Co-immobilized Phosphorylated Cofactors and Enzymes as Self-Sufficient Heterogeneous Biocatalysts for Chemical Processes

Article abstract of DOI:10.1002/anie.201609758

Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co-immobilizing phosphorylated cofactors (PLP, FAD⁺, and NAD⁺) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self-sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD⁺-dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.

Full text of DOI:10.1002/anie.201609758

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Communications
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International Edition: DOI: 10.1002/anie.201609758
German Edition: DOI: 10.1002/ange.201609758
Biocatalysis
Co-immobilized Phosphorylated Cofactors and Enzymes as Self-
Sufficient Heterogeneous Biocatalysts for Chemical Processes
Susana Velasco-Lozano, Ana I. Benꢀtez-Mateos, and Fernando Lꢁpez-Gallego*
Abstract: Enzyme cofactors play a major role in biocatalysis,
as many enzymes require them to catalyze highly valuable
reactions in organic synthesis. However, the cofactor recycling
is often a hurdle to implement enzymes at the industrial level.
The fabrication of heterogeneous biocatalysts co-immobilizing
phosphorylated cofactors (PLP, FAD+, and NAD+) and
enzymes onto the same solid material is reported to perform
chemical reactions without exogeneous addition of cofactors in
aqueous media. In these self-sufficient heterogeneous biocat-
alysts, the immobilized enzymes are catalytically active and the
immobilized cofactors catalytically available and retained into
the solid phase for several reaction cycles. Finally, we have
applied a NAD+-dependent heterogeneous biocatalyst to
continuous flow asymmetric reduction of prochiral ketones,
thus demonstrating the robustness of this approach for large
scale biotransformations.
non-porous anionic exchangers has shown that the cofactor
re-utilization works in organic media[9] but fails in aqueous
media owing to the lixiviation of both NAD(P)H and
enzymes from the matrix.[10] The reason behind such lixivia-
tion is the formation of reversible ionic interactions between
the negatively charged phosphorylated cofactors and the
positive charges of the solid material, establishing an associ-
ation/dissociation equilibrium which permits the release of
cofactor molecules from the solid surface to the bulk.[11] In
contrast, if we ionically adsorb the cofactors on porous
materials, such adsorption is dynamic and allows that some
cofactors are associated to the solid surface enabling their
reutilization in aqueous media, while others are dissociated
(free) but confined into the porous space becoming available
for the enzymes. Therefore, the cofactors are continuously
shifting from the associated to the dissociated states generat-
ing an exchange between the enzyme active sites and the
carrier surface without being released to the bulk. Herein, we
harness such association/dissociation equilibrium within
a porous environment to fabricate self-sufficient heteroge-
neous biocatalysts capable of regenerating and retaining the
phosphorylated cofactors in the solid-phase for several
operational batch-cycles and in continuous processes in
aqueous media.
I
ndustrial biocatalysis is transforming chemical manufactur-
ing towards more sustainable and environmentally friendly
processes.[1] Some of the most interesting reactions in
industrial biocatalysis are catalyzed by cofactor-dependent
enzymes such as NADH-dependent reductases and oxidases,
PLP-dependent transaminases, and FAD+-dependent oxy-
genases.[2] Consequently, the regeneration and reutilization of
these expensive cofactors is a major requirement for the
implementation of enzymatic processes at the industrial
scale.[3] Hitherto, many systems based on enzymatic and
chemical reactions regenerate the cofactors allowing their use
in catalytic amounts, but still they must be exogenously
added. Inspired by nature, NAD(P)H and enzymes have been
co-immobilized on solid materials allowing their solid-phase
recycling and reusability for several operational cycles.[3a,4]
These heterogeneous systems, however, have shown several
drawbacks; the enzymatic activity towards the immobilized
cofactors is low,[5] the turnover numbers of the immobilized
cofactors are poor (ꢀ 1),[6] the re-usability of both enzymes
and cofactors is limited[5,7] and the fabrication of these
systems is hardly scalable.[8] However, the use of strong and
We first optimized the ionic adsorption of NAD+ on two
different anionic exchangers under different conditions[12]
(Supporting Information, Table S1). Under a low buffer
concentration (10 mm) and offering 100 mmolNADþ gꢁ1 at
pH 7.0, agarose microbeads activated with polyethyleneimine
25 kDa (Ag-GPEI) loaded 18 mmolNADþ gꢁ1 and retained 22%
of the immobilized cofactor after 5 wash cycles, whereas
agarose microbeads activated with triethyl amine (Ag-TEA)
loaded 11 mmolNADþ gꢁ1 and 100% of the cofactor was
lixiviated after the first wash (Supporting Information,
Table S1, Figure S1). Similar results were found when PEI
was irreversibly attached to other porous commercial carriers
(Purolite) (Purolite-GPEI) and agarose microbeads activated
with divinyl-sulfone (Ag-DVSPEI)[13] (Supporting Informa-
tion, Figure S2). Encouraged by these results, we expanded
this strategy to other phosphorylated cofactors, such as flavin
adenine dinucleotide (FAD+) and pyridoxal phosphate (PLP)
used by other industrially relevant enzymes.[2a,c,d,14] Figure 1A
shows that the absorption yield of PLP on Ag-GPEI was 1.8
and 4.8 times higher than FAD+ and NAD+, respectively, and
99% of immobilized PLP remained in the microbeads after 8
washes with low ionic strength buffer at pH 7, while 85% and
80% of the adsorbed FAD+ and NAD+ were lixiviated under
the same conditions, respectively (Figure 1B). Both cofactor
adsorption and lixiviation rely on the association/dissociation
equilibrium that governs the interactions between Ag-GPEI
[*] Dr. S. Velasco-Lozano, A. I. Benꢀtez-Mateos, Dr. F. Lꢁpez-Gallego
Heterogeneous biocatalysis group, CIC biomaGUNE, Edificio
Empresarial “C”
Paseo de Miramꢁn 182, 20009 Donostia (Spain)
E-mail: flopez.ikerbaske@cicbiomagune.es
Dr. F. Lꢁpez-Gallego
IKERBASQUE, Basque Foundation for Science
Bilbao (Spain)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201609758.
