Myoglobin-Catalyzed Efficient In Situ Regeneration of NAD(P)+ and

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Myoglobin-Catalyzed Efficient In Situ Regeneration of NAD(P)+ and Their Synthetic Biomimetic for Dehydrogenase-Mediated Oxidations Hao-Yu Jia, Minhua Zong, Gao-Wei Zheng, and Ning Li ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b04890 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 4, 2019

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ACS Catalysis

Myoglobin-Catalyzed Efficient In Situ Regeneration of NAD(P)+ and Their Synthetic Biomimetic for Dehydrogenase-Mediated Oxidations

Hao-Yu Jia†, Min-Hua Zong†, Gao-Wei Zheng‡, and Ning Li†*

†School of Food Science and Engineering, South China University of Technology, 381 Wushan Road, Guangzhou 510640, China ‡ 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 * Corresponding author. Dr. N. Li, Tel/Fax: +86 20 2223 6669; Email: [email protected]

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Abstract: Based on its catalytic promiscuity, wild-type myoglobin (Mb) from horse heart was used for in situ regeneration of oxidized nicotinamide cofactors. Mb proved to be a versatile catalyst, since it was capable of efficient oxidation of both natural cofactors NAD(P)H and their synthetic biomimetic BNAH with H2O2. Mb-catalyzed oxidation of reduced nicotinamide cofactors was promoted significantly in the presence of mediators such as scopoletin and acetaminophen. A cofactor total turnover number (TTN) up to 50 000 was obtained in glucose dehydrogenase-catalyzed oxidation of glucose coupled with Mb/scopoletin regeneration system. And approximately 4 moles of glucose were enzymatically oxidized with one mole of H2O2. Besides, this system is compatible well with various dehydrogenases. The desired products were afforded with the yields of 93-97% in dehydrogenase-catalyzed oxidations. A radical mechanism was proposed for Mb/scopoletin-catalyzed recycling of NAD(P)+. The Mb-based recycling system may create opportunities for academic and industrial biocatalytic applications. Keywords: cofactor regeneration, dehydrogenases, enzyme catalysis, enzyme catalytic promiscuity, oxidation


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Introduction Oxidation is an important reaction in synthetic chemistry. Biocatalytic oxidation has received growing interest as a green and clean alternative to chemical processes, because of many advantages such as mild reaction conditions, exquisite selectivity, high efficiency and environmental friendliness.1-3 Nicotinamide cofactor-dependent dehydrogenases (DHs) represent versatile catalysts for organic transformations, since they mediated the oxidation reactions as well as reductions. To make DH-catalyzed processes acceptable from an economical point of view, in situ regeneration of costly nicotinamide cofactors is necessary. In the last decades, in situ recycling of reduced nicotinamide cofactors (NAD(P)H) has been studied extensively, and a variety of methods have been established.4 On the contrary, efficient in situ regeneration systems for their oxidized forms (NAD(P)+) are so far less developed.5 Enzymatic approaches based on substrate- and enzyme-coupled systems are usually used for regenerating NAD(P)+, at the expense of a second substrate.4, 6 Recently, Angelastro et al. reported a glutathione-based recycling system in which glutaredoxin was combined with glutathione reductase for NADP+ regeneration, with a high total turnover number (TTN) for cofactor.7 Laccase/mediator systems were capable of transforming NAD(P)H into their oxidized forms with O2.8-11 Haas et al described a NAD(P)+ regeneration method by using of a quinone reductase and a quinone mediator.12 NAD(P)H oxidases (NOX, EC 1.6.3.X) are promising catalysts for NAD(P)+ regeneration,13 because of use of clean oxygen as the oxidant, generation of less organic wastes, and simple reaction setups. However, NOX are accompanied with some drawbacks such as high substrate


