Enabling industrial biocatalytic processes by application of silicone-coated enzyme preparations

Article abstract of DOI:10.1002/adsc.201100421

Composite particles of the commercial lipase preparation NZ435 and silicone (silcoat-NZ435) have recently been described as promising biocatalysts for synthetic use. In this study, their actual potential for enhanced performance in industrial applications was evaluated, focusing on scenarios where carrier disintegration and catalyst leaching constitute key limitations. All three investigated model reactions, the syntheses of myristyl myristate, poly(ethylene glycol) 400-coconut fatty acid monoester and ethylene oxide and propylene oxide copolymer (EO/PO)-oleic acid diester, were considerably improved in terms of the maximal number of reaction cycles performed with the same batch of catalyst, and consequently in terms of the obtainable product amount. The total turnover numbers (TTN) increased by a factor up to 50, making the realization of this type of reactions in an industrial process more feasible. The utility of silcoat-NZ435 for stereoselective transformations was demonstrated with the enantioselective acylation of 1-phenylethanol with vinyl butyrate, in which full retention of the excellent stereoselectivity of native NZ435 was observed. Moreover, it was demonstrated for the first time that the methodology by which silcoat-catalysts are obtained can be successfully transferred to alternative carriers and enzymes (e.g., protease, esterase and laccase), opening a broad field for the implementation and advancement of biocatalysis in industrial processes. Copyright

Full text of DOI:10.1002/adsc.201100421

FULL PAPERS
DOI: 10.1002/adsc.201100421
Enabling Industrial Biocatalytic Processes by Application of
Silicone-Coated Enzyme Preparations
R. Nieguth,a M. Eckstein,b L. O. Wiemann,a,c O. Thum,b,*
and M. B. Ansorge-Schumachera,*
a
Technische Universitꢀt Berlin, Institut fꢁr Chemie, Fachgebiet Enzymtechnologie (TC4), Str. des 17. Juni 124, 10623
Berlin, Germany
Fax : (+49)-30-314-22127; e-mail: m.ansorge@chem.tu-berlin.de
Evonik Goldschmidt GmbH, Goldschmidtstr. 100, 45127 Essen, Germany
b
Fax : (+49)-201-173-711658; e-mail: oliver.thum@evonik.com
Current address: Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Project Group BioCat,
c
Schulgasse 16, 94315 Straubing, Germany
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adcs.201100421.
Abstract: Composite particles of the commercial of this type of reactions in an industrial process more
lipase preparation NZ435 and silicone (silcoat- feasible. The utility of silcoat-NZ435 for stereoselec-
NZ435) have recently been described as promising tive transformations was demonstrated with the
biocatalysts for synthetic use. In this study, their enantioselective acylation of 1-phenylethanol with
actual potential for enhanced performance in indus- vinyl butyrate, in which full retention of the excellent
trial applications was evaluated, focusing on scenar- stereoselectivity of native NZ435 was observed.
ios where carrier disintegration and catalyst leaching Moreover, it was demonstrated for the first time that
constitute key limitations. All three investigated the methodology by which silcoat-catalysts are ob-
model reactions, the syntheses of myristyl myristate, tained can be successfully transferred to alternative
poly(ethylene glycol) 400-coconut fatty acid mono- carriers and enzymes (e.g., protease, esterase and
ester and ethylene oxide and propylene oxide co- laccase), opening a broad field for the implementa-
ACHTUNGTRENNUNGpolymer (EO/PO)-oleic acid diester, were considera- tion and advancement of biocatalysis in industrial
bly improved in terms of the maximal number of re- processes.
