The thermostability of C10 is enhanced by addition of Ca²⁺ and CR.

<p>Thermotolerance of the C10 chimera and parental enzymes in the absence of Ca²⁺ (A), presence of Ca²⁺ (B), presence of CR (C), and presence of both Ca²⁺ and CR (D). Purified cellulases (1μg) were pre-incubated in 50 mM sodium acetate buffer (pH 5.0) at different temperatures for 10 min with or without additional Ca²⁺ 0.05 mM or/and CR 6.25×10⁻³ mM. The residual enzyme activity was then determined on 1% PASC at pH 5.0 at their optimal temperature for 6 hrs. The 100% value of relative activity refers to the optimal activity of each enzyme without thermal treatment.</p

Informative subsets of parental, chimeric, and designed GH5 cellulases.

<p>Informative subsets of parental, chimeric, and designed GH5 cellulases.</p

Modeling of thermostability and thermoactivity of GH5 chimeras.

<p>(A) Stabilizing or destabilizing effects of 8 noncontiguous blocks, relative to P2 (BsCel5A) for the <i>T</i>₅₀ model. Blocks A, B, G and H of P1 are predicted to be stabilizing, but replacement of C, D, E and F had negative effects on thermostability. (B) Stabilizing or destabilizing effects of 8 noncontiguous blocks, relative to P2 for the <i>T</i>_A50 model. Blocks B, E and H of P1 are predicted to be stabilizing.</p

Structural comparison of C10 and P1 at N-termini.

<p>C10 undergoes additional interactions through R22 and K8. The nitrogen, oxygen and carbon atoms of C10 are colored blue, red and cyan, respectively, whereas P1 is illustrated with the carbon, oxygen and nitrogen atoms in green, red and blue, respectively. Grey dashes represent hydrogen bonds.</p

Crystal structure of chimera C10/crown ether complex.

<p>(A) The overall structure of C10 with HEPES, two CR molecules, and a calcium ion (ball-and-stick models). (B) Two C10 monomers (light green and light blue) from different unit cells are in contact with 2-fold symmetry. Two CR molecules interacting across different unit cells, CR^a and CR^b+, are shown as purple and green carbon atoms, respectively. Fo-Fc omit maps (orange) were calculated for the Ca²⁺ and CR molecules at 2 σ level. The calcium ion is bound to two water molecules, the backbone oxygen of G130 and the three carboxylate side chains of D168, D170 and N171, with an octahedral configuration. A CR molecule (CR^a) is bound to K257 and V15 from two different C10 monomers. The second CR molecule (CR^b) has a hydrophobic interaction with CR^a. (C) Comparison of GsCelA P1, BsCe5lA P2 (PDB: 3PZT) and chimera C10 structures. The catalytic domains are superimposed and colored in green, blue and orange, respectively. The N and C termini are also indicated. (D) β4α3 loop region comparison. GsCelA P1, BsCe5lA P2 and chimera C10 are colored as before. The D68 side chain atoms are hydrogen bonded to N70 and N107, while P69 has a hydrophobic interaction with I103. The chimera C10 protein residues are shown as stick models and carbon atoms are colored orange. (E) The Ca²⁺ binding sites in GsCelA P1, BsCe5lA P2 and chimera C10 are superimposed. The carbon atoms of GsCelA P1, BsCe5lA P2 and chimera C10 are colored in light green, light blue and orange, respectively. The Ca²⁺ atom from GsCelA P1 is depicted as a light magenta sphere.</p

Characterization of GH5 chimeras.

<p>(A) Activity profiles of GH5 parents and chimeras at high temperatures. (B) Residual activities (thermostability) of GH5 parents and chimeras after heat-treatment. One microgram of purified cellulases was pre-incubated in 50 mM sodium acetate buffer (pH 5.0) at different temperatures for 10 min. The residual enzyme activities were then determined on 1% PASC at pH 5.0 at their optimal temperature for 6 hrs. Relative activities were compared with the optimal activity of each enzyme without a pre-incubating treatment at 60°C. Some of the enzymes, e.g. C5, do not have the initial point (60°C) of 100% because their optimal activities are not at this reaction temperature and therefore have a slightly lower staring points in the analyses with 60°C pre-incubation temperature. (C) Acid stability of the parents and chosen chimeras. One microgram of purified cellulases was pre-incubated in 50 mM buffer with variable pH for 12 hrs. The residual enzyme activity was determined. Relative activities were compared with the optimal activity of each enzyme without a pre-incubating treatment at pH 5. (D) Long-term activity of the most stable chimera C10 compared with its parents in PASC hydrolysis. One microgram of purified cellulases plus 0.5 μg Novo-188 (β-glucosidase, Novozyme) was added into 1% PASC at 50°C, pH 5.0 for 6, 12, 24, 36, 48, and 60 hrs.</p

Sequence alignment and noncontiguous blocks of the cellulases GsCelA and BsCelA.

<p>(A) The C10 chimera sequence is aligned with P1 and P2 parental sequences. Residues in the α-helices and β-strands are indicated by red cylinders and blue arrows, respectively. P1 is the catalytic core of thermostable GsCelA from <i>Geobacillus</i> sp. 70PC53, and P2 is the catalytic core of <i>Bacillus subtilis</i> 168 BsCel5A. The asterisks indicate catalytic acids and bases. (B) Noncontiguous blocks in the designed GH5 chimera. The eight noncontiguous blocks indicated by colored regions were calculated using the SCHEMA program at = 16.25 and = 47. Amino acids that are the same in both parent enzymes are shown in grey.</p

Profile of SCHEMA block E of GsCelA.

<p>(A) Thermostabilities of parent enzymes, chimeras and mutants are highlighted for block E. (B) Thermal denaturation curves of P2 (without E loop) and C13 (P2 with additional E loop). (C) Thermal denaturation curves of P2 and C13 in the presence of 0.6 mM CMC (1% w/v).</p