Encapsulation of Crosslinked Penicillin GAcylase Aggregates in Lentikats: Evaluationof a Novel Biocatalyst in Organic Media
Lorena Wilson,1 Andres Illanes,2 Benevides C. C. Pessela,2 Olga Abian,2
Roberto Fernandez-Lafuente,1 Jose M. Guisan1
1Departamento de Biocatalisis, Instituto de Catalisis,Campus UAMCantoblanco, 28049 Madrid, Spain; telephone: +34-91-585-4809;fax: +34-91-585-4760; e-mail: [email protected] of Biochemical Engineering, Universidad Catolica de Valparaıso,Valparaıso, Chile
Received 15 October 2003; accepted 13 January 2004
Published online 15 April 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20107
Abstract: The encapsulation of crosslinked enzyme aggre-gates (CLEA) of penicillin G acylase into a very rigid poly-mericmatrix basedonpolyvinyl alcohol (LentiKats) hasbeenused successfully to improve the inadequate mechanicalproperties of CLEA. This encapsulation decreased CLEAactivity byonly around40%.Ascompensation, a significantimprovement in the stability of the CLEA in the presence oforganic solvents was detected. This could be related to thehighly hydrophilic environment inside the LentiKats bio-catalysts: Partition experiments showed that the concen-tration of dioxane inside LentiKats was lower than in thereaction medium. In fact, thermal stability was about thesame as in the corresponding CLEA. This permitted greatimprovement in the reaction rate for thermodynamicallycontrolled synthesis of a model antibiotic (using phenyl-acetic acid and 7-amino-deacetoxycefalosporanic acid).Even more importantly, yields could be improved by usingLentiKats-encapsulated CLEA, very likely by a favorableproduct/substrate partition. Thus, this very simple tech-nique not only provides an efficient technique for solving themechanical stability problemassociatedwithCLEA,but alsogreatly improves the behavior of CLEA in organic media.B 2004 Wiley Periodicals, Inc.
Keywords: LentiKats; entrapment; immobilization; poly-vinyl alcohol; hydrogel; crosslinked enzyme aggregates;penicillin acylase; enzymes in organic solvents
INTRODUCTION
One of the challenges of using enzymes for organic synthesis
is to produce biocatalysts that are strong enough to cope with
the harsh conditions of nonaqueous media, which are often
required for such reactions (Klibanov, 2001). Crosslinked
enzyme crystals (CLEC) (Noritomi et al., 1998) and cross-
linked enzyme aggregates (CLEA) (Broun, 1977; Cao et al.,
2003; Tyagi et al., 1999) have been found to be powerful
catalysts in organic media. The former have the drawback of
cost, mainly because very pure enzyme preparations are
required for the crystallization step.This is certainlynot so for
CLEA, where themain drawback is the difficulty in handling
and recovery of biocatalyst particles due to their low
mechanical resistance. Khare et al. (1991) tried to solve this
problem by aggregation of proteins inside polymeric beads
(i.e., Sephadex).
Lens-shaped polyvinyl alcohol (PVA) hydrogel particles
(LentiKats) are very promising as matrices for biocatalysis
because of their good mechanical properties and adequate
geometry in terms of mass transfer and ease of separation
(Jekel et al., 1998). LentiKats have been used successfully
for cell immobilization (Durieux et al., 2000; Lozinsky
and Plieva, 1998). However, enzymes are too small to be
retained effectively and therefore some kind of supra-
molecular coupling is required, such as complexation of
the enzyme with polyelectrolytes (Czichocki et al., 2001;
Groger et al, 2001). Use of CLEA is very promising for the
creation of a supramolecular protein structure, which is
explored in this work for the enzyme penicillin acylase.
Penicillin acylase (penicillin amidohydrolase, E.C.3.5.1.11)
is a remarkably versatile enzyme that not only catalyzes the
hydrolysis of h-lactam antibiotics, which is its present
industrial application (Bruggink et al., 1998; Parmar et al.,
2000), but also, under the proper reaction conditions, can
catalyze reverse reactions of syntheses to yield semi-
synthetic h-lactam antibiotics (Arroyo et al., 2003; Wegman
et al., 2001) and many other reactions of organic synthesis
(Basso et al., 2001; Ebert et al., 1996; Fernandez-Lafuente
et al, 1998; Fite et al., 1997; Illanes and Fajardo, 2001;
Rocchietti et al., 2002; Roche et al., 1999; Stambolieva et al.,
1998; van Langen et al., 2000).
