Enzyme-catalyzed laurolactam synthesis via intramolecular amide bond formation in aqueous solution

Article abstract of DOI:10.1002/adsc.201100396

Lactam formation from ω-aminocarboxylic acids is thermodynamically unfavored in aqueous solution and therefore hard to achieve. In the present work ω-laurolactam hydrolases from Acidovorax sp. T31 and Cupriavidus sp. U124 were investigated regarding their potential to catalyze lactam formation. Both enzymes are known to hydrolyze laurolactam to 12-aminododecanoic acid. The ω-laurolactam hydrolase genes were expressed in Escherichia coli BL21 (DE3) and the catalytic activity of the respective proteins was investigated. As expected from thermodynamics, only laurolactam hydrolysis but not 12-aminododecanoic acid cyclization was observed in whole-cell biotransformations and cell extract assays. The utilization of 12-aminododecanoic acid methyl ester, as an activated form of 12-aminododecanoic acid, resulted in intramolecular amide bond formation with the product laurolactam. Maximum laurolactam formation rates of 13.5 and 14.3 U g _CDW^-1 and molar yields of 11.5% and 13.0% were achieved in biotransformations at pH 10 with recombinant E. coli harboring the ω-laurolactam hydrolase from Cupriavidus sp. U124 and Acidovorax sp. T31, respectively. Furthermore, it was shown that under the harsh reaction conditions applied, the utilization of whole-cell biocatalysts enables 17.2-fold higher laurolactam formation activity in comparison to free enzymes in solution. This study shows that hydrolase-catalyzed laurolactam synthesis can be achieved in aqueous solution by selection of an appropriate substrate and reaction pH. Copyright

Full text of DOI:10.1002/adsc.201100396

FULL PAPERS
DOI: 10.1002/adsc.201100396
Enzyme-Catalyzed Laurolactam Synthesis via Intramolecular
Amide Bond Formation in Aqueous Solution
Nadine Ladkau,a Inna Hermann,a Bruno Bꢀhler,a and Andreas Schmida,*
a
Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund
University, Emil-Figge-Str. 66, 44227 Dortmund, Germany
Fax : (+49)-231-755-7382; e-mail: andreas.schmid@bci.tu-dortmund.de
Received: May 16, 2011; Revised: August 8, 2011; Published online: September 5, 2011
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adcs.201100396.
À1
Abstract: Lactam formation from w-aminocarboxylic lactam formation rates of 13.5 and 14.3 UgCDW and
acids is thermodynamically unfavored in aqueous so- molar yields of 11.5% and 13.0% were achieved in
lution and therefore hard to achieve. In the present biotransformations at pH 10 with recombinant E.
work w-laurolactam hydrolases from Acidovorax sp. coli harboring the w-laurolactam hydrolase from Cu-
T31 and Cupriavidus sp. U124 were investigated re- priavidus sp. U124 and Acidovorax sp. T31, respec-
garding their potential to catalyze lactam formation. tively. Furthermore, it was shown that under the
Both enzymes are known to hydrolyze laurolactam harsh reaction conditions applied, the utilization of
to 12-aminododecanoic acid. The w-laurolactam hy- whole-cell biocatalysts enables 17.2-fold higher laur-
drolase genes were expressed in Escherichia coli olactam formation activity in comparison to free en-
BL21 (DE3) and the catalytic activity of the respec- zymes in solution. This study shows that hydrolase-
tive proteins was investigated. As expected from catalyzed laurolactam synthesis can be achieved in
thermodynamics, only laurolactam hydrolysis but not aqueous solution by selection of an appropriate sub-
12-aminododecanoic acid cyclization was observed in strate and reaction pH.
whole-cell biotransformations and cell extract assays.
The utilization of 12-aminododecanoic acid methyl
ester, as an activated form of 12-aminododecanoic Keywords: hydrolase; kinetically controlled amide
acid, resulted in intramolecular amide bond forma- synthesis; lactam formation; laurolactam; whole-cell
tion with the product laurolactam. Maximum lauro- biocatalysis
Introduction
C16PyCl,I in stoichiometric amounts, and intermolecu-
lar dimerization or polymerization reactions. Another
Intermolecular amide bond formation can be per- approach to synthesize C6 to C13 ring lactams from
formed in chemical or enzymatic reactions and is well corresponding w-aminocarboxylic acids utilizes chem-
established for the synthesis of medium size pep- ical solid-phase peptide formation.[6] However, the re-
tides.[1–4] In contrast, there are only few chemical or quirement for toxic solvents and protective groups
enzymatic approaches describing an intramolecular makes the process laborious and environmentally un-
amide bond formation, e.g., for the synthesis of lac- friendly.
