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d-Allose has attracted a great deal of attention in recent years due to its many pharmaceutical activities, which include anti-cancer, anti-tumor, anti-inflammatory, anti-oxidative, anti-hypertensive, cryoprotective, and immunosuppressant activities. d-Allose has been produced from d-psicose using d-allose-producing enzymes, including l-rhamnose isomerase, ribose-5-phosphate isomerase, and galactose-6-phosphate isomerase. In this article, the properties, applications, and metabolism of d-allose are described, and the biochemical properties of d-allose-producing enzymes and their d-allose production are reviewed and compared. Moreover, several methods for effective d-allose production are suggested herein. [ABSTRACT FROM AUTHOR]

Retinal, a precursor of vitamin A, has been used in foods, cosmetics, pharmaceuticals, nutraceuticals, and animal feed additives. Carotenoid 15,15′-oxygenases, including β-carotene 15,15′-oxygenases from mammalians, chickens, fruit flies, zebrafishes, the uncultured marine bacterium, and the fungus Fusarium fujikuroi, and apo-carotenoid 15,15′-oxygenases from cyanobacteria produce retinal from carotenoids. In this article, the biochemical properties, reaction mechanism, and substrate specificity of carotenoid oxygenases are reviewed, along with a description of the enzymatic biotransformation of carotenoids to retinal. Retinal producing methods using metabolically engineered cells and uncharacterized proteins are suggested. [ABSTRACT FROM AUTHOR]

Galacto-oligosaccharides have become the focus of a great deal of attention in the field of functional foods, owing to their known health benefits and potential to improve the quality of many foods. Because of these properties, they are currently used as low-calorie sweeteners in fermented milk products, confectioneries, breads, and beverages. In this article, the characterization and product inhibition of many microbial β-galactosidases with transgalactosylation activities, their galacto-oligosaccharide production from lactose, and the application of galacto-oligosaccharides are reviewed. A method for increasing galacto-oligosaccharide production by reducing product inhibition properties via the immobilization and structure modification of the enzymes is also introduced herein. [ABSTRACT FROM AUTHOR]

A recombinant β-galactosidase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 211 U mg−1 by using heat treatment and His-trap affinity chromatography. The native enzyme was an 80-kDa trimer with a molecular mass of 240 kDa. Maximum activity was observed at pH 6.0 and 80ºC, and the half-life at 70ºC was 48 h. The enzyme exhibited hydrolytic activity for p-nitrophenyl-β- d-galactopyranoside ( pNPGal), oNPGal, or lactose, whereas no activity for p-nitrophenyl-β- d-glucopyranoside ( pNPGlu), oNPGlu, or cellobiose. The catalytic residues E150 and E311 of β-galactosidase from C. saccharolyticus were completely conserved in all aligned glycoside hydrolase family 42 β-galactosidases. The results indicated that the enzyme was a β-galactosidase. Galactose uncompetitively inhibited the enzyme. Glucose inhibition of the enzyme was the lowest among β-galactosidases. When 50 g l−1 galactose was added, the enzyme activity for pNPGal was reduced to 26%. When 400 g l−1 glucose instead of galactose was added, the activity was reduced to 82%. When adding galactose (200 g l−1), only 14% of the lactose was hydrolyzed after 180 min. In contrast, the addition of glucose (400 g l−1) did not affect lactose hydrolysis, and more than 99% of the lactose was hydrolyzed after 120 min. [ABSTRACT FROM AUTHOR]

d-Tagatose has attracted a great deal of attention in recent years due to its health benefits and similar properties to sucrose. d-Tagatose can be used as a low-calorie sweetener, as an intermediate for synthesis of other optically active compounds, and as an additive in detergent, cosmetic, and pharmaceutical formulation. Biotransformation of d-tagatose has been produced using several biocatalyst sources. Among the biocatalysts, l-arabinose isomerase has been mostly applied for d-tagatose production because of the industrial feasibility for the use of d-galactose as a substrate. In this article, the characterization of many l-arabinose isomerases and their d-tagatose production is compared. Protein engineering and immobilization of the enzyme for increasing the conversion rate of d-galactose to d-tagatose are also reviewed. [ABSTRACT FROM AUTHOR]

