Characterization of the novel xylanase from the thermophilic Geobacillus thermodenitrificans JK1

Thermophilic strain JK1 was isolated from compost using xylan as a single carbon source. On the basis of 16S rRNA gene phylogenetic analysis and spo0A gene sequence similarity analysis, strain JK1 was identified as Geobacillus thermodenitrificans strain. During the exponential culture growth, the strain JK1 was found to produce the single xylan degrading enzyme ??45 kDa in size. Xylose was not an inducer of this xylanase. Cloning, expression and characterization of the recombinant xylanase were performed. Xylanase of G. thermodenitrificans JK1 was cellulase-free; pH and temperature optimums were found to be 6.0 and 70°C, respectively. The metal ions Na⁺, K⁺, Ca²⁺, and Co²⁺ showed partial inhibition of the activity, while Mn²⁺ had slight stimulating effect on the enzymatic activity. Recombinant xylanase was thermostable over the temperature range of 55?C70°C. It presented the highest stability after incubation at 55°C for 60 min showing 84% residual activity. 50% residual activity was revealed after incubation at 60°C for 60 min as well as at 65 and 70°C for 30 min. Results of the thermostability experiments showed xylanase of JK1 having quite low thermostability when compared with the respective enzymes of the other geobacilli.     

Specific binding of dexamethasone to plasma membranes from skeletal muscle

A procedure was developed to isolate plasma membranes from rabbit skeletal muscle. K⁺-dependent phosphatase activity was used as marker enzyme for plasma membranes and was determined in the presence of CHAPS (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate), a zwitterionic detergent. Ca²⁺-ATPase and succinate dehydrogenase activities were used as marker enzymes for sarcoplasmic reticulum and mitochondria, respectively. Electron-microscopy revealed that plasma membranes were in the form of vesicles. Significant proteolysis of membrane proteins was observed during extraction, which was inhibited by EGTA and 20 mM molybdate. SDS-polyacrylamide gel electrophoresis revealed the disappearance of an intense 96 kDa protein band when membranes were purified in the absence of EGTA and molybdate. Specific binding sites for [³H]dexamethasone were identified in plasma membranes after freezing and incubation with CHAPS. Dithiothreitol was essential for steroid binding and ATP increased it. Under standardized assay conditions, binding was complete with 50 min à 37°C. No binding occurred at 0°C, nor if EGTA and molybdate were absent from the extraction medium.     

Metabolic differences inBacillus stearothermophilus grown at 55°C and 37°C

Summary Bacillus stearothermophilus was adapted to grow at 55°C and 37°C in a complex medium with almost equivalent yields in cell mass. In both temperature ranges the maximum specific growth rates (μ_max) were identical. Cellular extracts of this bacterium showed remarkable differences in the activity levels of several enzymes, depending on the respective growth temperature. High activities of glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase were observed in bacteria from thermophilic cultures (55°C) and the respiratory quotient exceeded 1.0. Under anaerobic conditions at 55°C μ_max was the same as in aerobic cultures. No alcohol dehydrogenase was detected in cells from mesophilic cultures (37°C), however, and the level of glyceraldehyde-3-phosphate dehydrogenase was also extremely low under mesophilic conditions. Succinate dehydrogenase and isocitrate dehydrogenase activity appeared to be higher in bacteria grown at 37°C; the resspiratory quotient was always lower than 1.0. At 37°C, acetoin formation was observed regularly, a fermentation product which was never detected in 55°C-cultures. Under anaerobic conditions at 37°C a very low growth rate was found. When adapted to grow at 37°C or 55°C,B. stearothermophilus is apparently able to use different catabolic systems.     

