Lysis of Yeast Cell Walls

Abstract

Bacillus circulans WL-12, when grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, produced five β-glucanases. Two β-(1→3)-glucanases (I and II), which are lytic to yeast cell walls, were isolated from the culture liquid by batch adsorption on yeast glucan, and separated by chromatography on hydroxylapatite. Lytic β-(1→3)-glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic β-(1→3)-glucanase I on laminarin was 4.1 U per mg of protein. The enzyme moved as a single protein with a molecular weight of 40000 during sodium dodecylsulfate electrophoresis in slab gels. It was specific for the β-(l→3)-glucosidic bond but the enzyme did not hydrolyze laminaribiose. Hydrolysis of laminarin went through a series of oligosaccharides, and laminaribiose and glucose accumulated till the end of the reaction. A small amount of gentiobiose was also produced from laminarin. Products from yeast cell walls and yeast glucan included laminaripentaose, laminaritriose, laminaribiose, glucose, and gentiobiose, but no laminaritetraose was detected. This glucanase has an optimum pH of 5.5. Laminarin hydrolysis followed Michaelis-Menten kinetics. A ^Km of 0.105 mg of laminarin per ml and a V of 5.6 micro-equivalents of glucose released/min per mg enzyme protein were calculated. The enzyme required no metal ions. The lytic β-(1→3)-glucanase I is a powerfully lytic enzyme, completely solubilizing the glucan of yeast cell walls. Synergism with lytic β-(1→3)-glucanase could be observed during yeast glucan hydrolysis. Lytic β-(1→3)-glucanase II could be further purified by carboxymethyl-cellulose and diethylamino-ethyl-agarose chromatography. At this stage lytic β-(1→3)-glucanase II was still contaminated with some non-lytic β-(1→3)-glucanase that could not be removed by any further chromatographic steps. The enzyme has an optimum pH of 6.5 to 7; its properties were not studied further.