Characterization of an ethanol‐tolerant 1,4‐β‐xylosidase produced by Pichia membranifaciens

Aims: The purification and biochemical properties of the 1,4‐β‐xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol‐tolerant 1,4‐β‐xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G‐100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS‐PAGE. The activity of 1,4‐β‐xylosidase was optimum at pH 6·0 and at 35°C. The activity had a K_m of 0·48 ± 0·06 mmol l^?1 and a V_max of 7·4 ± 0·1 μmol min^?1 mg^?1 protein for p‐nitrophenyl‐β‐d ‐xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4‐β‐xylosidase from P. membranifaciens to be used in the bioethanol production process.     

Hydrolytic properties of a thermostable α‐l‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Aims: To characterize of a thermostable recombinant α‐l ‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l ‐arabinose. Methods and Results: A recombinant α‐l ‐arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi‐Trap anion exchange chromatography with a specific activity of 28·2 U mg^?1. The native enzyme was a 58‐kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α‐l ‐arabinofuranosidases were completely conserved in α‐l ‐arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5·5 and 80°C with a half‐life of 49 h at 75°C. Among aryl‐glycoside substrates, the enzyme displayed activity only for p‐nitrophenyl‐α‐l ‐arabinofuranoside [maximum k_cat/K_m of 220 m(mol l^?1)^?1 s^?1] and p‐nitrophenyl‐α‐l ‐arabinopyranoside. This substrate specificity differs from those of other α‐l ‐arabinofuranosidases. In a 1 mmol l^?1 solution of each sugar, arabino‐oligosaccharides with 2–5 monomer units were completely hydrolysed to l ‐arabinose within 13 h in the presence of 30 U ml^?1 of enzyme at 75°C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino‐oligosaccharides of α‐l ‐arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l ‐arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino‐oligosaccharides performed by thermostable α‐l ‐arabinofuranosidase.     

Molybdate reduction by Pseudomonas sp. strain DRY2

Aims: To isolate and characterize a potent molybdenum‐reducing bacterium. Methods and Results: A minimal salt medium supplemented with 10 mmol l^?1 molybdate, glucose (1·0%, w/v) as a carbon source and ammonium sulfate (0·3%, w/v) as a nitrogen source was used in the screening process. A molybdenum‐reducing bacterium was isolated and tentatively identified as Pseudomonas sp. strain DRY2 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny. Strain DRY2 produced 2·4, 3·2 and 6·2 times more molybdenum blue compared to Serratia marcescens strain DRY6, Enterobacter cloacae strain 48 and Eschericia coli K12, respectively. Molybdate reduction was optimum at 5 mmol l^?1 phosphate. The optimum molybdate concentration that supported molybdate reduction at 5 mmol l^?1 phosphate was between 15 and 25 mmol l^?1. Molybdate reduction was optimum at 40°C and at pH 6·0. Phosphate concentrations higher than 5 mmol l^?1 strongly inhibited molybdate reduction. Inhibitors of electron transport system such as antimycin A, rotenone, sodium azide and cyanide did not inhibit the molybdenum‐reducing enzyme activity. Chromium, copper, mercury and lead inhibited the molybdenum‐reducing activity. Conclusions: A novel molybdenum‐reducing bacterium with high molybdenum reduction capacity has been isolated. Significance and Impact of the Study: Molybdenum is an emerging global pollutant that is very toxic to ruminants. The characteristics of this bacterium suggest that it would be useful in the bioremediation of molybdenum pollutant.     

6‐Methyl 3‐chromonyl 2,4‐thiazolidinedione/2,4‐imidazolidinedione/2‐thioxo‐imidazolidine‐4‐one compounds: novel scavengers of reactive oxygen species

