Immobilized hydroxynitrile lyase: A comparative study of recyclability

Article abstract of DOI:10.1002/cctc.201300892

Hydroxynitrile lyase from cassava, Manihot esculenta, (MeHNL) catalyzes the formation of (S)-cyanohydrins from HCN and aldehydes or ketones. Four differently immobilized MeHNLs were prepared: by noncovalent immobilization (celite R-633), covalent immobili

Full text of DOI:10.1002/cctc.201300892

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DOI: 10.1002/cctc.201300892
Immobilized Hydroxynitrile Lyase: A Comparative Study of
Recyclability
[a]
[a]
[b]
[a]
Guzman Torrelo,* Nelleke van Midden, Radek Stloukal, and Ulf Hanefeld*
Hydroxynitrile lyase from cassava, Manihot esculenta, (MeHNL)
catalyzes the formation of (S)-cyanohydrins from HCN and al-
dehydes or ketones. Four differently immobilized MeHNLs
were prepared: by noncovalent immobilization (celite R-633),
covalent immobilization (cross-linked enzyme aggregates,
CLEA), encapsulation [in a poly(vinyl alcohol) hydrogel Lenti-
kats] and a combination of the above, an unusual immobiliza-
tion, CLEA encapsulated in a poly(vinyl alcohol) hydrogel. A
comparative study of the recyclability of each immobilized
MeHNL was performed. Particular attention was paid to the
stability and activity of the new immobilized MeHNL–CLEA–
Lentikats and to the minimum enzyme loading required to
achieve high yields and enantiomeric excesses. MeHNL–CLEA
stability was slightly improved by encapsulation into Lentikats
and good recyclability rates at low enzyme loading were ob-
tained. However, MeHNL immobilized on celite R-633 exhibited
the best recyclability, giving >95% conversion and an enantio-
meric excess of 99% during 12 cycles.
Introduction
Chiral cyanohydrins are important synthetic intermediates for
many industrial products, owing to the fact that both function-
al groups of the cyanohydrins, the hydroxyl and cyanide
moiety attached to the same carbon, can easily be converted
into a wide range of other chiral products such as a-hydroxy
aldehydes and ketones, b-amino alcohols, a-fluoro cyanides,
taining a small amount of water), in which the racemic back-
ground reaction is suppressed.
The stability of enzymes in organic solvents can be strongly
enhanced by immobilization. Moreover, the catalyst can be fil-
tered off easily and no extraction step is required to recover
the product of the reaction from the liquid phase. Although
several immobilization methods have been employed for
[
1]
etc. One possibility of synthesizing chiral cyanohydrins is the
enantioselective addition of HCN to a prochiral aldehyde or
[4]
HNLs, each having their particular advantages, the reaction
conditions under which they have been tested were not
always identical. In addition, if immobilized HNLs are reused,
[2]
ketone with hydroxynitrile lyases (HNLs) as biocatalysts.
The enantiomeric purity of cyanohydrins obtained from the
single-aqueous-phase reaction catalyzed by HNLs is disturbed
by the nonenzymatic reaction that forms the undesired race-
mic products. The rate of this competing process depends on
the water content and the pH of the water phase in the reac-
[5]
enzyme recycling protocols are diverse, including washing
steps that can clearly effect enzyme activity and/or make the
process unviable on an industrial scale. This is to say often
high enzyme loadings are used masking enzyme deactivation
and/or enzyme deactivation is not mentioned.
[
3]
tion system. Different approaches were introduced to solve
this problem: 1) biphasic systems of buffer and immiscible or-
ganic solvent, which provides many advantages such as high
efficiency, prevention of substrate or product inhibitions, cost
effectiveness, and easy downstream processing because the
product is extracted into the organic phase while the enzyme
remains in the aqueous phase; 2) organic solvents (dry or con-
Previous studies on the syntheses of chiral cyanohydrins in
biphasic systems and organic solvents were performed on the
[6]
HNL from Manihot esculenta (MeHNL), but information on
enzyme recyclability is scarce. Moreover, the reaction condi-
tions are not comparable, that is, it is not easy to ascertain
which immobilization method is more plausible on an industri-
al scale. This HNL is particularly interesting because of its sta-
bility with respect to higher temperatures and low pH values
making it superior to the other HNLs with a/b-hydrolase
[
a] Dr. G. Torrelo, N. van Midden, Prof. U. Hanefeld
Gebouw voor Scheikunde, Biokatalyse
Afdeling Biotechnologie
[2b,7]
fold.
