Hydrolytic properties of crude alpha-L-rhamnosidases produced by several wild strains of mesophilic fungi

AIMS: Observation of the dependence of alpha-L-rhamnosidase activity on pH and temperature and the capability to hydrolyse concentrated naringin solutions and hesperidin suspensions of enzyme complexes produced by several fungi. METHODS AND RESULTS: The enzymes were produced by several wild strains of mesophilic fungi grown in liquid media containing rhamnose as sole carbon source. The properties and their ranges of values measured were as follows: (i) optimum pH, 3.5-6.5; (ii) optimum temperature, 50-65 degrees C; (iii) hydrolysis of supersaturated 100 g l(-1) naringin solutions, 45-100% and (iv) hydrolysis of hesperidin suspensions, 6-35%. CONCLUSIONS: Some alpha-L-rhamnosidase enzymes hydrolysed supersaturated naringin solutions with a high yield. The enzyme produced by Fusarium sambucinum 310 showed good activity even at pH 10. SIGNIFICANCE AND IMPACT OF THE STUDY: Crude enzymes with possible utilization as catalysts for the manufacture of hydrolysis products of the flavonoid glycosides were found.     

Thermal inactivation and product inhibition of Aspergillus terreus CECT 2663 alpha-L-rhamnosidase and their role on hydrolysis of naringin solutions

The kinetics of thermal inactivation of A. terreus alpha-rhamnosidase was studied using the substrate p-nitrophenyl alpha-L-rhamnoside between 50 degrees C and 70 degrees C. Up to 60 degrees C the inactivation of the purified enzyme was completely reversible, but samples of crude or partially purified enzyme showed partial reversibility. The presence of the product rhamnose, the substrate naringin, and other additives reduced the reversible inactivation, maintaining in some cases full enzyme activity at 60 degrees C. A mechanism for the inactivation process, which permitted the reproduction of experimental results, was proposed. The products rhamnose (inhibition constant, 2.1 mM) and prunin (2.6 mM) competitively inhibited the enzyme reaction. The maximum hydrolysis of supersaturated naringin solution, without enzyme inactivation, was observed at 60 degrees C. Hydrolysis of naringin reached 99% with 1% naringin solution, although the hydrolysis degree of naringin was only 40% due to products inhibition when the initial concentration of flavonoid was 10%. The experimental results fitted an equation based on the integrated Michaelis-Menten's, including competitive inhibition by products satisfactorily.     

Thermal Inactivation and Product Inhibition of Aspergillus terreus CECT 2663 α-L-Rhamnosidase and Their Role on Hydrolysis of Naringin Solutions

The kinetics of thermal inactivation of A. terreus α-rhamnosidase was studied using the substrate p-nitrophenyl α-L-rhamnoside between 50°C and 70°C. Up to 60°C the inactivation of the purified enzyme was completely reversible, but samples of crude or partially purified enzyme showed partial reversibility. The presence of the product rhamnose, the substrate naringin, and other additives reduced the reversible inactivation, maintaining in some cases full enzyme activity at 60°C. A mechanism for the inactivation process, which permitted the reproduction of experimental results, was proposed. The products rhamnose (inhibition constant, 2.1 mM) and prunin (2.6 mM) competitively inhibited the enzyme reaction. The maximum hydrolysis of supersaturated naringin solution, without enzyme inactivation, was observed at 60°C. Hydrolysis of naringin reached 99% with 1% naringin solution, although the hydrolysis degree of naringin was only 40% due to products inhibition when the initial concentration of flavonoid was 10%. The experimental results fitted an equation based on the integrated Michaelis-Menten's, including competitive inhibition by products satisfactorily.     

Substrate specificities in hydrolysis of polyhydroxyalkanoates by microbial esterases

Summary Enzymatic degradations of 5 different polyhydroxyalkanoates (PHA) were investigated at 37°C in the aqueous solutions (pH 7.4) containing different microbial enzymes of 16 lipases and 5 PHA depolymerases. The substrate specificities of microbial PHA depolymerases on hydrolysis of polyhydroxyalkanoates were distinguished from those of microbial lipases.     

