Factors altering the haemolytic power of crude venom from Aiptasia mutabilis (Anthozoa) nematocysts

The effect of different agents upon the haemolytic power of Aiptasia mutabilis crude venom was studied in human erythrocytes to determine its toxicity and stability. Nematocysts were isolated from acontia of the Anthozoan A. mutabilis and submitted to sonication for extracting crude venom. Aliquots of venom were tested in 0.05% erythrocyte suspensions in the presence of various factors such as proteases (papain, collagenase, trypsin, α-chymotrypsin); cations (Ca²⁺, Mg²⁺, Ba²⁺, K⁺ and Cu²⁺), osmotic protectants as polyethylenglycole (PEG) of different MW and antioxidant compounds (GSH, cysteine and ascorbic acid). Results demonstrate the dose–response of the haemolytic effect of A. mutabilis. Haemolysis by the crude venom was prevented by Ca²⁺, Ba²⁺ and Cu²⁺ treatment, and to a minor extent by Mg²⁺ and K⁺. Papain and PEG with a molecular mass exceeding 1000 Da also prevented haemolysis. These findings are consistent with a pore-forming mechanism of crude venom in erythrocytes rather than an oxidative damage at the employed doses.     

Cu2+, Co2+, and Mn2+ modify the gating kinetics of high-voltage-activated Ca2+ channels in rat palaeocortical neurons

The effects of three divalent metal cations (Mn2+, Co2+, and Cu2+) on high-voltage-activated (HVA) Ca2+ currents were studied in acutely dissociated pyramidal neurons of rat piriform cortex using the patch-clamp technique. Cu2+, Mn2+, and Co2+ blocked HVA currents conducted by Ba2+ ( IBa) with IC50 of approximately 920 nM, approximately 58 micro M, and approximately 65 micro M, respectively. Additionally, after application of non-saturating concentrations of the three cations, residual currents activated with substantially slower kinetics than control IBa. As a consequence, the current fraction abolished by the blocking cations typically displayed, in its early phase, an unusually fast-decaying transient. The latter phenomenon turned out to be a subtraction artifact, since none of the pharmacological components (L-, N-, P/Q-, and R-type) that constitute the total HVA currents under study showed a similarly fast early decay: hence, the slow activation kinetics of residual currents was not due to the preferential inhibition of a fast-activating/inactivating component, but rather to a true slowing effect of the blocker cations. The percent IBa-amplitude inhibition caused by Mn2+, Co2+, and Cu2+ was voltage-independent over the whole potential range explored (up to +30 mV), hence the slowing of IBa activation kinetics was not due to a mechanism of voltage- and time-dependent relief from block. Moreover, Mn2+, Co2+, and Cu2+ significantly reduced I(Ba) deactivation speed upon repolarization, which also is not compatible with a depolarization-dependent unblocking mechanism. The above results show that 1) Cu2+ is a particularly potent HVA Ca2+-channel blocker in rat palaeocortical neurons; and 2) Mn2+, Co2+, and Cu2+, besides exerting a blocking action on HVA Ca2+-channels, also modify Ca2+-current activation and deactivation kinetics, most probably by directly interfering with channel-state transitions.     

Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella: effect of various divalent cations on the lattice formation

The R-form lipopolysaccharide from Klebsiella pneumoniae strain LEN-111 (O3-:K1-), from which cationic material had been removed by electrodialysis, was previously shown to form a hexagonal lattice structure with the lattice constant of 14 to 15 nm when suspended in 50 mM tris(hydroxymethyl)aminomethane buffer at pH 8.5 containing 10 mM Mg2+. Under this experimental condition, effects of other divalent metal cations on the hexagonal assembly of the electrodialyzed LPS were compared with that of Mg2+. The Zn2+, Hg2+, Cu2+, and Ni2+ could produce essentially the same hexagonal lattice structure with the lattice constant of 14.5 to 15.0 nm as that formed with Mg2+. The Cd2+, Co2+, and Fe2+ produced the hexagonal lattice structure with the lattice constant of 15.5 to 16.0 nm, and Ba2+, Sr2+, and Ca2+ produced that with the lattice constant of 18 to 19 nm. In addition, the hexagonal lattice structures formed with the latter three cations were less orderly than those formed with the other cations. When the higher concentrations of Ba2+, Sr2+, and Ca2+ were used, the lattice constants were not shortened. The length of lattice constants of the hexagonal lattice structures formed with the divalent cations did not relate to the quantity of the cations bound to the LPS. Among the divalent cations tested, Hg2+ was bound to the LPS in the smallest amount (its atomic ratio to P, 0.07), and Zn2+ and Fe2+ were bound in very large amounts (their atomic ratios to P, 2.94 and 8.28, respectively).     

