1098 Protein & Peptide Letters, 2009, 16, 1098-1105 0929-8665/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd. Invertase from Hyper Producer Strain of Aspergillus niger: Physiochemi- cal Properties, Thermodynamics and Active Site Residues Heat of Ioniza- tion Habibullah Nadeem, Muhammad Hamid Rashid * , Muhammad Riaz, Bibi Asma, Muhammad Rizwan Javed and Raheela Perveen Enzyme Engineering Lab., National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan Abstract: Here we report for the first time heat of ionization of invertase (E.C.3.2.1.26) active site residues from hyper- producer strain of Aspergillus niger (34.1 U ml -1 ), along with its physiochemical properties, kinetics and thermodynamics of stability-function. The Invertase showed great potential for industry as being highly efficient (k cat = 24167 s -1 at 65 °C, pH 5.0) and stable (half life= 12 h at 56°C). Keywords: Thermostability, Enthalpy, Entropy, Free energy, Ionization energy. INTRODUCTION Invertase ( -fructofuranosidase: E.C.3.2.1.26) catalyse the hydrolysis of sucrose into glucose and fructose. It is one of the most widely used enzymes in the food industry, espe- cially in the preparation of jams and candies [1], where fruc- tose is preferred over sucrose because it is sweeter and does not crystallize easily. Using waste biomass to produce biofuel (ethanol) can reduce the use of fossil fuels, reduce greenhouse gas emis- sions and reduce pollution and waste management problems [2]. Biologically alcohols are produced by the action of mi- crobes and enzymes through the fermentation of sugars. Ethanol can be used in petrol engines as a replacement for gasoline; it can be mixed with gasoline to any percentage. Gasoline with ethanol added has higher octane, hence the engine can typically burn hotter and more efficiently. Molas- ses, a sub-product in sugar production is rich in sucrose and has been used as a carbon source in the production of - fructofuranosidase by Saccharomyces cerevisiae [3]. Inver- tases have high potential to be used in biofuel production because they have a tendency to enhance the glucose content in molasses, which is considered to be an excellent ethanol feedstock. Invertase has been mainly produced from Saccharomyces cerevisiae and commercial fungal strains of the Aspergillus genus, such as A. niger, A. oryzae and A. ficcum [4]. Asper- gillus niger is a filamentous, non-pathogenic fungus that belongs to the “Generally Recognized As Safe (GRAS)” microbial organisms, which are used for the production of enzymes [5]. Although there are many reports on isolation and characterization of invertases from various microbes but on an average less than 1% of the potential microbes have *Address correspondence to this author at the Principal Scientist, National Institute for Biotechnology and Genetic Engineering, P.O. Box 577, Jhang Road, Faisalabad, Pakistan; Fax: +92-041-2651472; E-mails: hamidcomboh@gmail.com; mhrashid@nibge.org been identified [6]. So the need to isolate and identify organ- isms, which are either hyper-producers and/or sufficiently robust to withstand conditions of the intended application and/or are producers of novel enzymes is highly significant. This current report deals with the purification, kinetic and thermodynamic properties of a highly efficient and stable - fructofuranosidase from a local, hyper-producing strain of A. niger. We considered that this biocatalyst has great potential to be used in the food industry and in biofuel production. MATERIALS AND METHODS Microorganism A pure local strain of Aspergillus niger was obtained from NIBGE, Faisalabad. The culture was maintained on potato dextrose agar (PDA) plates and slants as described earlier [7]. All chemicals were purchased from MP Biomedi- cals, USA. Inoculum Preparation & Biomass Estimation For inoculum preparation a loop full of spores from a 5 to 6 days old A. niger slant was transferred into a 250 ml Er- lenmeyer flask containing 50 ml of sterile Vogel’s medium and 8-10 acid washed glass beads were added. The composi- tion of the growth medium was (gl -1 ): glucose (20.0), triso- dium citrate (2.5), KH 2 PO 4 (5.0), NH 4 NO 3 (2.0), (NH 4 ) 2 SO 4 (4.0), MgSO 4 .7H 2 O (0.2), peptone (2.0), microelement solu- tion (10 ml) and vitamin solution (5.0 ml). The pH of the medium was adjusted to 5.0 using 1M HCl/1M NaOH. The flask was incubated at 30 °C on an orbital shaker at 150 rpm for 48h to get a homogenous cell suspension. The biomass content of the inoculum was estimated on the basis of absorptiometry (cells turbidity) by measurement of the light transmitted at 610 nm [8]. The standard curve for biomass estimation was prepared as follows: 50 ml culture was harvested and centrifuged at 15,300 g at 4 °C for 15