Heterogeneous Asymmetric Oxidation Catalysis Using Hemophore Hasapf. Application in the Chemoenzymatic Deracemization of Sec-alcohols with Sodium Borohydride

This study aims to demonstrate the coordination of oxygen regarding the hemophore HasApf expressed by Escherichia coli cells, which appears to create an unlikely oxygen-activating system in HasA due to the already-coordinated iron. In the asymmetric oxidation of rac-1-(6-methoxynaphthalen-2-yl)ethanol (rac-1) using dissolved oxygen, the signals at g-values of 2.8, 2.22, and 1.72 in the electron spin resonance (ESR) spectra disappeared in conjunction with the promotion of oxoferric (Fe III´O–O ´) species in the distal site. These results suggest that the iron of porphyrin/Fe may be oxidized in water, leading to exhibition of greater asymmetric oxidation activity in the promotion of oxoferryl (Fe IV =O) species. A ketone (~50% chemical yield) produced from (R)-(´)-sec-alcohol can be desymmetrized by NaBH 4 in aqueous medium at 40 ˝ C (>99% enantiomer excess, ee, >90% chemical yield) in the absence of NAD(P). Therefore, HasA can be regenerated via successive asymmetric catalytic events through an incorporated iron electron-transfer system in the presence of oxygen: Fe II + O 2 Ñ Fe III´O–O ´ Ñ Fe IV =O (oxidizing rac-1) Ñ Fe II + H 2 O. This process is similar to a Fenton reaction. The use of a HasA-catalytic system with an incorporated redox cofactor for asymmetric oxidation overcomes the apparent difficulties in working with pure dehydrogenase enzyme/redox cofactor systems for biotransformations.