Cross-linked enzyme aggregates (CLEAs) are considered
Technische Universiteit Delft
Julianalaan 136, 2628 BL Delft (Netherlands)
Fax: (+31)15-278-1415
E-mail: G.TorreloVilla@tudelft.nl
U.Hanefeld@tudelft.nl
[8]
a more viable alternative for industrial immobilization and
CLEAs of the HNL from Manihot esculenta (MeHNL) are com-
mercially available biocatalysts for the enantioselective synthe-
sis of (S)-cyanohydrins. Though a PaHNL–CLEA exhibited good
recyclability, no recycling studies using MeHNL–CLEA can be
found in the literature. On the other hand, poly(vinyl alcohol)
hydrogels (Lentikats) of PaHNL have been reported as efficient
[
b] Dr. R. Stloukal
LentiKat’s a.s.
Pod Vinicꢀ 83
4
71 27 Strꢁ zˇ pod Ralskem (Czech Republic)
[5a]
Fax: (+420)255-710-699
and robust catalysts for the synthesis of (R)-mandelonitrile,
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but the encapsulation of a MeHNL into Lentikats has not been
tested yet.
Another successful immobilization technique is the immobi-
lization of enzymes on Celite. A comparative study of a MeHNL
immobilized on Celite and cross-linked crystals of MeHNL was
[
5b]
reported. The MeHNL–Celite exhibited low operational sta-
bility, losing all the activity after 5 batches, although the use of
dry acetone to wash the enzyme several times between batch-
es could explain these results, because it could remove the en-
trapped water in the immobilized enzyme and successive stud-
ies have shown the importance of this water in enzyme
[
6c,9]
stability.
Herein, to compare the stability and recyclability in the
enantioselective synthesis of (S)-mandelonitrile by MeHNL, four
differently immobilized MeHNLs were prepared: by noncova-
lent immobilization (Celite R-633), covalent immobilization
Figure 1. Recycling of the MeHNL–CLEA (5 mg, 15 U) in seven successive
mandelonitrile synthesis reactions. Conditions: benzaldehyde (1 mmol) in
(CLEA), encapsulation (in Lentikats), and a combination of the
1.5m HCN solution in buffer-saturated MTBE (2 mL); reaction time 4 h.
above, an unusual immobilization, CLEA encapsulated in
a poly(vinyl alcohol) hydrogel. Our attention focused on immo-
bilization strategies and reaction protocols by which the HNL
can be recycled efficiently.
tion before the addition of new reagents). Furthermore, the
catalyst loss is likely to accumulate with each cycle. In all cases,
the enantiomeric excess of the product was higher than 98%.
Three different enzyme washing protocols were tested to
evaluate the effect on enzyme activity over 5 cycles. If the
MeHNL–CLEA was washed with pure MTBE and dried between
each cycle, a rapid loss of activity was observed and the
MeHNL–CLEA gave only 44% conversion after the fourth cycle
and 32% conversion after the fifth (Figure 2). In this case, the
Results and Discussion
Recyclability of MeHNL–CLEA
The synthetic potential of the MeHNL–CLEA in organic solvents
[
5c,10]
was previously described,
although the reuse was not
studied thoroughly. Recyclability of the MeHNL–CLEA was,
therefore, studied herein on a laboratory scale by reusing the
catalyst in seven successive reactions of benzaldehyde
(1 mmol) and HCN (3 equiv., Scheme 1) with a low enzyme
Scheme 1. MeHNL-catalyzed hydrocyanation of benzaldehyde.
loading (5 mg, 15 U) in buffer-saturated methyl tert-butyl ether
(MTBE, citrate phosphate buffer 50 mm, pH 5.5). The enzyme
loading was chosen such that full conversion was reached but
a possible loss of activity would also be immediately visible.
The successive reactions were performed by allowing each re-
action cycle to proceed for 4 h, after which the reaction solu-
tion was replaced with a fresh solution of the reagents in
buffer-saturated MTBE. The reactions were shaken because we
found that magnetic stirring can damage the CLEAs. The
MeHNL–CLEA was not washed between cycles. The first three
cycles reached 98–95% conversion (Figure 1). In the next
cycles, however, a successively decreasing reactivity of approxi-
mately 10% was observed. In addition to deactivation, a possi-
ble reason for the decrease is catalyst loss rather than actual
loss in reactivity (centrifugation and removal the reaction solu-
Figure 2. Recycling of the MeHNL–CLEA (5 mg, 15 U) in five successive man-
delonitrile synthesis reactions, including washing of the enzyme between
cycles. Gray: wash with pure MTBE (1 mL); gray cross-hatched: wash with
buffer-saturated MTBE (1 mL); black: wash with 50 mm citrate phosphate
buffer (1 mL), pH 5.5. Conditions: benzaldehyde (1 mmol) in 1.5m HCN solu-
tion in buffer-saturated MTBE (2 mL); reaction time 4 h.
