High Yield Production of a Soluble Bifidobacterial β‑Galactosidase (BbgIV) in E. coli DH5α with Improved Catalytic Efficiency for the Synthesis of Prebiotic Galactooligosaccharides Ali Osman,* ,† George Tzortzis, ‡ Robert A. Rastall, † and Dimitris Charalampopoulos † † Department of Food and Nutritional Sciences, The University of Reading, PO Box 226, Whiteknights, Reading, Berkshire, RG6 6AP, United Kingdom ‡ Clasado Ltd., 5 Canon Harnett Court, Wolverton Mill, Milton Keynes, MK12 5NF, United Kingdom ABSTRACT: The bifidobacterial β-galactosidase (BbgIV) was produced in E. coli DH5α at 37 and 30 °C in a 5 L bioreactor under varied conditions of dissolved oxygen (dO2) and pH. The yield of soluble BbgIV was significantly (P < 0.05) increased once the dO2 dropped to 0-2% and remained at such low values during the exponential phase. Limited dO2 significantly (P < 0.05) increased the plasmid copy number and decreased the cells growth rate. Consequently, the BbgIV yield increased to its maximum (71-75 mg per g dry cell weight), which represented 20-25% of the total soluble proteins in the cells. In addition, the specific activity and catalytic efficiency of BbgIV were significantly (P < 0.05) enhanced under limited dO2 conditions. This was concomitant with a change in the enzyme secondary structure, suggesting a link between the enzyme structure and function. The knowledge generated from this work is very important for producing BbgIV as a biocatalyst for the development of a cost- effective process for the synthesis of prebiotic galactooligosaccharides from lactose. KEYWORDS: β-galactosidase, Bif idobacterium, protein expression, dissolved oxygen, prebiotic galactooligosaccharides, specific activity ■ INTRODUCTION β-Galactosidases (EC 3.2.1.23) are ubiquitous enzymes in nature. According to the International Union of Biochemistry and Molecular Biology (IUBMB), β-galactosidases catalyze the hydrolysis of terminal nonreducing β-D-galactose residues in β- D-galactosides. One of the preferred substrates for this enzyme is the disaccharide lactose. The application of β-galactosidase in the hydrolysis of lactose into its monosaccharide components, glucose and galactose, is a well-established industrial process which increases the sweetness of food products containing lactose and helps making dairy nutrients available to lactose intolerant individuals. 1 More recently, the use of β-galactosidase to catalyze the synthesis of galactooligosaccharides (GOS) from lactose through transgalactosylation reactions has become a process of great interest, due to the emerged health benefits associated with the consumption of prebiotic GOS as functional food ingredients. 2,3 β-Galactosidases from various sources such as Lactobacillus, Bif idobacterium, Aspergillus and Kluyveromyces species have been used as either whole cell or single enzyme biocatalysts to conduct GOS synthesis reactions. Compared to whole cells, the use of free β-galactosidases usually increases GOS synthesis reaction rates and leads to obtaining the maximum GOS yield in shorter reaction times. 4 Additionally, the use of free β-galactosidases ensures that transgalactosylation can be better controlled compared to the use of whole cells, especially when whole cells contain multiple β-galactosidases which differ in their biochemical characteristics and their ability to conduct GOS synthesis. 4,5 However, the use of native β- galactosidases as biocatalysts in GOS synthesis requires the isolation of the enzyme either from the producing micro- organism or from the culture medium. In both cases, the isolation process is tedious and costly as bacterial β- galactosidases, in particular, are naturally found in low quantities. This represents the major hurdle in using free β- galactosidases as biocatalysts in GOS synthesis reactions. Recombinant DNA technology offers the possibility to produce high yields of the β-galactosidase of interest using suitable expression hosts, such as Escherichia coli. Compared to native β- galactosidases, the use of recombinant β-galactosidases offers various advantages such as the large scale production, the ease of purification due to high expression yields and the possible improvement in enzyme activity and stability. 6 In addition to the expression yield of the β-galactosidase of interest, the solubility and the enzyme characteristics such as its activity, stability and catalytic efficiency are additional important parameters that influence the potential of using recombinant β-galactosidases as biocatalysts in GOS synthesis reactions. These parameters are likely influenced by a variety of factors related to the used vector, the host strain, the host-vector relationship, the folding of the expressed enzyme and the fermentation conditions such as pH, temperature, aeration rate, dissolved oxygen and type of cultivation. 7-11 From our previous research, it was demonstrated that GOS can be efficiently synthesized using whole cells of Bif idobacte- rium bif idum NCIMB 41171. 12-14 It was also reported that this bacterial strain contains four β-galactosidases, i.e., BbgI, BbgII, BbgIII and BbgIV, 15 of which BbgIV was shown to be the best enzyme that converts lactose into GOS. 4,16 Consequently, increased industrial attention has been given to the production Received: November 17, 2012 Revised: January 30, 2013 Accepted: February 5, 2013 Published: February 5, 2013 Article pubs.acs.org/JAFC © 2013 American Chemical Society 2213 dx.doi.org/10.1021/jf304792g | J. Agric. Food Chem. 2013, 61, 2213-2223