Production of l-rhamnulose,a rare sugar,from l-rhamnose using commercial immobilized glucose isomerase

Abstract

A commercial immobilized d-glucose isomerase from Streptomyces murines (Sweetzyme) was used to produce l-rhamnulose from l-rhamnose in a packed-bed reactor. The optimal conditions for l-rhamnulose production from l-rhamnose were determined as pH 8.0, 60?°C, 300?g L^?1 l-rhamnose as a substrate, and 0.6?h^?1 dilution rate. The half-life of the immobilized enzyme at 60?°C was 809?h. Under the optimal conditions, the immobilized enzyme produced an average of 135?g L^?1 l-rhamnulose from 300?g L^?1 l-rhamnose after 16 days at pH 8.0, 60?°C, and 0.6?h^?1 dilution rate, with a productivity of 81?g/L/h and a conversion yield of 45% in a packed-bed reactor.     

Optimization of the 503 antigen induction strategy of Leishmania infantum chagasi expressed in Escherichia coli M15

Abstract

Leishmaniosis is a complex of diseases that can be fatal, if not given proper attention. Despite its relevance in the public health system, there is no vaccine capable of preventing the disease in humans so far and its treatment is expensive and aggressive to human health. The present study aims to optimize the induction parameters of the 503 Leishmania i. chagasi antigen expressed in recombinant Escherichia coli M15. The induction at different cell densities was evaluated in order to analyze the influence of the induction time on the yield of the protein of interest. In this segment, lactose and isopropyl-β-d-thiogalactopyranoside (IPTG) were used as inducer molecules, using various concentrations: 0.1?g/L, 1.0?g/L, and 10?g/L for lactose and 20?μM, 100?μM, 500?μM, and 1000?μM for IPTG. The results presented that the concentration of IPTG that obtained the higher antigen levels was that of 100?μM (0.087?g/L), a 10-fold lower concentration than was being previously used in this type of system and for lactose, it was 1?g/L (0.016?g/L). Thus, the induction with 100?μM allowed obtaining the antigen with a concentration 5.6 times higher than the lactose induction maximum concentration.     

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase from Corynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10^?2mm and 10^?1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.     

Studies on d-Glucose-isomerizing Activity of d-Xylose-grown Cells from Bacillus coagulans,Strain HN-68

d-Glucose-isomerizing enzyme has been extracted in high yield from d-xylose-grown cells of Bacillus coagulans, strain HN-68, by treating with lysozyme, and purified approximately 60-fold by manganese sulfate treatment, fractionation with ammonium sulfate and chromatography on DEAE-Sephadex column. The purified d-glucose-isomerizing enzyme was homogeneous in polyacrylamide gel electrophoresis and ultracentrifugation and was free from d-glucose-6-phosphate isomerase. Optimum pH and temperature for activity were found to be pH 7.0 and 75°C, respectively. The enzyme required specifically Co⁺⁺ with suitable concentration for maximal activity being 10^?3 m. In the presence of Co⁺⁺, enzyme activity was inhibited strongly by Cu⁺⁺, Zn⁺⁺, Ni⁺⁺, Mn⁺⁺ or Ca⁺⁺. At reaction equilibrium, the ratio of d-fructose to d-glucose was approximately 1.0. The enzyme catalyzed the isomerization of d-glucose, d-xylose and d-ribose. Apparent Michaelis constants for d-glucose and d-xylose were 9×10^?2 m and 7.7×10^?2 m, respectively.     

Radiation Chemistry of Foods

An intermediate radical, ?H₂OH, was produced in aqueous methanol solution containing nitrous oxide by γ-irradiation. Yields of ethylene glycol and formaldehyde, the major and the minor product from ?H₂OH, respectively, changed on the addition of some solutes. Cysteine lowered the both product yields to zero even at a low concentration of 5 × 10^?5m. Oxygen of low concentrations (2.5~7.5 × 10^?5 m) changed effectively the major product from ethylene glycol to formaldehyde. k _(CySH+?H2OH)/k_(O2+?H2OH) was calculated as 0.5.

Ascorbic acid (5 × 10^?5 m) lowered ethylene glycol yield to 48%, cystine (10^?3m) to 15%, methionine (10^?3m) to 31%, histidine (10^?3m) to 42%, tryptophan (10^?3m) 46%, tyrosine (10^?3m) to 77%, phenylalanine (10^?3m) to 73%, hypoxanthine (10^?3m) to 37%, adenine (10^?3m) to 52%, uracil (10^?3m) to 20%, thymine (10^?3m) to 10%, cytosine (10^?3 m) to 49%, rutin (10^?3m) to 23%, pyrogallol (10^?3m) to 41%, and gallic acid (10^?3m) to 78% of the control. These results suggest that the reactions of the secondary radicals such as ?H₂OH perform an important role in material change of foods irradiated with γ rays.     