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.     

米氏凯伦藻溶血毒素的溶血反应特征

探讨了温度、pH值、二价阳离子等对米氏凯伦藻(Karenia mikimotoi Hasen)溶血毒素溶血活性的影响,分析了米氏凯伦藻溶血毒素的溶血反应特征.结果表明,实验室培养米氏凯伦藻的溶血活性约为64.69±6.43 HU L-1,单个藻细胞的溶血活性为6.17±0.61×10-6 HU;在实验温度(0～37℃)下,溶血活性随温度的增加而增加;pH6.0时的溶血活性最高;Cu2+、Mg2+、Mn2+、Ca2+、Co2+、Zn2+和Hg2+等对米氏凯伦藻的溶血活性的影响不同.离子浓度为5 mmol/L时,Hg2+的抑制作用最强.高浓度Hg2+对红细胞的集合效应不但阻止了Hg2+进入血细胞诱导的溶血作用,而且阻止了毒素对细胞膜的破坏,但这种抑制作用可被EDTA消除.     

Effects of divalent cations on the E-4031-sensitive repolarization current, I(Kr), in rabbit ventricular myocytes.

The effects of divalent cations on the E-4031-sensitive repolarization current (I(Kr)) were studied in single ventricular myocytes isolated from rabbit hearts. One group of divalent cations (Cd2+, Ni2+, Co2+, and Mn2+) produced a rightward shift of the I(Kr) activation curve along the voltage axis, increased the maximum I(Kr) amplitude (i.e., relieved the apparent inward rectification of the channel), and accelerated I(Kr) tail current kinetics. Another group (Ca2+, Mg2+ and Sr2+) had relatively little effect on I(Kr). The only divalent cation that blocked I(Kr) was Zn2+ (0.1-1 mM). Under steady-state conditions, Ba2+ caused a substantial block of I(K1) as previously reported. However, block by Ba2+ was time dependent, which precluded a study of Ba2+ effects on I(Kr). We conclude that the various effects of the divalent cations can be attributed to interactions with distinct sites associated with the rectification and/or inactivation mechanism of the channel.     

Effects of divalent cations and pH on amiloride-sensitive Na+ fluxes into luminal membrane vesicles from pars recta of rabbit proximal tubule.

The effect of Ca2+, Cd2+, Ba2+, Mg2+ and pH on the renal epithelial Na(+)-channel was investigated by measuring the amiloride-sensitive 22Na+ fluxes into luminal membrane vesicles from pars recta of rabbit proximal tubule. It was found that intravesicular Ca2+ as well as extravesicular Ca2+ substantially lowered the channel-mediated flux. Amiloride sensitive Na+ uptake was nearly completely blocked by 10 microM Ca2+ at pH 7.4. The inhibitory effect of Ca2+ was dependent on pH. Thus, 10 microM Ca2+ produced 90% inhibition of 22Na+ uptake at pH 7.4, and only 40% inhibition at pH 7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over the range from 7.0 to 7.4. All the cations Ca2+, Cd2+, Ba2+ except Mg2+ inhibited the 22Na+ influx drastically when added extravesicularly in millimolar concentrations. The cations Cd2+, Ba2+ and Mg2+ in the same concentrations intravesicularly inhibited the 22Na+ influx only slightly. A millimolar concentration of Ca2+ intravesicularly blocked the amiloride-sensitive 22Na+ flux completely. The data indicate that Ca2+ inhibits Na+ influx specifically by binding to sites composed of one or several deprotonated groups on the channel proteins.     

