abstract
Aqueous biphasic systems (ABSs) are an interesting alternative for separating industrial enzymes due to easy scale-up and low operational cost. The proteases of Pseudomonas sp. M211 were purified through ABS platforms formed by polyethylene glycol (PEG) and citrate buffer salt. Two experimental designs 2(3) + 4 were performed to evaluate the following parameters: molar mass of PEG (M-PEG), concentration of PEG (C-PEG), concentration of citrate buffer (C-Cit), and pH. The partition coefficient (K), activity yield (Y), and purification factor (PF) were the responses analyzed. The best purification performance was obtained with the system composed of M-PEG = 10,000 g/mol, C-PEG = 22 wt%, C-Cit = 12 wt%, pH = 8.0; the responses obtained were K = 4.9, Y = 84.5%, PF = 15.1, and tie-line length = 52.74%. The purified proteases of Pseudomonas sp. (PPP) were used to obtain hydrolysates of Lupinus mutabilis (Peruvian lupin cultivar) seed protein in comparison with the commercial protease Alcalase (R) 2.4L. A strong correlation between hydrolysis degree and radical scavenging activity was observed, and the highest antioxidant activity was obtained with Alcalase (R) (1.40 and 3.47 mu mol Trolox equivalent/mg protein, for 2,2 '-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and oxygen radical absorbance capacity, respectively) compared with PPP (0.55 and 1.03 mu mol Trolox/mg protein). Nevertheless, the IC50 values were lower than those often observed for antioxidant hydrolysates from plant proteins. PEG/citrate buffer system is valuable to purify Pseudomonas proteases from the fermented broth, and the purified protease could be promising to produce antioxidant protein hydrolysates.
keywords
LIQUID-LIQUID-EXTRACTION; 2-PHASE SYSTEMS; ANTIOXIDANT PEPTIDES; PARTITION BEHAVIOR; SERINE-PROTEASE; IDENTIFICATION; OPTIMIZATION; EQUILIBRIUM; TEMPERATURE; SEPARATION
subject category
Biotechnology & Applied Microbiology; Food Science & Technology
authors
Pillaca-Pullo, OS; Intiquilla, A; Santos, JHPM; Sanchez-Moguel, I; Brandelli, A; Zavaleta, AI
our authors
João Henrique Picado Madalena Santos
PhD Studentacknowledgements
Consejo Nacional de Ciencia y Tecnologia, Grant/Award Number: 007-2014-FONDECYT; Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Grant/Award Number: 306936/2017-8; Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Grant/Award Number: 2018/25994-2; Fundacao para a Ciencia e a Tecnologia, Grant/Award Number: SFRH/BD/102915/2014