Reactivity of easily removable

protecting groups for glycine in

peptide synthesis using papain
as catalyst
M. Fité,* G. Alvaro,* P. Clapés,† J. López-Santin,* M. D. Benaiges,* and
G. Caminal*

*Department d’Enginyeria Quı́mica, Laboratori Associat al CNB, Facultat de Ciències,

Universitat Autònoma de Barcelona, Bellaterra, Spain; and †Unitat de Quı́mica i Bioquı́mica de
Proteı̈nes, Centre d’Investigació i Desenvolupament C.S.I.C., Barcelona, Spain

The reactivity of enzymatically removable protecting groups in peptide synthesis (phenyl acetyl and mandelyl) has
been studied for the papain-catalyzed condensation between different N-a protected esters of glycine and
H-Trp-OBzl. These protecting groups have also been compared with Z and Boc usually employed in chemical and
enzymatic peptide synthesis. PhAc-Gly-OCam and Mand-Gly-OBzl gave good yields (89 –90%) using papain
deposited onto celite in saturated ethyl acetate and with 0.2% (v/v) of buffer content, respectively. The above acyl
donors gave similar synthetic yields than Z-Gly-OCam and higher than Boc-Gly-OCam derivatives. All these
enzymatic synthesis reactions have been performed with the nucleophile as limiting reagent. In all cases, the final
yields were influenced by secondary reactions of the dipeptide product leading to its hydrolysis or additional
H-Trp-OBzl condensation; thus, it has been necessary to determine the reaction conditions which minimize the
undesired by-products. © 1998 Elsevier Science Inc.

Keywords: Papain; enzymatic peptide synthesis; n-a protecting groups; immobilized PGA

Introduction fragment condensation when there are labile groups that

Chemical and enzymatic peptide synthesis require the use of
conveniently protected amino acids to drive reactions to-
ward the target products. The use of protecting groups easy
to introduce and remove is an interesting goal1 which
greatly simplifies obtaining the final free peptide; moreover,
protected amino acids or peptides could present different
solubilities as well as reactivities depending on the nature of
the protecting group.2–3
In previous work,4 we studied the use of penicillin G
acylase (PGA) to introduce phenylacetic (PhAc) and man-
delic (Mand) acids in amino acids and peptides as protecting
groups. Although these protecting groups introduce racem-
ization when the synthesis is performed chemically,5 they
could be interesting in enzymatic synthesis mainly in
Address reprint requests to Dr. G. Caminal, Universitat Autonoma de
Barcelona, Laboratori Associat al CNB, Dept. of d’Enginyeria Quimica,
Facultat de Ciencies, 08193 Bellaterra, Spain
Received 18 June 1997; revised 5 February 1998; accepted 17 February
1998
could be affected by the usual deprotecting technology.
The above concepts have been applied in the context of
the total enzymatic synthesis of CCK-8, the biologically
active carboxyl-terminal octapeptide of cholecystokinin (H-
Asp-Tyr(HSO3)-Met-Gly-Trp-Met-Asp-Phe-NH2), a poten-
tial therapeutic agent of gastrointestinal function.6 In the
proposed scheme,7 the final step is the fragment condensa-
tion between Asp-Tyr-Met and Gly-Trp-Met-Asp-Phe-NH2;
therefore, glycine has to be deprotected before the final step
in the synthesis. The first group we employed was benzy-
loxycarbonyl (Z), widely used either in enzymatic and
chemical synthesis. One disadvantage was that the target
peptide has two methionine residues which made the final
deprotection steps by means of catalytic hydrogenation
rather difficult. Alternatively, the Z group was deprotected
by liquid hydrogen fluoride (HF), an efficient but dangerous
and difficult to scale-up method.
To circumvent these problems with the Z group and to
develop a simple process to obtain CCK-8, different n-a
protecting groups were tested in the condensation reaction
between R1-Gly-OR2 and H-Trp-OBzl. n-a-Phenylacetyl

