[ 5 5 ] E N Z Y M E S T A B I L I Z A T I O N BY C R O S S - L I N K I N G 6 1 5

Thermodenaturation of Native, Amidinated, and Cross-Linked P-2-O

Native and cross-linked enzymes exhibit the same thermodenatura- tion kinetics (Fig. 11). The amidinated P-2-O preparations, however, show considerably greater thermostability. Apparently, amidination pro- vides more opportunities for hydrogen bondings and hydrophobic interac- tions which may enhance the thermostability of the enzyme. The chemi- cal modification of P-2-O with higher levels of amidination results in enzyme preparations that are I0 times more thermostable than native P-2-O. There are two effects on modification: (1) there is an elimination of the fast inactivation pattern of normal proteolyzed P-2-O; and (2) there is a 5-fold deceleration of the principal cause of thermal inactivation.

Acknowledgment

We thank our colleagues Mark Pemberton and Mike Kunitani for supplying us with purified P-2-O and glucosone, and S. Daniell for carrying out the cross-linked catalase immobilization.

[55] S tab i l i za t ion o f E n z y m e s b y I n t r a m o l e c u l a r

C r o s s - L i n k i n g Us ing B i func t iona l R e a g e n t s

By KAREL MARTINEK and V. P. TORCHILIN

The problem of enzyme stabilization has received considerable atten- tion in recent years. 1-7 Enzyme immobilization has been used most fre- quently to solve the problem of enzyme stabilization. However, other methods have been suggested as well. 3 For example, enzyme stabilization has been achieved after (1) addition of low molecular weight compounds to enzymes free in solution, (2) chemical modification of enzymes by substitution with low molecular weight compounds, and (3) use of bifunc- tional reagents to produce enzymes containing artificial intramolecular cross-links. These methods are desirable in particular when the presence

t K. Martinek, A. M. Klibanov, and I. V. Berezin, J. Solid-Phase Biochem. 2, 343 (1977). 2 A. M. Klibanov, Anal. Biochem. 92, 1 (1979). 3 V. P. Torchilin and K. Martinek, Enzyme Microb. Technol. 1, 74 (1979). 4 R. D. Schmid, Adv. Biochem. Eng. 12, 41 (1979). 5 K. Martinek and V. V. Mozhaev, Enzyme Eng.-Future Directions, p. 3 (1980). 6 A. M. Klibanov, Biochem. Soc. Trans. 11, 19 (1983); Science 219, 722 (1983). 7 V. V. Mozhaev and K. Martinek, Enzyme Microb. Technol. 6, 49 (1984).

Copyright © 1988 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 137 All rights of reproduction in any form reserved,

616 TECHNIQUES AND ASPECTS OF ENZYMES AND CELLS [55]

of a support may decrease both the binding capacity and the reactivity of the enzyme. Also, in medical therapy applied enzyme must in many cases interact with receptors or other components of cellular membranes. In this instance, a support may change the key pathways dramatically.

In this chapter, stabilization of enzymes through intramolecular cross- linking will be discussed in detail. The principles of intramolecular cross- linking are shown schematically in Fig. 1. This approach is based on diminishing the polypeptide entropy which is the principal thermody-

!

I L

@ ...... @ - - ®

2

VERY SLOW

2

SLOW I t ,

3

5

1

3

A

FIG. 1. General scheme of enzyme stabilization effected by intramolecular cross-linking. (A) 1, Native oligomeric enzyme; 2, reversibly dissociated subunits; 3, irreversibly dena- turated subunits; 4, cross-linked enzyme; 5, irreversibly denaturated cross-linked enzyme. (B) 1, Native monomeric enzyme; 2, denaturated enzyme; 3, cross-linked enzyme.