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specificity toward NADH or NADPH, significantly decreased activities under slightly alkaline conditions and limited stability (due to interfacial inactivation resulting from bubbling aeration), which may be the major barriers for their application in organic synthesis. P450 BM3 monooxygenase was presented as a NAD(P)H oxidase for the regeneration of NAD(P)+.14 In addition to enzyme catalysis, chemical,15, electrochemical17 and photochemical methods18,

19

16

were developed for NAD(P)+

recycling. Generally, only proof-of-concept use of these NAD(P)+ regeneration systems was demonstrated in dehydrogenase-catalyzed oxidations. And examples of their applications under industrially sound conditions remain scarce.14

Figure 1. Oxidized nicotinamide cofactors and their synthetic biomimetic Myoglobin (Mb), an oxygen storage protein, has an iron porphyrin IX (heme) cofactor which is shared by hemoproteins such as hemoglobin (Hb), horseradish peroxidase (HRP), cytochrome C (Cyt C), and P450s. Previously, it was found that wild-type Mb showed promiscuous, albeit low, peroxidase20-22 and nitrite reductase activities.23, 24 In addition, rational redesign of Mb received considerable attention by protein engineering and replacement of the native heme with functionalized metalloporphyrins.25-28 It not only led to significantly improved activities, but also created new enzymes with


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interesting catalytic activities for synthetic applications.26 For example, peroxidase activities of Mb were enhanced significantly by site-directed mutation,29-31 and using artificial cofactors.32,

33

A variety of engineered Mb variants were exploited for

abiological carbene and nitrene transfer reactions34-39 as well as C-H hydroxylation.40 Recently, we found that Hb containing a heme cofactor showed a promiscuous activity capable of oxidizing NAD(P)H with H2O2.41 Based on this finding and inspired by the study conducted by Gröger and co-workers with a synthetic FeIII porphyrin,15 we reasoned that Mb was able to regenerate NAD(P)+ with H2O2. Indeed, hemoproteins such as Mb and Cyt C could catalyze the oxidation of NAD(P)H in the presence of H2O2. More interestingly, 1-benzyl-1,4-dihydropyridine-3-carboxamide (BNAH), a synthetic nicotinamide biomimetic, could be efficiently oxidized into enzymatically active BNA+ (Figure 1) by Mb. Enzymatic regeneration of oxidized cofactors was enhanced markedly in the presence of external mediators. Besides, this enzymatic regeneration system combined with glucose dehydrogenase (GDH) exhibited potent application potential in large-scale synthesis, evidenced by a cofactor TTN as high as 5 × 104. This TTN value is higher than those of most the existing NAD(P)+ regeneration systems.42-44 More importantly, this cofactor regeneration system is highly atomeconomical, since approximately 5 moles of NAD(P)H are oxidized per mole of H2O2. Results and Discussion Hemoprotein-catalyzed oxidation of reduced cofactors


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100

Conversion (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 60 40 20

BNAH NADPH NADH

0 Mb

HRP Hb Hemoproteins

Cyt C

Figure 2. Hemoprotein-catalyzed oxidation of NAD(P)H and BNAH. Reaction conditions for NAD(P)H oxidation: 1 mM NAD(P)H, 10 mM H2O2, 1 mg/mL hemoproteins (59 μM Mb; 16 μM Hb; 23 μM HRP; 83 μM Cyt C), 150 μL phosphate buffer (50 mM, pH 7 for Cyt C and HRP; pH 8 for Mb and Hb), 30°C, 12 min; reaction conditions for BNAH oxidation: 0.5 mM BNAH, 10 mM H2O2, 0.5 mg/mL hemoproteins (30 μM Mb; 8 μM Hb; 12 μM HRP; 42 μM Cyt C), 3 mL phosphate buffer (50 mM, pH 7 for Cyt C and HRP; pH 8 for Mb and Hb) containing 2% DMSO, 30°C, 12 min.