tams. The formation of lactams via intramolecular
Enzyme-catalyzed intramolecular amide formation
ring closure reaction of the corresponding w-amino- from w-amino esters can be an alternative to chemical
carboxylic acids is entropically unfavored and there- syntheses as described for g- and d-lactam synthesis
fore difficult to achieve.[5]
using porcine liver esterase in aqueous solution.[7]
Rico et al. reported the utilization of N-hexadecyl- Compared to chemical synthesis, the enzymatic reac-
2-chloropyridinium iodide (C16PyCl,I) as an activating tion takes place under mild reaction conditions with
agent to facilitate the ring closure reaction of 12-ami- reduced production of toxic waste.
nododecanoic acid to laurolactam.[5] Major disadvan-
Enzyme-catalyzed syntheses can either be carried
tages are the use of toxic compounds, such as 1,2-di- out with free enzymes or with whole cells containing
chloroethane or triethylamine, the application of the enzymes. Both approaches are widely used for the
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production of chemicals and pharmaceuticals.[2,3,8–10] tramolecular amide bond formation and thus to con-
The choice for one of these options depends on the vert 12-aminododecanoic acid to laurolactam serving
reaction itself, enzyme stability, and costs of enzyme as the model reaction (Scheme 1).
isolation and purification. Natural encapsulation
inside cells protects the enzyme from inactivating
agents or shear forces[2,3,11] and may increase enzyme
stability, even when the cell is metabolically inac-
tive.[12] However, diffusion of substrates over the cell
membrane may limit mass transfer in whole-cell sys-
tems, which often results in reduced catalytic activity
Scheme 1. Condensation reaction of 12-aminododecanoic
compared to free enzymes.[13]
acid to laurolactam catalyzed by an w-laurolactam hydro-
lase.
Recently, laurolactam hydrolysis activity has been
reported in several soil bacteria, and w-laurolactam
hydrolases were identified as responsible enzymes.[14]
In cell-free extracts of recombinant E. coli JM109 har-
The catalytic behavior of the w-laurolactam hydro-
boring the w-laurolactam hydrolases from either lases was evaluated following the equilibrium con-
Acidovorax sp. T31 or Cupriavidus sp. U124, 12-ami- trolled and kinetically controlled reaction concepts,
nododecanoic À1acid formation rates of 2.77 and which originally have been developed for peptide syn-
1.24 Umgprotein were observed, respectively.[15]
thesis[16,17] (Scheme 2). The pH optimum of the lactam
In this study, these w-laurolactam hydrolases were formation reaction was investigated using whole cells
investigated regarding their potential to catalyze in- and cell extracts.
Scheme 2. Equilibrium (a) and kinetically controlled (b) peptide synthesis and their time dependent yield (c). Adapted from
Wegmann et al. and Heyland et al.[9,10]
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Enzyme-Catalyzed Laurolactam Synthesis via Intramolecular Amide Bond Formation
Results and Discussion
increasing the pH of the reaction medium. However,
a pH>8 generally results in w-laurolactam hydrolase
deactivation.[14] To find
compromise between
Construction and Characterization of Recombinant
a
E. coli Strains
enzyme deactivation at higher pH and providing a
sufficient amount of non-protonated amino species
In order to achieve and evaluate the expression of the for the reaction, biotransformations were performed
w-laurolactam hydrolase genes from Acidovorax sp. at pH 10. However, laurolactam formation was also
T31 and Cupriavidus sp. U124 in recombinant E. coli not observed in whole-cell biotransformations at
BL21 (DE3), the expression vectors pCom10_T31 pH 10.
and pCom10_U124 were constructed and biocatalytic
Amide bond formation also depends on the concen-
activities for laurolactam hydrolysis were determined. tration of the uncharged carboxylic acid group of the
Laurolactam hydrolysis was tested in biotransforma- substrates.[16] Since about 99% of the carboxylic acid
tions using whole cells or crude extracts.
groups of 12-aminododecanoic acid are present as
Compared to E. coli BL21 (DE3) (pCom10_U124), anions at pHꢀ7.4 (Marvin Software, ChemAxon Ltd.
E. coli BL21 (DE3) (pCom10_T31) showed about Budapest, Hungary), the formation of the acyl-
1.4- and 1.2-fold higher 12-aminododecanoic acid for- enzyme is inhibited and thus the ring closure reaction
mation rates in vivo and in vitro, respectively to the lactam is hindered.
