Original Research Paper Production of an extracellular phytase from a thermophilic mould Humicola nigrescens in solid state fermentation and its application in dephytinization Anju Bala, Sapna, Jinender Jain, Amit Kumari, Bijender Singh n Laboratory of Bioprocess Technology, Department of Microbiology, Maharshi Dayanand University, Rohtak 124001, Haryana, India article info Article history: Received 10 May 2014 Received in revised form 18 June 2014 Accepted 9 July 2014 Keywords: Phytic acid Phytase Solid state fermentation Humicola nigrescens Flour abstract In view of increasing demand of phytase, it is essential to produce and characterise cost-effective phytase from a new microbial source. Solid state fermentation (SSF) is the promising technology for commercial enzyme production with lower production cost. Among six isolates of thermophilic moulds, Humicola nigrescens was selected as a potent phytase producer on the basis of zone of hydrolysis. The mould produced phytase (48.28 U/g DMB) optimally in SSF using wheat bran at 45 1C, pH 5.0 after 2 days with substrate to moisture ratio of 1:2.5. Supplementation of wheat bran with sucrose and ammonium sulphate further enhanced phytase production. Phytase of H. nigrescens was partially purified 3.3-fold using ammonium sulphate precipitation. The partially purified enzyme was optimally active at pH 5.0 and 50 1C. The partially purified enzyme resulted in dephytinization of wheat and gram flour at 50 1C with the concomitant liberation of inorganic phosphate. Based on its biochemical properties and ability to dephytinize flours, phytase of H. nigrescens could be a potential candidate for food and feed industries. & 2014 Elsevier Ltd. All rights reserved. 1. Introduction Phytase (myo-inositol hexakisphosphate 3-phosphorylase, EC 3.1.3.8) is used as important feed additive to increase the avail- ability of organic phosphate and other nutritionally important minerals for monogastrics by the hydrolysis of phytic acid (myo-inositol hexakisdihydrogen phosphate), an anti-nutritional factor present in most of the cereal and legume-based diets (Vohra and Satyanarayana, 2003; Vats and Banerjee, 2004; Singh and Satyanarayana, 2006). Since phytin is not metabolised in the intestinal tract of monogastric animals that consume cereals and legumes, much of the phosphate passes through the animal excreta which leads to pollution in aquatic bodies due to eutro- phication (Singh and Satyanarayana, 2006, 2011). The commer- cially available phytase is produced by Aspergillus ficuum and A. niger strains by submerged fermentation (SmF). The cost of commercial phytase supplementation is about $2–$3/metric ton of feed (Bogar et al., 2003a,b). A more economical alternative method for the production of this enzyme would be solid-state fermentation (SSF) (Pandey et al., 1999, 2001). Filamentous fungi have been employed for the production of phytases in SSF (Pandey et al., 1999, 2001). Attention is being focused on the utilisation of agricultural crops and their residues for phytase production in SSF (Sabu et al., 2002). SSF has been employed for phytase production by filamentous fungi such as Aspergillus ficuum (Bogar et al., 2003a), Mucor racemosus (Bogar et al., 2003b) Thermo- ascus aurantiacus (Nampoothiri et al., 2004), A. oryzae (Sapna and Singh 2014), Rhizomucor pusillus (Chadha et al., 2004) and Sporo- trichum thermophile (Singh and Satyanarayana, 2006, 2008). Phytase may be produced directly using SSF by filamentous fungi on the selected animal feeds, and the crude product may be mixed in feed rations as a value-added supplement (Bogar et al., 2003a,b). The fungal product contains not only phytase, but also accessory enzymes, fungal proteins, and organic acids that increase feed digestibility and access to phytate in plant cells (Pandey et al., 1999, 2001). Recently research work on thermophilic fungi has been encour- aged because of the secretion of novel enzymes with high temperature optima, stability in acidic pH and a long shelf-life, which are desirable characteristics of an ideal phytase employed as additive/supplement in the diets of monogastric animals (Singh et al., 2011; Singh and Satyanarayana, 2011). In the present investi- gation, phytase production by Humicola nigrescens BJ82 was optimised in SSF followed by its partial purification and character- isation. The applicability of the enzyme was also studied in the dephytinization of commonly used food ingredients. To the best of our knowledge, this is the first report on phytase production by a thermophilic mould H. nigrescens. Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/bab Biocatalysis and Agricultural Biotechnology http://dx.doi.org/10.1016/j.bcab.2014.07.002 1878-8181/& 2014 Elsevier Ltd. All rights reserved. n Corresponding author. Tel.: þ91 1262 393587; fax: þ91 1262 274640. E-mail address: ohlanbs@gmail.com (B. Singh). Please cite this article as: Bala, A., et al., Production of an extracellular phytase from a thermophilic mould Humicola nigrescens in solid state fermentation and its application in dephytinization. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.07.002i Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