Metagenomic cloning and characterization of Na+/H+ antiporter genes taken from sediments in Chaerhan Salt Lake in China

A metagenomic library containing 8,000 clones was constructed by using genomic DNA obtained from Chaerhan Salt Lake in northwest China. Three Na⁺/H⁺ antiporters, C4-NhaG, C47-NhaG and C49-NhaG that grouped to the NhaG family, were screened and cloned from this metagenome by complementing Escherichia coli strain KNabc (ΔnhaA ΔnhaB ΔchaA) in medium containing 0.2 M NaCl. The three putative Na⁺/H⁺ antiporters were membrane proteins with 10, 11 and 11 transmembrane segments, respectively. They enabled E. coli KNabc to grow in medium containing 0.2–0.6 M Na⁺ or 7–14 mM Li⁺. Everted membrane vesicles prepared from E. coli KNabc cells carrying C49-NhaG exhibited Na⁺/H⁺ and Li⁺/H⁺ antiport activities.     

Application of protease from Nocardiopsis sp. as a laundry detergent additive

The application of protease as a laundry detergent additive from a newly isolated Nocardiopsis sp., isolated from a soil sample collected in Northeast Brazil is reported. The optimal pH and temperature for protease activity were pH 10.5 and 50 °C, respectively. The enzyme was stable in a long-term incubation, showed 73.5% of initial activity at pH 10.5 and 61.7% at pH 12.0 for 120 min. Approximately 60% of initial activity remained after 120 min at 50 °C or after 30 min at 80 °C. Almost 87% of enzyme activity was retained in the presence of 10% (v/v) of peroxide at 40 °C, after 1 h. The protease also was stable in the presence of oxidants and surfactants such as SDS, saponin, Tween 20 and Tween 80 after 30 min. In the presence of Omo^®, the enzyme retained 64% of its activity at 40 °C for 1 h. An increase in the proteolytic activity (6–17%) was observed with K⁺, Na⁺, and Mg⁺⁺ ions. At pH 8.0, the protease hydrolysed casein maximally (50 U/mg).     

Use of low temperature and high K+ incubation media for in vitro tissue preparation for X-ray microanalysis

Incubation of tissue slices in physiological buffers gives rise to significant changes in the intracellular ion concentrations, which may disturb subsequent X-ray microanalysis. In the present study it was attempted to design incubation conditions that retain the in vivo conditions better. The following variables were investigated: (1) exchange of Na⁺ in the incubation medium for K⁺, and exchange of Cl^–for the less permeable gluconate anion; (2) incubation at 4°C rather than at 37°C; and (3) addition of dextran to the incubation medium. Brief exposure (a few seconds) of liver slices to a buffer causes changes in the intracellular Na, Cl and K concentrations, depending on the ionic composition of the buffer. Incubation in a normal physiological (high NaCl) buffer at 37°C results in a further increase of Na and Cl and a further decrease in K in liver cells. The changes reach a maximum at 30 min and the concentrations then remain stable throughout a 2-h incubation. Incubation in sodium gluconate medium or addition of dextran to the physiological buffer somewhat reduces the changes in the intracellular ion composition (compared to the standard physiological incubation medium). Incubation in potassium gluconate medium results in a decrease in cellular Na and an increase in K. Quantitative morphological studies show that tissue oedema is observed to the same extent in hepatocytes incubated in sodium gluconate, potassium gluconate and physiological buffer containing 10% dextran. However, these buffers cause significantly less cell oedema than the physiological (high NaCl) buffer. Incubation of liver, cerebral cortex or submandibular gland slices in physiological (high NaCl) solutions at 4°C for 4 h caused a more extensive increase in Na⁺ and decrease in K⁺ than incubation at 37°C for 2 h. This suggests inhibition of the Na⁺, K⁺-ATPase under these conditions. As compared to incubation at 37°C for 2 h, tissues incubated in potassium gluconate buffer at 4°C for 4 h have a cellular K concentration closer to the in situ value. Cholinergic stimulation of tissue slices from cerebral cortex and submandibular gland at room temperature for 1 min shows the best physiological response in tissue slices preincubated at 4°C for 4 h in high KCl, potassium gluconate and high NaCl, in this order. The response can, however, only be seen, when cholinergic stimulation is carried out in a standard physiological buffer with a high NaCl concentration. It is concluded that in vitro storage of tissue for X-ray microanalysis is best carried out at 4°C in a solution with a high K⁺ concentration.     