The benefits of antioxidants on human health are usually ascribed to their potential ability to remove reactive oxygen species providing protection against oxidative stress. In this paper the free radicals scavenging activities of nine 6‐methyl 3‐chromonyl derivatives (CMs) were evaluated for the first time by the chemiluminescence, electron paramagnetic resonance, spin trapping and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) methods. The total antioxidant capacity was also measured using a ferric‐ferrozine reagent. Compounds having a hydrogen atom at the N3‐position of the β‐ring were effective in quenching CL resulted from the KO₂/18‐crown‐6‐ether system (a source of superoxide anion radical, ) in a dose‐dependent manner over the range of 0.05–1 mmol/L [IC₅₀ ranged from 0.353 (0.04) to 0.668 (0.05) mmol/L]. The examined compounds exhibited a significant scavenging effect towards hydroxyl radicals (HO^? HO^?), produced by the Fenton reaction, and this ranged from 24.0% to 61.0%, at the concentration of 2.5 mmol/L. Furthermore, the compounds examined were also found to inhibit DPPH^? and this ranged from 51.9% to 97.4% at the same concentration. In addition, the use of the total antioxidant capacity assay confirmed that CM compounds are able to act as reductants. According to the present study, CM compounds showed effective in vitro free radical scavenging activity and may be considered as potential therapeutics to control diseases of oxidative stress‐related etiology. Copyright © 2013 John Wiley & Sons, Ltd.     

Metabolic pathway of α‐ketoglutarate in Lactobacillus sanfranciscensis and Lactobacillus reuteri during sourdough fermentation

Aim: To identify metabolites of α‐ketoglutarate (α‐KG) in Lactobacillus sanfranciscensis and Lactobacillus reuteri in modified MRS and sourdough. Methods and Results: Lactobacillus sanfranciscensis and L. reuteri were grown with additional α‐KG in mMRS and in wheat sourdough. In mMRS, α‐KG was used as an electron acceptor and converted to 2‐hydroxyglutarate (2‐OHG) by both organisms. Production of 2‐OHG was identified by high performance liquid chromatography (HPLC) and confirmed by gas chromatography (GC). Crude cell extracts of L. sanfranciscensis and L. reuteri grown with or without α‐KG exhibited OHG dehydrogenase activity of 6·3 ± 0·3, 2·3 ± 0·9, 1·2 ± 0·2, and 1·1 ± 0·1 mmol l^?1 NADH (min x mg protein)^?1, respectively. The presence of phenylalanine and citrate in addition to α‐KG partially redirected the use of α‐KG from electron acceptor to amino group acceptor. In wheat sourdoughs, α‐KG was predominantly used as electron acceptor and converted to 2‐OHG. Conclusions: Lactobacillus sanfranciscensis and L. reuteri utilize α‐KG as electron acceptor. Alternative use of α‐KG as amino group acceptor occurs in the presence of abundant amino donors and citrate. Significance and Impact of the Study: The use of α‐KG as electron acceptor in heterofermentative lactobacilli impacts the formation of flavour volatiles through the transamination pathway.     

Increased synthesis of α‐tocopherol,paramylon and tyrosine by Euglena gracilis under conditions of high biomass production

Aims: To analyse the production of different metabolites by dark‐grown Euglena gracilis under conditions found to render high cell growth. Methods and Results: The combination of glutamate (5 g l^?1), malate (2 g l^?1) and ethanol (10 ml l^?1) (GM + EtOH); glutamate (7·15 g l^?1) and ethanol (10 ml l^?1); or malate (8·16 g l^?1), glucose (10·6 g l^?1) and NH₄Cl (1·8 g l^?1) as carbon and nitrogen sources, promoted an increase of 5·6, 3·7 and 2·6‐fold, respectively, in biomass concentration in comparison with glutamate and malate (GM). In turn, the production of α‐tocopherol after 120 h identified by LC‐MS was 3·7 ± 0·2, 2·4 ± 0·1 and 2 ± 0·1 mg [g dry weight (DW)]^?1, respectively, while in the control medium (GM) it was 0·72 ± 0·1 mg (g DW)^?1. For paramylon synthesis, the addition of EtOH or glucose induced a higher production. Amino acids were assayed by RP‐HPLC; Tyr a tocopherol precursor and Ala an amino acid with antioxidant activity were the amino acids synthesized at higher concentration. Conclusions: Dark‐grown E. gracilis Z is a suitable source for the generation of the biotechnologically relevant metabolites tyrosine, α‐tocopherol and paramylon. Significance and Impact of the Study: By combining different carbon and nitrogen sources and inducing a tolerable stress to the cell by adding ethanol, it was possible to increase the production of biomass, paramylon, α‐tocopherol and some amino acids. The concentrations of α‐tocopherol achieved in this study are higher than others reported previously for Euglena, plant and algal systems. This work helps to understand the effect of different carbon sources on the synthesis of bio‐molecules by E. gracilis and can be used as a basis for future works to improve the production of different metabolites of biotechnological importance by this organism.     