Penicillin acylase in the form of CLEC is already in the
market, and CLEA of penicillin acylase have been produced
at the laboratory scale and tested for the synthesis of
h-lactam antibiotics (Cao et al., 2001). In this work, the
B 2004 Wiley Periodicals, Inc.
Correspondence to: R. Fernandez-Lafuente
Contract grant sponsors: Spanish CICYT; Program of International
Cooperation CSIC (Spain); CONICYT (Chile); AECI
Contract grant numbers: PPQ 2002-01231 A; COST 840
preparation of LentiKats from CLEA of penicillin acylase is
reported. This encapsulation may solve the problem
associated with the poor physical properties of the final
biocatalyst. Moreover, the stability of CLEA in organic
media can potentially be increased even further by creating
a more hydrophilic microenvironment surrounding the
enzyme, such as that provided by PVA within LentiKats.
This may promote a certain partition of the hydrophobic
organic solvent molecules away from the biocatalyst, in a
manner similar to when using polymeric hydrophilic
polymers (Abian et al., 2001).
MATERIALS AND METHODS
Materials
Soluble enzyme Penicillin G acylase (PGA-SE) from
Escherichia coli (with a purity of only 10%) and 7-amino-
deacetoxycephalosporanic acid were kindly provided by
Antibioticos SA (Leon, Spain). Sodium borohydride and
phenylacetic acid were from Sigma, and polyethylene-
glycol 600 (PEG) was from Merck. Glutaraldehyde solu-
tion was from Fluka. All other reagents were of analytical
grade. LentiKat Liquid and LentiKat Printer, used to
form identical lens-shaped LentiKats containing CLEA,
were from GeniaLab (Braunschweig, Germany). PGA
immobilization on glyoxyl-agarose (GA), was carried out
as previously described using an enzyme loading of
200 IUH/mL (Fernandez-Lafuente et al., 1998). Immobi-
lized PGA (Fluka) was also used (PGA-Fluka) under assay
conditions of 50 IUH/mL.
Assays
Enzyme activity of hydrolysis was determined using a
pH-stat (Model DL50, Mettler-Toledo) to titrate the H+
produced by the hydrolysis of 10 mM Penicillin G in 0.1 M
sodium phosphate (pH 8) at 25jC, using 50 mM NaOH as
titrant. One international unit of hydrolytic activity of PGA
(IUH) was defined as the amount of enzyme that could
hydrolyze 1 Amol of Penicillin G per minute under the
conditions just described.
Enzyme activity of synthesis was determined as the
initial rate of synthesis of deacetoxycephalosporin G
(DCG) at 4jC, from 12.5 mM 7-amino-deacetoxycephalo-
sporanic acid (7-ADCA) and 12.5 mM phenylacetic acid
(PAA), in a medium composed of 75% (v/v) dioxane and
25% (v/v) 100 mM phosphate buffer (pH 7.0). The reaction
mixture was gently stirred and samples were taken at
different timepoints; the mixture was then dissolved in 25%
(v/v) acetronitrile in 10 mM phosphate buffer (pH 3.0)
and residual substrates and product assayed by high-
performance liquid chromatography (HPLC). One interna-
tional unit of synthetic activity of PGA (IUS) was defined
as the amount of enzyme that synthesizes 1 Amol of DCG
per minute under the aforementioned conditions.
Protein was determined according to the method
described by Bradford (1976).
Preparation of Biocatalysts
CLEA
CLEA of penicillin acylase were prepared by adding 20 mL
of PEG to 10 mL of PGA solution (purity 10%) under
strong agitation. Glutaraldehyde (2 mL) was then added
to crosslink the enzyme precipitate. The reaction volume
was then duplicated by adding 100 mM sodium bicarbo-
nate solution (pH 10) and 60 mg of sodium borohydride.
After 15 min, 60 mg more of sodium borohydride was
added and, 15 min later, the CLEAs produced were washed
repeatedly with 100 mM sodium phosphate buffer (pH 7.0)
and centrifuged.
CLEA into LentiKats
LentiKats were produced according to the protocol from
GeniaLab (Lentikats Tips & Tricks, Braunschweig; Ger-
many), as reported previously (Jahnz et al., 2001).
