Chemical derivatives of Pseudomonas aeruginosa elastase showing increased stability Christine Besson,* Guy Favre-Bonvin,* Ciaran O’Fagain,+ and Jean Wallach*

*Laboratoire de Biochimie Analytique et de Synthtse Bioorganique, ICBMC, Universitt Claude Bernard-Lyon I, Villeurbanne, France; and Tichool of Biological Sciences, Dublin City University, Dublin, Republic of Ireland

Elastase from Pseudomonas aeruginosa has recently been used successfully for peptide synthesis. To improve its

performance we attempted to increase its catalytic stability by chemical modification. Two distinct sorts of amino

group-specific modifiers, dimethyl suberimidate and cyanuric chloride-activated polyethylene glycol (PEG).

gave a two-fold increase in catalytic stability at 70°C and greater degrees of stabilization at lower temperatures.

Suberimidate treatment seemed to act by intramolecular crosslinking, whereas the activated PEG gave rise to an

elastase-PEG conjugate. The thermal transition CT,,,) for suberimidate-treated elastase was unchanged from the native value of 72°C. PEG-conjugated elastase gave anomalous T,,, curves: therefore, a value could not be

determined. The lack of correspondence of catalytic stabilization with increased T,,, values is discussed.

Keywords: Enzyme stability; enzyme stabilization; chemical modification; Pseudomonas elastase; proteinase

Introduction

Pseudomonas aeruginosa is normally a harmless soil bac- terium, but it can act as an opportunistic pathogen of certain patients, notably those suffering from bums or cystic fibro- sis.’ In cystic fibrosis, the organism’s secreted elastase causes part of the pathogenic effect by breaking down the important elastin protein in lung connective tissue2 and var- ious other biologically active peptides and proteins. This elastase (E.C.3.4.21.11) has accordingly been well studied. It differs markedly from mammal elastases and is a zinc- containing endoproteinase with a molecular weight of 33,000 and a p1 of 5.9. Its optimum activity occurs at neu- tral pH. The active enzyme is secreted after proteolytic modification of the initial prepropeptide. Its activities on small synthetic peptides and on different elastins have been described elsewhere.3,4

The elastase structural gene has been isolated and

Address reprint requests to Dr. Wallach, Laboratoire de Biochimie Ana- lytique et de Synthkse Bioorganique, ICBMC, UniversitC Claude Bemard- Lyon I, Bltiment 303, 69622 Villeurbanne Cedex, France Received 1.5 December 1994.

cloned, leading to its full DNA and amino acid se- quences.5*6 Its three-dimensional X-ray structure is strik- ingly similar to that of the proteinase thermolysin from Ba- cillus thermoproteolyticus.7 Both proteins have a catalyti- cally essential zinc atom and an active-site histidine. Thermolysin, however, contains no cysteine, whereas the elastase has two disulphide bridges formed from its four cysteines.

Given appropriate conditions, proteinases can catalyze synthesis of peptide bonds as well as their hydrolysis. In this way, they can be used for peptide synthesis and semi- synthesis. A variety of proteinases has been used in this manner,8,9 including thermolysin. lo As opposed to thermol- ysin, P. aeruginosa elastase can cleave X-Tyr sequences, where X is any amino acid. ’ ’ This substrate specificity, together with its resemblance to thermolysin, makes the elastase an interesting candidate for enzymatic peptide syn- thesis. Successful synthesis of protected dipeptides using P. aeruginosa elastase has recently been described. i2 High concentrations of water-miscible organic solvents must be used in the reaction mixture, however, to shift the chemical equilibrium away from hydrolysis toward synthesis (as in other similar systemss.9). Use of these hydrophilic solvents often leads to loss of enzyme activity.13,14 Accordingly,

Enzyme and Microbial Technology 17:877-881, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0141-0229/95/$10.00 SSDI 0141-0229(94)00005-P