deactivation is probably owing to the fact that if solvent is
used for washing, some of the entrapped water will be
washed away from the MeHNL–CLEA, lowering the enzyme ac-
tivity. If the MeHNL–CLEA was washed with buffer-saturated
MTBE (citrate phosphate buffer 50 mm, pH 5.5) the loss of ac-
tivity was similar to that with the washing steps using pure
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MTBE during the first three cycles. However, the MeHNL–CLEA
gave 58% conversion after the fourth cycle and 49% after the
fifth. Compared to the first protocol, this lower deactivation
probably owes to suppression of the loss of the essential
water entrapped in the CLEA by use of a buffer-saturated sol-
vent. Finally, the MeHNL–CLEA was washed with citrate phos-
phate buffer 50 mm, pH 5.5. As the CLEA is not entirely stable
in buffer, a small amount of enzyme was withdrawn during
each washing step. Moreover, the CLEA took up part of the
buffer, making it difficult to obtain the CLEA with low water
content before starting a new cycle. Owing to this, the succes-
sive reactions revealed that the CLEA is surrounded by water
and the reaction conditions are similar to those of a biphasic
system. The loss of activity was similar to that with the wash-
ing step using pure MTBE, even slightly higher in the cycles 2
and 3.
Figure 3. Mandelonitrile synthesis reaction by PaHNL–Lentikats (1 g, 5 U)
after 144 h stirring ( ) and by fresh PaHNL–Lentikats (*). Conditions: citrate
phosphate buffer 50 mm (5 mL) pH 4.5, MTBE (3 mL), 1.5m HCN solution in
MTBE (2 mL), benzaldehyde (1 mmol).
&
Preparation of MeHNL–CLEA–Lentikats
The first encapsulation of a nonpurified PaHNL into Lentikats
[
5a]
was described by Grçger et al. The protocol to prepare an
efficiently entrapped HNL included a cross-linking process to
increase the molecular weight of the enzyme, because, accord-
ing to the authors, enzymes with a molecular mass lower than
Table 1. Enzyme activity recovered upon encapsulation in Lentikats ac-
cording to the activity test described in the Experimental Section.
MeHNL
CLEA–MeHNL
5
0000 Da are not restrained in the hydrogels. A combination
À1
À1
Enzyme stock solution [UmL ; Umg
]
453
207
13
3
–
50
of glutaraldehyde and chitosan gave the best results. Then, the
cross-linked enzyme was entrapped in a hydrogel matrix based
on poly(vinyl alcohol) and used for the synthesis of mandeloni-
trile in a biphasic system.
À1
Activity after purification [Umg
Activity recovery in Lentikats [%]
]
As PaHNL has a molecular mass 61 kDa, we reasoned that
the cross-linking process could be omitted if using the correct
poly(ethylene glycol)/poly(vinyl alcohol) ratio. Purified PaHNL
was encapsulated in a poly(vinyl alcohol) hydrogel and a leach-
ing experiment was performed. The PaHNL–Lentikats was
stirred for 144 h in a 50 mm citrate phosphate buffer at pH 4.5.
The buffer phase was used to conduct a standard activity test
for HNLs to check the presence of leached enzyme. No activity
was found. The PaHNL–Lentikats were stirred for 144 h and
fresh PaHNL–Lentikats were used separately to perform man-
delonitrile synthesis reactions in a biphasic system. Results of
these experiments are shown in Figure 3. Clearly, the PaHNL
maintained its original activity after 144 h of stirring.
MeHNL was encapsulated in a first trial without a previous
cross-linking step. 13% of the original activity was recovered
after the encapsulation in Lentikats (Table 1). This result is in
accordance with a previous study that demonstrated that im-
mobilization by this method yielded very little attached
[11]
enzyme. The same leaching experiment described previously
for the PaHNL was then performed with the MeHNL-Lentikats.
In this case, in contrast, the activity test using the reaction
buffer gave positive results. Similarly, the MeHNL–Lentikats
storage buffer was checked and enzyme activity was observed.
Sodium dodecyl sulfate (SDS) gel electrophoresis confirmed
the presence of free MeHNL outside the poly(vinyl alcohol).
Once leaching was confirmed, a cross-linking step to avoid
this leaching was introduced. An unusual immobilization,
MeHNL–CLEA encapsulated in a poly(vinyl alcohol) hydrogel,
was prepared. Three different amounts of MeHNL–CLEA were
encapsulated: 5, 10, and 15 milligrams per gram of poly(vinyl
alcohol). Approximately 50% of the original activity was recov-
ered in the Lentikats with MeHNL–CLEA contents of 5 and
A biphasic system with low-pH buffer combined with MTBE
was chosen to perform the synthesis of mandelonitrile, be-
cause the Lentikats tend to form aggregates in pure organic
solvents, resulting in a poor conversion of the substrates. The
organic layer in which most of the starting material and prod-
uct reside helps to suppress the racemic reaction, since no re-
action occurs in it. It does, however, introduce diffusion limita-
tions, which have to be overcome by rapid stirring.
