Enhancement of the thermostability and hydrolytic activity of GH10 xylanase by module shuffling between Cellulomonas fimi Cex and Thermomonospora alba XylA.

摘要

Two family GH10 endo-1,4- beta-xylanases (EC 3.2.1.8) with different thermal stabilities, Cex (optimum temp. 40 degrees C) from Cellulomonas fimi and XylA (optimum temp. 80 degrees C) from Thermomonospora alba, were used to construct a chimeric xylanase by module shuffling for investigating the structural determinants responsible for this difference. The parent genes were shuffled by crossovers at selected module borders using self-priming PCR. The shuffled construct, designated CXC-X4,5, was cloned and its nucleotide sequence was confirmed. The chimera CXC-X4,5 showed activity against 4-O-methyl-D-glucurono-D-xylan-Remazol Brilliant Blue R (RBB-xylan) and over-expressed as His-tag fusion proteins. The homogeneous chimeric protein CXC-X4,5 showed significantly improved thermal profiles (optimum temp. 65 degrees C) compared with those of Cex. This was apparently due to the influence of amino acids in modules M4 and M5 inherited from thermophilic XylA. Measured Km and kcat values for the substrate p-nitrophenyl- beta-D-cellobioside were closer to those of Cex; however, the Km and kcat values for the substrate p-nitrophenyl- beta-D-xylobioside were intermediate between those of the 2 parental xylanases. The ability of the chimeric enzyme to produce reducing sugar from xylan was enhanced in comparison with the parental enzymes. These results indicated that amino acid residues in modules M4 and M5 of XylA play an important role in determining enzyme characteristics such as thermal stability, and xylanases with improved properties may thus be prepared by manipulating this segment.