Stability of alanine aminotransferase is enhanced by chemical modification

Stability of alanine aminotransferase is enhanced by chemical modification Ciaran O'Fagain,* Richard O'Kennedy* and Cormac Kiltyt * S c h o o l o f Biological Sciences, Dublin City University, Dublin, ~re~and a n d ? Baxter-Dade AG, Duedingen, Switzerland

Alanine aminotransferase is a clinical diagnostic marker enzyme. It is often included in commercial control sera. The stabilization o f soluble alanine aminotransferase catalytic activity by chemical modification with b/s-/m/dates and with succinic anhydride is described. At 4°C, the b/s-/m/dates-modified enzyme is predicted to be ap to 43 times more stable, and the sttccinic anhydride-modified enzyme six times more stable, than the unmodified enzyme control. The degree o f stability was estimated by an accelerated storage protocol with experimental data being analyzed by a dedicated computer program. The applicability o f this methodology to studies o f protein stability is briefly discussed.

Keywords:Enzyme stabilization: protein modification:alanine an3inotransferase: his-/m/dales: succinic anhydride Introduction The enzyme alan/he aminotransferase (L-alanine:2oxo-glutarate aminotransferase, ALT, SGPT, E.C. 2.6.1.2) is one of the transaminases widely used as a clinical diagnostic marker. Together with its sister enzyme, aspartate aminotransferase (E.C. 2.6.1. I), its levels in serum act as a key indicator of liver and other functions in a variety of clinical states, particularly hepatitis and myocardial disease.J Commercial control sera containing known amounts of diagnostically important enzymes are used to validate the results obtained in clinical laboratories. Usually such sera offer many advantages compared with laboratory-prepared controls. 2 However, the stability of enzyme activities in such controls (whether liquid or rehydrated, freeze-dried) is limited. This can result in waste of expensive serum-based components. The time required for complete rehydration of freeze-dried controls can lead to delays in obtaining results, and complete rehydration is often not achieved. Use of a liquid control serum containing stabilized enzymes would help in eliminating or ameliorating these problems. A variety of methods for stabilizing enzyme catalytic activity have been reported, and these have been reviewed recently. 3-5 Such methods include addition of known stabilizing compounds, 6'7 enzyme immobilization, 8 cross-linking of the protein backbone, 9-12 modi-

Address reprint requests to Dr. O'Ffig(iinat the Schoolof Biological Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland Received 17 May 1990

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Enzyme Microb. Technol., 1991, vol. 13, March

f/cation of surface functional groups, j3,j4 and protein engineering by site-directed mutagenesis. ~5-~7 Not all of these approaches will be suitable for a particular enzyme or application. An enzyme in a diagnostic control, for example, must be in free solution (not immobilized), and addition of certain substances may result in unacceptable viscosity or inappropriate salts balance. We have subjected alanine aminotransferase to chemical modification with b/s-/m/dates and with succinic anhydride. We have compared the stabilities of the modified fractions with those of unmodified controls. Successful stabilization of soluble ALT was achieved using dimethylsuberimidate and succinic anhydride.

Materials and methods Beef heart alanine aminotransferase was a gift from Dr. A. Posner, Baxter-Dade, Miami 33152-0672, Florida, USA. Diethylaminoethyl- (DEAE-) and carboxymethyl- (CM-) celluloses were obtained from Whatman UK. Sephadex G-25 (T.M.) was obtained from Pharmacia. All chemicals used for ALT modification were supplied by Sigma. The FORTRAN program D E G T E S T used for analysis of results was kindly supplied by the National Institute for Biological Standards & Control, Potters Bar, Herts EN6 3QG, UK. The E N Z F I T T E R software package of R. J. Leatherbarrow was obtained from Biosoft/Elsevier, Cambridge CB2 1LA, UK. The gel densitometer GS 300 and computer software (GS 350) were obtained from Hoefer Scientific Instruments, San Francisco, California 94107, USA. ALT activity was measured by the continuous spectrophotometric IFCC (International Federation of Clin~ 1991