À1
10 mgg , respectively (Table 1), but only a 22% in the Lenti-
À1
kats with MeHNL–CLEA content of 15 mgg . In the last case,
According to the results obtained with the PaHNL, we pro-
ceeded to encapsulate the MeHNL in a poly(vinyl alcohol) hy-
drogel. At first glance, the low molecular mass of the MeHNL
the MeHNL–CLEA–Lentikats obtained were spindle-shaped and
tended to form aggregates. For that reason, the 5 and
À1
10 mgg MeHNL–CLEA–Lentikats were chosen for further ex-
(
28–30 kDa) seems to require the use of an initial cross-linking
periments. Owing to the diffusion limitations of the biphasic
system, the reaction time necessary to reach full conversion
was 8 h. This was also the case if the same number of enzy-
matic units (15 U) and amount of substrate as in the experi-
step to increase the enzyme size and avoid leaching, but this
enzyme forms trimers and tetramers in solution, giving molec-
[
2b]
ular masses higher than 60 kDa.
For that reason, purified
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ments with the MeHNL–CLEA were used. Clearly the extra im-
to the adsorbed enzymes and correspondingly excellent activi-
[12]
mobilization layer hindered diffusion.
ties inside the microaqueous reaction systems. To determine
an appropriate enzyme/support ratio, several ratios from 1:50
to 1:4 were tested. To obtain a high conversion (99%) and ee
value (99%) in 180 min, an enzyme/support ratio of 1:4 was
used in buffer-saturated MTBE for the enantioselective addition
of HCN to benzaldehyde. Prolonged reaction times (300 min)
did not reduce the ee of the mandelonitrile (data not shown).
Generally, enzyme immobilization facilitates filtration and re-
cycling of the biocatalyst. Therefore, a sufficiently strong bind-
ing of the biocatalyst to the support under the respective reac-
tion conditions is required. To analyze the potential leaching of
active MeHNL from the celite into the reaction medium, two
identical samples of celite–MeHNL were prepared and packed
into fine-woven nylon-mesh “tea bags”. Such tea bags allow
the simple removal of the biocatalyst from the reaction
medium. Subsequently, the synthesis of (S)-mandelonitrile was
monitored over 60 min in two parallel reactions; in one reac-
tion setup the MeHNL-celite was removed from the reaction
medium after 15 min. However, the conversion in both reac-
tions was monitored over 60 min. As demonstrated in Figure 5,
the continuous MeHNL-celite reaction displayed the expected
course of conversion, whereas the aborted reaction stopped
Recyclability of MeHNL–CLEA–Lentikats
As was previously mentioned, a tightly attached nonpurified
PaHNL in a poly(vinyl alcohol) hydrogel was prepared by
[
5a]
Grçger et al., and the enzyme could be recycled 20 times
without loss of activity or enantioselectivity. However, the in-
ability to see enzyme deactivation can be attributed to the
high enzyme loading (11 grams of immobilized enzyme, 440 U)
used in the recyclability study.
For the recyclability of the MeHNL–CLEA–Lentikats, our at-
tention focused on the minimum enzyme loading required to
achieve high yields and enantiomeric excesses, and at the
same time, the amount of enzyme that allows monitoring the
enzyme deactivation. Seven successive reactions of benzalde-
hyde (1 mm) and HCN (3 equiv.) were performed. The succes-
sive reactions were performed by allowing each reaction cycle
to proceed for 8 h, after which the organic phase was replaced
with a fresh solution of the reagents in MTBE, and the water
phase containing the Lentikats was reused. The first three
cycles reached almost full conversion (Figure 4) with >98% ee.
Figure 5. Test for leaching of MeHNL–celite into the reaction medium
Figure 4. Recycling of the MeHNL–CLEA–Lentikats (15 U) in seven successive
mandelonitrile synthesis reactions. Conditions: 50 mm citrate phosphate
buffer (5 mL) pH 4.5, MTBE (3 mL), benzaldehyde (1 mmol), and 1.5m HCN
solution in MTBE (2 mL); reaction time 8 h.
(
buffer-saturated MTBE). Standard reaction (*) and reaction in which the im-
mobilized MeHNL was removed from the reaction medium ( ).
&
In the fourth cycle, a slight decrease of the conversion and ee
value was observed. As in the case of the MeHNL–CLEA, in the
next cycles a decreasing reactivity of approximately 10% was
observed successively, and, additionally, the ee dropped by ap-
proximately 2–4%. Clearly, if the conversion is low, the ee
value drops, owing to the fact that the racemic-base-catalyzed
reaction takes place in the water phase. Notably, similar results
were obtained if the aqueous phase was replaced after each
cycle.
directly after removing the immobilized catalyst from the reac-
tion medium. This observation demonstrates that no leaching
of active catalyst occurs in the reaction system.
