The consensus concept for thermostability engineering of proteins: further proof of concept.
Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al. (2000)Protein Eng., 13, 49-57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus phytases-10 and -11. Since consensus phytase-10, again, was 7.4 degrees C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3 degrees C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5 degrees C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4 degrees C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.
Lehmann
Martin
M
Roche Vitamins AG, Department VFB, Building 203/112a, CH-4070 Basel, Switzerland. martin.lehmann@roche.com
Loch
Claudia
C
Middendorf
Anke
A
Studer
Dominik
D
Lassen
Søren F
SF
Pasamontes
Luis
L
van Loon
Adolphus P G M
AP
Wyss
Markus
M
D010832:6-Phytase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology; D015202:Protein Engineering; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D010832:6-Phytase