Volume 4 • Issue 1 • 1000125 Enz Eng ISSN: 2329-6674 EEG, an open access journal Research Article Open Access Holsbeeck et al., Enz Eng 2014, 4:1 DOI: 10.4172/2329-6674.1000125 Research Article Open Access Keywords: Escherichia coli; High-Cell-Density cultivation; L-Arabinose isomerase; D-tagatose; Alternative sweetener Abbreviations: L-AI: L-Arabinose Isomerase; HCDC: High- Cell-Density Cultivation; WT i : Wild Type enzyme produced with an inducible expression system; CIP: Center for Protein Engineering; IPTG: IsoPropyl β-D-1-TioGalactopyranoside; Tris: Tris (hydroxymethyl) Aminomethane; OD: Optical Density; DCW: Dry Cell Weight; HPLC: High-Performance Liquid Chromatography; DAD: Diode Array Detector; RI: Refractive Index Introduction Nowadays, the efcient production of recombinant proteins has become more and more important in biotechnology. Te combination of recombinant proteins and large-scale processes make it possible to produce these proteins in amounts exceeding greatly the availability in natural resources [1-4]. Te most commonly used host organism for recombinant protein production is still Escherichia coli because its genetic properties and physiological behaviour are well-known [1-3,5- 9]. Within this study, High-Cell-Density Cultivation (HCDC) using E. coli as host organism was evaluated in view of a good expression and production of the L-arabinose isomerase (L-AI) from Geobacillus stearothermophilus. Te L-AI enzyme converts D-galactose into D-tagatose. D-tagatose is a natural occurring bulk sweetener showing a wide range of healthy properties. Te sweetness and aroma profle of D-tagatose are similar to sucrose. Furthermore, D-tagatose possesses a low caloric value (1.5 kcal g -1 ), a low glycaemic index, prebiotic properties and shows non-cariogenic properties [10-17]. D-tagatose is present in apples, pineapples, oranges, sterilized cow milk etc. but not in sufcient amounts for commercialization [12,13]. For industrial implementation, a biochemical production of D-tagatose by means of an L-AI enzyme is preferred. However, a cost-efcient production of the L-AI enzyme is therefore required. An interesting approach for a cost-efcient production of recombinant proteins is High-Cell-Density Cultivation (HCDC) of the host organism. Tis method is especially interesting for E. coli, where recombinant proteins ofen accumulate intracellular [6]. In this case, the productivity is directly correlated with the fnal cell density [1]. Te advantages of HCDC are its reduced reactor volume, enhanced downstream processing, higher volumetric productivities, reduced waste water, less production costs and reduced investment in equipment [1,2]. However, several disadvantages are also encountered during HCDC such as the high initial concentration of medium components and low oxygen solubility. Furthermore, under oxygen limiting conditions and a high carbon concentration, by-products like acetate can be produced which inhibit growth [2-3,6]. In literature, the cultivation of E. coli for the production of the L-AI with a complex medium [18]. Te medium contained 3.0 g L -1 *Corresponding author: Ilse Van de Voorde, KU Leuven, Faculty of Engineering Technology, Department of Microbial and Molecular Systems (M²S), Cluster of Bioengineering Technology (CBeT), Laboratory of Enzyme, Fermentation and Brewing Technology, Technology Campus Ghent, Gebroeders De Smetstraat 1, B-9000 Ghent, Belgium, Tel: +32 (0)9 265 86 13; E-mail: ilse.vandevoorde@ kuleuven.be Received October 28, 2014; Accepted December 18, 2014; Published December 21, 2014 Citation: Holsbeeck MV, Tsakali E, Syryn E, Aerts G, Impe JV, et al. (2014) Overexpression of L-Arabinose Isomerase for Production of the Low-Calorie Bulk Sweetener D-Tagatose. Enz Eng 4: 125. doi:10.4172/2329-6674.1000125 Copyright: © 2014 Holsbeeck MV, 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 High-Cell-Density Cultivations show a huge potential to produce recombinant proteins to amounts greatly exceeding the availability in natural resources. An interesting example of a recombinant protein is an L-arabinose isomerase, which is able to convert D-galactose to the low-caloric and low-glycaemic bulk sweetener D-tagatose. Within this study, the L-arabinose isomerase from Geobacillus stearothermophilus was expressed intracellularly in Escherichia coli. The cultivation medium contained glucose, yeast extract and various macro- and micronutrients. The effect of air fow rate on E. coli growth and expression of L-arabinose isomerase was studied. After 52 hours, an Optical Density and Dry Cell Weight of 154 ± 4 and 54.8 ± 1.3 g L -1 were reached, respectively by regulating the air fow rate between 0.2 and 30 L min -1 . A corresponding L-arabinose isomerase activity of 6.99 ± 0.46 U mL -1 was reached. A drawback of High-Cell-Density Cultivation is the production of the by-product acetic acid which may inhibit growth. However, the acetic acid concentration was maintained as low as possible during fermentation to avoid inhibitory effects inherent to this compound. With the L-arabinose isomerase produced, a conversion percentage of 37.1 ± 1.5% was achieved, corresponding to 94.9 ± 3.7 g L -1 D-tagatose. Thus, the implementation of a High-Cell- Density Cultivation led to an effcient expression of the L-arabinose isomerase enzyme and D-tagatose production. Also the storage stability of the cells was investigated during several months at 4°C. A stable L-arabinose isomerase enzyme was noticed during at least 8 months storage at 4°C. Overexpression of L-Arabinose Isomerase for Production of the Low- Calorie Bulk Sweetener D-Tagatose Monika Van Holsbeeck 1,2 , Efstathia Tsakali 2,3 , Evelien Syryn 1 , Guido Aerts 1 , Jan Van Impe 2 and Ilse Van de Voorde 1 * 1 KU Leuven, Faculty of Engineering Technology, Department of Microbial and Molecular Systems (M²S), Cluster of Bioengineering Technology (CBeT), Laboratory of Enzyme, Fermentation and Brewing Technology, Technology Campus Ghent, Gebroeders De Smetstraat 1, B-9000 Ghent, Belgium 2 KU Leuven, Faculty of Engineering Science, Department of Chemical Engineering (CIT), Chemical and Biochemical Process Technology and Control (BioTeC), Willem de Croylaan 46, B-3001 Heverlee, Belgium 3 T.E.I. of Athens, Faculty of Food Technology and Nutrition, Department of Food Technology, Laboratory of Food and Industrial Microbiology, Agiou Spyridonos 12210 Egaleo, Athens, Greece E n z y m e E n g i n e e r i n g ISSN: 2329-6674 Enzyme Engineering