Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 β-fructofuranosidase

Abstract

A β-fructofuranosidase from Microbacterium saccharophilum K-1 (formerly known as Arthrobacter sp. K-1) is useful for producing the sweetener lactosucrose (4(G)-β-D-galactosylsucrose). Thermostability of the β-fructofuranosidase was enhanced by random mutagenesis and saturation mutagenesis. Clones with enhanced thermostability included mutations at residues Thr47, Ser200, Phe447, Phe470, and Pro500. In the highest stability mutant, T47S/S200T/F447P/F470Y/P500S, the half-life at 60 °C was 182 min, 16.5-fold longer than the wild-type enzyme. A comparison of the crystal structures of the full-length wild-type enzyme and three mutants showed that various mechanisms appear to be involved in thermostability enhancement. In particular, the replacement of Phe447 with Val or Pro induced a conformational change in an adjacent residue His477, which results in the formation of a new hydrogen bond in the enzyme. Although the thermostabilization mechanisms of the five residue mutations were explicable on the basis of the crystal structures, it appears to be difficult to predict which amino acid residues should be modified to obtain thermostabilized enzymes.

Recent novel applications of levansucrases

“…A dual-enzyme system containing a Z. mobilis lenvansucrase and cheap commercial glucose oxidase increases the efficiency of sucrose and lactose conversion to lactosucrose to 43.2 %, which is much higher than the efficiency obtained using the single enzyme (28.5 %) (Han et al 2009). The thermostability of lactosucrose-producing β-fructofuranosidase from M. saccharophilum K-1 has been remarkably enhanced through molecular modification (Ohta et al 2014). In addition, continuous lactosucrose production, resulting in higher productivity, has been performed using immobilized whole cells (Lee et al 2007a) and a simulated moving bed reactor (Kawase et al 2001).…”

Crystal structure of a β-fructofuranosidase with high transfructosylation activity from Aspergillus kawachii

“…1,7) A GH68 FFase from Microbacterium saccharophilum K-1 (MsFFase) catalyzes transfructosylation to produce lactosucrose (β-D-Galp-(1 → 4)-α-D-Glcp-(1 ↔ 2)-β-D-Fruf) from a mixture of sucrose and lactose. 8) GH32 FFases from Aspergillus niger ATCC 20611. 9) and Aspergillus japonicus CB05 (AjFFase).…”

Perspectives on the biotechnological production and potential applications of lactosucrose: A review

“…A mutagenesis approach applied to β-galactosidase from Geobacillus stearothermophilus resulted in a simultaneous improvement of the transglycosylation activity and reduction of hydrolysis to potential transglycosylation products (Placier, Watzlawick, Rabiller, & Mattes, 2009). Mutagenesis can also be useful to provide enzymes with enhanced thermostability, an important characteristic for the industrial use of enzymes (Ohta et al, 2014;Yuan et al, 2008), or to minimize the effect of some inhibitors, such as galactose (Hu, Robin, O'Connell, Walsh, & Wall, 2010) or glucose (Trollope, Nieuwoudt, Görgens, & Volschenk, 2014). Additionally, the heterologous expression of enzymes has been reported as a successful approach to achieve high levels of extracellular enzyme (Gimeno-Pérez, Linde, Fernández-Arrojo, Plou, & Fernandéz-Lobado, 2015;Oliveira, Guimarães, & Domingues, 2011) which clearly facilitate its recovery and further application.…”