Lipase-catalyzed acylation of naringin with palmitic acid in highly concentrated homogeneous solutions

Acylation reactions of naringin with palmitic acid were performed by a lipase after formation of highly concentrated homogeneous solutions. Their initial naringin concentration was 840–950 mM, which is 20–60 times greater than that in organic solvent media. The overall productivity of highly concentrated solutions was more than 15 times greater than those of organic phase media. The addition of DMSO (20–40%, w/w) to substrate mixtures lowered the melting temperature of a naringin–palmitic acid mixture (1:1 molar ratio) to about 40 °C. Reactions at 80 °C apparently followed Michaelis–Menten kinetics despite extremely high substrate concentrations. As the temperature increased from 60 °C to 80 °C, the apparent viscosity of the highly concentrated solution decreased remarkably from 4.31 Pa s to 0.063 Pa s. An activation energy of 7.65 kcal/mol obtained in a range of 60–75 °C suggests a diffusion-control. On the other hand, an activation energy of 17.09 kcal/mol in a range of 75–90 °C indicates a reaction-control. The highest product conversion yield of 33% (mol/mol) was obtained in a 10 h reaction at 80 °C. Addition of activated molecular sieves to the highly concentrated solution increased the product conversion yield by 7% (mol/mol), suggesting that the original equilibrium was disrupted by removing water and then a new equilibrium was reached.     

Pepstatin-sensitive proteolytic activity of sorghum seeds

Two novel proteinases were isolated from resting sorghum seeds and purified 100-fold. The activity of the purified enzymes was completely inhibited by pepstatin A and was unaffected by PMSF, leupeptin, EDTA and E-64 (L-trans-epoxysuccinyl leucylamino 4 guanidino butane), which indicates that they belong to the class of aspartic proteinases. SDS-PAGE and native-PAGE revealed a monomeric 29-kDa enzyme and a heterodimeric 61-kDa enzyme with two S-S linked subunits of 49 and 12 kDa. The proteases have maximum activity at 45 °C and pH 3.5, with haemoglobin as substrate. Activity at 60 °C is higher than at 30 °C.     

Growth conditions and temperature-dependent substrate specificity of two extremely thermophilic bacteria

Summary Conditions for cultivating two extremely thermophilic bacteria, isolated from the hot springs of Yellowstone National Park, are described. One of these strains, Thermus aquaticus, can be grown on either succinate or pyruvate as the best substrates at 78° C. Acetate, glucose, and sucrose can also be utilized at this temperature. The temperature optimum was found to be 70° C, but the bacterium can be adapted to grow on succinate or pyruvate at 80° C. The other strain, YT-G has its growth optimum at 80° C and the maximum temperature was found to be 84° C. At this temperature pyruvate is the only substrate which gives good results, while glucose cannot be used as a carbon source. At 70° C, however, the yields obtained with glucose as a substrate are better than those with pyruvate at 80° C.Experiments with C¹⁴-labelled glucose have shown that the inability to utilize glucose at 80° C is not due to an inactivation of the initial steps of the glycolytic pathway. Phosphorylated sugars and a compound corresponding to -glycerophosphate were found to be formed, the latter being accumulated as a side product of normal glycolysis. The enzymes leading to this product, and those which are involved in the conversion of pyruvate were found to be functioning at 80° C, while intermediate enzymes of the glycolytic pathway are assumed to be less heat resistant, thus blocking the utilization of glucose at this temperature. The ability of strain YT-G to grow on glucose is, however, promptly resumed if the temperature is lowered.Lysozyme treatment was found to lead to a complete conversion of T. aquaticus cells to spheroplast while cells of strain YT-G are only slightly altered by this procedure.     

