Light-Driven Enzymatic Decarboxylation of Dicarboxylic Acids

Article abstract of DOI:10.1002/open.202100039

Photodecarboxylase from Chlorella variabillis (CvFAP) is one of the three known light-activated enzymes that catalyzes the decarboxylation of fatty acids into the corresponding C1-shortened alkanes. Although the substrate scope of CvFAP has been altered by protein engineering and decoy molecules, it is still limited to mono-fatty acids. Our studies demonstrate for the first time that long chain dicarboxylic acids can be converted by CvFAP. Notably, the conversion of dicarboxylic acids to alkanes still represents a chemically very challenging reaction. Herein, the light-driven enzymatic decarboxylation of dicarboxylic acids to the corresponding (C2-shortened) alkanes using CvFAP is described. A series of dicarboxylic acids is decarboxylated into alkanes in good yields by means of this approach, even for the preparative scales. Reaction pathway studies show that mono-fatty acids are formed as the intermediate products before the final release of C2-shortened alkanes. In addition, the thermostability, storage stability, and recyclability of CvFAP for decarboxylation of dicarboxylic acids are well evaluated. These results represent an advancement over the current state-of-the-art.

Full text of DOI:10.1002/open.202100039

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Light-Driven Enzymatic Decarboxylation of Dicarboxylic
Acids
Yong-Yi Zeng,[a] Lan Liu,[a, b] Bi-Shuang Chen,*[a, c] and Wuyuan Zhang[d, e]
Photodecarboxylase from Chlorella variabillis (CvFAP) is one of
the three known light-activated enzymes that catalyzes the
decarboxylation of fatty acids into the corresponding C1-
shortened alkanes. Although the substrate scope of CvFAP has
been altered by protein engineering and decoy molecules, it is
still limited to mono-fatty acids. Our studies demonstrate for
the first time that long chain dicarboxylic acids can be
converted by CvFAP. Notably, the conversion of dicarboxylic
acids to alkanes still represents a chemically very challenging
reaction. Herein, the light-driven enzymatic decarboxylation of
dicarboxylic acids to the corresponding (C2-shortened) alkanes
using CvFAP is described. A series of dicarboxylic acids is
decarboxylated into alkanes in good yields by means of this
approach, even for the preparative scales. Reaction pathway
studies show that mono-fatty acids are formed as the
intermediate products before the final release of C2-shortened
alkanes. In addition, the thermostability, storage stability, and
recyclability of CvFAP for decarboxylation of dicarboxylic acids
are well evaluated. These results represent an advancement
over the current state-of-the-art.
1. Introduction
CÀ C bond-forming reactions have been combined with redox
enzymatic biotransformation,[5] (3) Combination of photo-/
electro-(chemistry and catalysis) and biocatalysis, which is the
most attractive approach to promote electron transfer, thus
enables the development of green and sustainable chemistry.[6]
However, most of the examples combing photo-/electro-and
biocatalysis are focusing on the cofactor recycling or cofactor
replacement with light irradiation.[7–10] Notably, examples of
photoenzymes by direct light activation are quite rare but
desirable.
So far, there are only three photoenzymes known today.
Protochlorophyllide oxidoreductase is the first example of light-
driven enzymes which was described in 2015 and able to
catalyze the reduction of the C17–C18 double bond of
protochlorophyllide to yield the desired product of
chlorophyllide.[11] The formation of chlorophyllide is the chal-
lenging step in the chlorophyll biosynthetic pathway.[12] As
protochlorophyllide oxidoreductase is light-dependent, it pro-
vides essential evidences for the combination of light-activated
chemical and biological catalysts. Another known blue-light-
activated enzyme is the flavin-dependent DNA-repair enzymes
(photolyase), which repairs ultraviolet-induced DNA damage
during the process of producing cyclobutene pyrimidine dimers
and pyrimidine-pyrimidone photoproducts with the overall
quantum yield.[13] Very recently, a unique photoenzyme (CvFAP)
was discovered from the microalga Chlorella variabilis and
shown to catalyze the free fatty acids into the corresponding
(C1-shortened) alkanes via decarboxylation by Beisson and the
co-authors.[14,15] CvFAP represents an advancement over the
current state-of-the-art photoenzyme and must be regarded as
door-openers for yet-to-be-expected next steps in organic
synthesis.
Cascade reactions combining chemo- and biocatalysis are
regarded as the most efficient and universal systems to foster
significant collaborations between chemists and enzymologists.
In the past decade, cascade reactions have been indeed
frequently used in biocatalysis together with different types of
chemical catalysis for successful performance of multistep
syntheses.[1,2] Bornscheuer and the co-workers first subdivided
the most intensively studied chemo-enzymatic strategies into
three concepts:[3] (1) combination of (transition) metalcatalysis
and biocatalysis, which have almost reached the “mature
technology” stage, especially in the cases of dynamic kinetic
resolution,[4] (2) combination of organo- and biocatalysis, which
is complementary, and has been used in a range of unique
synthetic cascades. For example, asymmetric organocatalytic
[a] Y.-Y. Zeng, Prof. Dr. L. Liu, Dr. B.-S. Chen
School of Marine Sciences
Sun Yat-Sen University
Zhuhai 519082 (P. R. China)
E-mail: chenbsh23@mail.sysu.edu.cn
[b] Prof. Dr. L. Liu
Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai)
Zhuhai 519082 (P. R. China)
[c] Dr. B.-S. Chen
Guangdong Provincial Key Laboratory of Marine Resources and Coastal
Engineering
Zhuhai 519082 (P. R. China)
[d] Prof. Dr. W. Zhang
Tianjin Institute of Industrial Biotechnology
Chinese Academy of Sciences
West 7th Avenue, Tianjin 300308 (P. R. China)
[e] Prof. Dr. W. Zhang
National Technology Innovation Center of Synthetic Biology
32 West 7th Avenue, Tianjin 300308 (P. R. China)
© 2021 The Authors. Published by Wiley-VCH GmbH. This is an open access
article under the terms of the Creative Commons Attribution Non-Com-
mercial License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited and is not used for
commercial purposes.
Indeed, following the footsteps of this contribution, several
studies on the application/catalytic mechanism of CvFAP have
been performed. For example, Hollmann and co-workers fully
assessed the preparative potential of CvFAP for the decarbox-
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ylation of fatty acids.[16,17] Later, CvFAP was used for the
production of chiral amines, α-hydroxy acid, and alkenes in
different laboratories.[3,18–22] Ma et al. expanded the substrate
scope to ‘real’ substrates of commercially available oils with a
bienzymatic cascade comprising a hydrolase and CvFAP.[23]
Asymmetric catalysis by an engineered CvFAP was described
recently for the kinetic resolution of α-functionalized carboxylic
acids[24] and phosphinothricin.[25] Nevertheless, information on
the mechanism of CvFAP-catalyzed decarboxylation are very
limited.[26,27] Very recently, the pathway about the synthesis of
alka(e)nes in Yarrowia lipolytica by CvFAP was reported.[28] It is
surprising that the preferred substrate for CvFAP is acyl-CoAs,
rather than free fatty acids.
These reports on light-dependent CvFAP encouraged us to
utilize it for the conversion of dicarboxylic acids, which still
represents a chemically very challenging reaction. Although the
substrate scope of CvFAP has been altered by protein
engineering[21,24] and decoy molecules,[17] it is still limited to
mono fatty acids. Therefore, it is of high interest to probe
whether the conversion of dicarboxylic acids to alkanes can be
accomplished by CvFAP and how broadly it is applicable. The
aim of this study was to demonstrate for the first time that
dicarboxylic acids can be converted by the newly discovered
photoenzyme CvFAP, enlarging the substrate scope of CvFAP.
41% was observed (Table 1). An increase of the cell concen-
tration to 300 mg/mL gave no significant change in the
conversion, revealing that 200 mg/mL might be a suitable cells
concentration. By increasing the substrate concentrations of
hexadecanedioic acid (1a) to 5 mM, 10 mM, 13 mM, an almost
linear increase in product formation was detected (Table 1).