action cycles performed with the same batch of cata-
lyst, and consequently in terms of the obtainable Keywords: Novozym 435; polyether ester; process
product amount. The total turnover numbers (TTN) optimization; silicone; stabilization; total turnover
increased by a factor up to 50, making the realization number
Introduction
(NZ435; Novozymes, Denmark), a highly active com-
mercial preparation of lipase B from Candida antarc-
In recent years, the implementation of biocatalyzed tica immobilized on a macroporous acrylic polymer
reactions into the production of fine and specialty resin[1] by adsorption through mainly hydrophobic in-
chemicals has become increasingly important for the teractions.[2] It is used for industrial-scale synthesis of
chemical industry. This mainly results from the often various fine chemicals[3] as well as specialty chemicals
high chemo-, regio-, and stereoselectivity of biocata- such as ceramides and emollient esters for cosmetic
lysts under mild reaction conditions (room tempera- formulations.[4] Unfortunately, its applicability is cur-
ture, neutral pH) yielding high quality products with- rently limited to the conversion and production of
out extensive energy input and downstream process- compounds with low to medium viscosity and none to
ing. Nevertheless, unless very high priced intermedi- only slightly surface-active properties. This obviates
ates or active compounds (e.g., for pharmaceuticals) the effective production of many cosmetic com-
are produced, an economically efficient process often pounds, for example, when diglycerol[5], polyglycerol
requires low costs for the applied biocatalyst.
or other polyols like R-butylglucoside[6] are involved.
A particularly versatile and successful biocatalyst
Viscosity restricts the application of NZ435 because
with regard to technical synthesis is Novozym 435 of the maximum possible pressure drop over the
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Enabling Industrial Biocatalytic Processes by Application of Silicone-Coated Enzyme Preparations
length of the fixed-bed reactor, which is typically used Results and Discussion
with this enzyme preparation.[7,8] Transfer of NZ435
to a stirred-tank reactor (STR) as the standard tech- Myristyl myristate, polyACHTNUGTRNEUNG(ethylene) glycol 400-coconut
nology for conversion and production of highly vis- fatty acid monoester and EO/PO-oleic acid diester
cous compounds, however, is prohibited due to the are important examples for industrial commodity
disintegration of the enzyme carrier by strong shear products with uses in the cosmetic, pharmaceutical
forces. Alternative reactor concepts such as bubble and food industries.[18,19,20,21] Their biocatalyzed synthe-
columns which provide proper mixing while exhibit- sis can be accomplished with NZ435.[18,22] but suffers
ing only low mechanical forces[8] can prevent this de- from the previously described disintegration of the
struction, but at low production volumes, the imple- catalyst carrier in STR or from enzyme leaching in
mentation of a novel technology is usually not justi- the presence of surface active reactants, respectively.
fied. Surface-active compounds, on the other hand,
Here, it was found that both disadvantages can
promote leaching of enzyme adsorbed on a carrier,[9] indeed be effectively overcome by implementation of
a phenomenon frequently observed.[8,10] The applica- silcoat-NZ435 into the production processes in place
bility of NZ435 can only be extended when these re- of native NZ435. In all three systems, this leads to a
strictions are overcome.
clear improvement of catalyst recyclability and ac-
We recently reported the development of a novel cordingly to an astonishingly increased total turnover
enzyme preparation with considerably improved sta- in terms of product formation per mass of catalyst
bility against enzyme leaching and carrier disintegra- (mproduct/mNZ435). Thus, process performance is consid-
tion.[11] This involves the combination of native erably advanced.
NZ435 with up to 54% (w/w) silicone to form distinct
composite particles with a residual activity of approxi-
mately 60% of the initial NZ435 activity towards the Increased Stability in STR
lab-scale synthesis of propyl laurate.[12] Promising fea-
tures of this approach with regard to industrial imple- Various emollient esters for the cosmetic industry are
mentation are the non-toxicity, low price and large- currently successfully produced on a ton-scale by use
scale availability of silicone as coating material, the of a fixed-bed reactor.[3] With this reactor set-up, the
established polymer formation via hydrosilylation and immobilized enzyme can be re-used for many cycles
the possibility to adapt the physicochemical proper- as no mechanical stress is exerted; the lipophilicity of
ties of the material to specific needs by varying the the products prevents desorption of the catalyst from
polymer chain length, the degree of cross-linking and the carrier. However, to allow the syntheses of a
the number of modifications.[14] Thus, it is clearly ad- larger product variety, multi-purpose reactor set-ups
vantageous over the reported alternative entrapment like STR are favorable. The performance of silcoat-
of carrier-bound enzymes in sol-gel,[13] which in addi- NZ435 in such a reactor was evaluated at the well
tion form a brittle material with only low stability studied esterification of myristyl myristate as a model
against carrier disintegration in standard industrial re- system (Figure 1) and compared to native NZ435.
