Novel microbial lipases: catalytic activity in reactions in organic media

2,000 microbial strains were isolated from soil samples and tested to determine their lipolytic activity by employing screening techniques on solid and in liquid media. Culture broths were initially tested with 1,2-O-dilauryl-rac-glycero-3-glutaric acid-resorufinyl ester, a chromogenic substrate specific for lipases. Fourteen lipase-producing microorganisms were selected and their taxonomic identification was carried out. Hydrolysis of tributyrin or olive oil and the esterification of oleic acid with heptanol were selected to preliminary evaluate the catalytic activity of these lipases. All the selected lipases catalysed this esterification reaction with good yields. Resolution of (R,S)-2-(4-isobutylphenyl) propionic acid, (R,S)-1-phenylethanol, (R,S) 1-phenylethylamine and of (R) or (S) glycidol were performed to evaluate the stereoselectivity of these novel enzymes as biocatalysts in reactions in organic media. Lipases from the fungi Fusarium oxysporum and Ovadendron sulphureo-ochraceum gave the best yields and enantioselectivities in the resolution of racemic ibuprofen and 1-phenylethanol. Several lipases displayed a high stereoselectivity in the resolution of chiral amines by an alcoxycarbonylation reaction.     

A new method to determine optimum temperature and activation energies for enzymatic reactions

A new method for determination of the optimum temperature and activation energies based on an idea of the average rate of enzymatic reaction has been developed. A mathematical model describing the effect of temperature on a dimensionless activity for enzyme deactivation following the first-order kinetics has been derived. The necessary condition for existence of the function extreme of the optimal temperature has been applied in the model. The developed method has been verified using the experimental data for inulinase from Kluyveromyces marxianus.     

Assessment of two immobilized lipases activity and stability to low temperatures in organic solvents under ultrasound-assisted irradiation

Both stability and catalytic activity of two commercial immobilized lipases were investigated in the presence of different organic solvents in ultrasound-assisted system. In a general way, for Novozym 435, the use of ethanol as solvent led to a loss of activity of 35% after 10 h of contact. The use of iso-octane conducted to a gradual increase in lipase activity in relation to the contact time, reaching a maximum value of relative activity of 126%. For Lipozyme RM IM, after 5 h of exposure, the enzyme presented no residual activity when ethanol was used as solvent. The solvents tert-butanol and iso-octane showed an enhancement of about 20 and 17% in the enzyme activity in 6 h of exposure, respectively. Novozym 435 and Lipozyme IM presented high stability to storage after treatment under ultrasound-assisted system using n-hexane and tert-butanol as solvents.     

A method to measure antioxidant activity in organic media: application to lipophilic vitamins

The 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS(.+)) can be generated by the enzymatic system formed by hydrogen peroxide and horseradish peroxidase in an organic medium. The ABTS radical is easily generated in acidified ethanol medium in about 100 s with a stability of 1.7 x 10(-3) (-deltaabs/min) monitored at 730 nm. Other organic solvents, such as methanol or acetone, have lower radical generation times but the radical is less stable. The addition of Trolox or a lipophilic antioxidant such as alpha-tocopherol or beta-carotene produces a decrease in absorbance that can be used to estimate antioxidant capacity. Using a spectrophotometric end-point method and microplate-reader equipment, we have developed a method that estimates the antioxidant activity of different lipophilic vitamins. The use of Trolox as an antioxidant standard led to a limit of detection of 0.08 nmoles and limit of quantitation of 0.28 nmoles, while similar values were obtained for alpha-tocopherol and beta-carotene. The relative antioxidant activity values obtained by different antioxidants showed that alpha-tocopherol has a similar antioxidant potential to Trolox and that beta-carotene has 2.6 times the antioxidant potential of Trolox. In our opinion, this method can be useful for estimating the antioxidant activity in lipophilic samples and as a complement to other methods that measure antioxidant activity in aqueous media.     

A simple method to determine the water activity of ethanol-containing samples

The water activity (aw) of microbial substrates, biological samples, and foods and drinks is usually determined by direct measurement of the equilibrium relative humidity above a sample. However, these materials can contain ethanol, which disrupts the operation of humidity sensors. Previously, an indirect and problematic technique based on freezing-point depression measurements was needed to calculate the aw when ethanol was present. We now describe a rapid and accurate method to determine the aw of ethanol-containing samples at ambient temperatures. Disruption of sensor measurements was minimized by using a newly developed, alcohol-resistant humidity sensor fitted with an alcohol filter. Linear equations were derived from aw measurements of standard ethanol-water mixtures, and from Norrish's equation, to correct sensor measurements. To our knowledge, this is the first time that electronic sensors have been used to determine the aw of ethanol-containing samples. Copyright 1999 John Wiley & Sons, Inc.     

