Production of glucose isomerase by different strains ofStreptomyces

Summary The glucose isomerase activity ofStreptomyces haeochromogenes strains 1 and 2 varies considerably with the assay conditions (pH, glucose concentration,etc.). Nine other species of streptomyces were tested under conditions optimal forS.phaeochromogenes 2. The highest enzyme activity was found inS.nigrificans 3014.     

Characterization of the purified multifunctional cellulase component of Penicillium funiculosum

Summary The endoglucanase component (CMCase I) ofPenicillium funiculosum cellulase was purified to apparent homogeneity by ultrafiltration and gel chromatography. It consists of a single polypeptide chain with a molecular weight of 56000 and is a glycoprotein. Viscometric and end-product analysis revealed the randomness of enzyme action. Multifunctional characteristic of CMCase I was studied with various carbohydrate substrates.NCL Communication No.: 4307     

Optimization of growth conditions for the induction of tyrosine decarboxylase inStreptococcus faecalis

Summary Growth conditions were investigated for optimal tyrosine decarboxylase (EC 4.1.1.25) activity in acetone dried cells ofStreptococcus faecalis. A growth pH of 6.0 was found optimal for enzyme induction. The enzyme was also shown to be growth-associated which indicates that continuous fermentation is preferable for optimal process productivity.     

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.     

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.     

Some characteristics of the cellulase of Aspergillus candidus

Summary The effects of pH, temperature and substrate concentration on the cellulase (B-1,4-endoglucanase) activity ofA. candidus were studied. Maximum enzyme activities were obtained when the concentration of the substrate (CMC) was 6 mg per ml, at pH 4, and temperature 50 C. The enzyme retained 85% of its original activity under optimal conditions of pH and temperature after 36 hours of incubation. The Km constant of the reaction was calculated as 2.3 mg of CMC per ml and the energy of activation of the enzyme as 7.92 k cal per mole.     

Simultaneous saccharification and protein enrichment fermentation of sugar beet pulp

Summary A product with 40 % protein content was obtained from sugar beet pulp (1.25–2.0 mm) in 48 h one stage (simultaneous) saccharification/fermentation process under optimized conditions using a specific enzyme mixture andCandida tropicalis strain, also saving about 40 % enzymes in comparison to a 2-stage process.     

Glyoxalase-I activity and cell cycle regulation in yeast

The status of glyoxalase-I was explored in exponentially growing and G₁ arrested temperature sensitive (ts) cell division cycle (cdc) mutants of Saccharomyces cerevisiae. It was observed that the specific activity of this enzyme was correlated with overall growth status. The activity was high in actively growing cells and was low in G₁ arrested cells. Specific activities of glyoxalase-I were also low in G₁ arrested prolonged stationary phase (PSP) cells of S. cerevisiae and Candida albicans. The activity of glyoxalase-I recovered when G₁ arrested S. cerevisiae (ts) cells were allowed to regrow under permissive conditions. Results demonstrate that although glyoxalase-I activity is a good indicator of cell growth status, it is not involved in cell cycle regulation of this eukaryotic organism.     

Enzyme activity engineering based on sequence co-evolution analysis

The utility of engineering enzyme activity is expanding with the development of biotechnology. Conventional methods have limited applicability as they require high-throughput screening or three-dimensional structures to direct target residues of activity control. An alternative method uses sequence evolution of natural selection. A repertoire of mutations was selected for fine-tuning enzyme activities to adapt to varying environments during the evolution. Here, we devised a strategy called sequence co-evolutionary analysis to control the efficiency of enzyme reactions (SCANEER), which scans the evolution of protein sequences and direct mutation strategy to improve enzyme activity. We hypothesized that amino acid pairs for various enzyme activity were encoded in the evolutionary history of protein sequences, whereas loss-of-function mutations were avoided since those are depleted during the evolution. SCANEER successfully predicted the enzyme activities of beta-lactamase and aminoglycoside 3′-phosphotransferase. SCANEER was further experimentally validated to control the activities of three different enzymes of great interest in chemical production: cis-aconitate decarboxylase, α-ketoglutaric semialdehyde dehydrogenase, and inositol oxygenase. Activity-enhancing mutations that improve substrate-binding affinity or turnover rate were found at sites distal from known active sites or ligand-binding pockets. We provide SCANEER to control desired enzyme activity through a user-friendly webserver.     

