, FeCI, COCI, NiCI, CUCI, ZnCI, Pb (CH,COO), a
+++
++++ +++i
++++ i f
+++
++ ++
+++
++ ++ ++
++ ++ ++ ++ ++
Symbols for gel strength correspond to respective load for gel-crush as follows:
-
(+) 100 to -200 g/cm2; (++) 200 to 500 g/cm*; (+++) 500 to -1000 g/cm2; (+ +) lo00 to 1500 g/cm2; (-) no gelation. Gelation was carried out as described
++
-
in the Material and Methods section.
of S . phaeochromogencs cells, very active preparations were obtained by using potassium chloride or hexamethylenediamine, and the use of an inhibitor of glucose isomerase such as calcium chloride or ferric chloride led to low activity as in the case of E. coli cells. Preparation of Various Shapes of Immobilizd Microbial Cells
Whole cells of E. coli having aspartase activity were immobilized into a k-carrageenan gel lattice in various shapes such as cube, bead, or membrane, and their enzyme activities were compared (Table 111). As shown in Table 111, some differences were observed between the enzyme activities of the different immobilized preparations. This may be due to the difference of the respective surface area of the preparations because the surface area of the preparations varies with shapes of gel as shown in Table 111.
I703
ENZYME IMMOBILIZATION USING CARRAGEENAN TABLE I. (Continued f r o m previous p a g e . ) Condition for gelation
Gel strengtha
4) Contact with amines
i) Aliphatic diamine Methylenediamine Eth ylenediamine Hexamethylenediamine Octamethylenediamine ii) Aromatic diamine p-Phenylenediamine iii) Amino acids and their derivatives Agmatine Histamine L-Ornithine L-Lysine GHydroxyl-L-iysine L-Lysine hydroxamate L-Histidine hydroxamate DL-Histidine hydrazide L-Tryptophan hydroxamate S-2-Aminoethyl-~-cysteine 5 ) Contact with water-miscible organic solvents Methanol Ethanol Acetone
+++ +++ ++++ ++++ +++ ++++ ++++ ++ ++ +++ +++
+++
++++
++ +++ ++ ++ ++
Itnmobilization of Various Enzymes arid Microbial Cells Immobilization of three kinds of enzymes and four kinds of microbial cells into a k-carrageenan cubic-type gel lattice was investigated using potassium chloride as a gel-inducing reagent, and the enzyme activities were compared with those of the respective native enzymes or intact cells (Table IV). As shown in Table IV, the activities of the obtained immobilized enzymes and microbial cells were high. However, the yield of their activities was around 50%. This may be due to diffusion limitation of substrate and/or product in the gel matrix. Hurdening Treatment for Stabilization of Immobilized Microbial Cells
In order to increase the operational stability of immobilized preparations obtained by using k-carrageenan, those were treated with
46.7 38.2
3.6 44.5 50.0 39.8 57.3
30360 24860
2340 28930 32520 25840 37260
(%)
4280 2480 300 2420 496 I960 I380 4216
glucose isomerase activity (pmol/hr/g cells)
54.1 31.3 3.8 30.6 6.3 24.7 17.4 53.3
(%)
activity yield
Immobilized cells obtained after activation by incubating with a substrate solution at 37°C for 24 hr. On the activation procedure see Ref.
5% absolute
0.3 M
Concentration
activity yield
S. phaeochromogenes
65000 and 7910 pmoVhr/g cells, respectively.
5 . Aspartase activity after sonication of intact cells of E. coli and glucose isomerase activity of intact cells of S . phaeochrornogenes were
a
KCI MgCI, CaCI, Ba-acetate FeCI, NH,CI NH,-fumarate Hexameth ylenediamine Polyeth yleneimine Acetone
Gel-inducing reagent
aspartasea activity (pmol/hr/g cells)
E . coli
TABLE I1 Immobilization of Whole Cells of E . coli and S . phaeochromogcwes into k-Carrageenan Bead-Type Gel Lattice Using Various Gel-Inducing Reagents
1
*r
rn
>
1
4 0 P
ENZYME IMMOBILIZATION USING CARRAGEENAN
I705
TABLE 111 Aspartase Activities of Immobilized E. coli Cells Formed in Various Shapes and Their Activity Yields Surface area of gel (cm*/g cells)
Shape of gel Bead (diameter = 3 mm) Cube (3 x 3 x 3 mm) Membrane (I x 10 x 10 mrn)
124 125 150
Aspartase activitya (pmoVhr/g cells)
Activity yield (%)
30360 32 130 41 120
46.7 49.4 63.3
a After activation by the same condition given in Table 11. Aspartase activity affer sonication of intact cells used was 65000 pmoUhr/g cells.