[55] ENZYME STABILIZATION BY CROSS-LINKING 617

namic quantity stabilizing the denatured form. 8 In 1967 Hartman and W o l d 9 introduced the use of bifunctional reagents in protein chemistry. Then, Husain and Lowe l° used protein cross-linking with a bifunctional reagent as a means to study the tertiary structure of an enzyme molecule consisting of a single polypeptide chain. In addition, this technique has been applied to exploring the quaternary structure of oligomeric enzymes by Davies and Stark.l~ Since the publication of these pioneering works cross-linking of proteins has become a widely used technique.~2-21 Proce- dures have been developed for attachment of DNA and RNA molecules to proteins 22 with the aid of cross-linking methodology. Immunoanalysis and radioactive labeling have been used for identifying proteins in cross- linked protein complexes, 23 and, recently, the use of the cross-linking approach in fundamental studies in biochemistry has been reviewed. 24

Cross-Linking Reagents

There are now many bifunctional compounds available for cross-link- ing of proteins. 12-2~ Examples of such reagents are dialdehydes, diimido esters, diisocyanates, and bisdiazonium salts. Moreover, diamines such as H2N(CH2)nNH2 may be used for cross-linking of protein carboxyl groups, if the latter have been preactivated by treatment with carbodi- imide. 25 Likewise, diacids such as HOOC(CH2)nCOOH (after their preac- tivation with carbodiimide) could be used for cross-linking of protein

8 p. j . Flory, J. Am. Chem. Soc. 78, 5222 (1956). 9 F. C. Hartman and F. Wold, Biochemistry 6, 2439 (1967).

10 S. S. Husain and G. Lowe, Biochem. J. 103, 855 (1968). ii G. E. Davies and G. R. Stark, Proc. Natl. Acad. Sei. U.S.A. 66, 651 (1970). lz H. Fasold, J. Klappenberger, and H. Remold, Angew. Chem., Int. Ed. Engl. 10, 795

(1971). ~3 F. Wold, this series, Vol. 25, p. 623. 14 O. R. Zaborsky, Enzyme Eng. 1, 211 (1972). ~5 R. E. Peeney, G. Blankenborn, and H. B. F. Dixon, Adv. Protein Chem. 29, 135 (1975). 16 R. Uy and F. Wold, in "Biomedical Applications of Immobilized Enzymes and Proteins"

(T. M. C. Chang, ed.), p. 15. Plenum, New York, 1976. 17 K. Peters and F. M. Richards, Annu. Rev. Biochem. 46, 523 (1977). 18 R. B. Freedman, Trends Biochem. Sci. (Pers. Ed.) 4, 193 (1979), 19 M. Das and F. Fox, Annu. Rev. Biophys. Bioeng. 8, 165 (1979). 10 T. H. Ji, this series, Vol. 91, p. 580. 21 K.-K. Han, C. Richard, and A. Delacourte, Int. J. Biochem. 16, 129 (1984). 22 K. C. Smith, in "Aging, Carcinogenesis and Radiation Biology," p. 67. Plenum, London,

1976. 23 S. K. Sinha and K. Brew, J. Biol. Chem. 256, 4193 (1981). 24 K. Martinek and V. V. Mozhaev, Adv. Enzymol. 57, 179 (1985). z5 V. P. Torchilin, A. V. Maksimenko, A. M. Klibanov, I. V. Berezin, and K. Martinek,

Biochim. Biophys. Acta 522, 277 (1978).

618 TECHNIQUES AND ASPECTS OF ENZYMES AND CELLS [55]

amino groups. 26 Both diamines and diacids are commercially available and relatively inexpensive, factors that are of prime importance in bio- technology. In addition, application of heterobifunctional cross-linking reagents 13,2°,2j offers the possibility of increasing the number of cross- links by reacting with different functional groups of the protein to be modified.

Photochemical activation provides another possibility in the use of cross-linking reagents 27 (for reviews, see Refs. 20, 21, and 28). Since cross-linking requires reaction with at least two functional groups, proba- bly differing in chemical reactivity and/or spatial location, better control over the cross-linking reaction might be obtained in a stepwise cross- linking approach. This is possible if the reagent contains both a chemi- cally reactive group and a light-activatable (photochemical) group (or two photochemical groups showing no overlap in their photoactivation spectra). 29

Cleavable cross-linking reagents useful in some situations contain in the molecule a chemical bond that can be split readily under mild condi- tions (e.g., under mild oxidation or reduction conditions)3°; for reviews, see Refs. 17-21. Also, water-insoluble (hydrophobic) cross-linking re- agents have been used to modify membrane proteins. 17,~8