Introduction
A review covering the well-known alcohol dehydrogenase enzyme system that incorporates redox cysteine disulfide bonds, redox zinc, and a redox cofactor has already been published [1]; moreover, the high cost and instability of the redox cofactors in enzymatic synthesis justifies efforts to regenerate them [2].However, the use of a HasA hemophore incorporating an iron electron-transfer system for the asymmetric oxidation (with oxygen) of secondary alcohols in organic synthesis has not yet been examined as a heterogeneous enzyme catalysis reaction [3].However, it is generally accepted that the system of hemophore HasA secreted by host ABC transporters [4] enables heme uptake across the cell outer membrane [5] and spontaneously transforms it into the HasR receptor at the heme-binding site [6].Thus, the use of a porphyrin/Fe holoprotein (e.g., HasA) system counteracts the apparent difficulties in working with pure dehydrogenase enzymatic/redox-cofactor systems for microbial biotransformations [7].One of the key structural features that makes cytochrome P450 a good system to activate oxygen and catalyze oxidation reactions is that its heme iron is coordinated by a cysteine ligand in the proximal site [8] and the distal site is open for oxygen binding [9].For all structures wherein a ligand of hemophore HasA serves as a coordinating ligand, the ligand in the proximal site is a histidine and the distal site of the heme is coordinated by a tyrosine [10].This set of ligands appears to make the heme in HasA an unlikely oxygen-activating system because of the already-coordinated iron (Scheme 1) [11].HasA serves as a coordinating ligand, the ligand in the proximal site is a histidine and the distal site of the heme is coordinated by a tyrosine [10].This set of ligands appears to make the heme in HasA an unlikely oxygen-activating system because of the already-coordinated iron (Scheme 1) [11].
Scheme 1.The structural representation of hemophore HasA for asymmetric oxidation.
The development of deracemization methods, which yield a single stereoisomer from a racemate, is an important topic in organic synthesis [12].The deracemization methods are classified according to the stereochemical course of enzymatic and chemical reactions [13], and special emphasis has been devoted to the theoretical background of the one-pot, single-step deracemization of sec-alcohols [14].The primary feature of deracemization via bio-or chemo-catalysts or combinations thereof is its applicability to secondary alcohols [15].An important topic in research related to such catalytic processes is the development of new biocatalysts, irrespective of whether the application of the HasA kinetic resolution to the chemoenzymatic methods involves a one-pot protocol (Scheme 2) [16].Over the past decade, the use of biomaterials as plant catalytic systems incorporating redox cofactors for asymmetric oxidation reactions has been investigated [17,18].Particularly, the redox protein eluted from pea protein (PP) encapsulated with calcium alginate gel (PP gel) is available for synthesis [19] and the enantiomeric resolution of m-and p-substituted racemic aryl methyl carbinols [20].Specifically, membrane-bound enzymes (MEs) are activated by a buffered glycine reaction solution (pH 9.0-10.0)[21].When eluted from encapsulated PP, under aeration, ME can be applied to turnover kinetic resolutions; e.g., MEs can be used to synthesize (S)-(+)-1 ((S)-naproxen precursor [22]) via a polyethylene glycol (PEG) 4000-coated ME [23].Moreover, an iron electron-transfer system can be incorporated as an oxygen-driven catalytic system for asymmetric oxidation [7].The exact nature of the species engaged in the key reaction has been demonstrated to be consistent with that of a heme-binding protein [24].An N-terminal sequence comparison also provides 93% similarity with a 20.853 kDa hemophore HasApf gene product (Pseudomonas fluorescens Pf-5) [25].Therefore, these features are regenerated by successive asymmetric catalytic events using an incorporated iron electron-transfer system in the presence of oxygen [26].This process is similar to that utilized by the oxygen-driven cytochrome P450: cysteine-Fe II + O2→ Fe III -O-O − → Fe IV =O (oxidizing rac-1 or -2) → Fe II + H2O [7].
Two notable issues are whether oxygen can bind at a distal site because the HasA hemophore is typically coordinated by histidine (proximal) and tyrosine (distal) [27] and whether the cyclic deracemization can occur via these proteins for oxygen activation [28].Therefore, we conducted The development of deracemization methods, which yield a single stereoisomer from a racemate, is an important topic in organic synthesis [12].The deracemization methods are classified according to the stereochemical course of enzymatic and chemical reactions [13], and special emphasis has been devoted to the theoretical background of the one-pot, single-step deracemization of sec-alcohols [14].The primary feature of deracemization via bio-or chemo-catalysts or combinations thereof is its applicability to secondary alcohols [15].An important topic in research related to such catalytic processes is the development of new biocatalysts, irrespective of whether the application of the HasA kinetic resolution to the chemoenzymatic methods involves a one-pot protocol (Scheme 2) [16].HasA serves as a coordinating ligand, the ligand in the proximal site is a histidine and the distal site of the heme is coordinated by a tyrosine [10].This set of ligands appears to make the heme in HasA an unlikely oxygen-activating system because of the already-coordinated iron (Scheme 1) [11].
