Cationic polyelectrolytes-lipases complexes formation as tool for recovery of these enzymes from their natural sources

In the present paper the formation of complexes between positively charge polyelectrolyte (polyethyleneimine and chitosan) and Candida rugosa lipase from a crude extract and porcine lipase from pancreas commercial homogenate preparations were analyzed. The solubility of lipases-cationic polyelectrolytes formation was dependent on: polyelectrolyte densities electrical charge, polyelectrolyte and enzyme concentrations and salts present in the solution. The lipase was recovered from the non-soluble complex by adding of NaCl at a given pH. Although the polyelectrolytes did not affect lipase biological activity, both of them produced good enzyme recovery (>90%); however, purification factors were low. This methodology appears to be a good previous prepurification and concentration method, using, low-cost polymers, allows the design of a purification method where the protein of interest is present in a large volume with respect to the small amount of polyelectrolyte added.     

Kinetic behavior of phosphotriesterase and its non-covalent complexes with polyelectrolyte in systems with polar and non-polar organic solvents

Soluble phosphotriesterase from E. coli DH5 together with E. coli DH5 cells with the phosphotriesterase activity were co-immobilized into poly(vinyl alcohol) (PVA) cryogel and studied in water/organic systems with polar and non-polar organic solvents. The phosphotriesterase activity was competitively inhibited by polar organic solvents. The inhibition constant correlated with the dielectric constant () of the solvent. The rate of the enzyme-catalyzed reaction in biphasic non-polar solvent/water systems was independent of water/organic ratio and the hydrophobicity of the solvent. Formation of the non-covalent complexes with polyelectrolytes was suggested to enhance the resistance of the phosphotriesterase towards inactivation by organic solvents in their homogeneous mixtures with water.     

A simple activity assay for thrombin and hirudin

Cloning of the thrombin cDNA has made it possible to study thrombin function by site-directed mutagenesis. Quantitative results from studies of thrombin mutants are often hindered by difficulties in assaying the enzyme activity. The high enzyme concentrations required for activity determination by standard methods limit their usefulness to thrombin mutants that cannot be readily produced in large quantities. We have developed a novel method using the synthetic substrate S-2238 and hirudin, a tight-binding inhibitor of thrombin, that allows for the active-site titration of thrombin at concentrations as low as 20 pM, with an error of 5%. In addition, hirudin activity can be determined by this method to concentrations as low as 40 pM, with an error of 5%.     

Lysophospholipase activity in rat brain subcellular fractions

Lysophospholipase activity in brain subcellular fractions was measured by the release of myristic acid from 1-myristoylglycerophosphocholine or through the formation of [³²P]glycerophosphocholine from [³²P]lysophosphatidylcholine. Although the lysophospholipase activity was highest in microsomes, considerable enzyme activity was also found in other subcellular membrane fractions. The pH optimum for the microsomal enzyme was around 7, whereas the synaptosomes and non-synaptic plasma membranes exhibited a pH maximum around 8. Although the enzyme did not require divalent cations for activity, divalent cations (1 mM) such as Hg²⁺, Cu²⁺, and Zn²⁺ inhibited potently the enzyme activity. Enzyme activity was also partially inhibited by both saturated and polyunsaturated fatty acids (25–200 M), and the inhibition seemed to be greater in the membrane than in the cytosolic fractions. Ionic detergents such as deoxycholate and taurocholate inhibited the lysophospholipase. On the other hand, the effect of Triton X-100 was biphasic, i.e., stimulation at concentrations below 100 g/mg protein and inhibition at higher concentrations. Addition of cholesterol (50–250 g/ml), but not cholesteryl esters, also potently inhibited enzyme activity. The presence of active lysophospholipase(s) in brain is probably an important mechanism for preventing unnecessary accumulation of lysophospholipids which may exert a deleterious effect on the membranes because, of their detergent properties.     

Stabilization and activation of alpha-chymotrypsin in water-organic solvent systems by complex formation with oligoamines

Formation of enzyme-oligoamine complexes was suggested as an approach to obtain biocatalysts with enhanced resistance towards inactivation in water-organic media. Complex formation results in broadening (by 20-40% v/v ethanol) of the range of cosolvent concentrations where the enzyme retains its catalytic activity (stabilization effect). At moderate cosolvent concentrations (20-40% v/v) complex formation activates the enzyme (by 3-6 times). The magnitude of activation and stabilization effects increases with the number of possible electrostatic contacts between the protein surface and the molecules of oligoamines (OA). Circular dichroism spectra in the far-UV region show that complex formation stabilizes protein conformation and prevents aggregation in water-organic solvent mixtures. Two populations of the complexes with different thermodynamic stabilities were found in alpha-chymotrypsin (CT)-OA systems depending on the CT/OA ratio. The average dissociation constants and stoichiometries of both low- and high-affinity populations of the complexes were estimated. It appears that it is the low-affinity sites on the CT surface that are responsible for the activation effect.     

