Protein‐Engineering Study of Contribution of Conceivable D‐Serine Residues to the Thermostabilization of Ovalbumin under Alkaline Conditions

Abstract

During storage of shell eggs, an egg-white protein, ovalbumin is converted into a molecular species with higher thermostability (Delta T(m)=8 degrees), which is named as S-ovalbumin. Since the pH of egg white is raised to 9.5 in that process, particular chemical modifications could occur. Although any evidence for relationship between chemical modifications and the thermostabilization had not been established, our X-ray crystallographic model for S-ovalbumin demonstrated that three serine residues (S164, S236, and S320) might be in D-configuration. Then, whether these serine residues contribute to the thermostabilization in S-ovalbumin is investigated by protein-engineering approach. By alkaline treatment, S236G mutant was converted to a more thermostable form, as well as ovalbumin has been converted to S-ovalbumin. Hence, the Ser236 is regarded to have no contribution to S-ovalbumin formation. On the contrary, S164G and S320G did show accelerated conversion to thermostable forms in comparison with ovalbumin or wild-type recombinant protein. When these residues were replaced with valine, which has a slower racemization rate, Delta T(m) after alkaline treatment had become half of that for the wild-type recombinant ovalbumin. Therefore, the process of S-ovalbumin formation is deduced to consist of two steps, which is closely related to the two serine residues.

Storage induced conversion of ovalbumin into S-ovalbumin in eggs impacts the properties of pound cake and its batter

“…Later, it was discovered that there are in fact two intermediate forms which both have one isomerized serine, i.e. Ser-164 or Ser-320, while Sovalbumin contains both serines in their isomerized form (Ishimaru, Ito, Tanaka, & Matsudomi, 2010;Takahashi, Maeda, Yamasaki, & Mikami, 2010).…”

Transition of serine residues to the d-form during the conversion of ovalbumin into heat stable S-ovalbumin

“…It has been suggested that the S-ovalbumin contains d-Ser residues and these residues may contribute the thermostability of the protein [14,15]. We demonstrated the presence of d-Ser residues in ovalbumin using a technique combining deuterium labeling of amino acids and LC-MS/MS.…”

“…All mutant plasmids were prepared using the QuickChange PCR mutagenesis kit (STRATA-GENE, CA, USA), as described previously [15].…”

“…These serine residues are exposed to the molecular surface, and analysis of ovalbumin mutants suggested that the Ser164 and Ser320 residues contribute to the thermostability of the protein [14,15]. However, it was not chemically verified that S-ovalbumin contains d-Ser residues.…”

Accelerated identification of serine racemase inhibitor from Centella asiatica

“…For SR inhibition assay using eluted fractioned, 5 µl and 7 µl of eluted fraction from pull down assay was used. For SR inhibition assay using purified compound madecassoside (MDS), 5,10,20,30,40,50,80, 100 µM of MDS was used. IC50 value for madecassoside was calculated using GraphPad Prism 7.…”