Dehydrogenase-Catalyzed Oxidation of Furanics: Exploitation of Hemoglobin Catalytic Promiscuity

Article abstract of DOI:10.1002/cssc.201701288

The catalytic promiscuity of hemoglobin (Hb) was explored for regenerating oxidized nicotinamide cofactors [NAD(P)⁺]. With H₂O₂ as oxidant, Hb efficiently oxidized NAD(P)H into NAD(P)⁺ within 30 min. The new NAD(P)⁺ regeneration system was coupled with horse liver alcohol dehydrogenase (HLADH) for the oxidation of bio-based furanics such as furfural and 5-hydroxymethylfurfural (HMF). The target acids (e.g., 2,5-furandicarboxylic acid, FDCA) were prepared with moderate-to-good yields. The enzymatic regeneration method was applied in l-glutamic dehydrogenase (DH)-mediated oxidative deamination of lglutamate and for l-lactic-DH-mediated oxidation of l-lactate, which furnished α-ketoglutarate and pyruvate in yields of 97 % and 81 %, respectively. A total turnover number (TTON) of up to approximately 5000 for cofactor and an E factor of less than 110 were obtained in the bi-enzymatic cascade synthesis of α-ketoglutarate. Overall, a proof-of-concept based on catalytic promiscuity of Hb was provided for in situ regeneration of NAD(P)⁺ in DH-catalyzed oxidation reactions.

Full text of DOI:10.1002/cssc.201701288

Accepted Article
Title: Exploitation of catalytic promiscuity of hemoglobin for NAD(P)+
in situ regeneration in dehydrogenase-catalyzed oxidation of
furanics
Authors: Hao-Yu Jia, Min-Hua Zong, Hui-Lei Yu, and Ning Li
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10.1002/cssc.201701288
ChemSusChem
COMMUNICATION
Exploitation of catalytic promiscuity of hemoglobin for NAD(P)+ in
situ regeneration in dehydrogenase-catalyzed oxidation of
furanics
Hao-Yu Jia,[a] Min-Hua Zong,[a] Hui-Lei Yu,[b] and Ning Li*[a]
regeneration.[2b, 8c] A number of laccase-mediator systems (LMSs)
Abstract: In this work, catalytic promiscuity of hemoglobin (Hb) was
was reported for in situ regeneration of NAD(P)+.[9] NAD(P)H
explored for regeneration of oxidized nicotinamide cofactors
(NAD(P)+). With H2O2 as oxidant, Hb was able to efficiently oxidize
oxidases represented a promising type of biocatalysts for
10]
NAD(P)H oxidation.[8b,
Hollmann et al. reported simple and
NAD(P)H into NAD(P)+ in 30 min. The new NAD(P)+ regeneration
system was coupled with horse liver alcohol dehydrogenase (HLADH)
for the oxidation of bio-based furanics such as furfural and 5-
hydroxymethylfurfural (HMF). The target acids (e.g., 2,5-
furandicarboxylic acid, FDCA) were afforded with moderate to good
yields. In addition, the enzymatic regeneration method was applied in
L-glutamic DH-mediated oxidative deamination of L-glutamate and L-
lactic DH-mediated oxidation of L-lactate, which furnished α-
ketoglutarate and pyruvate, respectively, with the yields of 97% and
81%. The total turnover number (TTN) up to approximately 5000 for
cofactor and the E factor of less than 110 were obtained in the bi-
enzymatic cascade synthesis of α-ketoglutarate. Overall, a proof-of-
concept based on catalytic promiscuity of Hb was provided for in situ
regeneration of NAD(P)+ in DH-catalyzed oxidation reactions.
