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Platycodon grandiflorum (balloon flower) root (Platycodi radix, PR) is used as a health supplement owing to its beneficial bioactive properties. In the present study, the anti-inflammatory, antioxidant, and whitening effects of deglycosylated platycosides (saponins) from PR biotransformed by pectinase from Aspergillus aculeatus were investigated. The bioactivities of the platycosides improved when the number of sugar moieties attached to the aglycone platycosides was decreased. The deglycosylated saponins exhibited higher lipoxygenase inhibitory activities (anti-inflammatory activities) than the precursor platycosides and the anti-inflammatory compound baicalein. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the pectinase-treated PR extract was higher than that of the non-treated PR extract. The trolox-equivalent antioxidant capacity (TEAC) assay showed improved values as the saponins were hydrolyzed. The tyrosinase inhibitory activities (whitening effects) of deglycosylated platycosides were higher than those of the precursor platycosides. Furthermore, 3-O-β-D-glucopyranosyl platycosides showed higher anti-inflammatory, antioxidant, and whitening activities than their precursor glycosylated platycosides. Therefore, 3-O-β-D-glucopyranosyl platycosides may improve the beneficial effects of nutritional supplements and cosmetic products.

Compound K (C-K) is one of the most pharmaceutically effective ginsenosides, but it is absent in natural ginseng. However, C-K can be obtained through the hydrolysis of protopanaxadiol-type ginsenosides (PPDGs) in natural ginseng. The aim of this study was to obtain the high concentration of food-available C-K using PPDGs in Korean ginseng extract by an extracellular enzyme from Aspergillus niger KACC 46495. A. niger was cultivated in the culture medium containing the inducer carboxymethyl cellulose (CMC) for 6 days. The extracellular enzyme extracted from A. niger was prepared from the culture broth by filtration, ammonium sulfate, and dialysis. The extracellular enzyme was used for C-K production using PPDGs. The glycoside-hydrolyzing pathways for converting PPDGs into C-K by the extracellular enzyme were Rb1 → Rd → F2 → C-K, Rb2 → Rd or compound O → F2 or compound Y → C-K, and Rc → Rd or compound Mc1 → F2 or compound Mc → C-K. The extracellular enzyme from A. niger at 8.0 mg/ml, which was obtained by the induction of CMC during the cultivation, converted 6.0 mg/ml (5.6 mM) PPDGs in Korean ginseng extract into 2.8 mg/ml (4.5 mM) food-available C-K in 9 h, with a productivity of 313 mg/l/h and a molar conversion of 80%. To the best of our knowledge, the productivity and concentration of C-K of the extracellular enzyme are the highest among those by crude enzymes from wild-type microorganisms.

d-Tagatose, a low-calorie functional sweetener, is biotransformed from lactose via galactose. The discovery of an enzyme with 4-epimerization activity toward d-fructose to produce d-tagatose has be...

Platycosides, saponins contained in balloon flower, which have been used as food health supplements for respiratory diseases, have diverse pharmacological effects. Platycosides exhibit better pharmacological activity by hydrolyzing their own sugars. However, to date, there have been no studies on the production of deglucosylated platycodin D suitable for food applications. In this study, Pluszyme 2000P, which was derived from Aspergillus niger, a food-grade microorganism, was used to completely convert platycoside E into deglucosylated platycodin D. For an efficient and economical production of deglucosylated platycodin D, the productivity was improved approximately 2.4 times by application of high hydrostatic pressure and the discarded balloon flower leaf was used as a substrate. As a result, deglucosylated platycodin D was produced with the highest concentration (3.49 mg/mL) and productivity (581.7 mg/L/h) reported so far. Our results contribute to functional saponin production and the related food industries., Platycoside; Pluszyme 2000P; Food additive; Aspergillus niger; Balloon flower; Medicinal herb.

Rare sugars are regarded as functional biological materials due to their potential applications as low-calorie sweeteners, antioxidants, nucleoside analogs, and immunosuppressants. D-Allose is a rare sugar that has attracted substantial attention in recent years, owing to its pharmaceutical activities, but it is still not widely available. To address this limitation, we continuously produced D-allose from D-allulose using a packed bed reactor with commercial glucose isomerase (Sweetzyme IT). The optimal conditions for D-allose production were determined to be pH 8.0 and 60°C, with 500 g/L D-allulose as a substrate at a dilution rate of 0.24/h. Using these optimum conditions, the commercial glucose isomerase produced an average of 150 g/L D-allose over 20 days, with a productivity of 36 g/L/h and a conversion yield of 30%. This is the first report of the successful continuous production of D-allose from D-allulose by commercial glucose isomerase using a packed bed reactor, which can potentially provide a continuous production system for industrial applications of D-allose.

To quantitatively hydroxylate 8S- and 10S-positions on polyunsaturated fatty acids by recombinant Escherichia coli cells expressing mouse arachidonate 8S-lipoxygenase (8S-LOX). Hydroxylated products gained from the conversion of arachidonic acid (20:4Δ5Z,8Z,11Z,14Z, AA), eicosapentanoic acid (20:5Δ5Z,8Z,11Z,14Z,17Z, EPA), and (22:6Δ4Z,7Z,10Z,13Z,16Z,19Z, DHA) by recombinant E. coli cells containing 8S-LOX from mouse were identified as 8S-hydroxy-5,9,11,14(Z,E,Z,Z)-eicosatetranoic acid (8S-HETE), 8S-hydroxy-5,9,11,14,17(Z,E,Z,Z,Z)-eicosapentanoic acid (8S-HEPE), and 10S-hydroxy-4,8,12,14,16,19(Z,E,Z,Z,Z,Z)-docosahexaenoic acid (10S-HDoHE), respectively. Under the optimal hydroxylation conditions of pH 7.5, 30 °C, 5% (v/v) ethanol, 15 g cells l−1, and 5 mM substrate, AA, EPA, and DHA were hydroxylated into 4.37 mM 8S-HETE, 3.77 mM 8S-HEPE, and 3.13 mM 10S-HDoHE for 60, 90, and 60 min, with 87, 75, and 63% molar conversions, respectively. To the best of our knowledge, this is the first quantitatively biotechnological production of 8S-HETE, 8S-HEPE, and 10S-HDoHE.

This study was conducted to characterize recombinant α-l-rhamnosidase from Chloroflexus aurantiacus and apply the enzyme in the production of isoquercitrin from rutin. The α-l-rhamnosidase from C. aurantiacus was cloned and expressed in Escherichia coli BL21 and purified as a soluble enzyme. α-l-rhamnosidase purified from C. aurantiacus has a molecular mass of approximately 105 kDa and is predicted to exist as a homodimer with a native enzyme of 200 kDa. The purified enzyme exhibited the highest specific activity for rutin among the reported isoquercitrin producing α-l-rhamnosidases and was applied in the production of isoquercitrin from rutin. Under the optimised conditions of pH 6.0, 50 °C, 0.6 U mL−1 α-l-rhamnosidase, and 30 mM rutin, α-l-rhamnosidase from C. aurantiacus produced 30 mM isoquercitrin after 2 h with a 100% conversion yield and productivity of 15 mM h−1. We achieved a high productivity of isoquercitrin from rutin. Moreover, these results suggest that α-l-rhamnosidase from C. aurantiacus is an effective isoquercitrin producer.

