P-Mannanase

Harjunpaa, Vesa. Enzymes hydrolysing wood polysaccharides. A progress curve study of oligosaccharide hydrolysis by two cellobiohydrolases and three P-mannanases. 1998.76 p. + app. 11 p. 

Beta-mannosidase and P-mannanase cleave the P-mannoside linkages in P-l,4-D-... 

Bacillus sp. AM-001 was cultivated aerobically under various conditions, and activities of P-mannanase in the culture broth and P-mannosidase extracted from the cells treated with 0.1% Triton X-100 were monitored. Both enzymes were formed when the bacterium was grown under alkaline conditions in the presence of optimal concentrations of 0.5% Na2C03 or 0.5-1.0% NaHCOa. Various carbohydrates were also tested and the best carbohydrate for enzyme production was konjak powder (1%... 

Three extracellular P-mannanases (M-1, M-II and M-III) were purifled by anunonium sulfate precipitation (80% saturation) followed by chromatography on a DEAE-Toyopearl 650 M column (4.6 x 35 cm) equilibrated and eluted with 0.01 M phosphate buffer (pH 7.0 ) and by a hydroxylapatite column (1.6 x 25 cm). As shown in Fig. 1, two active fractions (Fraction 1 and 2) were detected after hydroxylapatite chromatography. Each fraction 1 and 2 was applied onto a Sephacryl S-200 column (2.6 x 90 cm) equilibrated with 0.01 M phosphate buffer (pH 7.0) containing 0.1 M NaCl and eluted with the same buffer. Mannanase-I and -II were isolated from fraction 1 and mannanase-III was from fraction 2. 

Fig. 2. Effects of pH on Enzyme Activity. Symbols ( ), P-mannanase M-I ( ), P-mannanase M-II (A), P-mannanase M-III.

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. 

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,... 

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. 

AM-001, and mannanase B properties are similar to those of P-mannanase M-III. Furthermore, the Ouchterlony double diffusion test showed that these five enzymes gave fused precipitation lines. However, N-terminal amino acid sequences of the five mannanases determined by an automatic amino acid sequencer revealed that the N-terminal amino acid sequence from amino acid 1 (Asn) to 9 (Gin) of the Bacillus sp. AM-001 enzymes coincides with those from amino acid 4 (Asn) to 12 (Asn) of the R coll JMlOl (pMAH3) enzymes as shown in Fig. 4. This may reflect differences in the specificities of the signal peptidases of the two bacteria. 

Fig. 4 (continued). Nucleotide sequence of the Xbal-PstI fragment containing )0-mannanase gene(s). Symbols (B), N-terminal of three P-mannanases produced by alkaliphilic Bacillus sp. A-OOl (E) N-terminal of two )0-mannanases isolated from E. Coli carrying pMAH3 (MA), C-terminal end of the mannanase A (MB), C-terminal end of the mannanase B. 

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. 

In the following section of this paper, some aspects of the author s work on the characterisation and utilisation of p-mannanases will be described. 

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. 

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... 

Table I. Oligosaccharides produced on hydrolysis of carob galactomannan by p-mannanases...

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. 

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