user-friendly photocatalytic NAD(P)H oxidation systems by
combining flavin photocatalysts with bright white light or light
emitting diode (LED) light.[11] Recently, Ying and co-workers have
reported a highly active bridged flavinium catalyst for in situ
recycling of NAD(P)+; interestingly, the recycling system is not
light-dependent, and is enzyme-compatible.[12] A disulfide-driven
NADP+ regeneration system with high turnover number has been
described by Allemann’s group recently.[13]
Hemoglobin (Hb) is an iron porphyrin metalloprotein, which
acts as an oxygen carrier in biological systems.[14] Hb was seldom
used as biocatalyst in organic synthesis.[15] In this work, inspired
by Kumar’s work in which oxidative aromatization of 1,4-
dihydropyridines using human Hb and H2O2 was reported,[15b] we
report Hb from human and bovine as biocatalyst for oxidizing
NAD(P)H with H2O2, and thus construct a novel and efficient
NAD(P)+ regeneration system. To our knowledge, the
promiscuous function (NAD(P)H oxidation) of this metalloprotein
was uncovered for the first time. Enzyme promiscuity is of great
interest,[16] since it enable organic chemists to rapidly develop
new approaches to valuable chemicals. Hb is not only readily
available, but also is active in the presence of H2O2 and organic
solvents[15b], which may image its promising application potential
in organic synthesis. To continue our interest in biocatalytic
upgrading of bio-based platform chemicals,[17] the oxidation of
furfural catalyzed by horse liver alcohol dehydrogenase (HLADH),
a versatile biocatalyst,[2b] was used as a model reaction for
verifying the effectiveness of this in situ regeneration method
(Scheme 1). Although upgrading bio-based furanics has attracted
considerable interest recently,[18] biocatalytic approaches remain
limited.[19] So the present work may provide a new biocatalytic
method for upgrading furanics. In addition, the enzymatic in situ
regeneration system was applied for HLADH-catalyzed oxidation
of other alcohols and aldehydes as well as other DH-mediated
oxidation reactions.
Oxidation of alcohols and aldehydes constitutes one of the
most important reactions in organic synthesis. Significant
progress in chemically catalytic oxidation was made in the last
decades.[1] Biocatalytic oxidation is an attractive and promising
candidate to supplement and even substitute chemical methods,
because of many advantages such as high efficiency, mild
reaction conditions, excellent selectivity and environmental
friendliness.[2] Alcohol dehydrogenases (ADHs, E.C. 1.1.1.x) that
are nicotinamide cofactor-dependent are versatile biocatalysts,[3]
since they can catalyze the oxidation transformations for the
synthesis of acids[4] and lactones[5] as well as the reduction of
prochiral ketones to chiral alcohols.[6] For economic reasons and
to shift the unfavorable thermodynamic equilibrium, in situ
regeneration of oxidized nicotinamide adenine dinucleotide
(phosphate) cofactors (NAD(P)+) is necessary, yet challenging in
ADH-catalyzed oxidation reactions.[7]
In situ regeneration of NAD(P)+ has been studied extensively,
and a variety of regeneration routes has been constructed.[8]
Substrate-coupled and enzyme-coupled systems were the most
commonly used enzymatic approaches for NAD(P)+
[a]
[b]
H.-Y Jia, M.-H. Zong, N. Li
State Key Laboratory of Pulp and Paper Engineering, School of
Food Science and Engineering
South China University of Technology
381 Wushan Road, Guangzhou 510640, China
E-mail: lining@scut.edu.cn
Scheme 1. Oxidation of furfural by using HLADH and Hb
H.-L. Yu
State Key Laboratory of Bioreactor Engineering, Shanghai
Collaborative Innovation Center for Biomanufacturing
East China University of Science and Technology
130 Meilong Road, Shanghai 200237, China
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/cssc.201701288
ChemSusChem
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0.21
0.18
0.15
0.12
0.09
0.06
0.03
0.00
Furfural
Furfuryl alcohol
furioc acid
100
80
60
40
20
0
NADH
NADPH
0
5
10
15
20
25
30
0
10
20
30
40
50
60
Time (min)
Time (h)
Figure 1. Enzymatic oxidation of NAD(P)H by Hb from human (full symbols)
and bovine (open symbols) with H2O2. Reaction conditions: 1 mg/mL Hb,
approximately 0.2 mM NAD(P)H, 50 mM H2O2, 0.15 mL phosphate buffer (50
mM, pH 7.0), 35 °C
Figure 2. Time courses of HLADH-catalyzed furfural oxidation with Hb from
human (full symbols) and bovine (half symbols) and LMS- (open symbols)
coupled NAD+ regeneration systems. Reaction conditions: 10 mM furfural, 1 mM
NAD+, 1.5 U/mL HLADH, 1 mL phosphate buffer (50 mM, pH 7.0), 150 rpm,
30 °C; for Hb-catalyzed regeneration systems, Hb (1 mg/mL) and H2O2 (50
mM) were supplemented; for LMS, laccase (1 mg/mL) and acetosyringone
(0.2 mM) were supplemented.
