Journal of Molecular Catalysis, 22 (1983) 145 - 152 145

POLYETHYLENIMINE/SILICA GEL AS AN ENZYME SUPPORT

KUNIHIRO WATANABE* and G. P. ROYERt

Department of Biochemistry, Ohio State University, 484 West 12th Avenue, Columbus, OH 43210 (U.S.A.)

(Received January 16,1983)

Summary

We have devised a process to covalently bond polyethylenimine to the surface of chromatographic grade silica gel. Activation for enzyme immobili- zation is easily accomplished by treatment of the amine support with gluta- raldehyde. Enzyme conjugates were produced with lactate dehydrogenase, trypsin, and chymotrypsin. Relatively small differences in K, and pH depen- dence were observed when the bound and free forms of the enzymes were compared. All three immobilized enzymes exhibited excellent operational stability in a stirred, flow-through reactor.

Introduction

An ideal enzyme support could be described as hydrophilic, dimension- ally and chemically stable, economical, and resistant to microbial attack. Good accessibility and low diffusional resistance are other important charac- teristics. We are interested in inorganic-organic conjugates as catalyst sup- ports [ 1 - 31, and have carried out studies involving polyethylenimine (PEI) crosslinked on inorganic surfaces with glutaraldehyde. In this report we describe the use of an amine crosslinking reagent, namely Xama@, which is a polyfunctional aziridine. We have devised a one-step process for producing a PEI-Xama@/silica gel conjugate which is depicted schematically in Fig. 1. The crosslinking reagent, PEI and silica gel are mixed and dispersed in a stirred organic phase. The resulting conjugate has excellent handling proper- ties and it is easily activated for enzyme attachment.

Experimental

Materials PEI (M.W. 60 000,33% aq.) and Xama@-2 were purchased from Cordova

Chemical Co. Silica gel (Grade 923, 100 - 200 mesh) was supplied by W. R.

*Present address: Kyoto University, Department of Industrial Chemistry, Sakyo-ku Kyoto 606 (Japan)

*Author to whom correspondence should be addressed.

0304-5102/83/$3.00 @ Elsevier Sequoia/Printed in The Netherlands

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ON (CH, CHJ'JH, )” + k- "3

H’ 1 4 Silica gel

Crosslinked PEI layer on Silica Gel

‘Fig. 1. Production of PEI/silica gel. PEI is reacted with a polyfunctional aziridine, and the polymer is then deposited onto chromatographic grade silica gel to form a stable layer.

Grace. Rabbit muscle lactate debydrogenase and bovine a-chymotrypsin were obtained from Sigma Chemical Co. Bovine trypsin was purchased from Worthington Biochemicals.

Methods Preparation of PEUsilica gel PEI solution (100 g, 33%) and 40 ml of concentrated HCl were mixed

with cooling in a three-neck flask fitted with an overhead stirrer. Silica gel (200 g) and 1’70 ml of methanol were mixed and then added to the flask followed by 30 g of Xama-2 with vigorous stirring. The organic phase (800 ml petroleum ether, 200 ml corn oil, 5 g Span 80-premixed) was added and

‘the mixture was stirred at room temperature for 2 h. The PEI/silica gel was collected and washed as follows: two 500-ml portions of petroleum ether; two 500-ml portions of methanol; and water until a negative ninhydrin test was obtained.

Enzyme immobilization PEI/silica gel (1 g, dry) was treated with 20 ml of glutaraldehyde solu-

tion (2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4) for 2.5 h at room temperature. The support was suspended by rocking or with an over- head stirrer (use of a magnetic stirrer is less desirable). After the reaction period was complete, the support was collected by filtration and washed with the same phosphate buffer (250 ml).

For the coupling reaction, enzyme was dissolved in phosphate buffer (0.1 M, pH 7.4, 5 ml for each gram of support). LDH was used alone (5 mg/ 5 ml buffer). Trypsin (25 mg/5 ml buffer) was accompanied by the compe- titive inhibitor benzamidine*HCl (10 mM). Chymotrypsin (25 mg/5 ml buf- fer) was accompanied by the competitive inhibitor indole (1 mM). In each case, the activated support was suspended in the enzyme solution overnight at 4 “C. After washing with water to remove unreacted and loosely bound enzyme, the immobilized enzyme preparations were stored damp at 4 “C.

Enzyme assays LDH activity was followed spectrophotometrically by observing the

decrease in absorbance at 340 nm. The assay mixture contained the follow-

147

ing components: phosphate buffer (33 mM, pH 7.4), NADH (0.15 mM), pyruvate (0.75 mM). The reaction volume was 3 ml; the temperature was maintained at 25 “C. Stirring of the assay mixture within the cuvette was accomplished by the method described by Mort et al. [4]. The continuous- flow assays were carried out in a reactor constructed from a sintered glass funnel (1.6 X 5 cm) fitted with a water jacket. Agitation was provided by a light magnetic stirring bar. The substrate solution consisted of the following components: 33 mM phosphate, 2 mM mercaptoethanol, 0.3 mM NADH, 0.75 mM pyruvate. This solution was pumped through the reactor at a flow rate of 36 ml/h. The absorbance (340 nm) of the eluent was monitored.

