Characterization of three distinct forms of lipolytic enzyme in a commercialCandida lipase preparation

Summary Three distinct forms of lipolytic enzyme were identified in a commercialCandida lipase preparation. Two of these lipases (lipases A & C) were isolated and characterized. Lipase A had a higher optimal reaction pH and a better thermal stability than those of lipase C. Lipase A and C displayed different acyl chain length specificity on the lipolysis of p-nitrophenol esters.     

Partial purification and properties of proteases from fig (Ficus carica) callus cultures

Summary Cell culture of fig (Ficus carica) was evaluated as a source of thermostable cysteine proteases. Extracts of 28 day old callus contained a benzoyl-arginine-p-nitroanilide (BAPNA) hydrolase activity of 2200 U/g dry wt (49.5 U/mg protein) at 25°C. Thermal stability and thermal denaturation (at 70°C) properties showed some similarities to those of commercial ficin (Sigma). High performance gel filtration revealed the presence of a peak with BAPNA hydrolase and caseinolytic activity which matched commercial ficin. Covalent chromatography using Thiopropyl Sepharose 6B (Pharmacia) demonstrated that approx 50% of the total BAPNA hydrolase activity could be attributed to thiol-proteins.Abbreviations BAPNA benzoyl-arginine-p-nitroanilide - BAPNAse BAPNA hydrolase Contribution No. 147.     

Extracellular L-glutaminase production by marine bacteria

Summary Four species of bacteria which includedPseudomonas fluorescens,Vibrio cholerae andVibrio costicola were observed to produce glutaminase both as extracellular and intracellular fractions. Comparatively both the fractions were higher in mineral media supplemented with 1% glutamine than in nutrient broth added with or without glutamine. Extracellular glutaminase production was about 2.6–6.8 times greater than the intracellular production by all the tested strains.     

The isomaltulose synthesising enzyme ofSerratia plymuthica

Summary An intracellular enzyme was located inSerratia plymuthica which produced isomaltulose from sucrose. The enzyme was purified giving a preparation with a specific activity of 1,285. It has pH and temperature optima of 6.0 and 30°C, respectively. The enzyme was stable retaining 100% activity after 2 weeks at 30°C. It had an isoelectric point at pH 9.0, a Mr of 79,500 and the Km for sucrose was 65.3mM. The enzyme converted 40% (w/v) sucrose to isomaltulose with an efficiency of 87%.     

Immunological similarity of epoxide hydrolase activity in the mitochondrial and cytosolic fractions of mouse liver

The light and heavy mitochondrial fractions of mouse liver have relatively high levels of epoxide hydrolase (EH) activity when monitored with trans-stilbene oxide as substrate. Using double diffusion analysis and immunoprecipitation experiments it was shown that EH activity in the mitochondrial fractions is immunologically similar to cytosolic EH, but immunologically dissimilar from microsomal EH. The EHs in the mitochondrial and cytosolic fractions also have a similar pI.     

Ribosylation of 3-Methylguanine and the Relative Stability of its 7- and 9-α-D-Ribofuranosides

Abstract

Ribosylation of 3-methylguanine la was investigated by enzymatic and chemical methods. Compound la did not act as a substrate for purine nucleoside phosphorylase. N-2-Protected 3-methylguanines 4 and 6 underwent exclusive N-7 glycosylation by fusion and chloromercury methods to give 5 and 7. Fully acetylated 7-α-D-ribofuranoside 5 was also obtained by thermal transglycosylation of the corresponding 9-α-D-ribofuranoside 9. The reverse isomerization 5 → 9 did not occur. The differences in the relative stability towards acidic hydrolysis between 7- and 9-(α-D-ribofuranosyl)-3-methylguanines are distinctly higher than those described so far for the other 7-9 isomeric nucleosides.     

Enzymatic difference between laticifers and cultured cells of papaya

Cell suspension cultures of Carica papaya were found to produce a papain-like enzyme showing an amidase activity similar to papain. Experiments suggested that the enzyme was an SH-enzyme, but an immunological test indicated the absence of papain in cultured cells. The isoelectric point (4.3) of the enzyme of cultured cells was the same as that of the leaf extract, but was different from that of papain or other amidases in the latex of the fruit.     

Extracellular invertase from Aspergillus athecius: Isolation and immobilization

Summary A fungal strain isolated from soil and identified asAspergillus athecius, when grown on moistened wheat bran produced large amounts of extracellular invertase. Most of the invertase from the moldy bran was easily extracted by low ionic strength buffer (0.005 M, pH 5.7). The crude invertase immobilized on DEAE cellulose showed not only increased activity (45%) but also greater thermal and storage stability than the free enzyme. The free and the bound enzymes showed a temperature optimum of 50–55°C and a pH optimum of 5.7 and 4.8 respectively. The K_m app. of the bound enzyme was lower than that of the free enzyme.     