Milk-clotting protease production by <Emphasis Type="Italic">Nocardiopsis</Emphasis> sp. in an inexpensive medium

The milk-clotting and proteolytic activities of extracellular enzyme preparations from Nocardiopsis sp. were investigated under different culture conditions. A soybean flour medium was used, with concentrations of soybean flour and of glucose varying from 0.25 to 1% w/v and from 0 to 1% w/v, respectively. Growth was monitored with 2ml samples withdrawn from the culture medium at 8-h intervals, for determination of total protein, proteolytic activity, milk-clotting activity and sugar reduction. The best milk-clotting protease production, with a specific activity of 24.49U/mg at 40h, was obtained in the glucose-free medium containing soybean flour 1% w/v.     

Partial characterization of β-glucosidase activity produced by Kluyveromyces marxianus IMB3 during growth on cellobiose-containing media at 45°C

Summary We report- the partial characterization of a -glucosidase produced during growth of the thermotolerant yeast, K. marxianus IMB3 on lactose-containing media at 45°C. The enzyme had Km values of 1.1mM and 14.8mM for the substrates p-nitrophenyl--D-glucoside and cellobiose, respectively. The enzyme had a pH optimum of 5.5 and was optimally active at 50°C. It was stable up to 125 hours at 25°C and 35°, with half-lives of 45 hours and 2 hours at 45°C and 50°C, respectively. The enzyme was inhibited to varying degrees in the presence of metal ions and was completely inactivated by Hg²⁺. Ethanol concentrations [1–10% (v/v)] had little effect on activity. Glucose (20mM) caused inhibition when p-nitrophenyl--D-glucoside was used as substrate, whereas lactose at similar concentrations had no effect.     

Effect of L-arginine on Na+,K+-ATPase activity in rat aorta endothelium

The effect of L-arginine on the Na⁺,K⁺-ATPase activity in rat aorta endothelium was studied at its physiological concentrations in the range of 10^–6-10^–3 M. The enzyme activity was 35.5% increased by low concentrations of L-arginine (10^–5 M) and its activity was 32.3-37.1% decreased at the L-arginine concentrations of 10^–4-10^–3 M. A similar inhibition (by 34.5-42.8%) was also found in the presence of a NO-donor nitroglycerol (10^–4-10^–3 M). An optical isomer of L-arginine, D-arginine, at the concentrations of 10^–5 M also increased the enzyme activity by 37.1%, but its inhibiting effect was much less pronounced and was 15.7% at the D-arginine concentration of 10^–3 M. An inhibitor of NO-synthase, L-NAME (N^G-nitroarginine, methyl ester), failed to inhibit Na⁺,K⁺-ATPase. However, the presence of L-NAME abolished the inhibition of Na⁺,K⁺-ATPase by high concentrations of L-arginine. Thus, the effect of L-arginine on the endothelial Na⁺-pump depended on its concentration, and it is suggested that the enzyme inhibition by high concentrations of L-arginine should be associated with activation of the endogenous synthesis of NO.     

Glycosylated α-chymotrypsin as a catalyst for kyotorphin synthesis in water-organic media

-Chymotrypsin was covalently modified with cellobiose by chemical means. After adsorption on to a porous polyamide support, both the native and the glycosylated immobilized derivatives were used to synthesize a kyotorphin derivative (N-benzoyl-l-tyrosyl-l-argininamide) in acetonitrile/water. Glycosylated chymotrypsin gave a 125% increase in product formation (750 nmol mg^–1 catalyst in 3 h) at 60% (v/v) acetonitrile/water. Maximal protective effect of this glycosylation process was at 70% (v/v) acetonitrile/water, at which concentration the half-life of the glycosylated enzyme was 20-times longer than that of the native form (52 min and 2.8 min, respectively).     