Enzyme and Microbial Technology 23:199 –203, 1998

© 1998 Elsevier Science Inc. All rights reserved. 0141-0229/98/$19.00
655 Avenue of the Americas, New York, NY 10010 PII S0141-0229(98)00030-1
Papers
(PhAc), mandelyl (Mand), and t-butyloxycarbonyl (Boc)
glycine derivatives were studied and the reaction perfor-
mance of these groups was compared with the one obtained
using Z. The synthesis was performed under kinetic control
using different esters of these glycyl derivatives as acyl
donors.
Materials and methods
Materials
Papain (EC 3.4.22.2) from papaya latex (Type IV, 23 crystallized
and lyophilized powder, 10 –20 IU mg21 using benzyl arginine
ethyl ester (BAEE) as substrate); d(2)-mandelic acid and amino
acids: Gly-OMe z HCl and H-Trp-OBzl z HCl were purchased from
Sigma (St. Louis, MO). Gly-OBzl z HCl was obtained from
Bachem (Bubendorf, Switzerland). Phenylacetic acid was from
Fluka (Buchs, Switzerland). PhAc-Gly-OCam, Boc-Gly-OCam,
and Z-Gly-OCam were synthesized in our laboratory by a standard
procedure described by Martinez,8 PhAc-Gly-OMe and PhAc-Gly-
OBzl were synthesized as reported previously4 and 6-nitro-3-
(phenyl-acetamido) benzoic acid (NIPAB) was from Sigma. Semi-
purified extracts (containing 400 –500 IU ml21 extract) of
penicillin G acylase from E. coli (EC 3.5.1.11) were donated by
Antibioticos S.A. (León, Spain). The agarose gels 6BCL were a
gift of Hispanagar S.A. (Burgos, Spain). Celite-545 (particle size
20 – 45 mm) was from Fluka. EP-700 PA-6-Pulver (polyamide)
PA602 was donated by AKZO (Obernburg, Germany). Solvents
and other chemicals used in this work were analytical grade.
Enzyme immobilization
Papain was immobilized by deposition onto solid supports. The
enzyme (100 mg) was dissolved in 0.1 m boric acid-borax buffer
pH 8.2 (1 ml) and the support material (celite or polyamide) (1 g)
was added. After thorough mixing, the enzyme-support prepara-
tion was dried under vacuum for 24 h.
PGA was immobilized on agarose gels by multipoint covalent
attachment. The activation of agarose by preparation of glyoxyl-
agarose (agarose-O-CH2-CHO) was performed as described in the
literature.9 Immobilized PGA on agarose was prepared as report-
ed.10 PGA (;420 IU ml21 of crude extract) was immobilized on
gloxyl-agarose in 60 mm bicarbonate buffer containing 100 mm
phenylacetic acid at pH 10.1 and 25°C. After 3 h, the derivatives
were reduced with NaBH4 (1 mg ml21) for 30 min. Finally, the
immobilized preparation was washed with water and stored at 4°C.
(;470 IU ml21 agarose)
PGA test activity
The activity of immobilized PGA was measured using the sub-
strate NIPAB. The appearance of the reaction product was mea-
sured by UV-VIS detection at 405 nm.11 It was converted into
PGA international units (IU). One IU is defined as the amount of
PGA which hydrolyzes 1 mmol penicillin G min21 at 37°C and pH
7.5. Assays were performed in spectrophotometric cells provided
with a magnetic stirrer.
HPLC analysis
The amounts of substrates, products, and by-products produced in
the enzymatic reactions were determined by HPLC (Waters TM,
LC Module/plus) analysis using a LichroCART 250-4 HPLC
cartridge column (Lichrosphere 100, RP-18, 5 mm, 250 3 4 mm).
The solvent system was the following: solvent (A), 0.1% (v/v)
aqueous trifluoroacetic acid (TFA) and 0.007% (v/v) (TEA);
200 Enzyme Microb. Technol., 1998, vol. 23, August 15/September
solvent (B), 0.086% (v/v) TFA in CH3CN z H2O (4:1 v/v) and
0.07% (v/v) TEA.
A flow rate of 1 ml min21 was used and substrates/products
were detected by UV (254 nm). HPLC reaction monitoring:
gradient elution from 40 to 80% B in 24 min. K9(H-Trp-OBzl) 5
4.6; K9(PhAc-Gly-Trp-OBzl) 5 9.3; K9(PhAc-Gly-Trp-OH) 5
3.2; K9(PhAc-Gly-Trp-Trp-OBzl) 5 11.0; K9(Mand-Gly-Trp-
OBzl) 5 7.5; K9(Mand-Gly-Trp-Trp-OBzl) 5 9.4; K9(Boc-Gly-
Trp-OBzl) 5 10.0; K9(Boc-Gly-Trp-OH) 5 3.8; K9(Z-Gly-Trp-
OBzl) 5 10.8; K9(Z-Gly-Trp-OH) 5 3.6. (Capacity factor K9 5
(tr 2 t0/t0).
Enzymatic synthesis of dipeptide R1-Gly-Trp-OBzl
The synthesis of R1-Gly-Trp-OBzl was performed enzymatically
as follows. A 5 ml stoppered flask was prepared containing the
corresponding acyl donor (R1-Gly-OR2 where R1 5 Mand, PhAc,
or Boc and R2 5 Cam, Bzl, or Me) and nucleophile (H-Trp-OBzl)
in a molar ratio 2:1 dissolved in ethyl acetate (2 or 4 ml) with
different buffer content (boric acid-borax 0.1 m, pH 8.2) and
0.75% b-mercaptoethanol (v/v). The concentrations of acyl donor
and nucleophile were 80 mm/40 mm, respectively, except for the
experiments presented in Figures 1 and 2 and Table 2 which are at
40 mm/20 mm, respectively.
The reactions were started by adding 0.1 g of immobilized
catalyst (papain deposited onto polyamide or celite; 100 mg papain
g21 support). The flask was placed on a reciprocal shaker (200
rpm) thermostated at 25°C. The progress of all enzymatic reactions
was followed by HPLC. Samples (25 ml) were withdrawn from
each flask, acetic acid (10 ml) were added in order to stop the
reactions, and they were diluted in HPLC eluent before analysis.
Enzymatic synthesis of Mand-Gly-OBzl
Preparative synthesis was performed as follows. Mandelic acid
(1.901 g, 12.4 mmol) and H-Gly-OBzl (2.5212 g, 12.5 mmol) were
dissolved in 25 ml of biphasic system which consisted of a mixture
of ethyl acetate/200 mm citrate buffer in a 1:1 volume ratio. The
pH was adjusted to 6.5 and immobilized PGA (2,069 IU) was
added. The capped glass bottle was placed on a reciprocal shaker
(250 rpm) at room temperature.
The progress of the enzymatic reaction was followed by HPLC.
Samples (25 ml) were withdrawn from each phase of the reaction
medium and diluted in HPLC eluent before analysis.
Enzymatic synthesis of Mand-Gly-OMe
Preparative synthesis was performed as follows: mandelic acid
(2.2027 g, 14.5 mmol) and H-Gly-OMe (1,884 g; 15 mmol) were
dissolved in 30 ml of biphasic system as described above. The pH
was adjusted to 6.5 and immobilized PGA (1,330 IU) was added.
The progress of the enzymatic reaction was followed as
described before.
Results and discussion
Z-Gly-Trp-OBzl dipeptide was prepared in previous exper-
iments in high yield working with ethyl acetate at a w around
0.1 (0.2% v/v buffer content) as solvent with papain
adsorbed on polyamide as catalyst and a molar nucleophile
excess of two.
These conditions were the starting point to study the
behavior and reactivity of PhAc as the first protecting group
tested for PhAc-Gly-OR2 acyl donor where OR2 was methyl
ester (OMe), benzyl ester (OBzl), or carboxamidomethyl
ester (OCam). The acyl donor/nucleophile ratio was modi-

