A new method of isolation and a new form of transketolase from baker's yeast]

A new procedure for the isolation of homogeneous transketolase from baker's yeast based on the use of enzyme-specific antibodies immobilized on a insoluble matrix has been developed. The enzyme yield is 90% of its total content in the original yeast extract. The eluate from the immunocolumn was found to contain a previously unknown form of transketolase which represents an enzyme-RNA complex.     

Active subunits of transketolase from baker's yeast.

Transketolase from baker's yeast was covalently bound to Sepharose via one subunit. Storage in glycine buffer pH 11 entailled loss of half of the protein and a 50% decrease in the activity of the immobilized enzyme. Addition to the system of free subunits of transketolase doubled the amount of the protein attached to the matrix and restored the catalytic activity to the initial level. Thermoinactivation of the initial immobilized dimer of transketolase and the renatured dimer formed on reassociation of the immobilized subunits with the free ones was the same and considerably differed from the thermoinactivation of the immobilized subunits. The conclusion is made that the individual subunits of transketolase are catalytically active.     

Purification of transketolase from baker's yeast by an immunoadsorbent

Baker's yeast transketolase has been purified by immunoaffinity chromatography on specific TK antibodies covalently linked to CNBr-agarose. Affinity chromatography allows a 480-fold purification of TK from yeast extracts and about 80% recovery of the original activity. The isolated enzyme has a specific activity of 12-60 U/mg and during polyacrylamide gel electrophoresis performed at pH 8.9 migrates as two protein bands possessing a transketolase activity which corresponds to two isoforms of the enzyme.     

Molecular weight and tertiary structure of transketolase from baker's yeast]

Molecular weight of native apotransketolase from baker's yeast is found to be 159000 +/- 6000 by means of sedimentation equilibrium and sedimentation-diffusion rate. The enzyme in a relatively low concentration reversibly dissociates into two subunits with molecular weight of about 80 000 at pH 7.6 and 20 degrees C. The equilibrium constant of the reaction monomer-dimer is 4.4 . 10(3) M-1. A decrease of the temperature stimulates the association of monomers into dimer, while the shift of pH 7.6 into acid or alkaline region stimulates the dissociation process. Dissociation becames irreversible at pH less than 5 and greater than 10.5.     

Computer-aided baker's yeast fermentations.

The economics of yeast production depend heavily upon the cellular yield coefficient on the carbon source and the volumetric productivity of the process. The application of an on-line computer to maximize these two terms during the fermentation requires a continuous method of measuring cell density and growth rate. Unfortunately, a direct sensor for biomass concentration suitable for use in industrial fermentations is not available. Material balancing, with the aid of on-line computer monitoring, offers an indirect method of measurement. Laboratory results from baker's yeast production in a 14-liter fermentor (with a PDP-11/10 computer for on-line analyses) show this indirect measurement technique to be a viable alternative. From the oxygen uptake and carbon dioxide production data, gas flow rate, and ammonia addition rate, the cell density during the fermentation has been estimated and found to compare well with actual fermentation data.     

Disruption of baker's yeast by a new bead mill

Summary A continuous high-speed bead mill of novel design (Sulzer Annu Mill 01) was tested for cell disruption of baker's yeast as a model system. The efficiency of cell disruption was evaluated for the relative amount of released protein. The effects of rotation speed, cell concentration and flow rate of cell suspension on the cell disruption were investigated. The maximum yield of released protein was found to be 2.62 kg protein/L.h. This novel design appears to be more effective than existing commercially available mills.Notations Cs cell concentration (g packed yeast/L) - F flow rate of suspension, mL/min - F_R cumulative residence time distribution - N rotation speed of the rotor (rpm) - P number of passes of suspension through mill - R amount of protein released from cell, mg/g packed yeast - R_m maximum amount of protein released, mg/g packed yeast - t time, s - mean residence time, s