actors such as STR.
The esterification was performed in a stirred-flask
This study evaluates the actual performance of the reactor (SFR) on a lab scale, resembling a small-scale
composite catalysts, further denoted as silcoat-NZ435, equivalent of a multi-purpose STR. This was operated
in industrial synthesis, with a focus on scenarios at a stirring speed of 350 rpm (to mimic the specific
where carrier disintegration and catalyst leaching con- power input observed in large-scale STR)[23] under re-
stitute key limitations. As model systems, the synthe- action conditions (temperature, catalyst ratio, applica-
ses of the emollient ester myristyl myristate and the tion of vacuum for water removal) adopted from the
poly
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG(ether) esters EO/PO-oleic acid diester and poly- large-scale production of myristyl myristate in a fixed-
(ethylene) glycol 400-coconut fatty acid monoester bed reactor.[5,24] Each production cycle covered a reac-
were investigated with particular regard to recyclabili- tion time of 24 hours. Within this time, the reaction
ty and total turnover number (TTN) of the catalyst was run to completion (100% conversion).
under process relevant conditions. Furthermore, the
When native NZ435 was used in this reaction set-
applicability of silcoat-NZ435 to stereoselective syn- up, the catalytic performance of the enzyme was com-
thesis was evaluated in the kinetic resolution of rac-1- parable to that in the fixed-bed process[7] and full con-
phenylethanol as prevalent model substrate[15,16,17] and version of substrates within the preset time was easily
finally the transferability of the methodology for sil- obtained. This demonstrates the principal suitability
coat-catalyst preparation to alternative carriers with of this reactor type for the synthesis. However, severe
immobilized enzymes is demonstrated for the first damage to the enzyme carrier was apparent, which is
time.
in agreement with the previously reported disintegra-
tion of NZ435 under mechanical stress.[11,12] As a con-
sequence, during catalyst removal the filter was
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Figure 1), the same batch of silcoat-NZ435 can cata-
lyze even more reaction cycles to full conversion
when the preset reaction time of each cycle is in-
creased.
Thus, the performance of silcoat-NZ435 under the
harsh conditions exerted in an STR (in terms of me-
chanical stress), is comparable to native NZ435 in the
standard fixed-bed reactor.[25] This shows that silcoat-
NZ435 offers an economically feasible reaction set-up
in multi-purpose STR, which is not only applicable to
the synthesis of myristyl myristate as a model product,
but to a number of other products with industrial
relevance.
Increased Stability Towards Leaching
The esterification of PEG 400 with coco fatty acid to
the corresponding monoester was chosen as a model
system representing a relatively low viscous product,
which can easily be handled in a fixed-bed reactor.
However, the reaction mixture and the product itself
show a strong surface-active behavior, causing protein
desorption from the carrier surface. This leads to a
loss of enzyme activity in the course of the reaction
and particularly during recycling of the catalyst.
The performance of silcoat-NZ435 under these con-
ditions was investigated in a lab-scale fixed-bed reac-
tor. As mentioned before, this reactor type offers high
productivity at low shear forces, excluding mechanical
damage on the NZ435 carrier. An equimolar mixture
of PEG400 and fatty acid was pumped through a
fixed-bed of NZ435 and silcoat-NZ435, respectively.
