Production, purification and characterization of a thermostable ????-Mannanase from Aspergillus niger AD-01: potential application in aquaculture

The potential for microbial β-mannanase enzyme as a feed additive has attracted substantial interest from feed manufacturers as a means to improve animal performance. This necessitates production of β-mannanase from novel strains with desired characteristics for feed applications. A fungal strain isolated from garden soil was found to be capable of producing extracellular β-mannanase enzyme. The strain was identified as Aspergillus niger and nucleotide sequence has been submitted in NCBI database under accession number MN239884.Optimum enzyme production in terms of specific activity 12.49 U/mg of total protein was obtained at 30°C, pH=5.0, using 2% locust bean gum as carbon source and yeast extract as nitrogen source, after 5 days of incubation.Fungal crude enzyme was purified by 6 fold with 24% yield and specific activity of 78.07U/mg by two step purification i.e. ammonium sulfate precipitation and gel-filtration chromatography. Thermo stability studies revealed retention of 50% β-mannanase activity upto 40°C.Moreover enzyme remained stable the pH range of 4-8.β-mannanase activity remained stable in the presence of 7 different metals, however activity declined sharply in the presence of Hg2+ and Ba2+. Inhibition by Hg2+ suggests that enzyme contains an essential sulfhydryl group. No loss of enzyme activity was observed after incubating the enzyme with 1 M or 2 M NaCl, proteinase K or trypsin at 37°C and pH 6.0 for 1 h. The weakly acidic, low temperature active, protease resistant profile of this particular type of β-mannanase enzyme makes it a promising candidate for use as a feed additive for agastric fish in aquaculture industry.