Scheme 1.The structural representation of hemophore HasA for asymmetric oxidation.
The development of deracemization methods, which yield a single stereoisomer from a racemate, is an important topic in organic synthesis [12].The deracemization methods are classified according to the stereochemical course of enzymatic and chemical reactions [13], and special emphasis has been devoted to the theoretical background of the one-pot, single-step deracemization of sec-alcohols [14].The primary feature of deracemization via bio-or chemo-catalysts or combinations thereof is its applicability to secondary alcohols [15].An important topic in research related to such catalytic processes is the development of new biocatalysts, irrespective of whether the application of the HasA kinetic resolution to the chemoenzymatic methods involves a one-pot protocol (Scheme 2) [16].Over the past decade, the use of biomaterials as plant catalytic systems incorporating redox cofactors for asymmetric oxidation reactions has been investigated [17,18].Particularly, the redox protein eluted from pea protein (PP) encapsulated with calcium alginate gel (PP gel) is available for synthesis [19] and the enantiomeric resolution of m-and p-substituted racemic aryl methyl carbinols [20].Specifically, membrane-bound enzymes (MEs) are activated by a buffered glycine reaction solution (pH 9.0-10.0)[21].When eluted from encapsulated PP, under aeration, ME can be applied to turnover kinetic resolutions; e.g., MEs can be used to synthesize (S)-(+)-1 ((S)-naproxen precursor [22]) via a polyethylene glycol (PEG) 4000-coated ME [23].Moreover, an iron electron-transfer system can be incorporated as an oxygen-driven catalytic system for asymmetric oxidation [7].The exact nature of the species engaged in the key reaction has been demonstrated to be consistent with that of a heme-binding protein [24].An N-terminal sequence comparison also provides 93% similarity with a 20.853 kDa hemophore HasApf gene product (Pseudomonas fluorescens Pf-5) [25].Therefore, these features are regenerated by successive asymmetric catalytic events using an incorporated iron electron-transfer system in the presence of oxygen [26].This process is similar to that utilized by the oxygen-driven cytochrome P450: cysteine-Fe II + O2→ Fe III -O-O − → Fe IV =O (oxidizing rac-1 or -2) → Fe II + H2O [7].
Two notable issues are whether oxygen can bind at a distal site because the HasA hemophore is typically coordinated by histidine (proximal) and tyrosine (distal) [27] and whether the cyclic deracemization can occur via these proteins for oxygen activation [28].Therefore, we conducted Over the past decade, the use of biomaterials as plant catalytic systems incorporating redox cofactors for asymmetric oxidation reactions has been investigated [17,18].Particularly, the redox protein eluted from pea protein (PP) encapsulated with calcium alginate gel (PP gel) is available for synthesis [19] and the enantiomeric resolution of mand p-substituted racemic aryl methyl carbinols [20].Specifically, membrane-bound enzymes (MEs) are activated by a buffered glycine reaction solution (pH 9.0-10.0)[21].When eluted from encapsulated PP, under aeration, ME can be applied to turnover kinetic resolutions; e.g., MEs can be used to synthesize (S)-(+)-1 ((S)-naproxen precursor [22]) via a polyethylene glycol (PEG) 4000-coated ME [23].Moreover, an iron electron-transfer system can be incorporated as an oxygen-driven catalytic system for asymmetric oxidation [7].The exact nature of the species engaged in the key reaction has been demonstrated to be consistent with that of a heme-binding protein [24].An N-terminal sequence comparison also provides 93% similarity with a 20.853 kDa hemophore HasApf gene product (Pseudomonas fluorescens Pf-5) [25].Therefore, these features are regenerated by successive asymmetric catalytic events using an incorporated iron electron-transfer system in the presence of oxygen [26].This process is similar to that utilized by the oxygen-driven cytochrome P450: cysteine-Fe II + O 2 ÑFe III -O-O ´Ñ Fe IV =O (oxidizing rac-1 or -2) Ñ Fe II + H 2 O [7].Two notable issues are whether oxygen can bind at a distal site because the HasA hemophore is typically coordinated by histidine (proximal) and tyrosine (distal) [27] and whether the cyclic deracemization can occur via these proteins for oxygen activation [28].Therefore, we conducted experiments using electron spin resonance (ESR) spectroscopy and cyclic deracemization.This study aims to (1) demonstrate the coordination of oxygen by ESR evidence; (2) demonstrate the cyclic deracemization process using HasA and NaBH 4 ; and (3) present HasAs as a new asymmetric oxidation catalysis tool similar to a Fenton reaction.