Recyclability of MeHNL–Celite (R-633)
The same setup using MeHNL–celite packed into a tea bag
was applied to study the recyclability of the immobilized
[13]
enzyme preparation. These MeHNL tea bags were applied in
fifteen consecutive hydrocyanations of benzaldehyde, includ-
ing intermediate washing steps with pure MTBE. As demon-
strated in Figure 6, MeHNL-celite displayed excellent recyclabili-
ty. A moderate decrease in enzyme activity was observed after
12 cycles. Importantly, the stability of the enzyme was highly
Preparation of MeHNL–Celite (R-633)
Previous studies have demonstrated the moderate hydropho-
bicity of celite supports, which lead to a good water transport
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method, these features make the MeHNL–celite extremely at-
tractive for biocatalytic applications, in contrast with the results
found in the literature that mentioned a poor operational sta-
bility. It is important to perform the reactions in solvents with
a minimum amount of water, because the entrapped water
has a significant influence on the enzyme activity. Thus, almost
as important as the immobilization technique used is the use
of less aggressive washing protocols to keep this water, and
therefore, the enzymatic activity.
Experimental Section
Caution: All procedures involving hydrogen cyanide were per-
formed in a well-ventilated fume hood equipped with a HCN de-
tector. HCN-containing wastes were neutralized by using commer-
cial bleach and stored independently over a large excess of bleach
for disposal.
Figure 6. Recycling of the MeHNL–Celite (50 mg, 14 U) in fifteen successive
mandelonitrile synthesis reactions. Conditions: benzaldehyde (1 mmol) in
1.5m HCN solution in buffer-saturated MTBE (2 mL); reaction time 3 h.
dependent on the treatment between the cycles. The enzyme
had to remain in the organic phase and drying needed to be
avoided. Rapid deactivation was observed if the enzyme was
allowed to dry, most likely owing to the phase change. Howev-
er, the enzyme remained active for a long time if stored in or-
Enzymes
MeHNL was supplied by Jꢁlich Chemical Company (Codexis). The
enzymatic activities of commercial MeHNL was measured accord-
[14]
ing to reported literature procedures
and was found to be
À1
453 Uml . The commercial enzyme was purified to remove the
salts to achieve a final enzyme in a well-known solution, by using
the following procedure: 15 mL volumes of the enzyme solution
were added up to an Amicon Ultra Filter Device and placed
capped into a centrifuge rotor (3000ꢂg for several hours). The con-
centrated solutes were recovered with a pipette and few microli-
ters of 0.1m citrate phosphate buffer (pH 5.0) containing 0.15% of
NaCl were added. The crude, brown/yellow-colored, enzyme sus-
pensions (2.5 mL) were applied to the top of a PD-10 desalting
column (Sephadex G-25 medium) and eluted with 10 mm potassi-
um phosphate buffer (pH 6.0) and subsequently lyophilized. The
enzyme activity after the purification step was found to be
[
4]
ganic solvent, in line with earlier observations.
Conclusions
The use of hydroxynitrile lyase from cassava, Manihot esculenta,
covalently immobilized as cross-linked enzyme aggregates
(
MeHNL–CLEA) for the hydrocyanation of benzaldehyde in
monophasic buffer-saturated methyl tert-butyl ether is an effi-
cient alternative to the hydrocyanation reaction in aqueous
media at low pH. The biotransformation of benzaldehyde re-
sulted in almost complete enzymatic conversion with excellent
stereoselectivity. A further advantage of reaction systems with
minimal water content is the efficient suppression of the non-
catalyzed formation of racemic cyanohydrins and the better
solubility of aromatic substrates and products. MeHNL–CLEA
exhibits reasonable recyclability with low enzyme loading.
Washing steps between batches proved to be unnecessary.
MeHNL encapsulated in poly(vinyl alcohol) hydrogels (Lenti-
kats) was not stable without a previous cross-linking step.
However, if the MeHNL–CLEA was encapsulated, good stability
and storability were observed. Owing to diffusion limitations of
the biphasic system (although the same enzymatic units, 15 U,
and substrate amounts as in the experiments with the MeHNL–
CLEA were used), longer reaction times were necessary. A de-
crease in the ee values was observed if the conversion was
lower than 95%, as a result of the racemic-base-catalyzed reac-
tion that takes place in water. The encapsulation of the
MeHNL–CLEA in a poly(vinyl alcohol) hydrogel improved its
stability slightly. However, diffusion limitations of both reaction
systems differed significantly and prevented a direct compari-
son of the relative enzyme activities.
À1
À1
2
07 Umg . CLEAs of MeHNL (66 Ug ) were supplied by CLEA
Technologies.