fied in order to reach the complete consumption of H-Trp-
OBzl which is necessary because it could undergo second-
ary reactions in the next steps of the overall synthesis of
CCK-8. Experimentally, it was determined that 2:1 was a
good acyl donor/nucleophile molar ratio for this purpose.
Working with PhAc-Gly-OCam, it was necessary to
increase the buffer content in ethyl acetate until 1.5% in
order to get its total solubilization. For this reason, experi-
ments were performed at 0.2% and 1.5% of buffer content
in order to be compared.
The time evolution of the reaction products is presented
in Figure 1. The reaction products followed by HPLC were:
dipeptide (PhAc-Gly-Trp-OBzl), hydrolyzed dipeptide
(PhAc-Gly-Trp-OH), and a product identified by mass
spectrometry as PhAc-Gly-Trp-Trp-OBzl. The hydrolysis
of acyl donor (PhAc-Gly-OH) was not quantified, because
its evolution with time did not affect the calculations of the
peptide yield when acyl donor is in excess. On the other
hand, PhAc-Gly-OH can be easily separated from the
dipeptide product by liquid extraction. As can be seen, both
the acyl donors PhAc-Gly-OR2 and the product PhAc-Gly-
Trp-OBzl react very fast with the nucleophile in the pres-
ence of a-chymotrypsin, giving PhAc-Gly-Trp-Trp-OBzl or
Enzyme Microb. Technol., 1998, vol. 23, August 15/September
Using papain as catalyst: M. Fité et al.
Figure 1 Time evolution concentration of products
obtained in the reaction between 40 mM PhAc-Gly-
OR2 and 20 mM H-Trp-OBzl with papain onto poly-
amide (100 mg enzyme g21 support) in ethyl acetate
(R2: Me, Bzl, Cam)
PhAc-Gly-Trp-OH as the main product depending on the
buffer content.
In the two buffer contents studied, carboxamidomethyl
ester was the best acyl donor, giving the highest yields. The
same behavior has been observed when a-chymotrypsin
was used as catalyst in other peptide synthesis reac-
tions.12,13 In the reactions presented in Figure 1, product
yield (dipeptide concentration/limiting reagent initial con-
centration) was low, being the best yield 28.6% with 1.5%
buffer (conditions in that PhAc-Gly-OCam was totally
solubilized). Working with 0.2% buffer, the actual acyl
donor/nucleophile ratio in solution was lower than two
which implies that H-Trp-OBzl could be in excess and
enhanced the PhAc-Gly-Trp-Trp-OBzl synthesis rate. On
the other hand, working with 1.5% buffer, hydrolysis of the
product (PhAc-Gly-Trp-OH) was increased; nevertheless,
as mentioned above, the highest product yield was obtained.
Due to the significance of the buffer content in the
reaction performance, this parameter was studied using
PhAc-Gly-OCam as acyl donor. Papain was immobilized by
deposition onto polyamide or celite as a support in order to
compare two supports with different physical properties
(aquaphilicity, surface area, and pore diameter).
201
Papers
Figure 2 Maximum yield obtained in the condensation be-
tween 40 mM PhAc-Gly-OCam and 20 mM H-Trp-OBzl in ethyl
acetate at different buffer contents working with papain (100 mg
enzyme g21 support) onto celite or polyamide
Increasing the buffer content in the medium gave higher
yields with both supports (Figure 2). Up to the complete
solubilization of PhAc-Gly-OCam, the acyl donor/nucleo-
phile ratio is lower than two, enhancing the formation of
PhAc-Gly-Trp-Trp-OBzl as mentioned before. When the
buffer content is higher than 1.5%, the acyl donor/nucleo-
phile ratio is always the same (2:1). The increase in yield
might be attributed to the higher activity of the enzyme at
higher buffer contents in organic media. The obtained
results seem to indicate that an increase in buffer content
increased the PhAc-Gly-enzyme complex formation rate to
a greater degree than the PhAc-Gly-Trp-enzyme rate. These
results seem contradictory with previously published data14
where the yield increased when lower buffer content was
used. In those experiments, the acyl donor was the limiting
reagent and its hydrolysis increased with increasing buffer
content, decreasing product yield. In the present work, the
limiting reactant is the nucleophile, and the hydrolysis of the
