Regiospecificity of a novel bacterial lipoxygenase from Myxococcus xanthus for polyunsaturated fatty acids.

Lipoxygenase (LOX) is the key enzyme involved in the synthesis of oxylipins as signaling compounds that are important for cell growth and development, inflammation, and pathogenesis in various organisms. The regiospecificity of LOX from Myxococcus xanthus, a gram-negative bacterium, was investigated. The enzyme catalyzed oxygenation at the n-9 position in C20 and C22 polyunsaturated fatty acids (PUFAs) to form 12S- and 14S-hydroxy fatty acids (HFAs), respectively, and oxygenation at the n-6 position in C18 PUFAs to form 13-HFAs. The 12S-form products of C20 and C22 PUFAs by M. xanthus LOX is the first report of bacterial LOXs. The residues involved in regiospecificity were determined to be Thr397, Ala461, and Ile664 by analyzing amino acid alignment and a homology model based on human arachidonate 15-LOX with a sequence identity of 25%. Among these variants, the regiospecificity of the T397Y variant for C20 and C22 PUFAs was changed. This may be because of the reduced size of the substrate-binding pocket by substitution of the smaller Thr to the larger Tyr residue. The T397Y variant catalyzed oxygenation at the n-6 position in C20 and C22 PUFAs to form 15- and 17-hydroperoxy fatty acids, respectively. However, the oxygenation position of T397Y for C18 PUFAs was not changed. The discovery of bacterial LOX with novel regiospecificity will facilitate the biosynthesis of regiospecific­oxygenated signaling compounds.

Crystal structures of an atypical aldehyde dehydrogenase having bidirectional oxidizing and reducing activities.

Aldehyde dehydrogenases (ALDHs) are NAD(P)+-dependent oxidoreductases that catalyze the oxidation of a variety of aldehydes to their acid forms. In this study, we determined the crystal structures of ALDH from Bacillus cereus (BcALDH), alone, and in complex with NAD+ and NADP+. This enzyme can oxidize all-trans-retinal to all-trans-retinoic acid using either NAD+ or NADP+ with equal efficiency, and atypically, as a minor activity, can reduce all-trans-retinal to all-trans-retinol using NADPH. BcALDH accommodated the additional 2'-phosphate of NADP+ by expanding the cofactor-binding pocket and upshifting the AMP moiety in NADP+. The nicotinamide moiety in NAD+ and NADP+ had direct interactions with the conserved catalytic residues (Cys300 and Glu266) and caused concerted conformational changes. We superimposed the structure of retinoic acid bound to human ALDH1A3 onto the BcALDH structure and speculated a model of the substrate all-trans-retinal bound to BcALDH. We also proposed a plausible mechanism for the minor reducing activity of BcALDH. These BcALDH structures will be useful in understanding cofactor specificity and the catalytic mechanism of an atypical bacterial BcALDH and should help the development of a new biocatalyst to produce retinoic acid and related high-end products.

Structure-based prediction and identification of 4-epimerization activity of phosphate sugars in class II aldolases.

Sugar 4-epimerization reactions are important for the production of rare sugars and their derivatives, which have various potential industrial applications. For example, the production of tagatose, a functional sweetener, from fructose by sugar 4-epimerization is currently constrained because a fructose 4-epimerase does not exist in nature. We found that class II D-fructose-1,6-bisphosphate aldolase (FbaA) catalyzed the 4-epimerization of D-fructose-6-phosphate (F6P) to D-tagatose-6-phosphate (T6P) based on the prediction via structural comparisons with epimerase and molecular docking and the identification of the condensed products of C3 sugars. In vivo, the 4-epimerization activity of FbaA is normally repressed. This can be explained by our results showing the catalytic efficiency of D-fructose-6-phosphate kinase for F6P phosphorylation was significantly higher than that of FbaA for F6P epimerization. Here, we identified the epimerization reactions and the responsible catalytic residues through observation of the reactions of FbaA and L-rhamnulose-1-phosphate aldolases (RhaD) variants with substituted catalytic residues using different substrates. Moreover, we obtained detailed potential epimerization reaction mechanism of FbaA and a general epimerization mechanism of the class II aldolases L-fuculose-1-phosphate aldolase, RhaD, and FbaA. Thus, class II aldolases can be used as 4-epimerases for the stereo-selective synthesis of valuable carbohydrates.

