endo-1,4 β-Mannanase

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Introduction

β-mannanase (endo-1,4-β-mannan mannanohydrolase, EC 3.2.1.78) is responsible for the random hydrolysis of β-1,4 mannosidic linkages in mannan, glucomannan, galactomannan, galactomannan and galactoglucomannan. In daily life, Amophophallus konjac is a good source of glucomannan, and is a tuber vegetable widely consumed in some tropical countries such as Japan, Bangladesh and India. The characteristic of β-mannanase is to effectively utilize β-mannan from various plants as bioresources to produce valuable oligosaccharides. As the hydrolytic products, the oligosaccharides play a very important role in the growth of intestinal microorganisms, especially Bifidobacteria, which show beneficial effects on intestinal mucosal cells and other intestinal microflora, thereby increasing the fecal mass. Mannanase can also be used in industrial production processes, such as extracting vegetable oil from leguminous seeds, and helping the manufacturing process of instant coffee, chocolate, and cocoa liquor to reduce the viscosity of the extract. Mannanase can also prebleach of softwood pulp with the synergistic help of xylanase. Despite its high practical potential, the use of mannanase is still limited due to its low yield and high production cost.

Recently, more attention has been focused on the extraction and utilization of β-mannanase from various sources of plants, bacteria, fungi, actinomycetes, and molluscs. Comparative studies of β-mannanase isolated from bacteria showed that the quantitative production and hydrolysis efficiency of the enzyme differed from species to species, even among different strains of the same species. When comparing between species, when a large amount of enzyme is required, bacterial sources enzymes are preferred.  

Properties of the enzyme

The molecular masses of the enzyme purified from Bacillus subtilis KU-1 strain determined by gel filtration and SDS-PAGE were 40kDa and 39kDa, respectively. The enzyme shows the optimal activity at pH 7.0 and at 50-55°C, and is stable at 60°C for 1 hour. In addition, the enzyme still retains more than 80% of its maximum activity after being stored in NaAc buffer at room temperature (approximately 25°C) and pH 6.5 for 2 weeks. The enzyme had an isoelectric point of 4.5. According to the Lineweaver-Burk plot, the calculated Michaelis constant (K_m) and maximum reaction velocity (V_max) of the enzyme are 0.91% and 2935 U mg^-1, respectively. When the enzyme was incubated with a metal compound at a concentration of 1 mM for 24 hours, Hg²⁺, Ag²⁺, Cu²⁺, Mn²⁺ and Cr²⁺ showed a strong inhibitory effect on the enzyme activity.

After the enzyme is completely acid-hydrolyzed, the amino acid composition is determined by amino acid analyzer. The measured amino acid composition (% mol) of the enzyme is calculated as: Asx, 15.1; Glx, 11.4; Gly, 5.6; Ala, 6.7; Val, 2.2; 1/2Cys, 0.3; Met, 1.7; Ile, 4.2; Leu, 9.3; Phe, 5.1; Lys, 5.3; His, 2.7; Arg, 3.4; Pro, 6.3 and from the mean contents after 24, 48 and 72 h hydrolysis.

The enzyme hydrolyzes Konjac glucomannan, β-mannan, larchwood xylan, locust bean gum, guar gum, cellulose, and inulin with relative reactivity of 100, 106, 66, 58, 54, 13, and 27%, respectively. But it has no hydrolytic activity on other substrates such as oat, birchwood xylan, carrageenan, cyclodextrins (α, β, γ), pectin, dextran, yeast glucan, laminaran, and lichenan. etc. For all oligomeric products, mannose is found at the reducing end.