Design of mutants for enhanced thermostability of ß-glycosidase BglY from Thermus thermophilus.

Three design strategies, based on rational and semi-rational approaches, were employed to investigate the functional impact of thermostability-related amino acid substitutions in the ß-glycosidase BglY from Thermus thermophilus. Five beneficial mutations were identified, of which 1 mutation was located in the active cavity of the enzyme and contributed to the released substrate inhibition. Combining all 5 beneficial substitutions resulted in the mutant HF5 with a 4.7-fold increase in half-life, with thermal inactivation at 93 °C, and complete lack of substrate inhibition toward the substrate p-nitrophenyl-ß-D-glucopyranoside at lower reaction temperatures. The results of this study provide valuable information on amino acid substitutions related to thermostability and substrate inhibition of BglY.

Three amino acid changes contribute markedly to the thermostability of ß-glucosidase BglC from Thermobifida fusca.

Thermostability of ß-glucosidase was enhanced by family shuffling, site saturation mutagenesis, and site-directed mutagenesis. Family shuffling was carried out based on ß-glucosidase BglC from Thermobifida fusca and ß-glucosidase BglB from Paebibacillus polymxyxa with the help of synthetic primers. High-throughput screening revealed mutants with higher thermostability than both parental enzymes. The most thermostable mutant VM2 containing three key amino acid changes in L444Y/G447S/A433V had a 144-fold increase in half-life of inactivation as compared to the parental enzyme BglC. The mutant VM2 showed 28% and 94% increase in k(cat) towards p-nitrophenyl-ß-D-glucopyranoside (pNPG) and cellobiose, respectively. The mutant with enhanced stability would facilitate the recycle of ß-glucosidase in the bioconversion of cellulosic biomass.

Introduction of glycine and proline residues onto protein surface increases the thermostability of endoglucanase CelA from Clostridium thermocellum.

A saturation mutagenesis library was constructed at the position 329 of the endoglucanase CelA from Clostridium thermocellum based on previous results (Yi and Wu, 2010), and one mutation, S329G, was identified to contribute to the enhanced thermostability. The result inspired a rational design approach focusing on the introduction of Gly or Pro residue onto the protein surface, which led to the identification of two additional beneficial mutations, H194G and S269P. Combination of these three mutations resulted in a mutant with a 10-fold increase in half-life of inactivation (60 min) at 86°C without compromising activity compared with the wild-type. Its reaction temperature for maximum activity increased from 75 to 85°C. The results provide valuable thermostability-related structural information on this thermophilic enzyme.

Mutations from a family-shuffling-library reveal amino acid residues responsible for the thermostability of endoglucanase CelA from Clostridium thermocellum.

We constructed a library of chimeras from the major endoglucanase, CelA, of Clostridium thermocellum and a less stable endoglucanase CelB from Clostridium josui with multiple point mutations using low-fidelity family-shuffling method. Mutations that inactivated the enzyme were rapidly eliminated with high-throughput screening. The activities and thermostabilities of selected variants were evaluated, and four amino acid substitutions, K249R, P258S, S329N and E355G, were identified as having significant impact on the thermostability of CelA without affecting enzymatic activity. In the crystal structure of CelA, most of them are away from the activity cleft and are responsible for the stabilization of secondary structures.

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

Protein engineering of the thermostable xylanase XT6 from Geobacillus stearothermophilus was performed to obtain enzymes with improved thermal tolerance. Mutants producing such enzymes were obtained after several rounds of directed evolution using error-prone PCR and sequence family shuffling, in combination with a consensus-based semi-rational approach. The most thermostable mutant enzyme contained 13 amino acid substitutions and its half-life of inactivation was 52-fold of that of the wild-type. Its reaction temperature for maximum activity increased from 77 degrees C to 87 degrees C, and catalytic efficiency (k(cat)/K(m)) increased by 90%. The mutant is of potential interest for industrial applications.

[Selection and interaction of Ni2+ metal-binding peptides].

Ni2+ binding peptides were selected from phage random dodecapeptide library by metal affinity chromatography. After four rounds of biopanning, phage amplification and DNA sequencing, a group of peptide sequences were obtained. GenBank blast found no homogenous sequences, Clustal W analysis showed no motifs but they were really riched in histidines and contained di- or more histidines(his). Affinity assays of selected metal-binding phages for various metal-charged NTA resins and the experiments of E. coli suppression and detoxification gave positive results for Ni2+ binding peptides: strong affinities for Ni2+ were found for Ni2+ binding peptide displayed phages, as well as for other metals (Cu2+, Co2+, Zn2+, Cr2+, Cd2+); affinities of the binding peptides for Cu2+, Ni2+, Co2+ and Zn2+ were much higher than that of Cd2+ and Cr2+; in addition, Ni2+ binding peptides displayed phages had effects on E. coli as to enhance the tolerance and detoxification of E. coli for heavy metals when exposed to Ni2+ and Cd2+. The interactions of meal binding peptides for heavy metals were also disclosed by microscopic observation. The research offered great values for the study of the interaction between metals and peptides, as well as in other areas such as heavy metal bioremediation.