acyl donor does not influence yield.
The influence of the support on yield is shown in Figure
2. In all cases, the yield obtained working with celite was
higher than with polyamide, although both enzymatic prep-
arations contained the same amount of deposited papain.
The chemical nature of the support and its interaction with
the enzyme and/or substrate plays an important role. In this
case, celite, a support with less surface area but also less
aquaphilicity,14,15 increases synthetic yields around two
times compared with polyamide.
For the synthesis of the PhAc-Gly-Trp-OBzl, the best
conditions hence were: saturated ethyl acetate as solvent,
celite as support for papain immobilization, and PhAc-Gly-
OCam as acyl donor, obtaining an important enhancement
in the yield, reaching a value of 81.4%.
The reactivity of another n-protecting group, mandelic
acid, was also studied in this reaction. To obtain Mand-Gly-
OCam, the papain-catalyzed removal of the benzyl ester
from Mand-Gly-OBzl or Mand-Gly-OMe was performed
before the introduction of the carboxamidomethyl ester by a
202 Enzyme Microb. Technol., 1998, vol. 23, August 15/September
Table 1 Influence of N-a protecting group of Gly-OCam on
maximum product yield of the reaction between R1-Gly-OCam
or Mand-Gly-OBzl (80 mM) and H-Trp-OBzl (40 mM) with papain
onto celite (100 mg enzyme g21 support) in saturated ethyl
acetate
Substrate Yield (%) Time (min)
PhAc-Gly-OCam 81.4 5
Z-Gly-OCam 81.0 20a
Boc-Gly-OCam 73.9 160a
Mand-Gly-OBzl 89 40
R1: PhAc, Z, Boc
a
Experiments performed with 0.25 g of immobilized preparation
standard procedure described by Martinez et al.8 Unfortu-
nately, the final purification step has a very low yield, so
Mand-Gly-OCam synthesis was not competitive. Conse-
quently, we studied only Mand-Gly-OMe and Mand-Gly-
OBzl as acyl donors in the dipeptide reaction. Working with
methyl ester, the reaction rate was very slow and with
benzyl ester, synthetic yields of 89% (ethyl acetate 10.2%
buffer) and 86% (saturated ethyl acetate) were obtained. In
both buffer contents, the product yields obtained for the
mandelic protecting group are higher than those obtained
when phenylacetyl was used. With the mandelic acid group,
no significant variations with buffer content were observed.
The results indicate that Mand-Gly-Trp-OBzl is not a
good substrate for papain, and probably the free hydroxyl
group present in the mandelic acid molecule has a non-
favorable interaction with the enzyme, minimizing the
formation of Mand-Gly-Trp-OH or Mand-Gly-Trp-Trp-
OBzl; nevertheless, the hydrolysis product is detected,
being the only by-product formed.
In conclusion, mandelic acid could be a good protecting
group when the product obtained is the final product or
when the next step is not catalyzed by papain. In addition,
because of the presence of the free hydroxyl in mandelic
acid, the protected molecule is expected to be more soluble
in hydrophilic solvents. The yields obtained with Mand-
Gly-OBzl are very similar to the best ones obtained working
with PhAc-Gly-OCam.
For comparison, benzyloxycarbonyl and t-butyloxycar-
bonyl were tested as protecting groups of R1-Gly-OCam.
Table 1 shows the results obtained with those groups
together with phenylacetyl protecting the same ester and
mandelyl protecting Gly-OBzl ester. The experiments done
with Z and Boc groups were performed with 2.5 times more
immobilized enzyme than with Mand and PhAc groups, due
to slow rates with the former amount of enzyme.
Boc-Gly-OCam is the worst acyl donor tested, because it
conducted to the lower yield over a longer time. This
indicates its lower reactivity even using more enzyme.
The other groups tested gave similar results. It is difficult
to select one using only these criteria (yield and productiv-
ity). In the case of PheAc, the reaction is so fast that the
maximum yield presented corresponds to the first sample
taken out, so it is not certain that this value is the real
maximum. For this reason, a set of experiments were
performed at different enzymatic load using PhAc-Gly-
OCam as acyl donor. As mentioned in Materials and

