Immobilization-Stabilization of Thermolysin Onto ...

Biocatalysis and Biotransformation

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Immobilization-Stabilization of Thermolysin Onto Activated Agarose Gels J. M. Guisán, E. Polo, J. Aguado, M. D. Romero, G. Álvaro & M. J. Guerra To cite this article: J. M. Guisán, E. Polo, J. Aguado, M. D. Romero, G. Álvaro & M. J. Guerra (1997) Immobilization-Stabilization of Thermolysin Onto Activated Agarose Gels, Biocatalysis and Biotransformation, 15:3, 159-173, DOI: 10.3109/10242429709103507 To link to this article: http://dx.doi.org/10.3109/10242429709103507

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Biocafalysis and Blotransformation, Vol. 15, pp. 159-173 Reprints available directly from the publisher Photocopying permitted by license only

Amsterdam B.V. Published in The Netherlands by Harwood Academic Publishers Printed in India

IMMOBILIZATION-STABILIZATION OF THERMOLYSIN ONTO ACTIVATED AGAROSE GELS J.M. GUISAN~,E. POLO^, J. AGUADO~,*,M.D. ROMERO~, G. ALVAROb and M.J. GUERRAb alnstituto de Catalisis y Petroleoquimica, C.S.I.C. 28049 Cantoblanco, Madrid; bDepartamento Ingenieria Quimica, Facultad Ciencias Quimicas, U n i v e r s i d a d Complutense, A v d a . Complutense sjn. 28040 Madrid, Spain (Received 26 July 1996; In final form 12 February 1997) A method for immobilization-stabilization of thermolysin onto activated agarose gels is

reported based on the formation of covalent bonds between the enzyme and the support. All derivatives prepared retained 100%. of the enzymatic activity and they show higher stability than free thermolysin. The effect of different variables concerning the strength of the enzymesupport attachment on the stability of the immobilized thermolysin derivatives has been established under different inactivation conditions: presence of a water miscible solvent (DMF); stirred biphasic systems, 1,2-dichloroethane/acetatebuffer; acid conditions (pH = 3) as well as in the absence of calcium ions. The possible reactivation of the derivatives inactivated by the loss of calcium ions was also studied. Keywords: Thermolysin immobilization; Thermolysin stabilization; Agarose gels

INTRODUCTION Enzymatic catalysis in organic solvents is an active field of research, with new processes being continuously reported in literature. Although the use of organic solvents usually increases the product yield, few industrial processes are currently carried out in this way. The reason is that enzymes are very labile catalysts and can be strongly inactivated in the presence of organic solvents. Therefore, producing

* Corresponding author. 159

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enzymatic derivatives which are stable in organic solvents is critical for industrial applications. Protease catalyzed peptide synthesis has attracted much attention in the last two decades, due to its benefits as compared to classical chemical peptide synthesis. The reactions take place in milder conditions, their high regiospecificity minimizes the need of substrate protection, and it is possible to develop stereospecific synthesis of L-peptides from racemic mixtures (Oyama and Kihara, 1984; Kullmann, 1987; Gill et al., 1996). Thermolysin is a zinc metalloprotease with four calcium atoms per molecule of enzyme, responsible for its high thermal stability (Voordouw et al., 1976; Dahlquist et a]., 1976). This enzyme has been widely employed in peptide synthesis reactions (Kullmann, 1982; Cerovsky, 1986; Reslow et al., 1988; Sakina et al., 1988; Kitaguchi and Klibanov, 1989), mainly in the industrial scale production of Cbz-aspartame (Cbz-Asp-Phe-OMe) (Nakanishi and Matsuno, 1988). In most of these applications thermolysin was used in organic solvent-water mixtures, as well as in stirred biphasic reactors. The presence of organic solvents as well as the large interfacial areas in stirred systems lead to enzyme inactivation. Therefore, both types of reaction system are very damaging for enzyme stability (Khmelnitsky et al., 1988). In this research, different thermolysin derivatives were prepared by immobilization onto activated agarose gels by multipoint covalent attachment. Using similar methods we have been able to stabilize several interesting industrial enzymes (Guisan, 1988; 1991). In this paper, we have tried to apply these methods to thermolysin, in which calcium ions play a critical role in enzyme deactivation, to check whether multipoint covalent immobilization may also improve the stability of this complex enzyme. This has revealed a relationship between the rigidity, the loss of calcium ions and enzyme stability. The stability of these derivatives has been tested under different inactivation conditions, mainly in the presence of organic solvents. Finally, the reversibility of the enzyme inactivation caused by loss of calcium ions as well as the possibility of reactivation have been also studied.

