urification of Pseudomonas sp. proteases through aqueous biphasic systems as an alternative source to obtain bioactive protein hydrolysate

Author(s)

Pillaca-Pullo, Omar S. ^[1] ; Intiquilla, Arturo ^[1] ; Santos, Joao H. P. M. ^{[2, 3]} ; Sanchez-Moguel, Ignacio ^[2] ; Brandelli, Adriano ^[4] ; Zavaleta, I, Amparo

Affiliation

^[1] I, Univ Nacl Mayor San Marcos, Fac Farm & Bioquim, Lab Biol Mol, Lima - Peru

^[2] Univ Sao Paulo, Sch Pharmaceut Sci, Dept Pharmaceut Biochem Technol, Sao Paulo - Brazil

^[3] Univ Aveiro, CICECO Aveiro Inst Mat, Dept Chem, Aveiro - Portugal

^[4] Univ Fed Rio Grande do Sul, Dept Ciencia Alimentos, Lab Bioquim & Microbiol Aplicada, Porto Alegre, RS - Brazil

• Document type: Journal article
• Source: BIOTECHNOLOGY PROGRESS; v. 37, n. 3 APR 2020.
• Web of Science Citations: 0

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. (AU)

• FAPESP's process: 18/25994-2 - Development of novel platforms for PEGylation of proteins with therapeutic potential using microfluidics
• Grantee: João Henrique Picado Madalena Santos
• Support Opportunities: Scholarships in Brazil - Post-Doctoral