Thermal responses of mutant enzymes and temperature limits to growth

Summary The half-lives of fifty-two mutant forms of -galactosidase, in which serine had been substituted for the original amino-acid, were measured at 57°C. Sixty percent of the mutant enzymes had less than half the stability of the normal enzyme at high temperature but none had greater sensitivity to low temperature. A sample of these thermolabile enzymes showed unaltered activation energy. Enzymes with altered substrate affinity generally had reduced apparent heats of activation, decreased heats and entropies of association, and were thus relatively less sensitive to temperature change.The intracellular behaviour of thermolabile enzymes was examined by showing that substrate hydrolysis is the limiting step in lactose utilization, that the enzyme-substrate complex is more stable than the free enzyme and that binding of the enzyme to structures within the cell increases stability and lowers affinity. Amongst enzymes having half-lives less than about seven minutes at 47.75°C, there is a close relationship between enzyme lability and diminished growth rate on lactose at 44°C. The effect of mutation on the thermal behaviour of multi-enzyme systems and the relation of enzyme thermolability to temperature-induced growth deficiencies and temperature-dependent heterosis are discussed.     

Unexpected Differences between D- and L- Tyrosine Lead to Chiral Enhancement in Racemic Mixtures Dedicated to the memory of Prof. Shneior Lifson – A great liberal thinker.

We report here an unexpected difference in the solubilities of D- and L-tyrosine in water, which could be discerned by their rate of crystallization and the resulting concentrations of their saturated solutions. A supersaturated solution of 10 mM L-tyrosine at 20 °C crystallized much more slowly than that of D-tyrosine under the same conditions, and the saturated solution of L-tyrosine was more concentrated than that of D-tyrosine. Supersaturated solutions of 10 mM DL-tyrosine in water formed precipitates of predominantly D-tyrosine and DL-tyrosine, resulting in an excess of L-tyrosine in the saturated solution. The experimental setups were monitored independently by UV-absorption, radioactivity tracing, optical rotation and X-ray diffraction. The process of nucleation and crystallization of D- and L-tyrosine is characterized by an exceptionally high cooperativity. It is possible that minute energy differences between D- and L-tyrosine, originating from parity violation or other non-conservative chiral discriminatory rules, could account for the observations. The physical process that initiated chiral selection in biological systems remains a challenging problem in understanding the origin of life, and it is possible that chiral compounds were concentrated from supersaturated racemic mixtures by preferential crystallization.     

Purification and characterization of naringinase from a newly isolated strain of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 1344 for the transformation of flavonoids

Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg²⁺, SDS, p-chloromercuribenzoate, Cu²⁺ and Mn²⁺ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca²⁺, Co²⁺ and Mg²⁺ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg²⁺ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.     

Inherent toxicity of organ preservation solutions to cultured hepatocytes

Organ preservation solutions have been designed to protect grafts against the injury inflicted by cold ischemia. However, toxicity of University of Wisconsin (UW) solution during rewarming has been reported. Therefore, we here assessed the toxicity of UW, histidine-tryptophan-ketoglutarate (HTK), Euro-Collins, histidine-lactobionate (HL), sodium-lactobionate-sucrose and Celsior solutions in cultured hepatocytes under hypothermic (4 °C), intermediate (21 °C) and physiological (37 °C) conditions. Marked toxicity of UW, HTK, HL and Euro-Collins solutions was observed at both 37 and 21 °C. With the exception of UW solution, these solutions also increased cell injury during cold incubation (LDH release after 18 h at 4 °C: HTK 76 ± 2%, Euro-Collins 78 ± 17%, HL 81 ± 15%; control: Krebs-Henseleit buffer 20 ± 6%). Testing of individual components using modified Krebs-Henseleit buffers suggested that histidine and phosphate are responsible for (part of) this toxicity. These potential toxicities should be taken into account in the development of future preservation solutions.     