Overdominance interacts with linkage to determine the rate of adaptation to a new optimum

Overdominance, or a fitness advantage of a heterozygote over both homozygotes, can occur commonly with adaptation to a new optimum phenotype. We model how such overdominant polymorphisms can reduce the evolvability of diploid populations, uncovering a novel form of epistatic constraint on adaptation. The fitness load caused by overdominant polymorphisms can most readily be ameliorated by evolution at tightly linked loci; therefore, traits controlled by multiple loosely linked loci are predicted to be strongly constrained. The degree of constraint is also sensitive to the shape of the relationship between phenotype and fitness, and the constraint caused by overdominance can be strong enough to overcome the effects of clonal interference on the rate of adaptation for a trait. These results point to novel influences on evolvability that are specific to diploids and interact with genetic architecture, and they predict a source of stochastic variability in eukaryotic evolution experiments or cases of rapid evolution in nature.     

Growing media constituents determine the microbial nitrogen conversions in organic growing media for horticulture

Vegetables and fruits are an important part of a healthy food diet, however, the eco‐sustainability of the production of these can still be significantly improved. European farmers and consumers spend an estimated €15.5 billion per year on inorganic fertilizers and the production of N‐fertilizers results in a high carbon footprint. We investigated if fertilizer type and medium constituents determine microbial nitrogen conversions in organic growing media and can be used as a next step towards a more sustainable horticulture. We demonstrated that growing media constituents showed differences in urea hydrolysis, ammonia and nitrite oxidation and in carbon dioxide respiration rate. Interestingly, mixing of the growing media constituents resulted in a stimulation of the function of the microorganisms. The use of organic fertilizer resulted in an increase in amoA gene copy number by factor 100 compared to inorganic fertilizers. Our results support our hypothesis that the activity of the functional microbial community with respect to nitrogen turnover in an organic growing medium can be improved by selecting and mixing the appropriate growing media components with each other. These findings contribute to the understanding of the functional microbial community in growing media and its potential role towards a more responsible horticulture.     

A simple method to determine trypsin and chymotrypsin inhibitory activity

A colorimetric method for serine protease inhibition was modified using N-Acetyl-DL-Phenylalanine beta-Naphthylester (APNE) as the substrate and o-Dianisidine tetrazotized (oD) as the dye. The reaction generated a single peak absorbing at 530 nm for both trypsin and chymotrypsin. Standard curves with increasing enzyme concentrations showed strong linearity. A standard curve for the serine protease inhibitor, Bowman-Birk Inhibitor (BBI), has been made using this modified method. The IC50 for 3 U of trypsin was found to be 33 ng and the IC50 obtained for 3 mU of chymotrypsin was 53 ng. A recombinant BBI (rBBI) gene was constructed, cloned and expressed in the yeast Pichia pastoris. Evaluating samples of rBBI for protease inhibitory activity by the gel activity method failed to quantify the inhibitor amounts, due to high sensitivity for trypsin inhibition and low sensitivity for chymotrypsin inhibition. After development, the results could not be quantified, even to the extent that 1 microl of rBBI could not be detected with chymotrypsin inhibition. Therefore, a modified method for trypsin and chymotrypsin inhibition was used to evaluate the level of rBBI-expression for these same samples. The level of rBBI expression was calculated to be 50-56 ng/microl of media. These amounts fit into the range of values previously obtained by Western blot analysis. This modified method allows us to combine the sensitivity of the gel activity method with the quantification attributes of a Western blot. Thus, the modified method represents a significant improvement in speed, sensitivity and reproducibility over the gel activity method.     

