Isolation of mannan-protein complexes from viable cells of Saccharomyces cerevisiae X2180-1A wild type and Saccharomyces cerevisiae X2180-1 A-5 mutant strains by the action of Zymolyase-60,000.

The viable whole cells of Saccharomyces cerevisiae X2180-1A wild type and its mannan mutant strain S. cerevisiae X2180-1A-5, were treated with an Arthrobacter sp. beta-1,3-glucanase in the presence of a serine protease inhibitor, phenyl-methylsulfonyl fluoride. Fractionation of the solubilized materials of each strain with Cetavlon (cetyltrimethylammonium bromide) yielded one mannan-protein complex. Molecular weights of these complexes were almost the same as that of the mannoprotein of the mutant strain prepared by Nakajima and Ballou, which had a molecular weight of 133,000 and were approximately three times larger than those of the mannans isolated from the same cells by hot-water extraction. Each mannan-protein complex contained up to 2% glucose residue, which was not removed by specific precipitation with anti-mannan sera or by affinity chromatography on a column of concanavalin A-Sepharose. Treatment of these complexes with alkaline NaBH4 produced peptide-free mannan containing small amounts of glucose nearly identical to those of the parent complexes. The above findings provide evidence that the glucose residues exist in a covalently linked form to the mannan moiety. Fractionation of the mannan-protein complex of the S. cerevisiae wild-type strain by DEAE-Sephadex chromatography yielded five subfractions of different phosphate content, indicating that these highly intact mannan-protein complexes were of heterogeneous material consisting of many molecular species of different phosphate content.     

NO(2) Sensor which uses immobilized nitrite oxidizing bacteria

A biosensor consisting of immobilized nitrite oxidizing bacteria and an oxygen electrode has been developed for the amperometric determination of NO(2) (nitrogen dioxide) gas. The response time for the determination of NO(2) was within 3 min. A linear relationship was observed between the current decrease and the NO(2) concentration below 255 ppm. The minimum concentration for the determination of NO(2) was 0.51 ppm. The current decrease was reproducible within +/-4% of the relative error. The selectivity of the microbial sensor for NO(2) was satisfactory. The current output of the sensor was almost constant for more than 24 days and 400 assays.     

Steady-state kinetics and spectral properties of Corynebacterium sarcosine oxidase

The overall reaction kinetics of Corynebacterium sarcosine oxidase were investigated and the reaction was shown to follow a ping-pong, bi-bi mechanism with two substrates, sarcosine and molecular oxygen. Sarcosine analogs, such as acetate, propionate and methoxyacetate, were competitive inhibitors of the reaction. Acetate caused characteristic alterations in optical and circular dichroic spectra, indicating that the microenvironment of the substrate-binding region of the enzyme increased in hydrophobicity on binding with the substrate analog. The dissociation constants of the analogs calculated from the spectral changes were in agreement with the kinetic inhibition constants. Inorganic metallic ions were also inhibitory. Of interest was the finding that the inhibition by Hg2+ was proportional to the square of its concentration, which suggests that at least two sulfhydryl groups are related to the catalytic activity of the enzyme.