Enhancement of botrallin and TMC-264 production in liquid culture of endophytic fungus Hyalodendriella sp. Ponipodef12 after treatments with metal ions

Background: Hyalodendriella sp. Ponipodef12, an endophytic fungus from a poplar hybrid, was a high producer of botrallin and TMC-264 with various bioactivities. In this study, the influences of eight metal ions (i.e.,Mn2+,Na+, Mg2+,Zn2+,Cu2+,Fe2+,Fe3+ and Al3+) on botrallin and TMC-264 production in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 were investigated. Results: Three most effective metal ions (Zn2+,Cu2+ and Mg2+) along with their optimum concentrations were screened. The optimum addition time and concentrations of Zn2+,Cu2+ and Mg2+ were further obtained respectively for improving botrallin and TMC-264 production. The combination effects of Zn2+,Cu2+ and Mg2+ on the production of botrallin and TMC-264 by employing statistical method based on the central composite design (CCD) and response surface methodology (RSM) were evaluated, and two quadratic predictive models were developed for botrallin and TMC-264 production. The yields of botrallin and TMC-264, which were predicted as 144.12 mg/L and 36.04 mg/L respectively, were validated to be 146.51 mg/L and 36.63 mg/L accordingly with the optimum concentrations of Zn2+ at 0.81 mmol/L, Cu2+ at 0.20 mmol/L, and Mg2+ at 0.13 mmol/L in medium. Conclusion: The results indicated that the enhancement of botrallin and TMC-264 accumulation in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 by the metal ions and their combination should be an effective strategy.

Isolation and characterization of a metal ion-dependent alkaline protease from a halotolerant Bacillus aquimaris VITP4.

A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40°C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu­­­2+, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.

Effect of lithium metal on the chemical status of glutathione [GSH] present in whole blood [especially in plasma and cytosolic fraction in human blood]

Lithium remains a mainstay in the acute and prophylactic treatment of bipolar affective disorder. It is used in the augmentation of antidepressant treatment and, less frequently, in the augmentation of antipsychotic treatment of schizophrenia. It is reported to have specific anti-suicidal effects. Thus the effect of Lithium was interesting to study on the glutathione [GSH] level in vivo conditions. Ellman's method has been used to see the effect of lithium on glutathione [GSH] level in whole blood. The time dependent effect of Lithium on the chemical status of glutathione [GSH] was determined in the whole blood [Plasma and cytosolic fraction] of human. The concentration of Glutathione was drastically decreased. The decrease in the glutathione level was concentration and time of interaction dependent, probably due to oxidation of glutathione [GSH] to corresponding disulphide [GSSG]. In this paper the effect of Lithium on the Thiol/GSH level was discussed in vitro, which in principal may present a model of in vivo reaction

Partial purification and some properties of -amylase from Bacillus subtilis KIBGE-HAS.

An extracellular -amylase from Bacillus subtilis KIBGE-HAS was partially purified by ultrafiltration and ammonium sulphate precipitation with 19.2-fold purification and specific activity of 4195 U/mg. The enzyme showed relatively high thermostability and retained 62% of its activity when kept at 70°C for 15 min. -Amylase was highly stable at -18°C and loss of activity was very low during stability study. Metal ions like Mn2+, Ca2+, Co2+, K+, Mg2+, and Fe3+ activated the enzyme, while Hg2+ Ba2+, Cu2+, Na+ and Al3+ strongly inhibited the activity. The α-amylase was highly stable in various surfactants and detergents. In the presence of surfactants such as SDS and Triton X-100 the enzyme activity was found 2.9 and 1.8-fold higher respectively than control. The non-ionic detergents (Tween 20 and Tween 80) exhibited slightly inhibitory effect on the enzyme activity.

Optimization of physical parameters for lipase production from Arthrobacter sp. BGCC#490.