Butterworth-Heinemann

Chemically stabilized alanine aminotransferase: C. O'F~gMn et al. ical Chemistry) method. 18 Pyridoxai phosphate was included in the Tris-alanine buffer, pH 7.5, as an activator. Protein content was estimated by absorbance at 280 nm and also by the bicinchoninic acid method of Smith and co-workers.~9

A L T purification Purification of the e n z y m e was based on the method described by Jenkins and Saier 2° for pig heart ALT. The crude preparation of A L T was desalted on Sephadex G-25 followed by chromatography on DEAE-cellulose in 0.02 M Tris-HC1/0.01 M EDTA/0.02 M 2-mercaptoethanol, pH 7.0. The e n z y m e eluted very early in the 0-0.15 M NaCI gradient applied. Fractions containing active enzyme were pooled and their pH was carefully lowered to 5.5 by addition of 5 M HC1 before application to CM-cellulose equilibrated in 0.06 M acetate/0.01 M EDTA/0.02 M 2-mercaptoethanol, pH 5.5. A L T enzyme did not adsorb under these conditions. The pH of the pooled active fractions eluting from the column was adjusted to 7.5 and A L T was then concentrated using an Amicon YM-5 membrane. Polyacrylamide gel electrophoresis was performed by the method of Laemmli. 2~ Molecular weight standards used in the 10% polyacrylamide gel were, in decreasing order of molecular weight: myosin (205 k), beta-galactosidase (116 k), phosphorylase b (97.4 k), bovine albumin (66 k), egg albumin (45 k), and carbonic anhydrase (29 k).

Bis-imidates modification Modification with bis-imidates was based on the protocols of de Renobales and Welch 22 and Minotani and colleagues. 23 A L T (3 mg ml 1) in 0.2 M phosphate buffer, pH 8.0, was treated with bis-imidates of varying molecular length (dimethyladipimidate, 0.77 nm, dimethylpimelimidate, 0.92 nm, and dimethylsuberimidate, 1.1 nm) at final concentrations of not less than 10 mM. The reaction took place at 27°C for 1 h, after which time the reaction was terminated by the addition of 1 ml of 0.2 M Tris-HC1, pH 7.4.

Succinic anhydride treatment Succinic anhydride treatment was based on the procedures of Torchilin and colleagues 24 and of Hollecker and Creighton. :5 A l0 mg ml ~ stock solution of succinic anhydride was prepared and was immediately added to portions of A L T (3 mg ml J) in 0.2 M phosphate buffer, pH 7.7, so as to give final succinic anhydride concentrations of 50 and 1,000/zg ml J. The reaction was allowed to proceed for 1 h at 27°C, after which time the reaction was terminated by the addition of 1 ml of 0.2 M Tris-HCI, pH 7.0.

Accelerated storage testing and kinetic determinations Accelerated storage tests were set up and carried out according to the recommendations of Kirkwood 26 and

were analyzed by the method of K i r k w o o d and Tydeman 27 using the F O R T R A N programme D E G T E S T and by the E N Z F I T T E R software of R. J. Leatherbarrow. E n z y m e kinetic determinations were carried out in triplicate over a 30-fold range of substrate concentrations, and the E N Z F I T T E R computer program was used to fit results to the Michaelis-Menten equation.

Results A L T purification The DEAE- and CM-column procedures yielded an A L T preparation with specific activity of up to 9.678 nanokatals mg -I protein (0.581/xmol min J mg -1 protein), a sixfold purification of the crude starting preparation, with 73% recovery of activity. Table 1 records details of a typical purification. SDS gel electrophoresis indicated a major peak of 59 k corresponding to A L T subunit molecular weight (ALT functions as a dimer of 114 k). The e n z y m e was estimated to be 51% pure, based on densitometric peak areas.

