Immobilized tannin — a novel adsorbent for protein and metal ion

Immobilized t a n n i n - a novel adsorbent for protein and metal ion ICHIRO CHIBATA*, TETSUYA TOSA, T A K A O MORI, T A I Z O W A T A N A B E and N O B U Y U K I S A K A T A *Research and Development Headquarters and Research Laboratory o f Applied Biochemistry, Tanabe Seiyaku Co. Ltd, 1 6 - 8 9 , Kashima-3-chome, Yodogawa-ku, Osaka, Japan

Summary. In order to prepare an adsorbent suitable for protein adsorption, we chose tannin as a ligand and attempted to bind it covalently to a water-insoluble matrix. The most favourable adsorbent was obtained by binding tannin to aminohexylcellulose. This adsorbent is called immobilized tannin, and can adsorb proteins well. Further, since it is known that tannin binds with metal ions as well as proteins, the adsorption specificity o f immobilized tannin for metal ions was tested and immobilized tannin was found also to adsorb metal ions. This review summarizes the preparation method, characteristics and applications o f immobilized tannin. Keywords.. Immobilized tannin; protein adsorption; metal adsorp-

tion; immobilized enzyme

adsorbing proteins could be prepared, it would be very advantageous in many fields. To design this adsorbent, we chose tannin, which has been used as a protein-precipitating agent since ancient times, as a ligand for immobilization. Immobilized tannin prepared by chemically binding tannin to a water-insoluble matrix would be the most suitable and specific adsorbent for proteins. Thus, we have extensively investigated the covalent binding of tannin to water4nsoluble matrices. Further, as it is known that tannin binds with metal ions as well as proteins, the adsorption specificity of the resulting immobilized tannin for metal ions was also tested. This article discusses the preparation, characteristics and application of immobilized tannin for protein and metal ions.

Preparation of immobilized tannin Introduction

Selection of immobilized method

Since the early 1960s we have been studying techniques for the immobilization of enzymes and microbial ceils, and we have succeeded in the industrial application of immobilized enzymes for the continuous optical resolution of D L-amino acids 1-3 and immobilized microbial cell systems for the production of L-aspartic acid, 4-6 L-alanine 7-9 and L-malic acid. l°-~z On the basis of this background, we have expanded the utilization of immobilization techniques to the field of general ligand affinity chromatography for the separation, purification and recovery of proteins. Protein adsorbents include many inorganic compounds, such as alumina, charcoal, clays, glass and silica, and many organic compounds, such as starch, ionexchange celluloses and ion-exchange resins. These have been used for the recovery,la removal ~4 and purification ~s of proteins and for immobilization of enzymes. 16 However, they do not fully satisfy the basic requirements for these uses. For example, they are not specific for proteins but adsorb various organic and inorganic compounds together with the proteins. Accordingly, proteins recovered from aqueous solution by these adsorbents are usually contaminated with impurities other than proteins. Further, when these adsorbents are used for the removal of unfavourable proteins in aqueous solution, such as Japanese sake, beer and wine, various useful compounds related to taste, flavour and colour are also removed and the quality of the beverage is lowered. Therefore, we felt that if an adsorbent specifically

In order to prepare an effective adsorbent for protein, the immobilization method of tannin, type of matrix, chain length of spacer, kind of tannin, and so forth, were studied in detail (for a review see ref. 17). First, we attempted to prepare immobilized tannin by two methods, as shown in Figure 1. In Method A, water-

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Matrix

~

amino alkylation

A m i n o alk¥1 m a t r i x

I

activation by epichlorohydrin

Activated a m i n o a l k y l m a t r i x

coupling reaction

coupling - I ~ I m m o b i l i z e d t a n n i n ~"

reaction

Activated t a n n i n

A

I activation by BrCN I Tannin Figure 1 General method for immobilization of tanninJ 7 - - - Method A ; , Method B 0141 --0229/86/030130--07 $03.00 © 1986 Butterworth & Co. (Publishers) Ltd