The physical parameters for the production of thermostable, alkaline lipase from Arthrobacter sp. BGCC# 490 were optimized using response surface methodology (RSM), employing face centered central composite design (FCCCD). The design was employed by selecting pH, temperature and incubation period as the model factors and to achieve maximum yield, interaction of these factors was studied by RSM. A second-order quadratic model and response surface method showed that the optimum conditions for lipase production (pH 10.0, temperature 40oC and incubation period 48 h) resulted in 1.6-fold increase in lipase production (13.75 EUml-1), as compared to the initial level (8.6 EUml-1) after 48 h of incubation, whereas its value predicted by the quadratic model was 12.8 EUml-1. Lipase showed stability in the pH range 8-10 and temperature range 40-60oC, with maximum activity at pH 9.0 and temperature 50oC. Lipase activity was enhanced in the presence of K+, Ca2+ and Mg2+ ions, but inhibited by Hg2+ ions. The enzyme exhibited high activity in the presence of acetone, isopropanol and ethanol, but was unaffected by methanol. These properties suggest that the lipase may find potential applications in the detergent industry. The present work also demonstrated the feasibility of using experimental design tools to optimize physical parameters for lipase production by an indigenous Arthrobacter sp.

Effect of metal ions on growth of Pseudomonas aeruginosa and siderophore and protein production.

Pseudomonas aeruginosa (GRC1) isolated from potato rhizosphere, grew better on succinate medium than tryptic soy medium and produced hydroxamate type of siderophore in iron-deficient succinate medium. When the strain GRC1 was grown in the presence of different metal ion compounds, viz. ZnSO4, MnSO4, MnCl2 and FeCl3 at 6 and 12 microM concentrations individually, ZnSO4 (12 microM) promoted siderophore production but suppressed the growth and protein content of test organism. MnCl2 and FeCl3 (12 microM) enhanced the growth, whereas MnCl2 and MnSO4 (12 microM) induced protein contents of strain GRC1.

Preliminary studies on production of extracellular lipase by an isolate from soil : Aspergillus flavus oryzae.

In our routine screening programme, using agar diffusion assay method, lipolytic activity was detected around a colony of a fungus. The fungus was isolated from a soil sample which was brought from a location near oil-mill. This lipolytic fungus was then identified to belong to Aspergillus flavus oryzae. A medium was then formulated and optimized which would not only support good growth but also would yield good extracellular lipolytic activity. It was observed that a conventional carbohydrate-protein containing medium supported good growth of the fungus but moderate lipase activity, whereas, a hydrocarbon containing medium, although supported relatively less growth, yielded much more lipase activity.

Association of metal tolerance with multiple antibiotic resistance of enteropathogenic organisms isolated from coastal region of deltaic Sunderbans.

A survey of microorganisms was conducted in four coastal regions of the deltaic Sunderbans. Among ten different isolates, three were enteropathogenic. They were Vibrio cholerae non-O1 (CT+), enterotoxinogenic Escherichia coli (ETEC) and Pseudomonas aeruginosa. These enteropathogens were able to grow in the presence of zinc (Zn++), cadmium (Cd++), lead (Pb++), cobalt (Co++), copper (Cu++), nickel (Ni++) and silver (Ag+) up to 10 mM concentration. They also showed resistance against 5 to 10 antibiotics. Metal tolerance and drug resistance were not determined by plasmid(s). Synthesis of outer membrane protein among the marine isolates was higher in presence of metal. Enteropathogens isolated from the deltaic Sunderbans were well adapted for growth of the saline environment with higher concentrations of toxic metals.

Effect of trace metals on production of exoprotein and beta-lactamase by Staphylococcus aureus.

A minimum concentration of 0.01 mM of Mg2+ was essential for any growth of S. aureus. Of the trace metals, Mg2+ and Ca2+ were highly stimulatory for growth. All trace metals except Ni2+ in small concentrations were stimulatory for growth and exoprotein production. Amounts of exoprotein and beta-lactamase produced relative to the number of viable cells were maximum during the period of suboptimal proliferation of bacteria. Trace metals did not significantly influence the amount of beta-lactamase produced relative to exoprotein.