Opposing effects of two osmolytes – trehalose and glycerol – on thermal inactivation of rabbit muscle 6-phosphofructo-1-kinase

Trehalose and glycerol are known as good stabilizers of function and structure of several macromolecules against stress conditions. We previously reported that they have comparable effectiveness on protecting two yeast cytosolic enzymes against thermal inactivation. However, enzyme protection has always been associated to a decrease in catalytic activity at the stabilizing conditions i.e., the presence of the protective molecule. In the present study we tested trehalose and glycerol on thermal protection of the mammalian cytosolic enzyme phosphofructokinase. Here we found that trehalose was able to protect phosphofructokinase against thermal inactivation as well as to promote an activation of its catalytic activity. The enzyme incubated in the presence of 1 M trehalose did not present any significant inactivation within 2 h of incubation at 50 C, contrasting to control experiments where the enzyme was fully inactivated during the same period exhibiting a t_0.5 for thermal inactivation of 56± 5 min. On the other hand, enzyme incubated in the presence of 37.5% (v/v) glycerol was not protected against incubation at 50 C. Indeed, when phosphofructokinase was incubated for 45 min at 50 C in the presence of lower concentrations of glycerol (7.5–25%, v/v), the remaining activity was 2–4 times lower than control. These data show that the compatibility of effects previously shown for trehalose and glycerol with some yeast cytosolic enzymes can not be extended to all globular enzyme system. In the case of phosphofructokinase, we believe that its property of shifting between several different complex oligomers configurations can be influenced by the physicochemical properties of the stabilizing molecules. (Mol Cell Biochem 269: 203)     

Operational and storage stability of neutral β-mannanase from Bacillus licheniformis

The stability of neutral -mannanase from Bacillus licheniformis during operation and storage was investigated. The enzyme activity decreased by 70% with a hydrolysate of glucomannan at 20 g l^–1 over 30 min at 25 °C. In an enzymatic membrane reactor operated at 50 °C after 24 h, the loss of enzyme activities were 23% and 9% in the absence/presence of the substrate. The residual activities of the enzyme were 21% and 90%, respectively, when stored in 30% (v/v) glycerol solution and in solid state at 4 °C after one year.     

The effects of aluminium on nodulation and symbiotic nitrogen fixation in Casuarina cunninghamiana Miq.

In order to investigate the effects of Al on nodule formation and function in the Casuarina-Frankia symbiosis, inoculated plants were grown in sand culture at five nominal Al concentrations (0-880 M Al) at pH 4.0. There was an Al-free control at pH 6.0 to assess the effects of pH 4.0 treatments. Mean N concentration of nodules was significantly less at pH 4.0 (1.83%) than at pH 6.0 (2.01%). There were nodulated plants at all Al levels, though there were fewer nodulated plants at 440 and 880 M Al. Dry weights of nodules, shoots and roots were not reduced by Al concentrations at or below 220 M Al, but were decreased by Al concentrations at or above 440 M Al. Nodule weight expressed as a percentage of total weight did not differ significantly with respect to an Al-free control at pH 4. N concentrations of shoots and whole plants were significantly reduced at 440 M Al. Nodular specific acetylene reduction activity (ARA) did not differ significantly among Al treatments. However, N₂-fixation efficiency was decreased from 0.20 to 0.10 mg N fixed mg nodule dry weight^–1 at 880 M Al.     

Acid proteinase of hypothalamus

In a continuing study of the physiological role of protein breakdown in the hypothalamus, acid proteinase from bovine hypothalamus was purified about 1000-fold. The molecular weight of the enzyme was approximately 50,000. Maximal activity against hemoglobin was obtained at pH 3.2–3.5; serum albumin was split much more slowly. Hypothalamus acid proteinase was partially inhibited by -phenyl pyruvate, or benzethonium Cl, and was completely inhibited by low concentrations of pepstatin. This proteinase splits somatostatin, substance P, and analogs of substance P. The probable sites of enzyme action on these peptides were determined by the end group dansyl technique. The enzyme, most likely cathepsin D, may play an important role in the formation and breakdown of peptide hormones in the hypothalamus.     

