One-step purification and characterization of a haloprotease from Micrococcus sp. PC7 for the production of protein hydrolysates from Andean legumes.

The high content and quality of protein in Andean legumes make them valuable for producing protein hydrolysates using proteases from bacteria isolated from extreme environments. This study aimed to carry out a single-step purification of a haloprotease from Micrococcus sp. PC7 isolated from Peru salterns. In addition, characterize and apply the enzyme for the production of bioactive protein hydrolysates from underutilized Andean legumes. The PC7 protease was fully purified using only tangential flow filtration (TFF) and exhibited maximum activity at pH 7.5 and 40 °C. It was characterized as a serine protease with an estimated molecular weight of 130 kDa. PC7 activity was enhanced by Cu2+ (1.7-fold) and remained active in the presence of most surfactants and acetonitrile. Furthermore, it stayed completely active up to 6% NaCl and kept Ì´ 60% of its activity up to 8%. The protease maintained over 50% of its activity at 25 °C and 40 °C and over 70% at pH from 6 to 10 for up to 24 h. The determined Km and Vmax were 0.1098 mg mL-1 and 273.7 U mL-1, respectively. PC7 protease hydrolyzed 43%, 22% and 11% of the Lupinus mutabilis, Phaseolus lunatus and Erythrina edulis protein concentrates, respectively. Likewise, the hydrolysates from Lupinus mutabilis and Erythrina edulis presented the maximum antioxidant and antihypertensive activities, respectively. Our results demonstrated the feasibility of a simple purification step for the PC7 protease and its potential to be applied in industrial and biotechnological processes. Bioactive protein hydrolysates produced from Andean legumes may lead to the development of nutraceuticals and functional foods contributing to address some United Nations Sustainable Development Goals (SDGs).

Biochemical and biophysical characterization of lysozyme modified by PEGylation.

PEGylation is a strategy that has been used to improve the biochemical properties of proteins and their physical and thermal stabilities. In this study, hen egg-white lysozyme (EC 3.2.1.17; LZ) was modified with methoxypolyethylene glycol-p-nitrophenyl carbonate (mPEG-pNP, MW 5000). This PEGylation of LZ produced conjugates that retained full enzyme activity with glycol chitosan, independent of degree of enzyme modification; its biological activity with the substrate Micrococcus lysodeikticus was altered according to its degree of modification. The conjugate obtained with a low degree of mPEG-pNP/NH(2) modification was studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), demonstrating a spectral peak at m/z 19,988 Da with 77% of its original enzymatic activity. Spectroscopic studies of Fourier transform infrared (FTIR) and circular dichroism (CD) did not show any relevant differences in protein structure between the native and conjugate LZ. Studies of the effects of pH and temperature on PEGylated LZ indicated that the conjugate was active over a broad pH range, stable at 50 degrees C, and demonstrated resistance to proteolytic degradation.

Characterization of inducible lysozyme activity in the hemolymph of Rhodnius prolixus.

1. The characterization and partial purification of an induced lysozyme activity in the hemolymph of adult Rhodnius prolixus inoculated with Micrococcus lysodeikticus is described. 2. Little or no activity against M. lysodeikticus appeared in the first hours after inoculation, but the activity increased reaching a maximum 4 days later, which was maintained to day 12. 3. The activity was characterized as lysozyme on the basis of the following considerations: 1) pH optimum and thermostability at acidic pH; 2) rate of lysis negatively dependent on ionic strength; 3) binding to SP-Sephadex at pH 5.5; 4) apparent molecular weight of 15 kDal. 4. Crude or semi-purified enzyme preparations showed a high degree of stability during handling, freezing and thawing, and standing at 5 degrees C. 5. Incubation of abdominal fat bodies from treated insects resulted in the release of activity into the medium. 6. The relationship between induced lysozyme activity and its role as an insect defense mechanism is discussed.