Divalent cation conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum

The conduction properties of the alkaline earth divalent cations were determined in the purified sheep cardiac sarcoplasmic reticulum ryanodine receptor channel after reconstitution into planar phospholipid bilayers. Under bi-ionic conditions there was little difference in permeability among Ba2+, Ca2+, Sr2+, and Mg2+. However, there was a significant difference between the divalent cations and K+, with the divalent cations between 5.8- and 6.7-fold more permeant. Single-channel conductances were determined under symmetrical ionic conditions with 210 mM Ba2+ and Sr2+ and from the single-channel current-voltage relationship under bi-ionic conditions with 210 mM divalent cations and 210 mM K+. Single-channel conductance ranged from 202 pS for Ba2+ to 89 pS for Mg2+ and fell in the sequence Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. Near-maximal single-channel conductance is observed at concentrations as low as 2 mM Ba2+. Single-channel conductance and current measurements in mixtures of Ba(2+)-Mg2+ and Ba(2+)-Ca2+ reveal no anomalous behavior as the mole fraction of the ions is varied. The Ca(2+)-K+ reversal potential determined under bi-ionic conditions was independent of the absolute value of the ion concentrations. The data are compatible with the ryanodine receptor channel acting as a high conductance channel displaying moderate discrimination between divalent and monovalent cations. The channel behaves as though ion translocation occurs in single file with at most one ion able to occupy the conduction pathway at a time.     

Effect of cations and temperature on kinetics of desmin assembly.

Smooth-muscle desmin, which was isolated from avian gizzard, was purified and used to form reconstituted intermediate filaments. Filament assembly was done in the presence of physiological cations, Ca2+, Mg2+, Na+, and Na+ plus Mg2+, and with non-physiological cations Cu2+ and Ni2+. Assembly was done at 2 degrees, 22 degrees and 37 degrees C, and was monitored by absorbance and by electron microscopy. Absorbance increased most rapidly during the first 2-5 min and then increased at a slower rate with the physiological cations, but decreased after that time with the non-physiological cations. For each physiological cation, absorbance increased with increasing temperature. This was particularly evident with Ca2+, which produced the lowest absorbance at 2 degrees C and the highest at 37 degrees C. When ionic strength was comparable, filament-forming buffers that contained bivalent cations were associated with higher absorbance values. Filament diameters were significantly smaller 60 min after assembly initiation than after 5 min. Average filament diameters, when formed in the presence of Cu2+ or Ni2+, were 10% greater than in the presence of the physiological cations and did not show a consistent tendency to decrease as time increased. These results demonstrate the importance, not only of the pH and ionic composition of the filament-forming buffer, but also of the temperature and duration of dialysis for reconstitution of desmin filaments.     

A fast centrifuge method for nematocyst isolation from Pelagia noctiluca Forskal (Cnidaria: Scyphozoa).

Nematocyst isolation from surrounding tissue is an important step to characterize Cnidarian venom. Although several protocols have been used to extract venoms from cnidarian tissues, the complete isolation of nematocysts from tissue is still difficult. The goal of the present work was to evaluate the effectiveness of three different media, Percoll, Ficoll and Methylcellulose in isolating nematocysts from Pelagia noctiluca tentacles by centrifugation. The complete sedimentation of nematocysts and tissue fragments to the bottom of the test tubes was observed in Ficoll and Methylcellulose suspensions. The best result was obtained using a discontinuous density gradient of Percoll: three types of nematocysts were concentrated in three different fractions along the density gradient. Protein assay and preliminary chromatographic analyses confirmed these results.     

Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane

Several divalent cations were studied as agonists of a Ca2+-activated K+ channel obtained from rat muscle membranes and incorporated into planar lipid bilayers. The effect of these agonists on single-channel currents was tested in the absence and in the presence of Ca2+. Among the divalent cations that activate the channel, Ca2+ is the most effective, followed by Cd2+, Sr2+, Mn2+, Fe2+, and Co2+. Mg2+, Ni2+, Ba2+, Cu2+, Zn2+, Hg2+, and Sn2+ are ineffective. The voltage dependence of channel activation is the same for all the divalent cations. The time-averaged probability of the open state is a sigmoidal function of the divalent cation concentration. The sigmoidal curves are described by a dissociation constant K and a Hill coefficient N. The values of these parameters, measured at 80 mV are: N = 2.1, K = 4 X 10(-7) mMN for Ca2+; N = 3.0, K = 0.02 mMN for Cd2+; N = 1.45, K = 0.63 mMN for Sr2+; N = 1.7, K = 0.94 mMN for Mn2+; N = 1.1, K = 3.0 mMN for Fe2+; and N = 1.1 K = 4.35 mMN for Co2+. In the presence of Ca2+, the divalent cations Cd2+, Co2+, Mn2+, Ni2+, and Mg2+ are able to increase the apparent affinity of the channel for Ca2+ and they increase the Hill coefficient in a concentration-dependent fashion. These divalent cations are only effective when added to the cytoplasmic side of the channel. We suggest that these divalent cations can bind to the channel, unmasking new Ca2+ sites.     

Effects of bivalent cations on post-rest adaptation in guinea-pig heart muscle

In isolated papillary muscles of guinea-pig hearts, the inotropic effects of bivalent cations, Ca2+, Ba2+, Sr2+, and Ni2+, were investigated during post-rest adaptation in order to study their individual action on excitation-contraction coupling. Upon exposure to each cation studied, the force of contraction was transiently enhanced, whereas the steady state force was influenced differently: it increased with Ca2+, Ba2+ and Sr2+ and was depressed by Ni2+. The transmembrane action potentials (measured at 90% repolarization) were slightly prolonged by Sr2+ and even more by Ba2+, and were shortened by Ca2+ and Ni2+. After 10 min rest, the post-rest contractions consisted of a late peak (PII) that was enhanced in high Ca2+-solution an by Sr2+. Ni2+ and Ba2+ depressed PII and during adaptation to pre-rest controls an early peak of contraction (PI) prevailed. There was no simple relation between post-rest adaptation of force and the duration of action potential in the presence of the bivalent cations tested. During post-rest adaptation the two components of contraction can be separated. The results are interpreted in terms of a model of excitation-contraction coupling which derives Ca ions for contractile activation from two sources: transmembrane calcium influx and calcium release from cellular stores. From the different effects on post-rest adaptation it is concluded that the individual cations influence excitation-contraction coupling more specifically and not merely by "screening-off" the negative surface charges.     

Stringent requirement for Ca2+ in the removal of Z-lines and alpha-actinin from isolated myofibrils by Ca2+-activated neutral proteinase

Treatment of isolated myofibrils with Ca2+-activated neutral proteinase (CANP) results in specific removal of Z-line and of alpha-actinin. To investigate the ionic requirement for these processes, we measured Z-line removal by phase-contrast and interference microscopy and alpha-actinin removal by sodium dodecyl sulphate/polyacrylamide-gel electrophoretic analysis of myofibrillar proteins. The proteolytic digestion of native purified proteins was measured directly on polyacrylamide gels and by the fluorescamine technique. We found that the removal of Z-line and alpha-actinin as well as the release of proteolytic degradation products from isolated myofibrils by CANP occur only in the presence of Ca2+; Sr2+, Ba2+, Mn2+, Mg2+, Co2+ and Zn2+ are all ineffective. In contrast with this stringent requirement for Ca2+, the proteolytic activity of CANP measured with denatured casein, native and denatured haemoglobin, native actin and tropomyosin also occurs in the presence of other bivalent cations, in the following order: Ca2+ greater than Sr2+ greater than Ba2+. These data suggest that only Ca2+ can produce the conformational change in myofibrils that renders them susceptible to the action of CANP, whereas its proteolytic activity is stimulated by several bivalent ions.     