After full conversion within 24 h, the product was re-
moved and replaced by fresh substrate to start con-
secutive reaction cycles.
Figure 1. Top : Esterification of myristyl alcohol and myristic
acid. Middle: Initial enzyme activity within the recycling of
silcoat-NZ435. Bottom: Half-life times and TTN of NZ435
preparations.
As expected, both the carriers of native NZ435 and
silcoat-NZ435 remained mechanically unaffected
blocked by NZ435 fragments and abrasion, reducing throughout all operation cycles. Preliminary experi-
the filtration efficiency and preventing the recovery ments on the thermal stability of the enzyme prepara-
of catalyst. In the end, a TTN of 100 gMM/gNZ435 could tions, involving incubation in a non-leaching product
be obtained, a half-life time could not be determined. and without mechanical forces at different tempera-
In contrast, no abrasion was detectable when sil- tures (608C, 908C, 1108C), demonstrated that the
coat-NZ435 (silicone content 45% w/v) was subjected composite formation had no stabilizing effect in this
to the same conditions, which is consistent with the regard. Both catalysts, native NZ435 and silcoat-
previously reported enhanced mechanical stability of NZ435, were stable at 608C and lost 10–15% and 50–
this catalyst preparation.[11] Accordingly, product pu- 60% of initial specific activity at 908C and 1108C, re-
rification, catalyst recovery and recycling were easily spectively (see Supporting Information). Therefore,
accomplished, so proving an excellent applicability of changes in the activity of the catalyst beyond the
the system from a technical point of view.
magnitude of spontaneous deactivation can be as-
The same batch of silcoat-NZ435 ran each reaction signed solely to the leaching of the catalyst from the
cycle to completion within 23 days. Therefore, the carrier.
TTN in this system could be increased by a factor of
During recycling, native NZ435 rapidly lost activity
23 compared to the set-up with native NZ435, achiev- (Figure 2). In only three production cycles, the initial
ing a value of 2300 gMM/gNZ435 and a half-life time of activity was reduced to 40%, corresponding to a half-
23 days. Due to the very slow deactivation of the cata- life time of 2.2 days. This is in perfect agreement with
lyst under the investigated conditions (ki =0.037, the distinguished surface-active properties of the in-
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Enabling Industrial Biocatalytic Processes by Application of Silicone-Coated Enzyme Preparations
Figure 2. Top : Esterification of PEG 400 with coco fatty
acid. Middle: Initial enzyme activity during repetitive use in
a lab scale fixed bed reactor. Bottom: Half-life times and
Figure 3. Top : Esterification of EO/PO-polyether and oleic
acid. Middle: Initial enzyme activity during repetitive use in
a lab scale bubble column reactor. Bottom: Half-life times
and TTN.
TTN of NZ435 preparations.
vestigated substrate mixture and the produced mono-
ester, both having a strong ability for leaching the
enzyme off the carrier. The resulting TTN was about a bubble column reactor (BCR) is applied enabling
189 gmonoester/gNZ435 appropriate mixing, optimal water removal and re-
When recycling silcoat-NZ435 under the same con- duced mechanical stress on the enzyme carriers.[8]
ditions, no measurable loss in enzyme activity could The BCR was filled with the EO/PO-polyether and
.
be observed enabling the repeated production of oleic acid at a molar ratio of 1:2 and was operated at
monoester with one batch of enzyme for more than 708C. For mixing and removal of process water, a
18 days. This increased the TTN by a factor of 8 to stream of nitrogen was continuously passed through
TTN>1459 gmonoester/gNZ435
.
the reactor. Each production cycle covered a reaction
time of 24 hours. After each cycle, the catalyst was
separated from the product by filtration and was re-
used in consecutive cycles.
