Glucanase cell-wall fractionation

Two cell-wall fractions were obtained from mycelia of Neurospora crassa by differential extraction with alkali (soluble in 4%, and insoluble in 24%) on incubation with endo-(l - 3)-/3-D-glucanase, both gave473 D-glucose, laminara-biose and -triose, and 62-/3-D-glucosyllaminarabiose (118). exo-(1 - 3)-/3-D-Glucanase released D-glucose and gentiobiose. Structures were proposed for both fractions when this evidence and the results of methylation analysis were considered. 

The cell-wall j3-D-glucan fractions, insoluble in alkali and acid, from wild and from a mutant S. cerevisiae have been compared. Methylation analysis indicated more 1,3- and 1,3,6-linked D-glucosyl residues, and fewer 1,6-linked in the latter. The wild type was hydrolyzed less by exo-(l- 3)-/3-D-glucanase and more by endo-(l -> 6)-/3-D-glucanase than was the glucan from the mutant, consistent with the methylation data.4733... 

To test this possibility Rombouts and Phaff (94,95) followed the purification of the /3-glucanase complex of B. circulans grown on bakers yeast cell walls by two separate procedures. In addition to the usual tests for / -( 1 — 3) - and / -( 1 — 6)-glucanases by reducing group increase on their respective soluble substrates, laminarin and pustulan, each fraction from the fractionating colums, was also tested for lytic activity on a cell wall suspension. They made two additional improvements in enzyme purification. 

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