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 8-glucanases. Two /?-(1-+3)-glucanases (I and 11), 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 /?-( 1 + 3)-glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic P-( 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 p-(1+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 K, 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 p-(1 --f 3)-glucanase I is a powerfully lytic enzyme, completely solubilizing the glucan of yeast cell walls. Synergism with lytic /?-( 1 +6)-glucanase could be observed during yeast glucan hydrolysis.Lytic B-( 1 -+3)-glucanase I1 could be further purified by carboxymethyl-cellulose and diethylamino-ethyl-agarose chromatography. At this stage lytic P-( 1 --f 3)-glucanase I1 was still contaminated with some non-lytic P-(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.Bacillus circulans WL-12 was isolated as an organism capable of degrading yeast cell walls [l]. It was found that this bacterium produces a number of P-glucanases when grown on bakers' yeast cell walls or on alkali-insoluble bakers' yeast glucan [l -31.Fleet and Phaff [2] isolated both a /?-(1+3)-and a P-(1 +6)-glucanase in highly purified form from the culture liquid, and observed that the hydrolytic action of these enzymes on isolated yeast cell walls was negligible. Neither of the enzymes nor the combination of the two could lyse cell walls to an extent that clearing could be observed in a cell wall agar plate. Also, the enzymes were unable to hydrolyze the microfibrillar glucan network present in Succharomyces walls [4]. The enzymes were therefore considered to be non-lytic. Subsequently, Rombouts and Phaff [3] found that lytic enzymes, which were present in the culture liquid of Bacillus circulans WL-12, could be -~ Enzyme. Endo-l,3-/~'-glucanase (EC 3.2.1.6).isolated by adsorption onto alkali-insoluble yeast glucan. Fractionation of the lytic p...

Cloning of two genes from Bacillus circulans WL-12 which encode 1,3- -glucanase activity

“…It has been reported that B. circulans WL-12, depending on the growth substrate used as inducer, is capable of Various biochemical and physical properties of the producing up to six different 1,3-P-glucanases (Esteban cloned 1,3-#&glucanases were examined in order to et af., 1984; Kobayashi et al, 1974a, b; Rombouts & substantiate that the proteins produced from Phaff, 1976;Tanaka et al, 1978Tanaka et al, , 1981. Tanaka et al lpMON5401 and pMON5402 are distinct and to facili-(1978) proposed that the observed induction patterns are iiate comparison of these activities (Table 3) with the a reflection of the structures of the mixed-linkage glucans…”

“…L. Tobey-Fincher and R . L. Fuchs reasons : (1) 1,3-P-glucanases from B. circulans have exhibited hydrolysis of fungal cell walls (Horikoshi & Atsukawa, 1973;Kobayashi et al, 19746); (2) 1,3-pglucanases from B. circulans have been biochemically well characterized (Esteban et al, 1984;Kobayashi et al, 1974a;Rombouts & Phaff, 1976;Tanaka & Phaff, 1965); and (3) the gene(s) encoding 1,3-P-glucanase(s) from Bacillus species are likely to be expressed in E . coli (Borriss et al, 1985;Cantwell & McConnell, 1983).…”

.BETA.-Glucosidase and .BETA.-galactosidase from the periplasmic space of Rhizobium trifolii cells.

“…The molecular weight of ~3-glucosidase was estimated to be about 74,000. Lytic i3-1,6-glucanase and lytic 13-1,3-glucanase of Bacillus circulans WL-12 were reported to have a molecular weight of 54,000 and 40,000 respectively (26,27). The molecular weight of a r3-1,6-glucosidase from Flavobacterium was estimated to be 59,000 (28).…”

Purification and Some properties of Endo-β-1,6-Glucanase fromAcinetobactersp.

“…2 , 3) By contrast, only a few cases have been reported as to (3-1,6-glucanase acting on yeast glucan. 4,5) This paper describes the properties of (3-1,6-glucanase obtained from the culture filtrate of Acinetobacter sp.…”