Application in a More Complex System
Figure 3 illustrates the performance of native
The esterification of EO/PO-polyether with oleic acid NZ435 and silcoat-NZ435 over eight to twelve pro-
to the corresponding diester represents a slightly sur- duction cycles with regard to the residual activity at
face-active reaction medium (diester) with a high vis- the beginning of every run. Abrasion of the enzyme
cosity (Figure 3). Due to this viscosity, a fixed-bed re- carrier was neither observed for native NZ435 nor sil-
actor cannot be used to perform the reaction. Instead, coat-NZ435 in this reaction set-up. It can therefore be
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R. Nieguth et al.
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assumed that changes in the activity of the catalysts hibited no difference to the native NZ435. Combined
beyond the magnitude of spontaneous deactivation with the findings of Hoyos et al.[27] who entrapped
are solely induced by catalyst leaching from the carri- Lipase TL (Meito Sangyo, Japan) in pure silicone
er.
without affecting the stereoselectivity, this provides a
With native NZ435, a maximum of 8 production strong hint that composite particles obtained from sil-
cycles could be accomplished before the residual ac- icone have no influence on the given stereoselectivity
tivity decreased below 25%. In contrast, a residual ac- of an enzyme catalyst and can therefore also be effec-
tivity of 75% was still measured after 12 production tively applied to the synthesis of chiral compounds.
cycles when silcoat-NZ435 was introduced to the pro-
Based on the excellent results achieved with NZ435
cess. This is in agreement with the previously reported as well-established commercial available lipase prepa-
enhanced stability of silcoat-NZ435 against enzyme ration, a test for transferability of this methodology to
leaching.[11]
other biocatalysts is of great interest. For a proof of
While the native enzyme preparation achieved a principle study, other mostly commercially available
half-life time of only about 8 days, silcoat-NZ435 re- carrier-bound enzymes were used with the intention
vealed a half-life time up to 63 days. This corresponds of setting standardized starting positions for following
to an increase in TTN from 329 gdiester/gNZ435 to optimizations. In order to cover a variety of industrial
2588 gdiester/gNZ435 for native NZ435 and silcoat-NZ435, relevant enzyme classes, an additional lipase system, a
respectively. Thus, formation of silicone composites protease,[28] an esterase,[29] and a laccase[30] immobili-
can effectively prevent protein leaching from the car- sate were tested.
rier surface and allows broadening the product portfo-
The formation of catalytically active silicone com-
lio even for surface active and highly viscous reaction posites was attempted with LCAHN, a commercial al-
media.
ternative to NZ435, in which lipase B from C. antarc-
tica is adsorbed on a polystyrene carrier, a commer-
cial protease (alcalase, IMMALC350) on an acrylic
polymer, a commercial esterase (Esterase ERO) on
Eupergit and a laccase on poly(methyl methacrylate)
Stereoselective Synthesis and Transferability
Encouraged by the performance of silcoat-NZ435 in (“Flavostar”). Importantly, this was successful for all
the synthesis of the specialty esters in the previous the investigated materials, albeit with a rather diverse
sections, further experiments were performed to esti- ability for silicone uptake and large varieties in the
mate the potential of silicone coating as a general
method for the provision of advanced catalysts for
technical synthesis. On the background of the particu-
lar importance of enzyme catalysis in the production
of chiral intermediates, this involved an examination
of the impact of composite formation on the enantio-
selectivity of NZ435. Since it is known that the prop-
erties of the immobilized enzyme strongly depend on
the enzyme microenvironment, it should be verified
that no significant interactions between the inert hy-
Table 1. Residual activities of different silicone-deposited
enzyme preparations.
Enzyme
Carrier
Silicone
[%]
Residual
activity
[%]
Lipase[a]
Polystyrene
Acrylic polyme-
r[e]
Eupergit
PMMA
40
40
66
65
drophobic silicone matrix and the active site of the Protease[b]
enzyme occurs, which is for instance detectable in
Esterase[c]
Laccase[d]
50
44
26
5.4
changes of the enantioselectivity[26]. As a simple and
representative model system we have chosen the ki-
netic resolution of rac-1-phenylethanol (1) as the al-
cohol component and vinyl butyrate as the acyl donor
(Scheme 1) in a transesterification reaction. It yielded
an enantiomeric excess of >99% of (R)-a-methylben-
zyl butyrate (2) at full conversion (50%) and thus ex-
[a]
Esterification of lauric acid and 1-propanol.