Abstract  Sinorhizobiumsp., which can convertd-fructose intod-psicose, was isolated from soil. The optimal pH, temperature, and cell concentration ford-psicose production with the isolated strain were 8.5, 40?C, and 60 mg/ml, respectively. The toluene-treated cells showed 2.5- and 4.8-fold increases in thed-psicose concentration and productivity compared with untreated washed cells. Under the optimal conditions, the toluene-treated cells produced 37 gd-psicose/l from 70% (w/v) (3.9 M)d-fructose after 15 h. [ABSTRACT FROM AUTHOR]

The article focuses on the study of the biotransformation of soybean from isoflavones to hydroxyisoflavones and also mentions the analysis of variations in the primary and secondary metabolite in soybean during fermentation by Aspergillus oryzae. Topics include analysis of decrease in acetylglycosides and glycosides through mass spectrometry, identification of biochemical processes of fermented soybean by liquid chromatography-mass spectrometry and antioxidant activities of fermented soybean.

A new biotransformation process for the production of the flavor lactone was developed by using permeabilized Waltomyces lipofer, which was selected as an efficient ?-dodecalactone-producing yeast among 10 oleaginous yeast strains. The optimal reaction conditions for ?-dodecalactone production by permeabilizedW. lipofer cells were pH 6.5, 35°C, 200 rpm, 0.7 M Tris, 60 g/liter of 10-hydroxystearic acid, and 30 g/liter of cells. Under these conditions, nonpermeabilized cells produced 12 g/liter of γ-dodecalactone after 30 h, with a conversion yield of 21% (wt/wt) and a productivity of 0.4 g/liter/h, whereas permeabilized cells obtained after sequential treatments with 50% ethanol and 0.5% Triton X-100 produced 46 g/liter of γ-dodecalactone after 30 h, with a conversion yield of 76% (wt/wt) and a productivity of 1.5 g/liter/h. These values were 3.7- and 3.8-fold higher than those obtained using nonpermeabilized cells. These are the highest reported concentration, conversion yield, and productivity for the production of the bioflavor lactone. [ABSTRACT FROM AUTHOR]

Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase from Clostridium difficile (CDRPI), mannose-6-phos- phate isomerase from Bacillus subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcusfuriosus (PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphate-binding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccha- rides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosac- charides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, RI 92 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to a D-ribose isomerase, an L-ribose isomerase, and an L-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isonierases. [ABSTRACT FROM AUTHOR]

A triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans was obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) for l-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/liter l-ribose from 300 g/liter l-ribulose for 60 min, with a volumetric productivity of 213 g liter-1 h-1, which was 4.5-fold higher than that of the wild-type enzyme. The kcat/Km and productivity of the triple-site variant were approximately 2-fold higher than those of the Thermus thermophilus R142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A novel bacterial aldehyde dehydrogenase (ALDH) that converts retinal to retinoic acid was first identified in Bacillus cereus. The amino acid sequence of ALDH from B. cereus (BcALDH) was more closely related to mammalian ALDHs than to bacterial ALDHs. This enzyme converted not only small aldehydes to carboxylic acids but also the large aldehyde all-trans-retinal to alltrans- retinoic acid with NAD(P)+. We newly found that BcALDH and human ALDH (ALDH1A1) could reduce all-trans-retinal to all-trans-retinol with NADPH. The catalytic residues in BcALDH were Glu266 and Cys300, and the cofactor-binding residues were Glu194 and Glu457. The E266A and C300A variants showed no oxidation activity. The E194S and E457V variants showed 15- and 7.5-fold higher catalytic efficiency (kcat/Km) for the reduction of all-trans-retinal than the wild-type enzyme, respectively. The wild-type, E194S variant, and E457V variant enzymes with NAD+ converted 400 µMall-trans-retinal to 210 µMall-transretinoic acid at the same amount for 240 min, while with NADPH, they converted 400 µMall-trans-retinal to 20, 90, and 40 µM all-trans-retinol, respectively. These results indicate that BcALDH and its variants are efficient biocatalysts not only in the conversion of retinal to retinoic acid but also in its conversion to retinol with a cofactor switch and that retinol production can be increased by the variant enzymes. Therefore, BcALDH is a novel bacterial enzyme for the alternative production of retinoic acid and retinol. [ABSTRACT FROM AUTHOR]

The article discusses a study on 8-hydroxydaidzein, an isoflavonoids produced from daidzin, daidzein and soybean by microorganisms including Aspergillus saitoi, Arthrobacter and Aspergillus oryzae, isolated from fermented soybean foods. It mentions that culture conditions including temperature, pH and agitation speed of fermentor was optimized for converting daidzin to 8-hydroxydaidzein. It suggests an enzymatic method for the production of 8-hydroxydaidzein using Cytochrome P450.