Papers

proteinases to be used in peptide synthesis may need to be stabilized against denaturing influences, notably solvents. One can achieve protein stabilization by various strate- gies, l5 including immobilization,‘“‘8 protein engineer-

ing, 1p-22 and chemical modification. 22-25 Chemical modi- fication in free solution (as opposed to immobilization) of- fers some significant advantages-experimental protocols often relatively simple-and results are obtained quickly. 22 Information gained may be used for further rounds of chem- ical modification. l5 Modification in solution is especially suitable in this case, because the synthetic procedures em- ployed to date for elastase involve precipitation of the dipeptide product, allowing easy separation of product from catalyst and reactants. l2 Here we describe two distinct types of chemical modification of the P. aeruginosa elastase that have led to increased functional stability: one is with a crosslinker, the other with a bulky hydrophilic conjugate. Each modifier was chosen on the basis of previous literature reports, and we discuss our results in these contexts as well as in the present one.

Materials and methods

We obtained purified Pseudomonas aeruginosa elastase (E.C.3.4.21.11) from Nagase Co. (Osaka, Japan) and used it at concentrations of approximately 0.55-l mg ml-’ (17-30 pM) in 0.1 M phosphate, pH 8. Chemicals used for modification were supplied by Sigma; Sephadex was from Pharmacia (Orsay. France).

The reaction protocol for the imidoesters, dimethyl suberimi- date (DMS) (homobifunctional crosslinker), and methyl acetimi- date (analogous monofunctional reagent) was loosely based on that of De Renobales and Welch. 26 Each was prepared as a stock solution in 0.1 M phosphate buffer, pH 8, and was quickly added to the elastase solution to 10% (vol vol - ‘) of total volume so as to achieve a 170-fold molar excess of modifying reagent over elastase concentration. We added 10% vol of phosphate buffer to an elastase control. The reaction proceeded for 1 h at room tem- perature. Imidoesters are reported to have very short solution half- lives,*’ but despite this, 10% (vol voll’) vol of 0.1 M Tris-HCl, pH 8, was then added to all fractions to ensure that the reaction was terminated by an excess of amino groups.

The conjugation reaction with polyethylene glycol (PEG) was loosely based on Abuchowski et al. 28 Cyanuric chloride-activated polyethylene glycol 5,000 (PEG 5,000) and nonactivated PEG were each added as an accurately weighed portion of the solid chemical to 0.5 ml of elastase in phosphate buffer. Incubation was at room temperature for 1 h followed by addition of 10% (vol vol-‘) vol of Tris buffer, as before. Next, unreacted PEG was separated from elastase by centrifugal gel filtration.29 Centrifuga- tion occurred for 30 s at 1,000 ‘pm in a bench centrifuge through a column of Sephadex G2.5 (5ml bed vol) in a 5-ml polypropylene syringe barrel. Each Sephadex minicolumn had been previously equilibrated in Tris buffer and centrifuged to remove excess inter- stitial fluid.

The elastase catalytic activity was measured by the conducti- metric method developed and refined in Lyon, as described in detail elsewhere.3,30.31 Typically, 4 ml of reaction mixture in 5 mu Tris-HCl, pH 8.6, was added to a temperature-controlled (30°C) conductimetric cell (type MCCD; Solea Tacussel, Villeur- banne, France), and conductance was measured over 15 to 20 min on a conductimeter linked to a chart recorder. The probes for conductance measurements were two platinized platinum disks, sealed into glass tubes placed opposite each other on the cell wall.

The substrate for hydrolytic activity measurements was Z-Ala,- Phe-Ala-NH,. The final concentration of elastase in each assay mixture was in the range l-2 IIM. Catalytic stability was estimated by placing elastase derivatives in a water bath at different temper- atures (40, 50, 60. and 70°C) and withdrawing samples at various time intervals into ice. The rate of catalysis of each sample was then determined. Activities were expressed as a percentage of the initial time zero activity. Comparisons between experimental mea- surements were made with the Student t test.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli.32 Each gel included a range of molecular weight standards. Thermal transi- tion (T,,,) melting curves were determined at 28.5 nm on a Gilford 260 spectrophotometer equipped with thermoprogrammer 2527 (set range 25-80°C) and automatic cell changer 245lA.j’ Mi- crocuvettes used had a l-cm light path and 0.3-m] capacity. So- lutions were degassed before use to prevent air bubble formation. T,,, values were calculated by tangential methods from the exper- imental curves.

