Protein Thermostability: Mechanism and Control Through Protein Engineering

Chemical modification and protein engineering especially are now the useful tools for thermo-stabilizing proteins, and also for elucidating the mechanism of protein stability. The information on the mechanism so far accumulated indicate that a single or few amino acid replacement(s) in a protein is/are sufficient to enhance protein thermostability. Salt bridges inside protein molecule or decrease of internal or external hydrophobicity, respectively, may contribute to increased thermostability. However, generalized molecular reasons for protein thermostability and generalized methods for protein stabilization have not yet been proposed. Some of typical examples of the application of protein engineering to stabilize proteins are presented. They are based on information concerning the tertiary structure of the proteins or their related proteins. Even if such structural information is unavailable, one can replace amino acid(s) in a protein by mutagenesis of the gene coding for the protein via the application of chemicals to the gene (or the plasmid harbouring the gene) or organism. A promising strategy involving transfer of the identified gene into a thermophile and subsequent growth at higher temperatures (thermal adaptation) is described.     

Gln277 and Phe554 residues are involved in thermal inactivation of protective antigen of Bacillus anthracis

Protective antigen (PA) is the main component of all the vaccines against anthrax. The currently available vaccines have traces of other proteins that contribute to its reactogenicity. Thus, purified PA is recommended for human vaccination. PA loses its biological activity within 48h at 37 degrees C and its thermolability has been a cause of concern as accidental exposure to higher temperatures during transportation or storage could decrease its efficacy. In the present study, we have used protein engineering approach to increase the thermostability of PA by mutating amino acid residues on the surface as well as the interior of the protein. After screening several mutants, the mutants Gln277Ala and Phe554Ala have been found to be more thermostable than the wild-type PA. Gln277Ala retains approximately 45% and Phe554Ala retains approximately 90% activity, even after incubation at 37 degrees C for 48h while in the same period wild-type PA loses its biological activity completely. It is the first report of increasing thermostability of PA using site-directed mutagenesis. Generation of such mutants could pave the way for better anthrax vaccines with longer shelf life.     

Thermostability of β-xylosidase from Aspergillus sydowii MG49

Heating of Aspergillus β-xylosidase at 85°C ± 1°C and pH 5.5–6.0 (optimum for activity), causes irreversible, covalent thermoinactivation of the enzyme, involving oxidation of the thiol groups that are required for catalysis. Exogenous addition of cysteine, DTT, GSH and mercaptoethanol stabilizes the enzyme by extending its half-life. A similar effect is also exhibited by bivalent cations like Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺and Zn²⁺ while, on the other hand Cu²⁺ accelerates thermoinactivation. Chemical modification of crude β-xylosidase with cross-linking agents like glutaraldehyde or covalent immobilization to a nonspecific protein like gelatin and BSA also enhances enzyme thermostability. These results suggest that addition of thiols and bivalent metal ions to a crude β-xylosidase preparation or immobilization/chemical modification enhances its thermal stability, thus preventing loss of catalytic activity at elevated temperatures.     

Enhancement of lipase stability and productivity through chemical modification and its application to latex-based polymer emulsions

The primary focus of this research was to employ amino-group specific chemical modifications for improving the productivity and stability of two commercially produced lipases, Lipase-A from Candida antarctica (CAL_UM) and Greasex from Humicola lanuginosa (HLL_UM), for application in a latex-based paint formulation. The modified lipases showed higher percentage increase (benzoic anhydride-modified, HLL_BA, 150%; PEG-modified, HLL_PEG,162% at 75 °C) as well as higher absolute productivities 41, 50, 52 and 53 μmole substrate mg⁻¹ lipase for unmodified, CAL_PEG, HLL_PEG and HLL_BA, respectively at 37 °C. The half-lives of thermal inactivation for all modified variants were improved from 40 to 166% at 50, 60 and 70 °C relative to unmodified lipases. The higher thermal stability and catalytic efficiency (k_cat/K_m) with concomitant lower activity (k_cat) indicates that enhanced productivity is likely to be due to the modified enzymes being better able to resist thermal denaturation over the time course of the productivity experiments. Importantly, both lipases, CAL_BA (60%) and HLL_BA (55%) retained the highest activity in paint compared with CAL_UM (36%) and HLL_UM (39%) after 20 weeks incubation at 25 °C. The long term stability of the modified lipases illustrates their potential value for commercial paint and other industrial applications.     

Hyperactivation and thermostabilization of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> lignin peroxidase by immobilization in xerogels

This is a continuation of our previous paper on production of lignin peroxidase (LiP) by Phanerochaete chrysosporium in solid substrate fermentation (SSF) medium of corncobs. The enzyme was purified by ammonium sulphate precipitation and ion-exchange fast protein liquid chromatography. Maximum yield of LiP was 13.7 U/gds (units per gram dry substrate) after 5 days of SSF with 70% moisture and 20% (v/w) inoculum. The approximate molecular mass of purified LiP, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 38 kDa. The pH and temperature optima for the LiP were 4 and 40°C, respectively. Immobilization of LiP in hydrophobic xerogels caused hyperactivation of LiP and enhanced its thermostability properties. The K _M and V _max values for immobilized LiP were 10.56 mg/ml and 16.67 μmol/min (120.49 U/mg of protein) as compared to 13 mg/ml and 11.76 μmol/min (85 U/mg of protein), respectively, for free LiP using veratryl alcohol as substrate.