Method for producing iodine

This invention relates to the production of iodine. More particularly, the invention relates to the enzymatic oxidation of iodide to iodine and the recovery of said formed iodine.

This invention relates to the production of iodine. More particularly, the 
invention relates to the enzymatic oxidation of iodide to iodine and the 
recovery of said formed iodine. 
There is an increasing demand for increased supplies of iodine and its 
major derivatives, iodide salts. The consumption of iodine and iodide 
salts is distributed among several industrially significant applications: 
catalysts, animal feed additions, stabilizers (as in nylon precursors), 
inks and colorants, pharmaceuticals, sanitary and industrial 
disinfectants, film, and other uses. The development of an economical 
process for more efficient and expanded production of iodine would be of a 
valuable contribution to the chemical industry. 
A brief description (source: Ency. Chem. Tech., 2nd Edition, R. E. Kirk and 
D. F. Othmer, Wiley-Interscience, N.Y., N.Y., 1965-Vol. 7) of typical 
processes for the production of iodine from brines follows: 
"The first step in each of these (iodine production processes from brine) 
is the clarification of the brine to remove oil and other suspended 
material. In one process, a silver nitrate solution is added to 
precipitate silver iodide, which is filtered and treated with scrap iron 
to form metallic silver and a solution of ferrous iodide. The silver is 
redissolved in nitric acid for use in another cycle, and the solution is 
treated with chlorine to liberate the iodine. 
"The largest part of the U.S. production is as a by-product from Michigan 
natural brines. The iodine is recovered by a process resembling that for 
recovery of bromine from seawater. The brine, containing from 30-40 ppm 
iodine, is acidified with sulfuric acid and treated with a slight excess 
of chlorine to liberate the iodine. It is then pumped to a denuding tower 
in which it gives up its iodine to a counter-current stream of air. The 
air passes to a second tower where the iodine is absorbed by a solution of 
hydriodic and sulfuric acids. This solution is treated with sulfur dioxide 
to reduce the iodine to hydriodic acid, and part is drawn off to a reactor 
for recovery of iodine, while the remainder is recirculated to the 
absorption tower. The liquor in the reactor is treated with chlorine and 
the liberated iodine is settled, filtered, melted in a kettle under 
concentrated sulfuric acid, and cast into pigs. 
"The process used by the Chilean nitrate industry differs from the others 
since the iodine is present as iodate. The iodine is extracted from the 
caliche as sodium iodate and is allowed to accumulate in the mother 
liquors from crystallization of sodium nitrate until a suitable 
concentration, about 6 g/liter, has been attained. Part is then drawn off 
and treated with the exact quantity of sodium bisulfite solution required 
to reduce all of the iodate to iodide. 
EQU 2NaIO.sub.3 +6NaHSO.sub.3 .fwdarw.2NaI+3Na.sub.2 SO.sub.4 +3H.sub.2 
SO.sub.4 
"This mixture, now acid with sulfuric acid resulting from the oxidation of 
the sulfur dioxide, is treated with just sufficient fresh mother liquor to 
liberate all the iodine in accordance with the reaction. 
EQU 5NaI+NaIO.sub.3 +3H.sub.2 SO.sub.4 .fwdarw.3I.sub.2 +3Na.sub.2 SO.sub.4 
+3H.sub.2 O 
"An additional source of iodine is seaweed from which some of the Japanese 
production has been derived." 
It is an object of the present invention to provide an improved method for 
producing iodine. 
Another object of the invention is to provide a process for producing 
iodine which does not require the expensive and dangerous substance, 
chlorine, common to many present processes and which is energy-intensive 
to produce. 
Another object of the invention is to provide a method for producing iodine 
which may be conducted at ambient temperature and atmospheric pressure. 
Another object of the invention is to provide a method for producing iodine 
which minimizes waste disposal problems to provide both economic and 
ecological benefits. 
Another object of the invention is to provide an improved method for 
producing iodine wherein selectivity for iodine recovery, even in the 
presence of bromide and chloride, is provided. 