Immobilized tannin: I. Chibata et aL

insoluble matrix containing amino, carboxyl or hydroxyl group is activated by cyanogen bromide, epichlorohydrin or carbodiimide. Then, the activated matrix is reacted with diaminoalkane to prepare aminoalkyl matrix. Tannin is activated by cyanogen bromide and, finally, activated tannin and aminoalkyl matrix are coupled to produce immobilized tannin. In Method B, aminoalkyl matrix is further activated, and the resulting activated matrix is coupled with tannin to prepare immobilized tannin. In both methods, the selection of a suitable matrix was carried out for the preparation of immobilized tannin having high adsorption capacity for a protein, giucoamylase. As a result, Method B using alkali-treated cellulose as a matrix and epichlorohydrin as an activating reagent of matrix was found to give the best adsorbent for protein. Alkali treatment of cellulose, maceration, is effective for increasing the chemical groups that allow covalent attachment of the ligand to cellulose. 18 Further, the effect of chain length of the spacer on adsorption capacity of immobilized tannin for glucoamylase was studied. It is well known in affinity chromatography that accessibility of macromolecules towards the ligand increases with the extension of chain length of the spacer. As shown in Table 1, the adsorption capacity of immobilized tannin for protein increased with extension of chain length of the spacer and reached a plateau with diaminohexane. Therefore, we chose diaminohexane, which is commercially available at low cost, as spacer. Several kinds of tannins, such as Chinese gallotannin, nutsgalls-tannin and tannin of persimmon juice, were coupled to aminohexyl cellulose, and the adsorption capacity of immobilized tannins was compared. The type of tannin did not influence the adsorption capacity of immobilized tannin. As Chinese gallotannin is commercially available in pure form and at low cost, it was chosen as a ligand. Thus, we decided to prepare immobilized tannin by using alkali-treated cellulose as a matrix, epichlorohydrin as an activating reagent of the matrix, and Chinese gallotannin as a ligand.

Preparation o f immobilized tannin with epichlorohydrin The conditions for preparing immobilized tannin were investigated in detail and finally the optimum condition

was determined. 19'2° The method of preparing the immobilized tannin with epichlorohydrin was as follows. To 3 litres of distilled water, 200 g of cellulose (filter pulp No. 4, Toyo Roshi Co. Ltd, Tokyo) was suspended a n d the suspension cooled at 4°C. To the suspension, 3 litres 6 M sodium hydroxide was added. The mixture was gently stirred for 3 rain at 4°C, and then left to stand for 30 min at 4°C. To the alkali-treated cellulose, 12 litres distilled water was added, and the suspension was stirred for 30 rain at 60°C. To the suspension, 2 litres epichlorohydrin was added, and the mixture was stirred for 30 rain at 60°C. The activated cellulose was collected by filtration, and washed with 10 litres distilled water. The resulting cellulose was suspended in 8 litres 0.625% diaminohexane solution, and the mixture was stirred for 120 rain at 60°C. After the reaction, the aminohexyl cellulose was collected and washed with 10 litres water. The resulting aminohexyl cellulose was suspended in 10 litres 0.25 M sodium hydroxide; to the suspension 1 litre epichlorohydrin was added and the mixture stirred for 30 min at 60°C. The activated aminohexyl cellulose was washed with 10 litres water, and then suspended in 8 litres 3% Chinese gallotannin aqueous solution adjusted to pH 7.0 with sodium hydroxide and then 4.8 g sodium borohydfide was added. Nitrogen gas was bubbled into the suspension while the suspension was stirred for 150 rain at 45°C. After the reaction, the immobilized tannin was collected and washed with 10 litres water. Immobilized tannin was suspended in 4.5 litres 30% aqueous acetone, and the pH of the mixture was adjusted to 2.0 with 3 1~ hydrochloric acid. The resulting suspension was stirred for i0 rain at 25°C. Immobilized tannin was collected by filtration and washed with 10 litres water. After washing the immobilized tannin with aqueous acetone three times, immobilized tannin was washed with 20 litres water. The resulting immobilized tannin preparation was suspended in 5 litres 20% ethanol (adjusted to pH 2.5 with HC1). After filtration of the mixture, immobilized tannin was stored at 4 - 1 0 ° C under tight sealing. The preparation obtained was 1290 g wet weight (about 4 litres in volume), and contained 81% water and 4.75% Chinese gallotannin. The suggested structure of the immobilized tannin obtained is shown in Figure 2. The adsorption capacity of this immobilized tannin for a protein, pepsin, is about 200 mg per g of dry matter.