Results

Recoveries of initial elastase activity following dimethyl suberimidate treatment ranged at 68 to 91%. Recoveries following methyl acetimidate treatment were similar. No activity loss occurred in the case of elastase mixed with nonactivated PEG (because this fraction is in all respects identical with native elastase, it will not be considered fur- ther). Loss of initial activity was greater after reaction with activated PEG. Recoveries ranged at 53 to 67% (it is pos- sible that the bulky PEG molecule attached to the enzyme may sterically hinder access of the peptide substrate to the elastase active site, giving an apparently lower recovery). Each imidate derivative yielded a single sharp band of ap- parent molecular weight 33,000, identical to the native elastase, on SDS-PAGE. In contrast, activated PEG deriv- atives migrated as a diffuse mass rather than as a single band of altered molecular weight. A comparison of loss of elastase activity at 70°C for native, imidoester-treated and PEG-conjugated elastases, after 10 or 30 min of incubation, clearly indicates a significant stabilization of the enzyme by these chemical modifications (Table 1). Furthermore, the catalytic activity of the modified elastases at 70°C has twice that of the native.

Deactivation was also measured for DMS-treated en- zyme at four different temperatures (40, 50, 60, and 70°C) and compared with the native enzyme (Figure I). Loss of elastase activity against time fit well to first-order exponential equations. (Note that data at 70°C could also be fit to a straight-line function.) Statistical analysis of the data indicated that stabilization occurred at any temperature (P < .Ol).

The native elastase had a single sharp T, at 72°C. This was consistent with a simple two-step mechanism of un- folding. The same value occurred for all of the derivatives prepared except the activated PEG conjugate, for which T, could not be measured. Both imidoester derivatives some- times showed small perturbations in the region 53-57°C. These were generally of small amplitude, so that accurate measurement was difficult. No such perturbations were ob- served for the native elastase (Figure 2).

878 Enzyme Microb. Technol., 1995, vol. 17, October

Chemical derivatives of P. aeruginosa elastose: C. Bessor et al.

Table 1 Residual activity at 70°C of native elastase, imidoester-treated elastase, and polyethylene glycol-conjugated elastase after 10 and 30 min incubation

Incubation DMS-modified PEG-conjugated

time (min) Native enzyme (%) enzyme f%) enzyme (%)

0 100 100 100

10 (65.3 s 2.0) (80.9 k 2.3)b (85.5 2 14.8)NS

30 (32.9 t 3.2) (53.4 + 1O)C (57.7 + 0.2)b

aExperimental conditions are described in Materials and methods. NS, Nonsignificant bP < .05 CP < .Ol

Acetamidate-modified enzyme (%)

100 (78.3 + 3.7)= (51.2 2 0.2)b

Discussion

The results in Table 1 show that chemical modification in solution is a valid and significant means of stabilizing P. aeruginosa elastase. Reaction under mild conditions of pH and temperature gave acceptable recoveries of starting en- zyme activity (>53%) and a reproducible two-fold increase in the apparent half-life of catalytic activity at 70°C. Greater degrees of stabilization occurred at lower temperatures.

Migration of the various elastase forms on SDS-PAGE showed that modification with DMS under these conditions did not lead to intermolecular crosslinking, because no in- creases in molecular weight occurred. As a monofunctional, methyl acetimidate cannot crosslink the protein or cause oligomerization. Elastase conjugated with activated PEG, however, migrated abnormally and very diffusely on SDS- PAGE. Each PEG has a molecular weight of 5,000 so that even one PEG conjugated to elastase will greatly alter the enzyme’s mobility on SDS-PAGE. PEG itself has a hydro- philic, polyhydroxylated, expanded structure that may not