Patterns of heavy metal resistance in marine Pseudomonas MR1.

Pseudomonas strain MR1 isolated from the coastal waters of Bay of Bengal was found to resist Hg, As, Cd, Cu, Zn and Pb. It efficiently detoxified both organic and inorganic mercuric compounds to non toxic metallic mercury by an inducible enzyme, mercuric reductase. Its resistance to arsenic might be due to energy-dependent arsenate efflux system. Cadmium was detected intracellularly and in the surrounding medium. The bacterium accumulated copper and lead intracellularly.

In-vitro study on the effect of adenine nucleotides, heavy metals and ascorbic acid on the lysosomal enzymatic activity and release rotes in rat brain

The effects of multivalent cations such as Pb 2+, Zn 2+ and Fe 3+, adenosine triphosphate [ATP], adenosine diphosphate [ADP], and adenosine monophosphate [AMP] on the enzyme release and total activity from cerebral lysosomes isolated from adult albino rats were examined in vitro. The test compounds were used in two concentration levels of 10 -3 and 10 -5 M. The three selected marker lysosomal acid hydrolases are acid phosphatase [ACP], N-acetyl B-glucosaminidase [B-NAG], and N-acetyl galactosaminidase. The effect of ascorbic acid sole and combined with the multivalent cations in an equimolar concentration on these enzymes were also examined. The results of this study showed that, ascorbic acid and the cations inhibited the enzyme release by different susceptibility on the lysosomal membrane. The suppressive effect of cations was significantly affected by treatment of the lysosome with ascorbic acid. On the other h and, ATP and ADP resulted in a decrease of ACP and B-NAG, while there was no effect on N-acetyl galactosaminidase activity. The total enzymatic activity of ACP was more inhibited than B-NAG by 10 -5 M of adenine nucleotides, while N-acetyl galactosaminidase activity was not affected by these nucleotides. AMP has no effect on the enzyme release. In addition, the degree of inhibition on the enzyme activity and release rate was decreased in the presence of ascorbic acid. In some cases, the inhibitory effect of the cations was changed to increase in either total activity or enzyme release by ascorbic acid

Effect of divalent metal ions on soluble and membrane-bound alkaline phosphatase activity in suckling rat intestine.

EDTA treatment of intestinal brush border membranes (BBM) and epithelial cell supernatant completely inhibited alkaline phosphatase (AP) activity in suckling rat intestine. AP activity was fully regained upon dialysis of the preparations against Zn2+ and to a lesser extent against Co2+, Ca2+ and Mn2+ ions. Other metal ions (Cd2+ and Mg2+) tested were essentially ineffective in restoring the enzyme activity. Considerable differences were observed in kinetic characteristics of the membrane-bound and soluble AP activities in response to various metal ions. There were apparent differences in Km, Vmax, energy of activation (Ea) and thermal stability of the soluble and membrane-bound AP activities, after metal ion substitutions. Nearly 35% AP activity was solubilized on sodium dodecyl sulphate treatment of brush borders (membrane protein: detergent ratio 1:3; w/w). Dialysis of detergent solubilized BBM against different metal ions reconstituted AP activity in the particulate fraction: the order of effectiveness was Zn greater than Ca greater than Mn greater than Co. The kinetic properties of the reconstituted AP were essentially similar to the non-integrated enzyme activity in response to various divalent metal ions examined. But there were apparent differences in Km, Vmax, Ea and thermal stability of the reconstituted AP activity compared to native brush border enzyme. The results suggest the unique requirement of Zn ions for stability and catalytic activity of the soluble and membrane-bound AP activity in suckling rat intestine.