Table 1 Effect of chain length of spacer on adsorption capacity of immobilized tannin for glucoamylase. [Reproduced from Watanabe, T., Matsuo, Y., Mori,T.,Sano, R .,Tosa,T.and Chibata, I .J. Sofid-Phase Biochem. 1978,3,161 by permission of Plenum Publishing Corporation©]

Coupled amount (/~mol g-I of adsorbent) Diaminoalkane spacer

Diaminoalkane

Tannin a

Adsorption capacity for glucoamylase (mg protein m1-1 of adsorbent)

NH2 (CH2)2 N H~

281

158

16.0

N H 2 (CH~)~ NH~

--

175

17.2

NH= (CH 2)4NH~ NH~ (CH~)6 NH= NH 2 (CH~)~ NH2 b NH 2 (CH~)s NH= b NH~ (CH=)x0 NH2 b NH 2 (CH~)xi NH2 b

230 -241 196

236 277 286 255 265 271

21.3 48.8 45.6 51.2 47.9 49.7

aMolecular weight of tannin was taken as 2600 bDissolved in 50% ethanol Alkali-treated cellulose was aminoalkylated with cyanogen bromide, and the resulting aminoalkylated cellulose was coupled to BrCN-activated Chinese gallotannin. The resulting immobilized tannin was packed into a column (bed volume 1 ml) and the column was equilibrated with acetate buffer (pH 4.3, 1 mmho). Glucoamylase dissolved in the same buffer at a concentration of 0.1% was continuously passed through the column until the protein concentration in the column effluent became equal to that in the charged solution. Glucoamylase adsorbed to the column was eluted with sodium carbonate buffer (pH 10, 50 mmho) and determined

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131

Review O-Gel o

HO - ~ C O - O

OH

O

o I I

o ~

O-CH~-CH-CH2-N

H-(CH2)=-N H-CH~-CH-CHg-O.~

OH

OH

HO

OH

/

Gal

I Gal

\

/ C\,,O Figure 2

-CO-O

O / Gel

OH

0

HO

OH

Suggested structure of immobilized tannin

Characteristics o f i m m o b i l i z e d tannin

Adsorbent for proteins In order to develop the application of this immobilized tannin, we investigated the adsorption specificity, the factors influencing protein adsorption and desorption, and the physical properties of the adsorbent. We first examined the adsorption specificity of immobilized tannin, 17'21 as shown in Table 2. The results obtained show that various kinds of protein are more or less adsorbed to immobilized tannin, though the rate of the extent of adsorption of protein to the adsorbent is affected by the pH of the protein solution, and the optimum pH for adsorption depends on the kind of protein used. Some proteins, such as wheat glutenin, zein and gelatin, were

poorly adsorbed, even at the optimum pH. We also investigated the adsorption of various organic compounds, except for protein, and established that sugars, amino acids, peptides, nucleic acid-related compounds, organic acids and alkaloids are not adsorbed to immobilized tannin, as summarized in Table 3. These results suggest that immobilized tannin is a specific adsorbent for proteins, and this new adsorbent overcomes the disadvantages of conventional adsorbents for proteins, which are not specific for proteins. Protein concentration, incubation temperature, incubation time and salt concentration also largely influenced the rate of extent of adsorption of protein to immobilized tannin. 22 Adsorption rate increased at higher protein concentration and at lower temperature. Initial rapid

Table 2 Adsorption specificity of immobilized tannin for various proteins, [Reproduced from Watanabe, T., Mori, T., Tosa, T. and Chibata, I.Agric. Biol, Chem. 1981,45, 1001 by permission of the Agricultural Chemical Society of Japan©] Protein

Amount adsorbeda

Type

Specific

pH 2

pH 4

pH 7

pH 10

Albumin

Ovalbumin Bovine serum albumin Bovine e-lactoglobulin Bovine serum/3-globulin IV-1 Concanavalin A Wheat glutenin Zein b Salmon protamine Gelatin Bovine milk casein Soybean casein Bovine haemoglobin Gastric mucin c~-Amy lase G Iu coamy lase Lysozyme Pepsin Trypsin

---21.3 ------

5.4 8.7 18.9 24.0 5.0 12.3 13.8 9.6 9,1 42.9 34.8 0 27.6 69.1 26.4 0 76.5 0

49.5 58.8 75.0 83.4 15.1 8.4 55.9 22.2 52.2 97.5 44.6 58.8 43.3 51.2 38.4 34.1 11.4

