Isolation, puriwcation and characterization of a novel glucose oxidase from penicillium sp. cbs 120262 optimally active at neutral ph

Protein Expression and PuriWcation 51 (2007) 260–266 www.elsevier.com/locate/yprep

Isolation, puriWcation and characterization of a novel glucose oxidase from Penicillium sp. CBS 120262 optimally active at neutral pH
C. Simpson a,¤, J. Jordaan a, N.S. Gardiner a, C. Whiteley b
b

CSIR Biosciences, Modderfontein, Johannesburg 1645, South Africa Department of Biochemistry, Microbiology andBiotechnology, Rhodes University, PO Box 94, Grahamstown 6140, South Africa Received 13 June 2006, and in revised form 11 September 2006 Available online 1 October 2006

a

Abstract A novel glucose oxidase (GOX), a Xavoenzyme, from Penicillium sp. was isolated, puriWed and partially characterised. Maximum activities of 1.08 U mg¡1dry weight intracellular and 6.9 U ml¡1 extracellular GOX wereobtained. Isoelectric focussing revealed two isoenzymes present in both intra- and extracellular fractions, having pI’s of 4.30 and 4.67. GOX from Penicillium sp. was shown to be dimeric with a molecular weight of 148 kDa, consisting of two equal subunits with molecular weight of 70 kDa. The enzyme displayed a temperature optimum between 25 and 30 °C, and an optimum pH range of 6–8 for the oxidationof -D-glucose. The enzyme was stable at 25 °C for a minimum of 10 h, with a half-life of approximately 30 min at 37 °C without any prior stabilisation. The lyophilized enzyme was stable at ¡20 °C for a minimum of 6 months. GOX from Penicillium sp. Tt42 displayed the following kinetic characteristics: Vmax, 240.5 U mg¡1; Km, 18.4 mM; kcat, 741 s¡1 and kcat/Km, 40 s¡1 mM¡1. Stability at roomtemperature, good shelf-life without stabilisation and the neutral range for the pH optimum of this GOX contribute to its usefulness in current GOX-based biosensor applications. © 2006 Elsevier Inc. All rights reserved.
Keywords: Glucose oxidase; Penicillium; PuriWcation; Characterisation

Glucose oxidase (GOX1— -D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) has been puriWed from a range of diVerentfungal sources, primarily from the genera Aspergillus [1–3] and Penicillium [4–8]. GOX from Penicillium sp. are generally more advantageous than those that have been isolated from Aspergillus sp. since they have enhanced kinetic parameters for glucose oxidation [4,9]. GOX is a Xavoprotein which catalyses the oxidation of -D-glucose to D-glucono- -lactone and hydrogen peroxide, using molecularoxygen as the electron acceptor [3,10–12]. The reaction can be divided into a reductive and an oxidative step (Scheme 1). In the reductive-half reaction, GOX catalyses the oxidation of -D-gluCorresponding author. Fax: +2711 608 3020. E-mail address: csimpson2@csir.co.za (C. Simpson). 1 Abbreviations used: GOX, glucose oxidase; MEA, malt extract agar; CAT, catalase. 1046-5928/$ - see front matter ©2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2006.09.013
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cose to D-glucono- -lactone, which can in certain fungi such as Aspergillus sp. [13], be enzymatically (lactonase; EC 3.1.1.17) or spontaneously hydrolyzed to gluconic acid. Subsequently the Xavine adenine dinucleotide (FAD) ring of GOX is reduced to FADH2 [11]. In the oxidative half reaction the reduced GOX is re-oxidisedby oxygen to yield hydrogen peroxide. GOX has enjoyed large-scale technological application since the 1950’s [14], which includes the enzymatic determination of glucose with biosensor technology [15,16], for the production of gluconic acid and as a food preservative [10]. Implantable glucose sensors may Wnd signiWcant application for the monitoring of glucose in diabetics [17]. Enhancement of theproperties of GOX is still receiving attention [8], presumably due to the current and extensive applications base of this enzyme. This article describes the production, puriWcation and characterisation of a novel GOX from a Penicillium species.

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Scheme 1. Representation of the GOX reaction (adapted from [11])....