Figure 1 shows Hb-catalyzed oxidation of NADH and NADPH
with H2O2 as oxidant. It was found that both NADH and NADPH
could be transformed into the corresponding oxidized factors by
Hb, regardless of its sources. Satisfactory conversions (71-75%)
were obtained within 30 min in human Hb-catalyzed oxidation of
NADH and NADPH. In addition, bovine Hb that is more
inexpensive and readily available was found to be a good
biocatalyst for NAD(P)H oxidation. Based on changes in
NAD(P)H concentrations within initial 6 min, the specific activities
of Hb from human and bovine were calculated. The specific
activities of human and bovine Hb were 14.4 and 12.2 U/mg (1 U
corresponds to the amount of enzyme which oxidizes 1 nmol
NAD(P)H per minute at 35 °C and pH 7.0), respectively, in NADH
oxidation, while being 4.2 and 8.3 U/mg, respectively, in NADPH
oxidation. No oxidation of NAD(P)H occurred in the absence of
Hb and/or H2O2 (Figure S2). The NADH oxidation efficiency
obtained in this work was comparable to that of a flavin
photocatalyst under LED light.[11b] Recently, simple coenzyme
biomimetics have sparked renewed interest in redox
biocatalysis.[20] Unfortunately, Hb could not accept 1-benzyl-1,4-
dihydronicotinamide, a nicotinamide mimics, as substrate.
Hb-mediated NAD+ regeneration systems were combined
with HLADH for the oxidation of furfural into furoic acid (Figure 2).
The laccase-acetosyringone system was used as the control,
since this LMS was demonstrated previously to be one of the most
efficient LMSs for in situ regeneration of NAD(P)+.[9b] As shown in
Figure 2, furfural was transformed into furoic acid (as the major
product) and furfuryl alcohol, regardless of the NAD+ regeneration
systems. It is not surprising, since HLADH is capable of catalyzing
the oxidation of aldehydes as well as their reduction.[4a] Previously,
Hollmann and co-workers reported enantioselective oxidation of
aldehydes by using ADHs.[4b] Then, furfuryl alcohol was re-
oxidized into the desired product furoic acid. When LMS was used,
the substrate conversion reached 100% within 48 h. And furoic
acid was afforded in the yield of approximately 83% after 60 h,
along with the formation of around 15% furfuryl alcohol.
Interestingly, the better results were observed in the cases of Hb,
compared to the control. For example, furfural was used up within
36 h when human Hb-catalyzed NAD+ recycling system was
applied, and a much higher yield of furoic acid (92%) was
obtained after 48 h. Although the transformation rate of furfural by
HLADH/bovine Hb appeared to be slightly lower, the yield of furoic
acid (94%) remained good. It suggests that, like human Hb, Hb
from bovine is an efficient biocatalyst for in situ NAD+ recycling in
HLADH-catalyzed oxidation. Poor results were obtained in the
absence of HLADH, Hb or H2O2 (Table S1). For example, furoic
acid was furnished with low yields (30-35%), in spite of moderate
substrate conversions (58-60%), when Hb or H2O2 was absent.
The reason was that, in addition to oxidation, furfural reduction
occurred simultaneously in the absence of NAD+ in situ recycling
system, thus resulting in the formation of furfuryl alcohol
(approximately 23%) as the by-product. Also, it indicates that
NAD+ in situ regeneration is indeed due to the catalytic behavior
of Hb in the presence of H2O2. Almost no biotransformation of
furfural happened in the absence of HLADH. In addition, HLADH-
catalyzed furfural oxidation with stoichiometric NAD+ was
conducted as well (Figure S5). It was found that the results
including the reaction rates and the product yields were
comparable in HLADH-catalyzed furfural oxidation with
stoichiometric NAD+ and that coupled with the enzymatic NAD+ in
situ regeneration system. It indicates that H2O2 doesn’t exert a
significantly detrimental effect on the catalytic activity of HLADH.
In addition, the effects of pH and temperature on the the
activity of Hb (Figures S3 and S4) were studied when NADH and
NADPH were used as substrates, repectively. It was found that its
opitmal pH and temperature were 8.0 and 30 °C, respectively,
regardless of sources and substrates. It suggests that Hb-
mediated NAD(P)+ regeneration system may be well compatible
with HLADH-catalyzed oxidation, because HLADH showed a
good activity under the above conditions.[21]
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10.1002/cssc.201701288
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chemicals and fuels has attracted great interest.[18b, 18c, 24] Encouraged
by the above results, direct oxidation of HMF into 2,5-
Optimization of enzymatic furfural oxidation was conducted to
improve the synthesis of furoic acid (Table S2). The synthesis of
furoic acid was improved slightly when fed-batch addition of H2O2
or supplementing H2O2 after 24 h was conducted (Table S2,
entries 1 and 2). It is well known that the ‘total turnover number’
(TTN, defined as the total number of moles of product formed per
mole of cofactor during the course of a complete reaction)[8d] for
cofactor is one of the key factors affecting the practicability of a
cofactor regeneration method. Generally, TTNs should be up to
103-105, which makes a process economically viable depending
on the value of the product.[22] To obtain a satisfactory TTN, the
NAD+ concentration was reduced in HLADH-catalyzed oxidation
of furfural (Table S2, entries 4-6). A good yield (91%) was retained
when the NAD+ concentration decreased to 0.01 mM, and the
TTN for cofactor was significantly improved to approximately 910.