Properties of Tyrosinase from Pseudomonas melanogenum

The properties of the tyrosinase from Pseudomonas melanogenum was investigated with the crude enzyme preparation. Optimum temperature and pH of the enzyme were 23°C and 6.8, respectively. l-Tyrosine, d-tyrosine, m-tyrosine, N-acetyl-l-tyrosine and l-DOPA were utilized as a substrate by the enzyme. The value for Km obtained were as follows: l-tyrosine 6.90 × 10^?4 m, d-tyrosine 1.43 ×10^?3 m and l-DOPA 9.90 × 10^?4 m. The enzyme was inhibited by chelating agents of Cu²⁺ l-cysteine, l-homocysteine, thiourea and diethyl-dithiocarbamate and the inhibition was completely reversed by the addition of excess Cu²⁺ From these results it is concluded that the enzyme is a copper-containing oxidase.     

Properties and Sugar Components of Asterosaponin A Isolated from Starfish

The starfish saponin previously reported by the present authors as the toxic principle of Asterias amurensis Lutken was found to be separable into six components. The main component was designated asterosaponin A. It shows an ultraviolet absorption at 244 mµ indicative of a heteroannular diene. The infrared spectrum shows a band at 1640 cm^?1. Acid hydrolysis of asterosaponin A yields an aglycone, one molecule of sulfuric acid, and each two molecules of d-quinovose and d-fucose. In addtion to the above sugars, d-galactose and d-xylose were isolated from the hydrolyzates of the crude saponin mixture.     

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

Amino Acid Metabolism in Microorganisms

3-Methylthiopropylamine (MTPA) formation from l-methionine in Streptomyces sp. K37 was studied in detail. The reaction was confirmed to be catalyzed by the decarboxylase of l-methionine. The properties of the enzyme were studied in detail using acetone dried cells or cell-free extract. The enzyme was specific for l-methionine. Pyridoxal phosphate stimulated the reaction and protected the enzyme against heat inactivation. The optimum pH for the reaction was 6.0~8.0 and the optimum temperature was about 40°C. Carbonyl reagents (10^?2~10^?3 m) inhibited the reaction completely, and silver nitrate and mercuric chloride (10^?3~10^?4 m) markedly inhibited the reaction. Km value for the reaction was 1.21 × 10^?5 m. l-Methionine assay using the decarboxylase was attempted and was found to be applicable to practical use.     

Crystalline d-Mannitol:NAD Oxidoreductase from Leuconostoc mesenteroides

The crystalline d-mannitol dehyrogenase (d-mannitol:NAD oxidoreductase, EC 1.1.1.67) catalyzed the reversible reduction of d-fructose to d-mannitol. d-Sorbitol was oxidized only at the rate of 4% of the activity for d-mannitol. The enzyme was inactive for all of four pentitols and their corresponding 2-ketopentoses. The apparent optimal pH for the reduction of d-fructose or the oxidation of d-mannitol was 5.35 or 8.6, respectively. The Michaelis constants were 0.035 m for d-fructose and 0.020 m for d-mannitol. The enzyme was also found to be specific for NAD. The Michaelis constans were 1 × 10^?5 m for NADH₂ and 2.7 × 10^?4 m for NAD.     

Bacterial Racemization of α-Amino-ε-caprolactam

1. Several bacteria were isolated from soil which grew on both d- and l-aminolactam and whose cells had an activity to racemize them. They were identified as Achromobacter obae nov. sp., Achr. cycloclastes, Alcaligenes faecalis and Flavobacterium arborescens.

2. Racemization of d- and l-aminolactam was investigated using the lyophilized cells of Achr. obae nov. sp. The optimum pH value of the reaction was about 8.0. The racemizing activity was completely inhibited by 10^?4 m hydroxylamine, and the inhibition was removed by 10^?4 m pyridoxal phosphate. Five percent d- and l-aminolactam solutions were completely racemized with a concomitant slight formation of l-lysine.     

Metabolic syndrome is associated to high-sensitivity cardiac troponin T elevation

Introduction: Metabolic syndrome (MetS) and high-sensitivity cardiac troponin T (hs-TnT) are associated with higher risk for cardiovascular diseases (CVD). Our aim was to assess the relation between hs-TnT elevation and MetS in a general population sample.