Reactions of Cross-Linking Reagents with Proteins

The reaction of a bifunctional reagent with an enzyme can in principle yield three different types of products: (1) a one-point modified enzyme, (2) an intramolecular cross-linked enzyme, and (3) an intermolecular cross-linked enzyme (see Fig. 2). The yields of one-point modification and intramolecular cross-linked products will depend on the length of the bifunctional reagent used and the distance between the functional groups on the protein to be modified. To increase the number of intramolecular cross-links in a protein molecule (and hence to decrease the degree of one-point modification) one can: (1) choose an optimal length of the cross- linking molecule25.26; (2) premodify the protein by substituting the protein surface with additional reactive groups25'31; (3) exploit the potentially re-

26 V. P. Torchilin, V. S. Trubetskoy, and K. Martinek, J. Mol. Catal. 19, 291 (1983). 27 j. R. Knowles, Acc. Chem. Res. 5, 155 (1972). 28 p. Guire, this series, Vol. 44, p. 280. 29 p. Guire, in "Enzyme Technology and Renewable Resources," p. 55. Univ. of Virginia,

Charlottesville, Virginia, 1976. 30 R. R. Taraut, A. Bollen, T. Sun, J. W. B. Hershey, J. Sundberg, and L. R. Pierce,

Biochemistry 12, 3266 (1973). 31 V. P. Torchilin, A. V. Maksimenko, V. N. Smirnov, I. V. Berezin, and K. Martinek,

Biochim. Biophys. Acta 568, 1 (1979).

[55] ENZYME STABILIZATION" BY CROSS-LINKING 619

Y Y - - X \ / ) ~ - ~ ONE-POINT MODIFICATION

/ ~ " X - Y Y

ENZYME t

"E t ~"a~ + v - - ~ --- x - ~ k ~ # t , j - x - v - - - v - x - ~ - x ~'~J-J,~'X BIFUNCTIONAL ~ ~P" ~.~ J

ACTIVESI.E O . O . - ' , . K I N G

CROSS-LINKING

FIG. 2. Possible reactions of bifunctional reagents with enzymes.

versible character of chemical cross-linking by applying a mixture of bi- functional reagents of different chain lengths. 31 This means that in the course of the reaction the protein molecule itself wil l"select" intramolec- ular cross-linking in preference to one-point modification.

Furthermore, the probability of intermolecular cross-linking may be reduced by decreasing the enzyme concentration in the reaction medium. Alternatively, to suppress intermolecular cross-linking the protein could be attached to a solid support through a cleavable spacer arm, prior to cross-linking. After cross-linking, the spacer containing, for example, a disulfide linkage, is cleaved by reducing the S-S bond with thiol re- agents. 32,33 In addition, a light-initiated heterobifunctional reagent can be used for cross-linking, resulting in no intermolecular side reactions. 27 In this case, first the bifunctional reagent reacts chemically at one end of the molecule, and then, after illumination of the premodified protein, it reacts photochemically at the other end of the cross-linking reagent (containing a diazo or an azide group). On illumination, a highly reactive carbene or nitrene is produced, reacting with the closest C - H linkage of the protein.

Thermostabilization of ot-Chymotrypsin by Intramolecular Cross-Linking

Succinylation of a-Chymotrypsin, a-Chymotrypsin (EC 3.4.21.1) is succinylated according to the method of Goldstein. 34 a-Chymotrypsin

32 G. P. Royer, S. Ikeda, and K. Aso, FEBS Lett. 80, 89 (1977). 33 S. Pillai and B. K. Bachhawat, J. Mol. Biol. 131, 877 (1979). 34 L. Goldstein, Biochemistry 11, 4072 (1972).

620 TECHNIQUES AND ASPECTS OF ENZYMES AND CELLS [55]

(900 mg) is dissolved in 30 ml of 0.2 M phosphate buffer, pH 7.7. Succinic anhydride (300 mg) is then added in small portions while keeping the enzyme solution in the cold (4 °) and maintaining the pH at 7.7. Under these conditions over 80% of available amino groups (14-15) of the en- zyme are succinylated. 25 The reaction mixture is then passed through a column (2.6 × 60 cm) packed with Sephadex G-50 (Pharmacia). (The column is preequilibrated with 10 mM KCI.) The elution rate is 1.5 ml/ min. The succinylated o~-chymotrypsin preparation shows both catalytic activity and thermostability that are comparable to the same properties of the native enzyme. 25

