Purification and characterisation of two extremely halotolerant xylanases from a novel halophilic bacterium.
The present work reports for the first time the purification and characterisation of two extremely halotolerant endo-xylanases from a novel halophilic bacterium, strain CL8. Purification of the two xylanases, Xyl 1 and 2, was achieved by anion exchange and hydrophobic interaction chromatography. The enzymes had relative molecular masses of 43 kDa and 62 kDa and pI of 5.0 and 3.4 respectively. Stimulation of activity by Ca(2+), Mn(2+), Mg(2+), Ba(2+), Li(2+), NaN(3) and isopropanol was observed. The K(m) and V(max) values determined for Xyl 1 with 4- O-methyl- d-glucuronoxylan are 5 mg/ml and 125,000 nkat/mg respectively. The corresponding values for Xyl 2 were 1 mg/ml and 143,000 nkat/mg protein. Xylobiose and xylotriose were the major end products for both endoxylanases. The xylanases were stable at pH 4-11 showing pH optima around pH 6. Xyl 1 shows maximal activity at 60 degrees C, Xyl 2 at 65 degrees C (at 4 M NaCl). The xylanases showed high temperature stability with half-lives at 60 degrees C of 97 min and 192 min respectively. Both xylanases showed optimal activity at 1 M NaCl, but substantial activity remained for both enzymes at 5 M NaCl.
Wejse
Peter Langborg
PL
Department of Microbial Ecology, Institute of Biological Sciences, University of Aarhus, Ny Munkegade, Building 540, 8000 Aarhus, Denmark.
Ingvorsen
Kjeld
K
Mortensen
Kim Kusk
KK
D001419:Bacteria / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007525:Isoelectric Focusing; D008970:Molecular Weight; D011189:Potassium Chloride / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D014995:Xylosidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D012965:Sodium Chloride; D011189:Potassium Chloride; D014995:Xylosidases