Utilization of Integrative Technique for Partial Recovery of Proteases from Soil Microbes (original) (raw)
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Biotechnology and Applied Biochemistry, 2015
The partitioning of protease expressed by Penicillium fellutanum from Brazilian Savanna in a novel inexpensive and stable aqueous two-phase system (ATPS) composed of poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) was studied in this work using factorial design. The aqueous two-phase systems are formed by mixing both polymers with a salt (NaCl) and fermented broth of Penicillium fellutanum. The effects of molar mass (2000, 4000, and 6000 g.mol −1) and concentration (6, 8, and 10 wt%) of PEG and that of NaPA concentration (6, 8, and 10 wt%) on protease partitioning (K) at 25°C were studied. A twolevel factorial design (2 3) was implemented. The effect of Na 2 SO 4 concentration (5, 10, and 15 wt%) on the re-extraction of the enzyme was also analyzed. The partition coefficient K ranged from 77.51 to 1.21, indicating the versatility of the method. The re-extraction was achieved with the addition of 5% Na 2 SO 4 , allowing the partitioning of the protease to the upper phase, whereas total proteins were directed to the bottom phase. The results of partitioning using the PEG/NaPA/NaCl system and that of the subsequent re-extraction with Na 2 SO 4 suggest that this method can be used to purify proteases from fermented broth of Penicillium fellutanum.
Biotechnology and Applied Biochemistry, 2014
The partitioning of proteases expressed by Penicillium restrictum from Brazilian Savanna in an inexpensive aqueous two-phase system composed of poly (ethylene glycol) (PEG) and sodium polyacrylate (NaPA) was studied. The effects of PEG molecular weight and concentration, as well as NaPA concentration and the concentration of fermented broth on protease partitioning were studied. Partitioning into the top PEG-rich phase was increased in systems with smaller PEG-molecular weight, higher NaPA concentration and lower PEG concentration. For most systems studied, purification has been achieved by directing the biomolecule partition to the opposite phase of the other proteins, providing the enzyme purification. The highest partition coefficient was obtained using 20 wt% NaPA, 4 wt% PEG 2000 g mol −1 and 45 wt% fermented broth, leading to a purification factor of 1.98 and partition coefficient of 37.73. The system showed high mass balances and yield, indicating enzyme stability and applicability for industrial processes. The partitioning results using the PEG/NaPA/NaCl system show that this method could be used to purify or concentrate protease from fermented broth.
Process Biochemistry, 2014
The partitioning of proteases expressed by Penicillium restrictum from Brazilian Savanna in an inexpensive aqueous two-phase system composed of poly (ethylene glycol) (PEG) and sodium polyacrylate (NaPA) was studied. The effects of PEG molecular weight and concentration, as well as NaPA concentration and the concentration of fermented broth on protease partitioning were studied. Partitioning into the top PEG-rich phase was increased in systems with smaller PEG-molecular weight, higher NaPA concentration and lower PEG concentration. For most systems studied, purification has been achieved by directing the biomolecule partition to the opposite phase of the other proteins, providing the enzyme purification. The highest partition coefficient was obtained using 20 wt% NaPA, 4 wt% PEG 2000 g mol −1 and 45 wt% fermented broth, leading to a purification factor of 1.98 and partition coefficient of 37.73. The system showed high mass balances and yield, indicating enzyme stability and applicability for industrial processes. The partitioning results using the PEG/NaPA/NaCl system show that this method could be used to purify or concentrate protease from fermented broth.
Biotechnology Letters, 2009
Aspergillopepsin I, an acid protease, was purified using an aqueous two-phase system that comprised various combinations of polyethylene glycol (PEG), NaH2PO4 and NaCl. Partition of the enzyme depended upon the molecular mass of the PEG and the presence of NaCl. With PEG 1500, 4000 and 6000, the partition coefficients were increased by 1,500-, 1,800- and 560-fold compared to values without NaCl. The presence of NaCl (8.75%, w/w) increased purification by 3.8, 9.5 and 2.8 times into these respective PEGs. The optimal aqueous two-phase system for acid protease purification was developed using response surface methodology. This system contained 17.3% of PEG 4000 (w/w), 15% NaH2PO4 (w/w) and 8.75% NaCl (w/w) and provided the best partition coefficient (Ke > 1,100) and yield over 99% in the same phase. The optimal ATPS purification factor of acid protease was over 5.
