Immobilization of proteases to porous chitosan beads and their catalysis for ester and peptide synthesis in organic solvents* Hideo Kise and Atsuhito Hayakawa Institute o f Materials Science, University o f Tsukuba, Tsukuba, Japan
c~-Chymotrypsin (CT), subtilisin BPN' (STB), and subtilisin Carlsberg (STC) were immobilized by adsorption to porous ehitosan beads (Chitopearl, CP). The immobilized enzymes showed higher catalytic activities than free enzymes for amino acid esterification in many hydrophilic organic solvents except for methanol and DMF. In ethanol, the initial rate of the esterification increased with water content, whereas in ethyl acetate, the maximum rate was obtained at 2%-3% water. CP-immobilized CT also catalysed transesterification of Ac-Tyr-OMe in ethanol and peptide synthesis in acetonitrile from Ac-Tyr-OH or its ethyl ester and amino acid amides. The immobilized enzymes are highly stable in organic solutions, and can easily be separated from the reaction solutions. Repeated esterifications of Ac-Tyr-OH in acetonitrile by a CP-immobilized CT gave almost constant yields of the ester for more than 3 weeks.
Keywords: Protease immobilization; porous chitosan beads; ester synthesis; peptide synthesis; organic solvent
Introduction Recently a great number of research studies have been performed on synthetic reactions by enzymes in organic solvents. One of the promising methods for biosynthetic reactions is the utilization of proteases or lipases in hydrophilic (water-miscible) organic solvents with restricted amounts of water. There are many advantages in employing cosolvent systems, such as high solubilities of both polar and nonpolar substrates, shift of t h e r m o d y n a m i c equilibria by controlling water content, and absence of diffusion limitation across water/ organic solvent interfaces. The main drawback of cosolvent systems is the possible inactivation of enzymes due to direct contact with organic solvents such as ethanol, i-6 H o w e v e r , recent studies have revealed that e n z y m e s can retain their activity at high concentrations of organic solvents and catalyse many synthetic reactions, including amino acid esterification, 7-9 transesterification, 1°-12 and peptide synthesis.13-16 Recent studies have also revealed that the activities
* Enzymatic reactions in aqueous-media. X. Address reprint requests to Dr. Kise at the Institute of Materials Science, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Received 7 August 1990; revised 18 December 1990 584
Enzyme Microb. Technol., 1991, vol. 13, July
of immobilized proteases in organic solvents are strongly dependent on the nature of the support material. lv'~8 Furthermore, it was reported that enzymes can be activated by complexation with polysaccharides such as chitin or chitosan, 19 or by lyophilizing from certain buffer solutions or solutions of specific amino acids. 13'2° These results indicate that the interactions of enzymes with support materials strongly affect the activity and stability of the e n z y m e s in hydroorganic reaction systems. In order to obtain e n z y m e preparations of high activity and easy handling, we studied the immobilization of proteases to commercially available porous chitosan beads, and we report here the results on the catalytic activity and the long-term stability of the immobilized enzymes for ester and peptide syntheses in organic solvents.
Materials and methods Materials
Bovine pancreatic ~x-chymotrypsin (CT) having a specific proteinase activity, with N-benzoyl-L-tyrosine ethyl ester, of 50 units (/~mol min -1 mg i) (pH 7.8 at 25°C) was purchased from Sigma Chemical Co. Subtilisin Carlsberg (type VIII, STC) and subtilisin B P N '
© 1991 Butterworth-Heinemann
Ester and peptide synthesis by immobilized proteases: H. Kise and A. Hayakawa (type XXVII, STB) having specific proteinase activities, with casein, of 11.6 and 6.8 units, respectively, were also purchased from Sigma. All the substrates were the products of Sigma, except for N-acetyl-Ltyrosine methyl ester, which was prepared from Nacetyl-L-tyrosine in methanol by the catalysis of dry hydrogen chloride and recrystallized from ethyl acetate. The alcohols and all the solvents were purchased from Nacalai Tesque, Inc., and dried on 3-A molecular sieves. Porous chitosan beads (Chitopearl BCW 3010), which are the products and generous gift of Fuji Spinning Co., have an average diameter of I mm, pore size of 0.1-0.2 ~m,, and specific surface area of 120-150 m 2 g-l. They are insoluble in and highly resistant to organic solvents.