Bis-imidates modification Recoveries of A L T activity from treatment with bisimidates were as shown in Table 2. The results of accelerated storage testing at 45°C, 37°C, 33°C, 27°C, and reference temperature 4°C are shown in Table 2. The loss of e n z y m e activity with time follows a first-order equation implying that a single molecular process is responsible for the loss of catalytic activity (see Figure 1). For the sake of clarity, only the dimethylsuberimidate-treated and untreated control ALTs are depicted in Figure 1. The modified e n z y m e obviously declines at a much slower rate than does the unmodified fraction. Values for the first-order rate constants, k, derived using E N Z F I T T E R are summarized in Table 2. There is a high standard error associated with the rate constant for dimethylpimelimidatetreated A L T at 45°C. The first-order fit was satisfactory at all other temperatures tested. The predicted activity losses and shelf-lives at 37°C, 20°C, and 4°C calculated by the D E G T E S T program 27 are shown in Table 3. "Shelf-life" was defined as the time required for activity to decrease to 90% of initial value. The shelf-lives at 4°C for the control and modified A L T s are predicted to be as follows: control, 116 days; dimethyladipimidate, 192 days; dimethylpimelimidate, 286 days; and dimethylsuberimidate, 5,000 days. The kinetic constants, gma x and K m, showed no significant alteration in value for the modified A L T s when compared with unmodified enzyme, gma x and K m were determined to be 44.9 p.kat 1-J and 0.29 mM, respectively, for 2-oxoglutarate and 44.6 p~kat 1-~ and 17.31 mM, respectively, for L-alanine. These K m values for the beef heart A L T contrast with previously reported values of 0.73 and 0.4 mM, respectively, for human ~8 and pig heart 2° A L T s on 2-oxoglutarate and 26.5 mM (human) and 28 mM (pig heart) on L-alanine. Both K m values for the beef e n z y m e are significantly lower than those for the human and pig enzymes.

Enzyme Microb. Technol., 1991, vol. 13, March

235

Papers Table 1

Purification of alanine aminotransferase

Stage Initial crude G-25 column DEAE-column CM-column

Volume (ml)

Activity a (nkat m1-1)

Total activity (nkat)

A280

Specific activity b

Purification (fold)

Recovery (%)

20 100 170 188

442 93 52 32

8,849 9,315 8,908 6,020

53.6 6.91 1.17 0.58

8.25 13.5 44.8 55.2

-1.6 5.4 6.7

100 105 101 68

n k a t m l ~ = #kat1-1 = 1 / 6 0 ( / ~ m o l m i n -~1 -~) b In this case, represents the value for activity (nkat ml 1) divided by A280 Accurate protein determination 19 after the CM-cellulose stage revealed a true specific activity of 5.16 nkat m1-1 for this preparation. For full experimental details see Materials and methods

Table 2 Values for first-order decay rate constant, k, at various temperatures for alanine aminotransferase modified with bis-imidates and with succinic anhydride First-order decay rate constant, k, at Enzyme modifier Control Adipimidate Pimelimidate Suberimidate Control Succinic anhydride, 50/~g ml -~ Succinic anhydride, 1,000/~g ml

Activity recovery

45°C

100% 96% 90% 88% 100% 99%

n.d. .144 -+ .014 .077 -+ .058 .116 -+ .035 n.d. n.d.

.080 .044 .042 .017 .131 .126

96%

.239 -+ .033

.065 -+ .003

37°C -+ .013 -+ .013 _+ .002 -+ .006 -+ .0006 -+ .013

33°C .067 .035 .028 .005 .073 .061

_+ -+ -+ ++_+

27°C

.019 .009 .007 .003 .008 .002

.031 _+ .007

.021 -+ .000 a .021 -+ .000 a .021 -+ .000 a n.d. n.d. .024 -+ .003 .012 _+ .001

n.d., Not determined The values displayed are means -+ standard error of the mean Units for the first-order decay rate constant, k, are days First-order decay rate constants were derived using ENZFITTER a No error was detected in these 27°C k values to three decimal places For full experimental details see Materials and methods (The ALT used in the bis-imidates and succinic anhydride experiments came from different preparations, hence the differences in values for the controls.)

