Innovative kinetic and thermodynamic analysis of a purified superactive xylanase from Scopulariopsis sp.
Two isoenzymes of endo-1,4-beta-xylanase (EC 3.2.1.8) from Scopulariopsis sp. were purified by a combination of ammonium sulfate precipitation, hydrophobic interaction, and anion-exchange and gel filtration chromatography. The native mol wts of the least acidic xylanase (LAX) and the highly acidic xylanase (HAX) were 25 and 144 kDa and the subunit mol wts were 25 and 36 kDa, respectively. The kcat values of LAX and HAX for oat-spelt xylan at 40 degrees C, pH 6.5, were 95,000 and 9900 min-1 and the Km values of LAX and HAX were 30 and 3.3 mg/mL. The thermodynamic activation parameters of xylan hydrolysis showed that the high activity of LAX when compared with HAX was not owing to a reduction in DeltaH# but was entropically driven. High-performance liquid chromatography analysis of the degradation products showed that LAX formed both xylotrioses and xylobioses, but HAX predominantly formed xylotrioses. The half-lives of LAX and HAX at 50 degrees C in 50 mM 2-N-morpholino ethanesulfonic acid (MES), pH 6.5 buffer were 267 and 69 min, respectively. Thermodynamic analysis showed that at lower temperatures, the increased thermostability of LAX (DeltaH#=306 kJ/mol) compared with HAX (DeltaH#=264 kJ/mol) was owing to more noncovalent surface interactions. At higher temperatures, LAX (DeltaS*=-232 J/[mol.K]) was more thermostable than HAX (DeltaS*=490 J/[mol.K]) owing to a more ordered transition-state conformation. An energy-activity diagram was introduced showing that kcat/Km does not successfully explain the true kinetic behavior of both xylanase isoenzymes. The simultaneously thermostable and highly active LAX could be utilized in biotechnological processes involving xylan hydrolysis.
Afzal
Ahmed Jawaad
AJ
Department of Molecular Biology, Southern Illinois University at Carbondale, Carbondale, IL 62901, USA. ajafzal@siu.edu
Ali
Sikander
S
Latif
Farooq
F
Rajoka
Muhammad Ibrahim
MI
Siddiqui
Khawar Sohail
KS
D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D013696:Temperature; D013816:Thermodynamics; D014990:Xylans / Q000737:chemistry / Q000378:metabolism
D007527:Isoenzymes; D014990:Xylans; D043364:Endo-1,4-beta Xylanases