Materials and methods: Individuals participating in an annual health survey program between 2010 and 2016 were included in the study. Blood samples including hs-TnT levels were collected. The study population was divided into three groups based on hs-TnT levels – undetectable (<5?ng/L), intermediate (5–14?ng/L) and elevated (>14?ng/L).

Results: A total of 5994 subjects were included in the study, the mean age was 48.5 and 4336 (72%) were males. Compared with subjects with undetectable hs-TnT the prevalence of MetS was higher in those with detectable and elevated levels – 392 (10%) vs. 270 (15%) and 51 (33%), respectively (p?<?0.001). In a multivariate model adjusted for age, gender and multiple co-morbidities, the number of MetS components and presence of MetS were significantly associated with an increased risk for detectable hs-TnT levels (OR?=?1.02 {for each component}; 95% CI [1.00–1.05], p?=?0.04) and (OR?=?1.13; 95% CI [1.07–1.2], p?<?0.001) respectively. Only the waist, glucose and hypertension components of the MetS were significantly associated with elevated troponin.

Conclusions: The MetS and its distinct components have a cumulative impact on hs-TnT levels in apparently healthy subjects.     

Inhibition of l-Alanine Adding Enzyme by Glycine

l-Alanine adding enzymes from Bacillus subtilis and Bacillus cereus which catalyzed l-alanine incorporation into UDPMurNAc were partially purified and the properties of the enzymes were examined. The enzyme from B. subtilis was markedly stimulated by reducing agents including 2-mercaptoethanol, dithiothreitol, glutathione and cysteine. Mn²⁺ and Mg²⁺ activated l-alanine adding activity and their optimal concentrations were 2 to 5 mm and 10 mm, respectively. The optimum pH was 9.5 and the Km for l-alanine was 1.8×10^?4m. l-Alanine adding reaction was strongly inhibited by p-chloromercuribenzoate and N-ethyl-maleimide. Among glycine, l- and d-amino acids and glycine derivatives, glycine was the most effective inhibitor of the l-alanine adding reaction. The enzyme from B. cereus was more resistant to glycine than that from B. subtilis. Glycine was incorporated into UDPMurNAc in place of l-alanine, and the Ki for glycine was 4.2×l0^?3m with the enzyme from B. subtilis. From these data, the growth inhibition of bacteria by glycine is discussed.     

Chelate-assisted phytoextraction of lead using Fagopyrum esculentum: laboratory vs. field experiments

Abstract

The development of more sustainable remediation techniques has been receiving greater attention, as an alternative to soil excavation plan in urban gardens. An in situ phytoextraction experiment with buckwheat (Fagopyrum esculentum) was performed with a 5?mmol kg^?1 citric acid (CA) application. Joint experiments under laboratory conditions were conducted using various cultivars of F. esculentum in two soils with a Pb contamination of either geogenic or anthropogenic origin and various chelate concentrations. Results show that a minimum dose of 50?mmol kg^?1 of CA is required to lower soil pH and raise the concentration of mobile Pb–CaCl₂ for both soils. Consequently, Pb shoot uptake is increased from 6.3 to 8.9 times depending on soil type. Phytoextraction efficiency is found to be 1.3 to 2.0 times higher in the anthropogenic contaminated soil than in the soil with geogenic Pb. A scale effect has also been identified since Pb root accumulation under laboratory conditions was 2.4 times higher than in the field experiment. Despite an increase in the Pb extraction rate with CA, buckwheat appears to lack the efficiency needed to remove Pb in moderately contaminated soils. The calculated remediation period would last 166?years to remove the mobile Pb fraction.     

Structures of the Oligosaccharides from the Enzymic Hydrolyzate of Rice-straw Arabinoxylan by a Xylanase of Aspergillus niger

A trisaccharide consisting of two d-xylose units and one l-arabinose unit, and a tetrasaccharide consisting of three d-xylose units and one l-arabinose unit were isolated from the hydrolyzate of rice-straw arabinoxylan by the xylanase I produced by Asp. niger.

The structures of the trisaccharide and the tetrasaccharide were determined to be 3¹-α-l-arabinofuranosylxylobiose ([α]_d^? 80°) and 3¹-α-l-arabinofuranosylxylotriose ([α]_d^? 84°), respectively, by chemical and enzymic methods.

According to the structures of two arabinose-xylose mixed oligosaccharides, it was shown that the rice-straw arabinoxylan is composed of chain of 1,4-linked βd-xylopyranose residues and some of xylose residues have side-chain of 1,3-linked α-l-arabinofuranose.     