Enzyme immobilization Typically, 100 mg of dry Chitopearl (CP) was added to a solution of 10 mg enzyme in 0.4 ml of water, and the mixture was stored at 4°C overnight. Then, the CPenzyme preparation was washed several times with water on a glass filter. The amount of immobilized enzyme was determined by a spectroscopic measurement (280 nm) of the filtrates for the enzyme, and the amount of water in the CP-enzyme was determined from the increase in the weight. Alternatively, and more conveniently, CT was adsorbed to CP by mixing dry CP with an enzyme solution in a specific amount of water. The mixture was kept standing for 1 h at room temperature, and the preparation was used directly for reactions in organic solvents.
Ester synthesis and transesterification To a CP-enzyme preparation described above, a solution of N-acetyl-L-tyrosine (Ac-Tyr-OH) (mostly 10 raM), 50 mg of acetanilide, which was an internal standard for HPLC analysis, and a specific amount of water in 20 ml of ethanol was added, and the mixture was incubated with constant reciprocal shaking (about 150 cycles min- ~) at 30°C. When organic solvents other than ethanol were used, solutions containing 10% (v/v) (1.7 M) ethanol were used. The amounts of Ac-Tyr-OH and its ethyl ester (Ac-Tyr-OEt) were determined with an HPLC (JASCO Tri Rotar SR-I or Shimadzu LC-6A) using JASCO Finepak SIL C18 or Shimpak C-18 columns eluted with water-acetonitrile (50/50 by volume). The reaction rate was calculated from the amount of Ac-TyrOEt formed after 10-60 rain reactions. Transesterification was carried out in a similar manner using Ac-TyrOMe as a substrate in ethanol. The operational stability of immobilized CT was estimated by measuring the ini tial reaction rate and ester yield after 24 h for repeated esterification reactions in ethanol; the reaction solution was replaced every 24 h by a fresh substrate (10 mM) solution containing 2.9% water.
Peptide synthesis To a solution of amino acid amide hydrohalide (AANH2HX, 0.12 retool) and triethylamine (0.12 mmol) in
0.3 ml of water, a solution of Ac-Tyr-OH or Ac-TyrOEt (0.1 mmol) and acetanilide (25 rag) in 10 ml of acetonitrile was added. The solution was then added to the immobilized enzyme, and the mixture was incubated and the product was analyzed as above. Authentic samples of Ac-Tyr-AA-NH 2 were used for HPLC calibration.
Adsorption of Ac-Tyr-OH and Ac-Tyr-OEt to CP A mixture of 0.3 ml of water and 50 mg of CP was kept standing at room temperature for 1 h. Then a solution of Ac-Tyr-OH or Ac-Tyr-OEt in 10 ml of ethanol or acetonitrile was added to the mixture, and the whole mixture was incubated with shaking at 30°C. After 0.5 and 24 h, the amounts of the adsorbed materials were determined by performing HPLC analyses of the solutions as above.
Results and discussion
Enzyme immobilization The porous chitosan bead (Chitopearl, CP) has a large surface area and strongly adsorbs many proteins from aqueous solutions. CP has been utilized as a support for enzyme immobilization in aqueous solutions, 21 but to our knowledge, no report has appeared on the catalysis of CP-immobilized enzymes in organic solvents. We obtained CP-CT preparations of different CT loadings by changing the concentration of CT in aqueous solutions in which CP was immersed. The amount of adsorbed CT increased with its concentration in the aqueous solutions up to 13% of the weight of dry CP. However, the comparison of the catalytic activities of CP-CT preparations of different CT loadings suggested that higher Ioadings led to the decrease in the activity of the enzyme. Therefore, most of the reactions were carried out using preparations of CT contents below 12% (w/w).
