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. 

Endoxylanase produced by a transformant strain of Escherichia coli has been shown to decrease the kappa number and increase the brightness of pulp in an enzymatic pretreatment followed by chemical extraction (27). Beta-xylosidase, on the other hand, had no effect in the treatment. The mannanase produced by Bacillus suhtilis was found to be equally effective as the xylanase. The specificities of mannanases produced by two different organisms, however, differed considerably 49). Enrichment of the culture broth of Streptomyces olivochromogenes, containing mainly endoxylanase, with different pure enzymes or enzyme mixtures acting on side chains and other hemicelluloses, resulted only in slight increases in brightness and in kappa number reduction (55). 

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. 

Polyacrylamide gel electrophoresis revealed that these three mannanases were electrophoretically homogeneous. The molecular weights estimated by SDS-PAGE... 

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,... 

Properties of a Mannanase Produced by E. co/i JMlOl carrying pMAH3... 

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. Nucleotide sequence of the Xbal-PstI fragment containing mannanase gene(s). Symbols (B), N-terminal of three -mannanases produced by alkaliphilic Bacillus sp. A-001 (E) N-terminal of two / -mannanases isolated from E. Coli carrying pMAH3 (MA), C-terminal end of the mannanase A (MB), C-terminal end of the mannanase B. (Continued on the next page.)...

Table IV. Properties of the Purified -mannanases from Bacillus sp. AM-001 and E. coli carrying pMAH3...

En me Treatment of LCCs. Purified Bacillus circulans xylanase (specific activity, 110 units/mg protein), purified Coriolus versicolor )9-mannanase (specific activity 179 units/mg protein), purified Thermoascus aurianticum /8-glucosidase (specific activity, 150 units/mg protein), and purified Schizophyllum commune acetyl xylan esterase (AXE specific activity, 180 units/mg protein) were used. Unit activity for xylanase and )9-mannanase was defined as the amount of enzyme catalyzing the release of 1 /xmol reducing power per minute at 50°C and pH 6.0 using oat spelts... 

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