(Table 1).
After complete consumption of laurolactam, the
yield of 12-aminododecanoic acid was greater than Laurolactam Synthesis Following a Kinetically
99%. Assuming a whole cell protein content of 55% Controlled Reaction Concept
of the bacterial cell dry mass,[18] the observed in vitro
À1
activities of 177.7Æ3.7 Ugprotein
and 149.7Æ According to the kinetically controlled reaction con-
À1
1.5 Ugprotein correspond well with the experimentally cept (Scheme 2), activated substrates can be used to
determined in vivo activities (Table 1). This indicates achieve protease-catalyzed peptide formation.[16,17]
that mass transfer of laurolactam over the cell mem- Activated substrates are w-aminocarboxylic acids
brane was not limiting. Wild-type E. coli BL21 (DE3) with a substituted carboxylic acid group such as, for
did not catalyze laurolactam hydrolysis (data not example, amides or esters, which provide good leaving
shown) confirming that laurolactam hydrolysis result- groups to form a covalent acyl-enzyme intermediate
ed from w-laurolactam hydrolase activity and not within serine or cysteine hydrolases. This intermediate
from intrinsic enzyme activities.
is attacked by a nucleophile and the acyl group is
transferred.[16,19] The investigated w-laurolactam hy-
drolases from Acidovorax sp. T31 and Cupriavidus sp.
U124 show 98% amino acid sequence homology to
the serine hydrolase NylA from Arthrobacter sp. and
contain the catalytic triad typical for serine hydrolas-
Laurolactam Synthesis from 12-Aminododecanoic
Acid
After the activity of w-laurolactam hydrolases in re- es.[14,20] Thus, following the kinetically controlled reac-
combinant E. coli BL21 (DE3) had been shown, 12- tion concept seemed to be a feasible approach for
aminododecanoic acid cyclization to laurolactam was laurolactam synthesis with the w-laurolactam hydro-
investigated. Laurolactam formation was not ob- lases.
served in biotransformations of 12-aminododecanoic
acid using whole cells and cell extracts at pH 7.4.
For this purpose, 12-aminododecanoic acid methyl
ester was chosen as activated substrate. Whole-cell
Following the concept of equilibrium controlled biotransformations were performed with E. coli BL21
peptide synthesis, amide bond formation can be pro- (DE3) (pCom10_T31) and E. coli BL21 (DE3)
moted by increasing the amount of non-protonated (pCom10_U124) at pH 7.4. Formation of laurolactam
amino acids in solution.[16,17,19] This can be achieved by was not detected. Only ester hydrolysis to 12-amino-
Table 1. 12-aminododecanoic acid formation activities and yields for laurolactam with recombinant E. coli BL21 (DE3).
BL21 (DE3) (pCom10_T31)
Initial rates[a] Acid yield[b]
BL21 (DE3) (pCom10_U124)
Initial rates[a] Acid yield[b]
À1
À1
In vivo
96.5Æ1.2 UgCDW
>99%
>99%
68.3Æ1.0 UgCDW
>99%
>99%
À1
À1
In vitro[c]
177.7Æ3.7 Ugprotein
149.7Æ1.5 Ugprotein
[a]
Initial rates are based on product formed within the initial 5 min of reaction.
Conversion of 1.5 mM laurolactam to 12-aminododecanoic acid within 60 min (in vivo) and 30 min (in vitro).
Specific activity is calculated based on whole-cell protein content.
[b]
[c]
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dodecanoic acid was observed. The iÀn1itial rates were BL21 (DE3) (pCom10_T31) and E. coli BL21 (DE3)
determined to be 80.3Æ5.6 U gCDW with E. coli (pCom10_U124) for 2 h and indeed resulted in lauro-
À1
BL21 (DE3) (pCom10_T31) and 84.2Æ1.9 UgCDW
lactam formation (Figure 1) as confirmed by GC-MS
with E. coli BL21 (DE3) (pCom10_U124). In control analysis (Supporting Information).
experiments with wild-type E. coli BL21 (DE3), ester
The substrate was completely converted to 12-ami-
hydrolysisÀ1 occurred at initial rates of 1.7Æ nododecanoic acid and laurolactam within 60 min.