Purification and characterization of thermostable pectate-lyases from a newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov.

A novel thermophilic spore-forming anaerobic microorganism (strain Ab9) able to grow on citrus pectin and polygalacturonic acid (pectate) was isolated from a thermal spa in Italy. The newly isolated strain grows optimally at 70°C with a growth rate of 0.23 h⁻¹ with pectin and 0.12 h⁻¹ with pectate as substrates. Xylan, starch, and glycogen are also utilized as carbon sources and thermoactive xylanolytic (highest activity at 70°–75°C), amylolytic as well as pullulolytic enzymes (highest activity at 80°–85°C) are formed. Two thermoactive pectate lyases were isolated from the supernatant of a 300-l culture of isolate Ab9 after growth on citrus pectin. The two enzymes (lyases a and b) were purified to homogeneity by ammonium sulfate treatment, anion exchange chromatography, hydrophobic chromatography and finally by preparative gel electrophoresis. After sodium dodecylsulfate (SDS) gel electrophoresis, lyase a appeared as a single polypeptide with a molecular mass of 135 000 Da whereas lyase b consisted of two subunits with molecular masses of 93 000 Da and 158 000 Da. Both enzymes displayed similar catalytic properties with optimal activity at pH 9.0 and 80°C. The enzymes were very stable at 70°C and at 80°C with a half-life of more than 60 min. The maximal activity of the purified lyases was observed with orange pectate (100%) and pectate-sodium salt (90%), whereas pectin was attacked to a much lesser extent (50%). The K _m values of both lyases for pectate and citrus pectin were 0.5 g·l⁻¹ and 5.0 g·l⁻¹, respectively. After incubation with polygalacturonic acid, mono-, di-, and tri-galacturonate were detected as final products. A 2.5-fold increase of activity was obtained when pectate lyases were incubated in the presence of 1 mM Ca²⁺. The addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These heat-stable enzymes represent the first pectate-lyases isolated and characterized from a thermophilic anaerobic bacterium. On the basis of the results of the 16S rRNA sequence comparisons and the observed phenotypic differences, we propose strain Ab9 as a new species of Thermoanaerobacter, namely Thermoanaerobacter italicus sp. nov. Received: May 25, 1997 / Accepted: June 5, 1997     

Purification and characterization of an extracellular cholesterol oxidase from a Bordetella species

A soil-isolated bacterium (strain B4) was identified as a species of Bordetella and deposited with the China General Microbiological Culture Collection (code, CGMCC 2229). The bacterium grew in a mineral medium, on cholesterol as a sole source of carbon and energy. Only one metabolite of cholesterol was accumulated in detectable amounts during the strain growth. It was identified as 4-cholesten-3-one. Cholesterol oxidase (COD) (EC 1.1.3.6), which catalyzes cholesterol into this metabolite, was evidenced from the strain. The conditions of the bacterium growth were optimized for extracellular enzyme production, which then reached around 1700 UL⁻¹ within 24 h culturing. The enzyme was purified from the spent medium of the strain to homogeneity on SDS-PAGE, and characterized. Its molecular mass, as estimated by this technique, was 55 kDa. COD showed an optimum activity at pH 7.0. It was completely stable at pH 5.0 and 4 °C for 48 h, and retained 80% at least of its initial activity at pH 4.0 or at a pH of 6.0–10.0. The optimum temperature for its reaction was 37 °C. The thermal stability of COD was appreciable, as 90% or 80% of its initial activity was recovered after 1 h or 2 h incubation at 50 °C. Ag⁺ or Hg⁺ at 1 mM, was inhibitor of COD activity, while Cu²⁺, at the same concentration, was activator. The COD K_m, determined at 37 °C and pH 7.0, was 0.556 mM. The enzyme was stable at pH 7.0 and 37 °C during 24 h mechanical shaking in the presence of 33% (v/v) of either of the solvents, dimethylsulfoxide, ethyl acetate, butanol, chloroform, benzene, xylene or cyclohexane.     