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towards both soluble and immobilized cofactors (Supporting
Information, Figure S3A). Using soluble NAD+, the KM of
soluble Cb-FDH was 12 times lower than its immobilized
form (Supporting Information, Figure S4). Unfortunately, the
apparent Michaelis–Menten constant could not be calculated
using the immobilized NAD+ because neither the soluble nor
the immobilized enzymes were saturated at any concentration
of immobilized cofactor. Under limiting NAD+ conditions
(166 mm in the bulk = 6.6 mmolcofactor gsupportꢁ1) where enzyme
activity linearly increases with the cofactor concentration, the
soluble Cb-FDH was 10 times less active towards the
immobilized cofactor than towards the soluble one. In
contrast, the immobilized enzyme was similarly active
towards both soluble and co-immobilized NAD+ under the
same conditions (Supporting Information, Table S3). Intro-
ducing those activity values in the corresponding Michaelis–
Menten equations, we estimated that apparent concentration
of immobilized NAD+ that was catalytically available for Cb-
FDH was 30 times higher when the enzyme was co-
immobilized with the cofactor than when the enzyme was
soluble (Supporting Information, Table S3). These results
suggest that both the co-immobilization of enzymes and
cofactors and the association/dissociation equilibrium of
NAD+ inside the porous microenvironment facilitate the
cofactor diffusion from the polymeric layer to the Cb-FDH
active sites.
Having now in hand a versatile and simple strategy to
incorporate phosphorylated cofactors within porous carriers,
we co-immobilized two different enzyme systems with their
corresponding cofactors (Table 1); system HB1 formed by
NAD+, alcohol dehydrogenase 2 from Thermus thermophi-
lus[15] (Tt-ADH2; Supporting Information, Figure S3B) and
Cb-FDH to carry out the asymmetric reduction of 1 to yield
(S)-2 and system HB2 formed by w-transaminase (commer-
cial source; w-TA) and PLP to catalyze the kinetic resolution
of rac-3 through S-selective deamination. We immobilized the
main enzymes (w-TA or Tt-ADH2) on agarose microbeads
activated with aldehydes (Ag-G),[16] followed by a polymeric
coating with PEI through the same amine–aldehyde chemis-
try,[17] and a final reduction step to turn the reversible PEI/
protein-agarose imines into irreversible secondary amines
(Figure 2A, step 1). Unfortunately, aldehyde immobilization
chemistry inactivated Cb-FDH (recycling enzyme) at the
Figure 1. A) Immobilization of phosphorylated cofactors on Ag-GPEI
(offered 100 mmolcofactor gsupportꢁ1). B) Residual NAD+ (dark gray bars),
FAD+ (light gray bars), and PLP (white bars) bound to Ag-GPEI after
washing treatments with 10 mm sodium phosphate at pH 7.
and each cofactor. The apparent dissociation constant (Kdapp
)
for PLP equilibrium is 26 and 6 times lower than for NAD+
and FAD+ equilibrium, respectively (Supporting Information,
Table S2), which supports the notion that the equilibrium of
the cofactor–PEI interactions favors 99% of PLP remaining
in the solid phase while FAD+ and NAD+ are significantly
lixiviated after 8 washes (Figure 1B).
Usually, enzymes display low activities towards reversible
or irreversible tethered cofactors on solid materials.[5,6,7b] To
test the performance of these reversibly immobilized cofac-
tors, we studied the steady-state kinetics of NAD+-dependent
formate dehydrogenase from Candida boidinii (Cb-FDH)
Table 1: Catalytic efficiency and reuse of different heterogeneous biocatalysts with soluble and immobilized cofactors.