Transferase reactions of the β-galactosidase from Streptococcus thermophilus

Summary The -galactosidase from Streptococcus thermophilus formed transferase products (including up to six disaccharides and two trisaccharides) during the hydrolysis of lactose to glucose and galactose. The extent of transferase products formed was dependent on the initial lactose concentration, reaching up to 40% of the total carbohydrate at 70% w/v lactose. At high lactose concentrations (40% w/v) trisaccharide transferase products were formed initially, followed by the appearance of disaccharide transferase products. In contrast, at low lactose concentrations (7.5 w/v), only traces of the trisaccharides were detected with disaccharides being the predominant transferase products. The disaccharide products accumulated to relatively high concentrations late in the overall hydrolysis of lactose, at both high and low initial lactose concentrations, while the trisaccharides peaked much earlier and were themselves subsequently hydrolysed prior to the complete disappearance of lactose. It was possible to study the hydrolysis of galactosyl lactose by the S. thermophilus -galactosidase using a semi-pure galactosyl lactose preparation containing 5% lactose. The hydrolysis of this trisaccharide occurred via at least four disaccharide intermidiates, which appeared chromatographically identical to the disaccharide transferase products formed during lactose hydrolysis. This suggests that the enzymic formation and subsequent hydrolysis of galactosyl lactose occurs via coincident reaction pathways. The initial rate of galactose over glucose formation during galactosyl lactose hydrolysis changed from a ratio of 3:1 at low (2–3% w/v) substrate concentrations to 1.5:1 at high (>20% w/v) concentrations. This indicates a shift in the preferred initial cleavage site from the galactose-galactose bond to the galactose-glucose bond.     

Nutritional status of Aureobasidium sp. ATCC 20524 for the production of β-fructofuranosidase

Sucrose at 10 to 20% (w/v) was the best carbon source for the production of -fructofuranosidase by Aureobasidium sp. ATCC 20524. At higher concentrations, it arrested growth. Glucose and fructose were also good carbon sources for the enzyme production. Yeast extract at 1.5 to 2% (w/v) was the best nitrogen source for the enzyme production and for cell growth. Addition of NaNO₃ (1 to 2%, w/v) and MgSO₄·7H₂O (0.5 to 1.5%, w/v) to the cultivation medium increased the intracellular enzymatic activity. The total enzymatic activity and cell growth reached 1.2×10⁴ U/flask and 2.5 g dry cell/flask, respectively after 48 h.Sachio Hayashi, Yoshihiko Shimokawa, Masaharu Nonoguchi, Yoshiyuki Takasaki and Kiyohisa Imada are with the Department of Industrial Chemistry, Faculty of Engineering, Miyazaki University, 1-1 Nishi, Gakuen Kibanadai, Miyazaki, 889-21 Japan. Hideo Ueno is with the Nippon Oligo Corporation, 588 Izumisawa, Jyohana-chyo, Tonami-gun, Toyama, 939-18, Japan.     

Bleach-stable, alkaline protease from Bacillus sp.

A bleach-stable, thermotolerant, alkaline protease for detergent formulation from a newly isolated Bacillus SB5 is reported. Most (85%) activity of the enzyme was retained in the presence of 10% (v/v) H2O2 and 1% SDS (w/v) at 40°C, after 1 h. The enzyme was optimal at pH 10 and 60°C to 70°C. Enzyme activity was enhanced 30 to 80% in presence of ionic and non-ionic detergents, surfactants and commercial detergents or bleach.     

An evaluation of phenylpropanoid metabolism during cold-induced sphagnorubin synthesis in Sphagnum magellanicum BRID

L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Sphagnum magellanicum BRID. is inhibited by t-cinnamic acid in vitro only at relatively high doses. In contrast, p-coumaric acid does not display an inhibitory effect in a comparable concentration range. Sphagnum acid, an endogenous cinnamic acid derivative of sphagna, strongly enhances PAL activity at certain concentrations. The involvement of the phenylpropanoid pathway in the biosynthesis of the main reddish-violet wall pigment of Sphagnum magellanicum (sphagnorubin) is studied at several metabolic levels. Extractable PAL activity rises in response to the stimulus of sphagnorubin synthesis (nightly application of low temperature). If the formation of sphagnorubin is blocked in vivo by the PAL-inhibitor L--aminooxy--phenylpropionic acid (AOPP), complementation of the mosses by p-coumaric acid is able to overcome partially the inhibition. The mechanism of PAL induction by nightly cold treatment is independent of soluble carbohydrates which concomitantly accumulate as a result of photosynthetic action. Suppression of the sugar formation by application of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) is contrasted with an enhancement of PAL activity above the level of the merely cold-treated plants. The fluctuations of the enzyme level are principally unaffected by a DCMU-treatment.Abbreviations L-AOPP L--aminooxy--phenylpropionic acid - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5.) - TAL L-tyrosine ammonia-lyase Dedicated to Professor H. Rudolph     