Preparation of Lentikats
Poly(ethylene glycol) (PEG, 3 g) was dissolved in demineralized
water (10 g). Then, poly(vinyl alcohol) (5 g) and demineralized
water (30 g) was added. The mixture was heated and stirred
during 20 min to reach the boiling point (98–98.58C). When the so-
lution became transparent, it was cooled down to 358C. The
enzyme solution [enzyme (75–225 mg)+0.5m sodium phosphate
buffer (3 g), pH 6.5] was added to poly(vinyl alcohol) gel (12 g). A
low-capacity laboratory device LentiPrinter was used for manufac-
turing of Lentikats biocatalyst. The poly(vinyl alcohol) and the
enzyme mixture were poured into a Petri plate. Delivery system
was first sunken into the poly(vinyl alcohol) mixture. Secondly,
drops of the poly(vinyl alcohol) and biomass mixture were printed
on a polyethylene drop plate. The drops of poly(vinyl alcohol) and
biomass mixture were dried at 30 to 358C to reduce the initial
weight of the biocatalyst by 25 to 30%. Collection and re-swelling
of the biocatalyst was performed in 0.5m phosphate buffer pH 6.5
within 20–40 min. After the production and collection, Lentikats
biocatalysts were placed into a fresh 0.5m phosphate buffer pH 6.5
(stabilized with 0.09% sodium azide) to be further stored in a refrig-
erator (48C) until the use.
MeHNL–celite exhibited excellent recyclability and storabili-
ty; together with the cheap, rapid, and simple preparation
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Preparation of Celite-immobilized HNL
Synthesis of (S)-mandelonitrile by MeHNL–Lentikats
Lyophilized MeHNL was re-dissolved in 50 mm potassium phos-
phate buffer (pH 6.0). Enzyme solution with an appropriate
enzyme concentration (enzyme/support ratio 1:4–1:100) was
added to the Celite support (Celite R-633). The preparation was
dried for at least 12 h under vacuum (20 mbar) in a desiccator over
silica gel and molecular sieves. HNL–Celite was transferred into
screw cap vials and stored at 48C.
Reactions were performed in a 25 mL two-neck flask with magnet-
ic stirring. Lentikats were added to 50 mm citrate phosphate buffer
(5 mL) pH 4.5. Then, a 3 mL volume of MTBE containing benzalde-
hyde (1 mmol) and 1,3,5-triisopropylbenzene (0.02 mmol, as an in-
ternal standard) was added and the reaction was started with 1.5m
HCN solution in MTBE (2 mL). Reaction was monitored by chiral
HPLC over 120–1440 min while the reaction flask was stirred at RT.
Chemicals
(
Æ)-Mandelonitrile (Sigma–Aldrich) was purified through column
Recycling studies
chromatography (PE/EtOAc 9:1/3:7) prior to use. Acetone cyanohy-
drin (Sigma–Aldrich) was distilled in the presence of 2% phospho-
ric acid prior to use and was stored under nitrogen at 48C. Benzal-
dehyde (Acros Organics) was always distilled prior to use and was
stored under nitrogen at 48C. Isopropanol and heptane (HPLC
grade) were purchased from Sigma–Aldrich. Petroleum ether and
ethyl acetate (technical grade) were purchased from VWR Interna-
tional. Methyl tert-butyl ether (MTBE, Acros Organics, 99.9% extra
pure) was used without further treatment unless otherwise speci-
fied. Aqueous buffers were prepared from analytical grade salts
and stabilized with 0.09% sodium azide.
MeHNL–CLEA: Seven consecutive hydrocyanation reactions were
performed over 240 min with 5 mg amounts of MeHNL–CLEA (ac-
tivity of the immobilized HNL=15 U), as described for the synthe-
sis of (S)-mandelonitrile by MeHNL–CLEA. The reactions were
stopped and the mixture was centrifuged 2 min at 13000 rpm. The
solvent was removed and the MeHNL–CLEA pellet was resuspend-
ed with fresh MTBE containing the substrates. Chiral HPLC was
used to monitor the reactions. Experiments were performed in du-
plicate.
MeHNL–CLEA-Lentikats: Lentikats (activity of the immobilized
HNL=15 U) were placed in a 25 mL two-neck flask. Seven consecu-
tive hydrocyanation reactions were performed over 480 min with
the MeHNL–CLEA-Lentikats, as described for synthesis of (S)-man-
delonitrile by Lentikats-MeHNL. The organic phase was removed
after each cycle and the aqueous phase containing the Lentikats
was reused. Fresh MTBE containing the substrates was added to
start the new cycle. Chiral HPLC was used to monitor the reactions.
Experiments were performed in duplicate.
