Production of D-Allose From D-Allulose Using Commercial Immobilized Glucose Isomerase.

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.

Radioisotope identification using an energy-weighted algorithm with a proof-of-principle radiation portal monitor based on plastic scintillators.

In this study, we validated the feasibility of an energy weighted algorithm that highlights a characteristic area including the Compton edge as a single peak in a proof-of-principle radiation portal monitor system with a plastic scintillator measuring 50 × 100 × 5 cm3. We measured the energy weighted spectra with steel shielding and the dynamic movements of the 137Cs and 60Co sources. The results showed that the peak locations of each source could be identified under shielded or dynamic motion conditions, each within a maximum difference of 0.08 MeV.

Cloning and characterization of α-L-rhamnosidase from Chloroflexus aurantiacus and its application in the production of isoquercitrin from rutin.

OBJECTIVE: This study was conducted to characterize recombinant α-L-rhamnosidase from Chloroflexus aurantiacus and apply the enzyme in the production of isoquercitrin from rutin. RESULTS: 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. CONCLUSIONS: 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.

Silver-Stained Fibrin Zymography: Separation of Proteases and Activity Detection Using a Single Substrate-Containing Gel.

Silver-stained fibrin zymography for separation of protease bands and activity detection using a single substrate gel was designed. The method takes advantage of the nano-scale sensitivity of both zymography and silver staining. After sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in a gel containing fibrin (protease substrate), the gel was incubated in enzyme reaction buffer and the zymogram gel was silver-stained. Bands with protease activity were stained with silver in clear areas where the protein substrate had been degraded. The molecular sizes of proteases were accurately determined.

Effect of high hydrostatic pressure treatment on isoquercetin production from rutin by commercial α-L-rhamnosidase.

OBJECTIVES: To optimize conversion of rutin to isoquercetin by commercial α-L-rhamnosidase using high hydrostatic pressure (HHP). RESULTS: The de-rhamnosylation activity of α-L-rhamnosidase for isoquercetin production was maximal at pH 6.0 and 50 °C using HHP (150 MPa). The enzyme showed high specificity for rutin. The specific activity for rutin at HHP was 1.5-fold higher than that at atmospheric pressure. The enzyme completely hydrolysed 20 mM rutin in tartary buckwheat extract after 2 h at HHP, with a productivity of 10 mM h(-1). The productivity and conversion were 2.2- and 1.5-fold higher at HHP than at atmospheric pressure, respectively. CONCLUSIONS: This is the first report concerning the enzymatic hydrolysis of isoquercetin in tartary buckwheat at HHP.

Validation of energy-weighted algorithm for radiation portal monitor using plastic scintillator.

To prevent illicit tracking of radionuclides, radiation portal monitor (RPM) systems employing plastic scintillators have been used in ports and airports. However, their poor energy resolution makes the discrimination of radioactive material inaccurate. In this study, an energy weight algorithm was validated to determine (133)Ba, (22)Na, (137)Cs, and (60)Co by using a plastic scintillator. The Compton edges of energy spectra were converted to peaks based on the algorithm. The peaks have a maximum error of 6% towards the theoretical Compton edge.

Production of d-psicose from d-fructose by whole recombinant cells with high-level expression of d-psicose 3-epimerase from Agrobacterium tumefaciens.

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.

Toxicity and dose determination of quillaja saponin, aluminum hydroxide and squalene in olive flounder (Paralichthys olivaceus).

Adjuvants are substances added to vaccines to enhance the immune response of a given antigen. Most of the adjuvants are toxic at certain doses, and toxicity varies in different species. Moreover, there are no standard dosage limits set for adjuvant use in fish vaccines. We evaluated the acute toxicity, serum enzymes (AST/ALT) indicating hepatic injury and histopathological changes due to intra-peritoneal administration of different concentrations of a panel of adjuvants including quillaja saponin, aluminum hydroxide, squalene emulsion and Freund's incomplete adjuvant (FIA) with a dose ranging study of saponin (500, 160, 50, 16 and 5µgfish(-1)), aluminum hydroxide (5000, 1600, 500, 160 and 50µgfish(-1)), squalene emulsion (20, 10 and 5%), and FIA to determine the acceptable dosage for vaccination in olive flounder (Paralichthys olivaceus) fingerlings measuring 4.66±0.41g, 8.47±0.42cm. Saponin was highly toxic with a LD50 of approximately 105µgfish(-1) (22.4mgkg(-1)) causing severe histological damage and AST level was high at dose above 16µgfish(-1) and ALT, specific for liver damage was high only at 160µgfish(-1) (11U/L) and was safe at 5µgfish(-1). Aluminum hydroxide was toxic at 5000µgfish(-1) and was acceptable at dose below 1600µgfish(-1) with moderate histology and AST/ALT levels similar with control. Squalene emulsion showed increased inflammation at 20% and 10% emulsions and the inflammatory response was mild at a concentration of 5% oil emulsion and AST/ALT levels being similar to control in 10% and 5% emulsions and elevated in 20% on both sampling days. FIA was not lethal, but induced severe inflammation at injection site and around blood vessels. In comparison to FIA, saponin found to be safe at dose of 5µgfish(-1), aluminum hydroxide below 1600µgfish(-1), and squalene at 5% emulsion and could be accepted for vaccination studies. These results provide an insight for the selection of safer dose of adjuvants for intra-peritoneal vaccination of olive flounder.

