Purification and properties of a new enzyme,propioin synthase in baker's yeast which forms propioin from propionaldehyde

The new enzyme, propioin synthase, concerned with the formation of propioin from propionaldehyde was purified 270-fold from the crude enzyme in a yield of 28% by protamine sulfate precipitation, ammonium sulfate fractionation and G-200 gel chromatography using citrate-phosphate buffer (0.1 M Na₂HPO₄–0.02 M citric acid, pH 6.8, containing 0.33 mM MgSO₄, 0.1 mM thiamine pyrophosphate, 2.5 mM MnSO₄ and 30 mM β-mercaptoethanol). The purified enzyme was homogeneous on disc gel electrophoresis. It was most active at pH 6.8–7.0 and 37°C, and stable at pH 7–8 and below 45°C. Its activity was enhanced by FeSO₄·7H₂O, MnSO₄, thiamine pyrophosphate, β-mercaptoethanol, MgSO₄, CaCO₃, and NaCl, and inhibited by AgNO₃, HgCl₂, CuSO₄, ZnSO₄, SnCl₂, NH₄Cl, (CH₃COO)₂Pb·3H₂O, iodoacetic acid, FeCl₃·6H₂O, and (NH₄)₂SO₄. Its molecular weight was 96,000 by sedimentation equilibrium, and 100,000 by Sephadex G-200 column chromatography.     

Characterization of a small proteolytic enzyme which lyses bacterial cell walls

Ensign, J. C. (University of Wisconsin, Madison), and R. S. Wolfe. Characterization of a small proteolytic enzyme which lyses bacterial cell walls. J. Bacteriol. 91:524-534. 1966.-An enzyme isolated from a myxobacter possesses both cell-wall lytic and proteolytic activity. The enzyme has been purified over 600-fold and is electrophoretically homogeneous upon cellulose acetate at several pH values and upon polyacrylamide gel columns. A single peak was obtained upon ultracentrifugation and density gradient centrifugation. Based upon Sephadex gel filtration, a molecular weight of 8,700 was determined for the enzyme. Albumin and casein were extensively degraded by the enzyme, with approximately one-third of the peptide bonds present in these proteins being hydrolyzed. The enzyme lyses cell walls by hydrolyzing peptide bonds in the glycosaminopeptide.     

Purification and some enzymatic properties of the chitosanase from Bacillus R-4 which lyses Rhizopus cell walls.

A strain of Bacillus sp (Bacillus R-4) produces a protease and a carbohydrolase both of which have the ability to lyse Rhizopus cell walls. Of the enzymes, the carbohydrolase has been purified to an ultracentrifugally and electrophoretically homogeneous state, and identified as a chitosanase. The enzyme was active on glycol chitosan as well as chitosan. Molecular weight of the purified enzyme was estimated as 31 000 and isoelectric point as pH 8.30. The enzyme was most active at pH 5.6 and at 40 degrees C with either Rhizopus cell wall or glycol chitosan as substrate, and was stable over a range of pH 4.5 to 7.5 at 40 degrees C for 3 h. The activity was lost by sulfhydryl reagents and restored by either reduced glutathione of L-cysteine. An abrupt decrease in viscosity of the reaction mixture suggested an endowise cleavage of chitosan by this enzyme.     

Preparation and evaluation of an enzyme which degrades yeast cell walls

Summary A method for the production and preparation of an enzyme which degrades yeast cell walls from a species of aRhizoctonia (tentatively identified asR. solani) was established on a commercial scale. The production of crude enzyme powder, having a lytic activity of 100 units/mg, in batches of 80 kg is feasible.The enzyme preparation was evaluated for industrial use. When yeast cells were treated with this enzyme, the digestibility of feed yeast was improved 1.4–2 fold in vitro; the efficiency of a mechanical disintegrator in extracting cellular substances was increased 35–50%; the release of soluble glucans having widely varying degrees of polymerization was induced; the extraction of cellular protein by alkali was facilitated 2–3 fold; an 80% release of cell-bound invertase was induced and 2–3 times more yeast extract could be prepared.Studies on Fungal Enzymes Active in Hydrolysing Yeast Cell Wall (VII)     

Purification and some properties of an esterase from yeast

An esterase was isolated and purified from baker's yeast by ammonium sulfate precipitation and column chromatographies on Sephacryl S-200, DEAE-Sephacel, chromatofocusing, and DEAE-Sephacel again. The molecular weight of the enzyme was approximately 84,000 on Sephadex G-100 and 40,000 by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis, suggesting a dimer for the activity. This enzyme hydrolyzed short-chain naphthyl esters and p-nitrophenyl esters, and its activity was strongly inhibited by mercuric compounds. The esterase appeared to be an arylesterase (EC 3.1.1.2) and its optimum pH was 8.0 at 30°C.     

Immunochemical properties of mannan-protein complex isolated from viable cells of Saccharomyces cerevisiae 4484-24D-1 mutant strain by the action of zymolyase

Viable cells of Saccharomyces cerevisiae 4484-24D-1 mutant strain were treated with an Arthrobacter sp. beta-1,3-glucanase, Zymolyase-60,000, in the presence of a serine protease inhibitor, phenylmethylsulfonyl fluoride. Fractionation of the solubilized materials with Cetavlon (cetyltrimethylammonium bromide) yielded a purified mannan-protein complex, which had a molecular weight of ca. 150,000, approximately three times higher than that of the mannan isolated from the same cells by the hot-water extraction method at 135 C. The amino acid composition of the mannan-protein complex was found to be very similar to that of the mannan-protein complexes of S. cerevisiae X2180-1A wild and S. cerevisiae X2180-1A-5 mutant strains, indicating the presence of large amounts of serine and threonine. It was unexpected that the antibody-precipitating activity of this complex against the homologous anti-whole cell serum was about twice as great as that of the mannan isolated by hot-water extraction. Treatment of this complex with 100 mM NaOH, hot water at 135 C, and pronase, respectively, gave degradation products having the same molecular weight and antibody-precipitating activity as those of the hot-water extracted mannan, allowing the assumption that the protein moiety participated in a large part of this activity.     

Acid phosphatase of the yeast Rhodotorula rubra. Purification and properties of the enzyme.

1. Acid phosphatase from the yeast Rhodotorula rubra was purified 44-fold. The purification procedure involved mechanical disruption of cells, precipitation with ethanol, chromatography on DEAE- and CM-cellulose. 2. The purified enzyme is homogeneous in polyacrylamide gels at pH 4.5, 9.5 and 8.4. Carbohydrate content accounts for 57% of the total weight. The optimum pH is at 4.0-4.6, and the enzyme is stable over pH range from 2.6 to 6.0. Full activity was retained on 60-min incubation at 50 degrees C, but it was reduced by half on 60-min incubation at 65 degrees C. 3. Specificity of the enzyme is fairly broad; monoesters of carbohydrates, and nucleosides and inorganic pyrophosphate can serve as substrates. Km was found to be 1 X 10(-4) M for p-nitrophenyl phosphate as a substrate. The enzyme is inhibited by molybdate, phosphate, arsenate and fluoride ions.