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Applied and Environmental Microbiology, May 2002, p. 2330-2336, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2330-2336.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of a Surface Protein from Lactobacillus fermentum 104R That Binds to Porcine Small Intestinal Mucus and Gastric Mucin

Maurilia Rojas,1,2* Felipe Ascencio,3 and Patricia L. Conway4

Universidad Autónoma de Baja California Sur,1 Centro de Investigaciones Biologicas de Baja California Sur, 23000 La Paz, Baja California Sur, México,3 Department of General and Marine Microbiology, Göteborg University, S-41390 Göteborg, Sweden,2 CRC Food Industry Innovation, School of Microbiology and Immunology, University of New South Wales, Sydney, New South Wales 2052, Australia4

Received 2 August 2001/ Accepted 14 February 2002

An adhesion-promoting protein involved in the binding of Lactobacillus fermentum strain 104R to small intestinal mucus from piglets and to partially purified gastric mucin was isolated and characterized. Spent culture supernatant fluid and bacterial cell wall extracts were fractionated by ammonium sulfate precipitation and gel filtration. The active fraction was purified by affinity chromatography. The adhesion-promoting protein was detected in the fractions by adhesion inhibition and dot blot assays and visualized by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and Western blotting with horseradish peroxidase-labeled mucus and mucin. The active fraction was characterized by estimating the relative molecular weight and by assessing the presence of carbohydrates in, and heat sensitivity of, the active region of the adhesion-promoting protein. The purified protein was digested with porcine trypsin, and the peptides were purified in a SMART system. The peptides were tested for adhesion to horseradish peroxidase-labeled mucin by using the dot blot adhesion assay. Peptides which bound mucin were sequenced. It was shown that the purified adhesion-promoting protein on the cell surface of L. fermentum 104R is extractable with 1 M LiCl and low concentrations of lysozyme but not with 0.2 M glycine. The protein could be released to the culture supernatant fluid after 24 h of growth and had affinity for both small intestinal mucus and gastric mucin. In the native state this protein was variable in size, and it had a molecular mass of 29 kDa when denatured. The denatured protein did not contain carbohydrate moieties and was not heat sensitive. Alignment of amino acids of the adhering peptides with sequences deposited in the EMBL data library showed poor homology with previously published sequences. The protein represents an important molecule for development of probiotics.

* Corresponding author. Mailing address: Universidad Autonoma de B.C.S., Apdo. Postal 19-B, 23000 La Paz, B.C.S., Mexico. Phone: 52 612 1280137. Fax: 52 112 1280137. E-mail: mrojas{at}uabcs.mx.

Applied and Environmental Microbiology, May 2002, p. 2330-2336, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2330-2336.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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