The S213C, I33L, and I33L S213C variants of d-psicose 3-epimerase from Agrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer of d-psicose. [ABSTRACT FROM AUTHOR]

In the fed-batch culture of glycerol using a metabolically engineered strain of Escherichia coli, supplementation with glucose as an auxiliary carbon source increased lycopene production due to a significant increase in cell mass, despite a reduction in specific lycopene content. l-Arabinose supplementation increased lycopene production due to increases in cell mass and specific lycopene content. Supplementation with both glucose and l-arabinose increased lycopene production significantly due to the synergistic effect of the two sugars. Cell growth by the consumption of carbon sources was related to endogenous metabolism in the host E. coli. Supplementation with l-arabinose stimulated only the mevalonate pathway for lycopene biosynthesis and supplementation with both glucose and l-arabinose stimulated synergistically only the mevalonate pathway. In the fed-batch culture of glycerol with 10 g l glucose and 7.5 g l l-arabinose, the cell mass, lycopene concentration, specific lycopene content, and lycopene productivity after 34 h were 42 g l, 1,350 mg l, 32 mg g cells, and 40 mg l h, respectively. These values were 3.9-, 7.1-, 1.9-, and 11.7-fold higher than those without the auxiliary carbon sources, respectively. This is the highest reported concentration and productivity of lycopene. [ABSTRACT FROM AUTHOR]

Ribose-5-phosphate isomerase (Rpi) catalyzes the conversion of d-ribose 5-phosphate (R5P) to d-ribulose 5-phosphate, which is an important step in the non-oxidative pathway of the pentose phosphate pathway and the Calvin cycle of photosynthesis. Recently, Rpis have been used to produce valuable rare sugars for industrial purposes. Of the Rpis, d-ribose-5-phosphate isomerase B from Clostridium thermocellum (CtRpi) has the fastest reactions kinetics. While Thermotoga maritime Rpi (TmRpi) has the same substrate specificity as CtRpi, the overall activity of CtRpi is approximately 200-fold higher than that of TmRpi. To understand the structural basis of these kinetic differences, we determined the crystal structures, at 2.1-Å resolution or higher, of CtRpi alone and bound to its substrates, R5P, d-ribose, and d-allose. Structural comparisons of CtRpi and TmRpi showed overall conservation of their structures with two notable differences. First, the volume of the CtRpi substrate binding pocket (SBP) was 20% less than that of the TmRpi SBP. Second, the residues next to the sugar-ring opening catalytic residue (His98) were different. We switched the key residues, involved in SBP shaping or catalysis, between CtRpi and TmRpi by site-directed mutagenesis, and studied the enzyme kinetics of the mutants. We found that tight interactions between the two monomers, narrow SBP width, and the residues near the catalytic residue are all critical for the fast enzyme kinetics of CtRpi. [ABSTRACT FROM AUTHOR]

putative ribose-5-phosphate isomerase (RpiB) from Streptococcus pneumoniae was purified with a specific activity of 26.7 U mg by Hi-Trap Q HP anion exchange and Sephacryl S-300 HR 16/60 gel filtration chromatographies. The native enzyme existed as a 96-kDa tetramer with activity maxima at pH 7.5 and 35°C. The RpiB exhibited isomerization activity with l-lyxose, l-talose, d-gulose, d-ribose, l-mannose, d-allose, l-xylulose, l-tagatose, d-sorbose, d-ribulose, l-fructose, and d-psicose and exhibited particularly high activity with l-form monosaccharides such as l-lyxose, l-xylulose, l-talose, and l-tagatose. With l-xylulose (500 g l) and l-talose (500 g l) substrates, the optimum concentrations of RpiB were 300 and 600 U ml, respectively. The enzyme converted 500 g l l-xylulose to 350 g l l-lyxose after 3 h, and yielded 450 g l l-tagatose from 500 g l l-talose after 5 h. These results suggest that RpiB from S. pneumoniae can be employed as a potential producer of l-form monosaccharides. [ABSTRACT FROM AUTHOR]