Carbodffmide Activation of Carboxyl Groups of ~-Chymotrypsin. ~-Chymotrypsin is treated with carbodiimide by a slightly modified ver- sion of the method described in Ref. 25. A solution (63 ml) containing ~-chymotrypsin (10 -6 M native or succinylated enzyme) is added to 7 ml of an aqueous solution containing 1-ethyl-3-(3-dimethylaminopropyl)car- bodiimide (EDC) (10 -2 M), and the mixture is left at a constant pH of 4.5 (using a pH-stat) for 1 hr at 20 °. Under these conditions 15 out of 17 exposed carboxyl groups of o~-chymotrypsin are modified. On treatment of a-chymotrypsin with carbodiimide, the relative catalytic activity of the enzyme drops 3-fold.

Reaction of Carbodiimide-Activated ~-Chymotrypsin with Diamines. The solution (10 ml) containing o~-chymotrypsin or succinylated o~-chymo- trypsin preactivated by carbodiimide treatment and 20 mM phosphate buffer (4 ml), pH 8.2, is added to a solution (1 ml) containing the amine reagent. The following amine concentrations are used: 10 mM hexa- methylenediamine and dodecamethylenediamine; 0.1 M ethylenediamine, tetramethylenediamine, and pentamethylenediamine; 10 v/v% of hydra- zine (or 1-amino-propan-3-ol). The reaction is carried out at pH 8.2 for 1 hr at 20 °.

Thermoinactivation. To a solution (10 ml) containing cross-linked ot-chymotrypsin (10 -6 M), 20 mM phosphate buffer (5 ml), pH 7.0, is added, and the mixture is left at 50 °. Aliquots (I ml) are withdrawn at certain time intervals, and the enzyme activity is determined.

Activity Measurements of Native and Modified Enzyme, The catalytic activity of the native and modified enzyme is measured in a Radiometer TTT-ld pH-stat (Radiometer) by determining the initial rates of hydroly- sis of 10 mM N-acetyl-L-tyrosine ethyl ester in 0.1 M KCI at pH 7.0, 20 ° (assay volume 10 ml).

Results. The rate of thermoinactivation of enzyme modified with diamines of different chain lengths showed a minimum in the inactivation curve (Fig. 3) when the cross-linking reagent contained 4 methylene

[ 5 5 ] E N Z Y M E S T A B I L I Z A T I O N BY C R O S S - L I N K I N G 621 05] 0.3

E 0.25 I o

Z o_ I---

o.ao Z o;

~ 0.15

O I-.- Z ~ 0.10 Z O o 3

,~ 0.05

0 4 8 12

ALKYL CHAIN LENGTH OF DIAMINES (n)

FIG. 3. Dependence of the first-order rate constant of thermoinactivation of cross-linked ~-chymotrypsin on the chain length of the diamine reagents used for cross-linking: curve 2, cross-linked native ~-chymotrypsin; curve 3, cross-linked succinylated c~-chymotrypsin; curve 1, thermostability of native and succinylated c~-chymotrypsin. From Torchilin et a l? s

groups. It is worth adding that intermolecular cross-links were not formed under the experimental conditions, and that the monofunctional cross- linking analog, 1-aminopropan-3-ol, caused a certain destabilization of the enzyme. On the basis of the above, it is suggested that intramolecular cross-links were formed in o~-chymotrypsin after treatment of the carbodi- imide-activated enzyme with 1,4-tetramethylenediamine. Premodification of the enzyme with succinic anhydride resulted in additional reactive carboxyl groups on the protein surface. It was found that cross-linked succinylated preparations showed an increased thermostability compared