Table 3

Values for predicted degradation rates and shelf-lives at various temperatures for alanine aminotransferase modified with bis-imidates and with succinic anhydride % Activity loss day -1 at Enzyme modifier

Control Adipimidate Pimelimidate Suberimidate Control Succinic anhydride, 50/~g m1-1 Succinic anhydride, 1,000/~g m1-1

Activity recovery 100% 96% 90% 88% 100% 99% 96%

37°C 8.0 3.7 3.2 2.0 8.4 9.3

-+ .01 -+ .01 -+ .01 -+ .01 _+ .02 -+ .07

4.8 -+ .15

20°C .90 .47 .36 .05 .60 .81

- .06 _+ .05 -+ .06 -+ .01 -+ .04 -+ .03

.18 + .02

.09 .05 .04 .002 .04 .06

4°C

Shelf-life at 4°C

-+ -+ 4-+ -+ -+

116 192 286 5,000 278 169

.01 .01 .01 .0001 .005 .004

.006 -+ .001

1,668

n.d., Not determined Predicted degradation rates and shelf-lives were calculated by the DEGTEST program The values displayed are means -+ standard error of the mean For full experimental details, see Materials and methods (The ALT used in the bis-imidates and succinic anhydride experiments came from different preparations, hence the differences in values for the controls)

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Enzyme Microb. Technol., 1991, vol. 13, March

Chemically stabilized alanine aminotransferase: C. O'F~gain

were: unmodified enzyme, 278 days; 50 ~g ml-~ anhydride, 169 days; and 1,000 ~g ml -I anhydride, 1,668 days. The 50 tzg ml-1 concentration has not stabilized ALT and may have marginally destabilized it.

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The chemical reactions studied (i.e. reaction with bisimidates and succinic anhydride) involve modification of free amino groups of the target protein. Bis-imidates react with protein amino groups with retention of the positive charge, 2s as depicted below, and bring about intra- or inter-chain cross-linking: + MeO-C=NH,

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Figure 2 37°C first-order decay curves for ALT treated with succinic anhydride. The ordinate shows catalytic activity expressed as a percentage of the activity at reference temperature of 4°C. Note that the ordinate figures must be multiplied by 100to obtain the true values, while abscissa values must be multiplied by 10. (rT) Control ALT; (11) succinic anhydride-treated ALT

Succinic anhydride treatment ALT was treated with succinic anhydride at final concentrations of 50 and 1,000/~g ml- J. Recoveries from this experiment are detailed in Tables 2 and 3. Succinic anhydride-modified ALT also follows a first-order decline in activity on accelerated storage at various temperatures, as depicted in Figure 2 and shown in Table 2. Figure 2 shows the decline in relative activity at 37°C with time for native ALT and for ALT treated with 1,000 tzg m1-1 succinic anhydride. The modified enzyme loses activity much more slowly than does the untreated control. It should be noted that only the higher concentration of succinic anhydride induced stabilization of the catalytic activity. The predicted shelf-lives for the native and modified enzymes at 4°C

+ MeOH

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CO~ It is clear that treatment of beef heart ALT with bisimidates can result in substantially stabilized catalytic activity. A "shelf-life" of up to 50-fold greater than that of the native enzyme was observed for ALT treated with dimethylsuberimidate. Dimethylpimelimidate stabilizes ALT catalytic activity to a much lesser extent than dimethylsuberimidate, while the additional stability due to dimethyladipimidate modification is only marginal. The degree of stabilization conferred by the bis-imidates used in this study seems to be related to the length of the bifunctional cross-linker, i.e. the greater the distance "bridged" by those bifunctional reagents employed here, the greater the stabilization factor achieved experimentally. It appears that succinic anhydride stabilization of ALT depends on there being a large excess of the reagent present. Since succinic anhydride is a functional group modifier and not a cross-linking reagent, it is not capable of forming oligomers or aggregates of enzyme protein. E n z y m e M i c r o b . T e c h n o l . , 1991, v o l . 13, M a r c h