0 0 5.1 27.5 4.1 4.8 94.0 2.4 31.2 28.1 16.4 25.4 0 0 80.2 0 11.7

Globulin

Glutelin Gliadin Protamine Scleroprotein Phosphoprotein Chromoprotein Glycoprotein Enzymec

a(amount of protein adsorbed/amount of protein used) X 100 bDissolved in 70% ethanol C ln the case of enzymes, experiments were carried out at an enzyme concentration of 1 mg ml -I Immobilized tannin (150 mg wet weight) prepared with epichlorohydrin was incubated with 10 ml of a buffer solution containing a protein (0,25mgm1-1 of buffer solution) for 10min at 5°C with shaking. After the reaction, the mixture was filtered and immobilized tannin was washed with 10 ml of the same buffer. The amount of proteins adsorbed on immobilized tannin was calculated from the difference between the amount of proteins in the initial solution and that in the filtrate and washings

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I m m o b i l i z e d tannin: I. Chibata et aL

adsorption of protein occurred and the maximum adsorption was achieved in 1-3 h. Further, the rate of extent of adsorption decreased at higher salt concentration. However, considerable amounts of protein were bound, even at higher salt concentration. These results indicate that various binding forces such as ionic, hydrophobic and hydrogen binding are involved in the binding of proteins to immobilized tannin. This can be understood from the chemical structure of tannin, which contains a large number of phenolic hydroxyl groups. On the basis of these results, desorption of protein from immobilized tannin was studied. As shown in Table 4, glucoamylase was not desorbed with water or with 0.5 M sodium chloride. Also, 0.001 M hydrochloric acid solution and 0.001 M sodium hydroxide were poor solvents for desorption, while, 0.1 M sodium chloride in 0.1 M sodium carbonate buffer, pH 10, 0.01 M hydrochloric acid solution, and 0.01 M sodium hydroxide were good solvents for desorption. The necessity of rather strong conditions for desorption of protein from the adsorbent suggests the occurrence of strong binding forces resulting from coTable 3 Summary of compounds not adsorbed to immobilized tannin. [Reproduced from Watanabe, T., Mori, T., Tosa, T. and Chibata, I. Agric. BioL Chem. 1981,45, 1001 by permission of the Agricultural Chemical Society of Japan©] Classification

Compounds tested

Sugars

Soluble starch, raffinose, lactose, maltose, sucrose, galactose, glucose, fructose, ribose, arabinose, mannose, xylose, sorbose, glucosamine

Amino acids and peptides

Alanine, aspartic acid, histidine, isoleucine, methionine, phenylalanine, tyrosine, Val-Leu-Ser-Ala, glutathione

Nucleic acid related compounds

Adenine, adenosine, AMP guanine, guanosine, GMP cytosine, cytidine, CMP uracil, uridine, UMP

Others

Caffeine, nicotine, fumaric acid, malic acid

Adsorption conditions were the same as in the case of Table 2 except concentration of compounds used for adsorption (0.1 mg ml -~ of buffer solution) Table 4 Desorption of glucoamylase from immobilized tannin. [Reproduced from Watanabe, T., Fujimura, M., Mori, T., Tosa, T. and Chibata, I. J. AppL Biochem. 1979, 1, 28 by permission of Academic Press, Inc.©]

Solvent for desorption

H 20 NaCI (0.5 M) NaCI (0.5 M, pH 10) NaCI (0.5 M) in Na2CO ~ (0.1 M, pH 10) NaOH (0.0001 M) NaOH (0.001 M) NaOH (0.01 M) HCI (0.001 M) HCI (0.01 M) HCI (0.1 M) Sodium acetate buffer (0,1 M, pH 5) Sodium phosphate buffer (0.1 M, pH 7) Sodium carbonate buffer (0.1 M, pH 10)

Recovery of protein (%) 0.0 12.5 27.4 91.5 10.5 26.1 87.6 6.2 86.6 92.9 0.4 0.9 1.7

Immobilized tannin (4g wet weight) was incubated with 500 ml of solution of glucoamylase (2 mg protein ml -~ , pH 4.3, conductivity 1 mmho) for 30 min at 25°C. The mixture was filtered and the immobilized tannin--enzyme complex was washed with water. Proteins of the complex (0.5 g) were then shaken for 20 min at 25°C with the solution indicated. Each mixture was filtered, and the amount of protein in the resulting filtrates was determined

operation of ionic, hydrophobic and hydrogen binding, as mentioned above. Thus, immobilized tannin can be easily regenerated by using an appropriate solvent for desorption, and reused.

