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To produce tagatose from fructose with a high conversion rate and to establish a high-yield purification method of tagatose from the reaction mixture. 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%. This is the first biotechnological production of tagatose from fructose and the first application of solvent recrystallization for the purification of rare sugars.

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 ( Bc ALDH) 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 Bc ALDH and human ALDH (ALDH1A1) could reduce all- trans -retinal to all- trans -retinol with NADPH. The catalytic residues in Bc ALDH 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 ( k cat / K m ) 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 Bc ALDH 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, Bc ALDH 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 Bc ALDH 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 Bc ALDH. 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.

Linoleate 13-hydratase from Lactobacillus acidophilus LMG 11470 converted linoleic acid to hydroxyl fatty acid, which was identified as 13S-hydroxy-9(Z)-octadecenoic acid (13-HOD) by GC-MS and NMR. The expression of linoleate 13-hydratase gene in Escherichia coli was maximized by using pACYC plasmid and super optimal broth with catabolite repression (SOC) medium containing 40mM Mg(2+). To optimize induction conditions, recombinant cells were cultivated at 37°C, 1mM isopropyl-β-d-thiogalactopyranoside was added at 2h, and the culture was further incubated at 16°C for 18h. Recombinant cells expressing linoleate 13-hydratase from L. acidophilus were obtained under the optimized expression conditions and used for 13-HOD production from linoleic acid. The optimal reaction conditions were pH 6.0, 40°C, 0.25% (v/v) Tween 40, 25gl(-1) cells, and 100gl(-1) linoleic acid, and under these conditions, whole recombinant cells produced 79gl(-1) 13-HOD for 3h with a conversion yield of 79% (w/w), a volumetric productivity of 26.3gl(-1)h(-1), and a specific productivity of 1.05g g-cells(-1)h(-1). To the best of our knowledge, the recombinant cells produced hydroxy fatty acid with the highest concentration and productivity reported so far.

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. Biotechnol. Bioeng. 2015;112: 2206–2213. © 2015 Wiley Periodicals, Inc.

A recombinant aldo-keto reductase (AKR) from Marivirga tractuosa was purified with a specific activity of 0.32 unit ml −1 for all- trans -retinal with a 72 kDa dimer. The enzyme had substrate specificity for aldehydes but not for alcohols, carbonyls, or monosaccharides. The enzyme turnover was the highest for benzaldehyde ( k cat = 446 min −1 ), whereas the affinity and catalytic efficiency were the highest for all- trans -retinal ( K m = 48 μM, k cat / K m = 427 mM −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, 10 mM NADPH, 1710 mg l −1 all- trans -retinal, and 3 unit ml −1 enzyme. Under these optimized conditions, the enzyme produced 1090 mg ml −1 all- trans -retinol, with a conversion yield of 64% (w/w) and a volumetric productivity of 818 mg l −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.

Hydroxy fatty acids are widely used in chemical, food, and cosmetic industries as starting materials for the synthesis of polymers and as additives for the manufacture of lubricants, emulsifiers, and stabilizers. They have antibiotic, anti-inflammatory, and anticancer activities and therefore can be applied for medicinal uses. Microbial fatty acid-hydroxylation enzymes, including P450, lipoxygenase, hydratase, 12-hydroxylase, and diol synthase, synthesize regio-specific hydroxy fatty acids. In this article, microbial fatty acid-hydroxylation enzymes, with a focus on region-specificity and diversity, are summarized and the production of mono-, di-, and tri-hydroxy fatty acids is introduced. Finally, the production methods of regio-specific and diverse hydroxy fatty acids, such as gene screening, protein engineering, metabolic engineering, and combinatory biosynthesis, are suggested.

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.

Hydroxy fatty acids (HFAs) such as 10-hydroxystearic acid (10-HSA) and 10-hydroxy-12(Z)-octadecenoic acid (10-HOD), which are similar to ricinoleic acid, are important starting materials and intermediates for the industrial manufacture of many commodities. Stenotrophomonas sp. KACC 91585, which was isolated from lake sediment, is an efficient bacterium for unsaturated fatty acid hydration that produces 10-HSA and 10-HOD from oleic acid and linoleic acid, respectively, with high conversion rates. The complete genome of this strain is 4,541,729bp with 63.83% GC content and devoid of plasmids. Sets of genes involved in the fatty acid biosynthesis and modification as well as modified lipids were identified in the genome, and these genes were concerned with HFA production. This genome sequence provides molecular information and elucidation for HFA production, and will be used as an efficient biocatalyst source for the biotechnological production of HFA.