Hydrolysis of ethyl N-acetylglycinate.
Hydrolysis of ethyl pentanoate.
[b]
[c]
[d]
Oxidation of 2,2’-azino-bis(3-ethylbenzthiazoline-6-sul-
phonic acid) (ABTS).
Scheme 1. Kinetic resolution of rac-1-phenylethanol by NZ435.
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Enabling Industrial Biocatalytic Processes by Application of Silicone-Coated Enzyme Preparations
neck, round-bottom flask. The solution was stirred with an
residual catalytic performance being observed
(Table 1).
Intermig mixer at a speed of 350 rpm. The reaction was
started by addition of biocatalyst (1% w/w) at 608C.
Vacuum was applied for water removal. Samples were with-
drawn from the round flask, acid values were measured to
follow the initial enzyme activity and the conversion of the
esterification. After 24 h and full conversion, the product
was removed by filtration and fresh substrate mixture was
added to the enzyme to start consecutive cycles.
For lipase B and alcalase a residual activity above
60 % was observed, which is in the same range as for
NZ435[12] and therefore offers good prospects for a
future application to technical synthesis. In contrast,
esterase and laccase revealed only low residual activi-
ty after transfer to a silcoat-catalyst. However, at a
closer look, this behavior can most probably be as-
signed to the rather hydrophilic substrates converted
by these enzymes, as their uptake must be considera-
bly restricted by the hydrophobic silicone matrix.
Thus, the results do not actually restrict the transfera-
bility of silcoat-catalyst formation to a broad range of
enzymes. For application to a broad range of reac-
tions, however, matrix design towards an increased
hydrophilicity must be considered and is currently
under investigation.
Esterification of PEG 400 and coco fatty acid (mono-
AHCTUNGTREGeNNUN ster) in fixed-bed reactor: Equimolar amounts of polyethy-
lene glycol 400 and coco fatty acid C8-18 (700 mmol) were
mixed at 608C in a round flask connected to a fixed bed
column filled with 1.5% NZ435 (weight% related to sub-
strates). The process was started by pumping the substrate
mixture through the fixed enzyme bed with a pump rate of
approx. 10 mL/min. Reaction water was continuously re-
moved by vacuum. Samples were withdrawn from the round
flask, acid values were measured to follow the initial
enzyme activity and the conversion of the esterification.
After 24 h, the reaction was stopped at full conversion, the
product was removed and consecutive cycles were started
with fresh substrate mixture.
Conclusions
Esterification of EO/PO polyether with oleic acid (di-
AHCTUNGTREGeNNUN ster) in bubble-column reactor: EO/PO and oleic acid
The use of silcoat-NZ435 overcomes some of the
most important limitations faced in the application of
biocatalysis under industrially relevant conditions.
The easily applicable silicone deposition opens the
way to use high-performance and therefore high-
priced biocatalyst preparations in a cost-effective and
highly economical manner. In addition, the possibility
to use these improved biocatalysts in an STR makes
accessible a broad range of potential products, such as
emulsifiers or rheological additives, which are current-
ly inaccessibly due to their high viscosities and surface
activity and the associated stability problems of
NZ435 or comparable enzyme preparations.
were mixed at a molar ratio of 1:2 (0.038 mol:0.076 mol)
and transferred to a bubble-column reactor having a diame-
ter of 3 cm. Mixing of the substrates and biocatalyst as well
as the removal of reaction water was performed with a con-
tinuous stream of 0.6 L/min nitrogen. The reaction was start-
ed by addition of biocatalyst (2% w/w). Samples were with-
drawn from the round flask; acid values were measured to
follow the initial enzyme activity and the conversion of the
esterification. After 24 h and full conversion, the product
was removed by filtration and fresh substrate mixture was
added to the enzyme to start consecutive cycles.
