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    • Circular dichroism CD provides useful information about prot

    2022-11-24

    Circular dichroism (CD) provides useful information about protein structure, the extent and rate of structural changes and ligand binding. CD is a form of light SC75741 synthesis spectroscopy that measures differences in the absorbance of right- and left-circularly polarized light (rather than the commonly used absorbance of isotropic light) by a substance. The application of CD spectra between 260 nm and approximately 180 nm can be used for determination of different secondary structural types: alpha helix, parallel and antiparallel beta sheet, turn, and others. Johnson, 1990, Woody, 1995 and Greenfield (2006) described in detail how this technique could be performed for determination of protein structure. CD is effective in the investigation of conformational changes in proteins that occur as a result of experimental parameters, such as pH, pressure, temperature and binding of ligands. CD analysis has been the subject of many studies and has generated important information about the effect of temperature (Balan et al., 2006, Thanassoulas et al., 2011), pH (Ramos, 2004, Ribeiro-Jr and Ramos, 2004), charge (Ramos et al., 1999, Regis et al., 2005) and ligands (Balan et al., 2006, Ramos et al., 2007) in the secondary and tertiary structure of proteins. Huang and Tanaka (2015) characterized two putative prolinases PepR1 and PepR2 from Lactobacillus plantarum WCFS1 and studied structural changes using CD spectroscopy. Their results indicated the existence of two isozymes with structural similarity but different catalytic properties. With regards to the effect of HPP on structural changes of proteins, only a limited number of papers are available in the literature. Alexandrakis, Katsaros, Stavros, Nounesis, and Taoukis (2016) investigated the combined effects of HP (500–900 MPa) and temperature (50–70 °C) on the activity and structural modifications of actinidin from kiwi fruit (Hayward var., A. chinensis) and reported reversible changes in its structure in the far- and near-UV spectra. Alexandrakis et al. (2014) correlated the effect of HPP on the activity and structure of pectinmethylesterases (PMEs) obtained from Valencia and Navel oranges. The results indicated that exposure to high pressure was likely to lead to a structurally molten globule-like state, where the protein maintained its secondary structure but the tertiary structure was affected substantially, which also impacted substrate–enzyme binding interactions and led to a reduction in enzyme activity. Menéndez, Schwarzenbolz, and Henle (2006) studied the effects of pressure (0.1–600 MPa) and heat treatment (40 °C) on the structure of microbial transglutaminase and reported loss of the tertiary structure after HPP at 600 MPa and a significant reduction in the alpha-helix content. Tedford, Smith, and Schaschke (1999) examined the combined effects of pressure and temperature on the structure of the milk protein, β-lactoglobulin. Their results indicated more significant effects on the tertiary structure than in the secondary structure of the protein. The main objective of the present research was to study systematically the effects of high pressure on the activity and structure of purified PepX aminopeptidase from S. thermophilus ACA DC 0022. Mathematical models have been used to describe pressure–temperature dependence of activation/inactivation rate constants of the enzyme (Katsaros et al., 2009). Such models can be a useful tool in designing and optimizing HP processes.
    Materials and methods
    Results and discussion
    Conclusions After a series of precipitation and chromatographic procedures, PepX was confirmed by SDS-page to have a molecular mass of 86 kDa. HPP resulted in an increase in purified PepX activity at moderate pressures (100–200 MPa) and temperatures (20–30 °C) but, under more intense processing conditions, inactivation of the enzyme was observed. Both activation and inactivation of the purified PepX were satisfactorily described by a first-order kinetic model. The purified PepX was found to be less sensitive to pressure treatment compared with the crude PepX. The far-UV CD spectra of PepX showed small secondary structure changes at 200 MPa but significant secondary structural changes for treatment at 450 MPa. The near-UV CD spectra of PepX samples showed significant tertiary structural changes for pressures greater than 100 MPa. High pressure treated proteins at 200 MPa appeared to retain their secondary structures but lost their tertiary structure. A further increase in pressure (450 MPa) resulted in denaturation, where both secondary and tertiary structures were severely affected. This study gives insight into structural modifications that at selected HPP and temperature conditions lead to enhanced activity, which has implications in ripening of white cheeses using S. thermophilus as the starter culture.