Encapsulation of CLEA into Lentikats was done as pre-
viously reported (Wilson et al., 2002). LentiKat Liquid and
a suspension of wet CLEA were mixed at a mass ratio of
4:1. The mixture was then fed to the LentiKat Printer where
small droplets were dripped over a plastic dish and exposed
to drying and stabilization to produce the encapsulated
CLEA (CLEA-LK).
Stability of Biocatalysts
Stability in Organic Solvent
Dioxane was selected as a suitable cosolvent to study bio-
catalyst stability because it is highly polar and deleterious
for penicillin acylase (Abian et al., 2001). To determine
inactivation in the presence of the organic cosolvent, the
biocatalysts were washed and equilibrated at 4jC with
75% (v/v) dioxane in 100 mM phosphate buffer (pH 7.0).
Periodically, samples were withdrawn and the residual
activity of synthesis was determined. Experiments were
performed in triplicate.
Thermal Stability
To determine thermal inactivation of the biocatalysts, the
previously described procedure was followed; this time,
however, after equilibration in 100 mM phosphate buffer
(pH 8.0), the temperature was raised to 50jC and the residual
hydrolytic activity of each sample was determined. Results
were adequately represented by a first-order mechanism
of inactivation, which was used to determine biocatalyst
half-life from extrapolation of the experimental data (Illanes
et al., 1996). Experiments were performed in triplicate.
WILSON ET AL.: EVALUATION OF A NOVEL BIOCATALYST IN ORGANIC MEDIA 559
Mechanical Resistance of LentiKats
Structural integrity of CLEA-LK in aqueous and organic
media was tested by incubation at 20jC for 50 days under
agitation at 40 rpm in a rotary shaker (Selecta, Movil-Rod).
Twenty grams of CLEA-LK was suspended in 200 mL
of either aqueous (100 mM phosphate buffer, pH 7.0) or
organic (50% [v/v] dioxane in 100 mM phosphate buffer,
pH 7.0) media, in a flask with a diameter of 10 cm.
Samples were taken at different intervals and filtered
through a metallic mesh. Residual activity was
measured for CLEA-LK and the filtrate to determine the
released CLEA to the medium from the LentiKats.
Experiments were performed in triplicate. Shape and
weight of the ‘‘Lents’’ were also evaluated.
Synthesis of DGC
The reaction was conducted using a batch reactor thermo-
stated at 4jC, with 12.5 mM 7-ADCA and 12.5 mM PAA
in a medium composed of 75% (v/v) dioxane and 25% (v/v)
25 mM phosphate buffer (pH 7.0). DCG was determined by
HPLC. Experiments were performed in triplicate.
RESULTS AND DISCUSSION
Preparation of Biocatalysts
The activity of the CLEA was around 350 IUH/g, bearing in
mind that we used a nonpurified PGA preparation (only
10% purity). The low purity of the sample showed that: (i)
the aggregation did not require use of purified proteins; and
(ii) the presence of a large amount of other nonactive
proteins could reduce the diffusion limitations (and thus
reduce volumetric activity). In fact, a yield close to 70%
was achieved during preparation of the CLEA.
These CLEA were encapsulated in LentiKats (20% in
weight). The activity of CLEA-LK was >40 IUH/g (bearing
in mind that 80% of the mass was wet PVA). The activity
recovery after trapping the CLEA in the LentiKats was
around 60%. This decrease in activity could be promoted
by diffusion restrictions within the LentiKats, although
some enzyme inactivation during the drying or stabilizing
process may have occurred. In fact, some activity could
have been recovered if the CLEA-LK were broken into
smaller fragments.
Stability of Biocatalysts
Stability in Organic Solvent
The inactivation courses in the organic medium of CLEA-
LK PGA and the corresponding CLEA, along with other
PGA preparations, is shown in Figure 1A.
Stability was greatly enhanced by encapsulation of the
CLEA; that is, the half-life of CLEA was only 8.9 h,
whereas CLEA-LK remained fully active after 300 h of
incubation at this high dioxane concentration. The prepa-
ration from Fluka was much less stable than the CLEA
preparation, whereas soluble enzyme was readily inacti-
vated under these conditions. The enzyme immobilized on
glyoxyl agarose (a derivative very stabilized via multi-
point covalent attachment) exhibited a stability slightly
higher than that of CLEA, but it clearly had less stability
than CLEA-LK.