Adsorbent for metal ion We also investigated the adsorption of metal ion to immobilized tannin. ~3 In order to clarify the adsorption specificity of immobilized tannin for various metal ions, the solution containing metal ion was passed through the column. From the results in Table 5, it is clear that immobilized tannin adsorbs iron, copper and lead ion well and, to a lesser extent, other metal ions. Adsorption specificity of immobilized tannin for varous kinds of cationic and anionic ions was also studied, and chlorine, sodium, calcium and fumarate ions were found to be poorly adsorbed. The effect of temperature and pH on the adsorption of metal ion on immobilized tannin was studied. The adsorption rate increased when the temperature was raised. This effect is opposite to the case of protein adsorption. This indicates that the adsorption mechanism is different for protein and metal ion adsorption. The adsorption rate also increased at higher pH. Desorption of meal ion adsorbed to immobilized tannin was investigated. For example, desorption of iron ion was carried out with aqueous hydrochloric acid, and the recovery of iron ion was almost 100% when 0.5 M hydrochloric acid was used as a solvent for desorption. Thus, regeneration can be carried out without loss of adsorption capacity.

Physical properties As immobilized tannin is a soft and compressible material made from fibrous falter pulp, a column volume packed with the adsorbent is significantly affected by the column diameter, the packing method and the flow rate. The pressure drop of an immobilized tannin column depends on the flow rate and the viscosity of fluid as shown in Figure 3, and the pressure drop is markedly increased at higher flow rates. In order to overcome the disadvantages of this compressible adsorbent, we studied flow kinetics in detail, and succeeded in designing a multi-stage column suitable for the use of the adsorbent in a column operation method. Further information on this topic can be found in ref. 24.

Application of immobilized tannin As mentioned above, immobilized tannin is a specific adsorbent for protein and metal ion and has some advantageous characteristics. We therefore tried to apply this adsorbent for various fields.

Recovery, separation and purification o f proteins Since immobilized tannin specifically adsorbs protein but not other organic and inorganic compounds, except metal ion, and the adsorbed protein is readily eluted, this adsorbent can be used for the recovery of protein from aqueous solution, such as culture broth, blood and urine. For example, we tried to recover hesperiginase from the culture broth of Aspergillus niger. This enzyme is used for removing bitter component of citrous fruit juice. When culture broth of pH 4.8 and 14 mmho was directly charged on the immobilized tannin column, both hesperiginase and undesired protein were adsorbed. On the other hand, from the results of another batchwise experiment, hesperiginase was found to be rather selectively adsorbed at pH 6 and 10

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133

Review Table 5

Adsorption specificity of immobilized tannin f o r various metal ions. [Reproduced from Chibata, I., Tosa, T., Mori, T., Watanabe, T., Yamashita, K. and Sakata, N. Enzyme Engineering 1982, 6 , 2 5 9 by permission of Plenum Publishing Corporation©] Concentration of metal ion (ppm) Fraction of column effluent (60 ml fraction) Metal compound

Charged solution

MnSO 4 Fe(N H 4 )2 (SO4)2 CoCI~

3.0 10.4

<0.05 7.5

0.08 10.3

Ni(CH~ COO) 2

9.5

5.7

9.0

8.7

CuSO 4 Zn (CH 3 COO)~

3.0 7.7

3.0

< 0.03 6.2

<0.03 6.0

HgCl=

1.9

0.81

2.4

-

-

Pb(CH3COO) =

8.4

<0.1

--

--

1

2

3.0

-

<0.1

3 2.9

4 3.1

3.2

0.08 -

0.08 -

< 0.03

Immobilized tannin (0.4 g dry weight) was packed into a column (bed volume 4 ml), and the solution containing a specified amount of metal ion was passed through the column at a f l o w rate of 60 ml h -~ and 25°C. The effluent was fractionated,and concentration of metal ion in the fraction was determined

mmho ionic strength. Thus, the culture broth was adjusted to the above conditions and passed through the immobilized tannin column. As shown in Figure 4, almost all the proteins except hesperiginase passed through the column. Hesperiginase was eluted with 0.02 M HC1, and the activity recovery was 92%. In order to confirm that immobilized tannin can be used for the separation of proteins, a model experiment for chromatographic separation of different kinds of protein was carried out.2° A mixture of trypsin, lysozyme and ovalbumin dissolved in pH 7.5 buffer was applied to the immo•bilized tannin column. After washing the column with the same buffer the adsorbed proteins were eluted with 0.05 M carbonate buffer, pH 9, and subsequently with 0.05 M acetate buffer, pH 4. Trypsin was not adsorbed on the column at neutral pH, whereas the other two proteins were adsorbed and eluted with alkaline buffer and the following acidic buffer without significant loss of protein. These results suggest that the separation and purification of proteins using immobilized tannin can be carried out by selecting suitable conditions for adsorption and desorption.