Four hemoproteins including Mb, HRP, Hb and Cyt C were examined for the oxidation of reduced nicotinamide cofactors (Figure 2). To improve the oxidation efficiency, optimization of the enzyme concentrations and pH was performed (Figure S1). The time courses of enzymatic oxidation of reduced cofactors were shown in Figure S2. It was found that both NADH and NADPH could be effectively oxidized by the four hemoproteins. Of the four hemoproteins tested, Mb exhibited the highest catalytic activities in the oxidation of NAD(P)H (Figure 2), in which the substrate conversions of approximately 70% were achieved within 12 min. A series of control experiments were conducted (Figure S3). Almost no NADH was oxidized in the absence of Mb and/or H2O2. It suggests that both Mb and H2O2 are required for the oxidation of NADH.


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Compared to Hb and HRP, higher catalytic efficiencies of Mb could be partially explained by its higher molar concentration (16-32 vs 59 μM). In addition, Mb also displayed better catalytic performances than Cyt C, although the molar concentration of the former was lower than that of the latter (59 vs 83 μM). In addition to a high molar concentration, there likely exist favorable interactions between Mb and NAD(P)H that contribute to its high catalytic efficiencies, since the interactions between enzyme and its substrate play a central role in enzyme catalysis. Recently, simple and inexpensive cofactor biomimetics have sparked renewed interest in redox biotransformations.45-47 In ene reductase-catalyzed reduction reactions, BNAH, one of the most widely used cofactor biomimetics in biocatalysis, outperformed the natural cofactors NAD(P)H in some cases.48 Regeneration of reduced nicotinamide biomimetics was fulfilled by using bio- and chemical catalysts.49-52 Herein, the oxidation of this synthetic mimic was conducted with H2O2 by hemoproteins (Figure 2). As shown in Figure 2, hemoproteins enabled to effectively oxidize BNAH, with the exception of Cyt C. More interestingly, the efficiencies of enzymatic BNAH oxidation appeared to be higher than those of the oxidation of its natural counterparts. The conversion was up to 92% in Mb-catalyzed BNAH oxidation (Figure 2). To date, limited methods have been reported for the oxidation of the cofactor biomimetics for biotransformations.53-56 The time course of enzymatic BNAH oxidation was monitored (Figure 3). The characteristic absorption peak of reduced cofactor analogue BNAH appeared at 361 nm, the intensity of which decreased gradually with the elongation of reaction periods. In contrast, the intensity of the peak at 265 nm which may be attributed


ACS Catalysis

to the characteristic absorption of its oxidized form (BNA+) increased. These results indicate occurrence of BNAH oxidation. To identify whether the produced BNA+ is enzymatically active, horse liver alcohol dehydrogenase (HLADH) and benzaldehyde were supplemented (Figure 3, inset), since this enzyme was reported to utilize BNAH for biocatalytic reduction.57 Significant increases in the intensity of the peak at 361 nm were observed upon addition of HLADH (Figure 3, inset). To exclude the interference of trace NAD(P)H present in HLADH, a control experiment where only the enzyme and substrate were added was conducted (Figure S4). No changes in the absorbance at 340 nm were observed. Hence, the findings demonstrate that the produced BNA+ is enzymatically active. 3 2 1 0

1.8

265 nm 361 nm

1.5

-1

Absorbance

Absorbance

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-2

1.2 0.9 0.6

0.0

-3

Adding HLADH

0.3 0

5

10

15

20

25

30

Time (h)

250

300

350

400

450

Wavelength (nm)

Figure 3. Mb-catalyzed oxidation of BNAH. Reaction conditions are the same as those in Figure 2. In the inset: 5 U HLADH and 10 mM benzaldehyde were supplemented at 14 min.