Comparitive study of hepatic coumarin hydroxylase [cyp2a-5] induction by heavy metal tin

The selective induction of the hepatic coumarin hydroxylase [Cyp 2a-5] elicited by the heavy metal tin [Sn C12] has been compared in male NMRI mice with that of the standard inducers, phenobarbitone [PB] and 3-methylcholanthrene [MC]. Although the standard inducers, PB and MC increase the microsomal specific content of cytochrome P 450 [P 450/mg microsomal protein], stannous chloride leads to a decrease in this concentration. On the other hand, regarding Cyp 2a-5 activity, ethoxycoumarin O-deethylase was increased to about 3.5-fold and coumarin 7-hydroxylase as much as 8-fold after pretreatment with stannous chloride. This-increase was much higher than that caused by PB, the only well known inducer of coumarin 7-hydroxylase and MC. However, the 2.2- or 2.4-fold increase in the N-demethylation of ehtylmorphine of benzphetamine, the model substrate used to visualize the latent PB-induction, by PB were insignificantly decreased after pretreatment by stannous chloride. The same is true when ethoxyresorufin O-deethylation, which would indicate MC-induction type was considered. Stannous chloride did not affect such activity whereas the MC increases it to about 11-fold. The data indicate that the heavy metal salt-stannous chloride causes induction of microsomal monooxygenase complex selective for coumarin 7-hydroxylase and 7-ethoxycoumarin O-deethylase and does not affect the rate of metabolism of model substrates, which would indicate a PB-or MC-type of induction

Antibiotic and heavy metal resistence associated to plasmids in clinical isolates of Klebsiella pneumoniae in Chile

Resistência a antibióticos e metais pesados associada a plasmídio em isolados clínicos de Klebsiella de origem hospitalar no Chile. Isolados clínicos de Klebsiella pneumoniae de origem hospitalar apresentaram resistência múltipla para antibióticos e metais pesados associada a presença de plasmídio

Association of R plasmids of Klebsiella pneumoniae with resistance to heavy metals, klebocin and beta lactamase production and lactose fermentation.

One hundred and fifty clinical isolates of Klebsiella pneumoniae were studied for beta lactamase production, Klebocin production and resistance to antibiotics and metals. 97.3 and 82.7 per cent strains produced beta-lactamase and klebocin respectively. Resistance to arsenite, antimony, tellurite, mercury and silver was observed in 91,66,6, 47.7, 44.8 and 33.3 per cent respectively and to ampicillin, tetracycline and nalidixic acid in 93.3,60, 41.3, 34.4, 28.2, and 1.3 per cent respectively. Forty eight antibiotic and metal resistant strains were then conjugated with Escherichia coli 711 F-Lac-Nxr. Transferable drug resistance, metal ion resistance, beta lactamase production, klebocin production and lactose fermentation was seen in 30, 21, 10, 11 and 5 isolates respectively.

Effect of selected metal ions on the motility and carbohydrate metabolism of ejaculated human spermatozoa.

Zinc, lead and cadmium in the form of chloride salts when added to a standard assay system containing 80 X 10(-6) ejaculated washed human spermatozoa caused a dose and duration-dependent inhibition of their motility. The activity of certain key enzymes of carbohydrate and energy metabolism, viz, glycogen phosphorylase, glucose-6-phosphatase, fructose-1, 6-diphosphatase, glucose-6-phosphate isomerase, amylase, Mg2+- dependent ATPase and lactic and succinic acid dehydrogenases were also found to be inhibited. The order of inhibitory effects of the heavy metals were zinc less than lead less than cadmium. The metal chelating agent, ethylene diamine tetra-acetic acid (EDTA, disodium salt) also interfered with the spermatozoal motility and inhibited the enzyme activities.

Effect of 'Arogyavardhini' against carbon tetrachloride induced hepatic damage in albino rats.

'Arogyavardhini'-an indigenous formulation was evaluated for its hepatoprotective activity in rats, using two models of carbon tetrachloride (CCl4) hepatic damage, one simulating vital hepatitis and the other simulating fatty change. The protective effect was assessed from serum aspartate transaminase (AST) and alkaline phosphatase levels and from histopathological changes in liver. The results revealed that 'Arogyavardhini' (5 mg/100g, PO daily) was effective in minimizing the changes in serum levels of AST and alkaline phosphatase induced by CCI. The protective effect was also evident on histopathological examination.