Allosteric regulation of cardiac sarcoplasmic reticulum Ca ATPase: a comparative study

Summary The mechanisms of allosteric regulation of the Ca-ATPases of cardiac and skeletal sarcoplasmic reticulum by ATP have been compared. Although both enzymes showed stimulation of ATPase activity by ATP, the cardiac enzyme did not show the plateau in ATPase activity at 10–100M ATP seen with the skeletal enzyme. Likewise the phosphoenzyme (EP) levels did not plateau with the cardiac enzyme as they did with the skeletal enzyme. The apparent negative cooperatively which was seen in the kinetics of ATP hydrolysis at low ATP concentrations was not due to negative cooperatively in substrate binding to either enzyme. The cardiac enzyme did show, however, much higher affinity for the ATP analog, AMPPCP, which helps explain how AMPPCP blocks ATPase activity in the cardiac enzyme and stimulates ATPase activity in the skeletal enzyme. Fluorescein isothiocyanate was used to determine if allosteric regulation takes place through site-site interactions in oligomers. The 1 to 1 ratio between AMPPCP binding sites and FITC binding sites eliminated allosteric regulation by effector sites in both enzymes. The allosteric mechanism which remained was one in which the active-site becomes an effector-site by the early departure of ADP in the reaction mechanism. The step stimulated by the binding of ATP at the active-site turned effector-site was a nonphosphorylated form of the enzyme in cardiac sarcoplasmic reticulum and a phosphorylated form in skeletal sarcoplasmic reticulum.Abbreviations AMPPCP Adenylyl Methylenediphosphonate - EGTA Ethyleneglycol Bis(amino-ethyl ether)-N,N,N,N Tetraacetic Acid - Pi Inorganic Phosphate - EP Phosphorylated Enzyme - FITC Fluorescein Isothiocyanate - MOPS 3-(N-morpholino)-Propanesulfonic Acid - v/EP ratio of calcium dependent ATPase activity to phosphoenzyme level - V initial rate of phosphoenzyme formation - LSSR Light Sarcoplasmic Reticulum - CSR Cardiac Sarcoplasmic Reticulum.     

Nitrate content and nitrate reductase activity in Rumex obtusifolius L.

Summary With Rumex obtusifolius L., the influence of some environmental conditions on nitrate uptake and reduction were investigated. Nitrate concentrations of plant material were determined by HPLC, the activity of nitrate reductase by an in vivo test. As optimal incubation medium, a buffer containing 0.04 M KNO₃; 0.25 M KH²PO₄; 1.5% propanol (v/v); pH 8.0 was found. Vacuum infiltration caused an increase of enzyme activity of up to 40%.High nitrate concentrations were found in roots and leaf petioles. Nitrate reductase activity of these organs, however, was low. On the other hand, the highest nitrate reductase activity was observed in leaf laminae, which contained lowest nitrate concentrations.In leaves, nitrate content and nitrate reductase activity exhibited inverse diurnal fluctuations. During darkness, decreasing activities of the enzyme were followed by increasing nitrate concentrations, while during light the contrary was true. In petioles diurnal fluctuations in nitrate content were observed, too. No significant correlations with illumination, however, could be found.Our results prove that Rumex obtusifolius is characterized by an intensive nitrate turnover. Theoretically, internal nitrate content of the plant would be exhausted within a few hours, if a supply via the roots would be excluded.     

A constitutive maltotetraose-producing amylase fromPseudomonas sp. IMD 353

Pseudomonas sp. IMD 353, secretes an extracellular maltotetraose-producing amylase. One of the most outstanding features of this enzyme is that it is produced constitutively (29 units/ml), using glucose (3%, w/v) as the carbon source. The amylase was purified to homogeneity and its enzymic properties examined. It had maxima for activity at pH 7.0 and 50°C, a relative molecular mass of 63,000 and an isoelectric point at pH 5.0. Specific amylase inhibitors, tendamistat and -amylase wheat inhibitor, activated the enzyme. Starch was hydrolysed from the non-reducing chain ends, by an endo-acting mechanism, producing maltotetraose in the -anomeric form. Yields of 65% (w/v), and 62% (w/v) were obtained on hydrolysis of starch (1%, w/v) and dextrin (15%, w/v), respectively. This enzyme failed to hydrolyse mmaltotetraose, even on prolonged incubation.