High-yield production of pure tagatose from fructose by a three-step enzymatic cascade reaction.

OBJECTIVE: To produce tagatose from fructose with a high conversion rate and to establish a high-yield purification method of tagatose from the reaction mixture. RESULTS: Fructose at 1 M (180 g l-1) was converted to 0.8 M (144 g l-1) tagatose by a three-step enzymatic cascade reaction, involving hexokinase, plus ATP, fructose-1,6-biphosphate aldolase, phytase, over 16 h with a productivity of 9 g l-1 h-1. No byproducts were detected. Tagatose was recrystallized from ethanol to a purity of 99.9% and a yield of 96.3%. Overall, tagatose at 99.9% purity was obtained from fructose with a yield of 77%. CONCLUSION: This is the first biotechnological production of tagatose from fructose and the first application of solvent recrystallization for the purification of rare sugars.

Crystallographic snapshots of active site metal shift in E. coli fructose 1,6-bisphosphate aldolase.

Fructose 1,6-bisphosphate aldolase (FBA) is important for both glycolysis and gluconeogenesis in life. Class II (zinc dependent) FBA is an attractive target for the development of antibiotics against protozoa, bacteria, and fungi, and is also widely used to produce various high-value stereoisomers in the chemical and pharmaceutical industry. In this study, the crystal structures of class II Escherichia coli FBA (EcFBA) were determined from four different crystals, with resolutions between 1.8 Å and 2.0 Å. Native EcFBA structures showed two separate sites of Zn1 (interior position) and Zn2 (active site surface position) for Zn2＋ ion. Citrate and TRIS bound EcFBA structures showed Zn2＋ position exclusively at Zn2. Crystallographic snapshots of EcFBA structures with and without ligand binding proposed the rationale of metal shift at the active site, which might be a hidden mechanism to keep the trace metal cofactor Zn2＋ within EcFBA without losing it. [BMB Reports 2016; 49(12): 681-686].

D-Allulose Production from D-Fructose by Permeabilized Recombinant Cells of Corynebacterium glutamicum Cells Expressing D-Allulose 3-Epimerase Flavonifractor plautii.

A d-allulose 3-epimerase from Flavonifractor plautii was cloned and expressed in Escherichia coli and Corynebacterium glutamicum. The maximum activity of the enzyme purified from recombinant E. coli cells was observed at pH 7.0, 65°C, and 1 mM Co2+ with a half-life of 40 min at 65°C, Km of 162 mM, and kcat of 25280 1/s. For increased d-allulose production, recombinant C. glutamicum cells were permeabilized via combined treatments with 20 mg/L penicillin and 10% (v/v) toluene. Under optimized conditions, 10 g/L permeabilized cells produced 235 g/L d-allulose from 750 g/L d-fructose after 40 min, with a conversion rate of 31% (w/w) and volumetric productivity of 353 g/L/h, which were 1.4- and 2.1-fold higher than those obtained for nonpermeabilized cells, respectively.

Alternative Biotransformation of Retinal to Retinoic Acid or Retinol by an Aldehyde Dehydrogenase from Bacillus cereus.

UNLABELLED: A novel bacterial aldehyde dehydrogenase (ALDH) that converts retinal to retinoic acid was first identified in Bacillus cereus The amino acid sequence of ALDH from B. cereus (BcALDH) was more closely related to mammalian ALDHs than to bacterial ALDHs. This enzyme converted not only small aldehydes to carboxylic acids but also the large aldehyde all-trans-retinal to all-trans-retinoic acid with NAD(P)(+) We newly found that BcALDH and human ALDH (ALDH1A1) could reduce all-trans-retinal to all-trans-retinol with NADPH. The catalytic residues in BcALDH were Glu266 and Cys300, and the cofactor-binding residues were Glu194 and Glu457. The E266A and C300A variants showed no oxidation activity. The E194S and E457V variants showed 15- and 7.5-fold higher catalytic efficiency (kcat/Km) for the reduction of all-trans-retinal than the wild-type enzyme, respectively. The wild-type, E194S variant, and E457V variant enzymes with NAD(+) converted 400 µM all-trans-retinal to 210 µM all-trans-retinoic acid at the same amount for 240 min, while with NADPH, they converted 400 µM all-trans-retinal to 20, 90, and 40 µM all-trans-retinol, respectively. These results indicate that BcALDH and its variants are efficient biocatalysts not only in the conversion of retinal to retinoic acid but also in its conversion to retinol with a cofactor switch and that retinol production can be increased by the variant enzymes. Therefore, BcALDH is a novel bacterial enzyme for the alternative production of retinoic acid and retinol. IMPORTANCE: Although mammalian ALDHs have catalyzed the conversion of retinal to retinoic acid with NAD(P)(+) as a cofactor, a bacterial ALDH involved in the conversion is first characterized. The biotransformation of all-trans-retinal to all-trans-retinoic acid by BcALDH and human ALDH was altered to the biotransformation to all-trans-retinol by a cofactor switch using NADPH. Moreover, the production of all-trans-retinal to all-trans-retinol was changed by mutations at positions 194 and 457 in BcALDH. The alternative biotransformation of retinoids was first performed in the present study. These results will contribute to the biotechnological production of retinoids, including retinoic acid and retinol.

