The effects of recombinant bovine interferon-alpha on fertility in ewes

Recombinant bovine interferon-alpha(I)1 (rBolFN-alpha) may be useful for enhancing fertility in sheep because it has extensive sequence homology with ovine trophoblast protein-1. To test the effectiveness of rBolFN-alpha, several experiments were performed in which bred females were given intramuscular injections of rBolFN-alpha around the time of maintenance of the corpus luteum. Treatment with rBolFN-alpha enhanced the fertility of ewes that were bred via natural service or embryo transfer of whole or demi-embryos. Interferon treatment was successful in enhancing lambing rate if injections were given twice daily from Days 11 to 18, 12 to 14, 12 to 15 or 12 to 16. Overall, the lambing rate for ewes bred via natural service was 94/126 (74.6%) for control ewes and 101/126 (80.2%) for rBolFN-alpha treated ewes. Litter size was not affected by treatment. Interferon treatment was not successful in increasing the lambing rate if given as a single injection on Day 12 or as a series of once-daily injections from Days 11 to 16. These results demonstrate that rBolFN-alpha can increase the lambing rate in ewes.     

NH(4)-Excreting Azospirillum brasilense Mutants Enhance the Nitrogen Supply of a Wheat Host

Spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense were selected on the basis of their excretion of NH(4). Two mutants exhibited no repression of their nitrogenase enzyme systems in the presence of high (20 mM) concentrations of NH(4). The nitrogenase activities of these mutants on nitrogen-free minimal medium were two to three times higher than the nitrogenase activity of the wild type. The mutants excreted substantial amounts of ammonia when they were grown either under oxygen-limiting conditions (1 kPa of O(2)) or aerobically on nitrate or glutamate. The mutants grew well on glutamate as a sole nitrogen source but only poorly on NH(4)Cl. Both mutants failed to incorporate [C]methylamine. We demonstrated that nitrite ammonification occurs in the mutants. Wild-type A. brasilense, as well as the mutants, became established in the rhizospheres of axenically grown wheat plants at levels of > 10 cells per g of root. The rhizosphere acetylene reduction activity was highest in the preparations containing the mutants. When plants were grown on a nitrogen-free nutritional medium, both mutants were responsible for significant increases in root and shoot dry matter compared with wild-type-treated plants or with noninoculated controls. Total plant nitrogen accumulation increased as well. When they were exposed to a N(2)-enriched atmosphere, both A. brasilense mutants incorporated significantly higher amounts of N inside root and shoot material than the wild type did. The results of our nitrogen balance and N enrichment studies indicated that NH(4)-excreting A. brasilense strains potentially support the nitrogen supply of the host plants.     

The hydrolysis of triglycerides by immobilized lipase in a hydrophilic membrane reactor

In the present article a method is described to immobilize lipase from Candida rugosa on a hollow fiber membrane, and the use of such a system for the hydrolysis of lipids is reported. The membranes were ENKA hydro philic Cuprophan((R))-type hollow fibers, having a large specific surface area. The immobilized lipase exhibited a high stability: the half-life time was 43 days at a temperature of 30 degrees C. Furthermore, it is proved that kinetic studies can be carried out with this system, operated in a batch or continuous mode. The relation between conversion rate and degree of hydrolysis was determined. On this basis, a dynamic model of the process was developed that describes the relation between reaction conditions and the conversion rate.     

Production of optically active 2,3-butanediol by Bacillus polymyxa

Bacillus polymyxa produces (R, R)-2,3-butanediol from a variety of carbohydrates. Other metabolites are also produced including acetoin, acetate, lactate, and ethanol. The excretion of each metabolite was found to depend on the relative availability of oxygen to the culture. When the relative oxygen uptake rate was high, enhanced yields of acetate and acetoin were noted. At an intermediate oxygen availability, the butanediol yield was maximal. When the availability of oxygen was more restricted, higher yields of lactate and ethanol occurred. The cells appeared to regulate themselves such that energy generation is optimal subject to the constraint that the cells do not produce more reducing equivalents than can be oxidized by the electron transport system. The dependence of each product yield on the relative oxygen availability was determined, and this knowledge was used to carry out a fed-batch fermentation that attained a final butanediol concentration of over 40 g/L in 50 h.     

