Permeabilization of yeast cells (Kluyveromyces fragilis) to lactose by cetyltrimethylammonium bromide

Summary Whole cells of lactose fermentingKluyveromyces fragilis had very low -galactosidase activity. Treating the yeast cells with a cationic detergent cetyltrimethylammonium bromide (0.1%) at 4°C for 5 mins increased the enzyme activity 480 fold. Detergent treated cells readily hydrolysed lactose present in milk and sweet whey and glucose produced was not further metabolized. These detergent permeabilized cells could be used to produce low lactose milk, in the utilization of whey and saccharification of lactose or whey for the production of alcohol.     

Immobilization of glucoamylase on naphthyl cotton cloth

Summary Aspergillus niger glucoamylase was adsorbed to -naphthyl cotton cloth by hydrophobic interaction. The adsorbed enzyme was cross-linked with glutaraldehyde. The immobilized glucoamylase exhibited greater pH dependence though the optimal pH did not change. The immobilized glucoamylase in a packed bed column completely hydrolysed 5% soluble starch at a specific velocity of about 4. Used naphthyl cloth could be regenerated by heating in 2 N NaOH at 100°C for 1 hour.     

Characteristics of yeast β-galactosidase immobilized on calcium alginate gels

Summary Saccharomyces anamensis having -galactosidase activity, has been immobilized in calcium alginate gel matrix that retained 78.6% enzyme activity to that of native cells. Optimum pH(7.0) was negligibly affected by immobilization. K_m values for immobilized and native cells were 119 mM and 102 mM respectively. Protective agents like dithioerythritol, bovine serum albumin, enhance the enzyme activity when added prior to immobilization. Immobilized cells can be stored in refrigeration(4°C) for 42 days without a significant loss of enzyme activity.     

Thermostable tryptophan synthase ofBacillus stearothermophilus expressed inEscherichia coli

Summary The tryptophan synthase genes,trpA andtrpB, from a moderate thermophile,Bacillus stearothermophilus IFO13737, were expressed efficiently inEscherichia coli. The recombinant tryptophan synthase amounted to 22% of the soluble cellular protein, and was purified to homogeneity by three steps. The enzyme is more thermostable thanE.coli tryptophan synthase, especially the subunit. The enzyme is also more resistant to sodium dodecylsulfate and methanol thanE.coli enzyme.     

The cell wall-associated inulinase of Kluyveromyces fragilis

The yeast Kluyveromyces fragilis (ATCC 12424) was grown on a 2% inulin-1% yeast extract medium for 36 h and subsequently fixed with 0.5% glutaraldehyde. The glutaraldehyde treatment did not affect the -fructofuranosidase (inulinase, EC 3.2.1.7) activity of the cells but it did make the cells resistant to chemical and physical treatments that normally release -fructofuranosidase from untreated cells. The enzyme in the treated cells exhibited K_m values for sucrose and raffinose identical to those obtained for the free enzyme. The cell wall of the treated cells exhibited the same diffusion properties for sucrose, raffinose, and inulin as those observed for untreated cells. The -fructofuranosidase was not bound covalently to the cell by the glutaraldehyde treatment. The results support the permeability barrier model for the enzyme retention in the yeast cell wall.     

Glutaraldehyde-fixation of yeast cells: Kinetics of a model system for enzyme cytochemistry