Ester synthesis and transesterification It is known that the catalytic activity of proteases in water-organic cosolvents is strongly dependent on water content. This is also the case for the esterification of Ac-Tyr-OH by free and CP-immobilized CT, as shown in Figure 1. No reactions occurred without water, and the reaction rate increased with water content. It can be seen that the catalytic activity of CT markedly increases by immobilization to CP. No reactions were detected in the absence of the enzyme, and this rules out the possibility of the catalysis by CP. The increase in reaction rate and ester yield by immobilization to CP is much larger than that with porous silica gel, controlled porous glass (CPG), or celite. Ig The reaction mixtures consist of clear solutions and hydrated CP containing the enzyme. An HPLC analysis revealed that a part of the substrate, Ac-Tyr-OH, is also adsorbed to CP, whereas the product, Ac-Tyr-OEt, is less strongly adsorbed to CP (Table 1). The result suggests
Enzyme Microb. Technol., 1991, vol. 13, July
585
Papers Table 1 Adsorption of Ac-Tyr-OH and Ac-Tyr-OEt to CP in organic solvents a
0.20 ~i-
0.16
£2
'~
Adsorbed (%) Solvent
0 12
EtOH
0.08
CH3CN
1
:. 0.04
Time (h)
Ac-Tyr-OH
Ac-Tyr-OEt
0.5 24 0.5 24
8.5 9.0 20 22
0.9 0.8 0.8 2.6
a Ac-Tyr-OH, Ac-Tyr-OEt 10 mM, H20 2.9%, CP 50 mg, organic solvent 10 ml, 30°C
0
2
4
6
8
10
i,;a; e r ~ i Figure 1 Effect of water content on initial reaction rate of AcTyr-OH esterification by free and CP-immobilized CT. Ac-Tyr-OH 10 mM, EtOH 20 ml, 30°C. (A) Free CT (10 mg); (A) immobilized CT (6 mg, CP 100 mg)
that Ac-Tyr-OH concentrates around the enzyme on CP, while the product is rapidly released from CP. Furthermore, since CP is a highly hydrophilic material, the local concentrations of water around the enzyme may also be higher than the bulk organic phase. Therefore, it may be assumed that CP enhances the reaction by providing the enzyme with enough water to activate it and by concentrating the substrates around the enzyme. The reactions exhibit Michaelis-Menten type kinetics, and the kinetic parameters obtained are summarized in Table 2. It is assumed that the esterification is the reverse process of ester hydrolysis, and the results indicate that the difference in the reaction rates by free and immobilized CT comes primarily from the difference in kcat. This may be reasonably interpreted by the above consideration on the activation of CT by immobilization. The immobilized CT retains high activities for AcTyr-OH esterification in many hydrophilic organic solvents, such as acetone, acetonitrile, THF, propylene carbonate, and 1,4-dioxane (data not shown). However, no reaction was detected in methanol and DMF.
Table 2
Kinetic parameters for Ac-Tyr-OH or Ac-Trp-OH esterification and Ac-Tyr-OMe transesterification in ethanol at 30°C
Reaction Esterification
Transesterification
586
CP-immobilized CT also exhibited high activity in ethyl acetate, which is less water-miscible and forms twophase reaction systems. Interestingly, in ethyl acetate, the reaction rate decreased at higher water contents, and the maximum rate (0.18/~mol min ~ mg CT-I at 30°C) was obtained with 2%-3% water. The result may be interpreted either by the decrease in the water-organic interface area or by partition of the product to the water phase at higher water contents. In nonpolar organic solvents, such as chloroform and toluene, most of the substrate was solubilized in the water phase, and the amounts of the product in the organic phase were negligibly small. Free and immobilized CT also catalyse transesterification of Ac-Tyr-OMe to Ac-Tyr-OEt in ethanol. The reactions are much faster than Ac-Tyr-OH esterification, as indicated by the values of k, Jk,n (which is equal to k:/K~) in Table 2. Since the esterification of Ac-Tyr-OH and the transesterification of Ac-Tyr-OMe in ethanol are considered to proceed via a common intermediate, Ac-Tyr-Enzyme, and the deacylation steps are identical, the difference in k.. is ascribed to the difference in k2; that is, acylation of the enzyme by Ac-Tyr-OMe may be much faster than by Ac-Tyr-OH. This may be responsible for the small increase in k,., for transesterification by immobilized CT compared to Ac-Tyr-OH esterification. Contrary to CT and subtilisin Carlsberg (STC), subtilisin BPN' (STB) is almost inactive for Ac-Tyr-OH esterification. 12 By immobilization to CP, however,
](cat
kcat/Km
Catalyst
H20 (%)
Km
Substrate
(mM)
(S 1)
(M-1 S-l)
Ac-Tyr-OH Ac-Tyr-OH Ac-Tyr-OH Ac-Tyr-OH Ac-Trp-OH Ac-Trp-OH Ac-Tyr-OMe Ac-Tyr-OMe
CT CT-CP STB-CP STC-CP CT CT-CP CT CT-CP
2.4 2.8 3.2 3.1 2.4 2.9 2.9 2.9
10 19 1.3 5.8 8.3 7.4 4.5 13
0.006 0.17 0.0098 0.015 0.0038 0.038 0.21 0.43
0.60 8.9 7.5 2.6 0.46 5.1 47 33
Enzyme Microb. Technol., 1991, vol. 13, J u l y
Ester and peptide synthesis by immobilized proteases: H. Kise and A. Hayakawa 100
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c~-Chymotrypsin (CT), subtilisin BPN' (STB), and subtilisin Carlsberg (STC) were immobilized by adsorption to porous ehitosan beads (Chitopearl, CP). The immobilized enzymes showed higher catalytic activities than free enzymes for amino acid esterification in many hydrophilic organic solvents except for methanol and DMF. In ethanol, the initial rate of the esterification increased with water content, whereas in ethyl acetate, the maximum rate was obtained at 2%-3% water. CP-immobilized CT also catalysed transesterification of Ac-Tyr-OMe in ethanol and peptide synthesis in acetonitrile from Ac-Tyr-OH or its ethyl ester and amino acid amides. The immobilized enzymes are highly stable in organic solutions, and can easily be separated from the reaction solutions. Repeated esterifications of Ac-Tyr-OH in acetonitrile by a CP-immobilized CT gave almost constant yields of the ester for more than 3 weeks.