Biochemical properties of retinoid-converting enzymes and biotechnological production of retinoids.

Retinoids are a class of compounds that are forms of vitamin A and include retinal, retinol, retinoic acid, and retinyl ester. Retinal is involved in visual cycle, retinol has anti-infective, anticancer, antioxidant, and anti-wrinkle functions, and retinoic acid is used to treat acne and cancer. Retinol, retinoic acid, and retinyl ester are used in cosmetic and pharmaceutical industries. In this article, the biochemical properties and active sites and reaction mechanisms of retinoid-converting enzymes in animals and bacteria, including retinol dehydrogenase, alcohol dehydrogenase, aldo-keto reductase, and aldehyde dehydrogenase, are reviewed. The production of retinoids, using retinoid-producing enzymes and metabolically engineered cells, is also described. Uncharacterized bacterial proteins are suggested as retinoid-converting enzymes, and the production of retinoids using metabolically engineered cells is proposed as a feasible method.

Unveiling of novel regio-selective fatty acid double bond hydratases from Lactobacillus acidophilus involved in the selective oxyfunctionalization of mono- and di-hydroxy fatty acids.

The aim of this study is the first time demonstration of cis-12 regio-selective linoleate double-bond hydratase. Hydroxylation of fatty acids, abundant feedstock in nature, is an emerging alternative route for many petroleum replaceable products thorough hydroxy fatty acids, carboxylic acids, and lactones. However, chemical route for selective hydroxylation is still quite challenging owing to low selectivity and many environmental concerns. Hydroxylation of fatty acids by hydroxy fatty acid forming enzymes is an important route for selective biocatalytic oxyfunctionalization of fatty acids. Therefore, novel fatty acid hydroxylation enzymes should be discovered. The two hydratase genes of Lactobacillus acidophilus were identified by genomic analysis, and the expressed two recombinant hydratases were identified as cis-9 and cis-12 double-bond selective linoleate hydratases by in vitro functional validation, including the identification of products and the determination of regio-selectivity, substrate specificity, and kinetic parameters. The two different linoleate hydratases were the involved enzymes in the 10,13-dihydroxyoctadecanoic acid biosynthesis. Linoleate 13-hydratase (LHT-13) selectively converted 10 mM linoleic acid to 13S-hydroxy-9(Z)-octadecenoic acid with high titer (8.1 mM) and yield (81%). Our study will expand knowledge for microbial fatty acid-hydroxylation enzymes and facilitate the designed production of the regio-selective hydroxy fatty acids for useful chemicals from polyunsaturated fatty acid feedstocks.

An amino acid at position 512 in ß-glucosidase from Clavibacter michiganensis determines the regioselectivity for hydrolyzing gypenoside XVII.