Biomass protein formation by filamentous fungi on woody substrates

Summary Growth and biomass protein formation by filamentous fungi grown on pretreated tropical woods of Mesta (Hibiscus cannabinus Linn.) and Subabul [Leucaena leucocephala (Lam.) de Witt] as well as their isolated hemicellulose and cellulose fractions have been studied. Penicillium janthinellum and Penicillium funiculosum produced a biomass having 20 to 30% crude protein when grown on either hemicellulose, while growth on pretreated (autoclaved in 1% NaOH) wood or isolated cellulose fractions was comparatively poor and crude protein content only 5 to 8% in the biomass.NCL Communication no.3550     

Stimulation of respiration and nitrogenase in bacteroids of Siratro (Macroptilium atropurpureum) by plant nodule cytosol

Nitrogenase activity (acetylene reduction) of isolated Siratro (Macroptilium atropurpureum) bacteroids was stimulated by addition of plant cytosol fractions which also preserved activity at high (up to 3%) O₂ tensions. These effects were not due to leghaemoglobin. Boiling removed some, but not all, of the protective capacity of the cytosol. Heat treated cytosol substantially stimulated the respiration of siratro bacteroids. Of a wide variety of compounds tested, only ascorbate could mimic the cytosol. Ascorbate was present in the cytosol fraction, in significant quantities. The effect of ascorbate was evident at low O₂ concentrations and in purified bacteroids, and was inhibited by cyanide. Siratro bacteroids appear to possess an oxidase which could serve a protective role in vivo.     

Immobilisation of bromoperoxidase from Corallina officinalis

Summary The vanadium-dependent bromoperoxidase from the macroalga Corallina officinalis was immobilised on a cellulose acetate support with retention of approaching 50% of the applied units of activity. The enzyme exhibited high thermal stability and retained activity in repeated use. The immobilised enzyme showed tolerance to organic solvents similar to that of the free enzyme in the case of methanol but differed for acetone and ethanol, and with the latter showed enhanced activity as the % by volume of the solvent was increased.     

Stability of poly(A)+RNA in nucleate and anucleate cells of Acetabularia

The stability of poly(A)⁺RNA was compared in nucleate and anucleate cells of Acetabularia. While in the absence of the nucleus poly(A)⁺RNA exhibits a pronounced stability, it is significantly less stable in the presence of the nucleus.     

Antiplasmodial activity of Artemisia annua plant cell cultures

Extracts of Artemisia annua cultures have been assessed for in vitro activity against the malarial parasite Plasmodium falciparum. Callus and suspension cells and medium were analysed and examined for their activity at different stages of growth and development. Time-course experiments were carried out to investigate the influence of various basal media, plant growth regulators and light on both growth and possible artemisinin production. Two active fractions were obtained but artemisinin was not detected.     

Extracellular xylanolytic enzymes ofPaecilomyces varioti

Summary Paecilomyces varioti produced an extracellular xylanase and B-xylosidase when cultured in a medium containing xylan and corn steep liquor. Xylose (2%, w/v) totally inhibited production of both enzymes. The enzymes were purified and both had a pH optimum of 4.0. The xylanase had a molecular weight of 20,000, an isoelectric point of 5.2 and was inactive on all substrates tested except xylan. The -xylosidase, a glycoprotein, had a molecular weight of 67,000, an isoelectric point of 4.0 and had highest activity on p-nitrophenyl--D-xyloside. The xylanase had a Km of 49.5 mg/ml for xylan and the -xylosidase had a Km of 5.4 mM for p-nitrophenyl--D-xyloside.     

Preparation and properties of glucose isomerase of Streptomyces kanamyceticus cells entrapped in calcium alginate

Summary The properties of glucose isomerase in native, heat-treated and immobilized cells of Streptomyces kanamyceticus after heat and mineral treatment have been compared. The optimum pH for glucose isomerase in native cells was shifted from 8.2 to 8.6 by heat treatment and immobilization. There is no change in the optimum temperature (90°C) for activity of the enzyme by the above treatment. Heat-treated cells and immobilized cells show greater pH and thermal stability of the enzyme. The Km values of the enzyme of native cells, heat-treated cells and immobilized heat-mineral-treated cells are 208 mM, 212 mM and 166 mM respectively; Mg⁺⁺ and Co⁺⁺ enhance the activity of isomerase in all cases.     

