Purificação parcial e caracterização de uma protease bacteriana e sua aplicação na produção de hidrolisados bioativos a partir de proteína isolada de soja

Abstract

Microbial diversity is a valuable resource for obtaining proteases which, depending on their properties, may be suitable for different biocatalytic processes. Protein hydrolysis is a relevant topic considering the bioactivities exhibited by protein hydrolysates, attributed to peptides released in this process. In this investigation, a protease from Bacillus sp. CL 18 was partially purified, characterized and used in the production of soy protein isolate (SPI) hydrolysates, which were subsequently evaluated for potential in vitro bioactivities. Partial purification was performed through ammonium sulfate precipitation (25-50% saturation) and gel-filtration chromatography. Chromatography fractions displaying proteolytic activity were pooled and used as partially purified protease (PPP; 1,565 U/mL). This process resulted in a purification factor of 60.7 fold (40,593 U/mg protein) with a 21.3% yield (32,150 U). PPP exhibited optimal activity at 55 °C (51-59 °C) and pH 8.0 (7.4-8.8). In the presence of ions (1 and 5 mM), activity was stimulated by Ca2+ (~7%), unaffected by Mg2+, but inhibited by Mn2+, Cu2+, Co2+, Fe2+ and Zn2+. Tween 20 and dimethyl sulfoxide did not influence the activity, which was inhibited by Triton X-100, sodium dodecyl sulfate (SDS) and β-mercaptoethanol. Inhibition by phenylmethylsulfonyl fluoride (PMSF) characterized PPP as a serine protease. Thermal inactivation followed first order kinetics. In the absence and presence of Ca2+ (5 mM), PPP half-life was 2.0 and 76.1 min at 60 °C, respectively, demonstrating the importance of Ca2+ for thermostability. PPP was able to hydrolyze casein > SPI >>> keratinous substrates. The hydrolysis of SPI (10 g/L) by the PPP (2% v/v = HPIS2; 4% v/v = HPIS4), performed at 55 °C, in the presence of 5 mM Ca2+ , for up to 5 h, resulted in increased concentrations of soluble proteins, reaching 4.8 (HPIS2) and 5.2 mg/mL (HPIS4) after 4 h, as compared to PIS (~1.95 mg/mL). Scavenging of 2,2'-azinobis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical by SPI (~17.7%) was increased to 69% after 4 h. Scavenging of radical 2,2-diphenyl 1-picrilhidrazil (DPPH) radical was increased to 39.8-42.2% after 4 h of hydrolysis, as compared to SPI (~27.4%). In comparison to SPI, increases in the reduction of Fe3+ have been demonstrated during hydrolysis; however, there were no consistent effects on Fe2+ chelation. It is indicated that the action of PPP released peptides with antioxidant capacity exerted by radical scavenging and electron transfer. Hydrolysates (4 h) inhibited angiotensin-converting enzyme (ACE) by 89.1% (HPIS4) and 65.6% (HPIS2), as compared to SPI (8-12%). Inhibition of the enzyme dipeptidyl peptidase IV (DPP IV) by SPI (4.1-5.2%) was also increased by hydrolysis, reaching 29.4-33.0% after 4 h. ACE and DPP IV inhibitions by the hydrolysates suggest the presence of peptides with antihypertensive and antidiabetic potentials, respectively. This PPP, active and stable at moderate conditions, can be used to produce bioactive hydrolysates from SPI. Both PPP and hydrolysates can find applications in food science and technology.