Mechanized techniques for the selective extraction of enzymes from plant epidermal glands

Many plant products are biosynthesized and accumulated in epidermal glands. For investigations on the metabolism of these compounds it is most convenient to obtain cell-free preparations enriched in gland contents. Two simple mechanized procedures have been developed for gently abrading the plant surface in order to efficiently extract glandular enzymes in high purity. These methods allow rapid processing of large quantities of plant material and yield extracts largely uncontaminated with materials from underlying tissue. The use of these procedures for isolating several enzymes of terpenoid metabolism is described. These techniques work especially well for microsomal enzymes and may be useful not only for enzymes found in epidermal glands but also for other enzymes localized in or near the epidermis. With simple modification, these procedures can be adapted for use with a variety of different types of plant tissues.     

Miniaturized enzyme production and development of micro-assays for cellulolytic and xylanolytic enzymes

Miniaturized fungal cultivation and enzyme assays were developed. Cultivation for enzyme production was performed in 50 mL conical tubes. In addition, the miniaturized enzyme assays reduced the amount of enzymes and reagents necessary. These procedures can be adopted in screening fungi to determine if they produce cellulolytic and xylanolytic enzymes.     

Purification and characterization of high Ca2+-requiring neutral proteases from porcine and bovine brains

Porcine and bovine brain high Ca2+-requiring neutral proteases were purified to homogeneity by the same isolation procedures, and their properties were compared. A high degree of similarity existed between the two proteases. The purification procedures included ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on phenyl-Sepharose CL-4B, second DEAE-cellulose chromatography, second phenyl-Sepharose CL-4B chromatography, and gel filtration on Ultrogel AcA 34. Both purified enzymes were composed of Mr 75,000 and 29,000 subunits, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both enzymes required 250 microM Ca2+ for half-maximal activity and 700 microM Ca2+ for maximal activity. Sr2+ and Ba2+, but not Mg2+ or Mn2+, also activated both enzymes but not as effectively as Ca2+. Both enzymes displayed maximum activity at pH 7.5-8.0. Leupeptin, antipain, and trans-epoxysuccinyl-L-leucylagmatine inhibited both enzymes. Neurofilament triplet proteins and microtubule-associated proteins were extensively hydrolyzed by both proteases, but tubulin and actin were not hydrolyzed. The amino acid compositions of the two proteases were very similar. Antisera against bovine brain protease cross-reacted with porcine brain protease when examined by immunoelectrotransfer blot techniques.     

Effect of various procedures of storing the fungus Rhizopus microsporus UzLT-1, a lipase producer

The effect of various storage procedures on the viability and synthesis of lipolytic enzymes by Rhizopus microsporus UzLT-1 was studied. The best procedures of producer storage proved to be regular passages and lyophilization. These procedures made it possible to maintain stable activity of lipolytic enzymes produced by the fungus for a long time. The fungal storage in vaseline oil or in a dried state was less effective due to significant losses of its lipolytic capacity.     

Aqueous two-phase extraction of plant enzymes from sources containing large amounts of tannins and anionic mucilages.

The isolation of plant enzymes is frequently hampered by the presence of phenolic compounds, pigments and mucilages. Recently, extraction procedures based on aqueous two-phase systems were used to overcome these problems. Two-phase systems have a great advantage in respect to yield, product purity and processing time. Two-phase systems may open many new avenues in research as well as for application of enzymes from plant material, especially making available enzymes of sources avoided till now, due to the difficulties to work with.     

A simple procedure for the isolation of seven abundant muscle enzymes

The present work describes procedures in which seven major muscle enzymes and serum albumin can be simultaneously isolated from chicken skeletal muscles. The seven enzymes isolated were: phosphorylase, enolase, creatine-P kinase, aldolase, glyceraldehyde-3-P dehydrogenase, phosphoglycerate mutase, and triose-P isomerase. The proteins isolated by these methods were judged to be greater than 97% pure on the basis of electrophoretic analysis in sodium dodecyl sulfate polyacrylamide gels. The procedure is applicable for isolation of the enzymes from large (greater than 100 g) or small (less than 0.5 g) amounts of muscle tissue and the entire procedure can be completed within two days. Particularly useful features of the procedures are: (1) preferential solubilization of the enzymes from myofibrils by extraction of muscle specimens in solutions of different ionic strength; (2) specific precipitation of phosphorylase, creatine-P kinase, and glyceraldehyde 3-Phosphate dehydrogenase from solutions of specified pH and degrees of ammonium sulfate saturation; and (3) an alternate method for isolation of glyceraldehyde-3-P dehydrogenase by specific elution of the enzyme from phosphocellulose columns with ATP. Because of the ease, rapidity, and reproducibility of the procedures, these methods may be useful for the routine isolation of the muscle enzymes in studies on biochemical regulation, as well as for obtaining large quantitites of the enzymes for structural analysis.     