The Platycodon grandiflorum root, Platycodi radix, a common vegetable, and its extract with glycosylated saponins, platycosides, have been used as food items and food health supplements for pulmonary diseases and respiratory disorders. Enzymes convert glycosylated saponins into deglycosylated saponins, which exhibit higher biological activity than glycosylated saponins. In this study, β-glucosidase from the hyperthermophilic bacterium Dictyoglomus turgidum converted platycosides in the Platycodi radix extract into deglucosylated platycosides. In addition, the enzyme completely converted platycoside E (PE), platycodin D3 (PD3), and platycodin D (PD) in Platycodi radix extract into deglucosylated platycodin D (deglu PD), which was first identified by nuclear magnetic resonance. The anti-inflammatory activities of deglu PD and deglucosylated Platycodi radix extract were higher than those of PE, PD3, PD, Platycodi radix extract, and baicalein, an anti-inflammatory agent. Therefore, deglucosylated Platycodi radix extract is expected to be used as improved functional food supplements.

Extract from balloon flower root (Platycodi radix) containing platycosides as saponins is a beneficial food additive and is used for their savory taste and the alleviation of respiratory diseases. Deglycosylated platycosides show greater pharmacological effects than glycosylated platycosides. However, there are no reports on the conversion of glycosylated platycosides into deapiosylated platycosides. In this study, we showed that the crude enzyme from Rhizopus oryzae, a generally recognized as safe (GRAS) fungus isolated from meju (fermented soybean brick), completely converted glycosylated platycosides in Platycodi radix extract into deapiosylated platycosides: deapiosylated platycodin D (deapi-PD), deapiosylated platycodin A (deapi-PA), deapiosylated polygalacin D (deapi-PGD), and deapiosylated platyconic acid A (deapi-PCA). Among these, deapi-PA and deapi-PCA were first identified using liquid chromatography/mass spectrometry. The anti-inflammatory and antioxidant effects of deapiosylated platycosides were greater than those of the precursor glycosylated platycosides. These deapiosylated platycosides could improve the properties of functional food additives.

Platycosides, saponins contained in balloon flower, which have been used as food health supplements for respiratory diseases, have diverse pharmacological effects. Platycosides exhibit better pharmacological activity by hydrolyzing their own sugars. However, to date, there have been no studies on the production of deglucosylated platycodin D suitable for food applications. In this study, Pluszyme 2000P, which was derived from Aspergillus niger, a food-grade microorganism, was used to completely convert platycoside E into deglucosylated platycodin D. For an efficient and economical production of deglucosylated platycodin D, the productivity was improved by application of high hydrostatic pressure and the discarded balloon flower leaf was used as a substrate. As a result, deglucosylated platycodin D was produced with the highest concentration and productivity reported so far. Our results contribute to functional saponin production and the related food industries.

D-Allose is a potential medical sugar because it has anticancer, antihypertensive, anti-inflammatory, antioxidative, and immunosuppressant activities. Allose production from fructose as a cheap substrate was performed by a one-pot reaction using Flavonifractor plautiiD-allulose 3-epimerase (FP-DAE) and Clostridium thermocellum ribose 5-phosphate isomerase (CT-RPI). The optimal reaction conditions for allose production were pH 7.5, 60°C, 0.1 g/l FP-DAE, 12 g/l CT-RPI, and 600 g/l fructose in the presence of 1 mM Co2+. Under these optimized conditions, FP-DAE and CT-RPI produced 79 g/l allose for 2 h, with a conversion yield of 13%. This is the first biotransformation of fructose to allose by a two-enzyme system. The production of allose by a one-pot reaction using FP-DAE and CT-RPI was 1.3-fold higher than that by a two-step reaction using the two enzymes.

Ginsenoside compound K is an essential ingredient in nutritional supplements, cosmetics, and traditional medicines. However, cultivation for the production of enzymes involved in ginsenoside biotransformation has not been attempted in a fermenter. The host strain Escherichia coli ER2566 and the constitutive pHCE vector were selected for the efficient production of β-D-glycosidase, and expression medium composition to produce Sulfolobus solfataricus β-glycosidase expressed in E. coli was optimized in flask and batch cultures. The total activity of β-Dglycosidase in fed-batch culture using a fermenter increased 14-fold before optimization. S. solfataricus β-D-glycosidase and Thermotoga petrophila α-L-arabinofuranosidase were produced in a fed-batch culture. These two enzymes completely converted protopanaxadiol-type ginsenosides in ginseng leaf extract obtained from discarded ginseng leaves as a renewable substrate to compound K. The effective bioprocess for compound K production developed here will contribute to the industrial biological production of compound K.

Aglycon protopanaxatriol (APPT) has valuable pharmacological effects such as memory enhancement and tumor inhibition. β-Glycosidase from the hyperthermophilic bacterium Dictyoglomus turgidum (DT-bgl) hydrolyzes the glucose residues linked to APPT, but not other glycoside residues. β-Glycosidase from the hyperthermophilic bacterium Pyrococcus furiosus (PF-bgl) hydrolyzes the outer sugar at C-6 but not the inner glucose at C-6 or the glucose at C-20. Thus, the combined use of DT-bgl and PF-bgl is expected to increase the biotransformation of PPT-type ginsenosides to APPT. We optimized the ratio of PF-bgl to DT-bgl, the concentrations of substrate and enzyme, and the reaction time to increase the biotransformation of ginsenoside Re and PPT-type ginsenosides in Panax ginseng leaf extract to APPT. DT-bgl combined with PF-bgl converted 1.0 mg/ml PPT-type ginsenosides in ginseng leaf extract to 0.58 mg/ml APPT without other ginsenosides, with a molar conversion of 100%. We achieved the complete biotransformation of ginsenoside Re and PPT-type ginsenosides in ginseng leaf extract to APPT by the combined use of two β-glycosidases, suggesting that discarded ginseng leaves can be used as a source of the valuable ginsenoside APPT. To the best of our knowledge, this is the first quantitative production of APPT using ginsenoside Re, and we report the highest concentration and productivity of APPT from ginseng extract to date.

The ginsenoside 20- O-β-glucopyranosyl-20( S)-protopanaxadiol, compound K, has attracted much attention in functional food, traditional medicine, and cosmetic industries because of diverse pharmaceutical activities. The effective production of compound K from ginseng extracts has been required. However, an enzyme capable of completely converting all protopanaxadiol (PPD)-type ginsenosides to compound K has not been reported until now. In this study, unlike other enzymes, β-glucosidase from Caldicellulosiruptor bescii was able to hydrolyze sugar moieties such as l-arabinofuranose as well as d-glucose and l-arabinopyranose as the C-20 outer sugar in ginsenosides. Thus, ginsenoside Rc containing l-arabinofuranose can be converted to compound K by only this enzyme. Under the optimized reaction conditions, the enzyme completely converted PPD-type ginsenosides in ginseng extracts to compound K with the highest productivity among the reported results. This is the first report of the enzyme capable of completely converting all PPD-type ginsenosides into compound K.