Purification and Some Properties of the Endo α-1,4 Polygalactosaminidase from Pseudomonas sp.

The endo α-1,4 polygalactosaminidase from Pseudomonas sp. 881 was purified from the culture nitrate by ethanol precipitation and sequential column chromatographies on CM-Sephadex C-25, Sephadex G-50 and Phenyl-Sepharose CL-4B. The purified enzyme was electrophoretically homogeneous and its molecular weight and isoelectric point were 31,000 and 6.7, respectively. The optimum pH and temperature for hydrolysis of polygalactosamine were 5.0 and 55°C, respectively. The enzyme was stable up to 45°C for 15min and from pH 4.0 to 7.6 at 37°C for 1 hr.

The Km value was 0.05% α-1,4 polygalactosamine and the V was 0.154μmol reducing sugar (galactosamine)/min/μg protein. This polygalactosaminidase was inhibited by Sn²⁺ , Fe²⁺ , Fe³⁺ , Hg²⁺, Cu²⁺ ions and SDS. The enzyme did not hydrolyze oligo galactosamines (n < tetramer) or N-acetyl-polygalactosamines. It acted only on oligo galactosamine (n > trimer) and polygalactosamine endogeneously so far tested.     

Greatly extended viability of rat brain storage vesicles in an intracellular medium based upon a non-permeant polyanion

The MgATP-stimulated accumulation of (-)-³H-nor- epinephrine (NE) by rat brain neuronal storage vesicles has been characterized in a new medium based upon polyacrylic acid (avg. MW 5,000). The medium allows careful regulation of K⁺ concentration (140 mM), has a large buffer capacity, and is non-permeant to membranes. Light scattering measurements have confirmed the osmotic stability of vesicles suspended in this medium. Vesicular accumulation of (-)-³ H-NE (Km 1 × 10^?6 M) in this system (37°) was examined under saturating (10^?5 M) and non-saturating (2 × 10^?7 M) concentrations of NE. At 10^?5 M NE, uptake saturated at 5 min and remained stable for periods up to one hour, with maximal uptake levels (pmol/mg protein) of 15.7±0.30 (37°), 3.0±0.49 (0°), 4.4±0.22 (reserpine pretreated $\text{in}$$\text{vivo}$) and 6.0±0.79 (without MgATP). At 2×10^?7 M NE uptake was biphasic with maximal uptake levels (pmol/mg protein) of 4.04±0.14 (37°), 0.19±0.01 (0°), 0.95±0.01 (reserpine) and 0.83±0.08 (without MgATP). Vesicle preparations refrigerated in this medium for 24 hrs displayed properties quite similar to those measured acutely (NE = 2.2x10^?7 M).     

Transport of l-arginine in brush border vesicles derived from rabbit kidney cortex

The uptake of l-arginine by brush border vesicles from rabbit kidney cortex was investigated at 37 °C and pH 7.5. The initial rate of uptake (15 s) was twice as fast in a highly purified brush border as in brush border contaminated by basal-lateral plasma membranes. The initial uptake in a mannitol medium can be best described as the sum of transfer by two systems with K_m values of 0.07 and 3.5 mm and V_max values of 1.5 and 8 nmol/mg protein × 15 s, respectively. For the inhibitors of l-[¹⁴C]arginine, uptake (15 s at two substrate concentrations of 0.1 and 2.5 mm in a mannitol medium) the following sequence of inhibitory strength was established: l-arginine, l-ornithine, l-cystine, l-lysine, d-arginine, and NaCl. When a vesicular membrane potential was induced transiently by a jump of the pH in the incubation medium from 5.9 to 7.5 or by an outward movement of K⁺ in the presence of gramicidin D, an overshoot of l-arginine uptake was observed. Initial uptake of l-arginine was slightly faster in the presence of a Na⁺ gradient (outside to inside) than under a K⁺ gradient. Both ion gradients reduced uptake as compared to the uptake in a mannitol medium. Uptake was also studied after the membrane potential was minimized by equilibrating the vesicles in a NaCl or KC1 medium in the presence of gramicidin D. Under these conditions, l-arginine uptake in the first 30 s was faster in the NaCl than in the KCl medium. These experiments indicate, beside a major ion-independent l-arginine transport, the presence of a transport stimulated by Na⁺ in isolated brush border vesicles.     

