Production of a Thermoresistant Alpha-galactosidase from Thermus sp. Strain T2 for Food Processing

Abstract

The gene of a thermostable alpha-galactosidase, aglA, from Thermus sp. strain T2 was sequenced, cloned and overexpressed in Escherichia coli (strain MC 2508. The purified enzyme proved to be quite thermostable, retaining high levels of activity even after incubation at 70°C. The optimal T was 65°C at pH 7. The highest activity was achieved at acid pH values, although good activity could be obtained in a broad pH range. Enzyme stability depends on the enzyme concentration at alkaline pH values, suggesting that under these conditions the subunit dissociation may be the first step in the inactivation of the enzyme. However, at pH 5 the enzyme stability becomes independent of the enzyme concentration. The enzyme also exhibited a quite broad specificity, although it shows the best activity with alpha derivatives of galactose, and was able to recognize very different substrates (alpha derivatives of mannose, xylose and maltose, and even beta galactose derivatives). There was no detectable activity against glucose derivatives.

Key Words:

• alpha-galactosidase
• Thermus
• purification

ACKNOWLEDGEMENTS

We thank Dr. Yoshinori Koyama (National Institute of Human Technology, Tsukuba, Japan) for his kind supply of the plasmid pOS105. This work was supported by grants O7G/002/2003, ALIBIRD-CM S-0505/AGR-0153 and AGR-O153/2005 from the Comunidad Autónoma de Madrid and the UE-Project (MATIONES G5RD-CT-2002–00752). We specially thank the Angolan Government by a Ph.D fellowship to Mr. Miguel Filho and a Ph.D fellowship from Universidad Industrial de Santander, Colombia for R.Torres.