ABSTRACT
In this study, reverse micellar extraction of papain model system was performed using cetyltrimethylammonium bromide (CTAB)/iso-octane/hexanol/butanol system to optimize the forward and back extraction efficiency (BEE). A maximum forward extraction efficiency of 55.0, 61.0, and 54% was achieved with an aqueous phase pH of 11.0, 150 mM CTAB/iso-octane and 0.1 M NaCl, respectively. Taguchi's orthogonal array was applied to optimize the pH of stripping phase, concentration of isopropyl alcohol (IPA) and potassium chloride (KCl) for maximizing BEE. The optimal levels of stripping phase pH, concentration of IPA and KCl were found to be 6, 20% (v/v), and 0.8 M, respectively. Under these optimal levels, the BEE was found to be 88% after which enzyme activity was recovered with 2.5-fold purification. Further optimization was performed using artificial neural network-linked genetic algorithm, where the BEE was improved to 90.52% with pH 6, IPA (%) = 19.938, and KCl (M) = 0.729.
Subject(s)
Carica/enzymology , Cetrimonium Compounds/chemistry , Detergents/chemistry , Micelles , Papain/isolation & purification , Carica/metabolism , Cations/chemistry , Cetrimonium , Neural Networks, Computer , Papain/metabolism , Salts/chemistryABSTRACT
An intracellular glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428 was isolated to apparent homogeneity. The homotetramer enzyme has a molecular mass of 144.4 kDa (MALDI-TOF MS) and an isoelectric point of approximately 8.4. The enzyme is very specific for its natural substrate, L-asparagine. The activity of L-asparaginase is activated by mono cations and various effectors including Na+, K+, L-cystine, L-histidine, glutathione and 2-mercaptoethanol whereas it is moderately inhibited by various divalent cations and thiol group blocking reagents. Kinetic parameters, Km, Vmax and kcat of purified L-asparaginase from P. carotovorum MTCC 1428 were found to be 0.657 mM, 4.45 U µg(-1) and 2.751×10(3) s(-1), respectively. Optimum pH of purified L-asparaginase for the hydrolysis of L-asparagine was in the range of 8.0-10.0, and its optimum temperature was found to be 40 °C. The purified L-asparaginase has no partial glutaminase activity, which can reduce the possibility of side effects during the course of anti-cancer therapy.
Subject(s)
Asparaginase/isolation & purification , Asparaginase/metabolism , Glutaminase/metabolism , Pectobacterium carotovorum/enzymology , Amino Acid Sequence , Asparaginase/chemistry , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Molecular Weight , Sequence Alignment , Sequence Analysis, Protein , Substrate Specificity , TemperatureABSTRACT
Glutaminase-free L-asparaginase is known to be an excellent anticancer agent. In the present study, statistically based experimental designs were applied to maximize the production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. Nine components of the medium were examined for their significance on the production of L-asparaginase using the Plackett-Burman experimental design. The medium components, viz., glucose, L-asparagine, KH2PO4, and MgSO(4).7H2O, were screened based on their high confidence levels (P<0.04). The optimum levels of glucose, L-asparagine, KH2PO4, and MgSO(4).7H2O were found to be 2.076, 5.202, 1.773, and 0.373 g L(-1), respectively, using the central composite experimental design. The maximum specific activity of L-asparaginase in the optimized medium was 27.88 U mg(-1) of protein, resulting in an overall 8.3-fold increase in the production compared to the unoptimized medium.
Subject(s)
Asparaginase/biosynthesis , Biotechnology/methods , Culture Media/chemistry , Pectobacterium carotovorum/enzymology , Antineoplastic Agents/metabolism , Asparaginase/chemistry , Asparagine/metabolism , Glucose/metabolism , Glutaminase/analysis , Models, Statistical , Pectobacterium carotovorum/growth & developmentABSTRACT
Biodegradation of pyrene by Mycobacterium frederiksbergense was studied in a two-phase partitioning bioreactor (TPPB) using silicone oil as non-aqueous phase liquid (NAPL). The TPPB achieved complete biodegradation of pyrene; and during the active degradation phase, utilization rates of 270, 230, 139, 82 mg l(-1)d(-1) for initial pyrene loading concentrations (in NAPL) of 1000, 600, 400 and 200 mg l(-1), respectively, were obtained. The degradation rates achieved using M. frederiksbergense in TPPB were much higher than the literature reported values for an ex situ PAH biodegradation system operated using single and pure microbial species. The degradation data was fitted to simple Monod, logistic, logarithmic, three-half-order kinetic models. Among these models, only exponential growth form of the three-half-order kinetic model provided the best fit to the entire degradation profiles with coefficient of determination (R2) value >0.99. From the experimental findings, uptake of pyrene by the microorganism in TPPB was proposed to be a non-interfacial based mechanism.
Subject(s)
Bioreactors , Mycobacterium/metabolism , Pyrenes/metabolism , KineticsABSTRACT
The engineering of enzymes with altered activity, specificity, and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. In vitro recombination techniques such as DNA shuffling, staggered extension process (StEP), random chimeragenesis on transient templates (RACHITT), iterative truncation for the creation of hybrid enzymes (ITCHY), recombined extension on truncated templates (RETT), and so on have been developed to mimic and accelerate nature's recombination strategy. This review discusses gradual advances in the techniques and strategies used for the directed evolution of biocatalytic enzymes aimed at improving the quality and potential of enzyme libraries, their advantages, and disadvantages.