Preparation and application of poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan hydrogels for immobilization of lipase

In the present of this study, two novel polymeric matrixes that are poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan was synthesized and applied for immobilization of lipase. For the immobilization of enzyme, two different immobilization procedures have been carried out via covalently binding and entrapment methods. On the free and immobilized enzymes activities, optimum pH, temperature, storage and thermal stability was investigated. The optimum temperature for free, covalently immobilized and entrapped enzymes was found to be 30, 35 and 30 degrees C, respectively. Optimum pH for both free and immobilized enzymes was also observed at pH 8. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for free and immobilized lipases. Furthermore, the reuse numbers of immobilized enzymes also studied. It was observed that after 40th use in 5 days, the retained activities for covalently immobilized and entrapped lipases were found as 39% and 22%, respectively. Storage and thermal stability of enzyme was also increased by as a result of immobilization procedures.     

Affinity chromatography of Anacystis nidulans ferredoxin nitrate reductase and NADP reductase on reduced ferredoxin-Sepharose.

The behavior of two ferredoxin-dependent enzymes—nitrate reductase and NADP reductase—fromAnacystis nidulans on a ferredoxin-Sepharose gel was examined. The oxidized gel-bound ferredoxin exhibited very low affinity for these enzymes but effectively bound both nitrate reductase and NADP reductase when reduced by dithionite. Selective procedures are described for the clution of each of these two enzymes from the reduced ferredoxin-Sepharose gel. These simple methods allow substantial purification of both enzymes.     

Carboxylesterases (EC 3.1.1). Purification and titration of chicken, sheep, and horse liver carboxylesterases.

Chicken, sheep, and horse liver carboxylesterases have been purified by procedures involving ammonium sulfate fractionation, ion-exchange chromatography and gel filtration on Sephadex. The actual yields of the procedures described were as follows: chicken, 1 g from 2 kg of liver powder (chloroform-acetone); sheep, 200 mg from 400 g of powder (chloroform-acetone); horse, 230 mg from 800 g of powder (acetone). The purified enzymes are free of non-carboxyl-esterase protein as shown by gel electrophoresis, although they do contain electrophoretic variants. The equivalent weight of the chicken enzyme is 67,000 based on titration with p-nitrophenyl diethyl phosphate or bis(p-nitrophenyl) phosphate, whereas those of the sheep and horse enzymes are similar to 69,500 and similar to 70,000, respectively, based on titration with p-nitrophenyl dimethylcarbamate.     

Phenylglyoxylate decarboxylase and phenylpyruvate decarboxylase fromAcinetobacter calcoaceticus

Phenylglyoxylate decarboxylase and phenylpyruvate decarboxylase were both purified from strains ofAcinetobacter calcoaceticus by very similar procedures involving ion-exchange chromatography, gel filtration, and hydrophobic chromatography. The enzymes were inducer- and substrate-specific, but in general had very similar properties. Both enzymes required thiamine pyrophosphate for activity. Phenylglyoxylate decarboxylase and phenylpyruvate decarboxylase both appeared to be tetrameric, with apparent subunit M_r values of 58,000 and 56,800 respectively.     

Study of lytic enzymes and prospects of their use

The paper reviews different procedures of preparation of lytic enzymes of microbiol origin, their properties and areas of application. It discusses the capacity of different microorganisms, actinomycetes and bacteria including (for instance, Actinomyces griseinus 11 and Bacillus subtilis 12), to produce lytic enzymes. The paper describes the conditions of disruption of yeast cells by lytic enzymes and demonstrates experimentally possible preparation of yeast lysates and protein isolates that can be used as food products.     

Simultaneous purification and characterization of glucokinase, fructokinase and glucose-6-phosphate dehydrogenase from Zymomonas mobilis.

The three enzymes glucokinase (EC 2.7.1.2), fructokinase (EC 2.7.1.4) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were isolated in high yield from extracts of Zymomonas mobilis. The principal steps in the isolation procedures involved the use of selected dye-ligand adsorbent columns, with affinity elution of two of the three enzymes. Glucokinase and fructokinase are dimeric proteins (2 X 33000 Da and 2 X 28000 Da respectively) and glucose-6-phosphate dehydrogenase is a tetramer (4 X 52000 Da). Some similarities in the structural and kinetic parameters of the two kinases were noted, but they have absolute specificity for their substrates. Fructokinase is strongly inhibited by glucose; otherwise non-substrate sugars had little effect on any of the three enzymes.     

Evaluation of the enzyme thermistor as a specific detector for chromatographic procedures

The successful application of a simple flow calorimeter, called the enzyme thermistor, to the specific monitoring of different enzymes in the eluants from some common chromatographic procedures is described. The enzyme thermistor enables a specific enzyme to be continuously determined even in optically dense solutions where spectrophotometric procedures cannot be used. The instrument can be operated either on-line or it can be used for discrete samples. The sensitivity is in the order of 0.1 IU/ml for most enzymes.     