A further object of the invention is to provide a method for producing 
iodine from relatively plentiful sources of iodide, such as brines and 
bitterns, rather than from less plentiful and more inconsistent sources 
such as seaweed and mining products. 
The ability of halogenating enzymes to catalyze the oxidation of iodide is 
well established. For example, phenols and proteins are iodinated by these 
enzymes. However, in these reactions, molecular iodine is not the 
preferred intermediate. In fact, many have questioned whether molecular 
iodine is even formed (B. Davidson, J. T. Neary, et al. Biochim Biophy 
Acta, 522, 318 (1978)). While others have recognized the catalytic ability 
of these enzymes, the invention of a process to produce iodine by this 
method has not been previously described. 
The method of the invention comprises providing a reaction mixture of pH 
buffered water, a halogenating enzyme, an oxidizing agent and a source of 
ionic iodide. The reaction is run in the absence of iodine acceptor 
substrates which allows the recovery of iodine from the reaction mixture. 
The starting material or source of ionic iodide may be any of a variety of 
brines or bitterns. The process may be used economically to recover iodine 
from natural brines, bitterns, salt lakes, and the like. 
The halogenating enzyme used in the reaction mixture may be in pure form, 
free or immobilized, or may be in microbial cells which produce the 
enzyme. Among the halogenases useful in the practice of this invention are 
those derived from the microorganism Caldariomyces fumago, seaweed, milk 
(lactoperoxidase), thyroid (thyroid peroxidase), leukocytes 
(myeloperoxidase) and horseradish (horseradish peroxidase). Sufficient 
water is employed in the reaction mixture to wet the enzyme and, in fact, 
may be the major solvent in the reaction mixture. 
The oxidizing agent employed in the reaction mixture may be of any suitable 
type, preferably hydrogen peroxide. The hydrogen peroxide need not be 
purified, but may be used in dilute form to reduce the cost of the 
hydrogen peroxide with respect to concentrated purified material. In 
addition, the use of dilute hydrogen peroxide increases the safety of 
usage and extends the life of the halogenating enzyme. The hydrogen 
peroxide may be added directly to the mixture in a single batch addition, 
or may be added in a continuous slow feed. Alternatively, the hydrogen 
peroxide may be generated as slow feed in situ by the use of a hydrogen 
peroxide-producing enzyme or chemical system. Such enzyme systems are well 
known in the art and include glucose oxidase in the presence of glucose; 
glucose-2-oxidase in the presence of glucose; methanol oxidase in the 
presence of methonal; D- and L-amino acid oxidases in the presence of D- 
and L-methionine; and diamine oxidases in the presence of histamine. 
Any or all of the enzymes or cells producing the enzymes in this invention 
may be used in either free or immobilized form. The processes for enzyme 
and cell immobilization are familiar to those skilled in the art and 
include reacting a solution of the enzyme or cells with one of a broad 
range of organic and inorganic supports. Included among these are 
polyacrylamide, ethylenemaleic acid copolymers, methacrylic-based 
polymers, polypeptides, styrene-based polymers, agarose, cellulose, 
dextran, porous glass beads, and aluminum or titanium hydroxide. Enzymes 
and cells in immobilized form have increased stability, extended 
usefulness, and recoverability. Reactions employing immobilized enzymes 
and cells may be run in columns or reaction tanks. 
The iodine recovery reaction of the invention is conducted within the pH 
range of from about 2 to about 8, which is enzyme-dependent. The pH of the 
reaction is maintained within the desired range by use of a buffering 
agent. Suitable buffers include sodium or potassium phosphate, gluconate, 
citrate, formate, and acetate-based systems. The reaction may be conducted 
in an aqueous medium. 
The production of iodine results during the course of the reactions which 
take place in the above reaction mixture. The iodine may be recovered by 
any convenient means, and may be produced either continuously or by batch 
processing. The reaction may be run in the presence of solvents which are 
immiscible in water and which can selectively extract the iodine as it is 
formed. Such solvents should, of course, have a non-detrimental effect on 
the enzymes used in the process. By extracting the iodine as it is formed, 
the use of such solvents will reduce the iodine toxicity to the enzyme and 
aid in the recovery of the free iodine. Among solvents that are suitable 
are benzene, toluene, xylene, mesitylene, ethyl acetate, ethyl ether, 
carbon tetrachloride and carbon disulfide. 
Another simple technique for continuous removal of iodine from the reaction 
is to bubble an inert gas, such as nitrogen, through the reaction mixture. 
The iodine is thus carried in the gas phase, is removed and concentrated 
from the gas stream by sublimation or other procedures known to the art. 
The spent inert gas is recycled for reuse. 
The reaction is preferably conducted in the temperature range of 15.degree. 
C. to about 50.degree. C., preferably about 20.degree. C. to about 
30.degree. C.

For purposes of further illustrating the invention, the following examples 
are set forth. These examples are not intended to limit the scope of the 
invention. 
EXAMPLE 1 
This example shows the pH range of activity for various halogenating 
enzymes useful in this process. 
Dilute hydrogen peroxide (0.044 mmoles; 273 .mu.g/ml final), potassium 
iodide (0.42 mmoles; 9730 .mu.g/ml final) and 0.1 M potassium phosphate 
buffer are mixed together to a final volume of 5 milliliters in a 100 
milliliter Pyrex flask at room temperature and room pressure. The 
halogenating enzyme is added. 
The halogenating enzymes are prepared as follows: 
Chloroperoxidase (CP). Mycelial pads of Caldariomyces fumago (ATCC 16373) 
are grown on potato agar slants as follows: Sliced potato (200 g) is 
cooked in distilled water (500 ml) for 40 minutes and then strained. A 
solution of glucose (21 g) and agar (20 g) in distilled water (500 ml) is 
added to the strained solution. The pH is adjusted to 6.8 and the volume 
is brought to 1 liter with distilled water. The medium is sterilized at 
121.degree. for 15 minutes. The organism is inoculated on the potato agar 
slants, produced in accordance with the above procedure, and is grown for 
about one week at room temperature. The organism is then used to inoculate 
the soybean-glucose medium (50 ml) prepared as follows: to 1 liter of 
distilled water are added extraction process soybean meal (30 g), glucose 
(30 g), and CaCO.sub.3 (7 g). The medium is sterilized at 121.degree. for 
30 minutes and is then inoculated with the organism after cooling. The 
organism is grown for 4-5 days on a rotary shaker at 25.degree.. 5 ml of 
this material is used to inoculate a 500 ml Erlenmeyer flask containing 
100 ml of a modified Czepek-Dox medium prepared by adding the following to 
1 liter of distilled water: NaNO.sub.3 (3 g), KH.sub.2 PO.sub.4 (1 g), KCl 
(0.5 g), MgSO.sub.4.7H.sub.2 O (10 mg) and glucose (40 g). The medium is 
sterilized at 121.degree. for 20 minutes prior to inoculation with the 
organism. The organism is grown under static conditions at room 
temperature 5-7 days. The black mycelial pads which form are collected, 
rinsed with distilled water, and stored in plastic bags in a freezer at 
-10.degree. for subsequent use. 
The halogenating enzyme is prepared by grinding 6 mycelial pads (prepared 
in accordance with the above procedures) with 60 g acid-washed sand and 60 
ml distilled water for 2 minutes in a Virtis 45 homogenizer. The 
homogenate is centrifuged while cold and the supernatant solution filtered 
through Whatman #1 paper at room temperature. The filtrate is concentrated 
about 10-fold using a rotary film evaporator at reduced pressure and 
temperature less than 35.degree.. The concentrate is chilled at 0.degree. 
in an ice bath, and prechilled (0.degree.) ethanol is added until 45% 
ethanol (v/v) is reached. The mixture is stirred vigorously for 15 
minutes, and then centrifuged at -10.degree. (at 15,000 g) with a 55-34 
rotor in a Sorval RC-5 Superspeed for 15 minutes. The black sediment is 
discarded. To the centrifugate, cooled at 0.degree., is added additional 
prechilled ethanol to give 65% ethanol (v/v). The mixture is slowly 
stirred for 30 minutes at 0.degree., and then centrifuged as before. The 
centrifugate is discarded and the precipitate containing the 
chloroperoxidase activity is dissolved in a minimum volume of 0.05 M 
potassium buffer (pH 7). The enzyme solution is stored at -20.degree.. The 
activity is measured as 80 monochlorordimedon units/ml. (Ref: Morris, D. 
R. and Hager, L. P., J. Biol. Chem. 241 1763 (1966). 20 units added to the 
reaction. 
Lactoperoxidase (LP). Purchased from Sigma Chemical Company (Catalogue 
#L-7129; activity of 470 purpurogallin units/ml). 200 units added to 
reaction. 
Seaweed Peroxidase (SWP). Coralina sp. obtained along the coast of La 
Jolla, California is ground in a Virtis 45 homogenizer for 5 minutes in 
distilled water. The homogenate is spun at 20,000 rpm for 20 minutes. The 
supernatant is decanted and saved. The pellet is resuspended in distilled 
water and recentrifuged. This supernatant and previous supernatant are 
combined. The solution is brought first to 33%, then to 55% saturation in 
ammonium sulfate. Centrifugation and separation of pellet is performed at 
each step. The 33%-55% pellet fraction is passed through a DEAE column 
using a 0.3 M to 1 M phosphate buffer (pH 6.0) gradient. The fraction 
which elutes at 1 M is dialyzed against 20 mM phosphate buffer (pH 6) 
overnight. This preparation is stored at -20.degree. until needed. The 
activity is measured as 2 monochlorodimedon units/ml. 1 unit is added to 
the reaction. 
Horseradish Peroxidase (HRP). Purchased from Sigma Chemical Company 
(Catalog #P-8250; activity of 165 purpurogallin units/mg solid). 500 units 
added to the reaction. 
The reaction mixtures are allowed to stand for 5 minutes and then 10 ml of 
chloroform is added to each one. The reaction mixtures are shaken and the 
violet coloration in the chloroform layer measured at 510 nm. Iodine 
standards are prepared by dissolving the appropriate amount of iodine 
(Baker Chemical Company, 99.9% pure) in chloroform. 
Product identity was confirmed by injecting 10 .mu.l of the reaction 
mixture into a Finnigan Model 4021 gas chromatograph/mass 
spectrometer/data system, equipped with a 6 foot by 1/4 inch coiled, glass 
column, packed with Tenax-GC (60/80 mesh). Carrier gas (helium) flow rate 
was set at 25 ml/minute. The column temperature was programmed from 
100.degree. C. to 250.degree. C. at a rate of 10.degree. C./minute. The 
mass spectrometer was set on electron impact ionization mode, 70 eV. 
Iodine elutes from the column near 200.degree. C. Iodine has a 
characteristic mass spectrum: 2 single peaks of high abundance, m/e 127 
and m/e 254. 
Variable conditions and results are set forth in Table I. 
TABLE I 
______________________________________ 
IODINE PRODUCED, mg** 
Non- 
enzy- CP LP SWP HRP 
pH matic* Enzymatic Enzymatic 
Enzymatic 
Enzymatic 
______________________________________ 
2.0 3.5 4.1 1.5 2.1 0.5 
3.0 3.5 5.1 3.9 2.3 5.5 
4.0 3.4 5.6 5.2 2.5 6.2 
5.0 2.8 6.8 6.8 3.4 7.0 
6.0 2.2 5.2 3.2 4.2 4.6 
7.0 1.9 0.7 0.7 0.5 2.1 
8.0 0.0 0.3 0.3 0.3 0.3 
______________________________________ 
*Spontaneous I.sub.2 production from I.sup.- under experimental 
conditions without added enzyme. 
**Under the experimental conditions, the maximum theoretical production o 
molecular iodine is 11.2 mg (reactions are H.sub.2 O.sub.2 limiting). 
Total iodine produced at a given pH is the sum of nonenzymatic plus 
enzymatic conversations. 
EXAMPLE 2 
The continuous production of iodine with chloroperoxidase is shown in this 
example. Chloroperoxidase is bound (immobilized) to calcium phosphate 
hydroxide (hydroxyapatite) and packed in a glass column (1.2 cm diameter 
by 10 cm height). The immobilized enzyme is prepared as follows: 
15 ml of chloroperoxidase solution (prepared according to the protocol 
described in Example 1) is diluted to 100 ml with 1 mM potassium phosphate 
buffer pH 5.0, and loaded onto the glass column containing 15 ml of 
hydroxyapatite. The column is then washed overnight with 500 ml of 10 mM 
potassium phosphate buffer pH 5.0 at 5.degree. C. The activity of the 
column is measured as 2235 monochlorodimedon units. 
A reaction mixture containing 100 ppm (0.6 mM) potassium iodide and 5 ppm 
(0.14 mM) hydrogen peroxide in 0.1 M potassium phosphate buffer pH 3.0 is 
passed through the column at a flow rate of 2 ml/minute. The column eluant 
is collected in fractions. These fractions are then extracted with 
chloroform and the violet coloration in the chloroform layer is measured 
at 510 nm. 
The following results are obtained: 
______________________________________ 
FRACTION [I.sub.2 ] 
______________________________________ 
COLUMN IN 2 .mu.g/ml 
COLUMN OUT 
1st 100 ml 24 .mu.g/ml 
2nd 100 ml 18 .mu.g/ml 
3rd 100 ml 16 .mu.g/ml 
______________________________________ 
Under the experimental conditions, the maximum theoretical production of 
iodine would be 37 .mu.g/ml, since the reactions are run under hydrogen 
peroxide limiting conditions. Over 50% of the theoretical yield is 
obtained. 
The molecular iodine can be recovered from the column eluant by standard 
techniques known to those skilled in this art. 
EXAMPLE 3 
The continuous production of iodine with seaweed peroxidase immobilized on 
hydroxyapatite is shown in this example. 
4 ml of Coralina sp. peroxidase supernatant (prepared according to the 
protocol in Example 1) is immobilized according to the procedure in 
Example 2, except the buffer is pH 6.0 instead of 3.0. 
The reaction is run according to the procedure in Example 2, except the 
buffer is at pH 6.0. 
The following results are obtained: 
______________________________________ 
FRACTION [I.sub.2 ] 
______________________________________ 
COLUMN IN 1 .mu.g/ml 
COLUMN OUT 
1st 100 ml 28 .mu.g/ml 
2nd 100 ml 29 .mu.g/ml 
3rd 100 ml 27 .mu.g/ml 
4th 100 ml 30 .mu.g/ml 
5th 100 ml 30 .mu.g/ml 
6th 100 ml 31 .mu.g/ml 
7th 100 ml 30 .mu.g/ml 
______________________________________ 
Over 80% of the theoretical yield was obtained. 
The molecular iodine can be recovered from the column eluant by standard 
techniques known to those skilled in this art. 
EXAMPLE 4 
The continuous production of iodine with seaweed peroxidase immobilized on 
glass beads is shown in this example. 
The immobilized seaweed peroxidase is prepared as follows: 
Glass beads (obtained from Sigma Chemical Company, PG-700-200) are 
activated by suspending 1 g of glass beads in 18 ml of deionized water. 2 
ml of 10% v/v .delta.-aminopropyltriethoxyl silane are added and the pH of 
the mixture is adjusted to 3-5 with 6 N HCl. The mixture is shaken at 
75.degree. C. for 2 hours. The glass beads are then vacuum dried overnight 
at 80.degree. C. 3.2 ml of purified Coralina sp. enzyme, prepared as in 
Example 1, and 50 mg of water soluble carbodiimide are added to the glass 
beads. The pH is adjusted to 4.5, and the mixture is then shaken at 
4.degree. C. overnight. The product--enzyme coated beads--is washed with 
water. The activity is measured as 2 monochlorodimedon units/g. of beads. 
Using 1 g of the seaweed peroxidase coated glass beads, the reaction is run 
according to the procedure in Example 3. 