The production of cellulase in a spouted bed fermentor using cells immobilized in biomass support particles

Continuous cellulase production by Trichoderma viride QM 9123, immobilized in 6 mm diameter, spherical, stainless steel biomass support particles, has been achieved using a medium containing glucose as the main carbon source. Experiments were carried out in a 10-L spouted bed fermentor. In this type of reactor-recycled broth is used to create a jet at the base of a bed of particles, causing the particles to spout and circulate. During the circulation, particles pass through a region of high shear near the jet inlet. This effectively prevents a buildup of excess biomass and thus enables steady-state conditions to be achieved during continuous operation. Continuous production of cellulase was achieved at significantly higher yield and productivity than in conventional systems. At a dilution rate of 0.15 h(-1) (nominal washout rate for freely suspended cells is 0.012 h(-1)), the yield of cellulase on glucose was 31% higher than that measured during batch operation, while the volumetric productivity (31.5 FPA U/L. h) was 53% greater than in the batch system. The specific cellulase productivity of the immobilized cells was more than 3 times that of freely suspended cells, showing that diffusional limitations can be beneficial. This offers significant opportunity for the further development of biomass support particles and associated bioreactors.     

Mode of action and properties of xylanase and beta-Xylosidase from Neurospora crassa

Extracellular beta-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) from culture filtrates of Neurospora crassa was purified to homogeneity by preparative isoelectric focusing followed by gel electrophoresis. The molecular weight of the purified xylosidase was 83,000 D and the K(m) on p-nitrophenyl-beta-D-xyloside was 0.047mM. The homogeneous xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) and beta-xylosidase showed differences in their mode of action towards xylooligosaccharides. The degree of hydrolysis of D-xylan by xylanase of N. crassa was 18%. Supplementation of beta-xylosidase from the same organism resulted in 48% hydrolysis. The synergistic effect was more pronounced, with the hydrolysis of 68%, when a homogeneous preparation of beta-xylosidase from Sclerotium rolfsii was added to the saccharification system.     

Purification and degradation of ribulose bisphosphate carboxylase from soybean leaves

A rapid method is described for the preparation of up to 500 milligrams of pure ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase) from 250 grams of field-grown soybean leaves. Leaves were extracted in 20 millimolar phosphate (pH 6.9) at 4°C, containing 4% (w/v) polyvinylpolypyrrolidone, 10 micromolar leupeptin, 1 millimolar phenylmethyl sulfonylfluoride, 1 millimolar diethyldithiocarbamate, 5 millimolar MgCl₂, 1 millimolar dithiothreitol, 0.2 millimolar ethylene-diaminetetraacetic acid, 50 millimolar 2-mercaptoethanol. The extract was incubated in the presence of 5 millimolar ATP at 58°C for 9 minutes, then centrifuged and concentrated. Sucrose gradient centrifugation into 8 to 28% (w/v) sucrose on a vertical rotor for 2.5 hours yielded pure enzyme with a specific activity of 1.1 to 1.3 micromoles per minute per milligram protein at pH 8.0, 25°C. Soybean plants of the same line grown (at 400 microeinsteins per square meter per second) in growth chambers yielded enzyme with a specific activity of 0.6 to 0.7 micromoles per minute per milligram protein. During prolonged purification procedures a proteolytic degradation of RuBP carboxylase caused complete loss of catalytic activity. Without destroying the quaternary structure of the enzyme, a 3 kilodalton peptide was removed from all large subunits before further breakdown (removal of a 5 kilodalton peptide) occurred. Catalytic competence of the enzyme was abolished with the loss of the first (3 kilodalton) peptide.     

Reaction kinetics of cephalexin synthesizing enzyme from Xanthomonas citri

In enzymatic synthesis of cephalexin from D-alpha-phenylglycine methyl ester (PGM) and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) using alpha-acylamino-beta-lactam acylhydrolase from Xanthomonas citri, it was found that this enzyme catalyzes all three reactions including PGM hydrolysis, cephalexin synthesis, and cephalexin hydrolysis. Based on our experimental results, a mechanistic kinetic model for cephalexin synthesizing enzyme system having acyl-enzyme intermediate was proposed. From this kinetic model, the reaction rate equations for three reactions were derived, and the kinetic parameters were evaluated. A good agreement between the simulation results and the experimental results was found.