Showing papers in "Applied Biochemistry and Biotechnology in 1986"

Liquid-liquid extraction of lactate dehydrogenase from muscle using polymer-bound triazine dyes.

Abstract: An extract from porcine muscle containing soluble enzymes has been partitioned between the two liquid phases of an aqueous, biphasic system consisting of dextran, polyethylene glycol, and water. The influence of polymer-bound triazine dyes (Cibacron blue F3G-A and Procion yellow HE-3G) on the partition of lactate dehydrogenase and total protein was studied. The effects of pH and concentrations of polymers and buffer on this so-called affinity partitioning were also determined. The two-phase systems were used in extraction procedures for purification of lactate dehydrogenase to a specific activity of 456–494 U (7.6–8.4 μkat) per mg protein. The use of these systems for extraction of enzymes in technical scale is discussed.

Laccases of Rigidoporus lignosus and Phellinus noxius

Abstract: A laccase that has been isolated previously (1) from the Basidiomycete,Rigidoporus lignosus, a white rot fungus of rubber tree, was used in the present study. When a thioglycolic lignin (TGL) was incubated in the presence of this enzyme, pronounced changes in the UV spectrum and size distribution of the substrate were observed. Sephadex gel filtration indicated that two types of reactions occurred: (1) A degradation of the polymer, as evidenced by the production of low-molecular-weight material; and (2) a condensation of some of the TGL molecules, as revealed by an increase in the fractions of higher molecular weight.

Enzymatic conversion of cellulosic materials to sugars and alcohol. The technology and its implications.

Abstract: This techno-economic study deals with the production of sugars and alcohols from cellulosic materials. It covers such key subjects as: potential raw materials; the state-of-the-art on production technologies; the economics of extant processes; and finally infers implications for developing countries from the foregoing.

Continuous production of L-aspartic acid

Abstract: For the continuous production ofl-aspartic acid from fumaric acid and ammonia by the action of aspartase, the enzyme extracted fromEscherichia coli orE. coli cells having high aspartase activity were immobilized by various methods.

Colorimetric Determination of Reactive Amino Groups of a Solid Support Using Traut's and Ellman's Reagents

Abstract: A simple colorimetric method has been developed for the quantitative determination of reactive amino groups of a solid support. The method calls for the use of a reagent that reacts specifically and facilely with amino groups and concurrently introduces free sulfhydryl groups. The latter can then be titrated colorimetrically by using the well-known Ellman’s reagent. Thus, an excess of 2-iminothiolane (Traut’s reagent) was reacted with amine-carrying solid supports. After removing unreacted 2-iminothiolane and keeping the resultant solid-phase sulfhydryl groups in a reduced state by using dithiothreitol, the solid supports, after being thoroughly washed, were then reacted with 5,5′-dithiobis-(2-nitrobenzoic acid) to quantify the sulfhydryl groups that were generated from reacting solid-phase amino groups with Traut’s reagents.

Specific hemoperfusion through agarose acrobeads

Abstract: Agarose acrobeads were produced by encapsulating polyacrolein microspheres (acrobeads) of 0.2 μm average diameter within an agarose matrix. Crosslinked agarose acrobeads of diameters ranging from 0.5 to 0.8 mm were found to be optimal spheres for specific hemoperfusion purposes. Agarose provides the biocompatibility and mechanical strength of the agarose acrobeads. Acrobeads contain a high aldehyde-group content through which various amino ligands, i.e., proteins, antigens, antibodies, enzymes, and so on, can be covalently bound in a single step under physiological pH (or other pH). Thus, antibodies, antigens, or toxic materials may be directly removed from whole blood by hemoperfusion. During in vitro and in vivo hemoperfusion trials, the content of erythrocytes, leukocytes, and thrombocytes was essentially unaltered. Likewise, a battery of the soluble blood components (Cl-, K+, Na+, Ca2+, PO 4 - ), total proteins, albumin, and C 4 ′ component of the complement cascade, as well as the enzymes SGOT, LDH, and alkaline phosphatase, remained constant within narrow limits during the hemoperfusion procedure. The chemical and physical structure of the beads is stable; neither acrolein nor bead fragments were detected in hemoperfusion trials. Similarly, leakage of antibody bound to the agarose acrobeads into the blood is insignificant. Thus far, we have demonstrated the efficacy of the crosslinked agarose acrobeads for extracorporeal removal of “unwanted” substances from whole blood in the following systems: (a) removal of specific antigens (digoxin or paraquat removal with antidigoxin or antiparaquat antibodies bound to the acrobeads, respectively), (b) removal of specific antibody (antiBSA) removal with BSA bound to the beads), (c) removal of immune complexes (BSA-antiBSA complex removal with C1q bound to acrobeads), and (d) removal of specific metals (removal of iron with deferoxamine bound to the agarose acrobeads).