Assay and properties of the hemolysis activity of pure venom from the nematocysts of the acontia of the sea anemone Aiptasia pallida

Pure venom from the acontial nematocysts of the sea anemone Aiptasia pallida was isolated and an assay for the hemolysis activity of the venom devised. The assay is rapid, sensitive, and reproducible. Venom concentrations as low as 0.1 μg protein/ ml were accurately assayed. The properties of the hemolysis activity were analyzed using techniques similar to those used to study enzyme-catalyzed reactions. The biochemical events underlying venom-induced lysis required the direct participation of millimolar levels of Ca²⁺. The slight variability of the apparent K_m for Ca²⁺ at different venom concentrations appeared to be due to the release of some material(s) from lysing cells. Both Sr²⁺ and Mg²⁺ weakly substituted for Ca²⁺. Inhibition of lysis by EDTA was reversed by Ca²⁺. Small monovalent cations, such as Na⁺ or K⁺, appeared to be involved in the venom-induced alteration of the red cell membrane so that lysis could occur. The venom's hemolysis activity was stabilized in solution only if the concentration of the venom proteins was high while also in the presence of at least the equivalent of 0.15 m NaCl.     

Purification and properties of phosphatidylglycerophosphate synthetase from mammalian liver mitochondria

The enzyme which catalyzes the synthesis of phosphatidylgly cerophosphate from an-glycerol-3-phosphated and cytidine diphosphate diacylglycerol was released from rat or pig liver mitochondrial membranes by extraction with Triton X-100 or Nonidet P-40. The detergent-extracted enzyme, like the activity of intact mitochondria, did not require added cations or lipids. The Triton extracts were fractionated by column chromatography on Bio-Gel A-1.5. The fractions obtained from the columns exhibited little activity in the standard assay system unless divalent cations were included. Additional stimulation (about twofold) was observed in the presence of added phospholipids. The cation requirement of the purified enzyme was relatively nonspecific with Mg2+, Ba2+, or Ca2+ providing maximal activity in the 10mM range. Either Mn2+ or Co2+ were stimulatory at somewhat lower concentrations but higher concentrations were inhibitory. Other cations such as Cd2+, Zn2+,Hg2+, or Cu2+ were ineffective as cofactors, and in the presence of Mg2+ inhibited the reaction at concentrations greater than 0.5 mM. The phospholipik stimulation was obtained specifically with phosphatidylethanolamines from natural or synthetic sources. Other diacylglycerophosphatides or lysophosphatides including lysophosphatidylethanolamine were ineffective.     

Divalent cation affinity sites in Paramecium aurelia

Sites with high calcium affinity in Paramecium aurelia were identified by high calcium (5 mM) fixation and electron microscope methods. Electron-opaque deposits were observed on the cytoplasmic side of surface membranes, particularly at the basal regions of cilia and trichocyst-pellicle fusion sites. Deposits were also observed on some smooth cytomembranes, within the axoneme of cilia, and on basal bodies. The divalent cations, Mg2+, Mn2+, Sr2+, Ni2+, Ba2+, and Zn2+, could be substituted for Ca2+ in the procedure. Deposits were larger with 5 mM Sr2+. Ba2+, and Mn2+ at ciliary transverse plates and the terminal plate of basal bodies. Microprobe analysis showed that Ca and C1 were concentrated within deposits. In some analyses, S and P were detected in deposits. Also, microprobe analysis of 5 mM Mn2+-fixed P. aurelia showed that those deposits were enriched in Mn and C1 and sometimes enriched in P. Deposits were seen only when the ciliates were actively swimming at the time of fixation. Locomotory mutants having defective membrane Ca-gating mechanisms and ciliates fixed while exhibiting ciliary reversal showed no obvious differences in deposition pattern and intensity. Possible correlations between electron-opaque deposits and the locations of intramembranous particles seen by freeze-fracture studied, as well as sites where fibrillar material associate with membranes are considered. The possibility that the action sites of calcium and other divalent cations were identified is discussed.     