A Pre-embedding Reaction Method for Localizing NADH Reductase and Succinic Dehydrogenase in Skeletal Muscle

Paraffin wax embedding methods suitable for demonstrating the distribution of enzyme activity in tissues sections are uncommon; most procedures rely on the use of frozen section techniques. This paper describes a system for demonstrating certain enzymes which involves incubation of the tissue with appropriate substrates before a Paramat wax embedding procedure. While it has distinct merits of its own, the procedure is eminently suitable for use where a cryostat is not available; it can also be readily applied to other enzymes and tissues.     

Enzymes go big: surface hydrolysis and functionalization of synthetic polymers

Enzyme technology has progressed from the biotransformation of small substrates to biotransformation of synthetic polymers. Important breakthroughs have been the isolation and design of novel enzymes with enhanced activity on synthetic polymer substrates. These were made possible by efficient screening procedures and genetic engineering approaches based on an in-depth understanding of the mechanisms of enzymes on synthetic polymers. Enhancement of the hydrophilicity of synthetic polymers is a key requirement for many applications, ranging from electronics to functional textile production. This review focuses on enzymes that hydrolyse polyalkyleneterephthalates, polyamides or polyacrylonitriles, specifically on the polymer surface thereby replacing harsh chemical processes currently used for hydrophilisation.     

The release and characterization of some periplasm-located enzymes of Pseudomona aeruginosa.

Pseudomonas aeruginosa (ATCC 9027) releases four periplasm-located enzymes, i.e., ribonuclease (EC 3.1.4.22; EC 3.1.4.23), alkaline phosphatase (EC 3.1.3.1), cyclic-2', 3'-phosphodiesterase (EC 3.1.4.d), and 5'-nucleotidase (EC 3.1.3.5) into the medium during growth. Ribonuclease and alkaline phosphatase are classed as enzymes which are readily extracted by osmotic shock and spheroplast formation whereas cyclic-2',3'-phosphodiesterase and 5'-nucleotidase are classed as enzymes which are not readily extracted by these procedures. In view of the relative ease of extraction of the former enzymes it is suggested that the lattter enzymes, cyclic-2',3'-phosphodiesterase and 5'-nucleotidase, are bound and located in the periplasm in a manner different to ribonuclease and alkaline phosphatase.     

Particulate and dissolved phosphorus forms in freshwater: composition and analysis

In recent research, particulate and dissolved phosphorus components have been separated and characterized on the basis of their physical and chemical properties and partly by their origins.Classical operationally defined monitoring variables (dissolved reactive phosphorus, dissolved unreactive phosphorus and particulate phosphorus) are not congruent with known specific physical or chemical components of phosphorus in natural waters or with their bioavailability.Physical isolation of true particles, colloids and molecules of various sizes is possible at present although it is not recommended for routine use.Chemical characterization of particulate phosphorus is performed mainly by sediment extraction procedures (specialized for inorganic species) and — to a lesser degree — by cell extraction procedures (specialized for organic compounds). The extraction procedures are similar and physical preseparation or alternative procedures (e.g. enzymatic assays) are essential.Smaller colloids and dissolved compounds are physically separated by column chromatography and are often chemically characterized by degradation on the addition of specific enzymes.     

High-level expression and one-step purification of cyclic amidohydrolase family enzymes

The cyclic amidohydrolase family enzymes, including hydantoinase, dihydropyrimidinase, allantoinase and dihydroorotase, are metal-dependent hydrolases and play a crucial role in the metabolism of purine and pyrimidine in prokaryotic and eukaryotic cells. With the increasing demand for the elucidation of enzyme structures and functions, along with industrial applications, the research on the family enzymes has recently been proliferating, but the related enzymes had been purified conventionally by multistep purification procedures. Here, we reported the expression in Escherichia coli cells of maltose-binding protein-fused family enzymes and their one-step purification. The expression levels of the fusion proteins account for 20-35% of the total protein in E. coli, allowing approximately 2-3 mg of the purified proteins by affinity chromatography to be obtained per 0.3 L of bacterial culture. As more promising results, their nascent biochemical properties, after the cleavage of the fusion proteins with Factor Xa, in terms of oligomeric structure, optimal pH, specific activity, and kinetic property, were also conserved as those from the native enzymes. The availability of the family enzymes to fusion strategy shows potential as a convenient procedure to recombinant protein purification and accelerates the structure-function study of the related family enzymes.     

Purification and properties of apple fruit malic enzyme

Malic enzyme was isolated and purified from mature apple fruits (Malus sylvestris, Miller) by utilizing procedures probably applicable to other soluble enzymes in this and similar tissues.