Keywords: Protease immobilization; porous chitosan beads; ester synthesis; peptide synthesis; organic solvent
Introduction Recently a great number of research studies have been performed on synthetic reactions by enzymes in organic solvents. One of the promising methods for biosynthetic reactions is the utilization of proteases or lipases in hydrophilic (water-miscible) organic solvents with restricted amounts of water. There are many advantages in employing cosolvent systems, such as high solubilities of both polar and nonpolar substrates, shift of t h e r m o d y n a m i c equilibria by controlling water content, and absence of diffusion limitation across water/ organic solvent interfaces. The main drawback of cosolvent systems is the possible inactivation of enzymes due to direct contact with organic solvents such as ethanol, i-6 H o w e v e r , recent studies have revealed that e n z y m e s can retain their activity at high concentrations of organic solvents and catalyse many synthetic reactions, including amino acid esterification, 7-9 transesterification, 1°-12 and peptide synthesis.13-16 Recent studies have also revealed that the activities
* Enzymatic reactions in aqueous-media. X. Address reprint requests to Dr. Kise at the Institute of Materials Science, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Received 7 August 1990; revised 18 December 1990 584
Enzyme Microb. Technol., 1991, vol. 13, July
of immobilized proteases in organic solvents are strongly dependent on the nature of the support material. lv'~8 Furthermore, it was reported that enzymes can be activated by complexation with polysaccharides such as chitin or chitosan, 19 or by lyophilizing from certain buffer solutions or solutions of specific amino acids. 13'2° These results indicate that the interactions of enzymes with support materials strongly affect the activity and stability of the e n z y m e s in hydroorganic reaction systems. In order to obtain e n z y m e preparations of high activity and easy handling, we studied the immobilization of proteases to commercially available porous chitosan beads, and we report here the results on the catalytic activity and the long-term stability of the immobilized enzymes for ester and peptide syntheses in organic solvents.
Materials and methods Materials
Bovine pancreatic ~x-chymotrypsin (CT) having a specific proteinase activity, with N-benzoyl-L-tyrosine ethyl ester, of 50 units (/~mol min -1 mg i) (pH 7.8 at 25°C) was purchased from Sigma Chemical Co. Subtilisin Carlsberg (type VIII, STC) and subtilisin B P N '
© 1991 Butterworth-Heinemann
Ester and peptide synthesis by immobilized proteases: H. Kise and A. Hayakawa (type XXVII, STB) having specific proteinase activities, with casein, of 11.6 and 6.8 units, respectively, were also purchased from Sigma. All the substrates were the products of Sigma, except for N-acetyl-Ltyrosine methyl ester, which was prepared from Nacetyl-L-tyrosine in methanol by the catalysis of dry hydrogen chloride and recrystallized from ethyl acetate. The alcohols and all the solvents were purchased from Nacalai Tesque, Inc., and dried on 3-A molecular sieves. Porous chitosan beads (Chitopearl BCW 3010), which are the products and generous gift of Fuji Spinning Co., have an average diameter of I mm, pore size of 0.1-0.2 ~m,, and specific surface area of 120-150 m 2 g-l. They are insoluble in and highly resistant to organic solvents.