0 40 80 120 Temperature

40°C

50°C

60°C

0 30 60

Figure 1 Deactivation of native and dimethyl suberimidate (DMSI-treated elastase at four different temperatures (40,50,60, and 70°C): W native elastase, 0 DMS-treated elastase. The ex- perimental conditions are described in Materials and methods

migrate “normally” on SDS-PAGE. This could explain the strange behavior observed. Alternatively, we may have pro- duced a population of elastases modified to different ex- tents, with different integral numbers of PEG molecules attached to the amino groups of elastase. Such a population, however, would be less likely to follow a single exponential loss of catalytic activity at high temperatures. The type of activated PEG used here could react with only one amino group; 28 therefore, elastase oligomerization or aggregation could not have occurred.

Native, suberimidate-modified, acetimidate-modified, and PEG-conjugated elastases each lose activity at 70°C according to a first-order exponential equation. Such a first- order process is consistent with the occurrence of a single molecular event (of whatever sort) leading to a catalytically inactive molecule. The modifications must postpone, limit, or prevent the occurrence of this process. T,,,, the midpoint temperature for denaturation unfolding, is generally re- garded as a robust and reliable index of protein stability. It may be directly measured by experiment and values com- pared, regardless of the underlying molecular process.34 If one can show independently that the denaturation of interest is two-state (i.e., that only folded and unfolded forms of the protein occur at equilibrium, with negligible amounts of intermediate forms) and reversible, T, values may be used to estimate parameters such as Gibbs energy changes (AG), and hence thermodynamic stability.35,‘”

Absorbance change I 0.02 I

Figure 2 Scheme of thermal denaturation of elastase and de- rivatives. (Left to right) Native elastase, dimethyl suberimidate- modified elastase and methyl acetimidate-modified elastase

Enzyme Microb. Technol., 1995, vol. 17, October 879

Papers

The modified elastases showed increased catalytic sta- bility at 70°C 6O”C, and 50°C but did not have increased T,,, values. At first this seems strange, but the contradiction is only on the surface. There are many indexes of protein stability.15 Some of these describe the molecular integrity of the protein under study. Here, our concern was mainly with the function of the elastase as a catalyst and less with its structural or folding integrity. We were more interested in the protein’s capabilities than in its intrinsic properties. For this reason we chose an appropriate stability index; namely, the persistence of catalytic function under stress conditions. It is considered to be an indication of long-term protein stability. 37 T in contrast relates to the sharp, usu- ally cooperative unf:lding of a protein’s tertiary structure that occurs at high temperature. 19.34

One can easily imagine that a relatively minor unfolding of an enzyme molecule not biophysically apparent (by spec- troscopy or calorimetry) may cause enough disruption of the active site to result in loss of activity. Such an idea can explain our findings. A minor molecular event occurring at temperatures below T,,,, resulting in a loss of elastase ac- tivity, is inhibited or delayed by chemical modification, but the point where major cooperative unfolding occurs has not changed. The anomalous thermal curves given by PEG- conjugated elastase from which no T,,, value could be de- termined may be due to ultraviolet absorption by the triazine ring of cyanuric chloride.

Many of the 20 amino acids found in proteins can take part in chemical reactions. Amino and thiol groups are es- pecially reactive. 24 Many types of protein modification re- agents, of varying degrees of side-chain specificity, are available. 38,39 Pseudomonas aeruginosa elastase has a total of 11 free amino groups (10 lysines plus the alanine N-ter- minus) but lacks free cysteines;5*6 therefore, amino-specific reagents may prove suitable. Imidoesters are not prone to side reactions with groups other than amino groups and cause little inactivation of the target protein2’ DMS is a bifunctional imidoester (or bis-imidate) that has been suc- cessfully used as an intramolecular crosslinker for protein stabilization.4w2 Methyl acetimidate is a monofunctional analogue with identical reaction chemistry. One may com- pare its stabilization effects (if any) on elastase with those of suberimidate. In this way, one may distinguish between stabilization due to genuine crosslinking and that brought about simply by amino group amidation.24 Enzyme-PEG conjugates are formed by reaction of PEG-linked cyanuric chloride with protein amino groups.28.43 Proteins modified in this way often show increased thermostability (probably due to steric hindrance of protein unfolding& or perhaps to perturbation of the bulk water structure in the region close to PEG45) and can dissolve more easily in organic sol- vents.43+’ The method has been applied to thermolysin spe- cifically for its use in peptide synthesis.43 This suggests that PEG conjugation may be useful for elastase also. We used phosphate buffer pH 8.0 in place of the usual tetraborate buffer pH 9.2, 28*43 because borate interferes with elastase assays. We also wished to use PEG under exactly the same conditions as for the imidodoesters. Both imidoesters and activated PEG were used at roughly a 200-fold molar excess with respect to concentration of the target elastase. This was to ensure that each reaction mixture contained an excess of