METHODS Materials Agarose gels 6B-CL and 1OB-CL, having an average particle size of 100 pm, were kindly donated by Hispanagar, S.A. Surface area and average

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A

pore diameter were 25 m2/ml gel and 800 for 6B-CL gels, and 50m2/ml gel and 400A for IOB-CL gels. Thennolysin from Bacillus thermoproteolyticus rokko (44 units/mg solid) and N-(3-[2-furyl]acryloyl)-glycyl-leucinamide (FAGLA) were purchased from Sigma Chemical Co. Analytical grades for other reagents and solvents were used. Assay of Thermolysin Activity

The hydrolysis of FAGLA, as substrate, was followed spectrophotometrically at 25°C using a Kontron Uvikon 900 spectrophotometer. For the standard assay 1 mM FAGLA solution was prepared in 50mM Tris buffer pH=7.5 containing 0.1 M NaCl and lOmM CaC12 (Feder, 1968). Assays using the above FAGLA solution, but without calcium chloride, were also carried out when studying the inactivation of thermolysin caused by loss of calcium ions. The assay was carried out in a 1 cm path length cell provided with magnetic stirring. The decrease of absorbance at 345 nm was monitored following the hydrolysis of substrate when samples of free or immobilized thermolysin were added to the cuvette.

Activation of Agarose Gels

Glyoxyl gels were prepared by etherification of agarose gels with 2,3epoxy- 1-propanol and further oxidation with sodium periodate as described previously (Guisan, 1988). Two activated gels were prepared from 6B-CL gel, containing 5 pmol aldehyde ml-' gel (about 1 aldehyde residue per 1000A2 of gel surface) and 75pmol aldehyde ml-' gel (18 aldehyde residues per 1000A2 of gel surface), respectively. Agarose gel 10B-CL was activated to 200pmol aldehyde ml-' gel (19 aldehyde residues per 1000A2 of gel surface).

Immobilization of Thermolysin onto the Activated Gels

An aqueous solution of thermolysin in 50mM borate buffer pH 10 containing lOmM CaC12 was mixed with the activated gel. At different times, samples of both liquid supernatant and suspension were withdrawn and their catalytic activity was assayed. In order to test the enzyme stability, a control of thermolysin was also prepared under the same immobilization conditions but in the absence of activated gel.

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After a predetermined contact time between the enzyme and the activated gel, solid NaBH4 was added to the immobilization mixture to a final concentration of 1 mgml-' and stirred at 25°C for 30min. Afterwards, derivatives were filtered and washed repeatedly with SO mM Tris buffer pH = 7.5 and distilled water, both containing 10 mM CaCl?.

Inactivation of Free and Immobilized Thermolysin Inactivation runs for free and immobilized thermolysin were carried out in different conditions. In each run, thermolysin was suspended in the inactivation medium. At different times, samples were withdrawn and assayed as described above. When inactivation experiments were carried out in the absence of calcium ions, the enzymatic activity was tested using the FAGLA standard assay without CaCl?.