1.1 UgCDW (data not shown). Thus, the hydrolysis of Molar laurolactam yields of 13.0 and 11.5% were ob-
12-aminododecanoic acid methyl ester at pH 7.4 can tained after 30 min with E. coli BL21 (DE3)
primarily be attributed to w-laurolactam hydrolase ac- (pCom10_T31)
and
E.
coli
BL21
(DE3)
tivities. (pCom10_U124), respectively, constituting a transient
Since the kinetically controlled approach did not maximum followed by a decrease due to secondary
result in laurolactam formation at pH 7.4, the pH of hydrolysis as expected for kinetically controlled
the reaction medium was adjusted to pH 10 to in- amide bond formation. Both strains showed similar
À1
crease the amount of non-protonated amine species initial laurolactam formation (14.4Æ0.7 UgCDW and
to 38% (Marvin Software, ChemAxon Ltd., Buda- 13.5Æ1.1 UgCDWÀ1, respectiÀv1ely) and ester hydrolysis
À1
pest, Hungary). Biotransformations of 12-aminodode- activities (63.2Æ2.3 UgCDW and 54.6Æ1.8 UgCDW
,
canoic acid methyl ester were performed with E. coli respectively). The balance of substrate and product
Figure 1. Substrate depletion and product formation patterns (A and C) and corresponding specific activities (B and D) ob-
served during whole-cell biotransformations of 12-aminododecanoic acid methyl ester with E. coli BL21 (DE3)
(pCom10_T31) (A and B) and E. coli BL21 (DE3) (pCom10_U124) (C and D) at pH 10. A cell concentration of
1.2 gCDW LÀ1 was used. The results shown were obtained from duplicate samples and were confirmed by repetition of the ex-
periment.
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Enzyme-Catalyzed Laurolactam Synthesis via Intramolecular Amide Bond Formation
concentrations was closed and no additional peaks activity to around 0.5Æ0.1 UgCDWÀ1. At pH 11, no
were observed by GC and HPLC analysis, clearly in- laurolactam formation was observed. Between pH 8
dicating that no product degradation by activities of and 9.5, maximum laurolactam yields of around 2–3%
host intrinsic enzymes occurred and no other side were observed after 5 min of biotransformation fol-
products such as intermolecular amides were formed. lowed by a decrease in laurolactam concentrations
In contrast to the biotransformation of laurolactam due to secondary hydrolysis. At pH 10, the non-pro-
at pH 7.4 (Table 1), the lactam was not completely hy- tonated substrate acted as an effective nucleophile in
drolyzed to 12-aminododecanoic acid. About 30 mM the formation of the lactam, whereas pH values
laurolactam remained after 2 h of biotransformation. higher than 10 drastically reduced the laurolactam
A pH-driven shift of the thermodynamic equilibrium yield, most likely due to enzyme inactivation. Conse-
to an equilibrium concentration of around 30 mM laur- quently, a pH value of 10 was found to be optimal for
olactam can be excluded as a reason for the lowered enzyme catalyzed laurolactam formation from 12-ami-
laurolactam degradation, as laurolactam formation nododecanoic acid using whole cells.
from 12-aminododecanoic acid was not detected at
pH 10. Hence, the reduced laurolactam hydrolysis
rates are most likely due to a loss of enzymatic activi- Whole-Cell Biocatalysis Enhances Enzyme
ty caused by the high pH applied.
Performance
At pH 10, wild-type E. coli BL21 (DE3) catalyzed
ester hydrolysis at a rate of 7.2Æ0.9 UgCDWÀ1. Lauro- To compare the catalytic performance of whole cells
lactam formation was not detected. As abiotic con- and free enzymes, 12-aminododecanoic acid methyl
trol, 1.0 mM 12-aminododecanoic acid methyl ester ester biotransformations were performed with cell ex-
was dissolved in the reaction buffer and shaken under tracts of E. coli BL21 (DE3) (pCom10_T31) at pH 10
biotransformation conditions. A spontaneous lauro- (Figure 3).
lactam formation was not detected. However, ester
Again, the substrate was completely converted to
hydrolysis to 12-aminododecanoic acid occurred at a 12-aminododecanoic acid and laurolactam within
rate of 0.8 mMminÀ1 (data not shown). Thus, laurolac- 60 min. Laurolactam was formed to a lower maximum
tam formation can be solely ascribed to w-laurolac- concentration of 65 mM (maximum yield of 6.3%)
tam hydrolase activity.
within 30 min and was only slightly degraded to
The pH value of the reaction medium obviously is 56 mM within 3 h. A maximum laurolactam formation
À1
a critical factor for enzyme-catalyzed laurolactam for- rate of 1.5Æ1.0 Ugprotein was obtained corresponding
mation. Hence, the pH dependency of whole cell-cat- to approximately 0.83 U gCDWÀ1. Ester hydrolysis to
alyzed laurolactam formation was investigated. Up to 12-aminododecanoic acid was observed at a maximum
À1
pH 10, laurolactam formation rates increased in cor- rate of 25.0Æ1.4 Ugprotein (ca. 13.8 UgCDWÀ1).