Enzymes and reactions
Specific activity [mmolmgꢁ1 minꢁ1 [d]
]
Yield [%][d]
TOF [minꢁ1
]
TTN [mol productmol NAD+ꢁ1 [e]
]
1.76ꢂ0.36[a]
1.19ꢂ0.25[b]
0.94ꢂ0.17[c]
100[a]
100[b]
100[c]
0.11[a]
10[a]
10[b]
40[c]
0.079[b]
0.064[c]
0.046ꢂ0.018[a]
0.005ꢂ0.002[b]
0.030ꢂ0.014[c]
50[a]
41[b]
46[c]
0.039[a]
0.012[b]
0.032[c]
5.0[a]
3.9[b]
16.8[c]
[a] Soluble enzymes and cofactor. [b] Soluble cofactor and immobilized enzymes. [c] Co-immobilized enzymes and cofactor. [d] Specific activity
(TtADH2 or w-TA) and yield in the first cycle. [e] TTN after 1 cycle for [a,b] and 4 batch cycles for [c]. TOF was calculated as mmol of product per mmol of
cofactor in one hour. For reaction conditions, see the Supporting Information, Figures S7–S9 and S11).
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Figure 2. A) Architecture of the self-sufficient heterogeneous biocatalyst. Cofactor association/dissociation equilibrium is depicted in the inset.
B) Spatial distribution of the self-sufficient heterogeneous biocatalysts (HB1 and HB2) by using fluorescence microscopy. Tt-ADH2 is labeled with
fluoresceine, Cb-FDH and w-TA are labeled with rhodamine, and NAD+ and PLP present autofluorescence. C) In operando analysis of the NADH
+
~
*
production within a single microbead of HB1 during the redox biotransformation with (green ) and without NAD recycling (red ). The
reaction was monitored and the average fluorescence was quantified by measuring the autofluorescence of NADH at 460 nm in 10 microbeads.
assayed conditions;[18] thereby this enzyme was co-immobi-
lized by ionic adsorption on the PEI-bed and subsequently
cross-linked with 1,4-butanediol diglycidyl ether to assure its
irreversible attachment (Supporting Information, Table S4;
Figure 2A, step 2).
NADH was not accumulated inside the particle as occurred in
the reaction only using formic acid. Then, we evaluated HB1
and HB2 as self-sufficient heterogeneous biocatalysts able to
catalyze their corresponding reactions without addition of
exogenous cofactors (Table 1). In batch reactions, the immo-
bilized Tt-ADH2 and w-TA presented lower specific activ-
ities than their soluble counterparts.
Finally, the corresponding cofactor is adsorbed to the
cationic bed upon the enzyme immobilization and cross-
linking (Figure 2A, step 3). In this architecture, both enzymes
and PEI are irreversibly bound to the agarose microbeads
leaving the enzymes surrounded by the cationic polymer with
the ionically adsorbed cofactor (Figure 2A). The different
enzymes were immobilized with yields ranging 79 to 100%
and recovering 14–100% of their specific activities upon the
immobilization process (Supporting Information, Table S4).
The co-immobilization of the cofactors (PLP and NAD+) with
the enzymes negligible affected their lixiviation pattern
(Supporting Information, Figure S5). We then studied the
spatial distribution of both autofluorescent cofactors and
fluorophore-labelled enzymes for HB1 and HB2 (Figure 2B).
In all cases, both enzymes and cofactors were homogeneously
co-localized across the porous surface of the agarose microbe-
ads which favors the cofactor shuttling between the enzymes
(Figure 2B; Supporting Information, Figure S6). To demon-
strate that the ionically adsorbed cofactors are recycled within
the solid phase and remain inside the porous structure, we run
the asymmetric reduction of 1 catalyzed by HB1 and
monitoring in operando the autofluorescence of NADH
within single-agarose microbeads under the microscope (Fig-
ure 2C). The in operando experiments demonstrate that the
immobilized redox cofactors are available for the immobi-
lized enzymes and remain inside the porous microbeads
during the reaction. When 1 and formic acid were present, the
In the asymmetric reduction of 1, HB1 exhibited a similar
conversion and reaction rate to the co-immobilized Tt-ADH2
and Cb-FDH with exogenous NAD+ (Supporting Informa-
tion, Figure S7). These reactions were run with 2 equivalents
of formic acid and 0.1 equivalents of either immobilized or
soluble NAD+ to enable the cofactor regeneration. A larger
excess of sodium formate (100 mm, 10 equivalents) reduced
the conversion (< 10%) because of the cofactor lixiviation
owing to the weakening of the ionic interactions (Supporting
Information, Figure S2B). The next step was testing the
reusability of this self-sufficient heterogeneous biocatalyst
without adding exogenous NAD+. HB1 was re-used for 4
batch cycles, achieving the maximum conversion in each cycle
and accumulating a total turnover number (TTN) of 40 (the
theoretical maximum; Supporting Information, Figure S8).