Tetradecane (2a) was obtained with a maximum concentration
of 10.28 mM. Notably, the enzyme reaction rate depended on
the concentration of hexadecanedioic acid (1a). However, when
the substrate concentration continued increasing to 15 mM, a
slight decrease in product formation was found, indicating that
13 mM was the maximum substrate concentration for LCDAs
decarboxylation. Since the time course, pH and temperature
profile of CvFAP had been described previously for the
decarboxylation of mono-fatty acids,[16] here we don’t want to
repeat again those studies. Besides, this study focused on the
evaluation of CvFAP as a photocatalyst for the decarboxylation
of LCDAs, to enlarge the substrate scope of CvFAP.
It is worth mentioning that background reactions were
performed in parallel with experiments of E. coli cells containing
an empty vector as catalysts, no CvFAP cells, heat-denatured
CvFAP cells. No background reaction was taking place, indicat-
ing that the decarboxylation of hexadecanedioic acid (1a) to
tetradecane (2a) was affected by the active CvFAP enzyme.
Thus, dicarboxylic acids were the suitable substrates for CvFAP.
We were pleased that the desired product tetradecane (2a)
could be obtained with a maximum concentration of 10.28 mM
when 13 mM of hexadecanedioic acid (1a) was loaded. There-
fore, the scalability of the developed reaction system was tested
by performing a preparative-scale synthesis. The reaction was
scaled up to 100 mL using hexadecanedioic acid (1a, 13 mmol)
to give the desired tetradecane (2a, 10.5 mmol) in a yield of
81%.
2. Results and Discussion
We started our investigation by evaluating the potential of
CvFAP as a photobiocatalyst for the decarboxylation of LCDAs
to the corresponding C2-shortened alkanes. As a result of its
rigid character and wide use as building blocks for polymers,
LCDA of C16 chain length (hexadecanedioic acid, 1a) was
chosen as the main test substrate.[29] According to the previous
studies,[15] although there were two variant CvFAP (long-length
CvFAP and short-length CvFAP) present in the microalga
Chlorella variabilis, only short-length CvFAP revealed a good
overproduction in Escherichia coli (E. coli). Thus, short-length
CvFAP was used for this study. After heterologous expression in
E. coli BL21 (DE3), the E. coli cells, the cell-free extract (CFE), as
well as the purified enzyme (after one-step purification with a
HisTrap affinity column) were all used for catalytic activity test.
Surprisingly, the overexpressed E. coli cells showed a significant
activity towards hexadecanedioic acid (1a), as compared to the
purified CvFAP or CFE. This is supported by the findings of
Scrutton and the co-workers, that “broken cell” operation often
leads to significant activity loss of CvFAP.[26] Methods to prevent
activity loss of CvFAP during purification step is of great
significance. Whereas, whole cells of overexpressed E. coli
(CvFAP@E.coli) were used for this study. In order to increase the
solubility of decarboxylated product of alkanes, the catalytic
reaction was performed in Tris-HCl buffer with DMSO as a
cosolvent (30%, v/v).
Table 1. Evaluation of the potential of CvFAP as a photobiocatalyst for
LCDAs decarboxylation.[a]
[Substrate]
[mM]
Catalyst
(wet cells)
[Product]
Yield
[%]
[mM][b]
1
5
200 mg/mL
200 mg/mL
200 mg/mL
200 mg/mL
200 mg/mL
E. coli cells containing an empty vector
0
0.41
2.88
7.80
10.28
10.03
–
41
58
78
79
68
–
10
13
15
13
13
13
–
–
–
–
denatured cells
The first experiment was performed with 1 mM of hexade-
canedioic acid (1a) and 200 mg/mL of wet cells under blue light
illumination for 6 h. The desirable product tetradecane (2a)
with a concentration of 0.41 mM, corresponding to a yield of
[a] Reaction conditions: [substrate]=given concentrations, [CvFAP wet
cells]=200 mg/mL, Tris-HCl buffer (pH 8.5, 100 mM), 30% DMSO, blue
°
light illumination at 30 C for 6 hours. [b] Product concentration was
determined by GC.