622 TECHNIQUES AND ASPECTS OF ENZYMES AND CELLS [55]

>: I - _s

_u i - J_J

(.3

UJ >

/ LIJ

A 2 0

I0

I i 0 I0 30

TIME, min

FIG. 4. (A) Thermoinactivation at 60 ° of native GAPDH (©) and of GAPDH cross-linked with oxalic acid ( I ) , succinic acid (&), glutaric acid ( I ) , adipic acid ([~), pimelic acid (A), and dodecandioic acid (<>). The inset shows the dependence of the half-life (~'1/2) of modified GAPDH on the number of methylene groups (n) of the diacid. (B) Densitometer traces for SDS-polyacrylamide gel electrophoresis of native GAPDH (1), GAPDH cross-linked with oxalic acid (2), GAPDH cross-linked with succinic acid (3), GAPDH cross-linked with glutaric acid (4), GAPDH cross-linked with adipic acid (5). Thirty micrograms of protein was applied to each gel. From Torchilin e t al . 26

with that of cross-linked unmodified enzyme (Fig. 3), indicating that a large quantity of cross-linkages had been formed (succinylation does not influence the thermostability of ot-chymotrypsin, see above). It was also found that for cross-linked succinylated a-chymotrypsin, the maximal stabilizing effect is produced not by 1,4-tetramethylenediamine but by the shorter reagent 1,2-ethylenediamine (Fig. 3). This fact is an additional indication that the surface of succinylated a-chymotrypsin globule is more "populated" with carboxyl groups than that of the native enzyme. Thus, premodification of the enzyme makes possible regulation of the stabilization effect both with respect to the degree and the optimal length of the cross-link.

[55] ENZYME STABILIZATION BY CROSS-LINKING 623

B

5

4

MIGRATION

FIG. 4B.

Thermostabilization of Glyceraldehyde-3-Phosphate Dehydrogenase by Intramolecular Cross-Linking

On heating or by action of a denaturant, oligomeric enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) are reversibly dissociated into subunits, leading to inactivation of the en- zyme. 35 The thermal stability of native GAPDH was studied and com-

35 R. Rudolph, I. Heider, and R. Jaenicke, Eur. J. Biochem. 81, 563 (1977).

624 TECHNIQUES AND ASPECTS OF ENZYMES AND CELLS [55]

pared with the thermostability of the enzyme modified with commercially available diacids such as HOOC(CH2)nCOOH (using reagents with n vary- ing from 0 to 10).

Experimental. On cross-linking two portions of solid carbodiimide (fi- nal concentration 2 x 10 -3 M) are added at 45-min intervals to an aqueous solution containing different cross-linking reagents (5 x 10 -4 M ) . HOW- ever, dodecandioic acid (5 x 10 -2 M) is activated in a solution containing dimethyl sulfoxide (DMSO) (1%, v/v). The reaction mixtures are allowed to incubate for 1.5 hr at pH 4.5, then the pH is increased to 8.2 and GAPDH is added to the reaction mixtures (final protein concentration 0.25 mg/ml). After reaction for 1.5 hr, the reaction is stopped by subject- ing the mixtures to gel chromatography on Sephadex G-50 (packed in a minicolumn that is placed in a centrifuge or by dialyzing the reaction mixtures prior to preparative electrophoresis. All experiments are per- formed 26 at 20 °. (The catalytic activity of the modified enzyme is found to be 20-40% of that of the native enzyme, depending on the bifunctional reagent used for cross-linking.)

In the thermoinactivation experiments, native or modified enzyme (2 × 10 -6 M ) in 50 mM phosphate buffer (pH 7.5) is incubated at 60 °. Samples are withdrawn at appropriate time intervals, and the enzyme activity is measured spectrophotometrically at 60 or 25 ° according to the assay method described in Ref. 36.

Analytical sodium dodecyl sulfate (SDS)-polyacrylamide gel electro- phoresis is performed as described by Laemmli. 37

Results and Discussion. In Fig. 4A it can be seen that intramolecularly cross-linked GAPDH is more stable at 60 ° than native enzyme. Figure 4A also shows that the degree of stabilization is dependent on the chain length of the bifunctional reagent used. Figure 4B shows the results of SDS-gel electrophoresis of various cross-linked preparations. By com- paring both figures it can be seen that the results of the SDS-polyacryl- amide gel electrophoresis agree well with the results of the thermoinac- tivation experiments. Both native enzyme and the enzyme treated with the shortest bifunctional reagent, oxalic acid (this cross-linked prepara- tion showed the same thermostability as native, untreated GAPDH), mi- grated in the gel as a single band corresponding to migration of the pro- moter of GAPDH (Fig. 4B). On the other hand, cross-linked enzyme preparations showing increased thermostability migrated as the dimer, trimer, tetramer, and/or higher oligomeric forms of the enzyme. Maximal thermostabilization was found when succinic acid (Fig. 4A) was used as a