Common responses of cultured soybean cells to 2,4-D starvation and fungal elicitor treatment

The patterns of in vivo protein synthesis in soybean cell suspensions were compared by polyacrylamide gel electrophoresis after the cells had been submitted to different stress conditions : treatment with Phytophthora megasperma (Pmg) cell wall elicitors, 2,4-D starvation and heat shock (HS) temperatures. Changes in protein synthesis patterns induced after elicitation of cell suspensions or after infection of soybean hypocotyls by Pmg were found to be similar to changes brought about by auxin starvation of the cells. Changes common to both stress situations involve a prominent 17 kDa peptide family and 27, 29, 35 and about 45 kDa peptides. Moreover, defense reactions, i.e. glyceollin accumulation and synthesis of chalcone synthase (CHS) were also strongly stimulated in auxin-starved cells. On the contrary, although characteristic sets of low molecular weight heat shock (HS) proteins were synthesized by cells grown at 37°C, no clear similarity was observed with peptides characteristic of auxin-starved cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - Pmg Phytophthora megasperma Drechs f.sp.glycinea - HS heat shock - PR pathogenesis-related - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - IEF isoelectrofocusing - iP isoelectric point - kDa kilodalton - P17 17 kDa peptide group of soybean cells cultured in vitro - CHS chalcone synthase     

Solid state fermentation and fractionation of oat straw by Basidiomycetes

Summary Seventee white-rot and brown-rot fungi were screened for their ability to fractionate the lignocellulose structure of oat straw through the preferential attack of lignin or cellulose. Fermentations were carried out under solid-state conditions with 25 g quantities of straw. The fermented straw was analyzed for weight loss, Klason lignin loss and cellulase digestion. All the fungi attacked both lignin and carbohydrate fractions causing 3–28% weight losses and 26–34 g/100 g enzymatic digestibility. Polyporus tulipiferae, Phanerochaete chrysosporium and Polyporus sp. were tested for the effects of various nitrogen, phosphate and carbon levels, incubation temperatures and incubation time. The three fungi had different responses to these factors.     

Effect of NaCl on lipid content of plasma membranes isolated from roots and cell suspension cultures of the dicot halophyte Kosteletzkya virginica (L.) Presl.

Lipid concent was examined in plasma membrane fractions isolated by discontinuous sucrose density-gradient centrifugation from both salinized and unsalinized roots and cell suspension cultures of Kosteletzkva virginica (L.) Presl., seashore mallow, a halophytic dicot. The distribution of marker enzymes along the gradient indicated that plasma membranes of roots and cell cultures accumulated primarily at the 34%/45% interface. Total sterol and phospholipid content increased significantly in plants and cell suspensions grown on salinized nutrient media. In addition, K. virginica plasma membranes were constitutively rich in sterols, and a high sterol-to-phospholipid ratio was maintained or elevated under saline conditions. These results are discussed in relation to membrane composition as a mechanism involved in the cellularly based salt tolerance of K. virginica.     

Two-dimensional separation of chloroplast membrane proteins by isoelectri focusing and electrophoresis in sodium dodecyl sulphate

Proteins of chloroplast subfragments enriched in Photosystem I and Photosystem II electron flow activity have been analyzed by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, polyacrylamide gel isoelectric focusing (pH 5–7) was used in the presence of Triton X-100, followed at right angle by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Characteristic fingerprints were obtained for the Photosystem I and II fractions and a correlation between the major proteins separated by isoelectric focusing and the major polypeptides separated by undimensional SDS electrophoresis was established. Two dominant spots of 68 000 and 60 000 daltons appeared in the two-dimensional patterns of Photosystem I fractions $\text{p}\text{I}$ values about 5.6; two spots with molecular weights of 33 000 and 23 000 were characteristics for Photosystem II fractions $\text{p}\text{I}$ values about 5.3 and 6.3). Photosystem I fractions were furthermore characteristics by a series of spots in the 44 000–33 000 range $\text{p}\text{I}$ values from about 5.9 to 6.8). The two-dimensional system revealed that (a) several SDS-polypeptides have multiple forms differing in charge only, (b) some proteins separated by isoelectric focusing are resolved in the second dimensional into polypeptides of different size. The two-dimensional method combining Triton X-100 isoelectric focusing' and SDS electrophoresis provides a higher degree of resolution than either of the unidimensional methods thus allowing a detailed analysis of chloroplast membrane proteins.     

The human MSH5 (MutS Homolog 5) protein localizes to mitochondria and protects the mitochondrial genome from oxidative damage

MutS homologs play a central role in maintaining genetic stability. We show that MSH5 (MutS Homolog 5) is localized into the mitochondria of germ and somatic cells. This protein binds to mtDNA and interacts with the Twinkle helicase and the DNA polymerase gamma. hMSH5 stimulates mtDNA repair in response to DNA damage induced by oxidative stress. Furthermore, we observed a subsarcolemmal accumulation of hMSH5 in COX negative muscle fibers of patients presenting a mitochondrial myopathy. We report a novel localization for hMSH5 suggesting that this protein may have functions other than those known in meiotic recombination.