A simple method for the isolation of plant protoplasts

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO4), and a protectant (CaCl2 2H2O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.

Kinetics of enzymatic synthesis of geranyl butyrate by transesterification in various supercritical fluids

The transesterification of methyl butyrate, ethyl butyrate and butyl butyrate to geranyl butyrate was investigated in supercritical carbon dioxide. The effect of chain length of the butyrate on the rate of transesterification was investigated. The initial rates followed the trend: ethyl butyrate < butyl butyrate < methyl butyrate. The transesterification of butyl butyrate to geranyl butyrate in various supercritical fluids such as ethylene, methane, ethane was also examined. The initial rate of transesterification of butyl butyrate in different supercritical fluids followed the order: ScCO2 < ScC2H6 < ScC2H4 < ScCH4. The highest initial rate was obtained in supercritical methane and the reasons for this observation were proposed. The Ping-Pong Bi-Bi model with inhibition by both acid and alcohol was used to model the experimental data and determine the kinetics of the reaction. (C) 2010 Elsevier B.V. All rights reserved.

Enzymatic Degradation of Poly(soybean oil-g-methyl methacrylate)

This study discusses grafting of methyl methacrylate units from thepolymeric soybean oil peroxide to produce poly(soybean oil-graft-methyl methacrylate) (PSO-g-PMMA). The degradation of this copolymer in solution was evaluated in the presence of different lipases, viz Candida rugosa (CR), Lipolase 100T (LP), Novozym 435 (N435) and Porcine pancreas (PP), at different temperatures The copolymer degraded by specific chain end scission and the mass fraction of the specific product evolved was determined The degradation was modeled using continuous distribution kinetics to determine the rate coefficients ofmenzymatic chain end scission and deactivation of the enzyme The enzymes, CR. LP and N435 exhibited maximum activity for the degradation of PSO-g-PMMA at 60 degrees C, while PP was most active at 50 degrees C. The thermal degradability of the copolymer, assessed by thermo-gravimetry, indicated that the activation energy of degradation of the copolymer was 154 kJ mol(-1), which was lesser than that of the PMMA homopolymer.

Enzymatic conversion of anthranilic acid to catechol by chloroplast from the leaves of Tecoma stans

An enzyme system which converts anthranilic acid to catechol was detected in the leaves of Tecoma stans, and its properties studied. The system is present exclusively in the chloroplast fraction of the leaves. The optimum pH of the reaction is 5·2 and maximum activity was obtained with citrate-phosphate buffer. There was good stoichiometry between the amounts of anthranilic acid disappeared and the amounts of catechol and ammonia formed. The enzyme system showed an absolute requirement for oxygen and evidence was obtained for the probable participation of NADPH and FAD in the hydroxylation step. The optimum concentration of anthranilic acid was 10−4 M; at higher concentrations the reaction was inhibited to a considerable extent. Cyanide, pyrophosphate, and EDTA also caused inhibition indicating a requirement for metal ions.

A method for the preferential isolation of actinomycetes from soils

A successful plate-method for the preferential isolation of actinomycetes from soils is described. The principles underlying it are: (1) the inhibition of growth of non-sporulating bacteria by pre-incubation at a high temperature (110 C) for 10 min, and (2) limiting the spreading growth of sporeforming bacteria and fungi by the use of dried plates. The majority of the 191 species isolated by this method from 82 soil samples were shown to be pectinolytic.

Isolation of Salmonella typhimurium mutants affecting the plaque morphology of phage P22

Salmonella typhimurium mutants affecting the plaque morphology of P22 and other phages have been isolated. Using one such bacterial mutant phage mutants making turbid plaques have been isolated.

Isolation Of A Cytokinin-Binding Protein From Cucumis-Sativus Cotyledons

A cytokinin-binding protein which exists as monomer and dimer was isolated from cucumber (Cucumis sativus L. var Guntur) cotyledons by affinity chromatography on AH-Sepharose-pi6 Ap /s> column. The protein bound to [3H]-N6-(Δ2-isopentenyl) adenosine trialcohol. On Sephadex G-50 chromatography it gave 2 peaks corresponding to molecular weight 4000 and 8000 daltons. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis, it gave only one band with an apparent molecular weight of 4000 daltons.

Beta-turns in nascent procollagen are sites of posttranslational enzymatic hydroxylation of proline

The selective hydroxylation of proline residues in nascent procollagen chains by prolyl hydroxylase (EC 1.14.11.2) can be understood in terms of the conformational feature of the -Pro-Gly-segments in linear peptides and globular proteins. The folded beta-turn conformation in such segments appears to be the conformational requirement for proline hydroxylation. The available data on the hydroxylation of native and synthetic substrates of prolyl hydroxylase are explained on the basis of the extent of beta-turn formation in them. Taken in conjunction with the conformational features of the hydroxyproline residue, our results bring out the conformational reason for the posttranslational proline hydroxylation which, it is proposed, leads to the "straightening" of the beta-turn segments into the linear triple-helical conformation.

Rapid isolation of lysyl-tRNA synthetase from rat liver

The high molecular weight aminoacyl-tRNA synthetase complex (the 24S complex) was isolated from rat liver by ultracentrifugation. The lysyl-tRNA synthetase (E.C. 6.1.1.6) was selectively dissociated by hydrophobic interaction chromatography on 1,6 diaminohexyl agarose followed by hydroxylapatite chromatography and DEAE chromatography. The lysyl-tRNA synthetase dissociated from the 24S synthetase complex was purified approximately to 2700 fold with 14% yield.

Chemical approaches of penicillin allergy—III Isolation of the penicillin-free carrier receptor protein (CRP) on a polymeric 7-deoxy penicillin analogue template and its role in penicillin immunogeneses in rabbit and man

Specific penicillin-carrier receptor proteins (CRP) have been isolated from the sera of penicillin allergic rabbits and human subjects in the unconjugated native state in electrophoretically homogeneous form by employing a synthetic polymeric affinity template containing the 7-deoxy analogue of penicillin G. The synthesis of the 7-deoxy analogue has been described. In this affinity system the antipenicillin-antibody is desorbed by 0·9M thiourea and the CRP in 8M urea. The CRP after incubation with penicillin is converted into the full-fledged antigen. Studies on the origin of CRP and the nature of antibody as well as comparative studies on the properties of the rabbit antibody and those of antibodies elicited by a BSA-BPO conjugate are reported.