Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2.
An alkalophilic bacterium was isolated based on the potential of extra-cellular enzymes for bioscouring. The bacterium was identified as a new strain of Bacillus pumilus BK2 producing an extra-cellular endo-pectate lyase PL (EC 4.2.2.2). PL was purified to homogeneity in three steps and has a molecular mass of 37.3+/-4.8 kDa as determined by SDS-PAGE and an isoelectric point of pH 8.5. Peptide mass mapping by nano-LC-MS of PL revealed 15% homology with a pectate lyase from Bacillus sp. The pectate lyase exhibited optimum activity at pH 8.5 and around 70 degrees C in Tris/HCl buffer. It showed a half-life at 30 degrees C of more than 75 h. Stability decreased with increasing temperature, extremely over 60 degrees C. The enzyme did not require Ca2+ ions for activity, and was strongly inhibited by EDTA and Co2+. PL was active on polygalacturonic acid and esterified pectin, but the affinity showed a maximum for intermediate esterified pectins and decreased over a value of 50% of esterification. The best substrate was 29.5% methylated pectin. PL cleaved polygalacturonic acid via a beta-elimination mechanism as shown by NMR analysis. PL released unsaturated tetragalacturonic acid from citrus pectin and polygalacturonic acid, but did not show any side activities on other hemicelluloses. On polygalacturonic acid PL showed a Km of 0.24 gl(-1) and a vmax of 0.72 gl(-1)min(-1). The applicability of pectate lyase for the bioscouring process was tested on a cotton fabric. Removal of up to 80% of pectin was proven by means of ruthenium red dyeing and HPAEC (65%). Structural contact angle measurements clearly indicated the increased hydrophilicity of enzyme treated fabrics.
Klug-Santner
Barbara G
BG
Department of Environmental Biotechnology, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
Schnitzhofer
Wolfgang
W
Vrsanská
Maria
M
Weber
Jörg
J
Agrawal
Pramod B
PB
Nierstrasz
Vincent A
VA
Guebitz
Georg M
GM
D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003035:Cobalt / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D010449:Peptide Mapping; D011133:Polysaccharide-Lyases / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014783:Viscosity
D001426:Bacterial Proteins; D003035:Cobalt; D004492:Edetic Acid; D011133:Polysaccharide-Lyases; C023911:pectate lyase