The kinetics of glutaraldehyde inactivation of a protoplasmic (-fructofuranosidase) and an extracytoplasmic (acid phosphatase) enzyme inSaccharomyces rouxii cells were studied at pH 5.5 and 30°C. The effects of glutaraldehyde concentration (0.5–3%), pH value, and temperature were surveyed by varying the fixation conditions. Cells from 1- to 10-day cultures retained 50–75% of their acid phosphatase activity and 15–24% of their -fructofuranosidase activity after 1-h exposures to 0.5% glutaraldehyde. The surviving -fructofuranosidase activity remained physically cryptic and was revealed only after further membrane perturbation with ethyl acetate. This crypticity barrier disappeared after overnight incubation of the treated cells at 4°C, with or without added glutaraldehyde, during which time the enzyme was resistant to further inactivation. The velocity ratio for raffinose versus sucrose, as substrate, decreased in treated cells, and changes inV _max andK _m were indicative of frank destruction of some enzyme molecules as well as modification of survivors. A comparable set of changes was also generated by treating cell-free extract with glutaraldehyde. Glutaraldehyde (0.5%) killed all yeast cells at 30°C within 5 min; at 4°C survival rates were quite high—81% after 15 min and 65% after 1 h. The bearing of these examples of enzyme inactivation, permeability barrier abolition, and structural stabilization on the general problems of yeast cytochemistry is discussed.     

Synthesis of novel sugars,oligoglucosyl-inositols,and their growth stimulating effect forBifidobacterium

Summary Novel sugars, oligoglucosyl-inositols, which were synthesized using CGTase fromBacillus ohbensis, stimulated the growth ofBifidobacterium. The enzyme catalyzed transglucosylation from -1,4-maltodextrin (donor) tomyo-inositol (acceptor). Of donors examined, -cyclodextrin gave superior oligoglucosyl-inositol yield of 56.6% (w/w) based on the conversion ratio of incubated inositol. Maltosyl-inositol stimulated growth ofB. adolescentis by 194% when compared with glucose.     

Lactulose production by β-galactosidase in permeabilized cells of<Emphasis Type="Italic"> Kluyveromyces lactis</Emphasis>

Lactulose production from lactose and fructose was investigated with several commercial -galactosidases. The enzyme from Kluyveromyces lactis exhibited the highest lactulose productivity among the -galactosidases tested. The reaction conditions for lactulose production were optimized using cells that had been permeabilized by treatment with 50% (v/v) ethanol: cell concentration, 10.4 g l^–1; concentration of substrates, 40% (w/v) lactose and 20% (w/v) fructose; temperature, 60^°C; pH 7.0. Under these conditions, the permeabilized cells produced approximately 20 g l^–1 lactulose in 3 h with a lactulose productivity of 6.8 g l^–1 h^–1. These results represent 1.3- and 2.1-fold increases in lactulose concentration and productivity compared with untreated washed cells. This is the first reported trial of enzymatic synthesis of lactulose using permeabilized yeast cells.     

Regulation of Escherichia coli phosphofructokinase in situ

The activity of E. coli phosphofructokinase in situ has been studied in cells permeabilized to its substrates, products and effectors by a toluene-freezing treatment. The in situ enzyme exhibits moderate cooperativity in respect to F6P (n_H up to 2.0), rather low affinity for ATP (with Km up to 1 mM when saturated with F6P), activation by ADP, and inhibition, within the physiological range of concentrations, by high ATP and phosphoenolpyruvate. This behaviour of the enzyme in situ at concentrations of the effector metabolites as those reported in intact cells in glycolytic and gluconeogenic conditions could account for the changes of phosphofructokinase activity needed for metabolic regulation in vivo.     

Formation of a raw starch-hydrolyzing alpha-amylase by Clostridium 2021: Effect of carbon sources

Summary Clostridium 2021 was found to produce -amylase effective at hydrolyzing raw starch. Of the carbohydrates examined, starch at 3 % concentration was found to be the best carbon source for enzyme production. The products of -amylase action on starch were: maltose. glucose and higher dextrins.     

Adsorption of aspartase onto hydrophobic cloth for conversion of ammonium fumarate

Summary A simple aspartase assay was developed. Aspartase fromEscherichia coli Crooks strain was adsorbed to -naphthyl cotton cloth by hydrophobic interaction. The adsorbed enzyme did not desorb in 1 M ammonium fumarate. The adsorbed enzyme exhibited the same pH vs. activity curve as free enzyme and had a half life of approx. 40 weeks. A column packed with the adsorbed aspartase showed 100% conversion of 1 M ammonium fumarate at a space velocity of approx. 2.     