Some factors contributing to the survival of rapidly cooled plant cells

The stability of innocuous intracellularly frozen cells was found to be influenced remarkably by the surrounding solutions and the degree of prefreezing. In the cells suspended in isotonic or slightly hypertonic glucose solution, innocuous intracellular freezing readily occurred and these rapidly frozen cells remained stable even at −30 °C for 20 min, while cells prefrozen to −10 °C after suspending in water survived standing at −30 °C only for 5 sec at most. These facts suggest that when the innocuous intracellularly frozen cells are surrounded with concentrated sugar solutions, these cells remain far more stable than those surrounded with ice.     

Phase transitions as a function of osmotic pressure in Saccharomyces cerevisiae whole cells, membrane extracts and phospholipid mixtures

Fourier Transform Infrared spectroscopy (FTIR) was used to determine the phase transition temperature of whole Saccharomyces cerevisiae W303-1 A cells as a function of Aw in binary water-glycerol media. A phase transition occurred at 12 °C in water, at 16.5 °C at Aw=0.75, and at 19.5 °C at Aw=0.65. The temperature ranges over which transition occurred increased with decreasing Aw. A total lipid extract of the plasma membranes isolated from S. cerevisiae cells was also studied, with a phase transition temperature determined at 20 °C in pure water and at 27 °C in binary water-glycerol solutions for both Aw levels tested. The pure phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) and three binary mixtures of these phospholipids (percentage molar mixtures of DMPC/DMPE of 90.5/9.5, 74.8/25.2, and 39.7/60.3) were studied. For DMPC, there was no influence of Aw on the phase transition temperature (always 23 °C). On the other hand, the phase transition temperature of DMPE increased with decreasing Aw for the three aqueous solutions tested (glycerol, sorbitol and sucrose), from 48 °C in water, to 64 °C for a solution at Aw=0.67. For the DMPC/DMPE mixtures, transitions were found intermediate between those of the two phospholipids, and a cooperative state was observed between species at the gel and at the fluid phases.     

The Sugar Model: Catalytic Flow Reactor Dynamics of Pyruvaldehyde Synthesis from Triose Catalyzed by Poly-L-Lysine Contained in a Dialyzer

The formation of pyruvaldehyde from triose sugars was catalyzedby poly-L-lysine contained in a small dialyzer with a 100molecular weight cut off (100 MWCO) suspended in a much largertriose substrate reservoir at pH 5.5 and 40 °C. Thepolylysine confined in the dialyzer functioned as a catalyticflow reactor that constantly brought in triose from thesubstrate reservoir by diffusion to offset the drop in trioseconcentration within the reactor caused by its conversion topyruvaldehyde. The catalytic polylysine solution (400 mM, 0.35mL) within the dialyzer generated pyruvaldehyde with a syntheticintensity (rate/volume) that was 3400 times greater than that ofthe triose substrate solution (12 mM, 120 mL) outside thedialyzer. Under the given conditions the final yield ofpyruvaldehyde was greater than twice the weight of thepolylysine catalyst. During the reaction the polylysine catalystwas poisoned presumably by reaction of its amino groups withaldehyde reactants and products. Similar results were obtainedusing a dialyzer with a 500 MWCO. The dialyzer method ofcatalyst containment was selected because it provides a simpleand easily manipulated experimental system forstudying the dynamics and evolutionary development of confinedautocatalytic processes related to the origin of life underanaerobic conditions.     

Foraging profits and thoracic temperature of honey bees (Apis mellifera)