In addition, the initial reaction rates of hemoprotein-catalyzed oxidation of reduced cofactors were correlated with peroxidase activities of these hemoproteins (Figure S5), which were determined with 2,2’-azinobis(3-ethylbenzylthiozoline-6-sulfonate)


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(ABTS) and guaiacol as substrates, respectively (Table S1). Unfortunately, no correlations were observed (Figure S5). Although peroxidase activities of HRP were very high, it displayed relatively poor catalytic activities in the oxidation of reduced nicotinamide cofactors. It might be due to the fact that these reduced nicotinamide cofactors are not the natural substrates of HRP. In contrast, Mb exhibited satisfactory catalytic activities in cofactor oxidation, in spite of extremely low peroxidase activities. These results indicate that it seems to be infeasible to predict the catalytic performances of hemoproteins in the oxidation of reduced nicotinamide factors, based on their peroxidase activities with commonly used compounds (ABTS and guaiacol) as substrates. Moreover, Mb is a simple protein composed of 153 amino acids, and is an appropriate protein scaffold for function redesign and modification.25, 27 Therefore, Mb was used in the subsequent studies.

Effect of mediators on Mb-catalyzed oxidation of NADH 1.5 Control 0.1 mM scopoletin 0.4 mM acetaminophen -1 0.1 mM guaiacol k1: 0.28 min

1.2

ln[NADH]0/[NADH]t

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k1: 0.20 min

0.9

-1

k1: 0.15 min

0.6

k1: 0.12 min

0.3

-1

-1

0.0 0

1

2

3

4

5

Time (min)

Figure 4. Kinetics of Mb-catalyzed oxidation of NADH in the presence of mediators. Reaction conditions: 1 mM NADH, 10 mM H2O2, 1 mg/mL (59 μM) Mb, mediator of the designated concentration, 150 μL phosphate buffer (50 mM, pH 8), 30℃.


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Fundamental biochemical studies on biological H2O2-generating systems may date back to early 1950s,58,

59

in which peroxidase-catalyzed NAD(P)H oxidation is

involved.60 In addition, this enzymatic oxidation is significantly promoted in the presence of some biologically active phenols.61-64 Therefore, the effect of three commonly used phenols including scopoletin, acetaminophen, and guaiacol on Mbcatalyzed oxidation of NADH was investigated (Figure S6). As expected, these phenols that acted as mediators accelerated the enzymatic oxidation of NADH. The kinetics of the enzymatic oxidation of NADH were studied in the presence of an optimal concentration of mediator (Figure 4). As shown in Figure 4, the enzymatic oxidation of NADH followed pseudo-first order kinetics, which is in good agreement with a previous report.64 The apparent first order rate constants (k1) were determined from the slopes of the lines (Figure 4). The highest k1 value was found in the case of scopoletin, suggesting that it is the optimal mediator for NADH oxidation among three phenols tested. In addition, a significantly positive effect of scopoletin on Mb-catalyzed oxidation of NADPH and BNAH was also observed (Figure S7). Besides, the oxidation of NADH catalyzed by other hemoproteins was promoted remarkably in the presence of scopoletin (Figure S8). It is well known that phenols are natural substrates of peroxidases. Compared to NAD(P)H, therefore, these mediators could be more readily oxidized into phenoxy radicals. Phenoxy radicals were reported to be highly reactive, and they enabled to efficiently oxidize NAD(P)H.64, 65 Table 1 shows the apparent kinetic parameters in Mb-catalyzed oxidation of reduced


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cofactors in the presence of scopoletin. The KM values as well as kcat were in the same order of magnitude for the three cofactors. Based on the kcat/KM values, BNAH appeared to be a slightly better substrate for Mb than its natural counterparts. In BNAH oxidation, the catalytic efficiency of Mb was comparable to that of NOX from Lactobacillus pentosus.53

Table 1. Apparent kinetic parameters of Mb toward reduced cofactors in the presence of scopoletin Substrate

KM (mM)

kcat (min-1)

kcat/KM (min-1 mM-1)