hardening reagents such as glutaraldehyde and hexamethylenediamine. The results were shown in Table V. The aspartase activity of immobilized E . coli cells was somewhat reduced by this hardening treatment, but the operational stabilities were markedly enhanced. In the case of the glucose isomerase activity of immobilized S. phacochromogcnes cells, there was no noticeable reduction by this hardening treatment, and their stabilities were also markedly improved. TABLE IV Immobilization of Various Enzymes and Microbial Cells Enzyme activitya Immobilization Enzyme and microorganism (enzyme) Arninoacylase Aspartase Fumarase
E . coli (Aspartase) B. ammoniagenes (Fumarase) S . phaeochromogenes
Yield before
after
(%)
20 650 360
10 300 220
50.0 46.2 61.1
30400
46.8
65Wb 967Oe
58Wd
60.0
7910
4280
54.1
(Glucose isomerase) a
Enzyme: prnollhrlmg protein; microbial cells: pmoVhr/g cells. After sonication. In the presence of bile extract. After treatment with bile extract.
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TABLE V Hardening Treatment for Stabilization of Aspartase Activity of Immobilized E . coli Cells and Glucose Isomerase Activity of Immobilized S. phaeochromogenes Cells Hardening treatment/Reagent and final conc E . coli (Aspartase) None HMDA" - GAb (mM) (mM) 85.0 I .7 85.0 17.0 85.0 85.0 85.0 170.0 S . phaeochromogenes (Glucose isomerase) None HMDA - G A (mM) (mM) 20 10 a
Enzyme activity (prnol/hr/g cells)
Half-life at 37°C (day)
30400
50
27500 I9900 21400 I8300
686 443
4280
53
4380
289
7s 168
HMDA: Hexamethylenediamine GA: Glutaraldehyde.
Comparison of Activity Yield and Stability of Iinmobilizod PrcJparations Obtained by Carrageenan and Polyacrylamide Gel Methods
Activity yields and operational stabilities of immobilized enzymes and immobilized microbial cells prepared by using k-carrageenan (cubic-type) were compared with those of immobilized preparations obtained by using polyacrylamide gel (Table VI). As shown in Table VI, the activity yields of immobilized preparations obtained by the carrageenan method were similar to or higher than those of preparations obtained by the polyacrylamide gel method. In addition, the operational stabilities of the preparations obtained by the carrageenan method were enhanced by hardening with glutaraldehyde and hexamethylenediamine, and the hardened preparations were more stable than the corresponding preparations obtained by the polyacrylamide gel method.
29
E . coli
a
12
31
31
60
51
60
34
Hardening with glutaraldehyde and hexamethylenediamine.
53
60
10
75
289a
686a
120 150
I20
60
20 5
30
carrageenan
half-life (day) pol yacrylamide
59a
37
31 60
31
(“c)
temp
51
33a
46 69a
29 12
(Aspartase) S. phasockromogetic,s (Glucose isomerase) B. ammotiiagc,tws (Fumarase) B. Jmum (Fumarase)
50a
carrageenan
50
polyacrylamide
Enzyme activity yield (%)
Aminoacylase Aspartase Glucose isomerase
Enzyme and microorganism (enzyme)
Operational stability
TABLE VI Comparison of Activity Yield and Stability of Immobitized Enzymes and Immobilized Microbial Cells Prepared by Carrageenan and Polyacrylamide Gel Methods
4 0
I .
1708
TOSA ET AL.