Characterization of a recombinant mannobiose 2-epimerase from Spirochaeta thermophila that is suggested to be a cellobiose 2-epimerase.

A purified recombinant enzyme from Spirochaeta thermophila, that is suggested to be a cellobiose 2-epimerase, was a 47 kDa monomer with a specific activity of 29.2 U min(-1) for mannobiose. The epimerization activity of the recombinant enzyme for mannobiose was maximal at pH 7.0 and 60 °C with a half-life of 124 h. The enzyme exhibited a higher epimerization activity for mannose or the mannose moiety at the reducing end of ß- and α-1,4-glycosyl-mannose than for glucose or the glucose moiety of ß- and α-1,4-glycosyl-glucose. The enzyme was identified as a mannobiose 2-epimerase by evaluating its substrate specificity with not only glucose-containing sugars but also mannose-containing sugars. The activities of the reported cellobiose 2-epimerases from Caldicellulosiruptor saccharolyticus, Dictyoglomus turgidum and Ruminococcus marinus for mannobiose were higher than those for cellobiose, strongly suggesting that these enzymes are not cellobiose 2-epimerases but are mannobiose 2-epimerases.

A screening method for ß-glucan hydrolase employing Trypan Blue-coupled ß-glucan agar plate and ß-glucan zymography.

A new screening method for ß-(1,3-1,6) glucan hydrolase was developed using a pure ß-glucan from Aureobaisidum pullulans by zymography and an LB-agar plate. Paenibacillus sp. was screened as a producer a ß-glucan hydrolase on the Trypan Blue-coupled ß-glucan LB-agar plate and the activity of the enzyme was analyzed by SDS-ß-glucan zymography. The ß-glucan was not hydrolyzed by Bacillus spp. strains, which exhibit cellulolytic activity on CMC zymography. The gene, obtaining by shotgun cloning and encoding the ß-glucan hydrolase of Paenibacillus sp. was sequenced.

Antiobesity activity of fermented Angelicae gigantis by high fat diet-induced obese rats.

This study aimed to evaluate the effects of Monascus purpureus-fermented Angelicae gigantis Radix (FAG) on body weight gain, visceral fat accumulation, biochemical markers of obesity, and the mRNA expression levels of various genes involved in adipogenesis in a high-fat-diet (HFD)-induced rat model of obesity. Effect of nodakenin isolated from Angelicae gigantis on 3T3-L1 preadipocyte differentiation was also investigated in vitro. Male Sprague-Dawley rats were randomly divided into five groups (n = 6 per group) based on five dietary categories: HFD control, HFD + 2.5% (w/w) AG, HFD + 5% AG, HFD + 2.5% FAG, and HFD + 5% FAG. Present study investigated nodakenin isolated from AG and FAG roots by measuring fat accumulation in 3T3-L1 preadipocyte using Oil Red O staining. FAG administration effectively lowered the body weight gain, visceral fat accumulation, and hepatic and serum lipid and leptin concentrations in obese rats. In addition, FAG administration significantly reduced the mRNA expression levels of adipose tissue genes encoding adipocyte protein 2 (aP2), peroxisome proliferator-activated receptor γ 2 (PPARγ2), and CCAT/enhancer-binding protein α (C/EBPα) as compared with HFD group. Furthermore, nodakenin reduced the fat accumulation in differentiated 3T3-L1 adipocytes in a dose-dependent manner. FAG ameliorates HFD-induced obesity, probably by modulating multiple genes associated with adipogenesis in the visceral fat tissue of rats. Accordingly, fermented Angelicae gigantis may be an ideal candidate for obesity relief.