Large-scale recovery and purification of L-asparaginase from Erwinia carotovora.

Abstract: A large-scale process was developed to purify gram quantities of a therapeutic enzyme, L-asparaginase, from submerged cultures of Erwinia carotovora. Cells were harvested from 150 L of fermentation broth and washed. A cellular acetone powder was prepared and extracted with pH 9.5 borate buffer. After continuous centrifugation and filtration to remove cell debris, the acetone powder extract was adjusted to pH 7.7 and adsorbed onto a 16-L CM-Sepharose Fast Flow column, with a precolumn packed with Cell Debris Remover. The enzyme was desorbed from the catin-exchange column at pH 9.0 and further purified with an affinity column of L-asparagine Sepharose CL-4B. After dialysis-concentration to remove buffer salt, the enzyme was depyrogenated, formulated, sterile filled, and lyophilized as a single-dose final product. The final-product evaluation included analysis of the content of protein, sodium chloride, glycine, sodium, glucose hydrate, phosphate, and endotoxin, as well as reconstitution, potency, pH, specific activity, uniformity of fill, and sterility. The product was further subjected to visual examination, sodium dodecyl sulfate polyacrylamide gel electrophoresis, native gel electrophoresis, isoelectric focusing, amino acid analysis, N-terminal sequencing, peptide mapping, and immunological comparison.

Ethanol production by yeast cells immobilized in open-pore agar

Abstract: An open-pore agar matrix has been shown to be suitable for the entrapment of microbial whole cells required for use in reactions that involve cell growth and gas evolution. Beads of porous agar with entrapped yeast cells have been used for the continuous fermentation of sugar cane molasses to ethanol, without apparent bead rupture, even after periods of 3 mo of use. The agar gel does not erode during prolonged operation, unlike porous gelatin cross-linked with glutaraldehyde.

Immunoaffinity purification of subcellular particles and organelles

Abstract: The application of immunoaffinity techniques to subcellular fractionation is reviewed and the basic principles underlying the various methods that have been successfully employed, identified. The requirement for organelle-specific antigens and high-avidity antibodies is discussed, as is the widespread use of indirect immunoadsorbents. Approaches for the optimization of immunoaffinity-based subcellular fractionation are suggested.

An engineer looks at photosynthesis

Abstract: Little engineering information is available on which to base the design and analysis of photosynthetic microbial systems. Frequencyresponse analysis of algal systems supplements basic data and uncovers fundamental time constants for the rate-controlling steps. An engineering perspective of photosynthesis focuses on these bottleneck steps that most strongly influence process economics.

Lipid-polyamide—Polyethyleneimine microcapsules for immobilization of free cofactors and multienzyme system

Abstract: Lipid-polyamide membrane microcapsules containing multienzyme system convert ammonia into glutamate.l-Glutamic dehydrogenase, alcohol dehydrogenase, NAD, and alpha-ketoglutarate retained within the microcapsules do not leak out. This way the multienzyme system can recycle the free cofactor NAD and synthesize glutamate from external lipophilic ammonia diffusing into the microcapsules. By using polyethyleneimine instead of hemoglobin as microcapsule filler it is possible to obtain higher conversion rates and to increase the storage stability of the recycling activity.