modifier molecules with respect to the 1 I free amino groups occurring on each elastase molecule.5,6

The degree of catalytic stabilization achieved (about two-fold at 70°C) is relatively small. Stabilization factors in the range of lOO- to 1 ,OOO-fold have been reported for sur- face-hydrophilized derivatives of o-chymotrypsin and trypsin at 60°C) 46 but the chemical stabilization of these two enzymes had already been diligently studied for a long time. Despite this, our results show that P. aeruginosa elastase can be stabilized chemically for possible biotechnological applications and is promising for the future. Possibly. dif- ferent reaction conditions and/or different modifiers (bis- imidates of different chain length4-“) may result in further stabilized derivatives. Even for a protein as well understood as subtilisin BPN’, protein engineering of increasingly sta- bilized mutants has taken much effort (see O’Fagain and O’Kennedyi5 and compare other references2’.47,48). The usefulness of our elastase derivatives in peptide synthesis is now being explored, especially with respect to their kinetics of reaction (chemical modification may change a protein’s catalytic characteristics as well as increase its stability4”). Elastase may yet become an important tool for the peptide chemist.

Acknowledgments

C.O.F. thanks the following for travel and/or maintenance awards: Eolas/CNRS (Ireland-France Bilateral Scientific Exchange Scheme), the Embassy of France in Ireland, and Dublin City University.

References

1

2

3

4

5

6

Wretlind. B. and Pavlovskis, 0. R. Pseudomonas aeruginosa elastase and its role in Pseudomonas infections. Rev. Infecf. Dis. 1983, 5, S998-S1003 Pavlovskis, 0. R. and Wretlind, B. in Medical Microbiology, vol. 1 (Easman, C. S. F. and Jeljaszewicz, E., eds.). Academic Press, London, 1982. 97-128 Saulnier, J., Curtil. F. M., Duclos, M.-C. and Wallach, J. M. Elas- tolytic activity of Pseudomonas aeruginosa elastase. Biochim. Bio- phvs. Acta 1989. 995. 285-290 Saulnier. 1.. Rayssiguie A. M. and Wallach, J. M. Mecanisme d’action des proteinases de Pseudomonas aeruginosa. Path. Biol. 1990. 38, 968-974 Bever, R. A. and Iglewski, B. H. Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase struc- tural gene. J. Bacterial. 1988, 170, 4309-4314 Fukushima. J., Yamamoto, S.. Morihara, K., Atsumi, Y., Taken- chi. H., Kamamoto S. and Okuda. K. Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IF0 3455 elastase. J. Bacreriol. 1988, 171, 1698-1704 Thayer, M. M., Flaherty. K. M. and McKay, D. B. Three- dimensional structure of the elastase of Pseudomonas aeruginosa at I,5 A resolution. J. Biol. Chem. 1991, 266. 2864-2811 Morihara. K. Using proteases in peptide synthesis. Trends Biorech- nol. 1987. 5, 164-170 Jakubke, H. D. Enzymatic peptide synthesis. In The Peprides: Anal- ysis, Synthesis. Biology, vol. 9 (Udenfriend, S. and Meienhorf, J., eds.). Academic Press, New York, 1987, 103-165 Veera Reddy. A. Thermolysin: A peptide forming enzyme. Indian J. Biochem. Biophys. 1991, 28, lC-15 Saulnier, J., Rayssiguie, A. M., Duclos, M.-C. and Wallach, J. M. Hydrolysis of alanyl-containing tetrapeptides by Pseudomonas aeruginosa proteinases. Biochem. Sot. Trans. 1990, 18, 900-901

880 Enzyme Microb. Technol., 1995, vol. 17, October

Chemical derivatives of P. aeruginosa elastose: C. Bessor et al.

