A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-beta-xylanase II.
We have successfully engineered a disulphide bridge into the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase II (XYNII) by substituting Thr-2 and Thr-28 with cysteine. The T2C:T28C mutational changes increased the half-life in thermal inactivation of this mesophilic enzyme from approximately 40 s to approximately 20 min at 65 degrees C, and from less than 10 s to approximately 6 min at 70 degrees C. Therefore, the N-terminal disulphide bridge enables the use of XYNII at substantially higher temperatures than permitted by its native mesophilic counterpart. Altogether, thermostability increased by about 15 degrees C. The kinetic properties of the mutant XYNII were maintained at the level of the wild type enzyme. Our findings demonstrated that a properly designed disulphide bridge, here within the N-terminal region of XYNII, can be very effective in resisting thermal inactivation.
Fenel
Fred
F
Carbozyme Ltd., Keilaranta 16, 02150 Espoo, Finland. fred.fenel@carbozyme.fi
Leisola
Matti
M
Jänis
Janne
J
Turunen
Ossi
O
D019943:Amino Acid Substitution; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D024861:Genetic Enhancement / Q000379:methods; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D004220:Disulfides; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases