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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.

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.

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.

Additional file 1: Figure S1. SDS-PAGE analysis of purified enzyme. Lane 1 marker proteins; lane 2 DT-bgl (85 kDa); lane 3 β-glycosidase from P. furiosus (55 kDa); lane 4 β-galactosidase from C. saccharolyticus (79 kDa); lane 5 CB-bgl (53 kDa); lane 6 β-glycosidase from S. acidocaldarius (57 kDa); and lane 7 β-glycosidase from S. solfataricus (57 kDa). Figure S2. HPLC profiles obtained during the conversion of PPD-type ginsenosides in ginseng root extract to APPD by DT-bgl alone. Figure S3. HPLC profiles obtained during the conversion of PPD-type ginsenosides in ginseng root extract to APPD by a DT-bgl supplemented with DT-bgl and b P. furiosus β-glycosidase. Table S1. Contents of protopanaxadiol (PPD)- and protopanaxatriol (PPT)-type ginsenosides in ginseng root extract powder.

Additional file 1: Figure S1. Effects of a pH and b temperature on the activity of the wild-type (closed circle) and W361F (open circle) variant enzymes. Data represent the means from three separate experiments, and the error bars represent standard deviations. Figure S2. Hanes-Woolf plots a using wild-type enzyme and ginsenoside Rb1, b using wild-type enzyme and ginsenoside Rd, c using W361F variant enzyme and ginsenoside Rb1, and d using W361F variant enzyme and ginsenoside Rd. Figure S3. HPLC profiles for the conversion of ginsenoside Rb1 to compound K via ginsenoside Rd by the wild-type and W361F variant enzymes. The retention times for ginsenoside Rb1, Rd, and compound K were 10.2, 13.4, and 29.8 min, respectively. Table S1. Abbreviations for ginsenosides. Table S2. Relative activities of the wild-type and W361F variant enzymes for aryl-glycosides.

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.

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.

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.

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.

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.

A recombinant β-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 β-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.

Background: Neurally mediated syncope is the most common cause of syncope. Patients with neurally mediated syncope sometimes experience stress from recurrent syncopal episode. The aim of this study is to evaluate the degree of anxiety and depression in patients with neurally mediated syncope. Method: A total 87 of neurally mediated syncope patients (59.1% female, mean age 38.81±14.06) completed the hospital anxiety and depression scale (HADS) from January to December 2009. The patient were divided into tilt positive (n=66) and negative group (n=21). Other cause such as cardiogenic syncope and orthostatic hypotension were excluded from the study. HADS scores were divided into nonsignificant (0–7) and significant (8–21) to assess the degree of anxiety and depression. Result: There was no difference in gender and age between tilt positive (n=66) and negative group (n=21). Significant anxiety score was more frequently found in patient with tilt negative than positive group (42.9% vs 16.7%, P