DISCUSSION In a previous paper,s we found that k-carrageenan is a suitable polymer for immobilization of S . phaeochromogenes, which is a microorganism displaying glucose isomerase activity. In order to apply this polysaccharide, which is commercially available at low cost, to the immobilization of other microbial cells and enzymes, the detailed conditions for gelation of k-carrageenan were investigated. As a result, it was clarified that the polysaccharide is easily induced to the gel by contact with a solution containing one of a number of gel-inducing reagents such as metal ions, amines, and water-miscible organic solvents as shown in Table I. By using this property of k-carrageenan, the immobilization of enzymes and microbial cells was investigated. Advantages of this method are that the immobilization can be performed under very mild conditions and can be achieved without the use of chemicals that may change the structure of enzyme proteins or microbial cells. Accordingly, as shown in Table IV, the activities and yields of immobilized enzymes and microbial cells obtained by using potassium ion as a gel-inducing reagent were high. Therefore, if a suitable gel-inducing reagent is selected for immobilization of individual enzymes or microbial cells, a preparation of high enzyme activity and operational stability may be obtained. The pore size of this gel matrix is small enough to prevent higher-molecular-weight compounds, such as enzyme proteins, from leaking out from the gel lattice, although the lower-molecularweight substrates and products easily pass through the gel lattice. These immobilized enzymes and immobilized microbial cells were stable, and a column packed with them can be used for continuous enzyme reaction for a long period. If the operational stabilities of the immobilized enzymes and microbial cells are unsatisfactory, preparations of greater stability can be obtained by further treatment with hardening reagents such as glutaraldehyde and hexamethylenediamine. One further advantage of this method is that the various shapes of immobilized enzymes or immobilized microbial cells can be easily tailored for particular application purposes as shown in Table 111. In addition, if immobilized microbial cells, prepared by the method using various gel-inducing reagents, are suspended in physiological saline, the k-carrageenan gel is rapidly dissolved and a stable cell suspension is obtained. This is advantageous for investigating characteristics of microbial cells after immobilization. For example, it
ENZYME IMMOBILIZATION USING CARRAGEENAN
I709
can be easily detected using this k-carrageenan as a matrix whether microbial cells after immobilization are living or not. That is, immobilized cells are thoroughly washed with sterilized potassium chloride solution and are suspended in sterilized physiological saline to dissolve k-carrageenan gel. The resulting cell suspension is suitably diluted with the same saline and is inoculated on an agar plate containing nutrient medium. The number of living cells entrapped in the gel can be counted as colonies. In conclusion, this facile carrageenan method is applicable for immobilization of many kinds of enzymes and microbial cells. Moreover, as this method is capable of making various shapes of immobilized preparations, the design of a suitable reactor for a given application becomes feasible. The authors thank G6do Shusei Co. Ltd., Tokyo, Japan for providing S . phaeochromogrnes cells.
References I . K . Mosbach, Methods in Enzymokigy (Immobilized Enzymes) (Academic, New York, 1976), Vol. 44. 2. 1. Chibata and T. Tosa, Advances in Applied Microbictktgy (Academic, New York, 1977). Vol. 22, p. I . 3. T. Tosa, T. Mori, N. Fuse, and I. Chibata, Enzymologia. 31, 214 (1966). 4. I. Chibata, T. Tosa, T. Sato, T. Mori, and Y. Matuo, Proc. Int. Ferment. Symp., Ferment. Techno/. Today. 4, 383 (1972). 5. I. Chibata, T. Tosa, and T. Sato, Appl. Microbiol.. 27, 878 (1974). 6. T. Tosa, T. Sato, T. Mori, and I. Chibata, Appl. Microbiol.. 27, 886 (1974). 7. K . Yamamoto. T. Tosa, K. Yamashita. and I. Chibata, Eitr. J. Appl. MiCriib i ~ l . 3, . 169 (1976). 8. K. Yamamoto, T. Tosa, K. Yamashita, and I. Chibata, Biotwhno/. Bioeng.. 19, I101 (1977). 9. I. Takata, T. Tosa, and I. Chibata, J. Solid-Phase Biochem.. 2, 225 (1977). 10. T. Tosa, T. Sato, Y. Nishida. and I. Chibata, Biochim. Biophys. Acta. 483, 193 (1977). 1 1 . T. Tosa, T. Sato, K. Yamamoto, Y. Matuo, and I. Chibata, Biochim. Biophys. Acta, 334, I (1974). 12. Z . Dishe and E. Borenfreund. J. Biol. Chem., 92, 583 (1951).
Accepted for Publication November 6, 1978