Analysis of Catalytic Activity
Acid number (MM, PEDE, PEME): Acid numbers were
determined by titration using a 100 mM solution of KOH in
EtOH and phenolphthalein as indicator. Samples (0.5 g)
were dissolved in ethanol (50 mL) containing 1% (w/v) of
indicator.
Experimental Section
Materials
All reactants were obtained from Evonik Goldschmidt
GmbH (Germany) and Sigma Aldrich (Germany) and were
used without further purification. Novozym 435 and “Flavo-
star” Laccase were received from Novozymes A/S (Den-
mark), LCAHN from Sprin Technologies (Italy), Esterase
ERO from Fluka (Germany) and IMMALC350 from Chiral
Vision (Netherlands). silcoat-Catalysts were prepared as de-
Kinetic resolution of 1-phenylethanol: Lipase activity in
kinetic resolution was determined using racemic 1-phenyle-
thanol (0.15 mmol) in hexane (1.5 mL) and vinyl butyrate as
acyl donor (0.15 mmol). NZ435 (25 mg) and silcoat-NZ435
(50 mg) was added and the mixture was stirred at room tem-
perature. Samples were taken after 2 h and diluted in n-
hexane. Conversion and enantiomeric excess were deter-
mined by GC analysis using the chiral column FS Cyclodex
b I/P. The detailled GC program is given in the Supporting
Information. Retention times were determined by using the
commercially available racemic alcohol and the correspond-
ing ester as references. The retention time for (R)-a-methyl-
benzyl butyrate was determined by using enantiopure (R)-1-
phenylethanol as substrate in the transesterification reaction
with vinyl butyrate as acyl donor.
scribed in literature.[11]
.
General Set-Up for Repetitive Synthesis
In comparative studies between native and silcoat-NZ435,
the same amount of catalyst related to NZ435 was applied.
All calculations are referred to the amount of native biocat-
alyst included in the preparation.
Esterification of myristyl alcohol with myristic acid
(MM) in STR: Equimolar amounts of myristyl alcohol and
myristic acid (1.035 mol) were melted and filled in a three-
Esterification: Esterification activity of lipase was deter-
mined at the formation of propyl laurate. The reactions
were performed in sealed glass vessels containing lipase
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preparation (20 mg) and a solvent-free equimolar mixture of
lauric acid and 1-propanol (16 mmol) at 60 8C. Samples
(50 mL) were taken every 5 min over a time range of 25 min
and were diluted with decane (950 mL). The product was an-
alyzed by gas chromatography (Shimadzu 2010 equipped
with a BTX column from SGE) as described previously.[11]
Hydrolysis of ethyl N-acetylglycinate: For determination
of protease (alcalase) activity ethyl N-acetylglycinate
(3.4 mmol) was dissolved in phosphate-buffer (25 mM,
pH 7.5, 25 mL) containing 1% (w/w) Tween 80. The reaction
was started by adding the catalyst (100 mg) and performed
under stirring at 408C. Formation of acetic acid was deter-
mined by titration using a 100 mM solution of NaOH.
Hydrolysis of methyl pentanoate: Esterase activity was
determined with a solution of methyl pentanoate (3.8 mmol)
in phosphate-buffer (1 mM, pH 8, 25 mL). The reaction was
started by adding the catalyst (10 mg) and was performed
under stirring at room temperature. The formation of penta-
noic acid was determined by titration with a 100 mM solu-
tion of NaOH.
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Acknowledgements
This work was funded by the FNR e.V. (Gꢀlzow, Germany),
(FKZ: 22005507) and scientifically supported by the Cluster
of Excellence “Unifying Concepts in Catalysis” coordinated
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Adv. Synth. Catal. 2011, 353, 2522 – 2528