Isolation and characterization of butachlor‐catabolizing bacterial strain Stenotrophomonas acidaminiphila JS‐1 from soil and assessment of its biodegradation potential

Aims: Isolation, characterization and assessment of butachlor‐degrading potential of bacterial strain JS‐1 in soil. Methods and Results: Butachlor‐degrading bacteria were isolated using enrichment culture technique. The morphological, biochemical and genetic characteristics based on 16S rDNA sequence homology and phylogenetic analysis confirmed the isolate as Stenotrophomonas acidaminiphila strain JS‐1. The strain JS‐1 exhibited substantial growth in M9 mineral salt medium supplemented with 3·2 mmol l^?1 butachlor, as a sole source of carbon and energy. The HPLC analysis revealed almost complete disappearance of butachlor within 20 days in soil at a rate constant of 0·17 day^?1 and half‐life (t_½) of 4·0 days, following the first‐order rate kinetics. The strain JS‐1 in stationary phase of culture also produced 21·0 μg ml^?1 of growth hormone indole acetic acid (IAA) in the presence of 500 μg ml^?1 of tryptophan. The IAA production was stimulated at lower concentrations of butachlor, whereas higher concentrations above 0·8 mmol l^?1 were found inhibitory. Conclusions: The isolate JS‐1 characterized as Stenotrophomonas acidaminiphila was capable of utilizing butachlor as sole source of carbon and energy. Besides being an efficient butachlor degrader, it substantially produces IAA. Significance and Impact of the Study: The bacterial strain JS‐1 has a potential for butachlor remediation with a distinctive auxiliary attribute of plant growth stimulation.     

Scavenging of reactive oxygen species by N‐substituted indole‐2 and 3‐carboxamides

Indole‐2 and 3‐carboxamides (IDs) are proposed to be selective cyclooxygenase inhibitors. Since cyclooxygenase‐1 may be involved in reactive oxygen species (ROS) production, we hypothesize that these indole derivatives have antioxidative properties. We have employed chemiluminescence (CL) and electron spin resonance (ESR) spin trapping to examine this hypothesis. We report here the results of a study of reactivity of 10 selected indole derivatives towards ROS. The following generators of ROS were applied: potassium superoxide (KO₂) as a source of superoxide radicals (O₂^·?), the Fenton reaction (Co‐EDTA/H₂O₂) for hydroxyl radicals (HO^·), and a mixture of alkaline aqueous H₂O₂ and acetonitrile for singlet oxygen (¹O₂). Hydroxyl radicals were detected as 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) spin adduct, whereas 2,2,6,6‐tetramethyl‐piperidine (TEMP) was used as a detector of ¹O₂. Using the Fenton reaction, 0.5 mmol/L IDs were found to inhibit DMPO‐?H radical formation in the range 7–37%. Furthermore the tested compounds containing the thiazolyl group also inhibited the ¹O₂‐dependent TEMPO radical, generated in the acetonitrile + H₂O₂ system. About 20% inhibition was obtained in the presence of 0.5 mmol/L IDs. 1 mmol/L IDs caused an approximately 13–70% decrease in the CL sum from the O₂^·? generating system (1 mmol/L). The aim of this paper is to evaluate these indole derivatives as antioxidants and their abilities to scavenge ROS. Copyright © 2004 John Wiley & Sons, Ltd.     

Substrate specificity of a recombinant d‐lyxose isomerase from Serratia proteamaculans that produces d‐lyxose and d‐mannose

