PURIFICATION AND ANALYSIS OF A MEMBRANE-ASSOCIATED STARCH DEGRADING ENZYME FROM RUMINOCOCCUS AMYLOPHILUS

Authors: ANDERSON, KEVIN; XU, LIANG - MISSISSIPPI STATE

Submitted to: Archives of Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/15/2002
Publication Date: 9/1/2003
Citation: ANDERSON, K.L., XU, L. PURIFICATION AND ANALYSIS OF A MEMBRANE-ASSOCIATED STARCH DEGRADING ENZYME FROM RUMINOCOCCUS AMYLOPHILUS. ARCHIVES OF MICROBIOLOGY. 2003. v. 8. p. 269-276.

Interpretive Summary: Starch is a significant source of dietary carbohydrate for livestock. Therefore, studying how bacteria, such as Ruminobacter amylophilus, degrade starch will increase microbiologists' understanding of how bacteria breakdown starch in the digestive tract of animals. Previous research of R. anylophilus determined that its starch-degrading enzymes were apparently retained within the cell rather than being secreted as an extracellulare enzyme. Instead, this bacterium binds starch to the exterior of the cell and then transports the polysaccharide into the cell. To date, this mechanism of bacterial starch degradation has only been observed with gastrointestinal bacteria. Therefore, to gain a better understanding of this unique way for bacteria to degrade starch, we studied one of the cell-associated starch-degrading enzymes of R. amylohilus. Purification and analysis of this enzyme indicated that it was tightly bound to the cell's membrane, this enzyme may be responsible for the initial degradation of th starch molecule as it is transported across the membrane. This intricate association with the cell's membrane may also protect the enzyme from protein degrading conditions in the gastrointestional tract, yet still allow it to attack starch molecules as they are being transported into the cell. Since only a few bacterial neopullulanases have yet been found (and even fewer that are bound to the cell's membrane), the biochemical characteristics of the enzyme from R. amylophilus provide addtional information to microbiologists about this class of microbial enzymes, and how some gastrointestinal bacteria degrade dietary starch.

Technical Abstract: A membrane-associated enzyme that could hydrolyze amylose, glycogen, and pullulan was detected in the anaerobic bacterium, Ruminobacter amylophilus. This enzyme could not be removed from the membrane fraction with 1 M Nacl, but could be partially extracted from the membrane with 4% Triton X-100, indicating the enzyme had an intrinsic association with the membrane fraction. Electrophoretic separation of polypeptides contained in the 4% triton-soluble fraction revealed the presence of seven polypeptides. These polypeptides were then further sparated based upon their respective isoelectric focal points, and one amyloltic activity was detected, which had a pI 5.85-5.9. Electrophoresis of this amylolytic fraction revealed only one polypeptide with a molecular weight of 91.2 kDa (as determined by SDS-PAGE) and 91.3 kDa (as determined by nondenaturing PAGE). Soaking nondenaturing PAGE gels with a 1% solution of soluble starch followed by soaking with an iodine solution revealed this polypeptide degraded starch, confirming its amylolytic nature. Paper chromatographic anyalyis of hydrolytic end-products indicated that this enzyme hydrolyzed pullulan to produce panose, while amylose and glycogen were hydrolyzed to produce only maltose and maltotriose. This is consistent with the characteristics of a neopullulanase. Storage of the purified enzyme in 4% Triton X-100 increased its stability, further indicating its hydrophobic nature. The specific activity of the purified enzyme was 216.6 U/mg protein with a purification activity increase of 49.5 fold.