A simple and rapid chemical method for the determination of cephalosporins

Abstract: A simple and rapid chemical assay for cephalosporins is described. It is a simple modification of the colorimetric determination of penicillins in which the narrow spectrum β-lactamase (penicillinase) is replaced by a broad spectrum β-lactamase (cephalosporinase) produced byEnterobacter cloacae. The method can be used for assay of fermentation broths as well as for pure cephalosporins.

Simple enzyme reactors suitable for the byproduct-free preparation of the aglycones of naturally occurring glycosides under mild conditions

Abstract: Naringinase from Penicillium species—containing α-l-rhamnosidase and β-d-glucosidase activity—immobilized on silicate carriers can be used as a mild and effective means to cleave naturally occurring glycosides into their aglycones and sugar components. The procedure was tested with flavonoid-, anthraquinone-, and steroidglycosides. Since the solubility of such compounds is limited in aqueous solutions, a simple batch procedure has been developed to convert solid substrates suspended in only small amounts of buffer to the desired products with high yields.

Immobilized respiratory chain activities from Escherichia coli utilized to measure d and l-lactate, succinate, l-malate, 3-glycerophosphate, pyruvate, or NAD(P)H

Abstract: The respiratory chain (membranous, multienzymatic system) from Escherichia coli, was coimmobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde. The film was fixed onto an oxygen sensor. The enzyme electrode can be used for measuring NAD(P)H, D- and L-lactate, succinate, L-malate, 3-glycerophosphate, or pyruvate. The range of metabolites concentrations was from 1 to 50 mM. It was possible to discriminate between the different metabolites (if mixed): By inducing during bacterial growth the specific flavoproteins necessary for L-lactate, succinate, L-malate, and 3-glycerophosphate respirations. The constitutive activities are unaltered on glucose or glycerol, namely D-lactate, NAD(P)H, and pyruvate respiration. When intact bacteria were immobilized (with or without induction), D- and L-lactate, succinate, 3-glycerophosphate, and L-malate respiration were measured, no activities of pyruvate and NAD(P)H respiration were obtained. For these last activities, French press breakage (see section on Membrane Preparations) of bacteria prior to immobilization was necessary. Products of reactions can be used as enzyme inhibitors: Pyruvate inhibits D- and L-lactate; fumarate inhibits succinate, and oxaloacetate inhibits L-malate respirations. Heat denaturation of the bacteria at 55 degrees C for 1 h maintains full activity of succinate and pyruvate respiration. On the other hand, no activity of D- and L-lactate, L-malate, or NAD(P)H respiration was measurable. These enzyme electrodes have many applications in basic and applied research.

Enzymatic transformations of 3-chloroalanine into useful amino acids.

Abstract: The investigation of the combination of enzymatic and chemical synthetic processes for the production of useful compounds has been carried out. This review focuses on the enzymatic transformation of chemically synthesized 3-chloroalanine into useful amino acids.

Immobilization of pig liver microsomes. Stability of cytochrome P-450-dependent monooxygenase activities.

Abstract: Microsomes from pig liver were covalently coupled to Sepharose activated by CNBr and to Sephadex activated by 1,1'-carbonyldiimidazole. Microsomes were also entrapped inside Ca-alginate and kappa-carrageenan gels. The concentration of immobilized cytochrome P-450 was determined by CO-difference spectra. The activity of the monooxygenase system was demonstrated by the N-demethylation of aminopyrine, the O-demethylation of p-nitroanisole, and the hydroxylation of perhexiline maleate. Upon immobilization, a 30-40% and a 60-70% decrease in Vappmax for the O- and N-demethylations were respectively observed. The Vappmax values for the hydroxylation of perhexiline maleate were essentially the same for the different immobilized forms and for the freely suspended microsomal cytochrome P-450. Under storage at 4 degrees C, microsomes entrapped inside kappa-carrageenan and Ca-alginate were less stable than the free microsomes, whereas immobilization on CNBr-activated Sepharose improved the stability of the hepatic microsomal monooxygenase system at the same temperature. These types of immobilized microsomes have the advantage of being easily recovered and reused for other assays. Finally, microsomes entrapped inside kappa-carrageenan or Ca-alginate can be used to follow up the continuous metabolization of p-nitroanisole for several hours in a stirred-batch reactor.