To determine whether the hydrophilic environment with-
in the LentiKats promoted cosolvent exclusion from the
enzyme microenvironment, the dioxane partition between
the external medium and the LentiKats was studied. Ten
grams of LentiKats with 80% moisture was placed in the
same amount of pure dioxane and left at room temperature
for 3 h under agitation. Samples were taken at the beginning
and at the end of the incubation period and the amount of
Figure 1. (A) Stability of different PGA preparations in the presence of
75% (v/v) dioxane and 25% (v/v) 100 mM phosphate buffer (pH 7) at 4jC.(B) Thermal inactivation at 50jC in 100 mM phosphate buffer (pH 8.0).
(.) CLEA, (o) CLEA-LK, (n) PGA-Fluka, (X) glyoxyl-agarose PGA,
and (E) soluble PGA.
560 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 86, NO. 5, JUNE 5, 2004
dioxane in the external medium was determined by HPLC.
The dioxane concentration in the external medium at the
end of the experiment was 71% (v/v), which is considerably
higher than the dioxane concentration under nonpartition
conditions (55%). Longer incubation times did not show a
change in these percentages. Thus, a clear partition effect
of dioxane from the LentiKats was demonstrated; this
lowering in the solvent concentration promoted high
stabilization of the PGA (Fernandez-Lafuente et al.,
1996). Therefore, CLEA-LK is a biocatalyst well-equipped
to perform at high cosolvent concentrations, which is rel-
evant for the enzymatic production of semisynthetic
h-lactam antibiotics.
Thermal Stability
Figure 1B shows similar thermal inactivation profiles for
CLEA and CLEA-LK, with no significant differences in
thermal stability. Thermal inactivation was adequately
described by a first-order mechanism of inactivation, and
the extrapolated values of half-life for CLEA and CLEA-LK
were 9.7 h and 8.7 h, respectively. The half-lives of these
biocatalysts were about the same (in fact, somewhat lower
for CLEA-LK). This result is not surprising because there is
no reason to believe that CLEA encapsulation would
contribute to stabilization of the enzyme structure.
In this case, CLEA and CLEA-LK proved considerably
less stable than the GA preparation, yet much more stable
than the Fluka preparation or soluble PGA.
These results suggest that the stability of CLEA-LK in
organic media is a consequence of the hydrophilic micro-
environment that surrounds the protein enzyme rather than
a conformational effect. In contrast, multipoint covalent
attachment in GA stabilized the conformation of the en-
zyme, making it more rigid and thus more thermostable.
Mechanical Resistance of LentiKats
CLEA-LK was incubated for 50 days at 20jC under
agitation in aqueous medium and in the presence of 50%
dioxane. No protein or enzyme activity was detected out-
side the LentiKats, meaning that no leakage occurred
during the prolonged incubation. Neither fracture nor alter-
ation in LentiKat shape was observed. The stability of
CLEA-LK after such prolonged incubation was very high,
with a residual hydrolytic activity of >95% in both media.
This behavior makes CLEA-LK a very promising bio-
catalyst for reactor operation.
Synthesis of DCG
Synthesis of DCG under thermodynamic control with
CLEA-LK and the corresponding CLEA is presented in
Figure 2. Although the initial rates were quite similar for
both preparations (using similar weight of CLEA in both
cases), the final yield was significantly higher in CLEA-LK
than in CLEA (55% and 43%, respectively). This is a
promising result, suggesting that conversion in thermody-
namically controlled synthesis can be modified as a
consequence of the partition effect provoked by the hydro-
philic environment within the LentiKats. This is done by
producing a favorable partition of substrates (more hydro-
philic) to the surrounding enzyme and product (more
hydrophobic) removal from the enzyme. A more compre-
hensive study of the effect of LentiKats on partition of
substrates and products in different organic medium is
underway.
The operational stability of CLEA-LK was assessed by
performing five successive batches of DCG production.
Full activity was recovered in the different reaction cycles
and identical yields were maintained.
CONCLUSIONS
Encapsulation of PGA CLEA in PVA hydrogels produced
LentiKats biocatalyst having greatly improved mechanical
properties with respect to fragile, nonencapsulated CLEA.
