Evaluation of some enzymatic methods to measure the bio-activity of aquatic environments

Methods have been evaluated and standardised to measure the phosphatase, saccharase, amylase and lipase enzymatic activities in aquatic environments. The analytical reproducibility, the ecological applicability and the etiology of these activities have been investigated. The usefulness of the various enzymatic assays for routine analyses and for fundamental research of aquatic environments is discussed.     

The enzymatic activity from the sediment of the Gilau dam reservoir - Cluj county

The enzymological studies on the sediment of the accumulation lake that has the main purpose of supplying drinking water to the city of Cluj-Napoca and the nearby villages, were aimed at the comprehensive understanding of the complex processes that happen in these habitats of special significance. In the sediment samples the following enzymatic activities have been quantitatively determined: phosphatase, actual and potential dehydrogenase, catalase, urease and protease. Non-enzymatic catalytic activity was also measured. Based on the relative values for the enzymatic activities, the enzymatic indicator of the sediment quality (EISQ) was calculated (ranging from 0.1 to 0.7). The enzymatic activities have been qualitatively determined for maltase, saccharase, lactase, cellobiase, amylase, dextranase, levanase, cellulase and inulinase. The correlation between the enzymatic and bacteriologic potential was statistically calculated.     

Effect of glutaraldehyde and lead on the activity of hepatic glucose-6-phosphatase. A biochemical and cytochemical study.

The sensitivity of mouse liver glucose-6-phosphatase activity towards glutaraldehyde fixation has been analysed by biochemical and cytochemical means. The degree of enzymatic inhibition and various enzymatic properties have been studied. Several differences have been observed in the Km determination, the sensitivity to pH 5 and the activity related to pH between fixed and unfixed enzymes. The role of Pb++ ions in the cytochemical media has also been estimated. It is concluded that several enzymatic differences appear between fixed and unfixed enzymes and that the inhibition by Pb ions is dependent on the buffer and on the amount of substrate used.     

Enzymatic characteristics of ligninperoxidases from Penicillium citrinum, Fusarium oxysporum and Aspergillus terreus using n-propanol as substrate

The activities of ligninperoxidases from Penicillium citrinum MTCC 3565, Fusarium oxysporum MTCC 3379 and Aspergillus terreus MTCC 3374 have been assayed and the enzymatic characteristics like Km, pH and temperature optima using n-propanol as the substrate have been reported. The results suggest that n-propanol can substitute veratryl alcohol as substrate for assaying ligninperoxidase activities from different fungal strains, without affecting the enzymatic characteristics. The above strains were selected, as they were known to secrete ligninperoxidase in the liquid culture medium.     

Synthesis of the tripeptide glycyl-L-leucyl-L-phenylalanine and its analogs]

Peptide Gly-L-Leu-L-Phe and its derivatives were synthesized by the C-end elongation utilizing DCC/HOBT technique and by enzymatic route with the help of papain using esters of N-benzyloxycarbonyl-glycine and -L-leucine as acyl donors have been suggested. The chemical, similarly to the enzymatic, synthesis was not accompanied by racemization. Conditions for HPLC separation and preparative isolation of the enzymatic reaction products were developed.     

Economic evaluation of enzymatic hydrolysis of phenol-pretreated wheat straw

Experimental data from the enzymatic hydrolysis of phenol-pretreated Swedish wheat straw have been used to evaluate the cost fractions of capital and utility, enzyme, and raw material. Two different raw material prices and varying enzyme prices have been used. The evaluation is based on an empirical model for the enzymatic hydrolysis and a computer program where utility and equipment, enzyme, and raw material prices can be varied. The optimal residence time for the enzymatic hydrolysis is in the range of 70-110 h. A fed-batch procedure with substrate concentrations higher than 10% oven-dried material (ODM) and enzyme concentrations in the range (6-10) . 10(6) FPU/ton ODM should be used.     

Effect of ultraviolet rays on peroxidation and its regulation in the rat liver

The indirect effect of rat skin ultraviolet (UV) irradiation on lipid peroxidation and enzymatic systems of the liver has been studied. The processes of lipid peroxidation have been intensified after 72 hours of UV-irradiation, which is evidently due both to the activation of enzymatic system of initiation and propagation of lipid peroxidation and to the parallel decrease of the activity of enzymatic system regulation of given process in liver.     

Novel cofactor derivatives and cofactor-based models

In 1997 and the first half of 1998, numerous publications appeared reporting studies of cofactors and their analogues in classical model systems and in enzyme-catalyzed reactions directed at understanding the enzymatic reactions of their natural cofactors. Model systems based on flavins have provided new insights into enzymatic modulation of the flavin reduction potential, and enzymatic reactions of coenzyme A analogues and derivatives have been employed in several studies of coenzyme A utilizing enzymes. Coenzyme B₁₂ analogues have been utilized as alternate cofactors for B₁₂-utilizing enzymes, while pyrroloquinoline quinone esters and analogues have been employed in model studies of the reactions of quinoprotein-catalyzed reactions.     