Summary Body temperature and duration of foraging activities were affected by the concentration of sucrose solution imbibed. When experienced foragers of Apis mellifera arrived at a gravity feeder from the hive, thoracic temperature (T_TH) was independent of sucrose concentration (X = 36.3 °C). While imbibing 40% and 60% (g solute per g of solution) solutions bees maintained T_TH at approximately the same high level as upon arrival, but those imbibing 10%, 20%, and 30% solutions regulated T_TH lower (X = 33.5 °C). All bees departed the feeder for the hive at the same T_TH (X = 36.1 °C). Bees that imbibed 40% and 60% solutions sometimes immediately took flight after imbibition and averaged less than 15 s to takeoff. Time to takeoff was 2–3 times longer for bees that had imbibed 10% and 20% solutions because warmup preceded takeoff. The rate of energy expenditure at T_TH=36.3°C (at 40% and 60% solutions) was 20% greater than that at 33.3°C (at 10%, 20%, and 30% solution). Bees that fed on the highly concentrated solutions regulated T_TH so that rate of net energy gain was enhanced, but bees that fed on less concentrated solutions could have increased rate of net gain by maintaining a higher T_TH which would have reduced time required for takeoff. The latter bees lowered rate of expenditure of their limited energetic costs and thereby lowered short-term net profits in favor of improved long-term contribution to the colony.Abbreviations T _A ambient temperature - T _TH thoracic temperature     

Cryobiological properties of immature zebrafish oocytes assessed by their ability to be fertilized and develop into hatching embryos

As a step to develop a cryopreservation method for zebrafish oocytes, we investigated the cryobiological properties of immature oocytes at stage III by examining their ability to mature and to develop into hatching embryos after fertilization. When oocytes were chilled at −5 °C for 30 min, the maturation rate decreased, but the rates of fertilization and hatching were not significantly different from those of controls. When oocytes were exposed to hypotonic solutions for 60 min at 25 °C, the rates of maturation, fertilization, and hatching decreased in a solution with 0.16 Osm/kg or below. When oocytes were exposed to hypertonic solutions (containing sucrose) at 25 °C for 30 min, the maturation rate decreased in solution with 0.51 Osm/kg, whereas the hatching rate decreased with lower osmolality (0.40 Osm/kg). In an experiment on the toxicity of cryoprotectants (∼10%, at 25 °C), it was found that glycerol and ethylene glycol were toxic both by the assessment of maturation and hatching. Propylene glycol, DMSO and methanol were less toxic by the assessment of maturation, but were found to be toxic by the assessment of hatching. Methanol was the least toxic, but it was less effective to make a solution vitrify than propylene glycol. Therefore, a portion of methanol was replaced with propylene glycol. The replacement increased the toxicity, but could be effective to reduce chilling injury at −5 °C. These results clarified the sensitivity of immature oocytes to various cryobiological properties accurately, which will be useful for realizing cryopreservation of zebrafish oocytes.     

Effect of temperature on rice growth in nutrient solution and in acid sulphate soils from Vietnam

Climatic and soil factors are limiting rice growth in many countries. In Vietnam, a steep gradient of temperature is observed from the North to the South, and acid sulphate soils are frequently devoted to rice production. We have therefore attempted to understand how temperature affects rice growth in these problem soils, by comparison with rice grown in nutrient solution. Two varieties of rice, IR64 and X2, were cultivated in phytotrons at 19/21°C and 28/32°C (day/night) for 56 days, after 3 weeks preculture in optimal conditions. Two soils from the Mekong Delta were tested. Parallel with the growing experiments, these two soils were incubated in order to monitor redox potential (E _h ), pH, soluble Al and Fe, soluble, and available P. Tillering retardation at 20°C compared to 30°C was similar in nutrient solutions and in soils. The effect of temperature on increasing plant biomass was more marked in solutions than in soils. The P concentrations in roots and shoots were higher at 20°C than at 30°C, to such an extent that detrimental effect was suspected in plants grown in solution at the lowest temperature. The translocation of Fe from roots to shoots was stimulated upon rising temperature, both in solutions and in soils. This led to plant death on the most acid soil at 30°C. Indeed, the accumulation of Fe in plants grown on soils was enhanced by the release of Fe²⁺ due to reduction of Fe(III)-oxihydroxides. Severe reducing conditions were created at 30°C: redox potential (E _h ) dropped rapidly down to about 0 V. At 20°C, E _h did not drop below about 0.2 V, which is a value well in the range of Fe(III)/Fe(II) buffering. Parallel to E _h drop, pH increased up to about 6–6.5 at 30°C, which prevented plants from Al toxicity, even in the most acid soil. Phosphate behavior was obviously related to Fe-dynamics: more reducing conditions at 30°C have resulted in enhancement of available P, especially in the most acid soil.     