Ribose-5-phosphate isomerase from Clostridium thermocellum converted d-psicose to d-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in d-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for d-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency ( k/ K) of the R132E mutant for d-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of d-psicose to d-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min. [ABSTRACT FROM AUTHOR]

The production of compound K and aglycon protopanaxadiol (APPD) from ginsenoside Rd and ginseng root extract was performed using a recombinant β-glycosidase from Pyrococcus furiosus. The activity for Rd was optimal at pH 5.5 and 95°C with a half-life of 68 h at 95°C. β-Glycosidase converted Rb, Rb, Rc, and Rd to APPD via compound K. With increases in the enzyme activity, the productivities of compound K and APPD increased. The substrate concentration was optimal at 4.0 mM Rd or 10% ( w/ v) ginseng root extract; 4 mM of Rd was converted to 3.3 mM compound K with a yield of 82.5% (mol/mol) and a productivity of 2,010 mg l h at 1 h and was hydrolyzed completely to APPD with 364 mg l h after 5 h. Rb, Rb, Rc, and Rd at 3.9 mM in 10% ginseng root extract were converted to 3.1 mM compound K with 79.5% and 1,610 mg l h at 1.2 h and were hydrolyzed completely to APPD with 300 mg l h after 6 h. The concentrations and productivities of compound K and APPD in the present study are the highest ever reported. [ABSTRACT FROM AUTHOR]

An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for L-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu2+. Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of L-ribulose to L-ribose, a potential starting material for many L-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in L-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (kcat/Km) for L-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The kcat/Km of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported kcat/ The R142N mutant enzyme produced 213 g/liter L-ribose from 300 g/liter L-ribulose for 2 h, with a volumetric productivity of 107 g liter-1 h-1,which was 1.5-fold higher than that of the wild-type enzyme. [ABSTRACT FROM AUTHOR]

Ginsenosides are the principal components responsible for the pharmaceutical activities of ginseng. The minor ginsenosides, which are also pharmaceutically active, can be produced via the hydrolysis of the sugar moieties in the major ginsenosides using acid hydrolytic, heating, microbial, and enzymatic transformation techniques. The enzymatic method has a profound potential for ginsenoside transformation, owing to its high specificity, yield, and productivity, and this method is increasingly being recognized as a useful tool in structural modification and metabolism studies. In this article, the transformation methods of ginsenosides, the characterization of microbial glycosidases with ginsenoside hydrolyzing activities, and the enzymatic production of minor ginsenosides are reviewed. Moreover, the conversions of ginsenosides using cell extracts from food microorganisms and recombinant thermostable β- d-glycosidases are proposed as feasible methods for use in industrial processes. [ABSTRACT FROM AUTHOR]

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Penicillium pinophilum KMJ601 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, the maximal EG activity of 5.0 U mg protein−1, one of the highest levels among EG-producing microorganisms, was observed. The optimum temperature and pH for EG production were 28°C and 5.0, respectively. The increased production of EG by P. pinophilum in culture at 28°C was confirmed by two-dimensional electrophoresis followed by MS/MS sequencing of the partial peptide. A partial EG gene ( eng5) was amplified by degenerate polymerase chain reaction (PCR) based on the peptide sequence. A full-length eng5 was cloned by genome-walking PCR, and P. pinophilum EG was identified as a member of glycoside hydrolase family 5. The present results should contribute to improved industrial production of EG by P. pinophilum KMJ601. [ABSTRACT FROM AUTHOR]

The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co2+. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the D and L forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for L-ribulose among all pentoses and hexoses. Thus, L-ribose, as a potential starting material for many L-nucleoside-based pharmaceutical compounds, was produced at 213 glliter from 300-g/liter L-ribulose by mannose-6-phosphate isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter-1 h-1. [ABSTRACT FROM AUTHOR]

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of all-transretinal. The molecular mass of the native purified enzyme was ~64 kDa as a dimer of 32-kDa subunits. The Km, kcat, and kcat/Km values for β-carotene as substrate were 37 μM, 3.6 min-1, and 97 mM-1 min-1, respectively. The enzyme exhibited the highest activity for β-carotene, followed by β-cryptoxanthin, β-apo-4′-carotenal, α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal, β-apo-12′-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C35 seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a β-carotene 15,15′-dioxygenase. Although the Blh protein and β-carotene 15,15′-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian β-carotene 15,15′-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial β-carotene-cleaving enzyme. [ABSTRACT FROM AUTHOR]