relation to the pH (Figure 2). A further increase of
Whole-cell biotransformations and crude extract
the pH to 10.5 resulted in a severe drop of the specific assays show clearly different bioconversion character-
Figure 2. Specific laurolactam formation rates (A) and maximally achieved laurolactam yields (B) during whole-cell bio-
transformations of 1.5 mM 12-aminododecanoic acid methyl ester with E. coli BL21 (DE3) (pCom10_U124) and E. coli
BL21 (DE3) (pCom10_T31). Maximal yields were obtained after 5 min of biotransformation at 8ꢁpHꢁ9.5 and after 30 min
of biotransformation at pH 10 and 10.5.
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pared to respective rates obtained with whole cells by
translating the latter into corresponding in vitro
values assuming a whole-cell protein content of 55%
(Figure 4).
Using cell extracts, laurolactam formation was ob-
served in biotransformations at pH values between
7.4 and 10. Maximum specific activities of 13.9Æ
À1
0.64 Ugprotein were observed at pH 8 and decreased
with increasing pH, most likely due to w-laurolactam
hydrolase deactivation. At pHꢀ10.5, no laurolactam
was found. The enzyme appeared to be completely in-
activated by the high pH applied. This is in accord-
ance with results obtained by Asano and co-work-
ers.[14] Similar to whole-cell biotransformations, the
maximum laurolactam yield (6.3% after 30 min) was
found at pH 10. At pH values between 8 and 9.5,
lower laurolactam yields were obtained due to ester
and lactam hydrolysis at high rates.
Enzyme-catalyzed laurolactam formation depends
on several parameters: (i) the primary and secondary
hydrolysis rate (Scheme 2), (ii) the nucleophilic attack
of the non-protonated substrate, and (iii) enzyme de-
activation at high pH values. Using whole cells, the
hydrolysis rates (sum of primary and seÀc1ondary hy-
drolysis) increased from 84.2Æ1.9 UgCDW to 221.3Æ
À1
3.1 UgCDW when increasing the pH from 7.4 to 9.5.
At pH 10, the hydrolysis rates were drastically re-
À1
duced to a minimum of 63Æ2.3 UgCDW with the
non-protonated substrate acting as an effective nucle-
ophile. Thereby, the activity ratio of laurolactam syn-
thesis to overall hydrolysis increased to a maximum
of 26.4% (Figure 5). Such a high ratio was not found
using crude extracts, since significant enzyme deacti-
vation occurred at pH>8 and primary and secondary
hydrolysis took place at high rates (310.9Æ
9.7 UgproteinÀ1) at pH 8.
Figure 3. Substrate depletion and product formation pat-
terns (A) and corresponding specific activities (B) observed
during biotransformations of 12-aminododecanoic acid
methyl ester at pH 10 with crude extract of E. coli BL21
(DE3) (pCom10_T31). A protein concentration of 1.63 gLÀ1
was used. The results shown were obtained from duplicate
samples and were confirmed by repetition of the experi-
ment.
In whole cells, the w-laurolactam hydrolase seems
to be protected from inactivation at least for a certain
istics at pH 10. In contrast to the distinct laurolactam period of time. At pHꢁ10, the bacterial pH regula-
concentration maximum observed in whole-cell bio- tion system might have adjusted the intracellular pH
transformations, the lactam was only scarcely degrad- to a lower level and thus preserved enzymatic activity.
ed when cell extracts were applied. Maximally ach- For example, sodium proton antiporters (NhaA) or
ieved laurolactam yields were 2.1-fold lower. Further- multidrug resistance transporters (MdfA) are in-
more, maximum laurolactam and 12-aminododecanoic volved in pH homeostasis in E. coli.[21,22] However,
acid formation rates were lowered 17.2- and 4.6-fold, the application of an external pH higher than pH 9
respectively. The different extent to which these rates also raises the intracellular pH above pH 8,[22] which
were reduced may be explained by the fact that next generally results in deactivation of the w-laurolactam
to the w-laurolactam hydrolase, intrinsic enzymes also hydrolase.[14] Furthermore, enzyme inactivation might
catalyzed ester hydrolysis to 12-aminododecanoic have been counteracted by host intrinsic chaperones,
acid. This becomes obvious from the control experi- which are able to refold denatured enzymes to their
ments with wild-type cells described above.