A soluble chloroplast protein catalyzes ribulosebisphosphate carboxylase/oxygenase activation in vivo

Ribulosebisphosphate carboxylase/oxygenase (EC 4.1.1.39) (rubisco) must be fully activated in order to catalyze the maximum rates of photosynthesis observed in plants. Activation of the isolated enzyme occurs spontaneously, but conditions required to observe full activation are inconsistent with those known to occur in illuminated chloroplasts. Genetic studies with a nutant of Arabidopsis thaliana incapable of activating rubisco linked two chloroplast polypeptides to the activation process in vivo. Using a reconstituted light activation system, it was possible to demonstrate the participation of a chloroplast protein in rubisco activation. These results indicate that a specific chloroplast enzyme, rubisco activase, catalyzes the activation of rubisco in vivo.     

Presence of two subunit types in ribulose-1,5-bisphosphate carboxylase from blue-green algae.

Ribulose-1,5-bisphosphate car?ylase (E.C. 4.1.1.39) from 2 blue-green algae, Plectonema boryanum and Anabaena variabilis, was isolated by sucrose density gradient centrifugation. Both enzymes had a sedimentation value of about 18s, similar to that of Chromatium enzyme. The presence of two subunits (A, B) in the algal enzyme was demonstrated by Nadodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight of the two subunits was determined: for Plectonema A, 5.4 × 10⁴ and B, 1.3 × 10⁴ and Anabaena A, 5.2 × 10⁴ and B, 1.3 × 10⁴, respectively. The car?ylase reaction catalysed by the algal enzyme was similar to the higher plant enzyme in exhibiting the Mg²⁺-effect, the optimal pH shifting from alkaline to neutral by elevating the concentration of Mg²⁺ in the assay mixture. The rabbit antisera developed against the spinach ribulose-1,5-bisphosphate car?ylase and its catalytic oligomer exhibited significant inhibitory effects on the car?ylation reaction catalysed by the algal enzyme.     

Novel biotechnology process for the production of (+)-2-aminobutyrate

Summary The production of (+)-2-aminobutyrate was catalyzed by a transaminating enzyme with 2-ketobutyrate and alanine as the reactants. The required enzyme was obtained from a plasmid-bearingE. coli strain.     

Quinoprotein D-glucose dehydrogenases inAcinetobacter calcoaceticus LMD 79. 41: Purification and characterization of the membrane-bound enzyme distinct from the soluble enzyme

Acinetobacter calcoaceticus is known to contain soluble and membrane-bound quinoprotein D-glucose dehydrogenases while other oxidative bacteria such asPseudomonas orGluconobacter contain only membrane-bound enzyme. The two different forms were believed to be the same enzyme or interconvertible. Present results show that the two different forms of glucose dehydrogenase are distinct from each other in their enzymatic and immunological properties as well as in their molecular size.The soluble and membrane-bound glucose dehydrogenases were separated after French press-disruption by repeated ultracentrifugation, and then purified to nearly homogeneous state. The soluble enzyme was a polypeptide of 55 Kdaltons, while the membrane-bound enzyme was a polypeptide of 83 Kdaltons which is mainly monomeric in detergent solution. Both enzymes showed different enzymatic properties including substrate specificity, optimum pH, kinetics for glucose, and reactivity for ubiquinone-homologues. Furthermore, the two enzymes could be distinguished immunochemically: the membrane-bound enzyme is cross-reactive with an antibody raised against membrane-bound enzyme purified fromPseudomonas but not with antibody elicited against the soluble enzyme, while the soluble enzyme is not cross-reactive with the antibody of membrane-bound enzyme.Data also suggest that the membrane-bound enzyme functions by linking to the respiratory chain via ubiquinone though the function of the soluble enzyme remains unclear.     

