Action of P-mannanases

Assay and Purification of p-Mannanases. p-Mannanase can be specifically measured in crude enzyme mixtures with a dye-labelled carob galactomannan substrate (6), This substrate has a galactose content high enough (23%) to impart solubility to the substrate, but sufficiently low as not to interfere with the action of p-mannanases on the D-mannan backbone. The soluble dye-labelled substrate is incubated with enzyme preparation under controlled conditions and the reaction is terminated and unreacted dyed polysaccharide precipitated by ethanol addition. 

As described in the previous section, this strain produced significant amounts of three extracellular P-mannanases and a cell-associated P-mannosidase. The three P-mannanases differed in several enzymatic properties including optimum pH for enzyme action, optimum temperature, pH stability, thermal stability, isoelectric point and molecular weight. To elucidate the genetic basis for production of multiple forms,... 

Hydrolysis of mannan-type polysaccharides by P-mannanase is dependent on substitution on and within the main-chain as well as the source of the P-mannanase employed. Characterisation of reaction products can be used to define the sub-site binding requirements of the enzymes as well as the fine-structures of the polysaccharides. Action of c/xt/o-arabinanase and em/o-galactanase on arabinans and arabinogalactans is described. Specific assays for ndo-arabinanase and arabinan (in fruit-juice concentrates) are reported. 

P-Mannanases are generally prepared by conventional chromatographic procedures, which can lead to high yields of enzyme, but in some cases only a poor state of purity. Small quantities of highly purified p-mannanases have been prepared by substrate affinity chromatography of partially purified enzymes on a column of glucomannan immobilised on aminohexane Sepharose 4B (6). The action patterns of enzymes described in this paper were determined using enzymes purified by this latter procedure. 

The difference in action patterns between these p-mannanases is shown clearly by fractionating and characterising the reaction products (Table I) (5). These products are a consequence of the abihty of the enzyme to cleave in the vicinity of D-mannosyl residues substituted by D-galactose, as well as the length of the 1,4-P-D-manno-oligosaccharide chain required by the enzyme for binding. The favoured conformation of the (l-4)-p-D-linked mannan chain is a flat... 

---