The Use of Forced Protein Evolution to Investigate and Improve Stability of Family 10 Xylanases

Abstract

Metal ions such as calcium often play a key role in protein thermostability. The inclusion of metal ions in industrial processes is, however, problematic. Thus, the evolution of enzymes that display enhanced stability, which is not reliant on divalent metals, is an important biotechnological goal. Here we have used forced protein evolution to interrogate whether the stabilizing effect of calcium in an industrially relevant enzyme can be replaced with amino acid substitutions. Our study has focused on the GH10 xylanase CjXyn10A from Cellvibrio japonicus, which contains an extended calcium binding loop that confers proteinase resistance and thermostability. Three rounds of error-prone PCR and selection identified a treble mutant, D262N/A80T/R347C, which in the absence of calcium is more thermostable than wild type CjXyn10A bound to the divalent metal. D262N influences the properties of the calcium binding site, A80T fills a cavity in the enzyme, increasing the number of hydrogen bonds and van der Waals interactions, and the R347C mutation introduces a disulfide bond that decreases the free energy of the unfolded enzyme. A derivative of CjXyn10A (CfCjXyn10A) in which the calcium binding loop has been replaced with a much shorter loop from Cellulomonas fimi CfXyn10A was also subjected to forced protein evolution to select for thermostablizing mutations. Two amino acid substitutions within the introduced loop and the A80T mutation increased the thermostability of the enzyme. This study demonstrates how forced protein evolution can be used to introduce enhanced stability into industrially relevant enzymes while removing calcium as a major stability determinant.

Effect of Glycosylation and Additional Domains on the Thermostability of a Family 10 Xylanase Produced by Thermopolyspora flexuosa

“…The disulfide bridge between the N and C termini (located close to each other) has previously been demonstrated to increase the melting temperature (T m ) of a family 10 xylanase by 4°C (2,35). The thermostability increase achieved by the 6ϫHis tag and CBM in T. flexuosa XYN10A was at the same level (in the range of 3°C in the activity assays).…”

“…Cavity filling and stabilization of loops and N-and C-terminal regions are also important factors (2,35). By studying chimeric xylanase created by the shuffling of Thermotoga maritima xylanases A and B, it was observed that the N-terminal and C-terminal regions of the xylanase structure formed from the TIM barrel are important for high thermostability (20).…”

Exploring Multimodularity in Plant Cell Wall Deconstruction

“…8a). A structural calcium ion conferring stability to Cellvibrio japonicus Xyn10A has been reported (45), but that metal ion is bound by residues from loop L7 (PDB code 1W2P).…”

Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis

“…The majority of studies on the improvement of thermo-tolerance have involved wild-type xylanases with relatively low optimal temperatures of 50-60°C, and most of them belong to family-11 (http://www.cazy.org/) (Andrews et al, 2004;Miyazaki et al, 2006;Palackal et al, 2004;Xie et al, 2006). So far, the generation of thermostable xylanases has only been attempted by mutation of XynA from Thermomyces lanuginosus (Stephens et al, 2007(Stephens et al, , 2009, and the best of the mutants obtained had a half-life of inactivation at 70°C 2.4-fold of that of the wild-type.…”