Purifications of HasApf from PP
The purification of a HasApf from PP and the kinetic resolutions of rac-2 for each fraction are summarized in Scheme 3 [21]; method details are provided in the Experimental Section.The preparation procedure was previously reported elsewhere [23], and HasApf was demonstrated to be effectively purified with a supernatant 2 sample via ultrafiltration processes using a Vivaspin molecular-weight cutoff (MWCO) 10 kDa, which is a protein sample concentrator that can surprisingly yield a single band of HasApf [23].Table 1 and Scheme 3 also show that P. fluorescens Pf-5 is absent in the PP analysis for common bacteria (CFU/g), whereas aerobic spore-bearing bacteria (Geobacillus, Alicyclobacillus, Bacillus, Paenibacillus) and catalase-positive and Gram-positive cocci (Staphylococcus, Kocuria, Micrococcus) are detected [25].Therefore, we speculated that the existence of the hemophore may be due to broad acquisition by the plant (e.g., PP) rather than by bacterial contamination [25].

Identification of HasApf from Supernatant 2 Filtrated by Vivaspin
SDS-PAGE was performed on supernatant 2 after ultrafiltration with a Vivaspin 2 (MWCO 10 kDa) (10 µL) [21].Precise analysis of the N-terminal amino-acid sequence (protein sequencing) of the single band obtained via SDS-PAGE was accomplished using a PPSQ-21A protein sequencer (Shimadzu) [23].The obtained amino-acid sequence was acquired using BLAST to identify regions of local similarity between the sequences [25].As shown in Table 2, the amino-acid sequence (33 residues) of a single band was detected, and an N-terminal sequence comparison showed 93% similarity with HBP, a 20.853 kDa HasAp gene product (Pseudomonas fluorescens Pf-5, a plant commensal bacterium), and a similar E value (2 ˆ10 ´11 ).This result is consistent with both the structure of the hemophore in Scheme 1 and the molecular mass in Figure 1.No BLAST-hit data exemplifying a hemophore HasA gene were detected from the plant for the connection between HBP (P.fluorescens Pf-5) and PP, as shown in Table 2; nonetheless, in the context of a highly conserved structure (Scheme 1) resulting from the broad acquisition by PP, amino acid sequence alignments involving a large number of HasA proteins from different organisms show strict conservation of the histidine residue that coordinates the heme iron in the proximal side.Consequently, the ultrafiltrated supernatant 2 enables the kinetic resolution of rac-1 for asymmetric oxidation, although the oxidative reaction on HasApf appears difficult because of the lack of a cysteine ligand.The important consideration is whether oxygen can bind at the distal site to be activated if iron is already coordinated.SDS-PAGE was performed on supernatant 2 after ultrafiltration with a Vivaspin 2 (MWCO 10 kDa) (10 μL) [21].Precise analysis of the N-terminal amino-acid sequence (protein sequencing) of the single band obtained via SDS-PAGE was accomplished using a PPSQ-21A protein sequencer (Shimadzu) [23].The obtained amino-acid sequence was acquired using BLAST to identify regions of local similarity between the sequences [25].As shown in Table 2, the amino-acid sequence (33 residues) of a single band was detected, and an N-terminal sequence comparison showed 93% similarity with HBP, a 20.853 kDa HasAp gene product (Pseudomonas fluorescens Pf-5, a plant commensal bacterium), and a similar E value (2 × 10 −11 ).This result is consistent with both the structure of the hemophore in Scheme 1 and the molecular mass in Figure 1.
No BLAST-hit data exemplifying a hemophore HasA gene were detected from the plant for the connection between HBP (P.fluorescens Pf-5) and PP, as shown in Table 2; nonetheless, in the context of a highly conserved structure (Scheme 1) resulting from the broad acquisition by PP, amino acid sequence alignments involving a large number of HasA proteins from different organisms show strict conservation of the histidine residue that coordinates the heme iron in the proximal side.Consequently, the ultrafiltrated supernatant 2 enables the kinetic resolution of rac-1 for asymmetric oxidation, although the oxidative reaction on HasApf appears difficult because of the lack of a cysteine ligand.The important consideration is whether oxygen can bind at the distal site to be activated if iron is already coordinated.

HasApf Protein Expression and Purification
The HasApf coding sequence was amplified by a polymerase chain reaction with bacterial genomic DNA (P.fluorescens) and cloned into pET28a (Novagen) to confirm whether oxygen could bind at the distal site.The resultant plasmids were transformed into BL21(DE3) Escherichia coli cells, and the transformants were cultured through continuous shaking at 30 °C in Terrific Broth to which 50 μL/mL ampicillin was added.The cells were lysed by sonication, and the HasApf proteins were purified from the supernatant using a His-Tag purification column.As shown in Figure 1, although cytochrome P450 electron-transport chains corresponding to NADPH→FAD-containing reductase → iron-sulfur complex→P450 have been reported [21], the HasApf shows a single band at X a : may be Cys (C) but not detected, X b : many amino acids were detected.c YP 262445.1: the accession hit on the query sequence was limited between the query coverage (>93%) and E value (2e-11), a 20.853 Da.HasAp gene product [hemophore: Pseudomonas fluorescens Pf-5] from plant commensal bacteria, which can inhibit the rhizosphere and produce secondary metabolites that suppress soil-borne plant pathogens.Red amino acids indicate "hits" between Supernartant 2 and YP 262445.1 c .Blue squares indicates the heme-binding site: His-32 (bearing loop), Tyr-75 (axial heme ligand), and His-83 (hydrogen ligand).Squares indicate the Cys (C) or Met (M) including sulfur.

HasApf Protein Expression and Purification
The HasApf coding sequence was amplified by a polymerase chain reaction with bacterial genomic DNA (P.fluorescens) and cloned into pET28a (Novagen) to confirm whether oxygen could bind at the distal site.The resultant plasmids were transformed into BL21(DE3) Escherichia coli cells, and the transformants were cultured through continuous shaking at 30 ˝C in Terrific Broth to which 50 µL/mL ampicillin was added.The cells were lysed by sonication, and the HasApf proteins were purified from the supernatant using a His-Tag purification column.As shown in Figure 1, although cytochrome P450 electron-transport chains corresponding to NADPHÑFAD-containing reductase Ñ iron-sulfur complexÑP450 have been reported [21], the HasApf shows a single band at approximately 21 kDa [23]; SDS-PAGE provided no preliminary evidence of iron sulfate participating in the HasApf [25].