Aims: Characterization of substrate specificity of a d ‐lyxose isomerase from Serratia proteamaculans and application of the enzyme in the production of d ‐lyxose and d ‐mannose. Methods and Results: The concentrations of monosaccharides were determined using a Bio‐LC system. The activity of the recombinant protein from Ser. proteamaculans was the highest for d ‐lyxose among aldoses, indicating that it is a d‐ lyxose isomerase. The native recombinant enzyme existed as a 54‐kDa dimer, and the maximal activity for d‐ lyxose isomerization was observed at pH 7·5 and 40°C in the presence of 1 mmol l^?1 Mn²⁺. The K_m values for d ‐lyxose, d ‐mannose, d ‐xylulose, and d ‐fructose were 13·3, 32·2, 3·83, and 19·4 mmol l^?1, respectively. In 2 ml of reaction volume at pH 7·5 and 35°C, d ‐lyxose was produced at 35% (w/v) from 50% (w/v) d ‐xylulose by the d‐ lyxose isomerase in 3 h, while d ‐mannose were produced at 10% (w/v) from 50% (w/v) d ‐fructose in 5 h. Conclusions: We identified the putative sugar isomerase from Ser. proteamaculans as a d ‐lyxose isomerase. The enzyme exhibited isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left‐hand configuration. High production rates of d‐ lyxose and d ‐mannose by the enzyme were obtained. Significance and Impact of the Study: A new d‐ lyxose isomerase was found, and this enzyme had higher activity for d ‐lyxose and d ‐mannose than previously reported enzymes. Thus, the enzyme can be applied in industrial production of d ‐lyxose and d ‐mannose.     

Improvement of covalent immobilization procedure of β‐galactosidase from Kluyveromyces lactis for galactooligosaccharides production: Modeling and kinetic study

Galactooligosaccharides (GOS) are prebiotics produced from lactose through an enzymatic reaction. Employing an immobilized enzyme may result in cost reductions; however, the changes in its kinetics due to immobilization has not been studied. This study experimentally determined the optimal reaction conditions for the production of GOS from lactose by β‐galactosidase (EC 3.2.1.23) from Kluyveromyces lactis covalently immobilized to a polysiloxane‐polyvinyl alcohol (POS‐PVA) polymer activated with glutaraldehyde (GA), and to study the transgalactosylation kinetics. Yield immobilization was 99 ± 1.1% with 78.5 ± 2.4% enzyme activity recovery. An experimental design 24 with 1 center point and 2 replicates was used. Factors were lactose [L], enzyme concentration [E], pH and temperature (T). Response variables were glucose and galactose as monosaccharides [G1], residual lactose [Lac]r and GOS as disaccharides [G2] and trisaccharides [G3]. Best conditions were pH 7.1, 40 °C, 270 gL^?1 initial lactose concentration and 6 U mL^?1 enzyme concentration, obtaining 25.46 ± 0.01 gL^?1 yield of trisaccharides. Although below the HPLC‐IR detection limit, tetrasaccharides were also identified after 115 min of reaction. The immobilization protocol was then optimized by diminishing total reactant volumes : support ratio, resulting in improved enzyme activity synthesizing 43.53 ± 0.02 gL^?1 of trisaccharides and 13.79 ± 0.21 gL^?1 of tetrasaccharides, and after four cycles remaining relative activity was 94%. A reaction mechanism was proposed through which a mathematical model was developed and rate constants were estimated, considering a pseudo steady‐state hypothesis for two concomitant reactions, and from this simplified analysis, the reaction yield could eventually be improved. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1568–1578, 2017     

Free radical scavenging activity of novel thiazolidine‐2,4‐dione derivatives

Free radical activity towards superoxide anion radical (), hydroxyl radical (HO^?) and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) of a series of novel thiazolidine‐2,4‐dione derivatives (TSs) was examined using chemiluminescence, electron paramagnetic resonance (EPR) and EPR spin trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide (DMPO) was applied as the spin trap. Superoxide radical was produced in the potassium superoxide/18‐crown‐6 ether dissolved in dimethyl sulfoxide. Hydroxyl radical was generated in the Fenton reaction (Fe(II) + H₂O₂. It was found that TSs showed a slight scavenging effect (15–38% reduction at 2.5 mmol/L concentration) of the DPPH radical and a high scavenging effect of (41–88%). The tested compounds showed inhibition of HO^? ‐dependent DMPO‐OH spin adduct formation (the amplitude of EPR signal decrease ranged from 20 to 76% at 2.5 mmol/L concentration. Our findings present new group compounds of relatively high reactivity towards free radicals. Copyright © 2012 John Wiley & Sons, Ltd.     