Platycosides are the functional saponins present in balloon flowers that exert diverse biological effects, and which can be further improved by their deglycosylation. Deapiose-xylosylated platycodin D, which is absent in balloon flowers, can be generated only by cytolase PCL5 by acting on platycoside E. To improve cytolase PCL5-catalyzed production of deapiose-xylosylated platycodin D from platycoside E, we explored the use of high hydrostatic pressure (HHP). At an HHP of 150 MPa, the optimal temperature of cytolase PCL5 activity for converting platycoside E into deapiose-xylosylated platycodin D shifted from 50 to 55 °C, and increased the activity and stability of the enzyme by 5- and 4.9-fold, respectively. Under HHP, the enzyme completely converted 1 mM platycoside E into deapiose-xylosylated platycodin D within 4 h, with a 3.75-fold higher productivity than that under atmospheric pressure. Our results suggest that the application of HHP is a potential method for the economical production of platycosides and enzyme-catalyzed biotransformation of functional saponins.

20(S)-Protopanaxadiol (APPD) has potential uses in the pharmaceutical, cosmetic, and food industries because of its anti-stress, anti-fatigue, anti-cancer, anti-inflammatory, and anti-wrinkle properties. However, APPD production is difficult because β-glycosidases that convert the protopanaxadiol (PPD)-type ginsenoside compound K to APPD are rare. β-Glycosidase from Dictyoglomus turgidum (DT-bgl) has the highest specific activity for converting compound K to APPD, but exhibits no activity towards the α-l-arabinopyranoside moiety in compound Y. Therefore, β-glycosidase from Caldicellulosiruptor bescii (CB-bgl), which has a strong α-l-arabinopyranosidase activity, was used along with DT-bgl. The volumetric and specific productivities of the two-enzyme system for APPD using ginseng root extract were 38.4- and 38.7-fold higher, respectively, than those of β-glycosidase from Pyrococcus furiosus, which had the highest volumetric productivity previously reported, at the same enzyme and substrate concentrations. Thus, DT-bgl combined with CB-bgl completely converted PPD-type ginsenosides to APPD with the highest volumetric and specific productivities reported thus far. Electronic supplementary material The online version of this article (10.1186/s13568-017-0524-9) contains supplementary material, which is available to authorized users.

Activation of L-type voltage-dependent Ca2+ channels (VDCCL) by membrane stretch contributes to many biological responses such as myogenic contraction of arteries. However, mechanism for the stretch-induced VDCCL activation is unclear. In this study, we examined the hypothesis that caveolar remodeling and its related signaling cascade contribute to the stretch-induced activation of VDCCL in rat mesenteric arterial smooth muscle cells. The VDCCL currents were recorded with nystatin-perforated or with conventional whole-cell patch-clamp technique. Hypotonic (~230 mOsm) swelling-induced membrane stretch reversibly increased the VDCCL currents. Electron microscope and confocal imaging analysis revealed that both hypotonic swelling and cholesterol depletion by methyl-β-cychlodextrin (MβCD) similarly disrupted the caveolae structure and translocated caveolin-1 (Cav-1) from membrane to cytosolic space. Accordingly, MβCD also increased VDCCL currents. Moreover, subsequent hypotonic swelling after MβCD treatment failed to increase the VDCCL currents further. Western blotting experiments revealed that hypotonic swelling phosphorylated Cav-1 and JNK. Inhibitors of tyrosine kinases (genistein) and JNK (SP00125) prevented the swelling-induced facilitation of VDCCL currents. Knockdown of Cav-1 by small interfering RNA blocked both the VDCCL current facilitation by stretch and the related phosphorylation of JNK. Taken together, the results suggest that membrane stretch is transduced to the facilitation of VDCCL currents via caveolar structure-dependent tyrosine phosphorylation of Cav-1 and subsequent activation of JNK in rat mesenteric arterial myocytes.

The generally recognized as safe microorganism Corynebacterium glutamicum expressing Geobacillus thermodenitrificans d-galactose isomerase (d-GaI) was an efficient host for the production of d-tagatose, a functional sweetener. The d-tagatose production at 500 g/L d-galactose by the host was 1.4-fold higher than that by Escherichia coli expressing d-GaI. The d-tagatose-producing activity of permeabilized C. glutamicum (PCG) cells treated with 1% (w/v) Triton X-100 was 2.1-fold higher than that of untreated cells. Permeabilized and immobilized C. glutamicum (PICG) cells in 3% (w/v) alginate showed a 3.1-fold longer half-life at 50 °C and 3.1-fold higher total d-tagatose concentration in repeated batch reactions than PCG cells. PICG cells, which produced 165 g/L d-tagatose after 3 h, with a conversion of 55% (w/w) and a productivity of 55 g/L/h, showed significantly higher d-tagatose productivity than that reported for other cells. Thus, d-tagatose production by PICG cells may be an economical process to produce food-grade d-tagatose.

To increase the production of 10S-hydroxy-8(E)-octadecenoic acid from oleic acid by whole recombinant Escherichia coli cells expressing Nostoc punctiforme 10S-dioxygenase with the aid of a chaperone. The optimal conditions for 10S-hydroxy-8(E)-octadecenoic acid production by recombinant cells co-expressing chaperone plasmid were pH 9, 35 °C, 15 % (v/v) dimethyl sulfoxide, 40 g cells l−1, and 10 g oleic acid l−1. Under these conditions, recombinant cells co-expressing chaperone plasmid produced 7.2 g 10S-hydroxy-8(E)-octadecenoic acid l−1 within 30 min, with a conversion yield of 72 % (w/w) and a volumetric productivity of 14.4 g l−1 h−1. The activity of recombinant cells expressing 10S-dioxygenase was increased by 200 % with the aid of a chaperone, demonstrating the first biotechnological production of 10S-hydroxy-8(E)-octadecenoic acid using recombinant cells expressing 10S-dioxygenase.

Hydroxy fatty acids are used as precursors of lactones and dicarboxylic acids, as starting materials of polymers, and as additives in coatings and paintings. Stenotrophomonas nitritireducens efficiently converts cis-9 polyunsaturated fatty acids (PUFAs) to 10-hydroxy fatty acids. However, gene encoding enzyme involved in this conversion has not been identified to date. We purified a putative fatty acid double-bond hydratase from S. nitritireducens by ultrafiltration and HiPrep DEAE FF and Resource Q ion exchange chromatographies. Peptide sequences of the purified enzyme were obtained by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Sequence of the partial gene encoding this putative fatty acid double-bond hydratase was determined by degenerate polymerase chain reaction (PCR) based on the peptide sequences. The remaining gene sequence was identified by rapid amplification of cDNA ends using cDNA of S. nitritireducens as a template, and the full-length gene was cloned subsequently. The expressed enzyme was identified as an oleate hydratase by determining its kinetic parameters toward unsaturated fatty acids. S. nitritireducens oleate hydratase showed higher activity toward PUFAs compared with other available oleate hydratases. This suggested that the enzyme could be used effectively to convert plant oils to 10-hydroxy fatty acids because these oils contained unsaturated fatty acids such as oleic acid (OA) and linoleic acid (LA) and PUFAs such as α-linolenic acid and/or γ-linolenic acid. The enzyme converted soybean oil and perilla seed oil hydrolyzates containing 10 mM total unsaturated fatty acids, including OA, LA, and ALA, to 8.87 and 8.70 mM total 10-hydroxy fatty acids, respectively, in 240 min. To our knowledge, this is the first study on the biotechnological conversion of PUFA-containing oils to hydroxy fatty acids. Biotechnol. Bioeng. 2017;114: 74-82. © 2016 Wiley Periodicals, Inc.