Redistribution and recycling of internalized membrane in seminal vesicle secretory cells

This study was performed to clarify the fate of membrane constituents internalized from the apical domain in secretory cells, in particular their possible recycling and the compartments involved in it. Glycoproteins of the apical membrane of seminal vesicle secretory cells from guinea-pig were covalently labeled in vitro (0°C, 20 min) with ³H-galactose and the epithelium incubated for 15 min (37°C, first incubation) to allow endocytosis. The label which was not internalized was then exposed to enzymatic hydrolysis (0°C, 30 min) and the epithelium re-incubated to allow membrane movement for 15 and 30 min (37°C, 2nd incubation). After each step of the protocol, tissue pieces were fixed and processed for electron microscope autoradiography and the results studied by morphometric analysis. Following labeling, 99% of the silver grains were associated with the apical domain of the cell membrane (AD). After the 1st incubation at 37°C, 30° of the grains were inside the cells in association with the cytoplasmic vesicles (Cyt ves), secretory vacuoles (SV), Golgi vesicles (GV), Golgi cisternae (GC), multivesicular bodies (MVB), lysosomes (LYS), and the cell membrane basolateral domain (BLD). About 58% of non-internalized radioactivity was removed by hydrolysis. During the 2nd incubation at 37°C the concentration of label increased in BLD and LYS, decreased in SV and MVB, and fluctuated in GC, GV and AD. The distribution of grains observed at 15 min, as compared using the χ-square test, was highly significantly different from that expected without recycling. The results show that cell membrane glycoproteins internalized at the cell apex recycle back to the membrane apical domain and are consistent with the involvement of GC and SV in the recycling pathway. Membrane shuttle between the apical and basolateral domains of the cell membrane is also suggested by these observations.     

Activation of K-Cl Cotransport by Mild Warming in Guinea Pig Red Cells

Unidirectional, ouabain-insensitive K⁺ influx rose steeply with warming at temperatures above 37°C in guinea pig erythrocytes incubated in isotonic medium. The only component of ouabain-insensitive K⁺ influx to show the same steep rise was K-Cl cotransport (Q₁₀ of 10 between 37 and 41°C); Na-K-Cl cotransport remained constant or declined and residual K⁺ influx in hypertonic medium with ouabain and bumetanide rose only gradually. Similar results were obtained for unidirectional K⁺ efflux. Thermal activation of K-Cl cotransport-mediated K⁺ influx was fully dependent on the presence of chloride in the medium; none occurred with nitrate replacing chloride. The increase of K⁺ influx through K-Cl cotransport from 37 to 41°C was blocked by calyculin A, a phosphatase inhibitor. The Q₁₀ of K-Cl cotransport fully activated by hydroxylamine and hypotonicity was about 2. The time course of K⁺ entry showed an immediate transition to a higher rate when cells were instantly warmed from 37 to 41°C, but there was a 7-min time lag in returning to a lower rate when cells were cooled from 41 to 37°C. These results indicate that the steepness of the response of K-Cl cotransport to mild warming is due to altered regulation of the transporter. Total unidirectional K⁺ influx was equal to total unidirectional K⁺ efflux at 37–45°C, but K⁺ influx exceeded K⁺ efflux at 41°C when K-Cl cotransport was inhibited by calyculin or prevented by hypertonic incubation. The net loss of K⁺ that results from the thermal activation of isosomotic K-Cl cotransport reported here would offset a tendency for cell swelling that could arise with warming through an imbalance of pump and leak for Na⁺ or for K⁺. Received: 1 November 1997/Revised: 5 March 1998     

Serratia Protease

A strain of Serratia, isolated from an intestinal canal of a silkworm, produced a large quantity of protease. The enzyme was extracellular and was named Serratiopeptidase, tentatively. Protease production of this strain was over 3 times as much as that of Serratia marcescens which was known as a protease-producing organism. The highly purified enzyme was prepared from the culture supernatant through ammonium sulfate precipitation, acetone fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-75.

