Kinetic properties and contribution to cellulose saccharification of a cloned Pseudomonas beta-glucosidase.

The plasmid pND71, which encodes beta-glucosidase (cellobiase) activity, cloned from the cellulolytic Pseudomonad, PS2-2, was mobilized by conjugation into 10 Pseudomonas strains. The highest specific activity was produced by 17498 (pND71) and the properties of the enzyme produced from this transconjugant were studied. The enzyme was shown to be cell associated, to have a temperature optimum of 37 degrees C, a pH optimum of 7.0 and Km values of 1.33 and 2.94 mM for pNPG and cellobiose respectively. It was competitively inhibited by glucose, with a Ki of 30 mM. Evidence was obtained which suggested that the enzyme was produced constitutively and that synthesis was not repressed by glucose. When culture preparations were used in combination with Trichoderma reesei QM9414 and C30 enzyme preparations to saccharify cellulose, 17498 (pND71) was more effective than preparations of PS2-2 in acting synergistically with T. reesei to solubilize more carbohydrate and produce more glucose.

Investigations into the kinetics and stoichiometry of bacterial oxidation of covellite (CuS) using a polarographic oxygen probe.

"Oxygraph" apparatus was used to measure quantitatively the kinetics of oxidation of synthetic covellite (CuS) in the presence and absence of Thiobacillus species. The expected stoichiometric relationship between oxygen consumed and cupric sulphate produced was verified by atomic absorption assays of cupric ion and sulphate ion. Thiobacillus cultures markedly increased the oxidation rate. The dependence of each oxygen-uptake rate on oxygen concentration was also measured. Sterile controls and some bacterial cultures showed first-order kinetics while other cultures showed zero-order kinetics. Addition of biological inhibitors to reacting slurries revealed that cultures showing first-order kinetics did not oxidize CuS itself but merely oxidized elemental sulphur formed by non-enzymic oxidation of CuS. Cultures showing zero-order kinetics oxidized CuS in a way that resulted in all oxygen reduction being enzymic. This mechanism possibly involves the cyclic oxidation and reduction of soluble iron.

Pectinolytic activity of Saccharomyces fragilis cultured in controlled environments.

Seven of 30 yeast stock cultures, covering nine genera, and 13 of 39 yeasts isolated from grapes gave positive reactions when screened for pectinolytic activity on pectin gel plates. The seven stock cultures covered six species and four genera. Only one of the yeasts, Saccharomyces fragilis Y49, excreted discernible pectinolytic activity into the fluid of shake flask cultures; the activity was partially constitutive and was repressed by high oxygen tensions.

A thermodynamic assessment of possible substrates for sulphate-reducing bacteria.

A thermodynamic feasibility study was applied as a means of predicting suitable energy-yielding substrates for growth of sulphate-reducing microorganisms. The average free energy release per electron pair for a substrate-sulphate oxidoreduction may be more or less than the energy requirement for ATP synthesis from ADP and Pi. Substrates were divided into two groups on this thermodynamic basis and the division was shown to accord with previous experimental reports; those substrates which released an average of at least 8-4 kcal per electron pair (35-2 kJ per electron pair) were able to support growth whilst those releasing less than 8-4 kcal were unable to do so. It is proposed that the thermodynamic assessment could be applied to a wide range of possible substrates to predict the likelihood of their serving as sole substrates for growth of these organisms. The literature concerning the use of hydrocarbons by sulphate reducers is confused and indefinite, but inclines toward the idea that use of long-chain hydrocarbons by these organisms is possible. In contrast, however, thermodynamic analysis showed that the highest energy release is from the short-chain alkynes.