ABSTRACT
Microbial proteases are widely used as commercial enzymes, which have an active role in several industrial processes. The aim of this study was to investigate the production and properties of extracellular proteases from Barrientosiimonas sp. strain V9. The cultivation conditions for protease production were studied using different carbon and nitrogen sources. Maximum protease production was obtained in medium containing 25 g L-1 sucrose, 7 g L-1 KNO3, and initial pH 7.0 at 35 °C and 150 rpm during 72 h. Under these conditions, maximum proteolytic activity reached 1200 U mL-1. The enzyme extract showed optimum activity at 60 °C, pH 9.0, and was stable from 30 to 50 °C within a pH range from 4.0 to 10.0 and NaCl concentration up to 2.5 M. The enzyme was stable in the presence of EDTA, urea, Triton X-100 and laundry detergent (sodium lauryl sulfate as main component). The addition of 1% sodium dodecyl sulfate, Tween-80, or Tween-20 increased the activity by 183% and 119% respectively, while 2-mercaptoethanol reduced the activity to 71%. Casein zymogram analysis revealed three hydrolysis zones suggesting that Barrientosiimonas sp. V9 expresses proteases with molecular weights about 60, 45, and 35 kDa, which were inhibited in the presence of phenylmethylsulfonyl fluoride. Barrientosiimonas sp. V9 produces halotolerant serine proteases with great biotechnological potential.
Subject(s)
Actinobacteria/enzymology , Extremophiles/enzymology , Peptide Hydrolases/metabolism , Culture Media , Enzyme Stability , Hydrogen-Ion Concentration , Peptide Hydrolases/biosynthesis , Proteolysis , TemperatureABSTRACT
Erythrina edulis Triana ex Micheli is a protein-enriched legume traditionally used for both dietary and medicinal purposes. In this paper, protein concentrate was obtained from the seed flour. SDS-PAGE analysis revealed a high number and intensity of bands in the range between 10 and 90 kDa. Neutrase, Flavourzyme, and Alcalase were used to hydrolyze the protein concentrate at different times. By SDS-PAGE, the lower resistance of proteins to Alcalase action was observed, providing hydrolyzates with higher radical scavenging activity. The 120 min-hydrolyzate showed ORAC and TEAC values of 2.51 and 0.91 µmol Trolox equivalents/mg of protein, respectively. A fraction lower than 3 kDa and rich in hydrophobic and aromatic amino acids was demonstrated to be mainly responsible for the observed activity. E. edulis could be a new alternative in the formulation of functional foods not only for its high protein content but also for the potential biological properties of its hydrolyzates.
Subject(s)
Antioxidants/chemistry , Erythrina/chemistry , Peptides/chemistry , Plant Proteins/chemistry , Seeds/chemistry , KineticsABSTRACT
El objetivo de este estudio fue caracterizar bacterias halófilas con actividad amilolítica provenientes de las Salinas de San Blas-Junín, ubicadas en los Andes peruanos aproximadamente a 4100 m de altitud. Este estudio se realizó con 34 bacterias aisladas de muestras de suelos las cuales se cultivaron en agar agua de sales (SW) 5 % conteniendo extracto de levadura 0,5 % y almidón 1 %. El 41 % de bacterias mostró la capacidad de hidrolizar almidón, éstas fueron caracterizadas mediante pruebas fisiológicas y bioquímicas convencionales. Tres bacterias fueron Gram-negativas y once Gram-positivas. El 21 % (3/14) creció en un amplio rango de concentración de sales, entre 5 y 20 %. El 14 % (2/14) de las bacterias presentó actividad lipolítica, proteolítica y nucleolítica, y el 29 % (4/14), presentó actividad proteolítica y nucleolítica. Las bacterias se identificaron mediante los perfiles de restricción de los genes ribosómicos 16S amplificados, las enzimas usadas fueron Hae III, BstU I, Hinf I y Cfo I. Los genes ribosómicos 16S de siete bacterias que presentaron perfiles de ADN diferentes se amplificaron, secuenciaron y analizaron mediante programas bioinformáticos. Del análisis fenotípico y molecular de las 14 bacterias amilolíticas se obtuvieron dos grupos, uno perteneciente al género Halomonas (3) y el otro, al género Bacillus (11). Las bacterias amilolíticas caracterizadas podrían ser de potencial uso a nivel industrial.
The aim of this study was to characterize halophilic amylolytic bacteria from San Blas Salterns-Junin, located in the Peruvian Andes at approximately 4 100 m of altitude. This study was conducted with 34 bacteria isolated from soil samples which were cultured in salt water medium (SW) 5 % containing 0,5 % yeast extract and 1 % starch. It was found that 41 % were starch-degrading bacteria, which were further characterized with conventional physiological and biochemical tests. Three bacteria were Gram-negative and eleven Gram-positive. Also, 21 % (3/14) was able to grow in a wide range of saltconcentration from 5 to 20 %. We reported that 14 % (2/14) of bacteria had all lipolytic, proteolytic and nucleolytic activity, and 29 % (4/14) had both proteolytic and nucleolytic activity. Bacteria were identified by restriction 16S ribosomal genes profiles, enzymes used were Hae III, BstU I, Hinf I and Cfo I. 16S ribosomal genes of seven isolated wich showed different DNA profiles were amplified, partial sequenced and analyzed using bioinformatic programs. By both phenotypic and molecular analysis of 14 amylolytic bacteria two groups were obtained, one belonged to the genus Halomonas (3) and the other, to the genus Bacillus (11). The characterized amylolytic bacteria could have a potential industrial use.