The following results are obtained: 
______________________________________ 
FRACTION [I.sub.2 ] 
______________________________________ 
COLUMN IN 1 .mu.g/ml 
COLUMN OUT 
1st 100 ml 20 .mu.g/ml 
2nd 100 ml 21 .mu.g/ml 
3rd 100 ml 19 .mu.g/ml 
4th 100 ml 23 .mu.g/ml 
______________________________________ 
Over 50% of the theoretical yield is obtained. 
The molecular iodine can be recovered from the column eluant by standard 
techniques known to those in this art. 
EXAMPLE 5 
The continuous production of iodine with lactoperoxidase is shown in this 
example. 
0.5 ml of lactoperoxidase (bovine milk) bound to Sepharose (purchased from 
P-L Biochemicals, Inc; Catalogue #0723; 10 units triiodide activity total) 
is run under the same column reaction conditions as in Example 3. 
The following results are obtained: 
______________________________________ 
FRACTION [I.sub.2 ] 
______________________________________ 
COLUMN IN 1 .mu.g/ml 
COLUMN OUT 
1st 100 ml 35 .mu.g/ml 
2nd 100 ml 36 .mu.g/ml 
3rd 100 ml 35 .mu.g/ml 
______________________________________ 
Over 90% of the theoretical yield is obtained. 
The molecular iodine can be recovered from the column eluant by standard 
techniques known to those skilled in this art. 
EXAMPLE 6 
The continuous production of iodine with chloroperoxidase and with in situ 
generation of hydrogen peroxide is shown in this example. 
Chloroperoxidase and glucose oxidase are bound to hydroxyapatite and packed 
in a glass column (1.2 cm diameter by 10 cm height). The immobilized 
enzymes are prepared as follows: 
10 ml of chloroperoxidase solution (prepared according to the protocol in 
Example 1) and 5 ml of glucose oxidase, (purchased from Sigma Chemical 
Company; Catalog #G-6500), are diluted to 100 ml with 1 mM potassium 
phosphate buffer pH 5.0, and loaded onto the glass column containing 15 ml 
of hydroxyapatite. The column is then washed overnight with 500 ml of 10 
mM potassium phosphate buffer pH 5.0 at 5.degree. C. The activity of the 
column is measured as 1490 monochlorodimedon units of chloroperoxidase and 
7400 o-dianisidine units of glucose oxidase. 
A reaction mixture containing 100 ppm (0.6 mM) potassium iodide and 1800 
ppm (10 mM) .beta.-D-glucose in 0.1 M potassium phosphate buffer pH 4.4 is 
passed through the column at a flow rate of 2 ml/min. The column eluant is 
collected in fractions. These fractions are then extracted with chloroform 
and the violet coloration in the chloroform layer was measured at 510 nm. 
The following results are obtained: 
______________________________________ 
FRACTION [I.sub.2 ] 
______________________________________ 
COLUMN IN 0 .mu.g/ml 
COLUMN OUT 
1st 100 ml 49.6 .mu.g/ml 
2nd 100 ml 44.8 .mu.g/ml 
3rd 100 ml 48.8 .mu.g/ml 
______________________________________ 
Over 50% of the iodide is converted to iodine. In this experiment the 
production of iodine is limited by the in situ production of hydrogen 
peroxide. 
The molecular iodine can be recovered from the column eluant by standard 
techniques known to those skilled in the art. 
EXAMPLE 7 
The continuous production of iodine with seaweed peroxidase and with in 
situ generation of hydrogen peroxide is shown in this example. 
3 ml of Coralina sp. preparation (prepared according to protocol in Example 
1) and 5 ml of glucose oxidase, (purchased from Sigma Chemical Company; 
Catalog #G-6500) are immobilized on hydroxyapatite as in Example 6, except 
the buffer is pH 5.0 instead of 4.4. 
The activity of the column is measured as 51 monochlorodimedon units of 
seaweed peroxidase and 7400 0-dianisidine units of glucose oxidase. 
The reaction is run according to the protocol in Example 6, except the 
buffer is at pH 5.0. 
