Studies On The Acidic β-Mannanase From Aspergillus Niger LW-1 And Its Application For The Production Of Oligosaccharides From Konjac Glucomannan

β-Mannanase (β-1,4-D-mannan mannanohydrolase EC 3.2.1.78), which catalyzes the random cleavage ofβ-D-1,4-mannopyranosyl linkages within the main chain of galactomannan, glucomannan, galactoglucomannan and mannan , is a kind of hemicellulase. It can be produced mainly by various microorganisms.β-mannanase can be useful in several processes in the food, feed, textile, as well as in the pulp and paper industries. Despite having high practical potentialities, the use of mannanase is still limited due to low yields and high-production costs . The researches on theβ-mannanase from Aspergillus niger LW-1 were very important for the manufacture of functional konjak glucomannan oligosaccharides , which provid an important base for industrialized production and application.Multivariant statistical approaches containing Plackett-Burman design and response surface methodology were employed to evaluate the effects of several variables on solid state fermentation in flask with the strain Aspergillus niger LW-1. The optimum medium ingredients for acidicβ-mannanase production in flask were as follows: the proportion of wheat bran to soybean flour 7.5 :2.5 ,initial pH natural, the ratio of water to draw medium 1.5:1, 4% konjak powder, 0.3%KH2PO4, 5%steep liquor, 0.1% CaCl2, 0.1%MgSO4, 1% (NH4)2SO4(all related to dry medium). The cultivation was carried out at 32℃for 84h. With these conditions, the acidicβ-mannanase activity reached 24879 IU/g dry medium, being 3.27-fold as compared with control. It reaches the highest enzyme activity of all reports so far.Based on successful results of flask, acidicβ-mannanase scale-up production was carried out in koji plate(φ20 cm×4 cm).The optimal koji plate fermentation conditions were as follows: the ratio of water to dry medium 1.8:1, initial pH natural, inoculum size 10%(related to dry medium), dry medium content 150g/koji plate. With these conditions, the acidicβ-mannanase activity reached 22250 IU/g dry medium at 32℃for 72 h by adding water and turning koji.Two fractions A and B withβ-mannanase activity from solid-state fermentation culture were purified through sequential steps of buffer extraction, ammonium sulfate precipitation, Phenyl Sepharose CL-4B hydrophobic interaction chromatography (with stepwise gradient), Sephadex G-75 gel filtration , DEAE Sepharose Fast Flow ion-exchange chromatography and Phenyl Sepharose CL-4B hydrophobic interaction chromatography (with Linear gradient). Fraction A was similar to fraction B on molecular mass and hydrophobic property . After the steps the enzyme was purified by 38.6-fold with a recovery of 14.3%. The main fraction B was homogeneous as examined by SDS-PAGE and HPLC.The molecular mass of the purified enzyme was estimated to be 41 kD by Sephadex G-75 gel filtration and 40 kD by SDS-PAGE, which indicated that theβ-mannanase was a monomer. The isoelectric point was estimated to be 4.3 by IEF-PAGE. The enzyme was a glycoprotein with carbohydrate content of 21.7% by phenol-vitriol method. The composition of amino acids of the enzyme was analyzed. It showed that the 5 highest content of amino acids were Asp, Gly, Ser, Glu and Thr of all the amino acids. The content of acidic amino acid was higher than that of basic amino acid, indicating that the purified enzyme was an acidic protein.The enzymatic properties of the purifiedβ-mannanase were investigated. The optimal temperature for enzyme reaction was 70℃. The optimal pH was 3.5 at 50⁷0℃, which indicated that theβ-mannanase was an acidic enzyme. The enzyme was stable between pH5.0⁸.0 and below 60℃. The enzyme activity was stimulated by Zn²⁺,Ca²⁺,EDTA and partly inhibited by Mn²⁺,Fe²⁺,Pb²⁺,Sn²⁺,Fe³⁺,Cu²⁺,Al³⁺,Ag+. These properties were very similar to crude enzyme. The Michaelis constants(Km) of theβ-mannanase for locust bean gum and konjak gum were 5.1 mg/ml and 1.4 mg/ml, and maximum velocities(Vmax) for these saccharides were 1429μmol·min^-1·mg^-1 and 625μmol·min^-1·mg^-1, respectively. The result indicated that konjak gum was more suitable substrate than locust bean gum.The hydrolysate of locust bean gum galactomannan degraded by the purifiedβ-mannanase was analyzed by HPLC and HPLC-MS. Theβ-mannanase hydrolyzed locust bean gum galactomannan maimly to mannobiose and mannotriose. The content of mannobiose ,mannotriose and mannotetraose reached 49.75% of total hydrolysate. Above result was similar to many reports, indicating that the purifiedβ-mannanase was an endoenzyme.The hydrolysis conditions of konjak glucomannan were investigated with crude enzyme from Aspergillus niger LW-1. Under the conditions of konjak gum water solution 150 g/L,β-mannanase 50 IU/g konjak gum, hydrolytic temperature 50℃and hydrolytic time 6 h, the hydrolysate was mainly oligo-saccharides with only small traces of mono-saccharides analyzed by thin-layer chromatography and by HPLC. The content of oligo-glucomannan in hydrolysate was obviously increased through eliminating mono-saccharides with yeast fermentation.An unique enzymolysis process for konjak gum was obtained. Konjak gum concentration was obviously increased from 10³0 g/L to 150 g/L. This made it possible for industrialized production from konjak gum to mannooligosaccharide. So far no information is available on similar production process about konjak glucomannan.