The first and second preferred substrates of recombinant Escherichia coli cells expressing 10R-dioxygenase (PpoC) from Aspergillus nidulans and the purified enzyme were linoleic acid and α-linolenic acid, respectively. PpoC in cells showed higher thermal and reaction stabilities compared to purified PpoC. Thus, 10R-hydroxy unsaturated fatty acids were produced from linoleic acid, α-linolenic acid, and hempseed oil hydrolyzate containing linoleic acid and α-linolenic acid as substrates by whole recombinant cells expressing PpoC. The optimal reaction conditions for the production of 10R-hydroxy-8E,12Z-octadecadienoic acid (10R-HODE) were pH 8.0, 30 °C, 250 rpm, 5 % (v/v) dimethyl sulfoxide, 5 g l(-1) linoleic acid, and 60 g l(-1) cells in 100-ml baffled flask. Under these conditions, whole recombinant cells expressing PpoC produced 2.7 g l(-1) 10R-HODE from 5 g l(-1) linoleic acid for 40 min, with a conversion yield of 54 % (w/w) and a productivity of 4.0 g l(-1) h(-1); produced 2.2 g l(-1) 10R-hydroxy-8E,12Z,15Z-octadecatrienoic acid (10R-HOTrE) from 3 g l(-1) α-linolenic acid for 30 min, with a conversion yield of 72 % (w/w) and a productivity of 4.3 g l(-1) h(-1); and produced 1.8 g l(-1) 10R-HODE and 0.5 g l(-1) 10R-HOTrE from 5 g l(-1) hempseed oil hydrolyzate containing 2.5 g l(-1) linoleic acid and 1.0 g l(-1) α-linolenic acid for 30 min, with a conversion yield of 74 and 51 % (w/w), respectively, and a productivity of 3.6 and 1.0 g l(-1) h(-1), respectively. To the best of our knowledge, this is the first report on the biotechnological production of 10R-hydroxy unsaturated fatty acids.

Recombinant Escherichia coli cells expressing linoleate 13-hydratase from Lactobacillus acidophilus were permeabilized by treating with 0.2 M NaCl. The optimal conditions for the production of 13-hydroxy-9,15(Z,Z)-octadecadienoic acid (13-HODE) from α-linolenic acid by permeabilized recombinant cells were pH 6.0, 40 °C, 7.5 % (v/v) methanol, 60 g/l permeabilized cells, and 15 g/l α-linolenic acid. Under these conditions, permeabilized cells produced 7.5 g/l 13-HODE after 6 h, with a conversion yield of 50 % (w/w) and a volumetric productivity of 1.25 g/l/h. These values were 161 and 160 % of those obtained by nonpermeabilized cells, respectively. To the best of our knowledge, this is the first report on the process optimization for the biotechnological production of 13-HODE.

The specific activity of recombinant Escherichia coli cells expressing the double-site variant (I33L-S213C) d-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was highest at 24 h of cultivation time in Terrific Broth (TB) medium among the media tested. The contents of crude protein and DPEase in recombinant cells at 24 h were 37.0 and 8.6% (w/w), respectively, indicating that the enzyme was highly expressed. The reaction conditions for the production of d-psicose from d-fructose by whole recombinant cells with the highest specific activity were optimal at 60°C, pH 8.5, 4 g/l cells, and 700 g/l d-fructose. Under these conditions, whole recombinant cells produced 230 g/l d-psicose after 40 min, with a conversion yield of 33% (w/w), a volumetric productivity of 345 g/l/h, and a specific productivity of 86.2 g/g/h. These are the highest conversion yield and volumetric and specific productivities of d-psicose from d-fructose by cells reported thus far.

To identify novel fatty acid diol synthases, putative candidate sequences from Penicillium species were analyzed, and hydroxy fatty acid production by crude Penicillium enzyme extracts was assessed. Penicillium chrysogenum was found to produce an unknown dihydroxy fatty acid, a candidate gene implicated in this production was cloned and expressed, and the expressed enzyme was purified. The product obtained by the reaction of the purified enzyme with linoleic acid was identified as 8R,11S-dihydroxy-9,12(Z,Z)-octadecadienoic acid (8R,11S-DiHODE). The catalytic efficiency of this enzyme toward linoleic acid was the highest among the unsaturated fatty acids tested, indicating that this enzyme was a novel 8R,11S-linoleate diol synthase (8R,11S-LDS). A sexual stage in the life cycle of P. chrysogenum has recently been discovered, and 8R,11S-DiHODE produced by 8R,11S-LDS may constitute a precocious sexual inducer factor, responsible for regulating the sexual and asexual cycles of this fungus.

Platycosides, the saponins abundant in Platycodi radix (the root of Platycodon grandiflorum), have diverse pharmacological activities and have been used as food supplements. Since deglycosylated saponins exhibit higher biological activity than glycosylated saponins, efforts are on to enzymatically convert glycosylated platycosides to deglycosylated platycosides, however, the lack of diversity and specificities of these enzymes has limited the kinds of platycosides that can be deglycosylated. In the present study, we examined the enzymatic conversion of platycosides and showed that Cytolase PCL5 completely converted platycoside E and polygalacin D3 into deapiose-xylosylated platycodin D and deapiose-xylosylated polygalacin D, respectively, which were identified by LC-MS analysis. The platycoside substrates were hydrolyzed through the following novel hydrolytic pathways: platycoside E &rarr, platycodin D3 &rarr, platycodin D &rarr, deapiosylated platycodin D &rarr, deapiose-xylosylated platycodin D, and polygalacin D3 &rarr, polygalacin D &rarr, deapiosylated polygalacin D &rarr, deapiose-xylosylated polygalacin D. Our results show that cytolast PCL5 may have a potential role in the development of biologically active platycosides that may be used for their diverse pharmacological activities.

Diol synthase-derived metabolites are involved in the sexual and asexual life cycles of fungi. A putative diol synthase from Penicillium oxalicum was found to convert palmitoleic acid (16:1n-7), oleic acid (18:1n-9), linoleic acid (18:2n-6), and α-linolenic acid (18:3n-3) to 6S,8R-dihydroxy-9(Z)-hexadecenoic acid, 6R,8R-dihydroxy-9(Z)-octadecenoic acid, 6R,8R-dihydroxy-9,12(Z,Z)-octadecadienoic acid, and 6S,8R-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid, respectively, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy analyses. The specific activity and catalytic efficiency of P. oxalicum 6,8-diol synthase were the highest for 18:2n-6, indicating that the enzyme is a 6R,8R-linoleate diol synthase (6R,8R-LDS) with new regiospecificity. This is the first report of a 6R,8R-LDS. LDS is a fusion protein consisting of a dioxygenase domain at the N-terminus and a cytochrome P450/hydroperoxide isomerase (P450/HPI) domain at the C-terminus. The putative active-site residues in the C-terminal domain of P. oxalicum 6R,8R-LDS were proposed based on a substrate-docking homology model. The results of the site-directed mutagenesis within C-terminal P450 domain suggested that Asn886, Arg707, and Arg934, are catalytic importance and belong to the catalytic groove. Phe794 and Gln889 were found to be involved in the regiospecific rearrangement of hydroperoxide, while the F794E and Q889A variants of P. oxalicum 6,8-LDS acted as 7,8- and 8,11-LDSs, respectively. All these mutations critically affected the HPI activity of P. oxalicum 6R,8R-LDS.