active conformation.[23] However, the pH regulation
The results indicate that the w-laurolactam hydro- system of E. coli is compromised at pH>9 and cell
lase was deactivated at high pH. Hence, biotransfor- death occurs.[22,24] Small et al. investigated pH resist-
mations were performed at pH values ranging from ance of E. coli by determining the number of colony
7.4 to 11 to determine the pH dependency for lauro- forming units after incubation at pH 10.2. It was
lactam formation using cell extracts. Laurolactam for- shown that 15% of the cells survived for 2 h at
mation rates obtained with crude extracts are com- pH 10.2.[25] These observations and the results ob-
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Enzyme-Catalyzed Laurolactam Synthesis via Intramolecular Amide Bond Formation
Figure 4. Specific laurolactam formation rates (A) and molar laurolactam yields (B) observed during biotransformations of
1.5 mM 12-aminododecanoic acid methyl ester with whole cells of E. coli BL21 (DE3) (pCom10_T31) and respective cell ex-
tracts. The rates obtained from whole-cell biotransformations were translated into their corresponding in vitro values, assum-
ing a whole-cell protein content of 55%. Maximal yields were obtained after 5 min of biotransformation at 7.4ꢁpHꢁ9.5
and after 30 min of biotransformation at pH 10 and 10.5.
hydrolysis rates after 60 min observed during whole-
cell biotransformations at pH 10 (Figure 1).
Future work will focus on the application of a two-
liquid phase system, which represents a possible strat-
egy to enhance the product yield[26] while operating at
lower pH. By addition of a suitable organic phase, en-
abling the efficient in situ extraction of laurolactam,
but not of 12-aminododecanoic acid, the conversion
of the respective methyl ester may be directed to-
wards laurolactam formation at the same time pre-
venting laurolactam hydrolysis.
Conclusions
w-Laurolactam hydrolases were tested for laurolac-
tam formation from 12-aminododecanoic acid and 12-
aminododecanoic acid methyl ester in aqueous solu-
tion. Laurolactam formation from 12-aminododecano-
Figure 5. Ratios of initial activities for laurolactam forma-
ic acid could not be observed. However, applying the
tion and overall hydrolysis in biotransformations of 1.5 mM
concept of kinetically controlled peptide synthesis to
12-aminododecanoic acid methyl ester with whole cells of E.
intramolecular amide bond formation, laurolactam
coli BL21 (DE3) (pCom10_T31) and respective cell extracts
formation from 12-aminododecanoic acid methyl
in dependence of pH.
ester was achieved. A maximum yield for laurolactam
of 13.0% was obtained during whole-cell biotransfor-
tained in this study suggest that the w-laurolactam hy- mations using E. coli BL21 (DE3) (pCom10_T31) and
drolase was protected from rapid inactivation during the application of whole cells resulted in 17.2-fold
whole-cell biotransformations of 12-aminododecanoic higher laurolactam formation rates in comparison to
acid methyl ester at pHꢁ10, resulting in higher prod- free enzymes at pH 10. The w-laurolactam hydrolase
uct formation rates as compared to rates observed seemed to be protected in whole cells, whereas the
with cell extracts. However, cell viability is expected free enzyme was deactivated. Thus, kinetically con-
to decrease over time resulting in an intracellular al- trolled amide bond formation proved to be a promis-
kalization and thus in w-laurolactam hydrolase inacti- ing approach for lactam synthesis using recombinant
vation. This is supported by the reduced laurolactam cells.
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and delivered in the plasmids pBSK_T31 and pBSK_U124.
The plasmids pBSK_T31, pBSK_U124, and pCom10 were
digested with NdeI and SalI. The pCom10 vector (7649 bp)
and the pBSK-inserts (1500 bp) were isolated from agarose
gels[30] and purified with a peqGOLD Gel Extraction Kit
(PEQLAB Biotechnology GmbH, Erlangen, Germany).
After dephosphorylation of pCom10 and ligation with the
isolated pBSK_U124 or pBSK_T31 inserts, the DNA was
transformed into E. coli DH5a via electroporation. Success-
ful cloning yielding pCom10_T31 and pCom10_U124 was
verified by restriction and sequence analysis of the insert
region.
Experimental Section
Strains, Plasmids and Growth Conditions
Laurolactam was purchased from TCI Europe (Zwijndrecht,
Belgium) with a purity of >99%. 12-Aminododecanoic acid
methyl ester with a purity ꢀ99% and 12-aminododecanoic
acid with a purity of 96% were obtained from Evonik De-
gussa GmbH (Marl, Germany).
