Mutagenesis thermal stability

High temperatures can break native S-S bonds and form new S-S bonds which can lock the protein into a denatured eonfiguration [89]. Low pH, sodium dodecyl sulfate. Tween 80, chaotropie salts, and exogenous proteins have been used to protect proteins from thermal inaetivation [90]. Ethylene glycol at 30-50% was used to protect the antiviral activity of P-interferon preparations [91]. Human serum albumin was used in recombinant human interferon-Psei-n which resulted in increased thermal stability [62]. Water-soluble polysaeeharides sueh as dextrans and amylose [92], as well as point-specific (site-directed) mutagenesis [93] have also been used to increase thermal stability of therapeutie proteins and peptides. 

D. Sriprapundh, C. Vielle, and ). G. Zeikus, Molecular determinants of xylose isomerase thermal stability and activity analysis of thermoenzymes by site-directed mutagenesis, Prot. Eng. 2000, 33, 259-265. 

Fujii, K., Iiboshi, M., Yanase, M., Takaha, T., and Kuriki, T. 2006. Enhancing the thermal stability of sucrose phosphorylase from Streptococcus mutans by random mutagenesis. /. Appl. Glycosci., 53, 91-97. 

Site-directed mutagenesis was used to substitute the His residue in position 775 to Asn (His775Asn). This mutation improves the substrate specificity but simultaneously causes a decrease in thermal stability. Some regions of A. calcoaceticus that are responsible for EDTA resistance and for thermal stability were inserted into the E. coli gene by homologous recombination. 

In summary, there is no simple correlation between the occurrence of peripheral ion pairs and thermal stabilization. Even after carefiil selection of specific loci for site-directed mutagenesis on the basis of multiple sequence alignments and homology modeling, the results are not clearly predictable. If we recall that the overall stability of proteins is a minute difference between large contributions of attractive and repulsive forces and the corresponding entropy contributions, this result is not surprising. 

By random and site-directed mutagenesis, variants of the enzyme have been prepared with improved thermal stability. While the wild-type lipase showed a thermal denaturation temperature (I d) of 61 °C, several of the variants obtained with one or more amino acid substitutions in the vicinity of the N-terminal amino acid had a of 64—66°C. One of the variants showed improved CTR hydrolysis at pH 9 and 75°C, and its temperature optimum was determined at 65°C [1]. The thermostability of the enzyme could be further improved by site-specific mutagenesis using synthetic nucleotides [81], and variants with a of 71-72°C were obtained [101]. The optimized PET hydrolase could be employed for PET fabric treatments performed at 75°C, resulting in a de-pilling effect and improved water absorption. 

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