RESULTS AND DISCUSSION Immobilization of Thermolysin Thermolysin was immobilized, under different conditions (contact time and temperature), onto the three activated supports. The immobilization conditions as well as the results obtained are shown in Table I. The enzyme was completely immobilized on the activated supports after 2 h. except when the 5 pmol aldehyde ml-' gel was used as support (only 5% of the offered enzyme was insolubilized after 5h). In all cases the TABLE I Immobilization-stabilization of thermolysin under different conditions

0-5 M75-0.5 M75-1.0 M75-1.5 M75-2 M75-7 M200-7 M200-40

5 75 75 75 75 75 200 200

5 05 10

IS -1 7 7 7

25 25 25 35 25 25 25 40

5 38 65 93 100 100

I00 I00 100 I00 100

I00

I00

100

100

100


163

agarose derivatives retained 100% of the activity of the corresponding amount of soluble enzyme. When the 5pmol aldehyde ml-' gel, having a low density of active groups, was used as support, each enzyme molecule (4 M 40A) could only be attached by a single bond. The low immobilization yield reached (5%) seems to confirm the reversibility of the covalent bonds (Schiff's base) between the support and the enzyme molecule. These results are in good agreement with those previously reported for immobilization of trypsin (Blanco et al., 1989), penicillin G acylase (Alvaro et al., 1990) or a-chymotrypsin (Guisan et al., 1991) using the same activated supports. With the exception of the 0 - 5 derivative, immobilization experiments were carried out offering to the support a small amount of thermolysin (about 1 mg of the commercial preparation per ml of gel) in order to avoid internal diffusion problems during the activity assay. The absence of diffusional control can be assumed for two reasons: (a) immobilized derivatives retain 100% activity of the corresponding amount of soluble enzyme and (b) no increase in activity was observed after reduction of particle size of immobilized derivatives. In order to establish the maximum thermolysin loading capacity for the two agarose gels (6B-CL and 10B-CL), immobilization experiments were carried out by offering higher amounts of thermolysin to the supports. The maximum loading capacity was 36mg of commercial preparation of thermolysin per ml of 6B-CL gel. For 10B-CL gel, having a larger specific surface, the maximum loading capacity was 50 mg/ml gel.

Thermal Inactivation of the One-Point Derivative (0-5) in Conditions of Enzyme Aggregation Aggregation of proteins is promoted by intermolecular interactions among their hydrophobic residues. Insoluble aggregates are formed by denatured protein molecules as we have previously observed for free thermolysin at pH 8.0. Figure 1 shows the inactivation courses, for free thermolysin and 0 - 5 derivative, carried out at 50°C and pH 8.0. Under these experimental conditions, aggregation was observed for free thermolysin but not for the 0 - 5 derivative, the latter showing higher stability than free thermolysin. This demonstrates that inactivation caused by aggregation of free thermolysin is avoided. The immobilization of the enzyme onto the agarose gels hinders the interaction among hydrophobic residues, resulting in an increase in stability.

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0

50

01.

100

150

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ELAPSED TIME (hours)

F I G U R E I Inactivation courses at 50°C in Tris buffer 50mM pH = 8.0 with lOmM CaC12. Free thermolysin. A Derivative 0 - 5 .

Thermal Inactivation of the 0 - 5 Derivative in the Presence of a Water-Miscible Solvent

Figure 2 shows inactivation runs for both free thermolysin and the 0 - 5 derivative, carried out at 60°C in acetate buffer 0.1 M pH = 6.0 with lOmM CaClz in the presence of 20% of N,N-dimethyl formamide (DMF). These strong inactivation conditions were selected due to the high stability of the free thermolysin. This figure shows that the 0 - 5 derivative is only slightly more stable than the free enzyme, suggesting that the conformation of the enzyme has not been modified by one-point immobilization. An inactivation run of free thermolysin, under the same experimental conditions, but in the presence of 20mM Chz-phenylalanine is also presented in Fig. 2. This thermolysin inhibitor ( K , = 0.5 mM) (Kester and Matthews, 1977) was added in order to avoid enzyme inactivation by autolysis. The similarity among the inactivation curves of free thermolysin, with and without Cbz-phenylalanine, demonstrates that under these conditions, autolysis is not the inactivation mechanism. Inactivation of the 0 - 5 Derivative in the Presence of a Water-Immiscible Solvent

The effect of organic interfaces on the stability of both free thermolysin and the 0 - 5 derivative has been investigated. The experiments were carried