A recombinant ß-glucosidase from Clavibacter michiganensis specifically hydrolyzed the outer and inner glucose linked to the C-3 position in protopanaxadiol (PPD)-type ginsenosides and the C-6 position in protopanaxatriol (PPT)-type ginsenosides except for the hydrolysis of gypenoside LXXV (GypLXXV). The enzyme converted gypenoside XVII (GypXVII) to GypLXXV by hydrolyzing the inner glucose linked to the C-3 position. The substrate-binding residues obtained from the GypXVII-docked homology models of ß-glucosidase from C. michiganensis were replaced with alanine, and the amino acid residue at position 512 was selected because of the changed regioselectivity of W512A. Site-directed mutagenesis for the amino acid residue at position 512 was performed. W512A and W512K hydrolyzed the inner glucose linked to the C-3 position and the outer glucose linked to the C-20 position of GypXVII to produce GypLXXV and F2. W512R hydrolyzed only the outer glucose linked to the C-20 position of GypXVII to produce F2. However, W512E and W512D exhibited no activity for GypXVII. Thus, the amino acid at position 512 is a critical residue to determine the regioselectivity for the hydrolysis of GypXVII. These wild-type and variant enzymes produced diverse ginsenosides, including GypXVII, GypLXXV, F2, and compound K, from ginsenoside Rb1. To the best of our knowledge, this is the first report of the alteration of regioselectivity on ginsenoside hydrolysis by protein engineering.

Characterization of an omega-6 linoleate lipoxygenase from Burkholderia thailandensis and its application in the production of 13-hydroxyoctadecadienoic acid.

A recombinant putative lipoxygenase from Burkholderia thailandensis with a specific activity of 26.4 U mg(-1) was purified using HisTrap affinity chromatography. The native enzyme was a 75-kDa dimer with a molecular mass of 150 kDa. The enzyme activity and catalytic efficiency (k cat/K m) were the highest for linoleic acid (k cat of 93.7 s(-1) and K m of 41.5 µM), followed by arachidonic acid, α-linolenic acid, and γ-linolenic acid. The enzyme was identified as an omega-6 linoleate lipoxygenase (or a linoleate 13S-lipoxygenase) based on genetic and HPLC analyses as well as substrate specificity. The reaction conditions for the enzymatic production of 13-hydroxy-9,11(Z,E)-octadecadienoic acid (13-HODE) were optimal at pH 7.5, 25 °C, 20 g l(-1) linoleic acid, 2.5 g l(-1) enzyme, 0.1 mM Cu(2+), and 6% (v/v) methanol. Under these conditions, linoleate 13-lipoxygenase from B. thailandensis produced 20.8 g l(-1) 13-HODE (70.2 mM) from 20 g l(-1) linoleic acid (71.3 mM) for 120 min, with a molar conversion yield of 98.5% and productivity of 10.4 g l(-1) h(-1). The molar conversion yield and productivity of 13-HODE obtained using B. thailandensis lipoxygenase were 151 and 158% higher, respectively, than those obtained using commercial soybean lipoxygenase under the optimum conditions for each enzyme at the same concentrations of substrate and enzyme.

Characterization of alcohol dehydrogenase from Kangiella koreensis and its application to production of all-trans-retinol.

A recombinant alcohol dehydrogenase (ADH) from Kangiella koreensis was purified as a 40 kDa dimer with a specific activity of 21.3 nmol min(-1) mg(-1), a K m of 1.8 µM, and a k cat of 1.7 min(-1) for all-trans-retinal using NADH as cofactor. The enzyme showed activity for all-trans-retinol using NAD (+) as a cofactor. The reaction conditions for all-trans-retinol production were optimal at pH 6.5 and 60 °C, 2 g enzyme l(-1), and 2,200 mg all-trans-retinal l(-1) in the presence of 5% (v/v) methanol, 1% (w/v) hydroquinone, and 10 mM NADH. Under optimized conditions, the ADH produced 600 mg all-trans-retinol l(-1) after 3 h, with a conversion yield of 27.3% (w/w) and a productivity of 200 mg l(-1) h(-1). This is the first report of the characterization of a bacterial ADH for all-trans-retinal and the biotechnological production of all-trans-retinol using ADH.

Characterization of a F280N variant of L-arabinose isomerase from Geobacillus thermodenitrificans identified as a D-galactose isomerase.