The article presents a study regarding the production of a ginsenoside compound K obtained from ginseng root extract by utilizing a thermostable recombinant β-glycosidase from Sulfolobus solfataricus. It investigates the effects of the potential of hydrogen (pH), temperature and thermostability on the production, and the compound K-producing activity and ginsenoside-hydrolyzing activity of the β-glycosidase. It discusses the advantages of this new method of producing compound K.

Nitrilase from Rhodococcus rhodochrous ATCC 33278 hydrolyses both aliphatic and aromatic nitriles. Replacing Tyr-142 in the wild-type enzyme with the aromatic amino acid phenylalanine did not alter specificity for either substrate. However, the mutants containing non-polar aliphatic amino acids (alanine, valine and leucine) at position 142 were specific only for aromatic substrates such as benzonitrile, m-tolunitrile and 2-cyanopyridine, and not for aliphatic substrates. These results suggest that the hydrolysis of substrates probably involves the conjugated π-electron system of the aromatic ring of substrate or Tyr-142 as an electron acceptor. Moreover, the mutants containing charged amino acids such as aspartate, glutamate, arginine and asparagine at position 142 displayed no activity towards any nitrile, possibly owing to the disruption of hydrophobic interactions with substrates. Thus aromaticity of substrate or amino acid at position 142 in R. rhodochrous nitrilase is required for enzyme activity. [ABSTRACT FROM AUTHOR]

Abstract  A recombinant β-galactosidase from Sulfolobus solfataricus produced galactooligosaccharides (GOS) from lactose by transgalactosylation. The enzyme activity for GOS production was maximal at pH 6.0 and 85°C. The half-lives of the recombinant β-galactosidase at 70, 75, 80, 85, and 90°C were 700, 111, 72, 43, and 2.4 h, respectively, and its deactivation energy was 213 kJ mol−1. The optimal amount of enzyme for effective GOS production was 3.6 U of enzyme ml−1. GOS production increased with increasing lactose concentration, whereas the yield of GOS from lactose was almost constant. The rates of hydrolysis and transgalactosylation reactions increased with increasing temperature but the final concentration of GOS was maximal at 80°C. Under the conditions of pH 6.0, 80°C, 600 g lactose l−1, and 3.6 U enzyme ml−1, 315 g GOS l−1 were obtained for 56 h with a yield of 52.5% (w/w). The β-galactosidase from S. solfataricus produced GOS with the highest concentration and yield among thermostable β-galactosidases reported to date. [ABSTRACT FROM AUTHOR]

An isolated bacterium that converted unsaturated fatty acids to hydroxy fatty acids was identified as Stenotrophomonas nitritireducens by API analysis, cellular fatty acids compositions, sequencing the full 16S ribosomal ribonucleic acid, and evaluating its nitrite reduction ability. S. nitritireducens has unique regio-specificity for C16 and C18 cis-9 unsaturated fatty acids. These fatty acids are converted to their 10-hydroxy fatty acids without detectable byproducts. Among the cis-9-unsaturated fatty acids, S. nitritireducens showed the highest specificity for linoleic acid. The cells converted 20 mM linoleic acid to 13.5 mM 10-hydroxy-12( Z)-octadecenoic acid at 30°C and pH 7.5 with a yield of 67.5% (mol/mol). [ABSTRACT FROM AUTHOR]

This report describes the optimization of culture conditions for vancomycin production by Amycolatopsis orientalis KCCM-10836P, an identified high-vancomycin-producing strain (US11/712,494). Among the conditions tested, pH and the dissolved oxygen tension (DOT) were key factors affecting vancomycin production. When the pH and DOT were controlled at 7.0 and 20–30%, respectively, a dry-cell weight (DCW) of 62.0 g l−1 and a vancomycin production of 11.5 g l−1 were obtained after 120 h of batch culture, corresponding to a specific vancomycin content of 185.4 mg g-DCW−1. Vancomycin production was scaled up from a laboratory scale (7-l fermentor) to a pilot scale (300 l) and a plant scale (5,000 l) using the impeller tip velocity ( V tip) as a scale-up parameter. Vancomycin production at the laboratory scale was similar to those at the pilot and plant scales. [ABSTRACT FROM AUTHOR]

