This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vian, A.
Right arrow Articles by Cortés, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vian, A.
Right arrow Articles by Cortés, E.
Agricola
Right arrow Articles by Vian, A.
Right arrow Articles by Cortés, E.

 Previous Article  |  Next Article 

Appl Environ Microbiol, June 1998, p. 2187-2191, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Structure of the beta -Galactosidase Gene from Thermus sp. Strain T2: Expression in Escherichia coli and Purification in a Single Step of an Active Fusion Protein

Alejandro Vian,1 Alfonso V. Carrascosa,1 José L. García,2 and Estrella Cortés1,*

Department of Microbiology, Instituto de Fermentaciones Industriales (CSIC),1 and Department of Molecular Microbiology, Centro de Investigaciones Biológicas (CSIC),2 28006 Madrid, Spain

Received 29 December 1997/Accepted 30 March 1998

The nucleotide sequence of both the bgaA gene, coding for a thermostable beta -galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (Mr, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bgaA gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the beta -galactosidase of Bacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric beta -galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70°C and a Km value of 1.6 mM with o-nitrophenyl-beta -D-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70°C.

* Corresponding author. Mailing address: Department of Microbiology, Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain. Phone: 34-1-5622900. Fax: 34-1-5644853. E-mail: IFIIC04{at}FRESNO.CSIC.ES.

Appl Environ Microbiol, June 1998, p. 2187-2191, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Campuzano, S., Serra, B., Llull, D., Garcia, J. L., Garcia, P. (2009). Cloning, Expression, and Characterization of a Peculiar Choline-Binding {beta}-Galactosidase from Streptococcus mitis. Appl. Environ. Microbiol. 75: 5972-5980 [Abstract] [Full Text]  
  • Tanaka, T., Fukui, T., Atomi, H., Imanaka, T. (2003). Characterization of an Exo-{beta}-D-Glucosaminidase Involved in a Novel Chitinolytic Pathway from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1. J. Bacteriol. 185: 5175-5181 [Abstract] [Full Text]  
  • Pessela, B. C. C., Vian, A., Mateo, C., Fernandez-Lafuente, R., Garcia, J. L., Guisan, J. M., Carrascosa, A. V. (2003). Overproduction of Thermus sp. Strain T2 {beta}-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports. Appl. Environ. Microbiol. 69: 1967-1972 [Abstract] [Full Text]  
  • Hung, M.-N., Xia, Z., Hu, N.-T., Lee, B. H. (2001). Molecular and Biochemical Analysis of Two {beta}-Galactosidases from Bifidobacterium infantis HL96. Appl. Environ. Microbiol. 67: 4256-4263 [Abstract] [Full Text]  
  • BROWN, N. F., BEACHAM, I. R. (2000). Cloning and analysis of genomic differences unique to Burkholderia pseudomallei by comparison with B. thailandensis. J Med Microbiol 49: 993-1001 [Abstract] [Full Text]  
  • Sheridan, P. P., Brenchley, J. E. (2000). Characterization of a Salt-Tolerant Family 42 beta -Galactosidase from a Psychrophilic Antarctic Planococcus Isolate. Appl. Environ. Microbiol. 66: 2438-2444 [Abstract] [Full Text]  
  • Coombs, J. M., Brenchley, J. E. (1999). Biochemical and Phylogenetic Analyses of a Cold-Active beta -Galactosidase from the Lactic Acid Bacterium Carnobacterium piscicola BA. Appl. Environ. Microbiol. 65: 5443-5450 [Abstract] [Full Text]  
  • Fridjonsson, O., Watzlawick, H., Gehweiler, A., Rohrhirsch, T., Mattes, R. (1999). Cloning of the Gene Encoding a Novel Thermostable alpha -Galactosidase from Thermus brockianus ITI360. Appl. Environ. Microbiol. 65: 3955-3963 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»