IMMOBILIZATION O F THERMOLYSIN

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0 100

200

300

400

FIGURE 2 Inactivation courses at 60°C in acetate buffer 50mM pH=6.0 with lOmM 0 Free thermolysin with 20 mM Cbz-phenylalanine. A Derivative 0-5. CaC12 and 20% of N,N-dimethyl formamide. 0 Free thermolysin.

out in a biphasic system (acetate buffer 0.1 M pH 6.0, lOmM CaC12:1,2dichloroethane) stirred at 1000 rpm. A control experiment using free enzyme dissolved in aqueous buffer saturated with 1 ,2-dichloroethane was also carried out in order to establish the effect of dissolved solvent in the aqueous phase. The results obtained are shown in Fig. 3. The free enzyme loses all its activity in the stirred biphasic system in less than one hour whereas the same free enzyme retains 100% of its initial activity after two hours in the control experiment. This confirms that contact with the aqueous-organic interface is responsible for enzyme deactivation (Ghatorae et al., 1994). When contact with the interface is avoided by confining the enzyme inside a porous support, a high stabilization effect is obtained as observed in the inactivation run of the 0 - 5 derivative in the stirred biDhasic svstem.

Thermal Inactivation of Derivatives Immobilized by Multipoint Attachment Thermolysin has. nine amino groups (eight lysine residues and the amino terminal group) (Colman, Jansonius and Matthews, 1972). Multipoint covalent attachment between the enzyme and support can be achieved by using very activated agarose with a high density of aldehyde groups.

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FIGURE 3 Inactivation courses at room temperature in 1,2-dichloroethane/acetate buffer 50mM p H = 6 . 0 with lOmM CaCI2 biphasic system. Free thermolysin stirred at 1000rpm. 0 Blank of free thermolysin in the buffer saturated with I,?-dichioroethane. A Derivative 0 - 5 stirred at 1000rpm.

This multipoint attachment leads to a more rigid structure of the enzyme, hindering conformational changes and producing a high degree of stabilization for different enzymes (Guisan. 1988). The stability of four thermolysin-agarose derivatives (M75-2; M75-7; M200-7 and M200-40) immobilized by multipoint covalent attachment was studied under very strong deactivation conditions. . The plots of the inactivation courses for these derivatives are compared in Fig. 4 together with that for the one point derivative (0-5). The activity half life of the 0 - 5 derivative was 20 h, whereas the use of activated agarose with 75pmol aldehyde ml-' gel (M75-2 derivative prepared with a contact time of 2 h) had a threefold higher half life than the 0 - 5 derivative. Increase of the contact time during the immobilization step resulted in further stabilization as shown in Fig. 4 for the M75-7 derivative (contact time = 7 h) which retains about 50% of its initial activity after approximately 400 h. In order to further improve the stability of thermolysin, new derivatives were prepared using agarose gels with higher density of aldehyde groups (200 pmol aldehyde ml-.' gel) (M200-7 derivative) or increasing the immobilization temperature up to 40°C (M200-40). The results shown in Fig. 4 show that the stability of these derivatives was close to that of the M75-7


0

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200 300 ELAPSED TIME (hours)

FIGURE 4 Inactivation courses at 60°C in acetate buffer 50mM pH=6.0 with 1OmM A Derivative 0-5. 0 Derivative M75-2. 0 Derivative M75-7. Derivative M200-7. V Derivative M200-40.

CaC12 and 40% of N,N-dimethyl formarnide.

derivative. This may be explained by suggesting that in the M75-7 derivative, thermolysin has already saturated all its amino groups available for the covalent attachment. In view of these results, we selected the M200-7 derivative for further experiments due to its higher enzyme loading. Stability of the 0 - 5 and M200-7 Derivatives in the Presence of a High Concentration of Organic Solvent