Table 2 Influene of encymatic load on maximum product yield
on the reaction between PhAc-Gly-OCam (40 mM) and H.Trp-
OBzl (20 mM) with papain onto celite in saturated ethyl acetate
Enzymatic load
(mg enzyme g21 support) Yield (%) Time (min)
10 ND .60
15 90.6 30
30 87.5 10
60 87.4 8 techniques. Chem. Rew. 1994, 94, 911–937
100 81.4 ,5
Not determined, ND
methods, all experiments were made at 100 mg enzyme
g21 support, taking into account previous work with papa-
in16 in order to obtain high initial reaction rates.
In the case of PhAc-Gly-OCam, the reaction rate was so
fast that the consecutive reaction (product hydrolysis) was
significant when the first sample was taken, so we tested
lower enzymatic loads in order to get slower reaction rates
to determine the optimal time to stop the reaction.
In Table 2, the maximum yield obtained and the time to
reach it is presented for the different enzymatic loads. As
can be seen, the yields obtained are very similar but
chromatographic analysis showed a significant difference.
Up to 30 mg g21, the hydrolysis product appears when the
limiting reactant was totally consumed and then a maximum
yield of 90.6% is obtained. A 100% yield should be
expected, but adsorption on the support decreases the
maximum yield obtained in the liquid. From 30 mg g21 up
to 100 mg g21, the detection of hydrolysis product started
before the limiting reactant was completely exhausted.
Consequently in this reaction, the optimal load is 15 mg
enzyme g21 support, because the maximum yield is ob-
tained at a reasonable reaction time (30 min); moreover, to
work at a low enzymatic load is cheaper, which is interest-
ing from the process point of view.
In conclusion, PhAc-Gly-OCam and Mand-Gly-OBzl are
similar as acyl donors in this dipeptide synthesis and
slightly better than Z-Gly-OCam which is used at higher
enzymatic content than the others. In addition, both protect-
ing groups, PhAc and Mand, are easily removable enzymat-
ically, making these groups as good alternatives in enzy-
matic peptide synthesis.
Acknowledgments
This work was financially supported by the Spanish CICYT
(project BIO95-1083). The Dept. of Chemical Engineering
UAB is a member of the Centre de Referència en Biotec-
Enzyme Microb. Technol., 1998, vol. 23, August 15/September
Using papain as catalyst: M. Fité et al.
nologia de la Generalitat de Catalunya. M. Fité acknowl-
edges a grant from this Center. The authors express their
gratitude to Antibioticos S.A. for supplying penicillin G
acylase and Hispanagar S.A. for the agarose gels.
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