The double-site variant (C450S-N475K) L-arabinose isomerase (L-AI) from Geobacillus thermodenitrificans catalyzes the isomerization of D-galactose to D-tagatose, a functional sweetener. Using a substrate-docking homology model, the residues near to D-galactose O6 were identified as Met186, Phe280, and Ile371. Several variants obtained by site-directed mutagenesis of these three residues were analyzed, and a triple-site (F280N) variant enzyme exhibited the highest activity for D-galactose isomerization. The k cat/K m of the triple-site variant enzyme for D-galactose was 2.1-fold higher than for L-arabinose, whereas the k cat/K m of the double-site variant enzyme for L-arabinose was 43.9-fold higher than for D-galactose. These results suggest that the triple-site variant enzyme is a D-galactose isomerase. The conversion rate of D-galactose to D-tagatose by the triple-site variant enzyme was approximately 3-fold higher than that of the double-site variant enzyme for 30 min. However, the conversion yields of L-arabinose to L-ribulose by the triple-site and double-site variant enzymes were 10.6 and 16.0 % after 20 min, respectively. The triple-site variant enzyme exhibited increased specific activity, turnover number, catalytic efficiency, and conversion rate for D-galactose isomerization compared to the double-site variant enzyme. Therefore, the amino acid at position 280 determines the substrate specificity for D-galactose and L-arabinose, and the triple-site variant enzyme has the potential to produce D-tagatose on an industrial scale.

Molecular characterization of an aldo-keto reductase from Marivirga tractuosa that converts retinal to retinol.

A recombinant aldo-keto reductase (AKR) from Marivirga tractuosa was purified with a specific activity of 0.32unitml(-1) for all-trans-retinal with a 72kDa dimer. The enzyme had substrate specificity for aldehydes but not for alcohols, carbonyls, or monosaccharides. The enzyme turnover was the highest for benzaldehyde (kcat=446min(-1)), whereas the affinity and catalytic efficiency were the highest for all-trans-retinal (Km=48µM, kcat/Km=427mM(-1)min(-1)) among the tested substrates. The optimal reaction conditions for the production of all-trans-retinol from all-trans-retinal by M. tractuosa AKR were pH 7.5, 30°C, 5% (v/v) methanol, 1% (w/v) hydroquinone, 10mM NADPH, 1710mgl(-1) all-trans-retinal, and 3unitml(-1) enzyme. Under these optimized conditions, the enzyme produced 1090mgml(-1) all-trans-retinol, with a conversion yield of 64% (w/w) and a volumetric productivity of 818mgl(-1)h(-1). AKR from M. tractuosa showed no activity for all-trans-retinol using NADP(+) as a cofactor, whereas human AKR exhibited activity. When the cofactor-binding residues (Ala158, Lys212, and Gln270) of M. tractuosa AKR were changed to the corresponding residues of human AKR (Ser160, Pro212, and Glu272), the A158S and Q270E variants exhibited activity for all-trans-retinol. Thus, amino acids at positions 158 and 270 of M. tractuosa AKR are determinant residues of the activity for all-trans-retinol.

Expression, crystallization and preliminary X-ray crystallographic analysis of cellobiose 2-epimerase from Dictyoglomus turgidum DSM 6724.

Cellobiose 2-epimerase epimerizes and isomerizes ß-1,4- and α-1,4-gluco-oligosaccharides. N-Acyl-D-glucosamine 2-epimerase (DT_epimerase) from Dictyoglomus turgidum has an unusually high catalytic activity towards its substrate cellobiose. DT_epimerase was expressed, purified and crystallized. Crystals were obtained of both His-tagged DT_epimerase and untagged DT_epimerase. The crystals of His-tagged DT_epimerase diffracted to 2.6 Šresolution and belonged to the monoclinic space group P21, with unit-cell parameters a=63.9, b=85.1, c=79.8 Å, ß=110.8°. With a Matthews coefficient VM of 2.18 Å3 Da(-1), two protomers were expected to be present in the asymmetric unit with a solvent content of 43.74%. The crystals of untagged DT_epimerase diffracted to 1.85 Šresolution and belonged to the orthorhombic space group P212121, with unit-cell parameters a=55.9, b=80.0, c=93.7 Å. One protomer in the asymmetric unit was expected, with a corresponding VM of 2.26 Å3 Da(-1) and a solvent content of 45.6%.

Expression, crystallization and preliminary X-ray crystallographic analysis of alcohol dehydrogenase (ADH) from Kangiella koreensis.

Alcohol dehydrogenases (ADHs) are a group of dehydrogenase enzymes that facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD(+) to NADH. In bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD(+). The adh gene from Kangiella koreensis was cloned and the protein (KkADH) was expressed, purified and crystallized. A KkADH crystal diffracted to 2.5 Šresolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 94.1, b = 80.9, c = 115.6 Å, ß = 111.9°. Four monomers were present in the asymmetric unit, with a corresponding VM of 2.55 Å(3) Da(-1) and a solvent content of 51.8%.