The optimal temperature and pH for retinal production using metabolically engineered Escherichia coli in a 7-l fermentor were found to be 30°C and 7.0, respectively. The agitation speed was a critical factor for retinal production. The optimal agitation speed was 400 rpm (oxygen transfer coefficient, k(L)a, = 92 1/h) in batch culture and 600 rpm (k(L)a=148 1/h) in a fed-batch culture of glycerol. Span 80 was selected as a surfactant for retinal production in metabolically engineered E. coli because Span 80 had proven the most effective for increased retinal production among the tested surfactants. Under the optimal conditions in the fed-batch culture with 5 g/l Span 80, the cell mass and the concentration, content, and productivity of retinal were 24.7 g/l, 600 mg/l, 24.3mg/g-cells, and 18 mg l(-1)h(-1) after 33 h, respectively. They were 1.2-, 2.7-, 2.3-, and 2.7-fold higher than those in the fed-batch culture without Span 80, respectively. The concentration and productivity of retinal in this study were the highest ever reported. The hydrophilic portion of Span 80 (sorbitan) did not affect cell growth and retinal production, but the hydrophobic portion (oleic acid) stimulated cell growth. However, oleic acid plus sorbitan did not stimulate retinal production. Thus, Span 80, as a linked compound of oleic acid and sorbitan produced by esterification, proved to be an effective surfactant for the enhancement of retinal production.

A putative fatty acid hydratase from Stenotrophomonas maltophilia was cloned and expressed in Escherichia coli. The recombinant enzyme showed the highest hydration activity for oleic acid among the fatty acids tested, indicating that the enzyme is an oleate hydratase. The optimal conditions for the production of 10-hydroxystearic acid from oleic acid using whole cells of recombinant E. coli containing the oleate hydratase were pH 6.5, 35°C, 0.05% (w/v) Tween 40, 10 g l(-1) cells, and 50 g l(-1) oleic acid. Under these conditions, whole recombinant cells produced 49 g l(-1) 10-hydroxystearic acid for 4 h, with a conversion yield of 98% (w/w), a volumetric productivity of 12.3 g l(-1) h(-1), and a specific productivity of 1.23 g g-cells(-1) h(-1), which were 18%, 2.5-, and 2.5-fold higher than those of whole wild-type S. maltophilia cells, respectively. This is the first report of 10-hydroxystearic acid production using recombinant cells and the concentration and productivity are the highest reported thus far among cells.

A recombinant enzyme from Lysinibacillus fusiformis was expressed, purified, and identified as an oleate hydratase because the hydration activity of the enzyme was the highest for oleic acid (with a k (cat) of 850 min(-1) and a K (m) of 540 μM), followed by palmitoleic acid, γ-linolenic acid, linoleic acid, myristoleic acid, and α-linolenic acid. The optimal reaction conditions for the enzymatic production of 10-hydroxystearic acid were pH 6.5, 35 °C, 4% (v/v) ethanol, 2,500 U ml(-1) (8.3 mg ml(-1)) of enzyme, and 40 g l(-1) oleic acid. Under these conditions, 40 g l(-1) (142 mM) oleic acid was converted into 40 g l(-1) (133 mM) 10-hydroxystearic acid for 150 min, with a molar yield of 94% and a productivity of 16 g l(-1) h(-1), and olive oil hydrolyzate containing 40 g l(-1) oleic acid was converted into 40 g l(-1) 10-hydroxystearic acid for 300 min, with a productivity of 8 g l(-1) h(-1).