NADH NADPH BNAH

1.20 0.91 0.96

7.85 4.84 6.51

6.54 5.32 6.78

Reaction conditions: 0.1-1.5 mM cofactor, 10 mM H2O2, 1 mg/mL (59 μM) Mb, 0.1 mM scopoletin, 150 μL phosphate buffer (50 mM, pH 8), 30℃

Biocatalytic application of Mb/scopoletin system Table 2. Dehydrogenase-catalyzed oxidations a Entry

Substrate

Concentration

DH

H2O2 feeding

Time

Yield

TTN

(mM)

dosage

/total

(h)

(%)

for

(U/mL)

concentration

cofactor

(mM) b

(×104)

1

Glucose

100

60

50 × 3 / 150

36

98

1

2

Glucose

100

60

25 / 25

48

96

1

3

Glucose

250

60

50 × 4 / 200

48

99

2.5

4

Glucose

500

60

50 × 5 / 250

60

83

4

5

Glucose

500

100

80 × 5 / 400

60

97

5

6

Glucose

500

200

62.5 × 2 / 125

48

97

5

7c

L-

50

80

50 × 2 / 100

36

97

0.5

50

80

50 × 2 / 100

48

95

0.5

Glutamate 8c

L-Lactate


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9d

Furfuryl

10

2.5

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10 / 10

48

93

0.019

alcohol a:

General reaction conditions unless otherwise stated: designated concentrations of substrate, DH and H2O2, 0.01 mM NAD+, 1 mg/mL (59 μM) Mb, 0.1 mM scopoletin, 1.5 eq. CaCO3, 4 mL phosphate buffer (50 mM, pH 8), 30°C, 150 r/min. b: “H O feeding” represents the initial concentration × feeding batch; H O was 2 2 2 2 generally supplemented every 12 h unless otherwise stated. c: H O was supplemented every 24 h. 2 mL phosphate buffer (50 mM, pH 7) 2 2 d: Reaction conditions: designated concentrations of substrate and HLADH, 0.1 mM BNAH, 0.5 mg/mL (30 μM) Mb, 0.1 mM scopoletin, 10 mM H2O2, 2 mL phosphate buffer (50 mM, pH 8), 30°C, 150 r/min.

To verify the practicality of this new regeneration method in biotransformations, GDHcatalyzed oxidation of glucose was used as a model reaction, because of its high enzyme activity. The effect of H2O2 and mediators on the activity of GDH was evaluated prior to biocatalytic application of this regeneration system (Figure S9). As shown in Figure S9a, GDH retained approximately 82% of original activity when the H2O2 concentration was up to 100 mM. In addition, mediators also had no significantly detrimental effect on the GDH activity, and its relative activities were more than 80% when the mediator concentrations were less than 0.4 mM (Figure S9b). These findings plus optimal pH of 8 indicate that this regeneration method is compatible well with GDH. Comparison of various in situ NAD+ recycling systems was performed in GDHmediated oxidation of glucose (Table S2). The desired product gluconic acid was produced with a 93% yield with a Mb/scopoletin system, whereas low yields (37-47%) were achieved in the cases of Hb and a laccase/acetosyringone system. The proof-ofconcept use of the latter was proved in dehydrogenase-catalyzed oxidations.10, 41 In addition, gluconic acid was not observed in the controls (Table S2). Then, the