The purified enzyme moved homogeneously with a sedimentation constant, s_20,w of 3.8 S in ultracentrifugation and the molecular weight was determined to be 6.0 × 10₄ by the Archibald method. Determination of the ultraviolet absorption spectrum indicated the E^1%_{280 mμ,1 cm} was 13.0. Neither carbohydrate nor sulfur-containing amino acid was detected in the purified enzyme preparation. The enzyme showed maximal activity at pH 9.0 and at 40°C, and was stable under lower temperatures over the pH range from 5 to 10, whereas it was unstable at 37°C in alkaline conditions. The enzyme was completely inactivated by heating at 55°C for 15 min.     

Production and Properties of Alkaline Dextranase from a Newly Isolated Brevibacterium

About 500 strains of dextranase producing microorganisms were examined in detail for pH- activity and enzyme stability. A gram positive bacterium identified as belonging to the genus Brevibacterium was found to produce alkaline dextranase. Maximal dextranase synthesis was obtained when grown aerobically at 26°C for 3 days in a medium containing 1 % dextran, 2% ethanol, 1 % polypeptone and 0.05 % yeast extract together with trace amounts of inorganic salts.

Brevibacterium dextranase had an optimum pH of 8.0 for activity at 37°C and an optimal temperature at 53°C at pH 7.5. The enzyme was quite stable over the range of pH 5.0 to 10.5 on 24 hr incubation at 37°C, especially on alkaline pH. The enzyme was also heat stable at 60°C for 10 min.     

Regional and Subcellular Localization of Li+ and Other Cations in the Rat Brain Following Long-Term Lithium Administration

Abstract: Rats were given LiCl in their diet (40 mmol/kg dry weight) for at least 3 months to elucidate the regional and subcellular localization of Li⁺ in the brain as well as the effect of chronic lithium administration on the distribution of other cations. At steady-state the mean concentrations of Li⁺ were 0.66 mmol/kg wet weight in the whole brain and 0.52 mM in plasma. The tissue/plasma concentration ratio exceeded unity in all anatomical regions. No region showed excessive accumulation of Li⁺. Whole brain or regional contents of Na⁺ or K⁺ were unaffected by lithium treatment. Subcellular Li⁺ localization was demonstrated in nuclear, crude mitochondrial, and microsomal fractions of whole brain homogenate. Subfractionation of the crude mitochondrial fraction revealed energy-independent intrasynaptosomal and intramitochondrial Li⁺ and K⁺ localization at 0–4°C. Li⁺ administered in vivo disappeared within 10 min from synaptosomes incubated at 37°C. Li⁺ added in vitro at 1 mM attained a synaptosomal steady-state concentration within 30 min at 37°C. In control rats, synaptosomal concentrations and synaptosomal/medium concentration gradients of cations paralleled their respective in vivo concentrations and gradients. Lithium treatment caused synaptosomal depletion of K⁺ and Mg²⁺ and hence probably partial membrane depolarization. Addition of 1 mM Li⁺ in vitro also caused synaptosomal Mg²⁺ depletion. The results indicate that Li⁺ is “accumulated” in brain sediments and synaptosomes following its long-term treatment. The estimated intracellular and intrasynaptosomal Li⁺ concentrations are lower than predicted by passive distribution according to the Nernst equation, evidencing active extrusion of Li⁺.     