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

Pauchon, V., Besson, C., Saulnier, J. and Wallach. J. M. Peptide synthesis catalyzed by Pseudomonas aeruginosa elastase. Biorech-

nol. Appl. Biochem. 1993, 17, 217-221 Got-man, L.-A. and Dordick, J. S. Organic solvents strip water off enzymes. Biorechnol. Bioeng. 1992, 39, 392-397

Mozhaev. V. V., Khmelnitsky, Y. L., Sergeeva, M. V., Belova, A. B., Klyachko, N. L., Levashov, A. V. and Martinek, K. Cata- lytic activity and denaturation of enzymes in water/organic cosolvent mixtures. Eur. J. Biochem. 1989, 184, 597-602

O’Fagain, C. and O’Kennedy, R. Functionally stabilized proteins: A review. Biofechnol. Ad%,. 1991, 9, 351409 Mosbach, K. Immobilized enzymes and cells: Parts B, C. Methods Enzymol. ~01s. 135 and 136 (Mosbach, K.. ed.). Academic Press, New York. 1987. Martinek. K. and Mozhaev. V. V. Immobilization of enzymes, an approach to fundamental studies in biochemistry. In: Advances in Enzymology, vol. 57 (Meister. A.. ed.). John Wiley & Sons. New York. 1985, 179-249 Klibanov. A. M. Enzyme stabilization: A review. Anal. Biochem. 1979. 93. l-25 Nosoh. Y. and Sekiguchi. T. Protein Stabilify and Stabilization

Through Protein Engineering. Ellis-Horwood. Chichester. UK. 1991 Nosoh. Y. and Sekiguchi. T. Protein engineering for thermostabil- ity. Trends Biotechnol. 1990. 8, 16-20 Wells. J. A. and Estell. D. A. Subtilisin: An enzyme designed to be engineered. Trends Biochem. Sci. 1987, 13, 291-297 Profy. A.-T. and Schimmel. P. Complementary use of chemical modification and site-directed mutagenesis to probe structure- activity relationships in enzymes. Prog. Nucl. Acids Res. Mol. Biol. 1988.35. l-26 Mozhaev, V. V.. Martinek, K. and Berezin. 1. V. Structure- stability relationships in proteins: Fundamental tasks and strategy for the development of stabilized enzyme catalysts for biotechnology. CRC Crit. Rev. Biochem. 1988. 23, 235-281

Wong, S. S. and Wong, L.-J. C. Chemical crosslinking and the stabilization of proteins and enzymes. En:yme Microb. Technol. 1992. 14, 866874 Mozhaev, V. V.. Melik-Nubarov, N. S., Siksnis. V. and Martinek, K. Strategy for stabilizing enzymes. Part two: Increasing enzyme stability by selective chemical modification. Biocaralysis 1990. 3, 189-196 De Renobales. M. and Welch, W. Chemical crosslinking stabilizes the enzymatic activity and quatemary structure of formyltetrahydro- folate synthetase. .I. Biol. Chem. 1990, 255, 10460-10463 Ji. T. H. Bifunctional reagents. Mezhods Enz!mol. 1983. 91, 581S 609

Abuchowski. A.. Van Es. T.. Palczuk, N. C. and Davis, F. F. Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. J. Biol. Chem. 1977. 252, 3578-3581 Helmerhorst. H. and Stokes, J. E. Microcentrifuge desalting: A rapid. quantitative method for desalting small amounts of proteins. Anal. Biochem. 1980, 104. 13&135 Manika-Saizonou, B., Bostancioglu, K., Mealet, C., Favre-Bonvin. G. and Wallach, J. M. Comparison of different assay methods for determination of elastase activity. Anal. Left. 1985. 18, 901-913

