Date Published: June 18, 2016
Publisher: Springer Berlin Heidelberg
Author(s): Hemant Soni, Hemant Kumar Rawat, Brett I. Pletschke, Naveen Kango.
Aspergillus terreus FBCC 1369 was grown in solid-state culture under statistically optimized conditions. β-Mannanase was purified to apparent homogeneity by ultrafiltration, anion exchange and gel filtration chromatography. A purification factor of 10.3-fold was achieved, with the purified enzyme exhibiting specific activity of 53 U/mg protein. The purified β-mannanase was optimally active at pH 7.0 and 70 °C and displayed stability over a broad pH range of 4.0–8.0 and a 30 min half-life at 80 °C. The molecular weight of β-mannanase was calculated as ~49 kDa by SDS-PAGE. The enzyme exhibited Km and Vmax values of 5.9 mg/ml and 39.42 µmol/ml/min, respectively. β-Mannanase activity was stimulated by β-mercaptoethanol and strongly inhibited by Hg2+. The β-Mannanase did not hydrolyze mannobiose and mannotriose, but only mannotetraose liberating mannose and mannotriose. This indicated that at least four mannose residues were required for catalytic activity. Oligosaccharide with a degree of polymerization (DP) three was the predominant product in the case of locust bean gum (16.5 %) and guar gum (15.8 %) hydrolysis. However, the enzyme liberated DP4 oligosaccharide (24 %) exclusively from konjac gum. This property can be exploited in oligosaccharides production with DP 3–4. β-Mannanase hydrolyzed pretreated lignocelluloses and liberated reducing sugars (% theoretical yield) from copra meal (30 %). This property is an important factor for the bioconversion of the biomass.
In plant cell walls, hemicelluloses are the second most abundant carbohydrates after cellulose. Mannans are the second largest group of hemicelluloses after xylan, which appear predominantly in softwoods of gymnosperms and also form a minor component of hardwoods (Puls and Schuseil 1993). These are composed of β-linked mannose sugar-based backbones with variable degrees of side substitutions. These polysaccharides are renewable resources and their enzymatic conversion is of great interest in the field of lignocellulose biotechnology (Soni and Kango 2013). For the majority of bioconversion processes, mannans must be first converted to mannose or manno-oligosaccharides (MOS).
Thermotolerant fungus A. terreus FBCC 1369 produced 59 U/gds titer of β-mannanase under non-optimized conditions in solid-state culture on copra meal. Optimization of various parameters, viz. the particle size of substrate and carbon and nitrogen supplementation, was carried out. The smallest particle size of 0.5 mm supported the maximum β-mannanase production and is similar to the findings of a particle size of 0.6 mm being suitable for xylanase production by Sporotrichum thermophile (Sadaf and Khare 2014). Among the carbon supplements examined, pulverized cellulose (solka floc) supported higher yields of β-mannanase. Similar levels of induction were also observed in Myceliophthora fergusii MTCC 9293 (Maijala et al. 2012). Glucose and mannose supplementation clearly repressed β-mannanase production. Supplementation of complex galactomannans like guar gum and LBG also lowered the enzyme yield significantly.
The high β-mannanase yield on low-value copra meal, exclusive generation of DP 4 oligosaccharide from konjac gum, formation of partially hydrolyzed guar gum (PHGG) and a 30 min half-life at 80 °C make A. terreus β-mannanase an attractive enzyme for the nutraceutical, food and paper industries. Copra meal is rich in indigestible mannan, cannot be used directly as animal feed (poultry and pigs) and its disposal causes pollution. In the present study it is utilized as substrate for the production of mannanase. The residues left after SSF have reduced galactomannan content and can be used as feed for monogastric animals. This study provides a suitable valorization solution for the utilization and management of copra-oil industry waste which causes pollution.