International Journal of Biological Macromolecules 50 (2012) 432–437 Contents lists available at SciVerse ScienceDirect International Journal of Biological Macromolecules jo u rn al hom epa ge: www.elsevier.com/locate/ijbiomac Immobilization of -d-galactosidase from Kluyveromyces lactis on functionalized silicon dioxide nanoparticles: Characterization and lactose hydrolysis Madan Lal Verma a , Colin James Barrow a , J.F. Kennedy b , Munish Puri a,∗ a Centre for Biotechnology, Chemistry and System Biology (BioDeakin), Institute for Technology and Research Innovation (ITRI), Deakin University, Geelong 3217, Australia b Chembiotech Laboratories, Institute of Advanced Science and Technology, Kyrewood House Tenbury Wells, Worcestershire WR15 8SG, United Kingdom a r t i c l e i n f o Article history: Received 24 October 2011 Received in revised form 18 December 2011 Accepted 21 December 2011 Available online 2 January 2012 Keywords: Covalent binding FTIR SEM imaging Thermal stability Reusability Lactose hydrolysis a b s t r a c t -d-Galactosidase (BGAL) from Kluyveromyces lactis was covalently immobilized to functionalized sili- con dioxide nanoparticles (10–20 nm). The binding of the enzyme to the nanoparticles was confirmed by Fourier transform-infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Function- alized nanoparticles showed 87% immobilization yield. Soluble and immobilized enzyme preparation exhibited pH-optima at pH 6.5 and 7.0, respectively, with temperature optima at 35 and 40 ◦ C, respec- tively. Michaelis constant (K m ) was 4.77 and 8.4 mM for free and immobilized BGAL, respectively. V max for the soluble and immobilized enzyme was 12.25 and 13.51 U/ml, respectively. Nanoparticle immobilized BGAL demonstrated improved stability after favoring multipoint covalent attachment. Thermal stability of the immobilized enzyme was enhanced at 40, 50 and 65 ◦ C. Immobilized nanoparticle–enzyme con- jugate retained more than 50% enzyme activity up to the eleventh cycle. Maximum lactose hydrolysis by immobilized BGAL was achieved at 8 h. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Beta-d-galactosidase (-d-galactoside galactohydrolase, EC.3.2.1.23), most commonly known as lactase, is a hydrolytic enzyme that catalyses the breakdown of lactose into glucose and galactose [1,2]. The enzyme can be obtained from a variety of sources like microorganisms, plants, and animals. The use of beta-d-galactosidase as a therapeutic enzyme to hydrolyze lactose from milk products has become commonplace in alleviating the symptoms of lactose intolerance. Its medical properties are also utilized to treat infants with genetic deficiency of intestinal lactase, and it can also be used to prevent lactose crystallization in concentrated or frozen dairy products such as condensed milk and ice cream, thereby increasing consumer acceptance [3]. Beta-d-galactosidase has potential application in an array of fields such as the food industry, bioremediation, biosensory, diagnosis and treatment of various disorders [4]. In Australia, the dairy industry represents one of the most sig- nificant export industries, ranking third among rural food and value-added food industries. Each year, Australia’s dairy manu- facturers generate 3.3 million tonnes of whey, a by-product of ∗ Corresponding author at: BioDeakin (ITRI), Deakin University, Victoria, Australia. Tel.: +61 3 5227 2325; fax: +61 3 5227 2170. E-mail address: munish.puri@deakin.edu.au (M. Puri). the cheese industry, causing waste disposal and environmental problems [5,6]. About 9 L of whey stream is generated during the production of 1 kg of cheese, amounting to over 160 million tonnes of whey produced globally each year [7]. The disposal of whey remains a major problem for the dairy industry, where a relatively insignificant amount of whey is used for the production of protein concentrates or permeates, while a significantly larger fraction is disposed into water streams, resulting in severe water contamina- tion and in turn high biochemical and chemical oxygen demand (BOD/COD) and 5–6% dissolved solids [4]. Lactose use in nutritious products is also limited due to its low solubility, low sweetening ability and laxative properties [8]. The hydrolysis of lactose to glucose and galactose by BGAL is an important process in the food industry, due to the potentially beneficial effects on assimilating the foods containing lactose, as well as the technological and environmental advantages of indus- trial applications for what would otherwise be almost exclusively a waste product [9,10]. Treatment of cheese whey with BGAL increases its fermentability to ethanol, helping to reduce the pol- lution caused by large volumes of this by-product released into the environment [11]. While whey represents a challenge in terms of down-stream processing, it has recently been utilized for ethanol, exopolysaccharide and single cell protein production by employing BGAL [12]. Thus an enzyme assisted technology is required which may convert whey lactose into value added products and address bioremediation. 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.12.029