The Influence of Temperature on Metabolic and Cellular Protection of the Heart during Long-Term Ischemia: A Study Using P-31 Magnetic Resonance Spectroscopy and Biochemical Analyses

We have compared the influence of two different cold temperatures (below 10°C) for cardiac ischemia by measuring a large variety of hemodynamic and metabolic parameters during ischemia and reflow. Isolated isovolumic rat hearts were arrested with a preservation solution which was developed in our laboratory and then submitted to 5 h of cold storage (4°C, group I; and 7.5°C, group II) in the same solution. After an additional period of 50 min of ischemia at 15°C with intermittent cardioplegic infusion, hearts were reperfused for 60 min at 37°C. Function was assessed during the control period and reflow. High-energy phosphates and intracellular pH were followed by³¹P magnetic resonance spectroscopy. Analyses of metabolites and enzymes were performed by biochemical assays and HPLC in coronary effluents and in freeze-clamped hearts to assess cellular integrity. The energetic pool was better preserved at 4°C during ischemia (ATP at the end of 4°C ischemia, 59 ± 7% in group I vs 31 ± 5% in group II,P< 0.01) and reflow (P< 0.05) but membrane protection was higher when increasing the temperature to 7.5°C (reduction of creatine kinase leakage, 89 ± 16 IU/min in group I vs 51 ± 5 IU/min in group II,P< 0.05). As a result, functional recovery, represented by the rate pressure product, was higher in hearts preserved at 7.5°C (52 ± 6% recovery in group I vs 77 ± 7% in group II at the end of reflow,P< 0.05). Altogether, cold storage at 7.5°C provides a better protection than storage at 4°C.     

A continuous culture study of an obligately psychrophilic Pseudomonas species

Summary The optimum temperatures for growth and respiration of an obligately psychrophilic Pseudomonas spec. were 14°C and 23°C, respectively. The maximum temperature for growth was between 19 and 20°C. When cells were grown in a chemostat with lactate as the growth-limiting substrate at a specific growth rate of 0.05 hr^-1 over a temperature range of 5–19°C, it was found that RNA concentration was lowest at 14°C. At lower temperatures the cells compensated the decrease of reaction rates by increasing the concentration of RNA and of respiratory enzymes. A temperature raise above 14°C also increased cellular RNA, which probably counteracted an impairment of protein synthesis. Above 18°C the RNA increase ceased, resulting in a rapid decrease of protein synthesis, until between 19 and 20°C growth ceased entirely. Cells grown at 14°C showed a linear increase of RNA content and values with growth rate, when this was varied from 0.025 to the maximum value of 0.2 hr^-1.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday.     

Purification and properties of a xanthan depolymerase from a heat-stable salt-tolerant bacterial consortium

Summary A bacterial consortium (NRRL B-14401) resulting from soil enrichment growth on xanthan gum produces enzymes that can degrade xanthan gum in salt-containing solutions at temperatures up to 65°C. One component that cleaves the backbone linkages of both xanthan gum and carboxymethyl cellulose is called xanthan depolymerase. Two such depolymerase activities were isolated by high performance anion exchange chromatography, and their molecular weights determined by size exclusion chromatography to be 170 000 and 100 000 Da. The 170-kDa protein was purified and its properties studied. Sodium chloride and potassium chloride enhanced the hydrolysis of carboxymethyl cellulose, but decreased the rate of degradation of xanthan gum. The purified enzyme, which was optimally active at pH 6, was less stable to extremes of temperature than crude mixtures of cell-free culture broth; stabilized by i substrate it was active for more than 6 h at 50°C.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.