Strains and plasmids used in this work are listed in
Table 2. E. coli DH5a was used for cloning purposes and E.
coli BL21 (DE3) for recombinant gene expression and bio-
catalytic studies.
To obtain recombinant strains, plasmid DNA was intro-
duced into the strains via electroporation (2.5 kV, EquiBio
Easyjet Prima, Ashford, UK) and transformants were select-
ed via their antibiotic resistance.
Whole-Cell Biotransformation
Whole-cell biotransformations with resting, i.e., non-growing
but metabolically active, cells were performed to determine
w-laurolactam hydrolase activity. The specific activities are
given in units per gram cell dry weight (UgCDWÀ1), whereby
1 U describes the activity forming 1 mmol product per
minute. Cell concentrations were measured with a Libra S11
spectral photometer (Biochrom Ltd, Cambrigde, UK). A
5 mL LB preculture was inoculated with a single colony and
incubated at 378C for 8 h. Fifty milliliters M9* medium
were inoculated with 500 mL LB pre-culture and incubated
overnight at 308C. Two-hundred milliliters M9* were inocu-
lated with the M9* pre-culture to an optical density at
450 nm (OD450) of 0.2 (1 OD450 =0.166 gCDW LÀ1[33]). The cul-
ture was incubated at 308C. At an OD450 of 0.5, induction
with 0.025% (vol/vol) dicyclopropyl ketone was carried out
and the cultivation was continued for 5 h. Cells were har-
vested by centrifugation (15 min, 48C, 4,595ꢃg; Heraeus
Multifuge 1 S-R, Oberhausen, Germany) and resuspended
either in 50 mM potassium phosphate buffer (pH 7.4) or
100 mM sodium carbonate buffer (pH 8, 9, 10, 10.5, 11) con-
taining 1% glucose to a cell concentration of 1 gCDW LÀ1.
Glucose was added to enable metabolism activity, e.g., for
pH homeostasis. Aliquots of 1 mL were filled in Pyrex tubes
and shaken at 308C and 350 rpm. After 5 min of adaptation,
the reaction was initiated by the addition of either 1.5 mM
laurolactam (from a 100 mM stock solution in ethanol) or
1.5 mM 12-aminododecanoic acid methyl ester (from a
60 mM stock solution in ethanol) or 0.75 mM 12-aminodo-
decanoic acid (from a 50 mM stock solution composed of
50% (v/v) 0.1N HCl in acetone). The reaction was stopped
by addition of 1 mL ice-cold diethyl ether containing
0.2 mM dodecane as internal standard for gas chromatogra-
E. coli cells were grown either in lysogeny broth (LB)[30]
or M9* minimal medium[30,31] containing 9 g KH2PO4, 25.5 g
Na2HPO4
·
2
H2O, 1 g NH4Cl, 0.5 g NaCl, 0.49 g
MgSO4·7H2O, 5 g glucose, and 1 mL USFe trace element so-
lution[32] per liter. Where appropriate, 50 mgLÀ1 kanamycin
or 100 mgLÀ1 ampicillin were added. Solid media contained
1.5% (w/v) agar. Cultivation temperature was 30 or 378C as
indicated. Liquid cultures were incubated in tubes or in baf-
fled Erlenmeyer flasks in horizontal shakers at 200 rpm.
Stock cultures were prepared by addition of 200 mL 50% (v/
v) glycerol to 800 mL over-night grown LB cultures and
stored at À808C.
Construction of pCom10_T31 and pCom10_U124
Restriction enzymes, Fast APTM Thermosensitive Alkaline
Phosphatase, and T4 Ligase were purchased from Fermentas
(St. Leon-Rot, Germany) and used according to the suppli-
erꢂs protocols. Plasmid DNA was isolated with a peqGOLD
Miniprep Kit I (PEQLAB Biotechnology GmbH, Erlangen,
Germany) according to the supplierꢂs recommendations.
Genes encoding the w-laurolactam hydrolases of Acidovor-
ax sp. T31 (NCBI gene bank: AB444713) and Cupriavidus
sp. U124 (NCBI gene bank: AB444714) were designed as
follows: The gene sequence was not modified except for the
start codon GTG, which was changed to ATG. The triplet
CAT was introduced upstream of the gene sequence to form
a NdeI restriction site together with the start codon. An ad-
ditional multiple cloning site[29] containing a SalI site was in-
troduced downstream of the stop codon. The genes were
synthesized by ATG:biosynthetics (Merzhausen, Germany)
Table 2. E. coli strains and plasmids used in this study.