Characteristics of transmembrane proton transport in the cells of Lupinus polyphyllus

The aim of this work was to examine H⁺-ATPase hydrolytic activity and the stability of transmembrane electrochemical gradients in membrane vesicles isolated from seedlings and leaf cells of an invasive Washington lupine (Lupinus polyphyllus Lindl.) and compare them with non-invasive yellow lupine (Lupinus luteus L.). Temperatures of 25 and 30°C, keeping in mind possible climate warming, were used. For harder stress conditions, short term cold treatment (?8°C in vivo) was used. Plasmalemma-, tonoplast-, and endoplasmic reticulum-enriched membrane fractions were obtained from a sucrose density gradient and identified. Differences in ATPase hydrolytic activity were significant only between lupine species and were more obvious in plasmalemma-enriched fractions. Preincubation of seedlings and leaves at ?8°C for 15 min to 24 h showed that microsomic fraction membranes of invasive lupine were more stable (according to Na⁺-diffusive potential) at low temperature compared to yellow lupine ones. The level of transmembrane electrochemical potential, mainly evoked by ATP-dependent active proton transport, was almost equal in both lupine species. Supposedly, the cells of invasive lupine are able to maintain transmembrane electrochemical potential by the employment of lower hydrolytic activity of H⁺-ATPase, thus saving energy for growth.     

Permeability and photoxidative damage of membrane enzymes

The pattern of photodynamic damage of pig erythrocyte and rat brain microsomal ATPases and erythrocyte acetylcholinesterases has been studied, using rose bengal as photosensitizer. Of these enzymes the Na⁺-K⁺-Mg²⁺-ATPase, believed to be associated with active transport, is very much more sensitive to damage than are the Mg²⁺-ATPase and the erythrocyte acetylcholinesterase. Earlier photoxidative studies of the haemolysis of erythrocytes have shown that ion movements occur in two phases which are dependent on the duration of exposure to light and it is suggested that these are correlated with the differential sensitivity of these membrane enzymes. The ATPases of the brain microsomal preparation were more sensitive to photodynamic damage during preincubation at 0°C, i.e. in the absence of substrate. Raising the preincubation temperature to 37°C protected both enzymes, the inactivation of the Na⁺-K⁺-Mg²⁺-ATPase being markedly reduced. The presence of substrate during pre-incubation at 0°C also protects both enzymes, especially the Mg²⁺-ATPase. These interacting effects of temperature and substrate are compared with the known different temperature sensitivities of these two enzymes.     

Screening of filamentous fungi for lipase production: Hypocrea pseudokoningii a new producer with a high biotechnological potential

Abstract

Filamentous fungi isolated from soil samples were screened for extracellular lipase production. The best producer was Hypocrea pseudokoningii identified by taxonomical criteria, and by rDNA sequencing of the variable internal transcribed spacers (ITS I and II) and the intervening 5.8S gene. The fungus was grown in a complex medium supplemented with 1% Tween 80 and 0.2% yeast extract, for 4 days. The optimum pH for extracellular and intracellular lipases was 7.0 and 8.0, respectively. Both enzymes exhibited maximum activity at 40°C. Extracellular and intracellular lipase activities were highly stable in the pH range 3.0–8.0 at room temperature. The intracellular lipase was thermostable up to 60°C, for 15 min and the extracellular, for 107 min, at the same temperature. The intracellular lipase was stimulated by silver ions. Extracellular lipase was stable in organic solvents, such as DMSO, alcohols, acetone, and acetonitrile, for 24 hours. Lipase activity increased around 80% when detergents were added to the enzymatic assay, such as Tween 80, Triton X-100, and SDS.     

Synthesis of Analogs of Pestalotin

Starch-utilizing mutants of Escherichia coli which can grow well on starch or amylose as the sole carbon source were isolated. The maximal viable cell number of the starch-utilizing mutants on the polysaccharide media reached the same level (4 × 10⁹ cells/ml) as that with glucose medium after incubation for 24 hours at 37°C. The isolated mutants could produce more intracellular α-amylase than the wild-type strain, and the enzyme activity was detected in the extracellular fluid. Polyacrylamide gel electrophoresis showed that the intracellular and extracellular enzymes had similar electrophoretic mobilities. These observations suggested that the ability of growth on the polysaccharide media was due to the excreted α-amylase, which appeared to be identical with the intracellular enzyme.