Nonetheless, the TTN for Hb (approximately 140) remained low.
The yields (93-96%) could be improved slightly when higher
enzyme dosages were used (Table S2, entries 7 and 8).
furandicarboxylic acid (FDCA),
a versatile bio-based polymer
precursor,[25] by using HLADH (Table 1, entry 2 and Figure S3b) was
attempted, since a primary hydroxyl as well as a formyl group is
present in this platform chemical. Unfortunately, HLADH preferred
oxidizing formyl to primary hydroxyl of HMF, thus affording 5-
hydroxymethylfuran-2-carboxylic acid (HMFCA) as the major product
(Table 1, entry 2). Likewise, a small amount of HMF was reduced
simultaneously into 2,5-bis(hydroxymethyl)furan (BHMF). Then,
BHMF was re-oxidized into HMFCA (Figure S3b). Prolongation of
reaction period only resulted in the production of a small amount of
FDCA (7% at 96 h). HMFCA was a poor substrate of HLADH, since
HMFCA of approximately 24% was consumed after 72 h. However,
interestingly, FDCA was obtained as the major product, despite a low
yield (20%, Table 1, entry 3). With 2,5-diformylfuran (DFF) as
substrate, a moderate conversion (52%) was observed after 72 h
(Figure S3d), likely due to its significant inhibitory effect on ADH.[6, 26]
FFCA was furnished as the major product (38% yield) in the
enzymatic oxidation of DFF; Supplementation of fresh enzymes and
H2O2 into the reaction mixture led to continuous oxidation of substrate
DFF as well as the intermediate FFCA, which afforded FDCA with a
moderate yield of 54% (Table 1, entry 4). Besides, FFCA could be
transformed smoothly into FDCA, with the yield of 96% (Table 1, entry
5 and Figure S3e). In addition to furanics, both benzyl alcohol and
benzaldehyde were good substrates of HLADH as well; benzoic acid
was obtained with the yields of more than 95% in the two cases (Table
1, entries 6 and 7). Although good yields were obtained in most cases,
its catalytic efficiencies remained unsatisfactory. One of the reasons
for low catalytic efficiencies may be the negative effect of H2O2 on
biocatalysts. Replacement of H2O2 with urea-hydrogen peroxide
(UHP) adduct[27] may be a promising strategy for facilitating HLADH-
catalyzed oxidation of furanics, since UHP has the potential of
releasing H2O2 in a controlled manner, thus avoiding enzyme
deactivation caused by H2O2 as well as the need to add H2O2 slowly
to the reaction mixtures.[28]
Figure 3. Substrates tested in HLADH-catalyzed oxidation and target products
Table 1. HLADH-catalyzed oxidation of various alcohols and
aldehydes
Conv.
(%)
Yield
(%)
Entry
1
Substrate
Product
Time (h)
48
Furfuryl
alcohol
HMF
HMFCA
DFF
Furoic acid
100
98
Scheme 2. Enzymatic oxidation of L-glutamate and L-lactate using DH and Hb
2
HMFCA
FDCA
FDCA
FDCA
Benzoic
acid
60
72
108
60
100
24
79
81
20
54
96
3
4[a]
5
FFCA
100
Benzyl
alcohol
Benzalde
hyde
100
6
60
100
96
Pyruvate
Benzoic
acid
-Ketoglutarate
7
60
100
95
80
Reaction conditions: 10 mM substrate, 0.1 mM NAD+, 1 mg/mL
human Hb, 50 mM H2O2, 1.5 U/mL HLADH, 1 mL phosphate buffer
(50 mM, pH 7.0), 150 rpm, 30 °C; H2O2 (50 mM) was supplemented
after 24 h. [a]: 1 mg/mL Hb, 1.5 U/mL HLADH and 50 mM H2O2 were
supplemented after 72 h.
