BIOTECHNOLOGY AND BIOENGINEERING
`
`¥OL. XVII (1975)
`
`Immobilization off ~-Galactosldase in Collodion
`Microcapsules
`
`One of the more flexible and efficient techniques of enzyme immobilization is
`the microencapsulation procedure developed by Chang.1 Nylon microcapsules
`can be made by the interracial polymerization method, while collodion micro-
`capsules can be made by interracial precipitation. While the nylon micro-
`capsules have thinner membranes (~100 X), the interfacial polymerization
`procedure often leads to denaturation of the enzyme. This was found to be the
`case by 0stergaard and Martiny~ in their study of the mieroencapsulation of
`~-gatactosidaae (E.G. 3.2.1.23) from E. coll. In the present investigation, we
`have microencapsulated this enzyme by the milder interfacial precipitation
`method for collodion microeapsules and show that the enzyme maintains high
`catalytic activity as measured by the conversion of o-nitrophenyl-~-I)-galacto-
`pyranoside (ONPG) (Sigma Corp.) in packed beds. To encapsulate the enzyme,
`75 mg of E. coil ~-galactosidase (Worthington Biochemical) were dissolved in
`2.5 mi of a Tris-buffered hemoglobin solution made by dissolving 1 g of hemo-
`globin (Sigma) and 200 mg Tris base (Worthington) in 10 ml of distilled water.
`The enzyme solution was then placed in a 150 ml beaker with 25 ml of an organic
`solution made by mixing 100 ml of water-saturated ether (U.S.P.) and 1 ml of
`Span 85 (City Chemical Co.). The solutions are immediately stirred using a
`Fischer Jumbo magnetic stirrer at a speed setting of 7 with a 4 cm stirring bar.
`After 5 sec of stirring, 25 ml of a cellulose nitrate sotution (made by evaporating
`collodion (U.S.P., MGB Corp.) to 20% of it.~ original weight and replacing its
`original volume with ether) is added and stirring ~s continued for 1 inin. The
`solution is then kept at 4°C for 1 hr. After settling, the supernatant is removed
`and 30 ml of n-butyl benzoate (MCB Corp.) containing 0.3 ral of Span 85 is
`added to the microcapsuleu and stirred at a speed of 5 for 30 see. The recruiting
`suspension is allowed to stand uncovered in crushed ice for 1 hr. After settling,
`the supernatant is again removed and 25 ml of a dispersing solution containing
`12.5 ml of Tween 20 (Sigma) and 12.5 ml of H~O is added to the mierocapsules.
`The suspension is stirred for 30 sec at a speed setting of 10. The stirring rate is
`slowed to a setting of 5 and 225 ml of water are slowly added. This suspension
`is again allowed to settle, and the mierocapsuies are washed repeatedly with a
`0.9 wt % NaC1 solution (approximately 100 ml at a time). This is done until no
`trace of enzyme (measured by ONPG conversion) ~ detectable in the wash
`(~-~5 washings are required). Approximately 1 ml of microcapsules is obtained
`using this procedure, and approximately 49 mg of the enzyme is unbound. Thus,
`a concentration of enzyme eorr~pondlng to 26 mg/m] is finally encapsulated.
`This is almost exactly the concentration of enzyme initially dissolved in the
`hemoglobin (30 mg/ml). The encapsulated enzyme exhibited a value of kr ffi
`6.02 ~mol/mg enzyme o min for ONPG, equal to the turnover number for the
`free enzyme, indicating that no appreciable enzyme activity was lost during
`encapsulation.~ This turnover number was measured in a 0.1M phosphate
`buffer (pH 6.(}) at 25°C.
`
`(~) 1975 by John Wiley & Sons, Inc.
`
`617
`
`Akermin, Inc.
`Exhibit 1015
`Page 1
`
`

`

`618 BIOTECItNOLOGY AND BIOENGINEERING VOL. XVII (1975)
`
`C
`
`lO
`
`O8
`
`0.1
`
`L
`~0
`
`I I t I
`lOO
`40
`50
`80
`
`120
`
`Fig. 1. Ratio of outlet to inlet ONPG concentration in a 1.6 cm diameter
`column containing suspended microcapsutes: (O) 0.4 cm column, (B) 0.9 em
`column. Buffer: 0.1M phosphate, pH 6.6, temperature 25°C,
`
`The 1 ml of microcapsuMs was mixed thoroughly with 25 ml of washed
`Sepharose 4B gel (Phaxmacia), and the slurry poured into a 1.6 cm diameter
`column (Phaxmacia, Model K-16) at the same time that the phosphate buffer was
`being added. The gel settled quickly with an even distribution of microeapsules.
`The size of the bed could be controlled by the amount of slurry originally added.
`A 0.625M ONPG solution in the buffer (as described above) was then pumped
`through the top of the column at various flow rates (0.5-3 ml/min). The
`amount of product (o-nitrophenyl) formed was measured spectrophotometrlcally
`at 420 nm using a Hitachi Model 102 spectrophotometer. The results are
`shown in Figure 1 where the ratio (C/Co) of the outlet substrate concentration to
`the inlet concentration ks plotted as a function of residence time # = z/v where z
`is the column length and v is the fluid velocity. Column lengths were short
`(0.4 and 0.9 cm), and with a residence time of ~-~100 see, approximately 23%
`conversion was obtained. This is a high conversion, considering that only 0.006
`to 0.0i ml of mlcrocapsules were used in these experiments.
`This shows that the collodion microencapsutation procedure is a suitable
`technique for the insolubilization of fl-galactosidaae. Even though the membrane
`thickness of collodion microcapsules is greater than that of nylon microcapsules
`(400 ]k when compared to 100 !~),t the results of these experiments indicate that
`the membrane is still permeable to large molecules such as ONPG (reel wt = 301).
`Furthermore, the encapsulation procedure is milder than the interracial polymeri-
`zation method leading to nylon microcapsules, resulting in little enzyme de-
`activation.
`
`References
`
`I. T. M. So Chang, Artificial Cells, C. C. Thomas, Springfield, 1t1., 1972.
`2. J. C. W. 0stergaard and S. C. MurLiny, Bio~echnol. Bioeng., 15, 561 (1973).
`
`Akermin, Inc.
`Exhibit 1015
`Page 2
`
`

`

`COMMUNICATIONS TO THE EDITOR
`
`619
`
`3. D. T. Wadiak, Masters The.sis, Chemical Engineering Dept., Univ. of
`California, Dsvis, 1974.
`
`Dept. of Chemical Engineering
`University of Californis
`Davis, California 95616
`
`Accepted for Publication December 12, 1974
`
`Akermin, Inc.
`Exhibit 1015
`Page 3
`
`