A new method to determine some characters of vegetation structure

The ecological importance of both horizontal and vertical denseness of vegetation is discussed. Existing methods to analyse them are reviewed and a new method is proposed. Horizontal and vertical denseness are estimated, both per layer of 10 cm and cumulative. From these data the following parameters are calculated: degree of overlap between layers, plant area index per layer, and average inclination per layer. The values thus obtained are compared with values measured directly or calculated otherwise. Some examples are given to illustrate the method.Abbreviations LAI = Leaf Area Index - GAI = Green Area Index - PAI = Plant Area Index     

A new biophysical method to determine the gain of the acoustic trachea in bushcrickets

A method is described for measuring the gain (i.e., the change of amplitude and phase angle) for sounds that propagate to the internal surface of the tympana in ears working as pressure difference receivers. The gain of the acoustic trachea has been measured in two similarly sized and closely related species of bushcrickets, in which the acoustic spiracles and tracheae differ markedly in size. The amplitude part of the gain is much larger in the species with the larger acoustic spiracle, whereas the phase part is very similar in the two species. The method is compared with other methods, which in the past have been used for estimating the gain of sound pathways inside animal bodies.     

A new method to determine the electrical transfer function of the human thorax

Traditional analyses have assumed that cardiac electrical activity is reflected on the surface ECG without distortion as the signal passes through the body tissues. This study aims to explore the frequency dependence of thoracic attenuation of surface-recorded intracardiac electrical activity. Twenty patients (14 men, 55 +/- 15 yr of age) undergoing electrophysiological study were enrolled. Rectangular unipolar stimuli were applied from a catheter positioned in the right ventricular apical area and another in the posteroseptal area without contact with the myocardium. An orthogonal Frank-lead surface ECG and a unipolar intracardiac electrogram near the pacing site were recorded. Frequency domain characteristics of the signal-averaged pacing impulses were analyzed. Linear regression analysis showed significant frequency-dependent attenuation in the magnitude transfer functions (R(2) = 0.84-0.89, P < 0.0001) and good linear fit for the phase transfer characteristics (R(2) = 0.98-1.0, P < 0.0001). Age, physical dimension, and respiratory characteristics had significant effects on the magnitude and phase characteristics of the transfer functions. Application of models of the low- and high-slope transfer functions to signal-averaged ECGs from 33 subjects showed differences in the attenuation of P and T waves relative to the QRS.     

A new method to determine the oxygen concentration inside the sapwood of trees

Research into the short-term fluctuations of oxygen concentrations in tree stems has been hampered by the difficulty of measuring oxygen inside tissues. A new method, which is based on fluorescence quenching of a ruthenium complex in the presence of oxygen, has been applied to measure changes of oxygen concentration in the sapwood of trees. During a field day-course oxygen increased with the radiation load and fell during the night (in Fagus orientalis from 20.3% in the afternoon to 17.5% in the morning next day). In a greenhouse experiment the sapwood oxygen concentration of Laurus nobilis could be influenced by flooding the root system. The very fast response, high resolution (better than 0.1%), easy calibration, and dependence only on oxygen and temperature make the technique well suited for measurements of oxygen concentrations in the sapwood.     

A new method to determine mean adult height from incomplete follow-up data

BACKGROUND/AIM: In long-term growth studies with adult height (AH) as outcome, reporting is often required while data are incomplete because some participants have not yet reached AH whereas others might be lost to follow-up. Current practice is to analyze only participants who did reach AH, which can easily give biased results. We introduce a new method into the area of growth research. METHODS: We used the data of patients from a registration database and a growth study. The new method uses growth data in time intervals. The percentage of children still growing and the mean growth at each interval are used to determine mean AH. RESULTS: With the new method, estimated mean AHs had smaller bias and standard error than with commonly used methods. The method is not hampered by a correlation between AH and age at reaching AH, unlike methods merely using patients who have reached AH. CONCLUSION: In contrast to commonly used methods, the new method provides valid results on mean AH when complete actual measurements of AH are not (yet) available, provided that drop-out, if any, is not related to (disappointing) growth. As it also uses observed data of children with incomplete follow-up, the method employs the data more effectively.     

A new method for the cytochemical staining of cells immobilized in agarose

Summary A quick, simple and inexpensive technique utilizing an isotonic matrix of semi-solid agarose for immobilizing channel catfish (Ictalurus punctatus) peripheral blood leucocytes onto microscope slides before cytochemical staining is described. The technique was demonstrated to be superior to the more conventional means of immobilizing cells for procedures requiring either fixed (Sudan Black B and nonspecific esterase stains) or viable (Nitro-Blue Tetrazolium stain) cells. Although the agarose immobilization technique was developed with channel catfish leucocytes, its utility with other fragile cell populations seems likely.