Factors affecting glycerol production byPichia farinosa under alkaline conditions

Abstract: Glycerol is an important and valuable chemical that can be produced from renewable resources by fermentation. The desirable features of a successful process are high values of substrate conversions and high yields and concentrations of glycerol in the product broth, coupled with rapid fermentation cycles. Of the various osmophilic and nonosmophilic yeasts tested for their ability to produce glycerol in the presence and absence of steering agents (sodium sulfite or sodium carbonate), an osmophilic yeastPichia farinosa (ATCC 20210) was found to give attractive yields. Important variables influencing glycerol production by this strain under alkaline conditions using sodium carbonate have been investigated. A rapid fermentation (less than 120 h), coupled with high glycerol yields (45%), has been obtained.

Coupling capacity of solid-phase carboxyl groups: determination by a colorimetric procedure

Abstract: A simple colorimetric procedure for determining the coupling capacity of solid-supported carboxyl groups has been developed. The carboxyl groups of a solid support were coupled to cystamine at pH 4–4.5, using a water soluble carbodiimide 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiime as the condensing reagent. The solid-phase coupled disulfides were then reduced to sulfhydryl groups by treating the solid phase with dithiothreitol. For every one carboxyl group coupled with cystamine, one solid-phase sulfhydryl is introduced. After removing all of the reducing reagents by extensive washing, the sulfhydryl content, which is equivalent to the carboxyl groups of the gel, was quantified by using 5,5′-dithiobis-(2-nitrobenzoic acid), the Ellman’s reagent.

Single-enzyme-based amperometric assay for phosphate ion.

Abstract: An amperometric assay for phosphate ion, using a single enzymatic process, has been developed. The enzyme used was pyruvate oxidase, which catalyzes the oxidation of pyruvate in the presence of oxygen and phosphate ion. The products of the enzymatic reaction are acetyl phosphate, carbon dioxide, and hydrogen peroxide. The latter was monitored by means of a hydrogen peroxide electrode and an oxidase meter. Phosphate ion in the concentration range of 50–500 μM can be measured within 4 min. Anions, such as pyrophosphate, sulfate, nitrate, nitrite, and acetate, gave only marginal responses.

Effect of glutaraldehyde on the activity of some DNA restriction endonucleases.

Abstract: The effect of the bifunctional crosslinking reagent glutaraldehyde on the activity of the restriction enzymes Bam HI,Hind III, EcoRI, and Tthlll I was investigated. The four enzymes exhibited differential sensitivity to inactivation. Tthlll I was the most sensitive, with activity losses occurring at levels of 0.0025% and above.Hind III was the most stable of the four and remained fully active at concentrations as high as 0.075%. Addition of BSA to incubation mixtures generally had a stabilizing effect. Implications of these results for the design of glutaraldehyde-based methods for the immobilization of restriction endonucleases are discussed.

Oxygen limitation on L-serine production in a hollow-fiber bioreactor.

Abstract: Pseudomonas AM1 utilizes glycine and methanol to producel-serine aerobically(1). The consumption of methanol in this bioconversion is stoichiometrically in excess of L-serine production(2). Consequently, the oxygen requirement associated with L-serine production is higher than expected for the conversion from glycine.

Use of reflectance in soft laser densitometry in scanning opaque electrophoregrams. A high-resolution technique.