The hydrophilic environment of CLEA encapsulated into
LentiKats protected the enzyme from inactivation in or-
ganic media, but conferred no protection against heat
inactivation. These results are consistent with the proposed
mechanism of cosolvent exclusion from LentiKats particles,
which is further supported by the study on dioxane partition
between the external medium and the inside of the
LentiKats. This hydrophilic environment of LentiKats
favored the thermodynamically controlled synthesis
of deacetoxycephalosporin G in organic medium not
only by increasing synthesis rate, as expected, but also
increasing final equilibrium conversion, which could be
attributed to the favorable partition of the (more) hydrophilic
Figure 2. Synthesis of deacetoxycephalosporin G from 12.5 mM
7-ADCA and 12.5 mM PAA using different preparations of PGA at 4jCin a medium composed of 75% (v/v) dioxane and 25% 100 mM phosphate
buffer (pH 8.0). Two grams of CLEA (.) or 10 g CLEA-LK (o),
previously equilibrated with the reaction media, were mixed with 23 or
15 mL (respectively) of reaction media.
WILSON ET AL.: EVALUATION OF A NOVEL BIOCATALYST IN ORGANIC MEDIA 561
substrates into the enzyme hydrophilic environment
within the LentiKats and the (more) hydrophobic product
away from it.
The present results show that PGA CLEA LentiKats are
particularly suitable biocatalysts for the synthesis ofh-lactamantibiotics at high concentration of organic cosolvents,
conditions at which enzymes are usually unstable and poorly
active, although adequate for synthesis due to depressed
water activity and displacement of ionic equilibrium toward
nonionized reactive forms of substrates.
LentiKats, successfully used for cell immobilization, are
proposed as suitable matrices for the immobilization of
enzyme aggregates. Lower specific activities and eventual
diffusion restrictions are problems that remain to be solved
by optimizing enzyme immobilization procedures.
The authors thank Antibioticos SA (Leon, Spain) for the generous
supply of penicillin acylase. LentiKats Printer was kindly donated
by M. Schlieker and K.-D. Vorlop. We also thank Dr. A. Berenguer
for his valuable suggestions.
References
Abian O, Mateo C, Fernandez-Lorente G, Palomo J, Fernandez-Lafuente
R, Guisan J. 2001. Stabilization of immobilized enzymes against
water-soluble organic cosolvents and generation of hyperhydrophilic
microenvironments surrounding enzyme molecules. Biocatal Biotrans-
form 19:489–503.
Arroyo M, de la Mata I, Acebal C, Castillon M. 2003. Biotechnological
applications of penicillin acylases: State of the art. Appl Microbiol
Biotechnol 60:507–514.
Basso A, Biffi S, de Martin L, Gardossi L, Linda P. 2001. Quantitative
acylation of amino compounds catalysed by penicillin acylase in
organic solvent at controlled water activity. Croat Chem Acta 74:
757–762.
Bradford M. 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the protein-dye binding. Anal
Biochem 72:248–254.
Broun G. 1977. Chemically aggregated enzymes. In: Mosbach K, editor.
Methods in enzymology. New York: Academic Press. p 263–269.
Bruggink A, Roos E, de Vroom E. 1998. Penicillin acylase in the industrial
production of h-lactam antibiotics. Org Proc Res Devel 2:128–133.
Cao L, van Langen F, van Rantwijk F, Sheldon R. 2001. Cross-linked
aggregates of penicillin acylase: Robust catalysts for the synthesis of
h-lactam antibiotics. J Mol Catal B Enzym 11:665–670.
Cao L, van Langen L, Sheldon R. 2003. Immobilised enzymes: Carrier-
bound or carrier-free?. Curr Opin Biotechnol 14:1–8.
Czichocki G, Dautzenberg H, Capan E, Vorlop K. 2001. New and effective
entrapment of polyelectrolyte – enzyme complexes in LentiKats.
Biotechnol Lett 23:1303–1307.
Durieux A, Nicolay X, Simon J. 2000. Continuous malolactic fermentation
by Oenococcus oeni entrapped in LentiKats. Biotechnol Lett 22:
1679–1684.
Ebert C, Gardossi L, Linda P. 1996. Control of enzyme hydration in
penicillin amidase catalyzed synthesis of amide bond. Tetrahedr Lett
37:9377–9380.
Fernandez-Lafuente R, Rosell C, Guisan J. 1998. Modulation of the
properties of penicillin G acylase by acyl donor substrates during N-
protection of amino compounds. EnzymeMicrob Technol 22:583–587.