Thermolysin possesses four calcium ions in equilibrium with calcium ions present in the medium (Dahlquist et al., 1976). When the concentration of calcium ions in the medium is less than lOP3M, the enzyme is inactivated because of the progressive release of calcium ions from the thermolysin molecules to the liquid phase. However, in peptide synthesis reactions, the presence of a high concentration of miscible organic so1,vents is often required in order to achieve high synthetic yields. Under these conditions the calcium ions in the medium can precipitate, shifting the enzymemedium calcium equilibrium and reducing enzyme stability. This negative effect must be added to the enzyme denaturation caused by the organic solvent itself. Inactivation experiments for the 0 - 5 and M200-7 derivatives were earried out at room temperature in citrate buffer 50mM (a chelating agent

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for calcium) at pH = 6.0 in the absence of calcium ions with a concentration of 75% DMF. Figures 5(a) and 5th) show the inactivation courses of the two derivatives when the agarose gel/solvent ratios were 1 : 1 and 1 : 9, respectively. For the 1 : 1 ratio the 0 - 5 derivative loses all its activity in less than four hours while the M200-7 derivative retains 15% of its initial activity after 600h. For the 1 : 9 ratio the stability of both derivatives was markedly reduced. reaching 15% of the initial activity for 0 - 5 and M200-7 derivatives after 0.2 and 1.6 h, respectively. Therefore, with immobilized thermolysin the release of calcium ions also plays a definitive role in enzyme deactivation, particularly for lower gel/solvent ratios. However, for the 1 : 1 ratio, the release of calcium ions is less important due to the small volume of the liquid, and the main contribution to enzyme inactivation is due to denaturation caused by the organic solvent. Under these conditions, multipoint attachment leads to a higher degree of stabilization in the immobilized enzyme.

Stability of Thermolysin Derivatives under Acid Conditions The highest stability of free thermolysin is exhibited in the pH range from 5 to 11 (Ohta, 1967). In peptide synthesis reactions, pH values lower than 5

0

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ELAPSED TIME (hours) FIGURE 5(a) Inactivation courses at room temperature in citrate buffer 50mM with 75% of N,N-dimethyl formamide. Assay of hydrolysis of FAGLA without calcium, yel/solvent volume ratio 1 : I . A Derivative 0 - 5 . Derivative M200-7.


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FIGURE 5(b) Inactivation courses at room temperature in citrate buffer 50mM with 75% of N,N-dimethyl formamide. Assay of hydrolysis of FAGLA without calcium, gel/solvent volume ratio 1 : 9. A Derivative 0-5. +t Derivative M200-7.

are sometimes required to reach high yields. In order to study the stability of our derivatives (0-5 and M200-7) under acid conditions, inactivation experiments were carried out at 50°C in acetate buffer 0.1 M pH = 3.0, containing 10mM CaCI2. Figure 6 shows the remarkable stabilization effect obtained by multipoint attachment (the activity half-life increases from 0.8 h for 0 - 5 derivative to 19.5 h for the M200-7). In thermolysin, the calcium ions interact with carboxylate groups of the protein through salt bridges. Protonation of these carboxylate groups, under acid conditions, may produce the inactivation of the enzyme, due to the release of calcium ions to the medium. Multipoint attachment increases the rigidity of the structure of the enzyme and may restrict the release of calcium, thus reducing these denaturant effects.

Reactivation of Thermolysin Derivatives As mentioned above, a gradual inactivation of thermolysin occurs when the enzyme is placed in a calcium free medium. The reversibility of this inactivation, caused by the release of calcium ions has been studied in order to examine the possibility of enzyme reactivation. Comparative experiments were carried out by placing both, 0 - 5 and M200-7 derivatives, as well as free thermolysin in 50mM citrate buffer

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(pH = 4.0) without calcium. The enzymatic activity was tested immediately by using the FAGLA assay in the presence and absence of calcium chloride. The results obtained are shown in Table 11. When free thermolysin was dissolved in buffer without calcium and assayed also without calcium, the activity obtained was only 41% of the activity obtained if the enzyme was both suspended and assayed in the presence of CaC12. Total recovery of activity was not reached, even when the enzyme was assayed in the presence of l 0 m M calcium chloride. Only 74% of enzymatic activity referred to standard conditions was reached.

1

P 20- L .