Expression, crystallization and preliminary X-ray crystallographic analysis of aldehyde dehydrogenase (ALDH) from Bacillus cereus.

Aldehyde dehydrogenase (ALDH) catalyses the oxidation of aldehydes using NAD(P)(+) as a cofactor. Most aldehydes are toxic at low levels. ALDHs are used to regulate metabolic intermediate aldehydes. The aldh gene from Bacillus cereus was cloned and the ALDH protein was expressed, purified and crystallized. A crystal of the ALDH protein diffracted to 2.6 Šresolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 83.5, b = 93.3, c = 145.5 Å, ß = 98.05°. Four protomers were present in the asymmetric unit, with a corresponding VM of 2.55 Å(3) Da(-1) and a solvent content of 51.8%.

Molecular characterization of a thermostable L-fucose isomerase from Dictyoglomus turgidum that isomerizes L-fucose and D-arabinose.

A recombinant thermostable l-fucose isomerase from Dictyoglomus turgidum was purified with a specific activity of 93 U/mg by heat treatment and His-trap affinity chromatography. The native enzyme existed as a 410 kDa hexamer. The maximum activity for l-fucose isomerization was observed at pH 7.0 and 80 °C with a half-life of 5 h in the presence of 1 mM Mn(2+) that was present one molecular per monomer. The isomerization activity of the enzyme with aldose substrates was highest for l-fucose (with a k(cat) of 15,500 min(-1) and a K(m) of 72 mM), followed by d-arabinose, d-altrose, and l-galactose. The 15 putative active-site residues within 5 Å of the substrate l-fucose in the homology model were individually replaced with other amino acids. The analysis of metal-binding capacities of these alanine-substituted variants revealed that Glu349, Asp373, and His539 were metal-binding residues, and His539 was the most influential residue for metal binding. The activities of all variants at 349 and 373 positions except for a dramatically decreased k(cat) of D373A were completely abolished, suggesting that Glu349 and Asp373 were catalytic residues. Alanine substitutions at Val131, Met197, Ile199, Gln314, Ser405, Tyr451, and Asn538 resulted in substantial increases in K(m), suggesting that these amino acids are substrate-binding residues. Alanine substitutions at Arg30, Trp102, Asn404, Phe452, and Trp510 resulted in decreases in k(cat), but had little effect on K(m).

Characterization of a recombinant thermostable D-lyxose isomerase from Dictyoglomus turgidum that produces D-lyxose from D-xylulose.

A putative D-lyxose isomerase from Dictyoglomus turgidum was purified with a specific activity of 19 U/mg for D-lyxose isomerization by heat treatment and affinity chromatography. The native enzyme was estimated as a 42 kDa dimer by gel-filtration chromatography. The activity of the enzyme was highest for D-lyxose, suggesting that it is a D-lyxose isomerase. The maximum activity of the enzyme was at pH 7.5 and 75°C in the presence of 0.5 mM Co(2+), with a half-life of 108 min, K(m) of 39 mM, and k(cat) of 3,570 1/min. The enzyme is the most thermostable D-lyxose isomerase among those characterized to date. It converted 500 g D-xylulose/l to 380 g D-lyxose/l after 2 h. This is the highest concentration and productivity of D-lyxose reported thus far.

Quercetin production from rutin by a thermostable ß-rutinosidase from Pyrococcus furiosus.

Pyrococcus furiosus ß-glucosidase converted rutin to quercetin and rutinose disaccharide with a ratio of 1:1, with no glucose, L-rhamnose, and isoquercitrin, indicating that the enzyme is a ß-rutinosidase. The specific activity for flavonoid glycosides followed the order of isoquercitrin > quercitrin > rutin. The conversion of rutin to quercetin was optimal at pH 5.0 and 95°C in the presence of 0.5% dimethyl sulfoxide with a half-life of 101 h, a k(cat) of 1.6 min(-1), and a K(m) of 0.3 mM. Under the improved conditions, the enzyme produced 6.5 mM quercetin from 10 mM rutin after 150 min, with a molar yield of 65% and a productivity of 2.6 mM/h. This productivity is the highest reported thus far among enzymatic transformations.