Meltem AŞAN, Numan ÖZCAN

Expression of the b-(1,3-1,4)-Glucanase Gene in Streptococcus salivarius subsp. thermophilus

The purpose of this study was the transformation and expression of the b -(1,3-1,4)-glucanase (lichenase) gene in Streptococcus salivarius subsp. thermophilus to create a recombinant probiotic for poultry and improve the thermostability of the lichenase enzyme. The recombinant plasmid TL1R containing the b -(1,3-1,4)-glucanase gene was introduced into S. salivarius subsp. thermophilus by electrotransformation. The expressing of the b -(1,3-1,4)-glucanase gene in S. salivarius subsp. thermophilus was confirmed on lichenan plate, SDS-PAGE, and zymogram analysis. The b -(1,3-1,4)-glucanase enzyme expressed by S. salivarius subsp. thermophilus cells seemed to increase its capacity for thermoresistance and so it maintained its activity at 70 ºC for 15 min. In contrast, the enzyme produced by Lactococcus lactis and Escherichia coli cells easily ceased activity when exposed to the same temperature. The enzyme expressed by all the recombinant bacteria resisted denaturation and somehow remained soluble after heat treatment from 37 to 100 ºC for 15 min.

Expression of the b-(1,3-1,4)-Glucanase Gene in Streptococcus salivarius subsp. thermophilus

Keywords:

Streptococcus salivarius subsp. thermophilus, b -(1, 3-1, 4)-glucanase expression, thermostability,

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Stone, B.A., Clarke, A.E.: Chemistry and Biology of 1,3-β- Glucans. La Trobe University Press, Bundoora, Australia. 1992. 2.Planas, A.

: Bacterial 1,3-1,4-β-glucanases: structure, function

and protein engineering. Biochim. Biophys. Acta, 2000; 1543: 361-382. 3.

Cantwell, B.A., McConnell, D.J.: Molecular cloning and expression

of a Bacillus subtilisβ-glucanase gene in Escherichia coli. Gene,

Ekinci, M.S., McCrae, S.I., Flint, H.J.: Isolation and overexpression of a gene encoding an extracellular β-(1,3-1,4)- glucanase from Streptococcus bovis JB1. Appl. Environ. Microbiol., 1997; 63: 3752-3756.

Pitson, S.M., Seviour, R.J., McDougall, B.M.: Noncellulolytic fungal β-glucanases: their physiology and regulation. Enzyme Microb. Technol., 1993; 15: 178-192.

Chesson, A.: Feed enzymes. Anim. Feed Sci. Technol., 1993; 45: 65-79.

Fuller, R.: Probiotics in man and animals. J. Appl. Bacteriol., 1989; 66: 365-378.

Johri, T.S.: Dietary additives for enhancing nutritional value of feeds. In: Poultry Nutrition Research in India and Its Perspective. FAO Publications and Documents, 2005; 97-271.

Somkuti, G.A., Solaiman, D.K., Johnson, T.L., Steinberg, D.H.: Transfer and expression of a Streptomyces cholesterol oxidase gene in Streptococcus thermophilus. Biotechnol. Appl. Biochem., 1991; 13: 238-245.

Somkuti, G.A., Solaiman, D.K.Y., Steinberg, D.H.: Cloning of a tyrosinase gene in Streptococcus thermophilus. Biotechnol. Lett., 1993; 15: 773-778.

Solaiman, D.K.Y., Somkuti, G.A.: Expression of a rhodococcal indigo gene in Streptococcus thermophilus. Biotechnol. Lett., 1996; 18: 19-24.

Coderre, P.E., Somkuti, G.A.: Cloning and expression of the pediocine operon in Streptococcus thermophilus and other lactic fermentation bacteria. Curr. Microbiol., 1999; 39: 295-301.

Sambrook, J., Fritsch, E.F., Maniatis, T.: Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor, New York. 1989.

Birnboim, H.C., Doly, J.: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic. Acids Res., 1979; 7: 1513-1523.

Wei, M.Q., Rush, C.M., Norman, J.M., Hafner, L.M., Epping, R.J., Timms, P.: An improved method for the transformation of Lactobacillus strains using electroporation. J. Microbiol. Methods, 1995; 21: 97-109.

McIntyre, D.A., Harlander, S.K.: Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation. Appl. Environ. Microbiol., 1989; 55: 604-610.

Holo, H., Nes, I.F.: High-frequency transformation by electroporation of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl. Environ. Microbiol., 1989; 55: 3119-3123.

Teather, R.M., Wood, P.J.: Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol., 1982; 43: 777-780.

Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970; 227: 680-685.

Saul, D.J., Williams, L.C., Love, D.R., Chamley, L.W., Bergquist, P.L.: Nucleotide sequence of a gene from Caldocellum saccharolyticum encoding for exocellulase and endocellulase activity. Nucleic Acids Res., 1989; 17: 439.

Özcan, N., Cunningham, C., Harris, W.J.: Cloning of a cellulase gene from the rumen anaerobe Fibrobacter succinogenes SD35 and partial characterization of the gene product. Lett. Appl. Microbiol., 1996; 22: 85-89.

Ekinci, M.S.: Heterologous Expression of Genes in the Anaerobic Bacterium Streptococcus bovis. PhD Dissertation. The University of Aberdeen, Aberdeen, UK. 1997.