237

Papers The activities of native A L T and both types of stabilized, modified A L T follow a first-order decay curve and are consistent with the occurrence of a single molecular event that results in loss of activity. The observed first-order process is not altered by either of the modifications. T w o features of this increased thermal stability deserve c o m m e n t . First, the two modifications that have successfully stabilized A L T have opposing effects on the charge of the reacting amino groups: bis-imidates retain the amino positive charge, while succinic anhydride reverses it. Second, these successful modifications act on the target protein in two different ways: the imidates by cross-linking the protein backbone, 28 and the anhydride by modifying labile surface groups .25 It is n o t e w o r t h y that of the three bis-imidates used in this study, only dimethylsuberimidate induced a significant increase in stability. This is the longest of the three, bridging a distance of 1.1 nm (11 angstroms). Since these bifunctional c o m p o u n d s cross-link protein c h a i n s ] 8 it appears that the " b r i d g i n g " distance of dimethylsuberimidate at 1.1 nm is the only one long enough to cross-link A L T (in either an intrachain or interchain fashion) in such a way that thermal stability increased. The stability e n h a n c e m e n t following dimethylsuberimidate treatment m a y be due to rigidification of the protein b a c k b o n e by cross-linking, preventing unfolding and denaturation of the e n z y m e molecule. 29 The amino groups linked in this way are not involved in the catalytic process. Dimethylsuberimidate treatment stabilizes A L T approximately 43-fold at 4°C according to the D E G T E S T analysis of experimental results (116 days for the native enzyme, 5,000 days for suberimidate-treated, as shown in T a b l e 2). Anhydride addition must result in the modification of labile amino groups (possibly lysines) whose loss would cause denaturation but whose charge is unimportant either for structural integrity or catalysis. Succinic anhydride has, by the same D E G T E S T analysis, stabilized A L T by a factor of six (278 days for native and 1,668 days for 1,000/xg ml i anhydridetreated enzyme). Succinic anhydride appears to be not as successful a stabilizing agent for A L T as dimethylsuberimidate when e m p l o y e d as described herein. Both of these stabilizing modifications have the advantage of being carried out in one-step, procedurally simple reactions. Accelerated storage testing is used as an index of the stability of a substance. It assumes that decline of the c o m p o n e n t of interest is first-order and then plots the logarithm of the first-order rate constant, k, against the reciprocal of the corresponding temperature in kelvins (Arrhenius equation) to predict the active lifetime at t e m p e r a t u r e s of interest. 26'27 While it involves transformations of experimental data and careful attention to experimental design, 26 a suitable c o m p u t e r program for analysis is available and can be used on a personal computer. 27 Accelerated storage can thus give reliable values for relative stabilities at different temperatures of c o m p o n e n t s under test, in this case native and chemically modified enzyme. Such a methodology would

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Enzyme Microb. Technol., 1991, vol. 13, March

appear to be eminently suitable for assessing the stability of an e n z y m e destined for refrigerated storage as a biological standard or diagnostic control. Other stability indices have been used in protein structure and stabilization studies, such as resistance to denaturing salts (or urea) 3° or resistance to thermal denaturation at a single, greatly elevated t e m p e r a t u r e . 31'32 Such determinations are of t r e m e n d o u s usefulness in calculations of protein conformational stability, as reviewed recently by Pace. 33'34 Unlike these methods, however, accelerated storage testing allows determination of the duration of functional activity at t e m p e r a t u r e s of interest. The present work points towards the possibility of engineering a more thermostable A L T e n z y m e . If the modified protein were to be sequenced, the crosslinked residues m a y be identified and the codons corresponding to their positions m a y be altered in a c D N A copy to cysteines or other residues with the potential for easy cross-linking.

Acknowledgements We are grateful to Baxter-Dade AG, Duedingen, Switzerland, and to Eolas, the Irish g o v e r n m e n t ' s science and technology agency, for financial support. We thank the U K National Institute for Biological Standards and Control for supplying K i r k w o o d and T y d e m a n ' s D E G T E S T program.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14

