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.     

Production of dextranase byLipomyces starkeyi

Summary The optimal growth rate ofLipomyces starkeyi, with dextran as sole carbon source, was found within the pH range 2.5–4.0, and temperature between 25–30°C. This yeast was unable to grow above 33°C. Dextranase production optima paralleled growth optima, except at pH 2.5. Decrease in enzyme yield at this pH could not be attributed to poor yeast growth or enzyme stability.     

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.     

Activation of tubulinyl-tyrosine carboxypeptidase by spermine,spermidine and putrescine

Spermine, spermidine and putrescine produce dose dependent stimulation of the invitro tubulinyl-tyrosine carboxypeptidase. Maximal stimulation was obtained with spermine, spermidine or putrescine at 0.06 mM, 1 mM and 6 mM, respectively. At higher concentrations, the enzyme activity was inhibited. The enzyme was also activated by Mg⁺⁺; the concentration formaximal effect was 4–6 mM. The stimulation produced by optimal concentration of each amine was unaffected by Mg⁺⁺ up to 2 mM; higher concentration of Mg⁺⁺ showed inhibitory effect. At optimal Mg⁺⁺ concentration, the carboxypeptidase activity was inhibited by increasing amine concentration. The amines at 0.5 or 5 mM did not produce any effect on the incorporation of tyrosine catalyzed by tubulin tyrosine ligase.     

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.     

Characterization of a monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus (L.) G. Don

Conditions have been established for the optimization of the specific activity of a membrane-bound monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus. In time course studies, the hydroxylase and NADPH-cytochrome c reductase exhibited maximal activities 18–20 days after inoculation, i.e., during early stationary phase. By late stationary phase, enzyme activity had declined. In contrast an enzyme of primary metabolism achieved optimal specific activity by the 12th day and remained constant through day 26, synchronous with general growth. Effects of nutritional and hormonal factors on the specific activity of the hydroxylase and cell growth were evaluated. Inhibitors of hydroxylase activity were also assessed in vitro. A soluble form of the monoterpene hydroxylase has been detected in cultured cells possibly affording a useful source of this enzyme for further purification.     

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.     

Xylanase ofChaetomium cellulolyticum: Its nature of production and hydrolytic potential

Summary Maximum xylanase production byChaetomium cellulolyticum was obtained in the culture supernatant after 30 h of growth at 37°C in basal medium containing 1% xylan at pH maintained between 6.5 and 7.5. Addition of 0.05% Tween 80 to the medium increased the enzyme production considerably. Xylanase production was found to be growth associated. The optimal conditions for enzymatic hydrolysis of xylan were found to be pH 6.0 and 50°C. During enzymatic hydrolysis, xylose, xylobiose and other xylooligosaccharides were liberated from xylan. The pH values for xylanase production and for xylan hydrolysis were closely related to the utilization of hemicelluloses of aspen wood for fungal protein production by this organism as reported in our earlier work.     

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.     

Regulation of cell proliferation and morphogenesis by amino acids inBrassica tissue cultures and its correlation with threonine deaminase

The effect of amino acids has been investigated with respect to the capacity ofBrassica cultures to undergo proliferation and differentiation. Hormone medium without any amino acid resulted in 6% shoot formation. Addition of optimal concentrations of L-leucine and L-isoleucine enhanced shoot formation upto 30% and 60%, respectively. L-methionine, L-threonine and pyruvic acid supported only proliferation but no differentiation. Amino acids had a marked effect on the activity of enzyme threonine deaminase (TD), bothin vivo andin vitro. TD in proliferating callus cultures was 3-fold higher than in differentiating cultures. Amino acids which induced cell proliferation increased TD while those which supported differentiation repressed it. Amino acids which did not alter TD activity had no effect on morphogenesis. The results suggest that amino acids play a regulatory role inBrassica morphogenesis which can be correlated with the activity of threonine deaminase.     

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.     

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.     

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.     

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.     

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.     

Production of fructosyl transferase fromAureobasidium pullulans

Summary Conditions for the production of intracellular fructosyl transferase fromA. pullulans were investigated. Sucrose was an excellent carbon source, and there was a tendency for the enzyme production to be increased as sucrose concentration was increased. Both 0.5% phosphate and 2% sodium nitrate had positive effects on enzyme production. It was possible to increase the intracellular enzyme production up to 140% by increasing the concentration of magnesium sulfate from 0.05 % to 0.2%.     

Production of mold lactase

A process for production of mold lactase was developed. Tests were carried out in pilot and industrial scale with an Aspergillus niger strain selected after screening a number of molds.A computer coupled autoanalyzer system was used for monitoring enzyme formation in the pilot fermentor. Lactase production was investigated using different pH- and temperature-profiles. A. niger lactase has an acid pH optimum, a high temperature optimum and good stability. It does not require any metal ions. It is suitable for immobilization for hydrolysis of lactose in acid whey.Three-fold enhancement of lactase production was obtained by mutagenizing A. niger using NTG as mutagenic agent.The lactases produced by the mutants have the same pH and temperature optima and stability but the growth properties of the mutants were different from those of the original strain.Sufficient specific activity of the enzyme preparation for immobilization was obtained by purifying the enzyme by selective adsorption on Na-Ca-silicate.     

Immobilized Aspergillus sydowii produces xylanase

Summary Spores ofAspergillus sydowii, immobilized in 2.5% caleium alginate was used as inoculum in batch cultures for production of xylanase enzyme using xylan as the sole carbon source. Partially germinated mycelium from these entrapped spores produced significant amount of the enzyme in a short period of 24 hours and the same inoculum could be used repeatedly for at least 5 cycles with less than 10% loss of enzyme activity.     

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.     

Growth of the yeast Hansenula anomala with nitrate as sole source of nitrogen under aerobic and anaerobic conditions

Summary The oxygen requirement ofHansenula anomala growing in batch culture on nitrate as sole source of nitrogen was examined. An aeration rate of 0.03 vvm or a constant oxygen partial pressure of 0.01 bar is sufficient for optimal growth.