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Molecular basis for thermal properties of Streptomyces thermovulgaris fumarase C hinge at hydrophilic amino acids R163, E170 and S347

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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 thermostable fumarase C that is active and stable at high temperatures 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 combination 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.

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Acknowledgements

We would like to express our greatest gratitude to Dr. Doreen Tan Sian Hoong for reading and editing this manuscript and her valuable comments, which aided us in the preparation of this manuscript.

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  1. Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore, 117597, Singapore

    Wenjie Lin, Maurice Chan, Liuh-Ling Goh & Tiow-Suan Sim

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  1. Wenjie Lin
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  2. Maurice Chan
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  3. Liuh-Ling Goh
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  4. Tiow-Suan Sim
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Correspondence to Tiow-Suan Sim.

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Lin, W., Chan, M., Goh, LL. et al. Molecular basis for thermal properties of Streptomyces thermovulgaris fumarase C hinge at hydrophilic amino acids R163, E170 and S347. Appl Microbiol Biotechnol 75, 329–335 (2007). https://doi.org/10.1007/s00253-006-0822-7

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  • DOI: https://doi.org/10.1007/s00253-006-0822-7

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