Aglycon protopanaxatriol (APPT) has valuable pharmacological effects such as anti-inflammatory and anti-stress activities. However, the complete conversion of all typical glycosylated protopanaxatriol ginsenosides to APPT has not been achieved to date. β-Glycosidase from the hyperthermophilic bacterium Dictyoglomus turgidum (DT-bgl) hydrolyzes the glucose residues at C-6 and the inner glucose at C-20 in protopanaxatriol (PPT), but not the outer rhamnose residues at C-6. In contrast, β-glycosidase from the hyperthermophilic bacterium Pyrococcus furiosus (PF-bgl) hydrolyzes the outer rhamnose residue at C-6 but not the inner glucose residues at C-6 and C-20 in PPT. Thus, the combined use of DT-bgl and PF-bgl resulted in the complete the conversion of all typical glycosylated PPT ginsenosides, including R1, R2, Re, Rg1, Rg2, Rh1, Rf, F1, F3, and F5, to APPT. DT-bgl combined with PF-bgl completely hydrolyzed 1.0 mg ml−1 R1 and 1.0 mg ml−1 total PPT-type ginsenosides in Panax notoginseng root extract to 0.5 and 0.63 mg ml−1 APPT for 4 and 3 h, with molar conversions of 100% and productivities of 125 and 210 mg l−1 h−1, respectively. To the best of our knowledge, this is the first report of the complete conversion of all typical glycosylated PPT ginsenosides to APPT and the highest productivity of APPT obtained from ginseng extract achieved to date. Electronic supplementary material The online version of this article (10.1186/s13568-018-0543-1) contains supplementary material, which is available to authorized users.

A putative diol synthase from the fungus Glomerella cingulate was cloned and expressed in Escherichia coli. The putative diol synthase from G. cingulate was purified by His-Trap affinity chromatography with a specific activity of 0.87 U mg(-1), an eightfold purification, and a yield of 28%. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of 7,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid (7,8-DiHODE) per min. The purified enzyme was estimated as a 127-kDa tetramer with a molecular mass of 510 kDa by gel filtration chromatography. The enzyme converted linoleic acid to a product, identified as 7S,8S-DiHODE by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (k cat/K m) of 7,8-diol synthase from G. cingulate for the conversion of fatty acid to dihydroxy fatty acid followed the order linoleic acid > α-linolenic acid > oleic acid > palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). The activity of the enzyme for the conversion of 7,8-DiHODE from linoleic acid was maximal at pH 6.5, 40 °C, and 2.5% (v/v) dimethyl sulfoxide (DMSO). Under these conditions, 7,8-LDS from G. cingulate converted 1.0 mM linoleic acid to 0.62 mM 7,8-DiHODE for 30 min, with a conversion yield of 62% (mol/mol), via 8-hydroperoxy-9,12(Z,Z)-octadecadienoic acid (8-HPODE) as an intermediate. The accumulation of 8-HPODE was due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain.

Linoleate 13-lipoxygenase from Burkholderia thailandensis was expressed in Escherichia coli for the production of 13-hydroxyoctadecadienoic acid (13-HODE), an antiseptic emulsifier. Linoleate 13-lipoxygenase in cells had higher thermal stability than the purified enzyme. To increase 13-HODE production, recombinant cells were permeabilized by solvents, detergents, salts, and other chemicals. The enzymatic activity in cells was the highest for permeabilized cells treated with 0.5 M NaCl among the permeabilizers tested. The optimal reaction conditions for the production of 13-HODE from linoleic acid by permeabilized cells treated with 0.5 M NaCl were at pH 7.5, 25 °C, 20 g/l linoleic acid, 15 g/l cells, 0.15 mM Cu2+, and 6 % (v/v) methanol in a 100-ml baffled flask containing a 5-ml working volume with agitation at 200 rpm. Under these conditions, permeabilized cells produced 15.8 g/l 13-HODE after 30 min with a conversion yield of 79 % (w/w) and a productivity of 31.6 g/l/h. The conversion yield and productivity of permeabilized cells for 13-HODE production were higher than those of purified and crude enzymes as well as nonpermeabilized cells. Therefore, permeabilized cells were efficient biocatalysts for 13-HODE production. To the best of our knowledge, this is the first report of the production of 13-HODE using cells.

To improve the production of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid (HODA) from α-linolenic acid in Stenotrophomonas nitritireducens. Cells of the bacterium were permeabilized with 1.25% (v/v) methanol. The optimal conditions for HODA production by permeabilized cells were pH 7, 35 °C, 5% (v/v) DMSO, 50 g cells l−1, and 22.5 g α-linolenic acid l−1. Under these conditions, permeabilized cells produced 16.4 g HODA l−1 after 2 h, with a conversion yield of 73 % (w/w) and a volumetric productivity of 8.2 g l−1 h−1. These values were 153 and 230 % of the values for non-permeabilized cells This is the highest concentration and volumetric and specific productivities of HODA reported thus far.

A β-glucosidase from Gordonia terrae was cloned and expressed in Escherichia coli. The recombinant enzyme with a specific activity of 16.4 U/mg for ginsenoside Rb1 was purified using His-trap chromatography. The purified enzyme specifically hydrolyzed the glucopyranosides at the C-20 position in protopanaxadiol (PPD)-type ginsenosides and hydrolyzed the glucopyranoside at the C-6 or C-20 position in protopanaxatriol (PPT)-type ginsenosides. The reaction conditions for the high-level production of Rg3 from Rb1 by the enzyme were pH 6.5, 30°C, 20 mg/ml enzyme, and 4 mg/ml Rb1. Under these conditions, G. terrae β-glucosidase completely converted Rb1 and Re to Rg3 and Rg2, respectively, after 2.5 and 8 h, respectively. Moreover, the enzyme converted Rg1 to Rh1 at 1 h with a molar conversion yield of 82%. The enzyme at 10 mg/ml produced 1.16 mg/ml Rg3, 1.47 mg/ml Rg2, and 1.17 mg/ml Rh1 from Rb1, Re, and Rg1, respectively, in 10% (w/v) ginseng root extract at pH 6.5 and 30°C after 33 h with molar conversion yields of 100%, 100%, and 77%, respectively. The combined molar conversion yield of Rg2, Rg3, and Rh1 from total ginsenosides in 10% (w/v) ginseng root extract was 68%. These above results suggest that this enzyme is useful for the production of ginsenosides Rg3, Rg2, and Rh1.