Spectrophotometric assay of immobilized tannase

A procedure for the assay of immobilized tannase with Polyacrylamide gel, collagen and Duolite-S-762 as matrices is described. It is based on the spectrophotometric determination of gallic acid formed by the enzymatic hydrolysis of tannic acid. The kinetic parameters of the enzymatic reaction have been studied and an assay procedure has been formulated. This method appears to be much more accurate than those reported earlier.     

Novel carbon-carbon bond formations for biocatalysis

Carbon-carbon bond formation is the key transformation in organic synthesis to set up the carbon backbone of organic molecules. However, only a limited number of enzymatic C-C bond forming reactions have been applied in biocatalytic organic synthesis. Recently, further name reactions have been accomplished for the first time employing enzymes on a preparative scale, for instance the Stetter and Pictet-Spengler reaction or oxidative C-C bond formation. Furthermore, novel enzymatic C-C bond forming reactions have been identified like benzylation of aromatics, intermolecular Diels-Alder or reductive coupling of carbon monoxide.     

The Quasi-Equilibrium Assumption for Bi-Bi Ordered Bisubstrate Enzymatic Reaction. How to Discriminate the Mechanism Correctly

Application of the quasi-equilibrium assumption for the steady-state kinetics of bisubstrate irreversible enzymat- ic reactions in the case of ordered binding of substrates (Bi-Bi ordered mechanism) is considered. The necessary and suffi- cient conditions for application of the quasi-equilibrium assumption have been found and accuracy of this assumption has been numerically evaluated. The limitations on application of the quasi-equilibrium assumption have been shown and errors of its application have been analyzed. It is shown that possible discrimination of substrate binding order using asymmetrical expressions grounded on the quasi-equilibrium assumption is inconsistent because such asymmetrical expressions arise from incorrect application of the quasi-equilibrium assumption. Moreover, it has been proved in the general case that mecha- nisms generating such substrate-asymmetrical expressions for the steady-state rate of enzymatic reaction do not exist. The error source when using graphical interpretation for discrimination of mechanisms of bisubstrate enzymatic reactions has been determined. The strategy to avoid such errors is pointed out.     

Contributions of Microorganisms to Industrial Biology

Life on earth is not possible without microorganisms. Microbes have contributed to industrial science for over 100 years. They have given us diversity in enzymatic content and metabolic pathways. The advent of recombinant DNA brought many changes to industrial microbiology. New expression systems have been developed, biosynthetic pathways have been modified by metabolic engineering to give new metabolites, and directed evolution has provided enzymes with modified selectability, improved catalytic activity and stability. More and more genomes of industrial microorganisms are being sequenced giving valuable information about the genetic and enzymatic makeup of these valuable forms of life. Major tools such as functional genomics, proteomics, and metabolomics are being exploited for the discovery of new valuable small molecules for medicine and enzymes for catalysis.     

Dipeptide synthesis catalyzed by aminoacyl-tRNA synthetases from Bacillus stearothermophilus

A new approach to enzymatic peptide synthesis by using aminoacyl-tRNA synthetase (ARS) as a catalyst has been investigated. Four ARSs (AspRS, HisRS, LeuRS and TyrRS) have been purified from a thermophilic bacterium, Bacillus stearothermophilus. By using TyrRS as a catalyst, tyrosine and leucinamide were shown to be condensed in the presence of ATP to give tyrosylleucinamide. In this manner, all of the ARSs investigated catalyzed the peptide synthesis reactions. TyrRS did not have strict specificity for the amino acid derivatives used as substrates and even D-amino acids were incorporated into peptides fairly easily in this enzymatic reaction. Preparative scale synthesis of L-Tyr-L-LeuNH2 was carried out and from this the scope and limitation of this new enzymatic reaction as a tool to the peptide synthesis has been described.     

Enzymatic production and biological activities of chitosan oligosaccharides (COS): A review

Many researchers have focused chitosan as a source of potential bioactive material during past few decades. However, chitosan has several drawbacks to be utilized in biological applications, including poor solubility under physiological conditions. Therefore, a new interest has recently been emerged on partially hydrolyzed chitosan, chitosan oligosaccharides (COS). During the resent past, several technological approaches have been taken to prepare COS and, enzymatic preparation methods captured a great interest due to safe and non-toxic concerns. With time, new improvements were introduced to enzymatic production and presently it has been developed to a continuous production process. Many of the biological activities reported for COS, such as antimicrobial, anticancer, antioxidant, and immunostimulant effects are depend on their physico-chemical properties. In this review, we have summarized different enzymatic preparation methods of COS and some of their reported biological activities.     

Enzyme-based antifouling coatings: a review

A systematic overview is presented of the literature that reports the antifouling (AF) protection of underwater structures via the action of enzymes. The overall aim of this review is to assess the state of the art of enzymatic AF technology, and to highlight the obstacles that have to be overcome for successful development of enzymatic AF coatings. The approaches described in the literature are divided into direct and indirect enzymatic AF, depending on the intended action of the enzymes. Direct antifouling is used when the enzymes themselves are active antifoulants. Indirect antifouling refers to the use of enzymes to release an active biocide with AF activity. For direct AF, several patents have been granted, and a commercial product has been launched. However, the achievement of an efficient broad-spectrum AF coating based on a single or a few enzymes has not yet been achieved. An indirect AF coating is not yet available commercially. The technology is mainly limited by the instability of substrate supply, whether the substrates are found in the surrounding seawater or in the coating itself. Legislative issues regarding which part(s) of an enzyme system should be regarded as biocidal for product registration purposes are also considered. The above question currently remains unanswered for technologies utilising indirect enzymatic AF.     