Di‐2‐ethylhexyl phthalate (DEHP) exposure disturbs lipid metabolism in juvenile yellow catfish Tachysurus fulvidraco

This study was conducted to determine the mechanism by which di‐2‐ethylhexyl phthalate (DEHP) exposure influences lipid metabolism of juvenile yellow catfish Tachysurus fulvidraco. Fish were exposed to three DEHP concentrations (0, 0·1 and 0·5 mg l^?1 DEHP) for 8 weeks. Fatty acid synthase (FAS) activity significantly decreased with increasing DEHP concentrations, the highest value was in the Tween control group, whereas the lowest activities of carnitine palmitoyltransferase (CPT) and lipoprotein lipase (LPL) were in this group. The messenger (m)RNA levels of 6‐phospho‐gluconate dehydrogenase (6PGD), FAS and acetyl‐CoA carboxylase a (ACCa) significantly increased with increasing DEHP concentration, the highest values were in the 0·5 mg l^?1 DEHP group. The mRNA level of peroxisome proliferator‐activated receptor γ (PPARγ) was lower in Tween control than in fish exposed to 0·1 and 0·5 mg l^?1 DEHP. The highest mRNA level of ACCb was in the 0·1 mg l^?1 DEHP group. These results indicate that DEHP exposure can disturb lipid metabolism at the enzymatic and mRNA levels in Pelteobagrus fulvidraco.     

Intermittent lead‐induced stress on antioxidant enzyme activity and subcellular distribution of Pb in Pogonatherum crinitum seedlings

• Pogonatherum crinitum is a promising lead (Pb) hyperaccumulator due to its high Pb tolerance and accumulation ability. However, the mechanisms that support Pb accumulation and tolerance in P. crinitum are not yet clearly understood.
• An indoor hydroponic experiment was conducted by cultivating P. crinitum seedlings exposed to intermittent Pb stress for 60 days, divided into four stages (T1, T2, T3 and T4), with a 15‐day duration per stage. The following concentrations of Pb were used: 0, 500, 0, 500 mg·l^?1 and 0, 1000, 0, 1000 mg·l^?1). Antioxidant enzyme activity, Pb concentration and subcellular distribution of Pb were measured at each of the above stages.
• The results showed that superoxide dismutase (SOD) activity in shoots, and SOD, peroxidase (POD) and malondialdehyde (MDA) activity in shoots and roots significantly increased from T1 (no Pb stress) to T2 (Pb stress) in both 500 mg·l^?1 and 1000 mg·l^?1 treatments; however, no significant difference was noted between stages T3 (no Pb stress) and T4 (Pb stress). There was no obvious effect of Pb stress on catalase (CAT) activity in shoots and roots among different stages. The Pb concentration in shoots was up to 5090.90 mg·kg^?1 and 7573.57 mg·kg^?1, and the bioconcentration factor (BFC) was 10.18 and 7.57 for the 500 mg·l^?1 and 1000 mg·l^?1 treatments, respectively, which confirmed the Pb hyperaccumulator characteristics of P. crinitum. For plants under Pb stress, most of the Pb was fixed in the cell walls, with a smaller amount in leaves and root vacuoles.
• Both SOD and POD scavenging of reactive oxygen radicals and fixing and compartmentalisation of Pb in the cell wall might play important roles in detoxification of P. crinitum seedlings in response to Pb stress. There was no phased response of P. crinitum to intermittent Pb stress and the physiological response to Pb stress may be contiguous.

     

Development of a colorimetric colony‐screening assay for detection of defluorination by micro‐organisms

Aims: To develop a colorimetric colony‐screening assay to facilitate the isolation of micro‐organisms capable of defluorination. Methods and Results: A metal‐dye chelate, zirconium‐xylenol orange was used to detect fluoride ions released from a fluorinated substrate through microbial metabolism. Depolymerised zirconium reagent gave the greatest visual contrast for the presence of fluoride compared to more polymerised forms of zirconium reagent. The sensitivity of the assay was greatest when the molar ratio of depolymerised zirconium to xylenol orange was 1 : 2. Using depolymerised zirconium and xylenol orange (150 and 300 nmol l^?1 respectively), the assay could detect a fluoride application spot (5 mmol l^?1) containing 50 nmoles of fluoride ions. Most media constituents were well tolerated by the assay, although phosphate ions needed to be restricted to 0·1 g l^?1 and some proteins digest to between 1 and 5 g l^?1. A microbial enrichment culture growing on solidified medium containing 20 mmol l^?1 fluoroacetate was screened using the assay, and defluorinating bacteria belonging to the genus Burkholderia isolated. Conclusions: A method was developed that is sensitive, rapid and reliable for detecting defluorination by micro‐organisms growing on solidified medium. Significance and Impact of the Study: This method can be used to facilitate the isolation of micro‐organisms capable of defluorination.     

