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Applied and Environmental Microbiology, June 2009, p. 3447-3454, Vol. 75, No. 11
0099-2240/09/$08.00+0     doi:10.1128/AEM.02675-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of Rhamnosidases from Lactobacillus plantarum and Lactobacillus acidophilus{triangledown}

Jules Beekwilder,1* Daniela Marcozzi,1 Samuele Vecchi,1 Ric de Vos,1 Patrick Janssen,2 Christof Francke,3,4 Johan van Hylckama Vlieg,2,5,{dagger} and Robert D. Hall1,6

Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands,1 NIZO food research, P.O. Box 20, 6710 BA Ede, The Netherlands,2 TI Food and Nutrition, P.O. Box 557, 6700 AN Wageningen, The Netherlands,3 Center for Molecular and Biomolecular Informatics, NCMLS, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500HB Nijmegen, The Netherlands,4 Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands,5 Centre for BioSystems Genomics, P.O. Box 98, 6700 AA Wageningen, The Netherlands6

Received 23 November 2008/ Accepted 27 March 2009

Lactobacilli are known to use plant materials as a food source. Many such materials are rich in rhamnose-containing polyphenols, and thus it can be anticipated that lactobacilli will contain rhamnosidases. Therefore, genome sequences of food-grade lactobacilli were screened for putative rhamnosidases. In the genome of Lactobacillus plantarum, two putative rhamnosidase genes (ram1Lp and ram2Lp) were identified, while in Lactobacillus acidophilus, one rhamnosidase gene was found (ramALa). Gene products from all three genes were produced after introduction into Escherichia coli and were then tested for their enzymatic properties. Ram1Lp, Ram2Lp, and RamALa were able to efficiently hydrolyze rutin and other rutinosides, while RamALa was, in addition, able to cleave naringin, a neohesperidoside. Subsequently, the potential application of Lactobacillus rhamnosidases in food processing was investigated using a single matrix, tomato pulp. Recombinant Ram1Lp and RamALa enzymes were shown to remove the rhamnose from rutinosides in this material, but efficient conversion required adjustment of the tomato pulp to pH 6. The potential of Ram1Lp for fermentation of plant flavonoids was further investigated by expression in the food-grade bacterium Lactococcus lactis. This system was used for fermentation of tomato pulp, with the aim of improving the bioavailability of flavonoids in processed tomato products. While import of flavonoids into L. lactis appeared to be a limiting factor, rhamnose removal was confirmed, indicating that rhamnosidase-producing bacteria may find commercial application, depending on the technological properties of the strains and enzymes.

* Corresponding author. Mailing address: Plant Research International, P.O. Box 16, 6700AA Wageningen, The Netherlands. Phone: 31-317-480979. Fax: 31-317-418094. E-mail: jules.beekwilder{at}wur.nl

{triangledown} Published ahead of print on 3 April 2009.

{dagger} Present address: Danone Research, Gut and Microbiology Platform, R.D. 128, 91767 Palaiseau Cedex, France.

Applied and Environmental Microbiology, June 2009, p. 3447-3454, Vol. 75, No. 11
0099-2240/09/$08.00+0     doi:10.1128/AEM.02675-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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