Hydrogen cyanide 1.5–2m solution in MTBE: Sodium cyanide
(
4.9 g, 0.1 mol) was dissolved in a magnetically stirred mixture of
water (10 mL) and MTBE (25 mL) at 08C. The biphasic system was
stirred vigorously for 15 min and 30% aqueous HCl (10 mL) was
added slowly. This mixture was allowed to warm slowly to RT (at
least 25 min). The phases were separated and MTBE (7 mL) was
added to the organic layer. The combined organic phases were
stirred and residual water was separated. This procedure was re-
peated with another 7 mL volume of MTBE. The standard HCN so-
lution was kept over citrate phosphate buffer (50 mm, pH 5.5) in
the dark. Determination of HCN concentration was performed as
MeHNL-Celite: Celite containing MeHNL (activity of the immobi-
lized HNL=14 U) was sealed into an organic solvent-resistant, fine-
woven nylon mesh “tea bag” (nylon net, pore size 0.4 mm) for easy
removal from the reaction medium by filtration. Fifteen consecu-
tive hydrocyanation reactions were performed over 180 min with
the MeHNL–Celite tea bag, as described for the synthesis of (S)-
mandelonitrile by MeHNL-Celite. The tea bags were washed be-
tween each reaction cycle with pure MTBE without buffer to
remove remaining product and refresh the immobilizate. Care had
to be taken to ensure that the enzyme preparation did not dry
out. Chiral HPLC was used to monitor the reactions. Experiments
were performed in duplicate.
[15]
described in the literature.
Activity test for Lentikats biocatalyst
Activity test for Lentikats biocatalyst was conducted in 10 mL final
[14]
reaction volume. The standard activity test procedure for HNLs
was modified to increase the final volume to 10 mL. Lentikats were
added to a 50 mL three-neck flask containing 0.05m citrate/phos-
phate buffer pH 5.0 (7 mL) and 0.005m phosphate buffer pH 6.5
(
1 mL). The reaction medium was stirred with a magnetic stirrer in
a temperature-controlled silicone bath until the lentils separated
À1
(
208C). Mandelonitrile solution (2 mL, 0.06 molL ) in 0.003m cit-
rate phosphate buffer pH 3.5 was added to start the reaction. Sam-
ples were taken over 7 min and the absorbance was measured at
HPLC method
2
80 nm.
Samples from the reaction mixtures were mixed with heptane/iso-
propanol (95:5), filtered, and dried with anhydrous MgSO . Vol-
4
umes of 10 mL of the final clear solution were injected into an
HPLC (Waters). Analyses were performed on a column (Daicel 4.6ꢂ
Synthesis of (S)-mandelonitrile by MeHNL–CLEA and
MeHNL–Celite
2
50 mm 5m Chiralpak AD-H) coupled to a SpH 99 column thermo-
MeHNL–CLEA (5–10 mg) or MeHNL–Celite (30–100 mg) were added
to 1.5m HCN solution in MTBE (2 mL, saturated with buffer, see
above) containing benzaldehyde (1 mmol) and 1,3,5-triisopropyl-
benzene (0.01 mmol, as an internal standard) previously mixed
under a nitrogen atmosphere. The reaction was monitored by
chiral HPLC over 120–240 min while the reaction flask was stirred
at RT.
stat (Chrompack), a 515 HPLC pump (Waters), an autosampler (717,
Waters), and a UV/Vis detector SPD-10 A (Shimadzu). The column
temperature was maintained at 408C. HPLC methodology: mobile
phase: heptane/isopropanol=95:5 (0.1% trifluoroacetic acid); flow
rate: 1 mLmin ; detection UV wavelength: 254 nm. Retention
times: 5.0 min (benzaldehyde), 11.3 min ((S)-mandelonitrile),
12.6 min ((R)-mandelonitrile).
À1
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemCatChem 2014, 6, 1096 – 1102 1101

CHEMCATCHEM
FULL PAPERS
www.chemcatchem.org
Investigation of potential leaching of active MeHNL from
immobilizates
Keywords: aldehydes · cyanides · enzyme catalysis · gels ·
immobilization
1
) Two identical MeHNL–Celite samples, shrink-wrapped in a nylon-
mesh tea bag, were prepared. Each sample was used for addition
of HCN to benzaldehyde as described in the synthesis of mandelo-
nitrile by MeHNL–Celite. In one reaction, the tea bag containing
the immobilized MeHNL was inside the reaction medium for the
whole reaction time, whereas it was removed from the reaction
medium after 15 min in the parallel reaction. Both samples were
monitored over 60 min by chiral HPLC. After removal of the tea
bag, the reaction should be aborted if no active enzyme leaches
into reaction medium. 2) MeHNL–CLEA–Lentikats (1 g) was stirred
for 144 h under the same reaction conditions as described for syn-
thesis of mandelonitrile by MeHNL–Lentikats but without the sub-
strates. After removal of the organic phase, samples of the buffer
phase were withdrawn and concentrated under vacuum to remove
MTBE traces. Activity assays were performed according to reported
[
1] a) M. North, Tetrahedron: Asymmetry 2003, 14, 147–176; b) M. North,
Tetrahedron 2004, 60, 10383–10384.
[2] a) J. Holt, U. Hanefeld, Curr. Org. Synth. 2009, 6, 15–37; b) M. Dadashi-
pour, Y. Asano, ACS Catal. 2011, 1, 1121–1149; c) I. Hajnal, A. Łyskowski,
U. Hanefeld, K. Gruber, H. Schwab, K. Steiner, FEBS J. 2013, 280, 5815–
5828.