Metal-ion susceptibility of oral bacterial species

Aims: This study aimed to evaluate the effect of lead (Pb) on growth of bacterial species related to dental diseases in vitro. Methods and Results: The effects of lead acetate on representative species of the oral flora were examined at 0·1–10 mmol l^?1 and compared with the effect of silver nitrate and ferrous sulfate. The minimal inhibitory concentration of lead acetate was between 0·15 and 5 mmol l^?1 for the bacterial strains tested. The minimal bactericidal concentration of lead acetate for most oral species was detected in the range of 5–10 mmol l^?1. Silver nitrate at a concentration of 1·25 mmol l^?1 was sufficient to exhibit antibacterial activity against almost all bacteria tested. Ferrous sulfate had the lowest effect. Conclusions: The study indicated a general antimicrobial effect of lead on oral bacterial species in the range of 0·15–10 mmol l^?1. The toxicity of silver nitrate was the highest, whereas that of ferrous sulfate was the lowest. Gram‐positive species had a tendency to be less susceptible for metals than Gram‐negatives. Significance and Impact of the Study: The study shows that it is possible that microbiological changes may occur in the dental plaque in children because of toxic exposure of environmental lead.     

L-lactate dehydrogenase from leaves of Capsella bursa-pastoris (L.) Med.

By ammonium sulfate fractionation and gel filtration an enzyme preparation which catalyzed NAD⁺-dependent L-lactate oxidation (10^-4 kat kg^-1 protein), as well as NADH-dependent pyruvate reduction (10^-3 kat kg^-1 protein), was obtained from leaves of Capsella bursa-pastoris. This lactate dehydrogenase activity was not due to an unspecific activity of either glycolate oxidase, glycolate dehydrogenase, hydroxypyruvate reductase, alcohol dehydrogenase, or a malate oxidizing enzyme. These enzymes could be separated from the protein displaying lactate dehydrogenase activity by gel filtration and electrophoresis and distinguished from it by their known properties. The enzyme under consideration does not oxidize D-lactate, and reduces pyruvate to L-lactate (the configuration of which was determined using highly specific animal L-lactate dehydrogenase). Based on these results the studied Capsella leaf enzyme is classified as L-lactate dehydrogenase (EC 1.1.1.27). It has a K_m value of 0.25 mmol l^-1 (pH 7.0, 0.3 mmol l^-1 NADH) for pyruvate and of 13 mmol l^-1 (pH 7.8, 3 mmol l^-1 NAD⁺) for L-lactate. Lactate dehydrogenase activity was also detected in the leaves of several other plants.Abbreviation FMN flavin adenine mononucleotide     

Calcium‐dependent behavioural responses to acute copper exposure in Oncorhynchus mykiss

Using rainbow trout Oncorhynchus mykiss, the present study demonstrated that: (1) calcium (Ca) increased the range of copper (Cu) concentrations that O. mykiss avoided; (2) Ca conserved the maintenance of pre‐exposure swimming activity during inescapable acute (10 min) Cu exposure. Data showed that when presented with a choice of Cu‐contaminated water (ranging from 0 to 454 µg Cu l^?1) and uncontaminated water in a choice tank, O. mykiss acclimated and tested at low Ca concentration (3 mg Ca l^?1) avoided the 10 µg Cu l^?1 only. By contrast, O. mykiss acclimated and tested at high Ca concentration (158 mg Ca l^?1) avoided all the Cu concentrations ≥37 µg l^?1. The Cu avoidance was connected with increased spontaneous swimming speed in the Cu‐contaminated water. When subjected to inescapable Cu exposure (35 µg Cu l^?1), O. mykiss acclimated and tested at low Ca concentration reduced their spontaneous swimming speed, whereas no response was observed in O. mykiss acclimated and tested at high Ca concentration. Collectively, the data support the conclusion that in O. mykiss the behavioural responses to acute Cu exposure are Ca‐dependent.     