36 W. Ferdinand, Biochem. J. 92, 578 (1964). 37 U. K. Laemmli , Nature (London) 227, 680 (1970).

[55] ENZYME STABILIZATION BY CROSS-LINKING 625

. I00

g

~ 50

,,-I, er'

A

I ~ ~ ' ~ " ~ " ~ i= - I0 50 TIME, min

~ B

I0 50

TIME, min FIG. 5. (A) Thermoinact iva t ion at 60 ° of GAPDH reconst i tuted from dimers cross-

linked with succinic acid (O); native enzyme (0) . (B) Semilogarithmic plot of the data in (A). Activity measu remen t s were performed at 25 °. From Torchilin et al. ~6

cross-linking reagent. In agreement with this observation is the finding that the same cross-linked enzyme yielded several SDS-polyacrylamide gel bands corresponding to different cross-linked forms of the enzyme (Fig. 4B). Thus, it is concluded that the chain length of succinic acid closely matches the distance between amino groups (located on different subunits of the enzyme) participating in the cross-linking reaction.

An interesting cross-linking experiment is thermoinactivation of GAPDH reconstituted from isolated cross-linked dimer molecules (Fig. 5). The dimers were prepared from succinic acid-treated GAPDH after preparative electrophoresis of the cross-linked enzyme in 8 M urea. From kinetic analysis of the thermoinactivation of native GAPDH, it can be concluded that the thermoinactivation is a two-step process in which the first step is reversible. It is interesting to note that the first step of the inactivation process was absent in thermoinactivation experiments of GAPDH obtained from cross-linked dimers. It should also be added that cross-linked GAPDH undergoes unfolding without prior dissociation of the enzyme into subunits and therefore cannot be reactivated.

Conclusion. The thermoinactivation of oligomeric enzymes is sug- gested 38 to be a two-step process in which the first step is protein dissocia-

38 V. S. Trube t skoy and V. P. Torchilin, In t . J . B i o c h e m . 17, 661 (1985).

626 T E C H N I Q U E S A N D ASPECTS OF ENZYMES A N D CELLS [56]

tion into subunits and the second step is unfolding of the subunits (Fig. 1). Thus, by cross-linking of protein structures, the first step of the inactiva- tion process is prevented due to an increased barrier to enzyme disso- ciation.

[56] L o n g - T e r m S t a b i l i t y o f N o n g r o w i n g I m m o b i l i z e d Cel ls o f Clostridiurn acetobutylicurn C o n t r o l l e d b y t he I n t e r m i t t e n t

N u t r i e n t D o s i n g T e c h n i q u e

By LENA H,~GGSTROM and CECILIA FORBERG

Long-term stability in immobilized cell processes for continuous pro- duction of metabolites is an important factor in considering their practical applications. The problems encountered are different depending on the nature of the biological system, i.e., whether growing cells or nongrowing, but viable, cells are employed. The reactor design and the immobilization method also influence the stability of the process. This chapter focuses on nongrowing, but viable, cells of Clostridium acetobu- tylicum immobilized in alginate or adsorbed to beech wood shavings for the continuous production of acetone and butanol.

In any system where nongrowing cells are applied a loss of activity with time should be expected due to turnover of essential cell constitu- ents. Addition of nutrients will restore the microbial activity, however, in order to maintain the cells in the nongrowing state and at the same time keep a constant productivity, the distribution of nutrients is critical. A technique for control of the activity in nongrowing immobilized cells has therefore been developed.l

Intermittent Nutrient Dosing Technique

The intermittent nutrient dosing technique is based on the pulsewise addition of nutrients to the reactor, which otherwise is continuously fed only with a nongrowth production medium. The nongrowth medium lacks a utilizable nitrogen source and growth factors. In order to maintain the organism in an active but nongrowing state the nutrient supply should be sufficient to enable the organism to restore essential cell constituents but not rich enough for reproduction to proceed. The addition of nutrients can

C. F 6 r b e r g , S . - O . E n f o r s , a n d L . H / i g g s t r 6 m , Eur. J. Appl. Microbiol. Biotechnol. 17, 143 (1983).

Copyright © 1988 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 137 All rights of reproduction in any form reserved.