Likewise, HB2 catalyzed asymmetric deamination of rac-3
without exogenous addition of PLP and surprisingly the
reaction was 8-fold faster with a conversion 1.1-fold higher
than when exogenous PLP was added (Table 1; Supporting
Information, Figure S9). When HB2 was loaded with
> 20 mmolPLP gꢁ1, the cofactor seems to be more catalytically
available for the immobilized w-TA than the soluble PLP
whose diffusion across the cationic bed might be hindered
owing to the strong PLP-PEI interactions (Supporting
Information, Figure S10). Finally, HB2 was re-used for up to
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4 cycles accumulating a TTN of 16.8 when the theoretical
maximum accumulated TTN should be 40. This lower
accumulated TTN value was due to the operational inactiva-
tion of immobilized w-TA; similar results were obtained when
reusing the same immobilized enzyme using exogenous PLP
(Supporting Information, Figure S11), indicating that the
immobilization of the w-transaminase must be optimized.
Data from the two systems support that the cofactor
immobilization is a dynamic process where cofactor mole-
cules shift between the associated and dissociate state inside
the porous surface enabling both the regeneration and the
reutilization of the cofactors.
enzymes and phosphorylated cofactors within the same
porous microbead through an innovative architecture where
the enzymes and PEI are irreversibly bound to the solid
surface, and the negative charged cofactors are reversibly
adsorbed to the PEI through ion-exchange interactions. To
the best of our knowledge, we have successfully applied
a heterogeneous biocatalyst integrating NAD+ to asymmetric
reduction of ketones in continuous for first time. This pioneer
work contributes to one of the challenges of the modern
biocatalysis; the cofactor-free biotransformations mediated
by self-sufficient artificial metabolic cells without genomic
regulations.
The excellent batch performance of immobilized NAD+
motivated us to convert 1 into S-2 in a flow reactor without
addition of exogenous cofactor. We packed HB1 into
a column and fed the reactor with the reaction mixture at
different flow rates (10–200) mLminꢁ1 (Supporting Informa-
tion, Table S5). 50 mLminꢁ1 was the optimal flow to achieve
100% conversion with a productivity of 250 mm minꢁ1. Under
such conditions, the reactor was running for 92 hours achiev-
ing a conversion of over 90% that decayed to 79% after
107 hours of continuous operation. According to these data,
the immobilized NAD+ was efficiently re-cycled, accumulat-
ing a TTN of 85 after 107 hours (356 operational volumes)
without significant NAD+ lixiviation (Figure 3). Additionally,
the Tt-ADH2 in HB1 maintained its high enantioselectivity
during the entire biotransformation (ee > 99% of S-2), thus
facilitating the work-up for the isolation of the enantiopure
product. We isolated 31.4 mg of S-2 and confirmed its
enantiopurity by chiral-phase HPLC (Supporting Informa-
tion, Figure S13) and its purity and chemical structure by
NMR (Supporting Information, Figure S14). Finally, we
successfully reloaded the heterogeneous biocatalyst with
fresh cofactor recovering the maximum enzyme performance
for three reloading cycles (Supporting Information, Table S6).
In summary, we have introduced the successful fabrication
of self-sufficient heterogeneous biocatalysts integrating
Acknowledgements
We all wish to acknowledge the funds from COST action
CM103-System biocatalysis, IKERBASQUE, Marie-Curie
Actions (NANOBIENER project), Spanish Minister
MINECO (BIO2014-61838-EXP and BIO2015-69887-R).
We wish to thank Dr. Iranztu Llarena and Dr. Monica
Carril for their excellent technical support in the microscopy
determinations and in the purification and characterization of
the produced S-2, respectively.
Conflict of interest
The authors declare no conflict of interest.
Keywords: biocatalysis · cofactor regeneration ·
heterogenous catalysis · porous materials ·
protein immobilization
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Manuscript received: October 5, 2016
Final Article published: && &&, &&&&
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Communications
Biocatalysis
Flatmates: The co-immobilization of
enzymes and cofactors on the same
porous particles results in a self-sufficient
heterogeneous biocatalyst that performs
a chemical reaction without addition of
exogenous cofactors. These systems are
easily integrated into flow reactors as
packed columns for continuous bio-
transformations.
S. Velasco-Lozano, A. I. Benꢁtez-Mateos,
F. Lꢂpez-Gallego*
&&&& — &&&&
Co-immobilized Phosphorylated
Cofactors and Enzymes as Self-Sufficient
Heterogeneous Biocatalysts for Chemical
Processes
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