Fusarium moniliforme var. subglutinans was selected from among 100 strains of fungi for producing ginsenoside F(1) from ginsenoside Rg(1). The enzyme responsible was purified as a single 85 kDa band with a specific activity of 136 U mg(-1). It hydrolysed glucose-linked ginsenosides Rb(1), Rd and Rg(1) but not for other monosaccharide-linked ginsenosides, Rb(2), Rc, R(1), and Re. Under the optimum conditions of pH 6.0, 50 °C, 30 U l(-1) of enzyme, and 5 mg Rg(1) ml(-1), 4 mg F(1) ml(-1) was produced after 4 h, with a molar yield of 100% and a productivity of 1 g l(-1) h(-1). This represents the highest productivity and conversion yield of F(1) yet reported.

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.

Aspergillus oryzae KACC 40247 was selected from among 60 fungal strains as an effective 7,8,4'-trihydroxyisoflavone (8-hydroxydaidzein)-producing fungus. The optimal culture conditions for production by this strain in a 7-L fermentor were found to be 30 °C, pH 6, and 300 rpm. Under these conditions, A. oryzae KACC 40247 produced 62 mg/L of 8-hydroxydaidzein from soybean extract in 30 h, with a productivity of 2.1 mg/L/h. These are the highest production and productivity for 8-hydroxydaidzein ever reported. To increase production, several concentrations of daidzin and of daidzein as precursor were added at several culture times. The optimal addition time and concentration for daidzin were 12 h and 1,248 mg/L, and those for daidzein were 12 h and 254 mg/L respectively. Maximum production and productivity for 8-hydroxydaidzein with the addition of daidzein were 95 mg/L and 3.2 mg/L/h respectively, and those with the addition of daidzin were 160 mg/L and 4.4 mg/L/h respectively.

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 degrees C, and 1 mM Co2+ with a half-life of 40 min at 65 degrees C, K-m of 162 mM, and k(cat) 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.

A recombinant oleate hydratase from Lysinibacillus fusiformis converted ricinoleic acid to a product, whose chemical structure was identified as the novel compound 10,12-dihydroxystearic acid by gas chromatograph/mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance analysis. The reaction conditions for the production of 10,12-dihydroxystearic acid were optimized as follows: pH 6.5, 30 °C, 15 g l(-1) ricinoleic acid, 9 mg ml(-1) of enzyme, and 4 % (v/v) methanol. Under the optimized conditions, the enzyme produced 13.5 g l(-1) 10,12-dihydroxystearic acid without detectable byproducts in 3 h, with a conversion of substrate to product of 90 % (w/w) and a productivity of 4.5 g l(-1) h(-1). The emulsifying activity of 10,12-dihydroxystearic acid was higher than that of oleic acid, ricinoleic acid, stearic acid, and 10-hydroxystearic acid, indicating that 10,12-dihydroxystearic acid can be used as a biosurfactant.

L-Ribose is an important precursor for antiviral agents, and thus its high-level production is urgently demanded. For this aim, immobilized recombinant Escherichia coli cells expressing the L-arabinose isomerase and variant mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans were developed. The immobilized cells produced 99 g/l L-ribose from 300 g/l L-arabinose in 3 h at pH 7.5 and 60 °C in the presence of 1 mM Co(2+), with a conversion yield of 33 % (w/w) and a productivity of 33 g/l/h. The immobilized cells in the packed-bed bioreactor at a dilution rate of 0.2 h(-1) produced an average of 100 g/l L-ribose with a conversion yield of 33 % and a productivity of 5.0 g/l/h for the first 12 days, and the operational half-life in the bioreactor was 28 days. Our study is first verification for L-ribose production by long-term operation and feasible for cost-effective commercialization. The immobilized cells in the present study also showed the highest conversion yield among processes from L-arabinose as the substrate.

Optimal hydrolytic activity of β-glucosidase from Dictyoglomus turgidum for the ginsenoside Rd was at pH 5.5 and 80 °C, with a half-life of ~11 h. The enzyme hydrolysed β-linked, but not α-linked, sugar moieties of ginsenosides. It produced the rare ginsenosides, aglycon protopanaxadiol (APPD), compounds Y, and Mc, via three unique transformation pathways: Rb(1) → Rd → F(2) → compound K → APPD, Rb(2) → compound Y, and Rc → compound Mc. The enzyme converted 0.5 mM Rb(2) and 0.5 mM Rc to 0.5 mM compound Y and 0.5 mM compound Mc after 3 h, respectively, with molar conversion yields of 100 %.

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.