An H1004A-C1006S variant of Aspergillus nidulans diol synthase was identified as an 8-dioxygenase. Whole recombinant Escherichia coli cells expressing the double-site variant showed the highest activity for converting linoleic acid to 8-hydroperoxy-9,12(Z,Z)-octadecadienoic acid (8-HPODE), which in turn was reduced to 8-hydroxy-9,12(Z,Z)-octadecadienoic acid (8-HODE) by the addition of a reducing agent. The optimization of 8-HPODE production was performed by response surface methodology, and the optimal conditions were pH 8.3, 27.2 °C, 22.7% dimethyl sulfoxide, 27.0 g l−1 cells, and 16.5 g l−1 linoleic acid in a 100 ml baffled flask containing a 10 ml reaction mixture with agitation at 236 rpm. The reduction of 8-HPODE to 8-HODE was highest when Tris(2-carboxyethyl)phosphine hydrochloride (TECP-HCl) was added at a final concentration of 25 mM. Under the optimized reaction and reduction conditions, whole cells expressing H1004A-C1006S variant of A. nidulans diol synthase produced 5.0 g l−1 8-HODE for 1 h without the formation of di-hydroxy fatty acids. This is the first report of the biotechnological production of 8-HODE.

MK801 (dizocilpine), a phencyclidine (PCP) derivative, is a potent noncompetitive antagonist of the N-Methyl-D-aspartate receptor (NMDAr). Another PCP derivative, ketamine, was reported to block voltage-gated K+ (Kv) channels, which was independent of NMDAr function. Kv currents are major regulators of the membrane potential (E-m) and excitability of muscles and neurons. Here, we investigated the effect of MK801 on the Kv channels and E-m in rat mesenteric arterial smooth muscle cells (RMASMCs). We used the whole-cell patch clamp technique to analyze the effect of MK801 enantiomers on Kv channels and E-m. (+)MK801 inhibited Kv channels in a concentration-dependent manner (IC50 of 89.1 +/- 13.1 mu M. Hill coefficient of 1.05 +/- 0.08). The inhibition was voltage- and state- independent. (+)MK801 didn't influence steady-state activation and inactivation of Kv channels. (+)MK801 treatment depolarized E-m in a concentration-dependent manner and concomitantly decreased membrane conductance. (-)MK801 also similarly inhibited the Kv channels (IC50 of 134.0 +/- 17.5 mu M, Hill coefficient of 0.87 +/- 0.09). These results indicate that MK801 directly inhibits the Kv channel in a state-independent manner in RMASMCs. This MK801-mediated inhibition of Kv channels should be considered when assessing the various pharmacological effects produced by MK801, such as schizophrenia, neuroprotection, and hypertension. (C) 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V.

Ginsenoside compound K has been used as a key nutritional and cosmetic component because of its anti-fatigue and skin anti-aging effects. β-Glycosidase from Sulfolobus solfataricus (SS-BGL) is known as the most efficient enzyme for compound K production. The hydrolytic pathway from ginsenoside Rb1 to compound K via Rd and F2 is the most important because Rb1 is the most abundant component in ginseng extract. However, the enzymatic conversion of ginsenoside Rd to F2 is a limiting step in the hydrolytic pathway because of the relatively low activity for Rd. A V209 residue obtained from error-prone PCR was related to Rd-hydrolyzing activity, and a docking pose showing an interaction with Val209 was selected from numerous docking poses. W361F was obtained by rational design using the docking pose that exhibited 4.2-fold higher activity, 3.7-fold higher catalytic efficiency, and 3.1-fold lower binding energy for Rd than the wild-type enzyme, indicating that W361F compensated for the limiting step. W361F completely converted Rb1 to compound K with a productivity of 843 mg l−1 h−1 in 80 min, and showed also 7.4-fold higher activity for the flavanone, hesperidin, than the wild-type enzyme. Therefore, the W361F variant SS-BGL can be useful for hydrolysis of other glycosides as well as compound K production from Rb1, and semi-rational design is a useful tool for enhancing hydrolytic activity of β-glycosidase. Electronic supplementary material The online version of this article (doi:10.1186/s13568-017-0487-x) contains supplementary material, which is available to authorized users.

To characterize l-rhamnose isomerase (l-RI) from the thermophilic bacterium Clostridium stercorarium and apply it to produce d-allose from d-allulose. A recombinant l-RI from C. stercorarium exhibited the highest specific activity and catalytic efficiency (k cat/K m) for l-rhamnose among the reported l-RIs. The l-RI was applied to the high-level production of d-allose from d-allulose. The isomerization activity for d-allulose was maximal at pH 7, 75 °C, and 1 mM Mn2+ over 10 min reaction time. The half-lives of the l-RI at 65, 70, 75, and 80 °C were 22.8, 9.5, 1.9, and 0.2 h, respectively. To ensure full stability during 2.5 h incubation, the optimal temperature was set at 70 °C. Under the optimized conditions of pH 7, 70 °C, 1 mM Mn2+, 27 U l-RI l−1, and 600 g d-allulose l−1, l-RI from C. stercorarium produced 199 g d-allose l−1 without by-products over 2.5 h, with a conversion yield of 33% and a productivity of 79.6 g l−1 h−1. To the best of our knowledge, this is the highest concentration and productivity of d-allose reported thus far.

Whole cells of recombinant Escherichia coli expressing diol synthase from Aspergillus nidulans produced 5,8-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid from α-linolenic acid via 8-hydroperoxy-9,12,15(Z,Z,Z)-octadecatrienoic acid as an intermediate. The optimal conditions for 5,8-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid production using whole recombinant cells were exhibited at pH 7.0, 40 °C, and 250 rpm with 40 g/L cells, 12 g/L, α-linolenic acid, and 5 % (v/v) dimethyl sulfoxide in a 250-mL baffled flask containing 50 mL reaction solution. Under these conditions, whole recombinant cells produced 9.1 g/L 5,8-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid for 100 min, with a conversion yield of 75 % (w/w), a volumetric productivity of 5.5 g/L/h, and specific productivity of 137 mg/g-cells/h. As an intermediate, 8-hydroperoxy-9,12,15(Z,Z,Z)-octadecatrienoic acid was observed at approximately 1.4 g/L after 100 min. With regard to dihydroxy fatty acid production, this is the highest reported volumetric and specific productivities thus far. This is the first report on the biotechnological production of 5,8-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid.

The optimal conditions for the production of 5,8-dihydroxy-9(Z)-octadecenoic acid (5,8-diHOME) from oleic acid by whole recombinant Escherichia coli cells expressing diol synthase from Aspergillus nidulans were 40 °C, pH 7.5, 10 % (v/v) dimethyl sulfoxide, 35 g cells l(-1), and 12 g oleic acid l(-1) at 250 rpm in a 250 ml baffled flask. Under these conditions, whole recombinant cells produced 5.2 g 5,8-diHOME l(-1) together with 1 g l(-1) of the intermediate 8-hydroperoxy-9(Z)-octadecenoic acid (8-HPOME) after 60 min. This corresponded to a conversion yield of 43 % (w/w), a volumetric productivity of 5.2 g l(-1 )h(-1), and a specific productivity of 148 mg g cells(-1 )h(-1). This is the first report of the biotechnological production of 5,8-diHOME from oleic acid.

