Studies on pH and thermal deactivation of pectolytic enzymes from Aspergillus niger

Abstract: Departamento de Bioquimica e Microbiologia Instituto de Biociencias Universidade Estadual Paulista, UNESP, Avenida 24A, 1515, CEP 13506-900 Rio Claro, SP

Abstract: Kinetic properties and thermal inactivation of polygalacturonase (PG) assayed in commercial preparations (Rapidase C80, Pectinase CCM and Pectinex 3XL) were studied. The PG activities in all samples followed Michaelis–Menten kinetics and their catalytic efficiencies were calculated. The optimum pH for enzyme activity was 4.7 for Pectinex 3XL and 4.0 for both Rapidase C80 and Pectinase CCM. The PG optimum temperature lay within the range 50–55 °C. The activation energies of Rapidase C80, Pectinase CCM and Pectinex 3XL PG were 26.5, 45.6 and 4.2 kJ mol −1 , respectively. The thermal inactivation curves were not linear in the range 40–60 °C. The biphasic curves fitted to a two-fraction first-order model suggested the presence of two groups of PG isozymes in Rapidase C80 and Pectinase CCM. Half-life values demonstrated that Pectinase PG had the largest thermal stability. Additionally, thermodynamic activation parameters, such as Δ E # , Δ H # , Δ G # and Δ S # , were calculated.

Abstract: 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 × 103 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.

Abstract: Today pectinases are upcoming industrially important bacterial enzymes. It can be produced by a variety of microorganisms. These enzymes act on pectin, which is the major component of middle lamella in plant cell wall. Pectinolytic enzymes are classified according to their mode of attack on the galacturonan part of the pectin molecules such as protopectinases, esterase’s and depolymerases. As we know that microbial enzymes work depends up on the type of enzymes application, temperature, concentration, and pH and so on, therefore, pectinase enzyme also differentiated according to their physical and chemical factors too. The biochemical structures of pectinases include members of all the major classes and the structure–function relationship, studies of a few available complexes of pectinases with substrate/analogs could be considered as prototypes for related family member and the molecular characterization of pectinolytic enzymes is also well documented. Furthermore, it provides a bird’s eye view of the possible application of these enzymes in commercial sector.

Abstract: Exo-polygalacturonase enzyme produced by Aspergillus sojae ATCC 20235 was purified using three-phase partitioning (TPP), an emerging bio-separation technique where a single step as compared to the classical multi-step purification was used. Using this technique, crude enzyme solution (pH 6.6) saturated to 30% (w/v) with ammonium sulphate and with a crude extract to tert-butanol ratio of 1:1 (v/v) at 25 °C resulted in 25.5% recovery of exo-polygalacturonase with a 6.7-fold purification. The purified enzyme was characterized with respect to its activity and stability at various pH and temperature ranges. Optimum pH and temperature for maximum activity were determined as pH 4 and 55 °C. The enzyme was stable at both acidic and alkaline pH for 2 h at 30 °C. The thermal stability study showed that the purified enzyme had an inactivation energy of 68.41 kcal/mol and a half-life (t1/2) value of 3.6 h at 75 °C presenting a large thermal stability. The kinetic constants Km and Vmax using polygalacturonic acid as substrate were 0.75 g l−1 and 1.14 μmol min−1, respectively. SDS-PAGE profiling revealed that the purified exo-polygalacturonase had two bands with the molecular weights of 36 and 53 kDa. The enzyme was completely inhibited in the presence of Mn2+ and SDS and induced significantly by EDTA, glycerol and β-mercaptoethanol.

Abstract: Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins, a number of modified analytical procedures utilizing this reagent have been reported for the determination of proteins in serum, in antigen-antibody precipitates, and in insulin. Although the reagent would seem to be recommended by its great sensitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard to effects of variations in pH, time of reaction, and concentration of reactants, permissible levels of reagents commonly used in handling proteins, and interfering substances. Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.

Abstract: The calculation of absolute reaction rates is formulated in terms of quantities which are available from the potential surfaces which can be constructed at the present time. The probability of the activated state is calculated using ordinary statistical mechanics. This probability multiplied by the rate of decomposition gives the specific rate of reaction. The occurrence of quantized vibrations in the activated complex, in degrees of freedom which are unquantized in the original molecules, leads to relative reaction rates for isotopes quite different from the rates predicted using simple kinetic theory. The necessary conditions for the general statistical treatment to reduce to the usual kinetic treatment are given.

Abstract: Pectinases are one of the upcoming enzymes of fruit and textile industries. These enzymes break down complex polysaccharides of plant tissues into simpler molecules like galacturonic acids. The role of acidic pectinases in bringing down the cloudiness and bitterness of fruit juices is well established. Recently, there has been a good number of reports on the application of alkaline pectinases in the textile industry for the retting and degumming of fiber crops, production of good quality paper, fermentation of coffee and tea, oil extractions and treatment of pectic waste water. This review discusses various types of pectinases and their applications in the commercial sector.