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It‘’s known that wild-type CvFAP preferentially catalyze the
dicarboxylic acids into alkanes, heptadecanedioic acid (1f),
which was readily decarboxylated by CvFAP, was chosen as the
represented substrate. This is to answer a question: two
carboxylic acid group were decarboxylated simultaneously to
yield alkanes or mono-fatty acids were formed as intermediate
products. The reaction was carried out with 13 mM of
heptadecanedioic acid (1f) and 200 mg/mL of wet cells under
blue light illumination in Tris-HCl buffer (pH 8.5, 100 mM) for
the given time. As shown in Figure 1, it is clear that the
substrate heptadecanedioic acid (1f) decreased in concentra-
tions readily over time. Palmitic acid (1.05 mM) and trace
pentadecane were observed in the reaction mixture after
decarboxylation of C16~C18 mono-fatty acids, which is not
satisfying.[16] The substrate specificity of CvFAP for mono-fatty
acids decarboxylation has been improved by protein engineer-
ing and decoy molecules. Since the substrates LCDAs in this
study are different from the mono-fatty acids shown in previous
study[16] in terms of sources, application, chemical/physical
properties, we were interested in the scope of CvFAP used as
photocatalysts for decarboxylation of LCDAs to alkanes. Eight
further LCDAs structurally related to the main test substrate 1a
were tested. The results are summarized in Table 2.
Generally speaking, CvFAP showed a high activity towards
LCDAs of C14~C20, although there was no clear trend towards
conversions and chain length. Hollmann and co-workers
proposed that in CvFAP-catalyzed mono-fatty acids decarbox-
ylation, the difference in conversions of different substrates was
assigned to the distance of the carboxylate group from
substrate to the flavin cofactor. Mono-fatty acids with longer
chain-length are more easily bound to CvFAP’s access channel
and the reactive group (À COOH) are more easily exposure to
°
incubation at 30 C for 5 min, ruling out the simultaneous
decarboxylation of the two carboxylic acid moieties, and
validating our hypothesis. The amount of palmitic acid
(3.30 mM) formed in the reaction system was increasing almost
in a triple rate between 5 min to 15 min, with a little amount of
pentadecane (2f) (0.46 mM) detected. A clear decrease in the
concentration of palmitic acid was observed after incubation
from 30 min, while, the concentration of pentadecane (2f)
significantly increased at the meantime. Almost all the remain-
ing palmitic acid were completely converted into pentadecane
(2f) after 3 h, resulting in a final yield of 87%.
*
*
the cofactor (to yield a CH3COO ···FAD radical pair), resulting in
high conversion and initial rate. LCDAs are different from
mono-fatty acids, having two reactive groups of À COOH.
Whereas, taking the findings of Hollmann and co-workers and
our results in this study into account, it can be proposed that
dicarboxylic acids were decarboxylated by CvFAP with mono-
fatty acids as intermediate products. In mono-fatty acids
decarboxylation, full conversion was observed for 13 mM of
substrates in some cases. In LCDAs decarboxylation, 10.28 mM
of the desired products were obtained from 13 mM of
substrates, as one of the best examples. This phenomenon
might be explained by the fact that binding groups shifted
from hydrophobic group (À R) in mono-fatty acids to hydrophilic
group (À COOH) in LCDAs.
In order to further confirm there exists such a reaction
pathway, we docked the substrate (heptadecanedioic acid, 1f)
into the crystal structure of CvFAP (PDB:5NCC). The molecular
docking analyses (Figure 2) clearly showed that only one
carboxyl group is positioning to the active site in a correct
productive conformation. Combining the results obtained in
this study and mechanism of FAP-catalyzed decarboxylation
Further proof of the reaction pathway of LCDAs decarbox-
ylation was evidenced by the time course of the reaction. In
order to have a clear look at how CvFAP catalyze the
Table 2. Relative activity of CvFAP towards different LCDAs.[a]
substrate structure
[substrate]
[mM]
[product]
relative
activity [%]
[mM][b]
HOOCCH2 (CH2)12CH2COOH (1a)
HOOCCH2 (CH2)8CH2COOH (1b)
HOOCCH2 (CH2)9CH2COOH (1c)
HOOCCH2 (CH2)10CH2COOH (1d)
HOOCCH2 (CH2)11CH2COOH (1e)
HOOCCH2 (CH2)13CH2COOH (1f)
HOOCCH2 (CH2)14CH2COOH (1g)
HOOCCH2 (CH2)15CH2COOH (1h)
HOOCCH2 (CH2)16CH2COOH (1i)
13
13
13
13
13
13
13
13
13
10.28
~0.5
~0.5
8.74
5.24
8.64
10.28
7.61
4.73
100
~5
~5
85
51
84
100
74
46
[a] Reaction conditions: [substrate]=13 mM, [CvFAP wet cells]=200 mg/
mL, Tris-HCl buffer (pH 8.5, 100 mM), 30% DMSO, blue light illumination at
Figure 1. Photoenzymatic decarboxylation of heptadecanedioic acid (1f)
into pentadecane (2f), forming palmitic acid as an intermediate product.