Diol synthase from Aspergillus nidulans was cloned and expressed in Escherichia coli. Recombinant E. coli cells expressing diol synthase from A. nidulans converted linoleic acid to a product that was identified as 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The recombinant cells and the purified enzyme showed the highest activity for linoleic acid among the fatty acids tested. The optimal reaction conditions for the production of 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid using whole recombinant E. coli cells expressing diol synthase were pH 7.5, 35°C, 250 rpm, 5 g l(-1) linoleic acid, 23 g l(-1) cells, and 20% (v/v) dimethyl sulfoxide in a 250-ml baffled flask. Under these optimized conditions, whole recombinant cells expressing diol synthase produced 4.98 g l(-1) 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid for 150 min without detectable byproducts, with a conversion yield of 99% (w/w) and a productivity of 2.5 g l(-1) h(-1). This is the first report on the biotechnological production of dihydroxy fatty acid using whole recombinant cells expressing diol synthase.

15-Hydroxyeicosatetraenoic acid (HETE), as a mammalian biologically active metabolite, has anticarcinogenic effect. The conditions of producing 15-HETE from arachidonic acid by using soybean lipoxygenase were optimal at pH 8.5 and 20°C with 9 g/l arachidonic acid, 54.4 U/ml soybean lipoxygenase, and 4% methanol. Under these optimized conditions, the enzyme produced 9.5 g/l 15-HETE after 25 min, with a molar conversion yield of 99% and a productivity of 22.8 g l(-1) h(-1). To the best of our knowledge, this is the first biotechnological production of 15-HETE.

β-Glucosidase from Thermus thermophilus has specific hydrolytic activity for the outer glucose at the C-20 position in protopanaxadiol-type ginsenosides without hydrolysis of the inner glucose. The hydrolytic activity of the enzyme for gypenoside XVII was optimal at pH 6.5 and 90 °C, with a half-life of 1 h with 3 g enzyme l(-1) and 4 g gypenoside XVII l(-1). Under the optimized conditions, the enzyme converted the substrate gypenoside XVII to ginsenoside F2 with a molar yield of 100 % and a productivity of 4 g l(-1) h(-1). The conversion yield and productivity of ginsenoside F2 are the highest reported thus far among enzymatic transformations.

Platycodin D has diverse pharmacological activities. An efficient and economical mechanism for obtaining platycosides (platycodin D in particular) would be very useful. Balloon flower leaf extract (BFLE) was obtained by recycling leaves discarded from Platycodi radix production, as they have a high platycoside E content. A recombinant &beta, glycosidase from Caldicellulosiruptor owensensis was characterized and applied to BFLE for platycoside bioconversion. The enzyme specifically hydrolyzed the glucose residue at the C-3 position in platycosides and was suitable for platycodin D production. Under optimized reaction conditions, &beta, glycosidase from C. owensensis completely converted platycoside E from BFLE into platycodin D with the highest concentration and productivity reported so far. These results greatly improve the production process for deglycosylated platycosides.

The specific activity of a recombinant β-glucosidase from Pyrococcus furiosus for protopanaxatriol (PPT)-type ginsenosides followed the order Rf > R1 > Re > R2 > Rg2, which were converted to Rh1, Rg1, Rg1, Rh1, and Rh1, respectively. No activity was observed with Rg1 and Rh1. Thus, P. furiosus β-glucosidase hydrolyzed the outer glycoside at the C-6 position in PPT-type ginsenosides whereas the enzyme did not hydrolyze the inner glucoside at the C-6 position and the glucoside at the C-20 position. The activity for Rf was optimal at 95 °C, pH 5.5, 5 mM ginsenoside, and 32 U enzyme l−1. Under these conditions, P. furiosus β-glucosidase completely converted from R1 to Rg1 after 10 h, with a productivity of 0.4 g l−1 h−1 and completely converted Rf to Rh1 after 1.2 h, with a productivity of 2.74 g l−1 h−1.

The ginsenoside compound K has pharmaceutical activities, including anti-tumor, anti-inflammatory, anti-allergic, and hepatoprotective effects. To increase the production of compound K, the α-L-arabinofuranoside-hydrolyzing α-L-arabinofuranosidase (CS-abf) and/or the α-L-arabinopyranoside-hydrolyzing β-galactosidase from Caldicellulosiruptor saccharolyticus (CS-bgal) were mixed with the β-D-glucopyranoside-hydrolyzing β-glucosidase from Sulfolobus acidocaldarius (SA-bglu). The optimum conditions for the production of ginsenoside compound K from ginsenoside Rc or Rb₂, or from major protopanaxadiol ginsenosides in ginseng root extract were determined to be pH 6.0 and 75°C with 8 mg ml⁻¹ ginsenoside Rc, 8 mg ml⁻¹ Rb₂, or 10% (w/v) ginseng root extract; and 10.5 U ml⁻¹ CS-abf or CS-bgal supplemented with 4.5 U ml⁻¹ SA-bglu, or 10.5 U ml⁻¹ CS-abf and 10.5 U ml⁻¹ CS-bgal supplemented with 4.5 U ml⁻¹ SA-bglu, respectively. Under optimum conditions, ginsenosides Rc and Rb2, and major protopanaxadiol ginsenosides in ginseng root extract were completely converted to compound K after 12, 14, and 20 h, respectively, with the respective productivities of 388, 328, and 144 mg l⁻¹ h⁻¹. This is the first report of the complete conversion of major protopanaxadiol ginsenosides to compound K.

A novel β-glucosidase from Penicillium aculeatum was purified as a single 110.5-kDa band on SDS-PAGE with a specific activity of 75.4 U mg⁻¹ by salt precipitation and Hi-Trap Q HP and Resource Q ion exchange chromatographies. The purified enzyme was identified as a member of the glycoside hydrolase 3 family based on its amino acid sequence. The hydrolysis activity for p-nitrophenyl-β-D-glucopyranoside was optimal at pH 4.5 and 70 °C with a half-life of 55 h. The enzyme hydrolyzed exo-, 3-O-, and 6-O-β-glucosides but not 20-O-β-glucoside and other glycosides of ginsenosides. Because of the novel specificity, this enzyme had the transformation pathways for ginsenosides: Rb₁ → Rd → F₂ → compound K, Rb₂ → compound O → compound Y, Rc → compound Mc₁ → compound Mc, Rg₃ → Rh₂ → aglycone protopanaxadiol (APPD), Rg₁ → F₁, and Rf → Rh₁ → aglycone protopanaxatriol (APPT). Under the optimum conditions, the enzyme converted 0.5 mM Rb₂, Rc, Rd, Rg₃, Rg₁, and Rf to 0.49 mM compound Y, 0.49 mM compound Mc, 0.47 mM compound K, 0.23 mM APPD, 0.49 mM F₁, and 0.44 mM APPT after 6 h, respectively.