In this study, extracellular alkaline protease producing bacterial isolates EN-2 and EN-3 from the agricultural soil of C.R.C. Pantnagar were identified as Bacillus megaterium strains EN-2 and Bacillus subtilis strain EN-3 on the basis of 16S rDNA gene sequencing. During kinetic characterization, optimum pH for EN-2 and EN-3 protease activity was 10 and 9, respectively. While optimum temperature, for maximum protease activity in both isolates, was 50°C. The crude extracellular alkaline protease from isolates EN-2 and EN-3 were partially purified using ammonium sulphate fractionation and dialysis to 1.50 and 1.42 fold with 53.77% and 42% recovery respectively. The observed values of V max and K m for protease from isolate EN-2 were found to be 11.57 U/ml and 17.442 mg/ml, while for EN-3 protease these were 42 U/ml and 10.62 mg/ml, respectively. The partially purified enzyme from both bacterial strains was then immobilized in sodium alginate beads with maximum immobilization efficiency at 3% (w/w) and some change in their kinetic properties. The immobilized alkaline protease from EN-2 and EN-3 showed their maximum protease activity at pH 9 and 10, and temperature 60 and 50°C, respectively. Due to these properties, isolated extracellular alkaline proteases from the two strains are ideal choice for application in detergent formulation, leather and food industries.
Acta Periodica Technologica, 2008
The partitioning of acid protease produced by submerged fermentation of Penicillium roqueforti in the aqueous two-phase system polyethylene glycol/sodium phosphate was studied. In the system with 15% polyethylene glycol 4000/25% NaH 2 PO 4 favourable partition coefficient and the top phase yield were obtained, 2.651 and 56.51%, respectively. The purification of acid protease from crude enzyme was optimized by optimizing separation factor. Completely randomized factorial design was applied and the system 15.54% PEG 4000/20% NaH 2 PO 4 appeared to be optimal. Statistically calculated separation factor of 3.553, was similar to the experimentally obtained value (3.084), which indicates the possible application of the factorial design onto ATPS partition and purifycation.
Effect of Culture Conditions on Protease Production and Activity of Protease from Soil Borne Fungi
International Journal of Scientific Research in Environmental Sciences, 2015
Soil is the ideal habitat for many extracellular enzymes producing microorganisms therefore soil samples were collected from botanical garden and medicinal plant garden of Banasthli University, respectively and fungi were isolated by serial dilution agar plate technique. Fungal isolates were screened on gelatin agar plates in order to identify protease producers. For quantitative production of protease, 4 isolates from soil sample 1 and 4 isolates from soil sample 2 were selected based on results of screening. Four fungal isolates from soil sample 1 were named as BY-1, BY-2, BY-3 and BY-4, respectively. Four fungal isolates from soil sample 2 were named as BY-5, BY-6, BY-7 and BY-8, respectively. Without optimization, isolate BY-4 and BY-8 demonstrated maximum protease activity with 76.55±0.91 U/mL and 41.57±1.98 U/mL, respectively. Carbon source, nitrogen source, pH and incubation period for hyper producing fungal isolates (BY-4 and BY-8) were optimized. The optimized culture conditions for protease production from isolate BY-4 were found to be at pH 9.5 after 6 days of incubation using galactose as carbon source and casein as nitrogen source. Maximum protease activity was observed from isolate BY-8 at pH 6.0 after 6 days of incubation using starch as carbon source and casein as nitrogen source. After optimization, isolate BY-8 exhibited maximum protease activity (73.20 U/mL) with mannitol as carbon source and isolate BY-4 demonstrated highest enzyme activity (70.33 U/mL) with galactose as carbon source.
Optimization of Alkaline Protease production from bacteria isolated from soil
Protease producing bacteria were isolated from soil and were identified as Pseudomonas fluorescens, Bacillus subtilus, E.coli and Serratia marscens. Optimization of the fermentation medium for maximum protease production was carried out. The culture conditions like pH, temperature, carbon sources and nitrogen sources were optimized. The Optimum conditions for protease production were found to be 37 o C at pH 10 with Glucose as Carbon Source and Peptone as Nitrogen Source. Soycake and Calcium chloride stimulates the production of protease with 1ml of inoculums size for 48 hrs of incubation period with less concentration of EDTA. Among all studied bacterial isolates, the highest enzyme activity was observed in B. Subtilis. Sonia Sethi et al J. Microbiol. Biotech. Res., 2012, 2 (6):858-865 ______________________________________________________________________________ 859 Available online at www.scholarsresearchlibrary.com