Enzyme immobilization Typically, 100 mg of dry Chitopearl (CP) was added to a solution of 10 mg enzyme in 0.4 ml of water, and the mixture was stored at 4°C overnight. Then, the CPenzyme preparation was washed several times with water on a glass filter. The amount of immobilized enzyme was determined by a spectroscopic measurement (280 nm) of the filtrates for the enzyme, and the amount of water in the CP-enzyme was determined from the increase in the weight. Alternatively, and more conveniently, CT was adsorbed to CP by mixing dry CP with an enzyme solution in a specific amount of water. The mixture was kept standing for 1 h at room temperature, and the preparation was used directly for reactions in organic solvents.
Ester synthesis and transesterification To a CP-enzyme preparation described above, a solution of N-acetyl-L-tyrosine (Ac-Tyr-OH) (mostly 10 raM), 50 mg of acetanilide, which was an internal standard for HPLC analysis, and a specific amount of water in 20 ml of ethanol was added, and the mixture was incubated with constant reciprocal shaking (about 150 cycles min- ~) at 30°C. When organic solvents other than ethanol were used, solutions containing 10% (v/v) (1.7 M) ethanol were used. The amounts of Ac-Tyr-OH and its ethyl ester (Ac-Tyr-OEt) were determined with an HPLC (JASCO Tri Rotar SR-I or Shimadzu LC-6A) using JASCO Finepak SIL C18 or Shimpak C-18 columns eluted with water-acetonitrile (50/50 by volume). The reaction rate was calculated from the amount of Ac-TyrOEt formed after 10-60 rain reactions. Transesterification was carried out in a similar manner using Ac-TyrOMe as a substrate in ethanol. The operational stability of immobilized CT was estimated by measuring the ini tial reaction rate and ester yield after 24 h for repeated esterification reactions in ethanol; the reaction solution was replaced every 24 h by a fresh substrate (10 mM) solution containing 2.9% water.
Peptide synthesis To a solution of amino acid amide hydrohalide (AANH2HX, 0.12 retool) and triethylamine (0.12 mmol) in
0.3 ml of water, a solution of Ac-Tyr-OH or Ac-TyrOEt (0.1 mmol) and acetanilide (25 rag) in 10 ml of acetonitrile was added. The solution was then added to the immobilized enzyme, and the mixture was incubated and the product was analyzed as above. Authentic samples of Ac-Tyr-AA-NH 2 were used for HPLC calibration.
Adsorption of Ac-Tyr-OH and Ac-Tyr-OEt to CP A mixture of 0.3 ml of water and 50 mg of CP was kept standing at room temperature for 1 h. Then a solution of Ac-Tyr-OH or Ac-Tyr-OEt in 10 ml of ethanol or acetonitrile was added to the mixture, and the whole mixture was incubated with shaking at 30°C. After 0.5 and 24 h, the amounts of the adsorbed materials were determined by performing HPLC analyses of the solutions as above.
Results and discussion
Enzyme immobilization The porous chitosan bead (Chitopearl, CP) has a large surface area and strongly adsorbs many proteins from aqueous solutions. CP has been utilized as a support for enzyme immobilization in aqueous solutions, 21 but to our knowledge, no report has appeared on the catalysis of CP-immobilized enzymes in organic solvents. We obtained CP-CT preparations of different CT loadings by changing the concentration of CT in aqueous solutions in which CP was immersed. The amount of adsorbed CT increased with its concentration in the aqueous solutions up to 13% of the weight of dry CP. However, the comparison of the catalytic activities of CP-CT preparations of different CT loadings suggested that higher Ioadings led to the decrease in the activity of the enzyme. Therefore, most of the reactions were carried out using preparations of CT contents below 12% (w/w).