Asymmetric Oxidation of rac-1 with Bacterial HasApf
The time course of the asymmetric oxidation activity with a HasApf expressed (40 μM: e.g., 0.36 mg) utilizing substrate rac-1 (0.8 mM: e.g., 0.4 mg) in deionized water (5.0 mL) is summarized in Figure 2, where (S)-1, (R)-1, and the product ketone were monitored [21].A redox protein obtained from expressed HasApf can oxidize an enantiomer in rac-1; thus, the other enantiomer could be obtained with high enantioselectivity (i.e., 50% yield, 99% ee) although the activity of PP-HasApf (0.1 μM, which is equal to PEG-precipitate 2 (30 mg)) is more than 200 times greater than that of the expressed HasApf (40 μM), suggesting that a nonplanar oxoferryl (Fe IV =O) species might be formed during oxidation between 0 h and 24 h incubations (Figure 2) [23]: when the iron of porphyrin/Fe of PEG-precipitate 2 and oxygen perfectly coordinates in the process of PP-gel aerations, leading to exhibition of greater asymmetric oxidation activity for the promotion of a reactive oxygen species (Scheme 3), the expressed HasApf gets coordinated to the oxygen at the distal site.Moreover, no participation of iron(II) sulfate and/or H2O2 in HasApf was detected, and the kinetic resolution of rac-1 to (S)-(+)-1 (>99% ee, ~50% chemical yield) via the selective oxidation of (R)-(−)-1 to the corresponding ketone (Figure 2) was catalyzed [25].

Asymmetric Oxidation of rac-1 with Bacterial HasApf
The time course of the asymmetric oxidation activity with a HasApf expressed (40 µM: e.g., 0.36 mg) utilizing substrate rac-1 (0.8 mM: e.g., 0.4 mg) in deionized water (5.0 mL) is summarized in Figure 2, where (S)-1, (R)-1, and the product ketone were monitored [21].A redox protein obtained from expressed HasApf can oxidize an enantiomer in rac-1; thus, the other enantiomer could be obtained with high enantioselectivity (i.e., 50% yield, 99% ee) although the activity of PP-HasApf (0.1 µM, which is equal to PEG-precipitate 2 (30 mg)) is more than 200 times greater than that of the expressed HasApf (40 µM), suggesting that a nonplanar oxoferryl (Fe IV =O) species might be formed during oxidation between 0 h and 24 h incubations (Figure 2) [23]: when the iron of porphyrin/Fe of PEG-precipitate 2 and oxygen perfectly coordinates in the process of PP-gel aerations, leading to exhibition of greater asymmetric oxidation activity for the promotion of a reactive oxygen species (Scheme 3), the expressed HasApf gets coordinated to the oxygen at the distal site.Moreover, no participation of iron(II) sulfate and/or H 2 O 2 in HasApf was detected, and the kinetic resolution of rac-1 to (S)-(+)-1 (>99% ee, ~50% chemical yield) via the selective oxidation of (R)-(´)-1 to the corresponding ketone (Figure 2) was catalyzed [25].

Asymmetric Oxidation of rac-1 with Bacterial HasApf
The time course of the asymmetric oxidation activity with a HasApf expressed (40 μM: e.g., 0.36 mg) utilizing substrate rac-1 (0.8 mM: e.g., 0.4 mg) in deionized water (5.0 mL) is summarized in Figure 2, where (S)-1, (R)-1, and the product ketone were monitored [21].A redox protein obtained from expressed HasApf can oxidize an enantiomer in rac-1; thus, the other enantiomer could be obtained with high enantioselectivity (i.e., 50% yield, 99% ee) although the activity of PP-HasApf (0.1 μM, which is equal to PEG-precipitate 2 (30 mg)) is more than 200 times greater than that of the expressed HasApf (40 μM), suggesting that a nonplanar oxoferryl (Fe IV =O) species might be formed during oxidation between 0 h and 24 h incubations (Figure 2) [23]: when the iron of porphyrin/Fe of PEG-precipitate 2 and oxygen perfectly coordinates in the process of PP-gel aerations, leading to exhibition of greater asymmetric oxidation activity for the promotion of a reactive oxygen species (Scheme 3), the expressed HasApf gets coordinated to the oxygen at the distal site.Moreover, no participation of iron(II) sulfate and/or H2O2 in HasApf was detected, and the kinetic resolution of rac-1 to (S)-(+)-1 (>99% ee, ~50% chemical yield) via the selective oxidation of (R)-(−)-1 to the corresponding ketone (Figure 2) was catalyzed [25].and substrate rac-1 (0.8 mM) in deionized water as a solvent (5.0 mL); the absorbances of (S)-1 (--: 0.4 mM), (R)-1 (--: 0.4 mM), and the product ketone (--) were monitored.Legend: A nonplanar oxoferryl (Fe IV =O) species might be formed during oxidation between 0 h and 24 h incubations.Labelled arrows: 1.No oxidation activity was detected.2. After 24 h, greater asymmetric oxidation activity was detected in the promotion of a reactive oxygen species.*There are no participation of iron(II) sulfate and/or H 2 O 2 in HasApf biotransformation.