[
3] J. Langermann, J.-K. Guterl, M. Pohl, H. Wajant, U. Kragl, Bioprocess Bio-
syst. Eng. 2008, 31, 155–161.
[
[
4] U. Hanefeld, Chem. Soc. Rev. 2013, 42, 6308–6321.
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1969–1972; b) D. Costes, E. Wehtje, P. Adlercreutz, J. Mol. Catal. B 2001,
11, 607–612; c) A. Chmura, G. M. van der Kraan, F. Kielar, L. M. van Lan-
[14]
literature procedures by using the concentrated buffer samples.
SDS gel electrophoresis was performed to detect enzyme presence
and size (see “SDS polyacrylamide gel electrophoresis” below) and
protein concentration was checked (see “Protein quantification”
below).
gen, F. van Rantwijk, R. A. Sheldon, Adv. Synth. Catal. 2006, 348, 1655–
1661.
[6] a) D. Costes, E. Wehtje, P. Adlercreutz, Enzyme Microb. Technol. 1999, 25,
84–391; b) M. Persson, D. Costes, E. Wehtje, P. Adlercreutz, Enzyme
3
Microb. Technol. 2002, 30, 916–923; c) M. Paravidino, M. J. Sorgedrager,
R. V. A. Orru, U. Hanefeld, Chem. Eur. J. 2010, 16, 7596–7604.
[
7] J.-K. Guterl, J. N. Andexer, T. Sehl, J. von Langermann, I. Frindi-Wosch, T.
Rosenkranz, J. Fitter, K. Gruber, U. Kragl, T. Eggert, M. Pohl, J. Biotechnol.
SDS polyacrylamide gel electrophoresis
2
009, 141, 166–173.
8] a) L. Cao, L. v. Langen, R. A. Sheldon, Curr. Opin. Biotechnol. 2003, 14,
87–394; b) R. Schoevaart, M. W. Wolbers, M. Golubovic, M. Ottens,
[
Sample volumes of 10 mL were mixed with SDS gel loading buffer
3
(
10 mL) and heated up to 958C for 10 min. After 10 min samples
A. P. G. Kieboom, F. van Rantwijk, L. A. M. van der Wielen, R. A. Sheldon,
Biotechnol. Bioeng. 2004, 87, 754–762.
9] a) V. Lꢃonard-Nevers, Z. Marton, S. Lamare, K. Hult, M. Graber, J. Mol.
Catal. B 2009, 59, 90–95; b) P. Halling, Enzyme Microb. Technol. 1994,
were allowed to cool to RT. 10 mL volumes of the samples and the
standard, respectively, were applied in the gel sample pockets. A
gel Criterion Precast XT Bis-Tris/Tris-Acetate (4–12%) (Biorad) was
run at 180 V (constant V) for 45 min. 2-morpholinoethanesulfonic
acid (Biorad) was used as the running buffer. After complete run,
gel was stained (SimplyBlue SafeStain) for 1–2 h and then was de-
stained using water. Precision Plus Protein Prestained Standards
was used as protein size standard.
[
1
6, 178–206.
[
10] a) F. L. Cabirol, U. Hanefeld, R. A. Sheldon, Adv. Synth. Catal. 2006, 348,
1645–1654; b) F. L. Cabirol, A. E. C. Lim, U. Hanefeld, R. A. Sheldon, Org.
Process Res. Dev. 2010, 14, 114–118.
[
11] E. Capan, U. Jahnz, K.-D. Vorlop, Landbauforsch. Volk. 2002, 241, 151–
1
53.
12] a) E. Wehtje, P. Adlercreutz, B. Mattiasson, Biotechnol. Bioeng. 1990, 36,
9–46; b) E. Wehtje, P. Adlercreutz, B. Mattiasson, Biotechnol. Bioeng.
993, 41, 171–178.
[
3
1
Protein quantification
A BC assay protein quantification kit (Uptima) was used to measure
the protein concentration in buffer following the enhanced proto-
col (detection limits: 2–500 mgmL ).
[13] The tea bag methodology was tried with the CLEA-MeHNL but the
pore size of the tea bag was not small enough to retain the CLEA and
biocatalyst was found in the reaction medium a few minutes after start-
ing the reaction.
À1
[
14] U. Hanefeld, A. J. J. Straathof, J. J. Heijnen, Biochim. Biophys. 1999, 1432,
85–193.
15] L. M. van Langen, F. van Rantwijk, R. A. Sheldon, Org. Process Res. Dev.
003, 7, 828–831.
1
Acknowledgements
[
2
G.T. thanks the Fundaciꢂn Alfonso Martꢀn Escudero (Spain) for
a postdoctoral fellowship. The authors thank LentiKat’s a.s.
Received: October 21, 2013
(Czech Republic) for hosting G.T. for immobilization experiments.
Published online on January 21, 2014
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemCatChem 2014, 6, 1096 – 1102 1102