Characterization of a recombinant cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus and its application in the production of mannose from glucose.

A putative N-acyl-D-glucosamine 2-epimerase from Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was identified as a cellobiose 2-epimerase by the analysis of the activity for substrates, acid-hydrolyzed products, and amino acid sequence. The cellobiose 2-epimerase was purified with a specific activity of 35 nmol min(-1) mg(-1) for D-glucose with a 47-kDa monomer. The epimerization activity for D-glucose was maximal at pH 7.5 and 75°C. The half-lives of the enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 142, 71, 35, 18, and 4.6 h, respectively. The enzyme catalyzed the epimerization reactions of the aldoses harboring hydroxyl groups oriented in the right-hand configuration at the C2 position and the left-hand configuration at the C3 position, such as D-glucose, D-xylose, L-altrose, L-idose, and L-arabinose, to their C2 epimers, such as D-mannose, D-lyxose, L-allose, L-gulose, and L-ribose, respectively. The enzyme catalyzed also the isomerization reactions. The enzyme exhibited the highest activity for mannose among monosaccharides. Thus, mannose at 75 g l(-1) and fructose at 47.5 g l(-1) were produced from 500 g l(-1) glucose at pH 7.5 and 75°C over 3 h by the enzyme.

Toll-like receptors and interferon associated immune factors in viral haemorrhagic septicaemia virus-infected olive flounder (Paralichthys olivaceus).

Pattern recognition receptor (PRR) toll-like receptors (TLRs), antiviral agent interferon (IFN) and the effector IFN stimulated genes (ISGs) play pivotal role in antiviral innate immunity of a host. The present in-vivo experiment was conducted to investigate the role of these innate immune factors in early phase as well as during recovery of viral haemorrhagic septicaemia virus (VHSV) infection by quantitative real-time reverse transcriptase polymerase chain reaction. A less lethal VHSV infection was generated in olive flounder (Paralichthys olivaceus) and was sampled at 3, 6, and 12h post infection (hpi), and 1, 2, 4, and 7 days post infection (dpi). At 3 hpi, the VHSV N gene was detected in three out of five fish and all five fish showed a relative fold increase of TLR 2, TLR 7, interleukin 8 (IL 8), IFN regulatory factor 3 (IRF 3), IRF 7, and ISG 15. Viral copies rapidly increased at 12 hpi then remained high until 2 dpi. When viral copy numbers were high, a higher expression of immune genes IL 1ß, IRF 3, IRF 7, Type I IFN, ISG 15 and Mx was observed. Viral copies were drastically reduced in 4 and 7 dpi fish, and also the immune response was considerably reduced but remained elevated, except for ISG 15 which found equal to control in 7 dpi fish. A high degree of correlation was observed between immune genes and viral copy number in each of the sampled fish at 12 hpi. A fish with ascites sampled at 7 dpi displayed high viral copy but under-expressed immune genes except for Mx. When viral copies were high at 1 and 2 dpi, both TLR 2 and TLR 7 were down-regulated, perhaps indicating immune suppression by the virus. The quick and prolonged elevated expression of the immune genes indicates their crucial role in survival of host against VHSV.

Production of aglycon protopanaxadiol via compound K by a thermostable ß-glycosidase from Pyrococcus furiosus.

The production of compound K and aglycon protopanaxadiol (APPD) from ginsenoside Rd and ginseng root extract was performed using a recombinant ß-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. ß-Glycosidase converted Rb(1), Rb(2), 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(-1) h(-1) at 1 h and was hydrolyzed completely to APPD with 364 mg l(-1) h(-1) after 5 h. Rb(1), Rb(2), 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(-1) h(-1) at 1.2 h and were hydrolyzed completely to APPD with 300 mg l(-1) h(-1) after 6 h. The concentrations and productivities of compound K and APPD in the present study are the highest ever reported.

Characterization of a recombinant thermostable L: -rhamnose isomerase from Thermotoga maritima ATCC 43589 and its application in the production of L-lyxose and L-mannose.

A putative L-rhamnose isomerase (RhaA) from Thermotoga maritima was purified with a specific activity of 55 U/mg by His-Trap affinity chromatography. The native enzyme was estimated as a 46 kDa tetramer by gel filtration chromatography. The half-lives of the enzyme at 75, 80, 85, 90 and 95°C were 773, 347, 187, 118, and 65 h, respectively, indicating that it is the most thermostable of all RhaAs. Under the optimum conditions of pH 8.0, 85°C, and 1 mM Mn(2+), RhaA with 100 U enzyme/ml converted 500 L-xylulose/l to 225 g/l L-lyxose after 3 h, and converted 500 L-fructose/l to 175 g/l L-mannose after 5 h.

