Engineering increased thermostability in the thermostable GH-11 xylanase from Thermobacillus xylanilyticus.
Enzymatic hydrolysis constitutes an attractive strategy for biorefining of abundant, low-cost agricultural by-products such as wheat bran and straw. However, to adopt such an approach, efficient enzymes are required, in particular xylanases. To promote heat-induced disorganization of the complex cell wall network in wheat bran and thus increase enzymatic hydrolysis, we have attempted to improve the thermoresistance of a GH-11 xylanase that is already moderately thermostable. Using a previously described engineering strategy that involves the introduction of disulphide bridges, a mutant (Tx-xyl-SS3) displaying enhanced thermostability and thermoactivity was obtained. The half life at 70 degrees C (180 min) of Tx-xyl-SS3 is 10-fold greater than that of the wild type enzyme and its specific activity is almost doubled (3500 IU mg(-1)). Despite these improvements, Tx-xyl-SS3 was unsuitable for use at significantly higher reaction temperatures (i.e. 85-95 degrees C) and thus the initial objective of this study remained unaccomplished. However, unexpectedly even at the normal hydrolytic temperature (60 degrees C), Tx-xyl-SS3 was able to solubilize 50% of the wheat bran arabinoxylans, 10 points more than the wild type enzyme in parallel reactions. The data presented here show that this improvement is not directly linked to the increase in thermostability and/or thermoactivity, but rather to other unidentified changes to physico-chemical properties that may allow Tx-xyl-SS3 to better penetrate the cell wall network in wheat bran.
Paës
Gabriel
G
INRA-UMR FARE 614, 8, rue Gabriel Voisin, BP 316, 51688 Reims Cedex 2, France.
O'Donohue
Michael J
MJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D004043:Dietary Fiber / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000138:chemical synthesis; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000138:chemical synthesis; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D004043:Dietary Fiber; D050505:Mutant Proteins; D011994:Recombinant Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; D043364:Endo-1,4-beta Xylanases