Fernandez-Lafuente R, Rosell CM, Guisan JM. 1996. Dynamic reaction
design of enzymatic biotransformations in organic media: Equilibrium
controlled synthesis of antibiotics by penicillin G acylase. Biotechnol
Appl Biochem 24:139–143.
Fite M, Capellas M, Benaiges M, Caminal G, Clapes P, Alvaro G. 1997.
N-protection of amino acid derivatives catalyzed by immobilized
penicillin G acylase. Biocatal Biotransform 15:317–332.
Groger H, Capan E, Barthuber A, Vorlop KD. 2001. Asymmetric synthesis
of an (R)-cyanohydrin using enzymes entrapped in lens-shaped gels.
Org Lett 3:1969–1972.
Illanes A, Altamirano C, Zuniga M. 1996. Thermal inactivation of pen-
icillin acylase in the presence of substrate and products. Biotechnol
Bioeng 50:609–616.
Illanes A, Fajardo A. 2001. Kinetically controlled synthesis of ampicillin
with immobilized penicillin acylase in the presence of organic
cosolvents. J Mol Catal B Enzym 11:605–613.
Jahnz U, Wittlich P, Prusse U, Vorlop K-D. 2001. New matrices and
bioencapsulation processes. In: Hofman M, Thonart P, editors.
Engineering and manufacturing for biotechnology: Focus on bio-
technology. Dordrecht: Kluwer. p 293–307.
Jekel M, Buhr A, Willke T, Vorlop K. 1998. Immobilization of biocatalysts
in LentiKats. Chem Eng Technol 21:275–278.
Khare S, Vaidya S, Gupta M. 1991. Entrapment of proteins by aggregation
within Sephadex beads. Appl Biochem Biotechnol 27:205–216.
Klibanov A. 2001. Improving enzymes by using them in organic solvents.
Nature 409:241–246.
Klibanov A. 1997. Why are enzymes less active in organic solvents than in
water? Trends Biotechnol 15:97–101.
Lozinsky V, Plieva F. 1998. Polyvinilalcohol cryogels employed as
matrices for cell immobilization. 3. Overview of recent research and
developments. Enzyme Microb Technol 23:227–242.
Noritomi H, Koyama K, Kato S, Nagahama K. 1998. Increased thermo-
stability of cross-linked enzyme crystals of subtilisin in organic
solvents. Biotechnol Techniq 12:467–469.
Parmar A, Kumar H, Marwaha S, Kennedy J. 2000. Advances in enzymatic
transformation of penicillins to 6-aminopenicillanic acid. Biotechnol
Adv 18:289–301.
Rocchietti S, Urrutia A, Pregnolato M, Tagliani A, Guisan J, Fernandez-
Lafuente R, Terreni M. 2002. Influence of the derivative preparation
and substrate structure on the enantioselectivity of penicillin G
acylase. Enzyme Microb Technol 31:88–93.
Roche D, Prasad K, Repic O. 1999. Enantioselective acylation of h-aminoesters using penicillin G acylase in organic solvents. Tetrahedr
Lett 40:3665–3668.
Stambolieva N, Mincheva Z, Galunsky B. 1998. Kinetic comparison of
penicillin amidase transfer of nonspecific and specific acyl moieties to
7-ADCA. Biocatal Biotransform 16:225–232.
Tyagi R, Batra R, Gupta M. 1999. Amorphous enzyme aggregates:
Stability toward heat and aqueous–organic cosolvent mixtures.
Enzyme Microb Technol 24:348–354.
van Langen L, Oosthoek N, Guranda D, van Rantwijk F, Svedas V,
Sheldon R. 2000. Penicillin acylase catalyzed resolution of amines in
aqueous organic solvents. Tetrahedr Asymm 11:4593–4600.
Wegman M, Janssen M, van Rantwijk F, Sheldon R. 2001. Towards
biocatalytic synthesis of h-lactam antibiotics. Adv Synth Catal
343:559–576.
Wilson L, Illanes A, Abian O, Fernandez-Lafuente R, Guisan J. 2002.
Encapsulation of very soft cross-linked enzyme aggregates (CLEAs)
into very rigid LentiKats. FAL Agric Res 241:121–125.
562 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 86, NO. 5, JUNE 5, 2004
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