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

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Research Paper 01/09/2019
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Production, purification and characterization of a thermostable 𝛽-Mannanase from Aspergillus niger AD-01: potential application in aquaculture

Aneesa Dawood, Amina Zuberi, Kesen Ma, Muhammad Imran, Maliha Ahmed
Int. J. Biosci.15( 3), 314-327, September 2019.
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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.


Ademark P, Varga A, Medve J, Harjunpää V, Drakenberg T, Tjerneld F, Stålbrand H. 1998. Softwood hemicellulose-degrading enzymes from Aspergillus niger: purification and properties of a β-mannanase. Journal of Biotechnology 63, 199-210.

Arisan-Atac I, Hodits R, Kristufek D, Kubicek, CP. 1993. Purification, and characterization of a β-mannanase of Trichoderma reesei C-30. Applied Microbiology and Biotechnology 39, 58-62.

Burke R, Cairney J. 1997. Carbohydrolase production by the ericoid mycorrhizal fungus Hymenoscyphus ericae under solid-state fermentation conditions. Mycological Research 101, 1135-1139.

Cai H, Shi P, Luo H, Bai Y, Huang H, Yang P, Yao B. 2011. Acidic β-mannanase from Penicillium pinophilum C1: cloning, characterization and assessment of its potential for animal feed application. Journal of bioscience and bioengineering 112, 551-557.

Chantorn ST, Buengsrisawat K, Pokaseam A, Sombat T, Dangpram P, Jantawon K, Nitisinprasert S. 2013. Optimization of extracellular mannanase production from Penicillium oxalicum KUB-SN2-1 and application for hydrolysis property. Journal of Science and Technology 35, 17-22.

Chen X, Cao Y, Ding Y, Lu W, Li D. 2007. Cloning, functional expression and characterization of Aspergillus sulphureus β-mannanase in Pichia pastoris. Journal of biotechnology 128, 452-461.

Cheng L, Duan S, Feng X, Zheng K, Yang Q, Liu Z. 2016. Purification and characterization of a thermostable β-mannanase from Bacillus subtilis BE-91: potential application in inflammatory diseases. BioMed research international 2016.

Christgau S, Kauppinen S, Vind J, Kofod LV, Dalbøge H. 1994. Expression cloning, purification and characterization of a beta-1, 4-mannanase from Aspergillus aculeatus. Biochemistry and molecular biology international 33, 917-925.

Civas A, Eberhard R, Le Dizet P, Petek F. 1984. Glycosidases induced in Aspergillus tamarii. Secreted α-d-galactosidase and β-d-mannanase. Biochemical Journal 219, 857-863.

de Vries RP, Visser J. 2001. Aspergillus enzymes involved in degradation of plant cell wall polysaccharides. Microbiology Molecular Biology Review 65, 497-522.

Haltrich D, Nidetzky B, Kulbe KD, Steiner W, Župančič S. 1996. Production of fungal xylanases. Bioresource Technology 58, 137-161.

Huang S, Wang C, Zhang G, Ma L. 2007. Construction of a double functional recombinant strain of Pichia pastoris co-expressing phytase and mannanase and the enzymatic analyses. Wei sheng wu xue bao= Acta microbiologica Sinica 47, 280-284.

Jackson M, Fodge D, Hsiao H. 1999. Effects of beta-mannanase in corn-soybean meal diets on laying hen performance. Poultry Science 78, 1737-1741.

Kato K, Matsuda K. 1969. Studies on the chemical structure of konjac mannan: part I. Isolation and characterization of oligosaccharides from the partial acid hydrolyzate of the mannan. Agricultural and Biological Chemistry 33, 1446-1453.

Kote NV, Patil AGG, Mulimani V. 2009. Optimization of the production of thermostable endo-β-1, 4 mannanases from a newly isolated Aspergillus niger gr and Aspergillus flavus gr. Applied biochemistry and biotechnology 152, 213-223.

Laemmli U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature 227, 680.

Lawrence AA. 1973. Edible gums and related substances.

Luo H, Wang Y, Wang H, Yang J, Yang Y, Huang H, Yang P, Bai Y, Shi P, Fan Y. 2009. A novel highly acidic β-mannanase from the acidophilic fungus Bispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris. Applied microbiology and biotechnology 82, 453-461.

McCutchen CM, Duffaud GD, Leduc P, Petersen AR, Tayal A, Khan SA, Kelly RM. 1996. Characterization of extremely thermostable enzymatic breakers (α‐1, 6‐galactosidase and β‐1, 4‐mannanase) from the hyperthermophilic bacterium Thermotoga neapolitana 5068 for hydrolysis of guar gum. Biotechnology and Bioengineering 52, 332-339.