°
30 C for 6 hours. [b] Product concentration was determined by GC.
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pH 8.5) containing 30% DMSO, 0.2 g of wet cells, 13 mM
°
substrate and shaken at 30 C under blue light illumination for
6 h. At the end of the reaction, the cells were centrifuged,
washed twice with Tris-HCl buffer (100 mM, pH 8.5) and reused
for the next cycle under the same reaction setup. Whole cells of
overexpressed E. coli (CvFAP@E.coli) showed no significant
changes in activities at the first 3 cycles. From the fourth cycle,
enzyme activities started decreasing in a moderate rate, where-
as, the cells retained 20% of the initial activity after 9
consecutive cycles. Such rapid deactivation was most likely
owing to the massive losses of CvFAP@E.coli cells during
washing step. Only 50 mg cell were remaining in cycle 9.
Immobilization can strongly enhance the stability of CvFAP@E.-
coli cells, and make the catalyst be filtered off easily without
washing step, which is currently being implemented in our
laboratory.
Figure 2. Docking analysis of heptadecanedioic acid (1f) (in dark green) in
the active site of CvFAP.
available,[17,26,30] it can be noted that after binding in CvFAP’s
access channel, heptadecanedioic acid (1f) first positioned one
of the carboxylic acid groups towards FAD, to form HOOC
The thermostability of whole cells of CvFAP@E.coli was
characterized as well. The cells were first incubated at various
°
temperatures (30–70 C) for 1 h, respectively, prior to the
*
*
(CH2)15COO ···FAD radical pair. Consequently, the flavin adduct
(HOOC(CH2)15-FAD-) was protonated to release the intermediate
palmitic acid (HOOC(CH2)14CH3). The intermediate product
palmitic acid (HOOC(CH2)14CH3) was ready to enter the next
catalytic cycle, which was ended by the final release of alkane
product (2f).
addition to the reaction mixture. Then, the cells were
resuspended to 1 mL Tris-HCl buffer (100 mM, pH 8.5) contain-
ing 30% DMSO, 13 mM hexadecanedioic acid (1a). The reaction
°
mixture was shaken at 30 C under blue light illumination for
6 h. As shown in Figure 4, the cells of CvFAP@E.coli maintained
approximately 99% of their original activities after 1 h incuba-
°
°
The enzymatic conversion of LCDAs to alkanes was estab-
lished to provide evidence for the recycling of aliphatic
polyesters polymers. Thus, the process must be as much
efficient as possible, which mostly depended on the enzymes.
An enzyme’s operational stability and recyclability over a long
period of time are considered as the most important character-
istics for practical applications.[31,32] The higher stability and
recyclability that an enzyme exhibits, the more efficiently a
process can be run. Therefore, the thermostability, storage
stability and recyclability of CvFAP for the decarboxylation of
LCDAs were evaluated.
tion at 30 C or 40 C. However, when the temperature was
°
higher than 40 C, a significant decrease was observed in the
activity of the cells over a 1 h period. This is supported by the
findings of Beisson and co-workers, who showed that the
activity of recombinant CvFAP protein decreased sharply when
[13]
°
temperature rose above 35 C.
Methods to enhance the
thermostability of CvFAP@E.coli cells is of great significance,
which is also under currently under way in our laboratory.
