Enzyme-Kinetic Analogy for Yeast Flocculation Data

Abstract

Flocculation is defined as the formation of flocs from previously single cells. The flocculation sedimentation rate is independent of the much lower rate of normal sedimentation of the yeast that occurs concurrently with flocculation-controlled sedimentation to the bottom of the vessel. Ca2+ is essential for flocculation to occur (Mill, 1964; Rainbow, 1966), and cell walls from flocculent strains of yeast bound, on average, twice as much Ca2+ as did nonflocculent walls. The latter contained phosphomannan-proteins of lower phosphate content, suggesting that Ca2+-phosphate bridges formed between adjacent yeast cell walls were the cause of the floc formation observed with flocculent yeasts (Lyons & Hough, 1970, 1971). Most quantitative estimates of flocculating ability (flocculence) are based on the observation that the sedimentation rate under gravity of yeast is greatly increased after flocculation has occurred. Techniques for measuring flocculence based on estimation of flocculation-controlled sedimentation rate in a buffer solution have been devised (Burns, 1937; Jansen & Medlick, 1951 ; Helm et a[., 1953). In all of these cases the results were obtained solely from visual observation of yeast settling rate. Spectrophotometric methods for estimating flocculence have also been used (Gilliland, 1951; Woof, 1962; Chester, 1963; Mill, 1964; Greenshields et a[., 1972). The technique used in our study is based on that of Mill (1964). Yeast cells were prepared by centrifugation of a late-exponential culture of a pure strain of Saccharomyces cerevisiae grown in a 0.3 %-yeast-extract medium containing 2 % (w/v) of glucose. The yeast was washed twice in 5Om~-sodium acetate buffer, pH4.6, and resuspended in this buffer at 32C, with the appropriate quantity of CaCI,, at a concentration of 5mg dry wt. of yeast/ml. The yeast suspension was first shaken thoroughly, then the extinction of the undisturbed suspension was read in the EEl portable colorimeter [OB3 filter, 600650nm (6000-6500A) range] at various time-intervals (in an 8 cm-long tube filled with yeast suspension; the light-path traverses only the bottom 2.5cm of the tube, a volume of one-third of the total of 6ml). A graph of yeast (dry wt.) sediment, obtained from a prepared standard curve with known concentrations of uniform yeast suspension in the absence of Ca2+, was plotted against time. The slope of the steep linear rise lasting about Smin, immediately after an initial 30s lag, was taken as the flocculation-controlled sedimentation initial rate. We express this initial flocculation rate as mg/ml sedimented yeast concentration increase/min per mg of yeast in total original suspension. CaZ+ concentration was found to have a linear relationship to flocculation (in the range 5-75p~-Ca*+) up to a concentration of 7 5 p ~ C a ~ + , where a maximum rate VF(max.) is reached. (Ample Ca2+ was present even at 5 p~ to ensure essentially complete flocculation-controlled sedimentation within about 20min. It is the initial rate that is Ca2+dependent in this concentration range, where linearity of increase is maintained for about 5min followed by a progressively slower increase in weight of yeast settled, over the 5-20min period, as the concentration of yeast in suspension decreases in the top 5.5cm of the tube.) Such 'saturation kinetics' confirms the presence of specific binding sites, in the yeast cell wall, for Ca2+. Clearly at 75p~-Ca,+ these binding sites are saturated. We suggest that it is precisely because these sites are saturated at this Ca2+ concentration that we reach the maximum floc size, as a consequence of all the possible Ca2+-phosphate bridges being completed, and hence reach the maximum initial flocculation rate. Fig. 1 is a plot of the reciprocal of the initial flocculation rate against the reciprocal of [Ca2+] (Lineweaver-Burk-plot analogy). This linear relationship allows accurate determination of maximum initial flocculation rate, i.e. VF(max.)r and also of [Ca2+] required to reach half this maximum rate, i.e. KF. For this yeast VF(max.) the maximum initial rate is 0.22mg/ml sedimented yeast concentration increase/min per mg of yeast in total original suspension and KF is 2.4 x M for Caz+.