Trypsin was assayed titrimetrically in individual assays and spectro- photometrically in the continuous-flow reactor. Titrimetric assays were made with N-cr-benzoyl-L-arginine ethyl ester * HCl (BAEE) as substrate and the following conditions: 20 ml of 10 mM Tris-HCl (pH 8.0) containing 100 mM CaCl, and 10 mM BAEE; the temperature was maintained at 25 “C and the titrant was 0.1 N or 0.01 N NaOH. The continuous assays were carried out with the reactor described above, using N-o-tosyl-L-arginine methyl ester (TAME) as substrate and the following conditions: flow rate 36 ml/h, 10 mM Tris-HCl (pH 8.0) containing 0.5 mM TAME and 10 mM CaCl,. The absor- bance change at 247 nm was monitored.

Chymotrypsin was assayed titrimetrically in batch reactions and spec- trophotometrically in the continuous-flow reactor. In the pH-stat assays, the substrate was N-o-acetyl-L-tyrosine ethyl ester (ATEE). The conditions were as follows: 25 ‘C, 20 ml 10 mM Tris-HCl (pH 8.0), 100 mM CaCl, and 10 mM ATEE. A stock solution of ATEE was prepared with acetonitrile as sol- vent. The final concentration of acetonitrile in the reaction mixture was 5%. Continous-flow assays for chymotrypsin were performed as described above. The substrate was N-cx-benzoyl-L-tyrosine ethyl ester (BTEE). Conditions were as follows: 25 “C, 0.5 mM BTEE, 25% methanol, 50 mM Tris (pH 8) and 50 mM CaCl,. The rate of esterolysis was determined by following the change in absorbance at 256 nm.

Results and discussion

PEI-coated silica gel has excellent mechanical and chemical stability. It is easy to handle; slurries are readily produced but the support settles from suspension rapidly. The primary amine content is 0.21 meq/g. PEI content of the conjugate is about 10% by weight.

Lactate dehydrogenase, trypsin, and chymotrypsin were immobilized using glutaraldehyde coupling. Specific activities were as follows: LDH, 30 units/g; trypsin, 156 units/g (BAEE); chymotrypsin, 202 units/g (ATEE). These values are only approximated in that the ‘damp weight’ of the gel is used as the weight of conjugate. The three preparations were characterized with respect to pH-rate profile, Michaelis constant, and stability.

Perturbation of the pH-dependence of immobilized enzymes can be related to the microenvironmental change in the local pH compared to the

bulk pH and to the buildup of H+/OH- gradients as a result of the progress of the enzyme-catalyzed reaction [53. In the first case, a positively-charged support tends to shift the pH optimum to a lower value. The buildup of Hr OH- gradients can occur even with neutral supports, and can be decreased by high concentrations of buffer species. In the reactions shown below, protons are consumed (LDH) or produced (trypsin, chymot~ps~) in all cases:

LDH pyruvate C NADH + Hf - L-lactate + NAD+

HINR:H&+OEt - trypsin

NH H2O

H2NR:HdO- + H+

NH 0

cr

0

0 +

EtOH

+ EtOH

+ H+

The pH-rate profiles of the reactions catalyzed by the bound and free forms of LDH, trypsin, and chymotrypsin are shown in Figs. 2 - 4 respec- tively. It is ~media~ly apparent that large differences between the bound and free systems do not exist. The bound form of LDH exhibits slightly higher activity at low pH relative to the free enzyme. This observation could be explained by the microenvironmental effect produced by the positively- charged support. Profiles for bound and free forms of trypsin and chymo- trypsin are nearly identical. The proton production may balance the micro- environmental effect. In any event, under the conditions described here, large perturbations of pH-dependence are not brought about by the PEI/ silica gel carrier,

The general effects of immobilization on the Michaelis constant, K,, for an enzyme system may be described in terms of’diffusional limitations or partitioning of subtrate between the support and the bulk phase; a substrate can be repelled or attracted by the support. Generally Km values increase when enzymes are immobilized; in other words, the concentration of substrate in the

149

- --o- - BOUND

--c- FREE

--e- FREE

0 ‘, ’ .b- 4 6 8 IO

PH

I

4 6 8 IO

PH

Fig. 2. pH-rate profiles for the free and immobilized forms of lactate dehydrogenase.