Oleate hydratases (OhyAs) catalyze the conversion of unsaturated fatty acids to 10-hydroxy fatty acids, which are used as precursors of important industrial compounds, including lactones and ω-hydroxycarboxylic and α,ω-dicarboxylic acids. The genes encoding OhyA and a putative fatty acid hydratase in Stenotrophomonas maltophilia were identified by genomic analysis. The putative fatty acid hydratase was purified and identified as an oleate hydratase (OhyA2) based on its substrate specificity. The activity of OhyA2 as a holoenzyme was not affected by adding cofactors, whereas the activity of the original oleate hydratase (OhyA1) showed an increase. Thus, all characterized OhyAs were categorized as either OhyA1 or OhyA2 based on the activities of holoenzymes upon adding cofactors, which were determined by the type of the fourth conserved amino acid of flavin adenine dinucleotide (FAD)-binding motif. The hydration activities of S. maltophilia OhyA2 toward unsaturated fatty acids, including oleic acid, palmitoleic acid, linoleic acid, α-linolenic acid, and γ-linolenic acid, were greater than those of OhyA1. Moreover, the specific activity of S. maltophilia OhyA2 toward unsaturated fatty acids, with the exception of γ-linolenic acid, was the highest among all reported OhyAs. IMPORTANCE All characterized OhyAs were categorized as OhyA1s or OhyA2s based on the different properties of the reported and newly identified holo-OhyAs in S. maltophilia upon the addition of cofactors. OhyA2s showed higher activities toward polyunsaturated fatty acids (PUFAs), including linoleic acid, α-linolenic acid, and γ-linolenic acid, than those of OhyA1s. This suggests that OhyA2s can be used more effectively to convert plant oils to 10-hydroxy fatty acids because plant oils contain not only oleic acid but also PUFAs. The hydration activity of the newly identified OhyA2 from S. maltophilia toward oleic acid was the highest among the activity levels reported so far. Therefore, this enzyme is an efficient biocatalyst for the conversion of plant oils to 10-hydroxy fatty acids, which can be further converted to important industrial materials.

The reaction conditions for the production of 7S,8S-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid by recombinant Escherichia coli expressing 7,8-linoleate diol synthase from Glomerella cingulata were optimized using response surface methodology. The optimal reaction conditions were pH 7.0, 18.6 °C, 10.8% (v/v) dimethyl sulfoxide, 44.9 g/L cells, and 14.3 g/L linoleic acid, with agitation at 256 rpm. Under these conditions, recombinant cells produced 7,8-dihydroxy unsaturated fatty acids in the range of 7.0–9.8 g/L from 14.3 g/L linoleic acid, 14.3 g/L oleic acid, and plant oil hydrolysates such as waste oil and olive oil containing 14.3 g/L linoleic acid or oleic acid. To the best of the authors’ knowledge, this is the first report on the biotechnological production of 7,8-dihydroxy unsaturated fatty acids.

Hydroxy unsaturated fatty acids can be used as antimicrobial surfactants. 8,11-Linoleate diol synthase (8,11-LDS) catalyzes the conversion of unsaturated fatty acid to 8-hydroperoxy unsaturated fatty acid, and it is subsequently isomerized to 8,11-dihydroxy unsaturated fatty acid by the enzyme. The optimal reaction conditions of recombinant Escherichia coli expressing Penicillium chrysogenum 8,11-LDS for the production of 8,11-dihydroxy-9,12(Z,Z)-octadecadienoic acid (8,11-DiHODE), 8,11-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid (8,11-DiHOTrE), 8-hydroxy-9(Z)-hexadecenoic acid (8-HHME), and 8-hydroxy-9(Z)-octadecenoic acid (8-HOME) were pH 7.0, 25°C, 10 g/L linoleic acid, and 20 g/L cells; pH 6.0, 25°C, 6 g/L α-linolenic acid, and 60 g/L cells; pH 7.0, 25°C, 8 g/L palmitoleic acid, and 25 g/L cells; and pH 8.5, 30°C, 6 g/L oleic acid, and 25 g/L cells, respectively. Under these optimized conditions, the recombinant cells produced 6.0 g/L 8,11-DiHODE for 60 min, with a conversion of 60% (w/w) and a productivity of 6.0 g/L/h; 4.3 g/L 8,11-DiHOTrE for 60 min, with a conversion of 72% (w/w) and a productivity of 4.3 g/L/h; 4.3 g/L 8-HHME acid for 60 min, with a conversion of 54% (w/w) and a productivity of 4.3 g/L/h; and 0.9 g/L 8-HOME for 30 min, with a conversion of 15% (w/w) and a productivity of 1.8 g/L/h. To best of our knowledge, this is the first report on the biotechnological production of 8,11-DiHODE, 8,11-DiHOTrE, 8-HHME, and 8-HOME. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:390-396, 2017.

Pyrococcus furiosus β-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 β-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.

Compound K (C-K) is a crucial pharmaceutical and cosmetic component because of disease prevention and skin anti-aging effects. For industrial application of this active compound, the protopanaxadiol (PPD)-type ginsenosides should be transformed to C-K. β-Glycosidase from Sulfolobus solfataricus has been reported as an efficient C-K-producing enzyme, using glycosylated PPD-type ginsenosides as substrates. β-Glycosidase from S. solfataricus can hydrolyze β-d-glucopyranoside in ginsenosides Rc, C-Mc1, and C-Mc, but not α-l-arabinofuranoside in these ginsenosides. To determine candidate residues involved in α-l-arabinofuranosidase activity, compound Mc (C-Mc) was docking to β-glycosidase from S. solfataricus in homology model and sequence was aligned with β-glycosidase from Pyrococcus furiosus that has α-l-arabinofuranosidase activity. A L213A variant β-glycosidase with increased α-l-arabinofuranosidase activity was selected by substitution of other amino acids for candidate residues. The increased α-l-arabinofuranosidase activity of the L213A variant was confirmed through the determination of substrate specificity, change in binding energy, transformation pathway, and C-K production from ginsenosides Rc and C-Mc. The L213A variant β-glycosidase catalyzed the conversion of Rc to Rd by hydrolyzing α-l-arabinofuranoside linked to Rc, whereas the wild-type β-glycosidase did not. The variant enzyme converted ginsenosides Rc and C-Mc into C-K with molar conversions of 97%, which were 1.5- and 2-fold higher, respectively, than those of the wild-type enzyme. Therefore, protein engineering is a useful tool for enhancing the hydrolytic activity on specific glycoside linked to ginsenosides.

To produce δ-decalactone from linoleic acid by one-pot reaction using linoleate 13-hydratase with supplementation with whole Yarrowia lipolytica cells.Whole Y. lipolytica cells at 25 g l(-1) produced1.9 g l(-1) δ-decalactone from 7.5 g 13-hydroxy-9(Z)-octadecenoic acid l(-1) at pH 7.5 and 30 °C for 21 h. Linoleate 13-hydratase from Lactobacillus acidophilus at 3.5 g l(-1) with supplementation with 25 g Y. lipolytica cells l(-1) in one pot at 3 h produced 1.9 g l(-1) δ-decalactone from 10 g linoleic acid l(-1) via 13-hydroxy-9(Z)-octadecenoic acid intermediate at pH 7.5 and 30°C after 18 h, with a molar conversion yield of 31 % and productivity of 106 mg l(-1) h(-1).To the best of our knowledge, this is the first production of δ-decalactone using unsaturated fatty acid.