Abstract: The present report describes a new approach in soft laser scanning densitometry performed on opaque electrophoregrams. The method employs a light reflectance system instead of absorption, which is commonly used. The data of this preliminary study were obtained by directly scanning a photograph of an electrophoregram exhibiting viral proteins separated by SDS-PAGE. Comparison of the two methods clearly demonstrate that laser reflectance offers finer resolution than laser absorbance when opaque materials are scanned.

Detection and assessment of citrus tristeza virus antigens by computer-aided scanning densitometry

Abstract: Citrus tristeza virus (CTV) extracted from Etrog citron (C.medica L.) was immunoprecipitated. The immunoprecipitate was fractionated by SDS-PAGE and western blotted onto nitrocellulose. The CTV antigens were determined by immunoblot analysis using rabbit anti-CTV IgG, and the protein-band pattern exhibited on the nitrocellulose was assessed by soft-laser scanning densitometry. The densitometric tracing revealed the presence of bands that were not visible to the naked eye. Using the superimposition mode of the instrument, it was also revealed that the protein-band patterns of different CTV samples were not identical. Computer-aided soft-laser scanning densitometry proved to be a powerful approach in the detection and assessment of protein bands revealed on nitrocellulose immunoblots, which we were previously unable to do employing conventional methods.

Soft-laser scanning densitometry performed on a 35-mm slide illustrating SDS-PAGE separation of adenovirion polypeptides

Abstract: Virion polypeptides (35 S), methionine-labeled and purified by CsCl gradient centrifugation, were separated by SDS-gel electrophoresis Analysis of their band pattern was performed by scanning the images of the SDS-gels shown on a 35-mm slide The densitometric tracings revealed the presence of 17 protein bands, although only 15 of them were visible to the naked eye The high sensitivity and resolving capacities of the soft-laser scanning densitometer enabled us to detect trace amounts of protein bands separated in SDS-gels and to obtain a resolution compatible to that of electrophoresis Fourfold electronic amplification of the densitometric tracings, produced by a computer, generated new information regarding the pattern of the electrophoregram The facility to amplify peaks of importance is particularly advantageous when faint or overlapping protein bands revealed on a gel are assessed

Depletion of human lymphocytes from peripheral blood and bone marrow by affinity ligands conjugated to agarose-polyacrolein microsphere beads.

Abstract: Protein-A or goat anti-mouse-Ig (GAMIg) covalently bound to agarose-polyacrolein microsphere beads (APAMB) were employed for the removal of T cells from human peripheral blood leukocytes (PBL) and bone marrow (BM). The cell suspensions were treated with a monoclonal anti-T cell antibody (Leu-1) or monoclonal antilymphocyte antibody (CAMPATH-1) and passed through the conjugated APAMB columns. Cell separation efficacy was determined by assaying the number and function of T cells in the final cell preparation in comparison with a sample of unseparated cells. The number of cells that form rosettes (E-RFC) with sheep red blood cells (SRBC) in a sample of PBL treated with anti-Leu-1 antibodies and subsequently passed once through GAMIg-conjugated APAMB dropped from a range of 41.5-86.0% to a range of 1.6-13.3%. The in vitro response to concanavalin-A (Con-A) dropped to a range of 0.7-27.2% (GAMIg) and a range of 1.2-21.8% (protein-A column) of the response of untreated PBL. Treatment with CAMPATH-1 antibody and passage through a protein-A-conjugated APAMB reduced E-RFC from a range of 55.6-57.4% to a range of 3.2-3.9% and abolished the Con-A induced proliferative responsiveness to background levels. Treatment of BM cells with CAMPATH-1 and passage of the cells through either GAMIg or protein-A conjugated APAMB columns resulted in reduction of E-RFC from a range of 12.4-17.7% to a range of 0-1% and from a range of 17.7-19% to a range of 1.6-3.2%, respectively. Viability of BM precursors, determined by the CFU-GM assay in semisolid medium, was not affected by these cell separation procedures. The data suggest that protein-A or GAMIg-conjugated APAMB columns may be a useful tool for separation of BM cell suspensions into specific cell subsets that can be defined by monoclonal antibodies.