Fig. 3. pH-rate profiles for the free and immobilized fore of trypsin (BAEE).

r I I I I 1 100 -

--O- - BOUND

-c- FREE

4 6 8 IO

PH

Fig. 4. pH-rate profiles for the free and immobilized forms of chymotrypsin (ATEE).

bulk phase required to reach half-maximal velocity increases when enzyme is bound. We selected typical conditions which would be reasonable for a batch-type application of an enzyme bound to PEI/silica gel. Michaelis con- stants are shown in Table 1. The K, for pyruvate is not changed when LDH is bound to PEI/silica gel. This result probably reflects a compensation of two factors: attraction of the organic anion (decrease in K,) and diffusional limitation (increase in K,). NADH is a large anion. When LDH is bound to agarose, a near-neutral carrier, the K, for NADH increases by a factor of 30 [6]. In the case of PEI/silica gel (positively-charged) the increase is only a

150

TABLE 1

Michaelis constants of free and bound forms of LDH, trypsin and chymotrypsin

Lactate dehydrogenase Trypsin

pyruvate NADH BAEE

Chymotrypsin

ATEE

bound free

3.0 x 1O-4 M 5.8 x lo-’ M 1.2 x 1O-4 M 2.5 x 1O-3 M 3.0 x 1O-4 M 9.4 x 1O-6 M 3.6 x lo--’ M 3.6 x 1O-3 M

factor of six, which indicates attraction of the NADH to the matrix. BAEE is a cation and should be repelled by the matrix. This tendency along with the diffusional limitation would suggest an increase in K,, which is in fact ob- served. When trypsin in bound to agarose, the K, for BAEE increases by a factor of 100 [ 71. In the case of trypsin bound to PEI/silica gel, the ratio of K, (bound)/&, (free) is only 3.3. One could conclude that diffusional resistance is less important for PEI/silica gel as a carrier compared to agarose; the fact that the K, for chymotrypsin/ATEE is improved on coupling tends to support this conclusion. For the three enzymes tested, the relatively small changes in K, are encouraging, and suggest that the support would provide enzyme conjugates useful at low substrate concentration.

The stability of immobilized enzymes is a fundamental consideration. An enzyme support with all the aforementioned advantages which is not stable is of little value. We evaluated our enzyme conjugates for stability in the following way. The immobilized enzymes were assayed daily with the continuous-flow system described earlier. The total throughput volumes were: LDH, 10 liters over 90 days; trypsin, 13.5 liters over 85 days; chymo- trypsin, 10.5 liters over 55 days. The time courses for decay are shown in Figs. 5 - 7. The loss of activity of immoblized LDH occurred to the extent of 50% after 500 runs during an 8Oday period. Immobilized trypsin and chymo- trypsin showed excellent stability. After 300 runs over two months, 95% of the activity was retained in both cases. LDH is an intracellular enzyme with multiple subunits. As a protein, one would predict greater instability for LDH than for extracellular enzymes with compact structures containing mul- tiple disulfide bridges. When autolysis is prevented by immobilization, which appears to be the case here, trypsin and chymotrypsin are stable for long periods on PEI/silica gel.

In conclusion, the results presented here suggest that PEI/silica gel has promise as a enzyme support. In three cases, enzyme conjugates were pro- duced which were active, stable and easily handled in a CSTR-type reactor. Preliminary studies suggest that the material may serve as an enzyme support suitable for use in packed-bed reactors. We have shown in a related study that similar versions of PEI/silica are excellent chromatographic materials useful for nucleotide separation and purification of proteins [ 81.

151

0.6

-2 0.5

s d 0.4 a >

b 0.3 2

t, a 0.2

0.1

600

0 AVERAGE OF EACH DAY’S

0 TOTAL NUMBER OF RUNS 100

8 300

ii

f

200 2

d 6

100 t-

ok0 OLO 0 20 40 60 a0

DAYS DAYS

Fig. 5. Stability of lactate dehydrogenase/PEI/silica gel.

Fig. 6. Stability of trypsin/PEI/silica gel. The substrate was TAME.

- 300 8

is

is - 200 -J

z

-I

O- I I I 0 0 20 40 60

DAYS

Fig. 7. Stability of chymotrypsin/PEI/silica gel. The substrate was BTEE.

Acknowledgement

This work was supported by a grant from Dow Chemical Company, U.S.A.

152

References

1 G. P. Royer, Chemtech, (1974) 694. 2 W. E. Meyers and G. P. Royer, J. Am. Chem. SOL, 99 (1977) 6141. 3 D. Coleman and G. P. Royer, J. Org. Chem., 45 (1980) 2268. 4 J. S. Mort, D. K. K. Chang and W. W. C. Chan, Anal. Biochem., 52 (1973) 162. 5 G. P. Royer, Fundamentals of Enzymology, Wiley, New York, 1982, p. 188. 6 T. K. Lee, Ph. D. Thesis, Ohio State University, 1981, p. 105. 7 R. Uy, V. S. H. Liu and G. P. Royer, J. Solid-Phase Biochem., I (1976) 51. 8 K. Watanabe, W. S. Chow and G. P. Royer, Anal. Biochem., 127 (1982) 155.