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concentrations of GDH and NAD+ were optimized (Figure S10). As shown in Figure S10a, the optimal GDH concentration was 60 U/mL in the oxidation of 100 mM glucose. A good yield (96%) was obtained within 48 h when the NAD+ concentration was 0.01 mM (Figure S10b). In addition, the desired product was furnished with a moderate yield (75%) in the case of 0.005 mM NAD+. Effect of the H2O2 concentrations on the oxidation of glucose was studied (Figure S11). It was interestingly found that high yields (more than 96%) remained when the molar ratio of H2O2 to glucose as low as 1:4 was used (Figure S11 and Table 2, entries 2 and 6). It suggests that the mechanism of Mb/scopoletin-mediated NADH oxidation may resemble that of HRP/scopoletin-mediated NADPH oxidation.63 CaCO3 was added to neutralize the produced gluconic acid, since pH had a significant effect on the GDH activity (Figure S12). As shown in Table 2, the desired product was achieved with the yields of approximately 97% when the glucose concentration was up to 500 mM (Entries 5 and 6). The TTN for cofactor is defined as the total molar number of the product formed per mole of cofactor during the course of a complete reaction,66 which is a vital indicator for the practicability of a cofactor regeneration method. Generally, TTNs as high as 103-105 make a process economically viable, depending on the value of the product.67 It was interestingly found that the cofactor TTN was around 50 000 (Table 2, entries 5 and 6). Although it was lower than the TTN (500 000) of glutathionebased recycling system,7 our NAD(P)+ recycling method appeared to be advantageous over the latter in terms of the oxidant and atom economy (0.25 eq. H2O2 vs 2 eq. 2hydroxyethyl disulfide), and substrate concentration (glucose: 500 mM vs 5 mM). In


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addition, the method developed by us is applicable for the regeneration of the synthetic biomimetic BNA+. These results demonstrate potent potential of this Mb/scopoletin regeneration system in large-scale industrial production. Additionally, we sought to explore the applicability of this in situ NAD+ regeneration method in the enzymatic oxidation reactions catalyzed by other DHs including L-glutamic dehydrogenase (LgluDH), L-lactic dehydrogenase (L-lacDH) and HLADH. As shown in Table 2, entries 7 and 8, the desirable products (α-ketoglutarate and pyruvate) were afforded in good yields (95-97%). Moreover, the enzymatic in situ regeneration of the synthetic biomimetic BNA+ was applied in HLADH-catalyzed oxidation of furfuryl alcohol into 2-furoic acid (Table 2, entry 9). It was found that 2-furoic acid was produced smoothly in a 93% yield, and the TTN for cofactor was approximately 190. Nonetheless, no oxidized products were found in the absence of BNAH. These results also suggest that the oxidized product BNA+ is enzymatically active.

Mechanism of Mb/scopoletin-catalyzed NAD+ regeneration


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Scheme 1. Postulated reaction mechanism. MOH and MO● are the mediator scopoletin and its phenoxy radical, respectively.

Based on the results obtained in this work and previous work, a radical mechanism was proposed for Mb/scopoletin-catalyzed oxidation of NADH (Scheme 1). The Soret maximum was around 408 nm (Figure S13), indicating a typical hexa-coordinated ferric high-spin heme present in wild-type Mb.68 FeIII porphyrin (Por) present in Ferric Mb was rapidly transformed into a plausible heme-bound peroxide (FeIIIOOH Por) in the presence of H2O2, which was evidenced by the disappearance of the characteristic peaks of ferric Mb at 500 and 624 nm.69 It was reported that the O-O bond of FeIIIOOH Por could be cleaved both heterolytically and homolytically in wild-type Mb, resulting in the formation of extremely active ferryl species.68 In the homolytic route, Mb-II, a ferry