Shikimate pathway activity in shake and fermenter cultures of Cinchona succirubra

Cell suspension cultures of Cinchona succirubra were cultivated in shake cultures and for the first time in airlift fermenters. Under both conditions L-tryptophan exerts a stimulatory effect on alkaloid formation. In this context the regulatory pattern of some shikimate pathway enzymes was investigated in non-supplemented and tryptophan supplemented Cinchona cell cultures. A remarkable increase of tryptophan decarboxylase (TDC) activity was observed in Cinchona cells under the influence of tryptophan. Apparently, like in some other indole alkaloid producing cell cultures, a high TDC activity is a prerequisite for alkaloid formation. Growth pattern and some enzyme activities of C. succirubra fermenter cultures at controlled and non-regulated pH levels were followed. Optimum growth and alkaloid formation were recorded under non-regulated (normal) pH conditions.Abbreviations TDC tryptophan decarboxylase - try L-tyrosine - phe L-phenylalanine - DAHP 3-deoxy-D-arabino-heptulosonic acid-7-phosphate - trp L-tryptophan - E-4-P erythrose-4-phosphate - PEP phosphoenolpyruvate - MDH malate dehydrogenase - G-6-PDH glucose-6-phosphate dehydrogenase - 6-PG-DH 6-phosphogluconate dehydrogenase - Ch-mutase chorismate mutase - AS-synthase anthranilate synthase - n.d. not determined     

Identity of the HL-A common portion fragment and human beta2-microglobulin

Glycogen synthetase D from the 17,000 × g supernatant of a homogenate of human polymorphonuclear leukocytes has been purified to a specific activity of 7,4 units/mg protein in a single step, chromatography on Concanavalin A bound to agarose (Con A-Sepharose). The overall recovery of the enzyme was 66% and the entire procedure requires only 3–4 hours. After an in vitro D to I conversion, glycogen synthetase I was purified to a specific activity of 11,5 units/mg protein in a similar procedure.     

Characterization of a novel cellobiase fromBacillus subtilis and expression of its structural gene inEscherichia coli

Summary A strain ofBacillus subtilis was found to produce a cellobiase resistant to catabolic repression by glucose. When the structural gene encoding cellobiase was cloned and expressed inEscherichia coli, the enzyme produced was resistant to repression by glucose.     

Screening of yeast strains capable of hyperproducing amylolytic enzymes

Summary Fifteen strains of yeast, which produced an extracellular amylolytic enzymes, were isolated from nature. One of them produced more than 100 times the enzyme activity in comparison with the 14 strains and the extremely hyperproducing strain of yeast was identified asCandida sp. 347. Paper chromatograms of the amylolytic enzyme demonstrated activity of amyloglucosidase. The optimum pH for activity of the enzyme was 5.5–6.0 and optimum temperature was 60°C.     

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)     

Polynucleotide phosphorylase from plant cells

The isolation of polynucleotide phosphorylase (EC 2. 7. 7. 8) from suspension cultured plant cells of parsley (Petroselinum sativum) and from tomato seedlings (Lycopersicon esculentum) is described. The procedure includes an ultracentrifugation step, a glycerol density gradient centrifugation and preparative gel electrophoresis under nondenaturing conditions. Isoelectric focusing gives rise to a major component (pI ≈ 7.5) and to a minor one (pI ≈ 5). The enzyme contains five subunits with apparent M_r values of 160 000, 140 000, 70 000, 34 000 and 12 000, the 70 000-dalton one being a glycoprotein.     

Utilization of free and immobilized Desulfovibrio hydrogenase in hydrogen photoproduction : Coupling efficiency of cytochrome C3, ferredoxin and flavodoxin

Summary Hydrogen photoproduction has been achieved by coupling free or immobilized hydrogenases from Desulfovibrio species to illuminated chloroplasts through different electron mediators. Whereas D. gigas flavodoxin or ferredoxin I cannot directly mediate the electron flux from chloroplasts to hydrogenase, the addition of these mediators considerably enhances the H₂ photoproduction of a system including cytochrome C₃. These immobilized hydrogenases exhibit good stability under working conditions and can be re-used.