Ester synthesis and transesterification It is known that the catalytic activity of proteases in water-organic cosolvents is strongly dependent on water content. This is also the case for the esterification of Ac-Tyr-OH by free and CP-immobilized CT, as shown in Figure 1. No reactions occurred without water, and the reaction rate increased with water content. It can be seen that the catalytic activity of CT markedly increases by immobilization to CP. No reactions were detected in the absence of the enzyme, and this rules out the possibility of the catalysis by CP. The increase in reaction rate and ester yield by immobilization to CP is much larger than that with porous silica gel, controlled porous glass (CPG), or celite. Ig The reaction mixtures consist of clear solutions and hydrated CP containing the enzyme. An HPLC analysis revealed that a part of the substrate, Ac-Tyr-OH, is also adsorbed to CP, whereas the product, Ac-Tyr-OEt, is less strongly adsorbed to CP (Table 1). The result suggests
Enzyme Microb. Technol., 1991, vol. 13, July
585
Papers Table 1 Adsorption of Ac-Tyr-OH and Ac-Tyr-OEt to CP in organic solvents a
0.20 ~i-
0.16
£2
'~
Adsorbed (%) Solvent
0 12
EtOH
0.08
CH3CN
1
:. 0.04
Time (h)
Ac-Tyr-OH
Ac-Tyr-OEt
0.5 24 0.5 24
8.5 9.0 20 22
0.9 0.8 0.8 2.6
a Ac-Tyr-OH, Ac-Tyr-OEt 10 mM, H20 2.9%, CP 50 mg, organic solvent 10 ml, 30°C
0
2
4
6
8
10
i,;a; e r ~ i Figure 1 Effect of water content on initial reaction rate of AcTyr-OH esterification by free and CP-immobilized CT. Ac-Tyr-OH 10 mM, EtOH 20 ml, 30°C. (A) Free CT (10 mg); (A) immobilized CT (6 mg, CP 100 mg)
that Ac-Tyr-OH concentrates around the enzyme on CP, while the product is rapidly released from CP. Furthermore, since CP is a highly hydrophilic material, the local concentrations of water around the enzyme may also be higher than the bulk organic phase. Therefore, it may be assumed that CP enhances the reaction by providing the enzyme with enough water to activate it and by concentrating the substrates around the enzyme. The reactions exhibit Michaelis-Menten type kinetics, and the kinetic parameters obtained are summarized in Table 2. It is assumed that the esterification is the reverse process of ester hydrolysis, and the results indicate that the difference in the reaction rates by free and immobilized CT comes primarily from the difference in kcat. This may be reasonably interpreted by the above consideration on the activation of CT by immobilization. The immobilized CT retains high activities for AcTyr-OH esterification in many hydrophilic organic solvents, such as acetone, acetonitrile, THF, propylene carbonate, and 1,4-dioxane (data not shown). However, no reaction was detected in methanol and DMF.
Table 2
Kinetic parameters for Ac-Tyr-OH or Ac-Trp-OH esterification and Ac-Tyr-OMe transesterification in ethanol at 30°C
Reaction Esterification
Transesterification
586
CP-immobilized CT also exhibited high activity in ethyl acetate, which is less water-miscible and forms twophase reaction systems. Interestingly, in ethyl acetate, the reaction rate decreased at higher water contents, and the maximum rate (0.18/~mol min ~ mg CT-I at 30°C) was obtained with 2%-3% water. The result may be interpreted either by the decrease in the water-organic interface area or by partition of the product to the water phase at higher water contents. In nonpolar organic solvents, such as chloroform and toluene, most of the substrate was solubilized in the water phase, and the amounts of the product in the organic phase were negligibly small. Free and immobilized CT also catalyse transesterification of Ac-Tyr-OMe to Ac-Tyr-OEt in ethanol. The reactions are much faster than Ac-Tyr-OH esterification, as indicated by the values of k, Jk,n (which is equal to k:/K~) in Table 2. Since the esterification of Ac-Tyr-OH and the transesterification of Ac-Tyr-OMe in ethanol are considered to proceed via a common intermediate, Ac-Tyr-Enzyme, and the deacylation steps are identical, the difference in k.. is ascribed to the difference in k2; that is, acylation of the enzyme by Ac-Tyr-OMe may be much faster than by Ac-Tyr-OH. This may be responsible for the small increase in k,., for transesterification by immobilized CT compared to Ac-Tyr-OH esterification. Contrary to CT and subtilisin Carlsberg (STC), subtilisin BPN' (STB) is almost inactive for Ac-Tyr-OH esterification. 12 By immobilization to CP, however,
](cat
kcat/Km
Catalyst
H20 (%)
Km
Substrate
(mM)
(S 1)
(M-1 S-l)
Ac-Tyr-OH Ac-Tyr-OH Ac-Tyr-OH Ac-Tyr-OH Ac-Trp-OH Ac-Trp-OH Ac-Tyr-OMe Ac-Tyr-OMe
CT CT-CP STB-CP STC-CP CT CT-CP CT CT-CP
2.4 2.8 3.2 3.1 2.4 2.9 2.9 2.9
10 19 1.3 5.8 8.3 7.4 4.5 13
0.006 0.17 0.0098 0.015 0.0038 0.038 0.21 0.43
0.60 8.9 7.5 2.6 0.46 5.1 47 33
Enzyme Microb. Technol., 1991, vol. 13, J u l y
Ester and peptide synthesis by immobilized proteases: H. Kise and A. Hayakawa 100
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