Structure-based annotation of a novel sugar isomerase from the pathogenic E. coli O157:H7.

Prokaryotes can use a variety of sugars as carbon sources in order to provide a selective survival advantage. The gene z5688 found in the pathogenic Escherichia coli O157:H7 encodes a "hypothetical" protein of unknown function. Sequence analysis identified the gene product as a putative member of the cupin superfamily of proteins, but no other functional information was known. We have determined the crystal structure of the Z5688 protein at 1.6 A resolution and identified the protein as a novel E. coli sugar isomerase (EcSI) through overall fold analysis and secondary-structure matching. Extensive substrate screening revealed that EcSI is capable of acting on d-lyxose and d-mannose. The complex structure of EcSI with fructose allowed the identification of key active-site residues, and mutagenesis confirmed their importance. The structure of EcSI also suggested a novel mechanism for substrate binding and product release in a cupin sugar isomerase. Supplementation of a nonpathogenic E. coli strain with EcSI enabled cell growth on the rare pentose d-lyxose.

Substrate specificity of a recombinant D-lyxose isomerase from Providencia stuartii for monosaccharides.

The specific activity and catalytic efficiency (k(cat)/K(m)) of the recombinant putative protein from Providencia stuartii was the highest for D-lyxose among the aldose substrates, indicating that it is a D-lyxose isomerase. Gel filtration analysis suggested that the native enzyme is a dimer with a molecular mass of 44 kDa. The maximal activity for D-lyxose isomerization was observed at pH 7.5 and 45 degrees C in the presence of 1 mM Mn(2+). The enzyme exhibited high isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left-hand configuration, such as D-lyxose, D-mannose, L-ribose, D-talose, and L-allose (listed in decreasing order of activity). The enzyme exhibited the highest activity for D-xylulose among all pentoses and hexoses. Thus, D-lyxose was produced at 288 g/l from 500 g/l D-xylulose by D-lyxose isomerase at pH 7.5 and 45 degrees C for 2 h, with a conversion yield of 58% and a volumetric productivity of 144 g l(-1) h(-1). The observed k(cat)/K(m) (920 mM(-1) s(-1)) of P. stuartiid-lyxose isomerase for D-xylulose is higher than any of the k(cat)/K(m) values previously reported for sugar and sugar phosphate isomerases with monosaccharide substrates. These results suggest that the enzyme will be useful as an industrial producer of D-lyxose.

Mannose production from fructose by free and immobilized D-lyxose isomerases from Providencia stuartii.

A recombinant D-lyxose isomerase from Providencia stuartii was immobilized on Duolite A568 beads which gave the highest conversion of D-fructose to D-mannose among the various immobilization beads evaluated. Maximum activities of both the free and immobilized enzymes for fructose isomerization were at pH 7.5 and 45 degrees C in the presence of 1 mM Mn(2+). Enzyme half-lives were 14 and 30 h at 35 degrees C and 3.4 and 5.1 h at 45 degrees C, respectively. The immobilized enzyme in 300 g fructose/l (replaced hourly), produced 75 g mannose/l at 35 degrees C = 25% (w/w) yield with a productivity of 75 g mannose l(-1) h(-1) after 23 cycles.

Biotransformation of ginsenosides by hydrolyzing the sugar moieties of ginsenosides using microbial glycosidases.

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 beta-D-glycosidases are proposed as feasible methods for use in industrial processes.

Hydrophobicity of residue 108 specifically affects the affinity of human beta-carotene 15,15'-monooxygenase for substrates with two ionone rings.

The Lys residue at position 108 of human beta-carotene 15,15'-monooxygenase is located on the outside surface of the active tunnel of the enzyme. Hydrophobic mutations (K108F and K108L) at this position substantially decreased the affinity of the enzyme for substrates with ionone rings at both ends, such as alpha-carotene, beta-carotene, and beta-cryptoxanthine. In contrast, these mutations had little effect on the affinity of the enzyme for substrates with one ionone ring and one open-chain end, such as beta-apo-4'-carotenal and beta-apo-8'-carotenal. The residue 108 may be related to the indirect interaction with the second ionone ring of the substrates with two ionone rings.