01 0

I

I

I

15

30

45


FIGURE 6 Inactivation courses at 50°C in acetate buffer 50mM p H = 3 . 0 with l 0 m M CaCI2. A Derivative 0-5. x Derivative M200-7.

TABLE I1 Reactivation of free thermolysin and 0 - 5 and M200-7 derivatives previously incubated in a calcium free medium Conditions

Enzymutic Activity ( X ) Referred to standard conditions: medium and assay with 10 mM CaC12

Free thermolysin

Incubated without calcium Assay without calcium Incubated without calcium Assay with l0mM CaCI2

Derivative

Derivative

0-5

M200-7

41

62

60

14

100

100


I71

100

-s

80

f

60

Y

0

a

2 3 0

40

2

CC

20

0

0

50

100

150

H)


FIGURE 7 Reactivation of free thermolysin, 0-5 and M200-7 derivatives. Incubated at room temperature in citrate buffer 50mM pH =4.0without calcium ions. Free thermolysin. FAGLA assay without lOmM CaCI2. W Derivative 0 - 5 . FAGLA assay without calcium. A Derivative 0-5. FAGLA assay with lOmM CaCI2. 0 Derivative M200-7. FAGLA assay without calcium. A Derivative M200-7. FAGLA assay with lOmM CaCI2.

On the other hand, 0 - 5 and M200-7 derivatives retained higher activities than the free thermolysin when they were placed in a medium without calcium. Both of them showed 60% of the standard activity when the FAGLA assay was carried out in the absence of calcium. Using the FAGLA assay in the presence of calcium ions, both derivatives recovered 100% of the standard activity, demonstrating the reversibility of this type of inactivation for both derivatives. In order to ascertain whether to recover activity -in this way is maintained for longer periods of time, 0 - 5 and M200-7 derivatives, as well as free thermolysin were incubated at room temperature in a calcium free medium. At different times, the enzymatic activity was tested by using FAGLA assay in the presence and absence of calcium ions. The results are shown in Fig. 7. Free thermolysin loses all its activity in less than 5 h due to the enzyme autolysis caused by the absence of calcium (Dahlquist, Long and Bigbee, 1976). Obviously, this inactivation was irreversible and no activity was recovered. In the case of the derivatives, the reactivation feasibility observed initialy was progressively reduced with time but was always greater for the derivative M200-7 due to the rigidity obtained by multipoint attachment.

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CONCLUSIONS The method described in this paper for immobilization-stabilization of thermolysin leads to immobilized derivatives having 100°/~of activity. The use of agarose gels with a high surface density of aldehyde groups, as a support, leads to multipoint derivatives having a remarkable increase of stability compared to the free enzyme. The one-point derivative (0-5) was highly stabilized against enzyme aggregation in comparison to free thermolysin as immobilization avoids the intermolecular interactions which are responsible for enzyme aggregation. Moreover, when thermolysin is confined in the pores of the support, contact of the enzyme with the liquid-liquid interface of a biphasic system is hindered. Thus, in stirred biphasic systems, thermolysin is markedly stabilized by immobilization. In strongly inactivating conditions (in a free calcium medium with 75% DMF),the multipoint derivative (M200-7) was always more stable than the 0 - 5 derivative. The differences in stability between these two derivatives were higher when the inactivation was caused by the presence of DMF than when the inactivation is due to the loss of calcium ions. The immediate reactivation observed for both derivatives, by addition of calcium ions in the FAGLA assay, demonstrates that this deactivation, caused by loss of calcium ions, is reversible when the enzyme is immobilized. In contrast, inactivation of the free enzyme by the loss of calcium ions was irreversible, probably due to autolysis.