The following results are obtained: 
______________________________________ 
FRACTION [I.sub.2 ] 
______________________________________ 
COLUMN IN 0 .mu.g/ml 
COLUMN OUT 
1st 100 ml 16.0 .mu.g/ml 
2nd 100 ml 24.4 .mu.g/ml 
3rd 100 ml 16.0 .mu.g/ml 
______________________________________ 
Over 50% of the iodide is converted to iodine. In this experiment, the 
production of iodine is limited by the in situ production of hydrogen 
peroxide. 
The molecular iodine can be recovered from the column eluant by standard 
techniques known to those skilled in this art. 
EXAMPLE 8 
The simultaneous production/recovery of iodine in a repetitive batch, 
biphasic system is shown in this example. 
To a 50 ml Erlenmeyer flask are added 10 ml of organic solvent (chloroform, 
carbon tetrachloride, xylene or carbon disulfide); 1 ml of lactoperoxidase 
bound to Sepharose (purchased from P-L Biochemicals, Inc.; Catalog #0723; 
activity listed at 20 triiodide units); 1 ml of 0.1 M potassium phosphate 
buffer pH 5.0; and a stir bar. 
By use of a buret, a reaction mixture, which consists of 100 ppm (0.6 mM) 
potassium iodide and 7 ppm (0.2 mM) hydrogen peroxide in 0.1 M potassium 
phosphate buffer pH 5.0, is added to the flask at a rate of 5 ml/minute. 
The flask contents are stirred by use of a magnetic stirrer. After 30 ml 
of reaction mixture is added, the addition is stopped. The organic solvent 
layer is separated from the aqueous layer. This organic solvent layer is 
measured at 510 nm to determine the molecular iodine level produced. 
The aqueous layer is centrifuged at 1000 rpm to pellet the immobilized 
enzyme. The pellet is returned to the 50 ml Erlenmeyer flask, fresh 
organic solvent is added (10 ml) and another portion of reaction mixture 
is slowly added (30 ml total addition volume at a 5 ml/min rate). The two 
layers are separated, processed and the reaction procedure repeated 
several more times. 
The following results are obtained: 
______________________________________ 
IODINE PRODUCED, mg 
SAMPLE CHCl.sub.3 
CCl.sub.4 
Xylene CS.sub.2 
______________________________________ 
Control Batch* 
0 0 0 0 
REACTION 
1st Batch 1.2 1.5 1.5 1.5 
2nd Batch 1.4 1.5 1.5 1.4 
3rd Batch 1.5 1.5 
4th Batch 1.4 
5th Batch 1.5 
6th Batch 1.5 
______________________________________ 
*no enzyme added. 
Under experimental conditions, the maximum theoretical production of iodine 
would be 1.5 mg since the reactions were run under hydrogen peroxide 
limiting conditions. Up to 100% of the theoretical yield is obtained. 
The molecular iodine can be recovered from the organic solvents by standard 
techniques known to those skilled in this art. 
EXAMPLE 9 
The simultaneous production/recovery of iodine from iodide solutions high 
in other halides is shown in this example. 
The procedure of Example 8 is followed with the following modifications: 
(a) carbon tetrachloride is the organic solvent. 
(b) seawater bittern which contains 140 mg/ml chloride ion and 1 mg/ml 
bromide ion is used. Iodide ion is not present at a detectable level. To 
this bittern, potassium iodide (100 ppm, 0.6 mM; final) is added. 
The following results are obtained: 
______________________________________ 
SAMPLE IODINE PRODUCED, mg 
______________________________________ 
Control Batch* 0 
REACTION 
1st Batch 1.5 
2nd Batch 1.5 
______________________________________ 
*no enzyme added. 
Under experimental conditions, the maximum theoretical production of iodine 
would be 1.5 mg, since the reactions were run under hydrogen peroxide 
limiting conditions. 100% of the theoretical yield is obtained. 
The molecular iodine can be recovered from the organic solvent by standard 
techniques known to those skilled in the art. 
Various modifications of the invention will become apparent to those 
skilled in the art from the foregoing description. Such modifications are 
intended to fall within the scope of the appended claims.