Effect of Divalent Metal Ions on Glucoamylase Activity of Glucoamylase Isolated from Aspergillus niger Okwuenu PC, Agbo KU, Ezugwu AL, Eze SO and Chilaka FC Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria. Corresponding author: Okwuenu PC, Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria, Tel: +2348065251877; E-mail: prosperokwuenu@yahoo.com Received date: Jan 06, 2017, Accepted date: Jan 28, 2017, Published date:Jan 31, 2017 Copyright: © 2017 Okwuenu PC, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract This study investigates the production of glucoamylase from Aspergillus niger in a submerged fermentation process using amylopectin fractionated from guinea corn starch as the carbon source. This work also studies the effect of a few metal ions (Ca 2+ , Zn 2+ , Co 2+ , Fe 2+ , Mn 2+ and Pb 2+ ) concentration on the glucoamylase activity. A Fourteen day experimental study was carried out to determine the day of highest glucoamylase activity. Maximum glucoamylase activity was observed on day five of the submerged fermentation; hence, day five was mass produced. The specific activity for the crude enzyme was found to be 729.45 U/mg. The crude enzyme was purified to the level of gel filtration (using sephadex G-100) via ammonium sulphate precipitation. Ammonium sulphate saturation of 70% was found suitable to precipitate the enzyme. After ammonium sulphate precipitation and gel filtration, the specific activities were found to be 65.98 U/mg and 180.52 U/mg respectively. The glucoamylase activity was enhanced by 2 mM to 5 mM of Ca 2+ , Co 2+ , Fe 2+ , Mn 2+ and Zn 2+ but Pb 2+ had inhibitory effect on the enzyme. The Michaelis constant, Km and maximum velocity Vmax of the enzyme was obtained from the Lineweaver-Burk plot of initial velocity data at different substrate concentrations. They were found to be 770.75 mg/ml and 2500 µmol/min respectively, when using cassava starch as substrate. The enzyme glucoamylase is known to have useful applications in food processing industries and fermentation biotechnology. Keywords Glucoamylase; Enzyme; Aspergillus; Metal ions; Activity Introduction Glucoamylase (exo-1, 4-α-D-glucan-glucanohydrolase, EC 3.2.1.3) belongs to the most important catalytically active proteins having broad possibilities of technical use. It is an exo acting enzyme that yields ß-D-glucose from the non-reducing chain ends of amylose and amylopectin by hydrolyzing α-1, 4 linkages in a stepwise manner [1]. It also hydrolyses α-1, 6 and the rare α-1, 3 linkages although at much slower rate [2]. Generally, amylases, (that is α- amylases, β-amylases and glucoamylase) can be produced either by submerged fermentation (SmF) or solid state fermentation (SSF) procedures; however, the convectional amylase production is carried out by submerged fermentation [3] Glucoamylase production from microbial sources especially from Aspergillus niger is generally extracellular, and the enzyme can be recovered from culture fltrates. However, the extensive utilization of glucoamylase is obtained by using the fungus Aspergillus niger in enzyme production industries. Enzymes may require metal ions for their maximal catalytic activity and are termed as holoenzymes. Metals are responsible for right orientation of active site of holoenzymes. Metal binding to enzyme is one of the factors responsible for protein stabilization [4,5]. As a result of the important role of glucoamylase, it is highly desirable to increase or enhance the glucoamylase activity in other to improve its useful applications and utilization. Materials and methods Chemicals Bovine serum albumin (BSA), Sephadex G-100 was purchase from Sigma Chemical Company Limited (USA). Folin–Ciocalteau was purchased from Sigma-Aldrich (USA). Ammonium Sulphate and Tris HCL salt were purchased from British Drug House (BDH) Chemicals Limited (USA). All other chemicals used in this work were of analytical grade and were obtained from reputable sources. Collection of plant material and processing of the starch Plant material (guinea corn seed and cassava tuber) was purchased from the Ogige market in Nsukka, Enugu state, Nigeria, and its starch was processed as described by Agboola [6]. Fractionation of starch into amylopectin Te starch obtained from guinea corn was fractionated according to the method of Sobukola and Aboderin [7]. Isolation and identifcation of the glucoamylase producing fungi Glucoamylase producing fungi was isolated and stored according to the method described by Martin et al. [8] and the identifcation was carried out using the method of Barnett and Hunter [9]. Okwuenu et al., Ferment Technol 2017, 6:1 DOI: 10.4172/2167-7972.1000141 Research Article Open Access Ferment Technol, an open access journal ISSN:2167-7972 Volume 6 • Issue 1 • 1000141 F e r m e nt a ti o n T e c h n o l o g y ISSN: 2167-7972 Fermentation Technology