Stimulation and inhibition of photosystem II electron transport in cyanobacteria by ions interacting with the cytoplasmic face of thylakoids

The mechanism by which suspension medium ions regulate the rate of photoinduced electron transport across photosystem II was investigated with ion permeabilized cells of the cyanobacterium Anacystis nidulans. Electron transport was measured as the reduction of the electroneutral acceptor dichlorophenol indophenol, whose surface concentration is independent of electrostatic membrane potential. Potassium salts stimulate photoinduced electron transport at low concentrations and inhibit it at higher concentrations. No inhibition is observed when an antichaotropic anion is associated with potassium, while the inhibition is more severe the stronger the chaotropic character of the anion. Neutralization of the surface charge by potassium ions ligated to negatively charged membrane sites at the cytoplasmic side is a prerequisite for the expression of the chaotropic inhibition of photosystem II electron transport.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenol indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DPC 1,5-diphenyl carbazide - FeCN ferricyanide anion - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - PS photosystem - TEC³⁺ tris ethylene diamine cobalt cation     

Mutants ofStreptomyces hygroscopicus deregulated in amylase and α-glucosidase formation

Summary Two mutants, M36 and M39, of turimycin-producingS. hygroscopicus JA 6599/PR1 obtained by directed selection in a chemostat displayed altered pattern of amylase and -glucosidase production as revelaed by both constitutive enzyme formation and higher enzyme levels.     

Cellulose acetate entrapment of Escherichia coli on cotton cloth for aspartate production

Summary Aspartase containingEscherichia coli cells were entrapped in cellulose acetate aggregated on cotton cloth. The enzyme activity of the cloth was stabilized by treatment with polyethylenimine and alkaline glutaraldehyde in the presence of 0.1 M sodium dithionite. A column packed with the cloth segments catalyzed 100% conversion of 1 M ammonium fumarate to aspartic acid at a space velocity of 7/h. When the same cloth segments were stirred at 100 rpm in the substrate solution, the productivity of the cloth increased 2.5 times.     

Elevation of nucleotide pyrophosphatase activity in skin fibroblasts from patients with Lowe's syndrome

Undersulfation observed in the glycosaminoglycans synthesized by cultured skin fibroblasts from a Lowe's syndrome patient[Fukui, S. etal. (1981) J. Biol. Chem. 256, 10313–10318] was found to be caused by elevated degradation of 3′-phosphoadenosine 5′-phosphosulfate (PAPS). The enzyme involved in this degradation was then identified as an enzyme of nucleotide pyrophosphatase (EC 3.6.1.9) nature, cleaving the phosphosulfate linkage. The specific activities were 8 – 24 (mU/mg protein) in patients' fibroblasts, in contrast to 3 in normal and 5 – 14 in heterozygote cells. A possibility is discussed that the elevation of nucleotide pyrophosphatase activity is the primary genetic defect in Lowe's syndrome.     

Effect of glutaraldehyde on oligosaccharide production by β-galactosidase from Bacillus circulans

Summary The oligosaccharide-producing activity of -galactosidase-1, one of the isomers of -galactosidase (-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans was changed after immobilization onto porous silica gel (Merckogel) by crosslinkage with glutaraldehyde. The reason for this modification was studied by treating the free enzyme with glutaraldehyde. Glutaraldehyde of 0.025% to 3% modified 40% to 90% of the free amino groups with or without intermolecular crosslinking. The maximum yield of oligosaccharides increased from 12% to 40% depending upon degree of modification, while native enzyme gave only 6% trisaccharides during hydrolysis of 127 mM lactose. The K _m value for the enzyme treated with glutaraldehyde was also increased.     