E. coli strain/
Characterization
References
Plasmid
[27]
[28]
BL21 (DE3)
DH5a
FÀ, ompT, hsdSB (rBÀ mBÀ), l
ACTHNGUTERNNU(G DE3 [lacI lacUV5 T7 gene 1 Sam7 Dnin5])
FÀ, endA1, glnV44, thi-1, recA1, relA1, gyrA96, deoR, supE44, F80dlacZDM15 D(lacZYA-
À
argF)-U169, hsdR17 (rK mK+), lÀ
pBSK_T31
pBSK_U124
pCom10
P15A ori, rep, T7 promoter, Ampr, w-laurolactam hydrolase gene from Acidovorax sp. T31
this study
P15A ori, rep, T7 promoter, Ampr, w-laurolactam hydrolase gene from Cupriavidus sp. U124 this study
[29]
broad-host-ranged expression vector, alk promoter, Kmr
pCom10_T31
pCom10 with w-laurolactam hydrolase gene from Acidovorax sp. T31
this study
this study
pCom10_U124 pCom10 with w-laurolactam hydrolase gene from Cupriavidus sp. U124
2508
asc.wiley-vch.de
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2011, 353, 2501 – 2510

Enzyme-Catalyzed Laurolactam Synthesis via Intramolecular Amide Bond Formation
phy (GC) or 0.5 mL acetonitrile for reversed phase high per-
formance liquid chromatography (RP-HPLC) analysis.
HPLC analysis. The acetonitrile containing sample was vor-
texed for 1 min and centrifuged (15 min, 48C, 17,000ꢃg).
The clear supernatant was analyzed. A 20 mL sample was in-
jected onto a Luna C8(2) column (4.6ꢃ150 mm, 5 mm,
100 ꢄ; Phenomenexꢅ, Aschaffenburg, Germany) on a LaCh-
rome Eliteꢅ HPLC system (VWR-Hitachi, Darmstadt, Ger-
many), which was linked to a Corona charged aerosol detec-
tor (Dionex Softronic GmbH, Germering, Germany). The
column temperature was set to 408C. A flow rate of
0.8 mLminÀ1 was applied. The mobile phases consisted of
water containing 0.4% trifluoroacetic acid (TFA) (A), meth-
anol (HPLC-grade) containing 0.2% TFA (B) and acteoni-
trile (HPLC-grade) (C). The following profile was applied:
0–2 min 45% A and 55% C, 2–24 min linear gradient to
25% A, and 75% C, 24–29 min linear gradient to 2% A,
30% B, and 68% C, 29–30 min linear gradient to 2% A, and
98% C, and 30–33 min linear gradient to 45% A and 55%
C, which was kept for 2 min.
Enzyme Activity Assay
Cells were grown, induced and harvested as described for
whole-cell biotransformations. After harvesting, the cells
from a 200 mL culture were resuspended in 5 mL 100 mM
sodium carbonate buffer (pH 10) and disrupted by using a
French press (three passages at 800 psi; SLM-Aminco, Ro-
chester, NY, USA). Cell debris and non-lysed cells were re-
moved by centrifugation (17,000ꢃg, 20 min, 48C; Fresco
centrifuge, Heraeus, Oberhausen, Germany). The clarified
cell extract was diluted ten-times with 100 mM potassium
phosphate buffer (pH 7.4) or 100 mM sodium carbonate
buffer (pH 8, 9, 9.5, 10, 10.5, 11) containing 1% glucose and
used for activity assays. Aliquots of 1 mL of the diluted cell
extracts were filled in Pyrex tubes and shaken at 308C and
400 rpm. After 5 min of adaption, substrate was added to a
final concentration of 1.0 mM 12-aminododecanoic acid
methyl ester (from a 40 mM stock solution in ethanol) or
1.5 mM laurolactam (from a 60 mM stock solution in etha-
nol). The reaction was stopped by addition of 1 mL ice-cold
diethyl ether containing 0.2 mM dodecane as internal stan-
dard for GC analysis or with 0.5 mL acetonitrile or RP-
HPLC analysis. Protein concentrations were determined
using a commercially available Quick Start BradfordTM Pro-
tein Assay solution (Biorad, Munich, Germany) according
to the supplierꢂs protocol. Bovine serum albumin was used
as standard protein.
Acknowledgements
This study was supported by a grant provided by the Federal
Ministry of Education and Research (grant number:
0315205).
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