60
40
20
0
This bi-enzymatic parallel cascade system was applied for the
oxidation of other alcohols and aldehydes (Figure 3 and Table 1).
Time courses of enzymatic oxidation of these chemicals were
available as Figure S6. As shown in Table 1 (Entry 1), furfuryl alcohol
could be directly transformed into furoic acid; a good yield (98%) was
obtained after 48 h. The above results indicate that HLADH is able to
catalyze the oxidation of both primary hydroxyl and formyl on the furan
ring into carboxylic acid group. 5-Hydroxymethylfurfural (HMF), an
important bio-based platform chemical, can be obtained via
carbohydrate dehydration.[23] Recently, upgrading of HMF into useful
0
10
20
30
40
50
60
Time (h)
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Figure 4. DH-catalyzed synthesis of α-ketoglutarate and pyruvate. Reaction
conditions: 50 mM L-glutamate/L-lactate, 0.01 mM NAD+, 1 mg/mL human Hb,
50 mM H2O2, 40 U/mL L-gluDH/L-lacDH, 1 mL phosphate buffer (50 mM, pH
7.0), 150 rpm, 30 °C; H2O2 (50 mM) was supplemented after 24 h.
Experimental Section
General procedure for enzymatic oxidation of furanics
Typically, phosphate buffer (50 mM, pH 7.0; 1 mL) containing
furanics (10 mM), NAD+ (0.1 mM), Hb (1 mg/mL), H2O2 (50 mM), and
HLADH (1.5 U/mL) was incubated at 30 °C and 150 rpm; H2O2 (50
mM, 0.05 mmol) was supplemented after 24 h. Aliquots were
withdrawn from the reaction mixtures at specified time intervals and
diluted with the corresponding mobile phase prior to HPLC analysis.
The conversion was defined as the percentage of the consumed
substrate amount in the initial substrate amount. The yield was
defined as the percentage of the measured product amount in the
theoretical product amount based on the initial amount of substrate.
All the experiments were conducted at least in duplicate, and the
values were expressed as the means.
We sought to explore the applicability of this NAD+ in situ
regeneration method in other DH-catalyzed oxidation reactions
(Scheme 2). Figure 4 shows the time courses of enzymatic oxidation
reactions. α-Ketoglutarate that can be synthesized from L-glutamate
by using L-glutamic DH (L-gluDH)[29] has broad applications in food,
medicine and polymer industries.[30] Therefore, the coupled system of
L-gluDH and Hb was used for the synthesis of α-ketoglutarate (Figure
4). As shown in Figure 4, α-ketoglutarate was obtained with a good
yield (97%), under non-optimized conditions. It was worth noting that
the cofactor TTN was up to approximately 5000, which is much higher
than that (100) in a previous report.[29] In addition, the TTN for Hb was
around 770. Also, Hb-H2O2 was applied for NAD+ in situ regeneration
in L-lactic DH (L-lacDH)-mediated production of pyruvate from L-
lactate. Pyruvate is a useful building block for the production of L-
amino acids, cyanoacrylate adhesives, perfumes, food additives,
Acknowledgements
This research was financially supported by the State Key
Laboratory of Pulp and Paper Engineering (2017C01), and the
National Natural Science Foundation of China (21676103)..
dietary and weight control supplements, antioxidants, etc.[31]
A
satisfactory yield (81%) and an excellent selectivity (94%) were
achieved as well. As compared to MoO3/TiO2-based synthetic route
to pyruvate,[31] this biocatalytic approach appeared to be
advantageous in terms of mildness of reaction conditions (200 °C vs
30 °C) and selectivity (80% vs 94%).
Keywords: cofactor regeneration • enzyme catalysis • enzyme
promiscuity • oxidation • platform chemicals
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10.1002/cssc.201701288
ChemSusChem
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Entry for the Table of Contents
COMMUNICATION
Catalytic promiscuity. Catalytic
promiscuity of hemoglobin was
exploited for NAD(P)+ in situ
Hao-Yu Jia,[a] Min-Hua Zong,[a] Hui-Lei
Yu,[b] and Ning Li*[a]
regeneration. The new NAD(P)+
regeneration system was
Page No. – Page No.
Exploitation of catalytic promiscuity
of hemoglobin for NAD(P)+ in situ
regeneration in dehydrogenase-
catalyzed oxidation of furanics
successfully coupled with horse liver
alcohol dehydrogenase for
upgrading of bio-based furanics into
target acids, with good yields.
This article is protected by copyright. All rights reserved.