20 A .Q

E O

=" E e~

10

o

t~

O

0

5

10

Linear velocity (m h -1 ) Figure 3 Effect of flow rate and viscosity of fluid on pressure drop of i m m o b i l i z e d tannin column. 24 Viscosity: e, 1.0 cP (water); o, 1.7 cP (ethanol); o, 3.0 cP (sak$). Immobilized tannin (60 g dry weight) was packed into a column (bed volume 1400 ml). Water, ethanol and sak$ were passed through the column at 20, 25 and I 0 ~C, respectively

7

1.5 _~

10

-H20-='~LI~-O.O2M H C I ' ~ - ~ I ~ O . 1M HCI

6

Immobilization o f enzymes The immobilization of enzyme was carried out very easily by adding enzyme dissolved in an appropriate buffer to the immobilized tannin and shaking the mixture for 6 0 - 1 2 0 min at 25°C. As shown in Table 6, immobilized enzymes having relatively high activity were obtained. The strong binding force is considered to participate in the binding between enzyme and immobilized tannin. Thus, the immobilized enzyme is stable during continuous operation, even at high substrate concentration. 2s'26

1.0

-E5 E

64

Removal o f undesired protein in aqueous solution

E

~

o.5

).

e

2

< fi

10

15

20

Fraction

Purification of hesperiginase from culture broth. 24 o, hesperiginase; e, protein; . - . , pH. Immobilized tannin (0.4g dry weight) was packed into a column (bed volume 4 ml),and 100 ml of culture broth was passed through the column at a flow rate of 20 ml h -~ and 10°C. Elution was carried out with the specified eluant at the same conditions described above. Fractions were of 5 ml Figure

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The soluble proteins in sak~ (Japanese rice wine), beer, wine and fruit juice are denatured and gradually grow to large particles during storage, and the resulting turbidity lowers the quality of beverages. In sake, the turbid material is too small to remove by ordinary filtration. Soluble tannin of persimmon juice has been conventionally used for this purpose. However, these procedures are carried out in batch method and require much time and labour. In order to overcome these disadvantages, we tried to remove protein precursors of turbidity in sak6 by a continuous process using immobilized tannin. 19'27-29 Fresh sak6 was passed through a column packed with the immobilized tannin at a flow rate of SV=15 and 10°C for 30 h, and the

Immobilized tannin: I, Chibata et al. Table 6 Immobilization of various enzymes on immobilized tannin. [Reproduced from Watanabe, T., Fujimura, M., Mori, T., Tosa, T. and Chibata, I. J. Appl. Biochem. 1979, 1,28 by permission of Academic Press, Inc.©]

Activity of enzymea

Immobilized enzyme

Kind of enzyme

Used

Adsorbed

Activity a

Yield (%)

Aminoacylase /3-Galactosidase Glucose isomerase Nari ngi nase Papal n

16.0 92.0 292.0 46.0 120.0

9.0 92.0 125.6 45.4 64.0

2.3 37.5 89.8 26.1 20.0

25.6 40.8 71.5 57.5 31.3

a/imol min -1 Immobilized tannin (40 mg dry weight) was suspended in 2 ml enzyme solution. The suspension was shaken for 1 h at 25°C, then filtered. The resulting immobilized enzyme was thoroughly washed with water, and the activity of immobilized enzyme was determined by the appropriate method Table 7

Adsorption of various forms of iron by immobilized tannin 23 Concentration of iron ion (ppm) Fraction of column effluent (30 ml fraction)

Form of iron

Charged solution

1

2

3

4

FeS04 Fe(N H4 )2 (SO4)= FeCI 3 FeCI~ + EDTA FeCI 3 + Na-tartrate FeCI~ + Humic acids Ferrichrysin

10 10 10 10 10 10 0.5

< 0.02 <0.02 <0.02 <0.02 <0.02 <0.02 0.15

< 0.02 <0.02 <0.02 <0.02 < 0.02 <0.02 0.48

< 0.02 <0.02 >0.1 0.1 > 0.1 <0.02 0.45

>0.1 0.1 >0.1 -

-

0.1 -

Experimental conditions are the same as in the case of Table 5. Five times of weight of EDTA, Na-tartrate and humic acids were added to FeCI~, respectively

