BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Molecular basis for thermal properties of Streptomyces thermovulgaris fumarase C hinge at hydrophilic amino acids R163, E170 and S347 Wenjie Lin & Maurice Chan & Liuh-Ling Goh & Tiow-Suan Sim Received: 26 October 2006 / Revised: 17 December 2006 / Accepted: 19 December 2006 / Published online: 24 January 2007 # Springer-Verlag 2007 Abstract Industrially, the use of high temperatures (40– 60°C) in the L-malate production process could result in rapid inactivation of the mesophilic fumarases, warranting constant replenishment of the biocatalyst. Thus, a thermo- stable fumarase C that is active and stable at high temper- atures would be ideal. Biochemical studies using recombinant fumarase C from thermophilic Streptomyces thermovulgaris (stFUMC) indicated that it was optimally active at 50°C and highly stable even after 24 h of incubation at 40°C. The same gene from mesophilic Streptomyces coelicolor (scfumC) was also cloned and expressed as soluble proteins for comparison in thermal properties of both enzymes. In contrast to stFUMC, scFUMC exhibited a lower temperature optima of 30°C and was rapidly denatured at 50°C. The specific activity of stFUMC was also higher than that of scFUMC by 20-fold. After primary sequence comparison, three hydrophilic amino acid residues, R163, E170 and S347, were forged into the thermolabile scFUMC either singly or in combina- tion for the investigation of their contributions in the thermal properties of the mutant enzymes. Of the mutants studied, the A347S scFUMC mutant resulted in the highest increase in optimum temperature of 10°C and a fourfold enhancement in specific activity. G163R/G170E and G163R/G170E/ A347S scFUMC mutants are more thermostable than wild- type scFUMC. These findings support stFUMC as a highly efficient, thermostable fumarase C with industrial potential and suggest that R163, E170 and S347 are involved in the enhancement of thermal properties in fumarase C. Keywords Streptomyces . Site-directed mutagenesis . Thermostability . Hydrophilic residues . Fumarase C Introduction Fumarase (E.C 4.2.1.2) is involved in the tricarboxylic acid (TCA) cycle, where it catalyses the reversible hydration of fumarate to L-malate. Mammalian fumarases involved in the mitochondrial TCA cycle (Kanarek et al. 1964; Kobayashi et al. 1981; Sacchettini et al. 1986) have been studied together with those from prokaryotes (Flint et al. 1992; Goh et al. 2005) and archeabacteria such as Sulfolobus solfataricus (Puchegger et al. 1990) and Ther- mus thermophilus (Mizobata et al. 1998), for better understanding of the carbon metabolism in different environmental conditions and phylogenetic relationship between these proteins. Currently, mesophilic microorganisms with high fuma- rase activities, such as those of Lactobacillus brevis and Brevibacterium flavum, have been exploited industrially for the production of L-malate using fumarate as a substrate (Takata and Tosa 1993; Terasawa et al. 1990). The use of fumarase as a biocatalyst to produce L-malate is preferred over chemical or fermentation means as L-malate formed by chemical synthesis has to be separated from a racemic end- product mixture (Takata and Tosa 1993). More economical methods using the continuous reaction of immobilised microbial cells (Bressler et al. 2002) with high fumarase activity or with enzymes alone (Giorno et al. 2001) are being actively explored. In the production of L-malate by B. flavum, reaction temperatures are commonly set between 40 and 60°C to reduce the formation of succinic acid, an unwanted product (Terasawa et al. 1990). However, at these temperatures, the Appl Microbiol Biotechnol (2007) 75:329–335 DOI 10.1007/s00253-006-0822-7 W. Lin : M. Chan : L.-L. Goh : T.-S. Sim (*) Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore e-mail: micsimts@nus.edu.sg