15 16 17

Schwartz, M. K. Methods Enzymol. 1971, 17B, 866-875 Thuma, R. S., Giegel, J. L. and Posner, A. H. in Laboratory Quality Assurance (Howanitz, P. J. and Howanitz, J. H., eds.) McGraw-Hill, New York, 1987, pp. 101-123 Mozhaev, V. V., Berezin, 1. V. and Martinek, K. CRC Crit. Rev, Biochem. 1988, 23, 235-281 O'F~gfiin,C., Sheehan, H., O'Kennedy, R. and Kilty, C. Process Biochem. 1988, 23, 166-171 Shami,E. Y., Rothstein, A. and Ramjeesingh, M. Trends Biotech. 1989, 7, 186-190 Klibanov, A. M. Adv. Appl. Microbiol. 1983, 29, 1-25 Scopes, R. K. in Protein Purification: Principles and Practice Springer, New York, 1982, pp. 194-200 Mosbach, K. (ed.) Immobilized Enzymes and Cells. Part B. Methods. Enzymol. 135 Academic Press, New York, 1987 Mozhaev, V. V. and Martinek, K. Enzyme Microb. Technol. 1984, 6, 50-59 Trubetskoy, V. S. and Torchilin, V. P. Int. J. Biochem. 1985, 17, 661-663 Duncan, R. J. S., Weston, P. D. and Wrigglesworth, R. A. Anal. Biochem. 1983, 132, 68-73 Dziember-Gryszkiewicz, E., Maksimenko, A. V., Torchilin, V. P. and Ostrowski, W. S. Biochem. lnternat. 1983, 6,627-633 Melik-Nubarov, N. S., Mozhaev, V. V., Siksnis, S. and Martinek, K. Biotech. Lett. 1987, 9, 725-730 Mozhaev, V. V., Siksnis, S., Melik-Nubarov, N. S., Galkantaite, N. Z., Denis, G. J., Butkus, E. P., Zaslavsky, B. Yu., Mestechkina, N. M. and Martinek, K. Eur. J. Biochem. 1988, 173, 147-154 Wigley,D. B., Clarke, A. R., Dunn, C. R., Barstow, D. A., Atkinson, T., Chia, W. N., Muirhead, H. and Holbrook, J. J. Biochim. Biophys. Acta 1987, 916, 145-148 Leatherbarrow, R. J. and Fersht, A. R. Protein Eng. 1986, 1, 7-16 Wells,J. A. and Estell, D. A. Trends Biochem. Sci. 1987, 13, 291-295

Chemically stabilized alanine aminotransferase: C. O'F~g~in et al. 18 19

20 21 22

Bergmeyer, H. U. and Horder, M. Clin. Chim. Acta 1980, 105, 147F-172F Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. and Klenk, D. C. Anal. Biochem. 1985, 150, 76-85 Jenkins, W. T. and Saier, M. Methods Enzymol. 1970, 17A, 159-163 Laemmli, U. K. Nature 1970, 227, 680-685 de Renobales, M. and Welch, W. J. Biol. Chem. 1980, 255, 10460-10463

23 24

Minotani, N., Sekiguchi, T., Bautista, J. G. and Yokohama, N. Biochim. Biophys. Acta 1979, 581, 334-341 Torchilin, V. P., Maksimenko, A. V., Smirnov, V. N., Berezin, I. V., Klibanov, A. M. and Martinek, K. Bioehim. Biophys. Acta 1979, 567, 1-11

25 26 27 28 29 30 31 32 33 34

Hollecker, M. and Creighton, T. E. Biochim. Biophys. Acta 1982, 701, 395-404 Kirkwood, T. B. L. J. Biol. Stand. 1984, 12, 215-224 Kirkwood, T. B. L. and Tydeman, M. S. J. Biol. Stand. 1984, 12, 207-214 Ji, T. H. Methods Enzymol. 1983, 91, 580-609 Torchilin, V. P. and Martinek, K. Enzyme Microb. Technol. 1979, 1, 74-82 Gianfreda, L., Marucci, G. and Greco, G. Biotechnol. Bioeng. 1986, 28, 1647-1652 Cho, I. C. and Swaisgood, H. Biochim. Biophys. Acta 1974, 334, 243-256 Gekko, K. J. Biochem. 1982, 91, 1197-1204 Pace, C. N. Trends Biotechnol. 1990, 8, 93-98 Pace, C. N. Trends Biochem. Sci. 1990, 15, 14-19

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