Protein engineering of subtilisins to improve stability in detergent formulations

Enhancing the Thermal Tolerance and Gastric Performance of a Microbial Phytase for Use as a Phosphate-Mobilizing Monogastric-Feed Supplement

“…Because we selectively altered an aspect of the phytase phenotype, we were interested to determine whether the specificity, turnover, and effect of pH on activity were altered in the daughter enzyme. The appA-encoded enzyme has been shown to rapidly remove four phosphates from phytate and then to slowly remove the fifth phosphate, leaving monophosphate inositol (9,28). The kinetics of orthophosphate removal by Phy9X were compared to those for the appA-encoded enzyme, and the enzymes were shown to be identical in rate (specific activity of 1,700 U/mg) and for the removal of four phosphate groups (data not shown).…”

“…These mutations would not have been captured by a typical mutagenesis protocol such as error-prone PCR, which accesses, at random, one base change per codon. It has been noted that the stabilizing effects of point mutations may be cumulative (1,8,18,21,22,28). Adding the single point mutations identified through GSSM evolution created a phytase that retained the desired activity properties while increasing the thermal tolerance and gastric stability of the enzyme.…”

Insights into Thermal Stability from a Comparison of the Glutamate Dehydrogenases from Pyrococcus furiosus and Thermococcus litoralis

“…A natural extension of this argument is that, at any given temperature, improved packing within the structures of the enzymes, from hyperthermophiles leads to lower levels of thermal motion than would be observed in their mesophilic counterparts. Thirdly, the observed substitutions between thermococcal and pyrococcal GluDH are associated with a general decrease in the sulphur content in the more thermostable enzyme, which may be a mechanism whereby problems associated with the oxidation of sidechains can be reduced (von der Osten et al, 1993). However, a low cysteine content does not appear to be a universal feature of thermostable enzymes, as studies on the 5'-methylthioadenosine phosphorylase from SuEfolobus solfuturicus have noted the use of cysteine residues in the formation of multiple disulphide bonds which are thought to contribute to the protein thermostability (Cacciapuoti et al, 1994).…”

Inactivation of a solid-state detergent protease by hydrogen peroxide vapor and humidity

“…Significant effort was done to improve storage stability of detergent proteases by site-directed mutagenesis, where oxidation-labile residues were successfully replaced by more resistant amino acids. Although considerable improvement in Savinase ® storage stability was achieved (Von der Osten et al, 1993), these alterations in the enzyme structure resulted in substantial reduction of specific activity towards synthetic substrates compared to the wild type (Stabile et al, 1996). Another strategy to solve the problem is to physically "isolate" the enzymes from the detrimental causes.…”