80

60

40 }Figure 6 Flow diagram of continuous removal of iron ion from water using immobilized tannin. 1, Pump; 2, flow meter; 3, filter; 4, pressure gauge; 5, air drain; 6, column for immobilized tannin; 7, packed bed; 8, HCl tank for regeneration; 9, control panel

20 A

I 1

I 2

I 3

-

A

I 4

I 5

I 6

I 7

Storage period (months) Figure 5 Fining of sak~ with immobilized tannin. 17 o, Untreated sake; e, treated sake. Immobilized tannin (2 g wet weight) was packed into a column (bed volume 5 ml) and 2 I of fresh sak~ was passed through the column at a flow rate of 60 ml h -~ and 10°C. The treated sak~ and untreated sak~ were pasteurized at 65°C for 30 rain and their turbidities were measured periodically

treated sak~ was stored for a long time. As shown in Figure 5, the turbidity in sak6 treated with the immobilized tannin was low, and the increase of turbidity during storage was small compared with that in untreated sake. Further, there was no change in the components of sake, except for colour and turbidity, between treated and untreated sak& These results indicate that immobilized tannin is a

specific adsorbent for protein and can be effectively used as an alternative method for fining of sak&

Removal of iron from water for brewing Immobilized tannin adsorbs metal ions, especially iron ion. Thus, we attempted to remove traces of iron ion from water for brewing with immobilized tannin. It is important to remove iron completely from water used for brewing because trace amounts of iron lower the quality of sake, wine and beer. However, as the water for brewing contains several forms of iron, it has proved difficult to remove trace amounts of the various forms of iron by a single method. As shown in Table 7, immobilized tannin adsorbs almost all kinds of iron, i.e. chelated forms of iron, and absorbs low levels of iron, even of the order of ppb of ion. For example, the removal of iron ion was carried out according to the flow diagram shown in Figure 6. 3o

E n z y m e M i c r o b . T e c h n o l . , 1986, vol. 8, March

135

Review The results are shown in Table 8. The concentration of iron ion in the effluent was less than 10 ppb, even when 104 1 of water for brewing per litre of adsorbent was passed through the immobilized tannin column. Also, it was established that sak6 brewed by using this treated water was of a satisfactory quality. By this new system, removal of iron ion and regeneration of the immobilized tannin could be carried out automatically. Further, this system can be used to remove iron ion from sak& In a few cases, sak6 contains a yellow-coloured substance, ferrichrysin, which consists of a chelate compound of iron and cyclic peptide. Ferrichrysin lowers the quality of sak6. However, it proved difficult to remove ferrichrysin by ordinary methods. In order to apply this system to removal of ferrichrysin, adsorption conditions were studied. It was found that the immobilized tannin effectively adsorbs ferrichrysin under appropriate conditions, i.e. at higher temperatures. Thus, this immobilized

Table 8 Removal of iron ion from water for brewing and tap water by immobilized tannin 3° Concn of iron ion in effluent (ppb) a Volume of charged water (I/I of adsorbent)

Water for brewing

Tap water

500 2500 4500 5000 5500 8500 10000

<10 < 10 < 10 <10 < 10 < 10 < 10

<10 < 10 <10 < 10 <10 < 10 <10

aConcn of iron ion in charged water was 10--40ppb in brewing water and 80 ppb in tap water Water for brewing or tap water was passed through 60 I of the immobilized tannin column at a flow rate of 60001h 1 and 20°C. The effluent was fractionated, and concentration of iron ion in the fraction was determined

Figure 7 Apparatus for removing iron ion in brewing water. 60 I of immobilized tannin was packed in a column

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E n z y m e M i c r o b . T e c h n o l . , 1986, vol. 8, March

tannin was commercialized in 1981 by Tanabe Seiyaku Co. Ltd for application in the brewing field, and Figure 7 shows the apparatus for removal or iron ion. In conclusion, immobilized tannin is a superior adsorbent for protein and metal ion than conventional adsorbents. Also, this adsorbent is chemically and physically stable and safe, so the adsorbent is promising for recovery and removal of protein and metal ion in a variety o f fields, including the food, medical, cosmetic and chemical industries.

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