Kinetic and thermodynamic study of cloned thermostable endo-1,4-β-xylanase from Thermotoga petrophila in mesophilic host.
The 1,044 bp endo-1,4-β-xylanase gene of a hyperthermophilic Eubacterium, "Thermotoga petrophila RKU 1" (T. petrophila) was amplified, from the genomic DNA of donor bacterium, cloned and expressed in mesophilic host E. coli strain BL21 Codon plus. The extracellular target protein was purified by heat treatment followed by anion and cation exchange column chromatography. The purified enzyme appeared as a single band, corresponding to molecular mass of 40 kDa, upon SDS-PAGE. The pH and temperature profile showed that enzyme was maximally active at 6.0 and 95 °C, respectively against birchwood xylan as a substrate (2,600 U/mg). The enzyme also exhibited marked activity towards beech wood xylan (1,655 U/mg). However minor activity against CMC (61 U/mg) and β-Glucan barley (21 U/mg) was observed. No activity against Avicel, Starch, Laminarin and Whatman filter paper 42 was observed. The K(m), V(max) and K (cat) of the recombinant enzyme were found to be 3.5 mg ml(-1), 2778 μmol mg(-1)min(-1) and 2,137,346.15 s(-1), respectively against birchwood xylan as a substrate. The recombinant enzyme was found very stable and exhibited half life (t(½)) of 54.5 min even at temperature as high as 96 °C, with enthalpy of denaturation (ΔH*(D)), free energy of denaturation (ΔG*(D)) and entropy of denaturation (ΔS*(D)) of 513.23 kJ mol(-1), 104.42 kJ mol(-1) and 1.10 kJ mol(-1)K(-1), respectively at 96 °C. Further the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for birchwood xylan hydrolysis by recombinant endo-1,4-β-xylanase were calculated at 95 °C as 62.45 kJ mol(-1), 46.18 kJ mol(-1) and 44.2 J mol(-1) K(-1), respectively.
Institute of Industrial Biotechnology, GC University, Lahore 54000, Pakistan. ikmhaq@yahoo.com
Ikram
I
ul Haq
Zahid
Z
Hussain
Mahmood Ali
MA
Khan
Bushra
B
Muneer
Sumra
S
Afzal
Sana
S
Majeed
Fatima
F
Akram
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005784:Gene Amplification; D015870:Gene Expression; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D007094:Imides / Q000378:metabolism; D009025:Morpholines / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013816:Thermodynamics; D014990:Xylans / Q000378:metabolism*; D047071:beta-Glucans / Q000378:metabolism
D004269:DNA, Bacterial; D007094:Imides; D009025:Morpholines; C527904:N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide; D011994:Recombinant Proteins; D014990:Xylans; D047071:beta-Glucans; D043364:Endo-1,4-beta Xylanases