To investigate the storage stability of CvFAP, the cells of
°
CvFAP@E.coli were stored at room temperature (25 C) and
tested for the activity in decarboxylation of hexadecanedioic
acid (1a). Cells were withdrawn at various time intervals (1, 2, 3,
4, 5, 6, 7, 14 days) and added to 1 mL Tris-HCl buffer (100 mM,
pH 8.5) containing 30% DMSO, and 13 mM hexadecanedioic
Experiments were performed to examine the recyclability of
CvFAP for the decarboxylation of hexadecanedioic acid (1a) as
an example. For the results summarized in Figure 3, every
reaction was carried out in 1 mL of Tris-HCl buffer (100 mM,
Figure 4. Thermostability of CvFAP@E.coli cells for the decarboxylation of
hexadecanedioic acid (1a) to tetradecane (2a) under blue light illumination.
Cells were incubated at various temperatures for 1 h before substrate was
added to initiate reactions.
Figure 3. Repeated decarboxylation of hexadecanedioic acid (1a) to tetrade-
cane (2a) under blue light illumination by cells of CvFAP@E.coli. Relative
activities were determined by GC.
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acid (1a) to initiate the reaction. The reaction of the mixture
cells could be reused for 3 cycles without significant loss of
activity. After 9 consecutive cycles, the cells retained 22% of the
initial activity. In summary, the newly discovered photoenzyme
CvFAP was able to catalyze the decarboxylation of LCDAs to the
corresponding (C2-shortened) alkanes under mild conditions.
This approach will hopefully provide concepts and solutions in
reducing the need of petrol-based materials for the production
of aliphatic polyesters, making degradable and sustainable
plastics possible.
°
was then shaken at 30 C under blue light illumination for 6 h.
Gratifyingly, in each case, the cells maintained almost 100% of
the initial activities. That is, the CvFAP@E.coli cells were stable at
°
room temperature (25 C) within the measured periods
(2 weeks) without any activity loss (data not shown).
3. Conclusion
The annul global production of plastics has exceeded 359 mil-
lion tons; 90% of these are derived from fossil feedstock, while, Experimental Section
only ~14% are collected for recycling. This causes a huge waste
of fossil resources. Besides, most of the plastics are remarkably
persistent in the environment, thus becoming a critical environ-
ment threat to the ecological systems. Especially, the negative,
hazardous effects of microplastics have induced increasing
concerns since they can enter the food chain and impose
serious problems. Degradable plastics from renewable resour-
ces is one of the most potential alternatives. Nevertheless, there
are very few reports on the synthesis of degradable plastics
from bio-based materials. Having both degradable and sustain-
able properties in one type of plastics is still challenging. For
example, one of the most competitive biodegradable polymers
commercialized up to now is aliphatic polyesters, while, their
monomers (LCDAs) are mainly produced from petrochemical
alkanes by fermentation with C. tropicalis in China so far.
To date, numerous microorganisms and/or enzymes have
been identified to hydrolyzing aliphatic polyesters plastics into
monomers, LCDAs and diols. The development indeed could
provide a viable bioremediation strategy to recycle and reuse
plastic waste, however, the loop of the circular plastics
economy is not yet closing. If the starting material (petrochem-
ical alkanes) could be isolated and reused, this could signifi-
cantly reduce the consumption of petrol-based feedstock,
thereby contributing towards the concept of a circular degrad-
able plastics economy. To isolate alkanes from monomer
(LCDAs), the key step involves the decarboxylation of LCDAs,
which still represents a chemically very challenging reaction.