References Alvaro. G.. Ferninder-Lafuente, R. and Guisin. J.M. ( 1990) Immobi~ization-slabiiization of penicillin G-acylase from Ewherichia coli. Appl, Biochem. Biortvh.. 26, 181 -- 195. Blanco, R.M.. Calvete, J.J. and Guisin. J.M. (1989) Immobilization-stabiliLation of enzymes. Variables that control the intensity of the trypsin (amine)-agarose (aldehyde) multipoint attachment. E t q n i e Microh. Tectitiol., 11, 353-359. Cerovsky, V. (1986) Synthesis of pressionic acid by enzymatically catalyzed formation of peptide bonds. C ' o k t i o n C:cc/ioslovak Chum. Con7nirtn.,51, 1352- 1360. Colman, P.M.. Jansonius, J.N. cnd Matthew. B.W. (1972) The structure of thermolysin: an electron density map at 2.3 A resolution. J . Mol. Bid.. 70, 701 --724. Dahlquist, F.W.. Long. J.W. and Bigbee, W.L. (1976) Role of Calcium in the Thermal Stability of Thermolysin. Biochetn.. 15. 1103- 1 1 1 I . Feder. J. (1968) A spectrophotometric assav for neutral protease. Biocliem. BiopI7j~s.Res. C'otnrnuri..32. 326-332. Ghatorae, A.S.. Guerra. M.J.. Bell. G . and Halling, P.J. (1994) Immiscible organic solvent inactivation of urease. chymotrypsin. Iipase and ribonuclcase: Separation of dissolved solvent and interfacial effects. Biotech. Bioet7.q.. 44. 1355 1361. Gill, Y.. Lopez-Fandifio, R . . Jorba, X. and Vulfson, E.N. (1996) Biologically active peptidcs and enzymatic approaches to their production. En:iwtr Microh. Techno/., 18, 162 183.


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Guisan, J.M. (1988) Aldehyde-agarose gels as activated supports for immobilization-stabilization of enzymes. Enzyme Microb. Technol., 10, 375-382. Guisan, J.M., Bastida, A,, Cuesta, C., Fernandez-Lafuente, R. and Rosell, C.M., (1991) Immobilization-stabilization of a-chymotrypsin by covalent attachment to aldehyde agarose gels. Biotech. Bioeng., 38, 1144-1 152. Kester, W.R. and Matthews, B.W. (1977) Crystallographic study of the binding of dipeptide inhibitors to thermolysin: Implications for the mechanism of catalysis. Biochemistry, 16, 2506-2516. Khmelnitsky, Y.L., Levashov, A.V., Klyachko, N.L. and Martinek, K. (1988) Engineering biocatalytic systems in organic media with low water content. Enzyme Microb. Technol., 10, 710-724. Kitaguchi, H. and Klibanov, A.M. (1989) Enzymatic peptide synthesis via segment condensation in the presence of water mimics. J. Am. Chem. SOC.,111, 9272-9273. Kullmann, W. (1982) Protease-catalyzed peptide bond formation: Application to synthesis of the COOH-terminal octapeptide of cholecystokinin. Proc. Natl. Acad. Sci. USA, 79, 2840-2844. Kullmann, W. (1987) Enzymatic synthesis of biologically active peptides. In: Enzymatic peptide synthesis. (CRC Press Inc., eds.). Boca Raton, Florida, 61-80. Nakanishi, K. and Matsuno, R. (1988) Recent developments in enzymatic synthesis of aspartame. In: Food Biochemistry. Vol 2. (King, R.D. and Cheetham, P.S.I., eds.). Elsevier, London, 218-249. Ohta, Y. (1967) Thermostable protease from thermophilic bacteria. J . Biol. Chem., 242, 509515. Oyama, K. and Kihara, K . (1984) A new horizon for enzyme technology. Chemtech., 14, 100-105. Reslow, M., Adlercreutz, P. and Mattiasson, B. (1988) The influence of water on proteasecatalyzed peptide synthesis in acetonitrile/water mixtures. Eur. J. Biochem., 177, 313-318. Sakina, K., Kawazura, K., Morihara, K. and Yajima, H. (1988) Themolysin-catalyzed synthesis of peptide amides. Chem. Pharm. Bull., 36, 4345-4354. Voordouw, G., Milo, C. and Roche, R.S. (1976) Role of bound calcium ions in thermostable proteolytic enzymes. Separation of intrinsic and calcium ions contributions to the kinetic thermal stability. Biochemistry, 15, 3716-3724.