Isolation and characterization of actinomycetes from healthy goat faeces

Aims: To isolate and characterize actinomycetes with probiotic activities from healthy goat faeces. Methods and Results: Faecal actinomycetes were isolated by dilution methods and identified by 16S rRNA gene sequence analysis. The hydrolytic enzyme activities were analysed by clear zone formation. The antimicrobial activities and resistance to heavy metals were tested by growth inhibition methods. The isolates belong to a small group of actinobacterial genera, including Streptomyces, Nocardiopsis and Oerskovia. The Oerskovia was the most widely distributed genus among the cultures. The proportion of streptomycete‐like strains producing amylase or protease is significantly higher than those of other actinomycetes (P < 0·05). Compared with streptomycete‐like strains, a higher proportion of (α‐ or β‐) galactase‐producing other actinomycetes was found in goat faeces. More than 50% of streptomycete‐like strains showed activities against test fungi. Streptomycetes could tolerate 0·25 mmol l^?1 Cr₂O₇^2?, 2 mmol l^?1 Ni²⁺; however, other actinomycetes are liable to 40 mmol l^?1 Fe³⁺ and 0·25 mmol l^?1 Cr₂O₇^2? and resistant to 5 mmol l^?1 Ni²⁺ and 2 mmol l^?1 Cu²⁺. Conclusions: The different physiological characteristics of the actinomycetes suggested that the cooperation in the actinomycetes might be involved in their association with goat. Significance and Impact of the Study: Probiotic mixtures based on faecal actinomycetes showed potentials in animal production.     

Compound amino acids added in media improved Solanum nigrum L. phytoremediating CD-PAHS contaminated soil

Cd hyperaccumulator Solanum nigrum L. was a promising plant used to simultaneously remediate Cd-PAHs combined pollution soil through its extra accumulation capacity and rhizosphere degradation. This article compared the strengthening remediation role of cysteine (Cys), glycine (Gly) and glutamic acid (Glu) with EDTA and TW80. The results showed that the addition of 0.03 mmol L^?1 Cys, Gly, and Glu didn't significantly impact (p < 0.05) shoot biomass of S. nigrum, but obviously increased Cd concentration. Therefore, Cd capacity (µg pot^?1) in shoots of S. nigrum was significantly increased (p < 0.05) by 37.7% compared to the control without reagent added. At the meantime, the PAHs degradation ratio in rhizoshpere was increased by 34.5%. Basically, the improving role of Cys, Gly, and Glu was higher than EDTA and TW80. The main reasons of enhanced the accumulation of S. nigrum to Cd might lie in the addition of Cys, Gly, and Glu which reduced pH and increased extractable Cd concentration in rhizosphere and phytochelatines (PCs) concentration in leaves. As for the degradation of PAHs in rhizosphere, increased microorganism number might be play important role.     

A highly active phosphoglucomutase from Clostridium thermocellum: cloning, purification, characterization and enhanced thermostability

Aims: Discovery and utilization of highly active and thermostable phosphoglucomutase (PGM) would be vital for biocatalysis mediated by multiple enzymes, for example, high‐yield production of enzymatic hydrogen. Methods and Results: The thermophilic cellulolytic bacterium Clostridium thermocellum was hypothesized to have a very active PGM because of its key role in microbial cellulose utilization. The Cl. thermocellum ORF Cthe1265 encoding a putative PGM was cloned and expressed in Escherichia coli. The purified enzyme appeared to be a monomer with an estimated molecular weight of 64·9 kDa. This enzyme was found to be a dual‐specificity enzyme – PGM/phosphomannomutase (PMM). Mg²⁺ and Mn²⁺ were activators. Ser144 was identified as an essential catalytic residue through site‐directed mutagenesis. The k_cat and K_m of PGM were 190 s^?1 and 0·41 mmol l^?1 on glucose‐1‐phosphate and 59 s^?1 and 0·44 mmol l^?1 on mannose‐1‐phosphate, respectively, at 60°C. Thermostability of PGM at a low concentration (2 nmol l^?1, 100 U l^?1) was enhanced by 12‐fold (i.e. t_1/2 = 72 h) at 60°C with addition of bovine serum albumin, Triton X‐100, Mg²⁺and Mn²⁺. Conclusions: The ORF Cthe1265 was confirmed to encode a PGM with PMM activity. This enzyme was the most active PGM reported. Significance and Impact of the Study: This highly active PGM with enhanced thermostability would be an important building block for in vitro synthetic biology projects (complicated biotransformation mediated by multiple enzymes in one pot).