Mohamad SN, Ramanan RN, Mohamad R, Ariff AB. 2011. Improved mannan-degrading enzymes’ production by Aspergillus niger through medium optimization. New biotechnology 28, 146-152.

Mok C, Lee J, Kim B. 2013. Effects of exogenous phytase and β-mannanase on ileal and total tract digestibility of energy and nutrient in palm kernel expeller-containing diets fed to growing pigs. Animal feed science and technology 186, 209-213.

Naganagouda K, Salimath P, Mulimani V. 2009. Purification and characterization of endo-β-1, 4 mannanase from Aspergillus niger gr for application in food processing industry. J Microbiol Biotechnol 19, 1184-1190.

Odetallah N, Ferket P, Grimes J, McNaughton, J. 2002. Effect of mannan-endo-1, 4-beta-mannosidase on the growth performance of turkeys fed diets containing 44 and 48% crude protein soybean meal. Poultry science 81, 1322-1331.

Pettey L, Carter S, Senne B, Shriver J. 2002. Effects of beta-mannanase addition to corn-soybean meal diets on growth performance, carcass traits, and nutrient digestibility of weanling and growing-finishing pigs. Journal of animal science 80, 1012-1019.

Puchart V, Vršanská M, Svoboda P, Pohl J, Ögel ZB, Biely P. 2004. Purification and characterization of two forms of endo-β-1, 4-mannanase from a thermotolerant fungus, Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Biochimica et Biophysica Acta (BBA)-General Subjects 1674, 239-250.

Rashid JIA, Samat N, Yusoff WMW. 2011. Optimization of temperature, moisture content and inoculum size in solid state fermentation to enhance mannanase production by Aspergillus terreus SUK-1 using RSM. Pakistan Journal of Biologycal Science 14, 533-539.

Reese ET, Shibata Y. 1965. β-Mannanases of fungi. Canadian journal of microbiology 11, 167-183.

Regalado C, GarcíaAlmendárez BE, VenegasBarrera LM, TéllezJurado A, RodríguezSerrano G, HuertaOchoa S, Whitaker JR. 2000. Production, partial purification and properties of β‐mannanases obtained by solid substrate fermentation of spent soluble coffee wastes and copra paste using Aspergillus oryzae and Aspergillus niger. Journal of the Science of Food and Agriculture 80, 1343-1350.

Setati ME, Ademark P, van Zyl WH, Hahn-Hägerdal B, Stålbrand H. 2001. Expression of the Aspergillus aculeatus endo-β-1, 4-mannanase encoding gene (man1) in Saccharomyces cerevisiae and characterization of the recombinant enzyme. Protein expression and purification 21, 105-114.

Soni H, Rawat HK, Pletschke BI, Kango N. 2016. Purification and characterization of β-mannanase from Aspergillus terreus and its applicability in depolymerization of mannans and saccharification of lignocellulosic biomass. 3 Biotech 6, 136.

Sornlake W, Matetaviparee P, Rattanaphan N, Tanapongpipat S, Eurwilaichitr L. 2013. β‐Mannanase production by Aspergillus niger BCC4525 and its efficacy on broiler performance. Journal of the Science of Food and Agriculture 93, 3345-3351.

Wainø M, Ingvorsen K. 1999. Production of halostable β-mannanase and β-mannosidase by strain NN, a new extremely halotolerant bacterium. Applied microbiology and biotechnology 52, 675-680.

Wu G, Bryant M, Voitle R, Roland Sr D. 2005. Effects of β-mannanase in corn-soy diets on commercial leghorns in second-cycle hens. Poultry Science 84, 894-897.

Yamka R, Hetzler B, Harmon D. 2005. Evaluation of low-oligosaccharide, low-phytate whole soybeans and soybean meal in canine foods. Journal of animal science 83, 393-399.

Yiğit NO, Koca SB, Didinen BI, Diler I. 2014. Effect of β-mannanase and α-Galactosidase supplementation to soybean meal based diets on growth, feed efficiency and nutrient digestibility of rainbow trout, Oncorhynchus mykiss (Walbaum). Asian-Australasian journal of animal sciences 27, 700.

Youssef AS, El-Naggar M, El Assar S, Beltagy EA. 2006. Optimization of cultural conditions for β-mannanase production by a local Aspergillus niger isolate. International Journal of Agriculture and Biology 8, 539-545.

Zhao J, Shi P, Luo H, Yang P, Zhao H, Bai Y, Huang H, Wang H, Yao B. 2010. An acidophilic and acid-stable β-mannanase from Phialophora sp. P13 with high mannan hydrolysis activity under simulated gastric conditions. Journal of agricultural and food chemistry 58, 3184-3190.

Zou X, Qiao X, Xu Z. 2006. Effect of β-mannanase (Hemicell) on growth performance and immunity of broilers. Poultry science 85, 2176-2179.