Carotenoids are ubiquitous pigments synthesized by plants, fungi, algae, and bacteria. Industrially, carotenoids are used in pharmaceuticals, neutraceuticals, and animal feed additives, as well as colorants in cosmetics and foods. Scientific interest in dietary carotenoids has increased in recent years because of their beneficial effects on human health, such as lowering the risk of cancer and enhancement of immune system function, which are attributed to their antioxidant potential. The availability of carotenoid genes from carotenogenic microbes has made possible the synthesis of carotenoids in non-carotenogenic microbes. The increasing interest in microbial sources of carotenoid is related to consumer preferences for natural additives and the potential cost effectiveness of creating carotenoids via microbial biotechnology. In this review, we will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms with particular focus on the potential of Escherichia coli for improved carotenoid productivity. [ABSTRACT FROM AUTHOR]

The gene encoding β-carotene 15,15′-monooxygenase from Mus musculus (house mouse), which cleaves β-carotene into two molecules of retinal, was cloned and expressed in Escherichia coli. The expressed enzyme was purified by His-tag affinity and resource Q ion exchange chromatography columns to a final specific activity of 0.51 U mg−1. The optimum pH, temperature, substrate and detergent concentrations, and enzyme amount for effective retinal production were determined to be 9.0, 37°C, 200 mg l−1 β-carotene, 5% (w/v) Tween 40, and 0.2 U ml−1 enzyme, respectively. Under optimum conditions, the recombinant enzyme produced 72 mg l−1 retinal in a 15-h reaction time, with a conversion yield of 36% (w/w). The specific activity of the purified enzyme and retinal production obtained in the present study were the highest results ever reported. [ABSTRACT FROM AUTHOR]

This report describes the optimization of culture conditions for coenzyme Q10 (CoQ10) production by Agrobacterium tumefaciens KCCM 10413, an identified high-CoQ10-producing strain (Kim et al., Korean patent. 10-0458818, ). Among the conditions tested, the pH and the dissolved oxygen (DO) levels were the key factors affecting CoQ10 production. When the pH and DO levels were controlled at 7.0 and 0–10%, respectively, a dry cell weight (DCW) of 48.4 g l−1 and a CoQ10 production of 320 mg l−1 were obtained after 96 h of batch culture, corresponding to a specific CoQ10 content of 6.61 mg g-DCW−1. In a fed-batch culture of sucrose, the DCW, specific CoQ10 content, and CoQ10 production increased to 53.6 g l−1, 8.54 mg g-DCW−1, and 458 mg l−1, respectively. CoQ10 production was scaled up from a laboratory scale (5-l fermentor) to a pilot scale (300 l) and a plant scale (5,000 l) using the impeller tip velocity ( V tip) as a scale-up parameter. CoQ10 production at the laboratory scale was similar to those at the pilot and plant scales. This is the first report of pilot- and plant-scale productions of CoQ10 in A. tumefaciens. [ABSTRACT FROM AUTHOR]

The noncharacterized gene previously proposed as the D-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from D-fructose to D-psicose. The enzyme exhibited maximal activity at 50°C and pH 8.0 with Mn2+. The turnover number (kcat) and catalytic efficiency (kcat/Km) of the enzyme for D-psicose were markedly higher than those for D-tagatose, suggesting that the enzyme is not D-tagatose 3-epimerase but D-psicose 3-epimerase. The equilibrium ratio between D-psicose and D-fructose was 32:68 at 30°C. D-Psicose was produced at 230 g/liter from 700-g/liter D-fructose at 50°C after 100 min, corresponding to a conversion yield of 32.9%. [ABSTRACT FROM AUTHOR]

Two enzymes, L-arabinose isomerase and mannose-6-phosphate isomerase, from Geobacillus thermodenitrificans produced 118 g/liter L-ribose from 500 g/liter L-arabinose at pH 7.0, 70°C, and 1 mM Co2+ for 3 h, with a conversion yield of 23.6% and a volumetric productivity of 39.3 g liter-1 h-1. [ABSTRACT FROM AUTHOR]