Isolation of aClostridium thermocellum gene encoding a thermostable β-1, 3-glucanase (laminarinase)

Summary AClostridium thermocellum gene directing the synthesis of a thermostable -glucanase was localized on a 1.9-kb DNA fragment by subcloning intoEscherichia coli plasmid vectors. The enzyme was highly efficient in degrading glucans with alternating -1, 3- and -1,4-linkages such as lichenan and barley glucan. It was also active towards the -1, 3-glucan laminarin, but lacked activity on cellulosic substrates and -glucans. The enzyme was therefore classified as -1, 3-glucanase (laminarinase) and the corresponding gene was designatedlicA. With barley -glucan as substrate the enzyme had a pH optimum around pH 6.5 and a temperature optimum at 65°C. It was stable for several hours at 60°C in the absence of substrate.     

Photosynthetic ability in dark-grown Reboulia hemisphaerica and Barbula unguiculata cells in suspension culture

Suspension cultured cells of the liverwort, Reboulia hemisphaerica and of the moss, Barbula unguiculata were independently subcultured in the medium containing 2% glucose in the dark or in the light for more than one year, and the photosynthetic activities of the final cultures were determined. Throughout the culture period light-grown cells of both species contained high amount of chlorophyll (4 to 34 g mg^–1 dry weight) and showed a high photosynthetic activity (10 to 84 mol O₂ mg^–1 chlorophyll h^–1). Dark-grown cells of R. hemisphaerica showed the same level of chlorophyll content and photosynthetic O₂ evolving activity as light-grown cells. Although chlorophyll content in dark-grown B. unguiculata cells was ten-fold lower than that in light-grown cells, the photosynthetic activity of these dark-grown cells was higher than that of light-grown cells based on chlorophyll content.     

Induction of enzymes of phytoalexin synthesis in cultured soybean cells by an elicitor from Phytophthora megasperma f. sp. glycinea

The glucan elicitor from cell walls of the fungal pathogen, Phytophthora megasperma f. sp. glycinea, induced rapid but transient increases in enzyme activities of general phenylpropanoid metabolism (phenylalanine ammonia-lyase and 4-coumarate: CoA ligase) and of the flavonoid pathway (chalcone synthase) in cell suspension cultures of soybean (Glycine max). After transferring cells into fresh medium, two peaks of inducibility for the enzymes by elicitor were observed, one shortly after transfer (stage I), and one at the end of the linear growth phase (stage II). Only one of the two isoenzymes of 4-coumarate: CoA ligase (isoenzyme 2), for which a specific involvement in flavonoid biosynthesis has been postulated, was affected by the elicitor. For two of the induced enzymes, phenylalanine ammonia-lyase and chalcone synthase, the changes in activity at stage I were shown to be preceded by large changes in their rates of synthesis, as determined by in vivo labelling with [³⁵S] methionine and immunoprecipitation.Abbreviations Pmg Phytophthora megasperma f. sp. glycinea - glyceollin is a term used to designate the 3 isomers which accumulate in challenged soybean tissue (Moesta and Grisebach 1981b)     

Organellar DNA synthesis in permeabilized soybean cells

Summary Cultured cells of Glycine max (L.) Merr. v. Corsoy were permeabilized by treatment with L--lysophosphatidylcholine (LPC). The permeabilized cells were capable of uptake and incorporation of deoxynucleoside triphosphates into DNA. Incorporation of exogenous nucleotides into DNA was linear for at least 90 minutes and the initial rate of incorporation approached 50% of the theoretical in vivo rate of DNA synthesis. However, DNA synthesis in the permeabilized cells was unaffected by the potent DNA polymerase inhibitor, aphidicolin. Analysis of newly synthesized DNA by molecular hybridization revealed that only organellar DNA was synthesized by the permeabilized cells. The LPC treated cells were also permeable to a protein as large as DNase I. The permeabilized cells were capable of RNA and protein synthesis as indicated by incorporation of radiolabeled UTP and leucine, respectively, into acid-precipitable material.