Thus, a straightforward light-driven route for the decarbox-
ylation of LCDAs to alkanes was established, employing
recombinant cells of a recently discovered photodecarboxylase
from Chlorella variabillis (CvFAP@E.coli). CvFAP readily decar-
boxylated a series of LCDAs with a concentration of 13 mM into
the corresponding (C2-shortened) alkanes in good yields under
blue light illumination. The reaction was scaled up to a 100 mL
scale to determine the scalability of the light-driven enzymatic
decarboxylation of hexadecanedioic acid with CvFAP@E.coli,
and 10.5 mM of the desired product tetradecane were achieved
after 24 h. Our study suggested that mono-fatty acids were
formed as an intermediate product before the final release of
C2-shortened alkanes. Thermostabilities studies showed that
CvFAP@E.coli cells maintained 99% of the original activity
Materials and Methods
Vector pET28a(+) was purchased from Novagen (Merck Millipore,
Amsterdam, The Netherlands). All chemicals were purchased from
Sigma-Aldrich (Schnelldorf, Germany) and were used without
further purification unless otherwise specified. The culture media
components were obtained from BD (Becton, Dickinson and
Company, Breda, The Netherlands).
Conversion of substrates and yield of products were quantified by
GC using calibration lines. GC analysis of alkanes was followed with
a Scion GC 456 system equipped with an Agilent J&W GC Columns
(60 m×0.53 mm×2.5 μm) using N2 as the carrier gas. The following
conditions were used for the dicarboxylic acids/alkanes separation:
°
°
injector 260 C, detector (FID) 280 C, FID hydrogen 30 oxygen 300,
column flow 20 mL/min, maximum temp: 255 C, temperature
°
°
°
°
program: start 110 C, hold time 2 min, rate 25 C/min to 190 C
°
°
hold time 2 min, rate 30 C/min to 280 C hold time 5 min, rate
°
°
30 C/min to 310 C hold time 2 min. The retention time was shown
as below: n-tetradecane (2a)=8.78 min, n-decane (2b)=4.86 min,
n-undecane (2c)=5.85 min, n-dodecane (2d)=6.85 min, n-tride-
cane (2e)=7.86 min, n-pentadecane (2f)=9.65 min, n- cetane
(2g)=10.45 min, n-heptadecane (2h)=11.24 min, n- octadecane
(2i)=11.97 min.
Heterologous Expression
The recombinant plasmids were received from the laboratory of
Prof. Frank Hollmann as a gift.[22] The recombinant plasmids were
subsequently transformed into E. coli BL21 (DE3) cells. Expression
was performed in LB medium containing 50 μg/mL kanamycin at
°
30 C. When OD600 reached 0.5–0.6, the production of the recombi-
nant CvFAP was induced by addition of isopropyl thio-β-D-galacto-
side (IPTG) to a final concentration of 0.1 mM. For the determi-
nation of the optimal expression conditions, cultures were grown
°
after induction between 17 and 30 C, and assayed after a period of
2 days. E. coli pET28a(+) empty was cultivated and induced with
the same system as control experiment. Cells were harvested by
centrifugation (11000 g, 10 min, 4 C) and washed two times with
°
Tris-HCl buffer (pH 8.0, 50 mM, NaCl, 100 mM). Harvested cells were
°
stored at À 80 C. When needed, the wet pellets were freeze dried
overnight and collected as lyophilized cells.
General Procedure for Decarboxylation
Reactions were carried out in 5 mL screw-capped glass vials to
prevent evaporation of substrate/product. The blue light-driven
CvFAP-catalyzed decarboxylation reaction was carried out in a total
volume of 1.0 mL of Tris-HCl buffer (pH 8.5, 100 mM) containing
30% DMSO as a co-solvent at 30 C for 6 h. The system contains
200 μL 65.5 mM dicarboxylic acids (DMSO as solvent), 100 μL pure
°
during 1 h of incubation at 40 C, while lost almost all activities
°
when temperature rose above 50 C. CvFAP@E.coli cells retained
°
99% of the initial activity after storage at room temperature (~
°
25 C) for 14 days. Batch experiments showed that CvFAP@E.coli
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DMSO, 200 mg whole-cell CvFAP@E.coli mixed in 700 μL Tris-HCl
buffer (pH 8.5, 100 mM). The transparent glass bottle was sealed
and exposed to a blue LED light under slight magnetic stirring. The
final conditions of the reaction are: [dicarboxylic acids]=13.1 mM,
[CvFAP]=0.2 g/mL in Tris-HCl buffer (pH 8.5, 100 mM), 30% DMSO.
