Mannan mannanase

These properties of the enzyme are good for the production of D-mannose from P-mannan in the presence of the P-mannanase described above. 

E, co/i JMlOl (pMAH3) was grown aerobically in LB broth for 24 h at 37°C. The P-mannanase activity was located mainly in the periplasmic (53%) and intracellular (43%) fractions. No induction of the enzyme could be detected in the presence or absence of mannan, such as konjak, locust bean and copra, in the growth medium. 

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. 

The most characterised D-mannan degrading enzymes are the endo- -mannanases (5). These enzymes act on a range of 1,4-p-D-mannan-type polysaccharides including D-mannan, glucomannans (acetylated and deacetylated), galactomannans and galacto-glucomannans. The extent and... 

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. 

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... 

Figure 1. Schematic representation of subsite binding between / -mannanase and the (1— 4)-/ -D-mannan chain. (Reproduced with permission from reference 5. Copyright 1983 Elsevier Science Publishers).

Future research. From an industrial standpoint, the major requirements of p-mannanases are thermostability and the ability to hydrolyse crystalline and/or amorphous mannan to low DP oligosaccharides. Enzymes with such properties could find application in such processes as coffee extraction and in enzymic treatment of wood pulp. 

This enzyme [EC 3.2.1.78], also known as mannan endo-l,4-j8-mannosidase and endo-l,4-mannanase, catalyzes the random hydrolysis of l,4-j8-D-mannosidic linkages in mannans, galactomannans, glucomannans, and galacto-glucomannans. 

SOLUBILITY PRODUCT MANGANESE PEROXIDASE PEROXIDASE /3-MANNANASE MANNOSIDASES Mannan endo-1,4-j8-mannosidase, 13-MANNANASE Mannans,... 

Galactomannans and oligosaccharide fragments are susceptible to hydrolysis by a number of enzymes, including a-D-galactosidase90 (EC 3.2.1.22), /S-D-mannanase, exo-/3-D-mannanase,91 /3-D-mannosidase, and exo-/3-D-mannan mannobiohydrolase (EC 3.2.1.100).92 Early applications of enzymic... 

The structures of the cell-wall D-mannans of several other yeasts have been investigated381 by use of this exo-a-D-mannanase. Five were degraded to the (l->6)-a-D-mannan chain. Those which contained jB-linked d-mannosyl units or a-D-galactosyl groups in the side chains were not significantly hydrolyzed. However, removal, by partial hydrolysis with acid, of the a-D-galactosyl units from five galactomannans, and of /3-linked D-mannosyl units from three other D-mannans, rendered these polysaccharides partially susceptible to hydrolysis by exo-a-D-mannanase, consistent with an a-linked-D-mannan structure. 

Invertase from a Saccharomyces cerevisiae mutant could be separated into two fractions on the basis of solubility in ammonium sulfate.382 The soluble fraction reacted with endo-(l - 6)-a-mannanase, when it became insoluble. The results suggested that the insoluble fraction contained only the highly branched, core section, but the soluble fraction also had the (l->6)-a-D-mannan chain attached. 

Sprucewood holocellulose was treated with an endo-p-1,4-mannanase isolated from Aspergillus niger and an endo-/3-1,4-xylanase, two avicelases, and a cellobiohydrolase C isolated from Trichoderma viride. The mannanase hydrolyzed about a quarter of the mannan in 2-3 days without xylan or cellulose degradation. The xylanase hydrolyzed about half the xylan with 10% mannan solubilization. The three cellulases hydrolyzed up to 45% of the cellulose and 20% of the xylan, accompanied by 40-70% solubilization of the mannan. Combined xylanase-mannanase treatment hydrolyzed about half the xylan and mannan. Addition of mannanase to to cellulose-treated samples increased the degradation of the cellulose and mannan. Micromorphological studies of the variously treated specimens revealed a loss of substances in P/Slf T, and adjacent zones of S2 of the tracheid wall. 

The galactoglucomannan of the holocellulose was degraded by the mannanase. The reaction was slow, but conversion showed a steady increase. After 80 hr of incubation, about 25% of the mannan was transformed into water-soluble products (Table I, Column 10). This figure was calculated on the basis of the mannose, glucose, and galactose present in the acid hydrolysate (cf. Experimental). 

Cellobiose or other cellulose fragments were not found in the reaction solution, neither after the xylanase nor after the additional action of mannanase. The glucose present probably was derived from the mannan see above and Mannanase). 

Mannan fragments that were dissolved by the action of the cellulase and most of the mannan left in the tissue were hydrolyzed when mannanase was added. At the end of the experiment (80 hr 48 hr with only one of the three cellulases and 32 hr with cellulase + mannanase), about 75-95% of the mannan present in the starting material was hydrolyzed (Table III. Column 10). The calculation of these values was based on the mannose and galactose present in the hydrolysates and on the ratio mannose glucose = 3.3 1 and was related to the amount of sugars in the total hydrolysate of the starting material (cf. Experimental). 

The xylan degradation rates relative to those of mannan ranged from 0.3 to 0.4 at the end of the experiment (80 hr). The same values were obtained before the additional action of mannanase on the samples treated with one of the three cellulases for 48 hr (Table III, Column 14). The corresponding values relative to the cellulose degradation rates ranged from 0.5 to 0.7 for the samples treated with cellulase only (48 hr) and after combined cellulase-mannase action (Table III, Column 15). 

The properties of the enzymes used in this study have been described in former publications (10,11,15). Important for the following interpretation are their hydrolytic specificities. The xylanase did not hydrolyze either isolated mannans or celluloses—or only to a very small extent (10). The same is true for the mannanase with respect to xylans and celluloses (11,15). The avicelases, which were not purified to the same extent as the xylanase and mannanase, did not hydrolyze mannans, but they degraded xylans besides crystalline cellulose (10). Also, the highly purified cellobiohydrolase C (12) degraded xylan to some extent (Dr. E. K. Gum, Jr., personal communication). 

The experiments were repeated under similar conditions with the same and another mannanase fraction of Yamazaki s, with sprucewood holocellulose prepared in a slightly different way (19). After three weeks incubation, about 19% of the mannan was dissolved away, and the loci of hemicellulose removal were revealed by electron microscopy. In these experiments, the bulk of hemicelluloses was obviously removed by the buffer solution. In addition to mannose, glucose and galactose derived from mannan, xylose and glucose were found in the reaction solutions in appreciable amounts. 

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