Regulatory Properties of Chorismate Mutase from Corynebacterium glutamicum

Regulatory properties of chorismate mutase from Corynebacterium glutamicum were studied using the dialyzed cell-free extract. The enzyme activity was strongly feedback inhibited by l-phenylalanine (90% inhibition at 0.1~1 mm) and almost completely by a pair of l-tyrosine and l-phenylalanine (each at 0.1~1 mm). The enzyme from phenylalanine auxotrophs was scarcely inhibited by l-tyrosine alone but the enzyme from a wild-type strain or a tyrosine auxotroph was weakly inhibited by l-tyrosine alone (40~50% inhibition, l-tyrosine at 1 mm). The enzyme activity was stimulated by l-tryptophan and the inhibition by l-phenylalanine alone or in the simultaneous presence of l-tyrosine was reversed by l-tryptophan. The Km value of the reaction for chorismate was 2.9 } 10^?3 m. Formation of chorismate mutase was repressed by l-phenylalanine. A phenylalanine auxotrophic l-tyrosine producer, C. glutamicum 98–Tx–71, which is resistant to 3-amino-tyrosine, p-aminophenylanaine, p-fluorophenylalanine and tyrosine hydroxamate had chorismate mutase derepressed to two-fold level of the parent KY 10233. The enzyme in C. glutamicum seems to have two physiological roles; one is the control of the metabolic flow to l-phenylalanine and l-tyrosine biosynthesis and the other is the balanced partition of chorismate between l-phenylalanine-l-tyrosine biosynthesis and l-tryptophan biosynthesis.     

Purification and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (K_m=12.5 mM), N-acetyl (K_m=2.52 mM), N-propionyl (K_m=0.194 mM), N-butyryl (K_m=0.033 mM), and N-glycyl (K_m =1.11 mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg²⁺, Ni²⁺, Cu²⁺) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.     

The Studies on the Action of Yeast Hexokinase upon the α- and β-Diasteromers of d-Glucose

The yeast hexokinase is highly specific for α-isomer of d-glucose. The relative rate of phosphorylation of β-d-glucose, catalyzed by the purified yeast hexokinase, is observed to be 60~70 (α-d-glucose=100). The average Michaelis constants of yeast hexokinase are found to be 1.8 × 10^?4 and 2.4 × 10^?4 for α-d-glucose and (β-d-glucose respectively, therefore the difference between the two constants is considered to be negligible.     

Syntheses of Several β-l-Rhamnopyranosides

To investigate the substrate specificity of β-l-rhamnosidase, the following β-l-rhamnopyranosides were synthesized: 1-(β-l-rhamnopyranosyl)-dl-glycerol (1), methyl β-l-rhamnopyranoside (2), methyl 2-O-(β-l-rhamnopyranosyl)-β-d-glucopyranoside (3) and methyl 2-O-β(β-l-rhamnopyranosyl)-α-l-arabinopyranoside (4). The synthesis of 3 was performed using l-quinovose with neighboring group participation, which lead stereoselectively to the β-l-quinovoside. The 2-OH of the l-quinovo-unit was selectively deblocked, oxidized to the keto group, and then stereoselectively reduced, whereby 3 was produced.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

l-Glutamic acid was formed from d-, l-, and dl-PCA with cell-free extract of Pseudomonas alcaligenes ATCC-12815 grown in the medium containing dl-PCA as a sole source of carbon and nitrogen. The enzyme(s) involved in this conversion reaction was distributed in the soluble fraction within the cell and in 0.5 saturated fraction at the fractionation procedure with the saturation of ammonium sulfate. Optimum pH of this enzyme(s) lied at pH 8.5 and optimum temperature was 30°C. Cu (5 × 10^?3 m) inhibited the reaction considerably while Ca or Fe accelerated it. PALP (1×10^?3 m) also gave an enhanced activity to some extent. The enzyme preparation converted dextro-rotatory enan-thiomorph of PCA to its laevo-rotatory one which in turn was not converted to the opposite rotation direction by this enzyme. Furthermore, the preparation did not, if any, show d-glutamic acid racemase activity. Isotopic experiments with using dl-PCA-1-¹⁴C revealed that l-glutamic acid-1-¹⁴C was formed by the cleavage of –CO–NH– bond of pyrrolidone ring of PCA. It was concluded that dl-PCA when assimilated by the present bacterium is at first transformed to l-PCA by the optically isomerizing enzyme and subsequently is cleaved to l-glutamic acid probably by the PCA hydrolysing enzyme.