UV-Absorbances at Each of the HasApf Reaction Times
As shown in Figure 3, UV-vis absorption spectrophotometry (UV-3600 Plus) was performed, where the absorption at wavelengths of 410 nm (heme) and 350 nm (product ketone-2) was monitored as a function of time (0, 24, 48, and 72 h).These results indicate that, whereas the absorbance at 350 nm (product ketone) continuously increased, the absorbance at 410 nm (HasApf heme) minimally varied with reaction time.Therefore, the expressed HasApf was fairly stable in a water reaction tube without any cap at 40 ˝C.ESR analysis could therefore be conducted to determine whether oxygen could bind at the distal site and whether His/Tyr-heme remained in the six-coodinate low-spin state.

UV-Absorbances at Each of the HasApf Reaction Times
As shown in Figure 3, UV-vis absorption spectrophotometry (UV-3600 Plus) was performed, where the absorption at wavelengths of 410 nm (heme) and 350 nm (product ketone-2) was monitored as a function of time (0, 24, 48, and 72 h).These results indicate that, whereas the absorbance at 350 nm (product ketone) continuously increased, the absorbance at 410 nm (HasApf heme) minimally varied with reaction time.Therefore, the expressed HasApf was fairly stable in a water reaction tube without any cap at 40 °C.ESR analysis could therefore be conducted to determine whether oxygen could bind at the distal site and whether His/Tyr-heme remained in the six-coodinate low-spin state.absorption was monitored at 410 nm (heme) and 350 nm (product ketone-2) (H) at incubation times of 0, 24, 48, and 72 h.

ESR Spectra at Each of the HASApf Reaction Times
As shown in Figure 4, we further confirmed whether substantial differences exist in ESR spectra related to the oxygen-binding at the distal site at each oxidative kinetic resolution time (i.e., 0, 50, and 100 h).These results show that, whereas the 0 h incubation (a) may result in a six-coordinate low-spin state with His/Tyr coordinated to heme, 50 h incubation (b) appears to result in high-spin complexes with distinctive g′x ≅ g′y ≅ 6 and g′z = 2 [27,28]; the low-spin species in the high-spin complexes, where the concentrations of low-spin species ranged from approximately 10% to 30%, generated g-values of 1.56 ≤ gx ≤ 1.72, 2.21 ≤ gy ≤ 2.23, and 2.78 ≤ gz ≤ 2.80 [29,30].Furthermore, with 100 h incubation, peaks appeared at g = 4.3 and g = 2.0 in the ESR spectra of ultrafiltrated dried supernatant 2 with the promotion of oxoferryl (Fe IV =O), suggesting that the iron of porphyrin/Fe may be oxidized in water to be oxoferric (Fe III −O-O − ) species and that an oxidative iron could exhibit higher asymmetric oxidation activity in the promotion of oxoferryl (Fe IV =O) species.absorption was monitored at 410 nm (heme) and 350 nm (product ketone-2) (H) at incubation times of 0, 24, 48, and 72 h.