For the blank reaction the setup was the same but heat
illumination for 6 h. At the end of the reaction, cells were
°
centrifuged at 4 C, 13000 g for 10 min to be separated from the
reaction mixture, then washed twice by Tris-HCl buffer (pH 8.5,
100 mM), and resuspended in 1 mL of the same buffer containing
the same substrates. The reaction mixture (1 mL of supernatant
separated from cells) was saturated with NaCl and then extracted
with 2×0.5 mL of ethyl acetate by shaking for 5 min. The combined
organic phases were dried over Na2SO4 and crude samples were
analysed by GC.
°
denaturated cells (90 C, 30 min) were used. For work-up, the cells
were removed by centrifugation and 1 mL of the supernatant was
saturated with NaCl followed by extraction with 0.5 mL (×2) of
ethyl acetate by shaking for 5 min. The combined organic layer was
dried over Na2SO4 and measured with GC for yield.
Thermostability
CvFAP@E.coli cells were first incubated at various temperatures (20–
Evaluation of the Potential of CvFAP as a Photobiocatalyst for
Hexadecanedioic Acid (1a) Decarboxylation
°
50 C) for 1 h, prior to the addition to the reaction mixture. Then,
the cells (200 mg) were resuspended to 1 mL Tris-HCl buffer
(pH 8.5, 100 mM) containing 30% DMSO and 13 mM hexadecane-
Samples for the evaluation of the potential of CvFAP as a photo-
biocatalyst for hexadecanedioic acid decarboxylation were pre-
pared and analysed as described above in General procedure for
decarboxylation. Reactions were performed using hexadecanedioic
acid (1a) as a substrate with various concentrations ranging from 1
to 15 mM.
°
dioic acid (1a). The reaction mixture was shaken at 30 C under
blue light illumination for 6 h. Work-up and analysis were
performed as described above.
Storage stability
°
CvFAP@E.coli cells were stored at room temperature (~25 C) for
General Procedure for Substrate Screening
given periods and tested for the activity in decarboxylation of
hexadecanedioic acid (1a). 200 mg cells were withdrawn at various
time intervals (1, 2, 3, 4, 5, 6, 7, 14 days) and added to 1 mL Tris-HCl
buffer (pH 8.5, 100 mM) containing 30% DMSO and 13 mM
hexadecanedioic acid (1a). The reaction mixture was shaken at
Reactions were carried out as described in the General procedure
for decarboxylation using the same concentration (13 mM) for each
substrate. After extraction with ethyl acetate (2×0.5 mL) samples
were dried over Na2SO4 and crude samples were analysed by GC for
yield determination.
°
30 C under blue light illumination for 6 h. Work-up and analysis
were performed as described above.
Scale up Experiment
The experiment of decarboxylation of long chain dicarboxylic acids
to alkanes was scaled up from 1 mL to 100 mL. In this reaction,
20 mL of 65.5 mM DMSO containing hexadecanedioic acid (1a),
10 ml of pure DMSO, and 70 mL of Tris-HCl buffer (Ph 8.5, 100 mM)
containing 10.0 g of wet whole-cell CvFAP were added to the
transparent glass container. The container was added to the stir bar
Acknowledgements
This study was funded by Guangdong Basic and Applied Basic
Research Foundation (Grant No. 2019 A1515011905), and the
National Natural Science Foundation of China (Grant No.
41706148).
°
and sealed, stirred gently at 30 C, under blue light illumination
overnight (24 h). The final reaction conditions of this reaction were:
[hexadecanedioic acid]=13.1 mM, [wet whole-cell CvFAP]=0.1 g/
°
mL, 30 C. The workup was the same as described above for the
General procedure for decarboxylation.
Conflict of Interest
The authors declare no conflict of interest.
Time Study to Indicate the Presence of Mono-Acids
Samples for the time study were prepared and analysed as
described above in the General procedure for decarboxylation.
Sample was taken from the reaction mixture after given times and
measured by GC after proper workup.
Keywords: photobiocatalysis · decarboxylation · dicarboxylic
acids · alkanes · biodegradable plastic
Docking of Heptadecanedioic Acid (1f)
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Manuscript received: February 14, 2021
Revised manuscript received: April 6, 2021
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