Acid and alkaline phosphatase activities in the clam Scrobicularia plana: kinetic characteristics and effects of heavy metals

The acid and alkaline phosphatase activities of the clam Scrobicularia plana have been partially characterised in different organs and tissues (digestive gland, gills, foot, siphon and mantle) and the 'in vitro' effect of heavy metals on both types of enzymatic activity have been analysed. The optimal pH ranged between 4.0 and 5.5 for acid phosphatase activity and 8.5 and 9.5 for alkaline phosphatase activity. The apparent optimum temperature was in the 30-60 degrees range for acid phosphatase activity and in the 30-40 degrees C range for alkaline phosphatase activity. The effect of substrate concentration on enzymatic activities in the tissues showed a good fit to the Michaelis-Menten model. For both types of enzymatic activity, the highest values were found in the digestive gland. The effect of heavy metals was dependent on the tissue analysed. Mercury showed the highest inhibition in the organs/tissues and the parameters Km and Vmax were modified when the inhibitor concentration increased, thus indicating a mixed type of inhibition.     

Enzyme-based antifouling coatings: a review

Abstract

A systematic overview is presented of the literature that reports the antifouling (AF) protection of underwater structures via the action of enzymes. The overall aim of this review is to assess the state of the art of enzymatic AF technology, and to highlight the obstacles that have to be overcome for successful development of enzymatic AF coatings. The approaches described in the literature are divided into direct and indirect enzymatic AF, depending on the intended action of the enzymes. Direct antifouling is used when the enzymes themselves are active antifoulants. Indirect antifouling refers to the use of enzymes to release an active biocide with AF activity. For direct AF, several patents have been granted, and a commercial product has been launched. However, the achievement of an efficient broad-spectrum AF coating based on a single or a few enzymes has not yet been achieved. An indirect AF coating is not yet available commercially. The technology is mainly limited by the instability of substrate supply, whether the substrates are found in the surrounding seawater or in the coating itself. Legislative issues regarding which part(s) of an enzyme system should be regarded as biocidal for product registration purposes are also considered. The above question currently remains unanswered for technologies utilising indirect enzymatic AF.     

Effect of particle size on the enzymatic hydrolysis of polysaccharides from ultrafine lignocellulose particles

The efficiency of the enzymatic hydrolysis of wood polysaccharides ground into ultrafine particles (UFPs) has been investigated. The content of reducing sugars (RS’s) in powdered raw materials and the yield of sugars during enzymatic hydrolysis have been shown to depend on the particle size. Laser interference microscopy and dynamic light scattering studies have shown that increasing the grinding time from 20 to 40 min resulted in the formation of particles ranging from 2 to 200 nm in size. Enzymatic hydrolyzates of UFPs mostly contained glucose and galactose. The grinding intensity (mill rotation rate) and time had a significant effect on the extent of the enzymatic hydrolysis of wood.     

Effect of particle size on the enzymatic hydrolysis of polysaccharides from ultrafine lignocellulose particles

The efficiency of the enzymatic hydrolysis of wood polysaccharides ground into ultrafine particles (UFPs) has been investigated. The content of reducing sugars (RS's) in powdered raw materials and the yield of sugars during enzymatic hydrolysis have been shown to depend on the particle size. Laser interference microscopy and dynamic light scattering studies have shown that increasing the grinding time from 20 to 40 min resulted in the formation of particles ranging from 2 to 200 nm in size. Enzymatic hydrolyzates of UFPs mostly contained glucose and galactose. The grinding intensity (mill rotation rate) and time had a significant effect on the extent of the enzymatic hydrolysis of wood.     

Kinetic model of 1,3-specific triacylglycerols alcoholysis catalyzed by lipases

A new model of enzymatic 1,3-specific alcoholysis of triacylglycerols has been developed. The irreversibility of the acyl bounds cleavage in glycerides, a reversible monoglycerides isomerization and an irreversible enzyme deactivation have been assumed. The Ping Pong Bi Bi mechanism with competitive inhibition by alcohol has been applied to describe rates of acyl bonds cleavage. The enzymatic propanolysis and iso-propanolysis of triacetin and tricaprylin catalyzed by immobilized lipase B from Candida antarctica (Novozym 435) have been investigated to verify the model. Good agreement between experimental data and calculations has been obtained. It was shown that the rate of tricaprylin alcoholysis is higher than the triacetin alcoholysis and that the rate of iso-propanolysis reactions are higher than propanolysis. The irreversible enzyme deactivation affects the conversion of glycerides whereas the competitive alcohol inhibition may be neglected. Empirical correlations of rates for monoglycerides isomerization and enzyme deactivation have been proposed.