Engineering Hyperthermostability into a GH11 Xylanase Is Mediated by Subtle Changes to Protein Structure

Abstract

Understanding the structural basis for protein thermostability is of considerable biological and biotechnological importance as exemplified by the industrial use of xylanases at elevated temperatures in the paper pulp and animal feed sectors. Here we have used directed protein evolution to generate hyperthermostable variants of a thermophilic GH11 xylanase, EvXyn11. The Gene Site Saturation Mutagenesis TM (GSSM) methodology employed assesses the influence on thermostability of all possible amino acid substitutions at each position in the primary structure of the target protein. The 15 most thermostable mutants, which generally clustered in the N-terminal region of the enzyme, had melting temperatures (T m ) 1-8°C higher than the parent protein. Screening of a combinatorial library of the single mutants identified a hyperthermostable variant, EvXyn11TS , containing seven mutations. EvXyn11 TS had a T m ϳ 25°C higher than the parent enzyme while displaying catalytic properties that were similar to EvXyn11. The crystal structures of EvXyn11 and EvXyn11 TS revealed an absence of substantial changes to identifiable intramolecular interactions. The only explicable mutations are T13F, which increases hydrophobic interactions, and S9P that apparently locks the conformation of a surface loop. This report shows that the molecular basis for the increased thermostability is extraordinarily subtle and points to the requirement for new tools to interrogate protein folding at non-ambient temperatures.

Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell

“…From previous work in the literature, typical stabilizations obtained, in the first round of selection, is in the range of 2-5°C (refs [11][12][13][14] and the stabilization seen in our experiments is two to three times better than these (median stabilization is 9°C). The excellent performance of Hot-CoFi in identifying stabilizing mutants is likely to arise from the direct read-out of thermostability, as well as a relatively large screening capacity.…”

“…For instance, thermal stability of biocatalysts is of major importance for industrial processes, since performing enzymatic reactions at high temperature has many advantages 9,10 . Therefore, during development, considerable engineering efforts are carried out to stabilize enzymes to increased temperatures [11][12][13][14] .…”

Terminal Amino Acids Disturb Xylanase Thermostability and Activity

“…Deletion of the four C-terminal glycine-rich repeats and the domain increased enzyme thermostability, substrate binding affinity, and activity, respectively (28,30,32). Residue mutations happened generally in the N termini of the 15 most thermostable xylanases, showing that the N-terminal region was more susceptible to thermal unfolding (9). The five N-terminal mutations might confer structural stability, and hence, prevent the overall thermal unfolding of the mesophilic Streptomyces olivaceovirdis xylanase (34).…”

Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop

“…Using thermostable cellulases can increase the production efficiency in industries that prefer high-temperature reaction conditions, such as brewing and bioethanol production (Blumer-Schuette et al 2008;Szijártó et al 2011;Vieille and Zeikus 2001;Viikari et al 2007). To exploit the high catalytic efficiency at elevated temperatures, scientists in both academic and industrial organizations have searched for suitable enzymes by screening the unknown in nature or modifying the known in the laboratory (Dumon et al 2008;Dutta et al 2008;Hua et al 2010;Kapoor et al 2008;Sandgren et al 2003b;Yang et al 2010). In general, there are two strategies of enzyme modification: (1) directed evolution by randomly mutating the enzyme-encoding gene and subsequent selection for desirable properties and (2) rational engineering by specifically mutating the gene, based on the structural information of the enzyme (Bornscheuer and Pohl 2001;Böttcher and Bornscheuer 2010;Percival-Zhang et al 2006;Schülein 2000).…”