ESR Spectra at Each of the HASApf Reaction Times
As shown in Figure 4, we further confirmed whether substantial differences exist in ESR spectra related to the oxygen-binding at the distal site at each oxidative kinetic resolution time (i.e., 0, 50, and 100 h).These results show that, whereas the 0 h incubation (a) may result in a six-coordinate low-spin state with His/Tyr coordinated to heme, 50 h incubation (b) appears to result in high-spin complexes with distinctive g 1 x -g 1 y -6 and g 1 z = 2 [27,28]; the low-spin species in the high-spin complexes, where the concentrations of low-spin species ranged from approximately 10% to 30%, generated g-values of 1.56 ď g x ď 1.72, 2.21 ď g y ď 2.23, and 2.78 ď g z ď 2.80 [29,30].Furthermore, with 100 h incubation, peaks appeared at g = 4.3 and g = 2.0 in the ESR spectra of ultrafiltrated dried supernatant 2 with the promotion of oxoferryl (Fe IV =O), suggesting that the iron of porphyrin/Fe may be oxidized in water to be oxoferric (Fe III ´O-O ´) species and that an oxidative iron could exhibit higher asymmetric oxidation activity in the promotion of oxoferryl (Fe IV =O) species.
An oxidation/hydroxylation system that includes enzymatic detoxification involving the promotion of a reactive oxygen species via an iron electron-transfer system [31] is already well known: hydroxylation [32], epoxidation [33], and dehalogenation [34] using cytochrome P450 enzyme [35] and porphyrin-like complexes [36] such as N-bridged di-iron oxo phthalocyanine [37] associating cytochrome P450 and a monooxygenase enzyme that uses H2O2 or mCPBA as an oxidant and NAD(P) redoxin complex [38].Furthermore, the biological degradation of heme (iron-protoporphyrin) plays a variety of critical functions in living organisms [39]; an enzyme termed heme oxygenase (HO) has already been reported to catalyze the regiospecific conversion of heme into biliverdin, carbon monoxide (CO), and a free ferrous iron via three successive oxygenation reactions [40] (Scheme 4).The literature contains numerous reports exemplifying other types of heme degradation enzymes, e.g., the HutZ protein in the heme utilization (Hut) system [41,42] and Mycobacterium tuberculosis enzyme MhuD [43].Therefore, the results in the present work newly indicate that (1) oxygen will bind at the distal site for oxygen activation to be oxoferric (Fe III   Therefore, although further explorations of the efficiency, turnover number, or enzyme loading in comparison to other known systems for this or similar transformations are being actively pursued in this laboratory, this process would overcome the apparent difficulties in working with pure dehydrogenase enzymatic/redox-cofactor systems for biotransformation.The use of a heme-binding protein (HBP) as a HasA-catalytic system has been proposed for heterogeneous enzyme catalysis [44][45][46], where a redox cofactor is incorporated to perform asymmetric oxidation.In the future, bacterial HasAs are expected to become important biological catalysts for the Therefore, although further explorations of the efficiency, turnover number, or enzyme loading in comparison to other known systems for this or similar transformations are being actively pursued in this laboratory, this process would overcome the apparent difficulties in working with pure dehydrogenase enzymatic/redox-cofactor systems for biotransformation.The use of a heme-binding protein (HBP) as a HasA-catalytic system has been proposed for heterogeneous enzyme catalysis [44][45][46], where a redox cofactor is incorporated to perform asymmetric oxidation.In the future, bacterial HasAs are expected to become important biological catalysts for the synthesis of optically active alcohols using environmentally friendly systems that promote industrial sustainability.PP (10 g) was added to 200 mL 0.75% aqueous sodium alginate and encapsulated with CaCl 2 (500 mL, 39 g/L).The PP gel was air-exposed for 5 h, and the resulting HasA suspension was extracted at 40 ˝C with distilled water (200 mL) in a 500 mL Erlenmeyer flask through centrifugation at 150 rpm for more than 40 h.To produce AG-precipitate 2, the precipitate 1 (wet: 16 g) was centrifuged (at 10,000 rpm for 10 min), then dissolved in a 5% (v/v) PEG (M W : 1000/4000 = 2/1)/ 50 mM glycine-NaOH solution (pH 9.0, 100 mL) and stored for over 20 h to allow aggregation.After the storage step, the precipitate was freeze-dried under vacuum and crushed using a ball mill.PEG-precipitate 2 was prepared as follows: precipitate 1 (wet: 16 g) was dissolved in a 50 mM glycine-NaOH solution (pH 9.0, 100 mL), and PEG (M W 4000) (500 mg) was added.Finally, to produce the dried supernatant 2, precipitate 1 (wet: 16 g) was dissolved in a 50 mM glycine-NaOH solution (pH 9.0, 100 mL) for over 3 h and the resulting mixture was centrifuged at 10,000 rpm for 10 min.The resulting supernatant 2 was ultra-filtered with the Vivaspin 2-10 K and was subsequently freeze-dried under vacuum, dried at a temperature over 50 ˝C on a hot plate (As One: THI-1000) [´50 ˝C/10 Pa (1 h)Ñ5 ˝C/min Ñ +50 ˝C/10 Pa (22 h)], and crushed using a ball mill.

HasApf Protein Expression and Purification
The HasApf coding sequence was amplified by a polymerase chain reaction with bacterial genomic DNA (P.fluorescens) and cloned into pET28a (Novagen).The resultant plasmids were transformed into BL21(DE3) E. coli cells, and the transformants were cultured through continuous shaking at 30 ˝C in Terrific Broth containing 50 µL/mL ampicillin.At OD 600 = 1, 0.1 mM isopropyl β-D-1-thiogalactopyranoside was added, followed by incubation at 30 ˝C for an additional 16 h.The harvested cells were resuspended in 15 mM potassium phosphate solution containing 1 mM EDTA and 2 mM 2-mercaptoethanol.The cells were lysed by sonication, and the HasA proteins were purified from the supernatant using a His-Tag purification column.Further purification was performed using a Sephadex G-75 gel filtration column (GE Healthcare Japan Corp., Tokyo, Japan) or a Mono Q anion-exchange column (GE Healthcare Japan Corp. Tokyo, Japan) whenever necessary.The wild-type proteins were purified to the heme-loaded holoforms.

ESR Analyses and UV-vis Spectroscopy of Expressed HasApf
ESR analysis of expressed HasA dried on a hotplate was performed using a JES-FA200 ESR spectrometer (Japan Electron Optics Laboratory Co., Ltd., Tokyo, Japan) operated under a magnetic

Scheme 1 .
Scheme 1.The structural representation of hemophore HasA for asymmetric oxidation.

EF
Cycle No. for YP 262445.1 c Full length gene and protein sequence based on a BLAST query sequence analysis act gac tgg tcg gcc tac ttc ggc gac gtc 30.N H R P G E V V D G aac cac cgc cca ggc gaa gtg gtc gac ggc T N T G G F N P G P acc aac acc ggt ggc ttc aac ccg ggc ccg acc gcc agt gac gcg gcc ttc gtc gcc gac G N L H Y T L F S N ggc aac ctg cac tac acc ctg ttc agc aac P S H T L W G S V D ccg agc cac acc ctg tgg ggc tcg gtg gac 90.T I S L G D T L A G act atc tcc ctg ggc gac acc ctc gcc ggt G S G S N Y N L V S ggt tcg ggc agc aac tac aac ctg gtc agc Q E V S F T N L G L cag gaa gtc agc ttc acc aac ctg ggc ctc 120.N S L K E E G R A G aac agc ctg aag gaa gaa ggc cgt gca ggc E V H K V V Y G L M gaa gtg cac aag gtg gtc tac ggc ctg atg S gat gcc ctg ctc aag gcg atc gac cca S L S V N S T F D D agc ctg tcg gtg aac tcc acc ttc gac gac L A A A G V A H V N ctg gcc gct gct ggc gtt gct cac gtc aac 180.P A A A A A A D V G ccg gct gcc gca gcc gct gcc gat gtt ggc
[23]; SDS-PAGE provided no preliminary evidence of iron sulfate participating in the HasApf[25].
−O-O − ) species; (2) the iron of porphyrin/Fe and oxygen coordinates solely in water due to the promotion of oxoferryl (Fe IV =O) species; (3) ESR low-spin signals (g = 2.8, 2.22, and 1.72) disappear for oxygen activation with high-spin signal contamination; and (4) HasA can be used for cyclic deracemization in combination with NaBH4.

Scheme 4 .
Scheme 4. (a) The incorporated iron electron-transfer system of cytochrome P-450 in the presence of oxygen: Fe II + O 2 Ñ Fe III ´O-O ´Ñ Fe IV =O (oxidizing rac-1 or -2) Ñ Fe II + H 2 O.(b) The pathway of heme metabolism: either heme oxygenase enzymes (HO; E.C. 1.14.99.3; heme-hydrogen donor: oxygen oxidoreductase) catalyze the rate-limiting step in heme metabolism or MhuD catalysis does not involve verdoheme, which is the key intermediate of ring cleavage by HO.

Table 1 .
Relation of the amount of bacteria to the kinetic resolutions of rac-2 for each fraction.

Table 1 .
Relation of the amount of bacteria to the kinetic resolutions of rac-2 for each fraction.Process (each fraction) Materials Bacteria (CFU/g) Resolutions Outcomes rac-2/ catalyst

Table 2 .
Results of a BLAST query sequence analysis based on the N-terminal amino-acid sequence identified band from Supernatant 2.

Table 2 .
Results of a BLAST query sequence analysis based on the N-terminal amino-acid sequence identified band from Supernatant 2.

Table 3 .
Application of polyethylene glycol (PEG)-precipitate 2 to the cyclic deracemization in the presence of NaBH4.

Table 3 .
Application of polyethylene glycol (PEG)-precipitate 2 to the cyclic deracemization in the presence of NaBH 4 .