A study of the molecular mechanism of DNA interaction with divalent metal ions

The interaction of DNA with divalent metal ions: Ba2+, Mg2+, Mn2+, Ni2+, Cu2+ in solutions at different ionic strengths mu was investigated. The combination of following methods: flow birefringence, viscometry, UV-spectroscopy and circular dichroism made possible to follow the state of the secondary and tertiary structure of the DNA molecule during its interaction with ions. The presence of divalent ions in solution affects the hydrodynamic properties of DNA only at low mu. At high mu the difference in the action of mono- and divalent ions disappears. The persistence length of DNA does not change during the experiment. It is shown that the Mg2+ and Ba2+ ions interact only with phosphate groups of DNA but Mn2+, Ni2+, Cu2+ ions interact also with the nitrogen bases of the macromolecule.     

Purification and Properties of ent-Kaurene Synthase B from Immature Seeds of Pumpkin

ent-Kaurene synthase B (KSB) was purified 291-fold from a crude enzyme preparation from endosperm of pumpkin (Cucurbita maxima L.). Separation of ent-kaurene synthase A and KSB was achieved by hydrophobic interaction chromatography. The fractions containing KSB activity were further purified by diethylaminoethyl, phenyl, and hydroxyapatite column chromatography. Using sodium dodecyl phosphate-polyacrylamide gel electrophoresis, the purest enzyme preparation showed a major band at an apparent molecular mass of 81 kD. The amount of protein in this band was correlated with KSB activity after diethylaminoethyl and hydroxyapatite chromatography. The N terminus of the 81-kD protein was blocked. Therefore, the protein was partially digested with protease and the amino acid sequences of the resulting major peptide fragments were analyzed. A polyclonal antibody was raised against a synthetic peptide based on the longest peptide fragment combined with a keyhole limpet hemocyanin. The antibody recognized only the 81-kD denatured protein and not the native KSB. The properties of KSB were examined using the phenyl-purified enzyme preparation. The Km value for copalyl pyrophosphate was 0.35 [mu]M, and the optimal pH was 6.8 to 7.5. The KSB activity required divalent cations such as Mg2+, Mn2+, and Co2+, whereas Cu2+, Ca2+, and Ba2+ inhibited the activity.     

A phospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5: purification and characterization of conditions for optimal activity with an artificial substrate

Phospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5 was purified from buffered yeast extract culture supernate by ion-exchange chromatography followed by fractionation by manganous chloride and ammonium sulphate precipitation steps. Enzyme activity was assayed by hydrolysis of p-nitrophenylphosphorylcholine and confirmed by release of radioactivity from tritiated L-alpha-dipalmitoylphosphatidylcholine labelled in the methyl groups of choline. After SDS-PAGE, the purified preparation yielded a single band upon Coomassie-blue staining. This protein migrated with an apparent Mr of 50,000-54,000. Phospholipase C activity was maximal at pH greater than or equal to 8.4 and was enhanced in the presence of sorbitol and of several nonionic detergents but was eliminated by SDS. EDTA, Cu2+, Fe2+ and Zn2+ inhibited enzyme activity, whereas Ba2+, Ca2+, Co2+, Mg2+ and Mn2+ restored activity to EDTA-treated material. No haemolytic activity was demonstrated with the purified enzyme.     

Xylanase production by Burkholderia sp. DMAX strain under solid state fermentation using distillery spent wash

Xylanase production by a newly isolated strain of Burkholderia sp. was studied under solid state fermentation using anaerobically treated distillery spent wash. Response surface methodology (RSM) involving Box-Behnken design was employed for optimizing xylanase production. The interactions between distillery effluent concentration, initial pH, moisture ratio and inoculum size were investigated and modeled. Under optimized conditions, xylanase production was found to be in the range of 5200-5600 U/g. The partially purified enzyme recovered after ammonium sulphate fractionation showed maximum activity at 50 degrees C and pH 8.6. Kinetic parameters like Km and Vmax for xylan were found to be 12.75 mg/ml and 165 micromol/mg/min. In the presence of metal ions such as Ca2+, Co2+, Mn2+, Ba2+, Mg2+ and protein disulphide reducing agents such as beta-mercaptoethanol and dithiotheritol (DTT) the activity of enzyme increased, where as strong inhibition of enzyme activity was observed in the presence of Cu2+, Ag+, Fe2+ and SDS. The crude enzyme hydrolysed lignocellulosic substrate, wheat bran as well as industrial pulp.