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

Ballot, C., Manika-Saizonou, B., Mealet. C.. Favre-Bonvin. G. and Wallach, J. M. Conductimetric measurements of enzyme activities. Anal. Chim. Acra 1984. 163, 305-308

Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970. 277, 68t%685

Favre-Bonvin, G.. Bostancioglu. K. and Wallach, J. M. Ca2 + and Mg2+ protection against thermal denaturation of pancreatic elastase. Biochem. Inr. 1986, 13, 983-989

Tombs, M. P. Stability of enzymes. J. Appi. B&hem. 1985, 7, 3-24

Pace. C. N. Measuring and increasing protein stability. Trends Bio-

rechuol. 1990, 8, 83-98

Baldwin, R. L. and Eisenberg, D. Protein stability. In: Protein En-

gineering (Oxender, D. L. and Fox. C. F., eds). Alan R. Liss. New York, 1987. 127-148

Mozhaev. V. V. Mechanism-based strategies for protein thermosta- bilization. Trends Biotechnol. 1993. 11, 88-95

Lundblad, R. L. and Noyes. C. M. Chemical Reagenls for Protein Modification. CRC Press. Boca Raton. FL. 1984

Means, G. E.. and Feeney. R. E. Chemical modification of pro- teins: History and applications. Bioconj. Chem. 1990. 1, 2-12

Sheehan. H.. O’Kennedy. R. and Kilty. C. lnvestigation of the properties of bovine heart creatine kinase crosslinked with dimeth- ylsuberimidate. Biochim. Bioph.vs. Acra 1990. 1041, 141-145

OFtagain. C., O’Kennedy, R. and Kilty. C. Stability of alanine aminotransferase is enhanced by chemical modification. En-yrne Mi-

rrob. Technol. 199 1. 13. 236239

Shaked. 2. and Wolfe, S. Stabilization of pyranose 2-oxidase and catalase by chemical modification. Methods Encvmnl. 1988. 137. 599-615

Ferjancic, A.. Puigserver. A. and Gaertner, H. Unusual specificity of polyethylene glycol-modified thermolysin in peptide synthesis catalyzed in organic solvents. Biotechnol. Let?. 1988. 10. 101-106

Inada. Y. Takahashi. K.. Yashimoto, T.. Ajima, A., Matsushima. A and Saito, Y. Application of polyethylene glycol-modified en- zymes in biotechnological processes: Organic solvent-soluble en- zymes. Trends Biotechnol. 1986. 4, 190-194

Arakawa. T. and Timasheff, S. N. Mechanism of polyethylene gly- col interaction with proteins. Biochemistrv 1985. 24, 6756-6762

Mozhaev. V. V., Siksnia. V. A., Melik-Nubarov. N. S.. Galkan- mite. N. 2.. Denis. G. J., Butkus, E. P.. Zaslavsky. B., Yu. Mestechkina. N. M. and Martinek. K. Protein stabilization via hy- drophilization. Covalent modification of trypsin and a-chymotryp- sin. Eur. J. Biochem. 1988, 173. 147-154

Pantoliano. M. W., Ladner, R. C.. Bryan. P. N.. Rollence, M. L.. Wood, J. F. and Poulos. T. L. Protein engineering of subtilisin BPN’: Enhanced stabilization through the introduction of two cys- teina to form disulfite bond. Bioc~hc,mi.~rr> 1987. 26, 2077-2082

Ziyang Z.. Liu. J. L.-C., Dinterman. L. hl.. Finkelman, M. A. G.. Mueller, W.-T., Rollence. M. L.. Whitlow, M. C.. Chi-Huey W. Engineering subtilisin for reaction in dimethylformamide. J. Amer. Chem. SK. 1991. 113, 683-684

Tsuji. R. F. Enhanced enzymatic activity and stability of trypsin by reductive alkylation in solid phase. Biorechnol. Bioeng. 1990, 36, 1001--1005

Enzyme Microb. Technol., 1995, vol. 17, October 881