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heme (FeIV=O Por) Mb that is equivalent to compound II of peroxidase, was formed, due to the appearance of its characteristic absorption peaks at 546 and 580 nm (Figure S13). Mb-I, a ferry porphyrin cation radical (FeIV=O Por●+) was produced at the first step in the heterolytical route. However, Mb-I was not observed in this work, likely because of its rapid decay to Mb-II.68, 70 These active species were reduced sequentially into ferric state through the one-electron oxidation of the mediator scopoletin. The oxidation of scopoletin verified by the decreased intensity of its characteristic peak at 381 nm (Figure S14) led to the formation of the phenoxy radical MO●.63, 64, 71 Then, MO● rapidly oxidized NADH into NAD● (Figure S15). The active NAD● was converted into the oxidized cofactor NAD+ in the presence of O2, which was used by GDH for the oxidation of glucose. O2 may stem from the decomposition of H2O2 as well as from air. To verify this, the dissolved oxygen concentrations in the H2O2 solution were monitored in the presence and absence of Mb (Figure S16). Indeed, it was found that the dissolved oxygen concentrations significantly increased in the presence of Mb. Like catalase, Mb also has a heme cofactor; thus, it may have a low catalase activity. In addition, no significant differences in the substrate conversions or product yields were observed in enzymatic oxidation of NADH and glucose under aerobic and anaerobic conditions (Figure S17), because O2 required in the catalytic cycle under anaerobic conditions could derive from enzymatic decomposition of H2O2. O2 was simultaneously reduced into O2●-, which might be confirmed by the fact that the enzymatic NADH oxidation was promoted significantly in the presence of superoxide dismutase (SOD) (Figure S18). O2●- underwent spontaneous dismutation, thus producing H2O2 and O2, which re-


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entered into the NADH oxidation cycle. The oxidized NADH was correlated with the consumed H2O2 in the presence and absence of scopoletin (Figure S19). Figure S19 shows that the molar ratios of the oxidized NADH to the consumed H2O2 are approximately 5 and 4 in the presence and absence of scopoletin, respectively. Considering the decomposition of H2O2, the molar ratio of around 5 is in agreement with the above results that around 4 moles of glucose are enzymatically oxidized per mole of H2O2 (Table 2, entries 2 and 6). As shown in Scheme 1, 2 moles of NAD+ are produced with no net H2O2 consumption in the heterolytic route, while 1 mole of H2O2 is required for the production of 2 moles of NAD+ in the homolytic route. Therefore, we reason that approximately 60% and 40% of FeIIIOOH Por are cleaved heterolytically and homolytically, respectively, in the presence of scopoletin; thus, 5 moles of NAD+ are produced per mole of H2O2 in the catalytic cycle. Of course, more experiments should be conducted to verify this hypothesis. In the absence of mediators, the regeneration of NAD+ may occur with a similar radical mechanism (Scheme S1). In addition, the proportion between heterolysis and homolysis is approximately 1:1 in the absence of mediators. The observations indicate that the heme-bound peroxide prefers heterolysis to homolysis in the presence of mediators. In addition, it is worth noting that the protein radicals (e.g. peroxy radical) may be involved in the oxidation of NADH by Mb/scopoletin.72

Conclusions In summary, we have constructed a new and efficient enzymatic regeneration system


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for oxidized nicotinamide cofactors, based on the catalytic promiscuity of wild-type Mb. Mb is capable of effective oxidation of the natural cofactors NAD(P)H as well as the synthetic biomimetic BNAH with inexpensive and environmentally friendly oxidant H2O2. The enzymatic oxidation of reduced cofactors was enhanced significantly in the presence of mediators, likely due to the formation of highly active phenoxy radicals. Mb/scopoletin-based regeneration system exhibited good compatibility with DH-catalyzed oxidations. The desired products were obtained with almost quantitative yields in all cases. With GDH-catalyzed oxidation of glucose as a model reaction, a high TTN for cofactor was achieved by using the Mb/scopoletin system under industrially sound conditions, which may highlight its potent application potential in large-scale biotransformations. In addition, use of the synthetic biomimetic BNA+ that was in situ regenerated by Mb/scopoletin system was proven in HLADHcatalyzed oxidation of bio-based furfuryl alcohol.

Conflicts of interest The authors declare no conflict of interest.

Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website. Detailed experimental procedures, analytic methods and data, enzyme assay, the results in the controls, process optimization, and UV-visible spectra.


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Acknowledgements This research was financially supported by the National Natural Science Foundation of China (21676103), the Natural Science Foundation of Guangdong Province (2017A030313056), and the Science and Technology Project of Guangzhou City (201804010179).

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Myoglobin-Catalyzed Efficient In Situ Regeneration of NAD(P)+ and

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