Enzyme protein release from yeast

Enzyme-extracted mannoproteins from the yeast cell wall added at a dose of 25 g/hL, can reduce by half the bentonite dosage necessary for protein stabilization of a very hazy wine (Table 5.3). During lees autolysis, MP32 is released from the... 

Cell fractionation by mechanical rupture has already come under investigation. Two separate studies of mechanical rupture of yeast showed different rates of release for enzymes in different cell locations (13,14). Wall-linked and periplasmic enzymes were released relatively faster than total protein, soluble cytoplasmic enzymes at about the same rate, and the mitochondrial enzyme fumarase later than total protein (13). Proteolysis by the yeast s own enzymes was not found to be a problem. Activities of the released enzymes declined slowly or not at all when disruption was continued after the end of protein release, and the effect of shear was not separated from the effect of proteolysis. Shetty and Kinsella (15) also found a low rate of proteolysis after mechanical disruption, though thiol reagents added to weaken the cell walls before disruption caused an important increase in the extent of protein breakdown. 

The structured model is consistent with features of lytic enzyme action and yeast structure reported in the literature. The sequential removal of the two wall layers, followed by protoplast rupture, accurately describes the early lag in protein and carbohydrate release. The presence of residual solids at long reaction times was accounted for stabilization of protoplasts by substances released from lysed cells. The structured model can be used to estimate the effects of several process alternatives, as shown in a simulation of a process for recovery of site-linked enzymes from yeast. 

The studies of Reed and co-workers on the nature of protein-bound lipoic acid and its enzymatic release and reincorporation may be applicable to biotin-containing enzymes. It is pertinent to note that a conjugated form of biotin, biocytin, has been isolated from yeast autolyzate and identified as A -biotinyl-L-lysine (Wright et ah, 19r)2 Peck et al., 1952). Biotin is now known to be the prosthetic group of several carboxylases (see Ochoa and Kaziro, 1961, for a review of these enzymes). Although the nature of the moiety to which biotin is bound has not been established, it seems highly probable that it is the -amino group of a lysine residue. 

The antimicrobial action of silver products has been directly related to the amount and rate of silver released and its ability to inactivate target bacterial and fungal cells. In varions laboratory and clinical studies it has been found that metallic silver does not possess significant antimiCTobial potency, while silver ions are highly antimicrobial. The oligodynamic microbicidal action of silver compounds at low concentrations probably does not reflect any remarkable effect of a comparatively small number of ions on the cell, but rather the ability of bacteria, trypanosomes, and yeasts to take up and concentrate silver from very dilute solutions. Therefore bacteria killed by silver may contain 10 -10 Ag+ per cell, the same order of magnitude as the estimated number of enzyme-protein molecules per ceU. 

Another problem to be elucidated is the role of copper in cytochrome a. In the known copper enzymes such as tyrosinase and laccase, copper is an important component of the prosthetic group, but is released from the protein moiety by dialysis against potassium cyanide. In the case of cytochrome a, however, the mode of combination of copper must be different, since very little copper is released from the protein moiety by dialysis. The best known method of releasing the copper is by acid treatment. The role of copper in the electron trasnferring system is still obscure, though Cohen and Elvehjem (1934), Yoshikawa (1937), Schultze (1939, 1941), Gallagher et al. (1956), and Gubler et al. (1957) observed, from dietary experiments, that copper-deficient tissues and yeast have a low cytochrome oxidase activity and a decreased content of hemin a. 

DAAO is one of the most extensively studied flavoprotein oxidases. The homodimeric enzyme catalyzes the strictly stere-ospecihc oxidative deamination of neutral and hydrophobic D-amino acids to give a-keto acids and ammonia (Fig. 3a). In the reductive half-reaction the D-amino acid substrate is converted to the imino acid product via hydride transfer (21). During the oxidative half-reaction, the imino acid is released and hydrolyzed. Mammalian and yeast DAAO share the same catalytic mechanism, but they differ in kinetic mechanism, catalytic efficiency, substrate specificity, and protein stability. The dimeric structures of the mammalian enzymes show a head-to-head mode of monomer-monomer interaction, which is different from the head-to-tail mode of dimerization observed in Rhodotorula gracilis DAAO (20). Benzoate is a potent competitive inhibitor of mammalian DAAO. Binding of this ligand strengthens the apoenzyme-flavin interaction and increases the conformational stability of the porcine enzyme. 

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