Full Factorial Design (3 2) used to determine the influence of pH and temperature on optimum activity of lipase obtained through solid-state fermentation by Aspergillus niger (strain O-4) and results of lipolytic activity (U/g). Results of mean and standard deviation.The enzyme activity was determined using the method standardized by Burkert et al.
which is based on titration with NaOH of fatty acids released by the action of lipase in the extract on the triacylglycerols of olive oil emulsified in arabic gum. The following were added to 250 mL flasks: 2 mL buffer prepared according to the objective of the test, 5 mL of emulsion prepared with 75 mL of 7% arabic gum, and 25 mL of olive oil. Next, 1 mL of enzyme extract was added to this system and it was incubated at temperatures described in the experimental design for 30 min. After incubation, the reaction was stopped by adding 15 mL of acetone: ethanol: water (1: 1: 1) and the released fatty acids were titrated with a solution of 0.01 mol/L NaOH using phenolphthalein as indicator.
One unit of activity was defined as the amount of enzyme that releases 1 μmol of fatty acid per minute per mL of enzyme extract of submerged fermentation (1 U = 1 μmol/minmL) or per g of fermented brand (1 U = 1 μmol/ming) of solid-state fermentation, under the test conditions. Temperature Stability of Enzymatic ExtractsThermostability of lipases obtained through submerged and solid-state fermentation was measured by incubating the enzyme extract at 35°C to 90°C. Aliquots were periodically taken to measure lipolytic activity, using the optimum temperature and pH for enzyme activity, obtained as mentioned in Section, to each enzymatic extract. The experiments were duplicated.For the enzymes obtained through solid-state fermentation it was possible to calculate the Arrhenius thermal deactivation and activation energy for thermal destruction constants. Therefore, the data of enzymatic activity at each temperature tested were used to calculate the residual lipase activity over time. The constant of thermal deactivation ( ) at each temperature was calculated by linear regression of the data of Ln versus time, according to the Arrhenius kinetic model, considering that inactivation of the enzyme obeys first-order kinetics, as in the following. ConsiderAfter integration,Considering that the enzyme concentration is directly proportional to the enzymatic reaction speed,We get the following:From the thermal deactivation constants at each temperature, the half-lives ( ) were obtained which corresponds to the time required, at the temperature tested, so that 50% of the initial enzyme concentration is inactivated:The activation energy for thermal destruction of the enzyme was calculated from.
The value of was obtained from the inclination of the regression line of ln versus: where = enzyme concentration, = residual activity of the enzyme, = time (min), = thermal deactivation constant, A = Arrhenius factor (depending, among other things, on the contact area), = activation energy, R = ideal gases constant (8.314 J mol −1K −1), and T = absolute temperature (K). PH Stability of Enzymatic ExtractsThe effect of pH on the stability of enzymes obtained through submerged fermentation was determined by treating 1 mL of enzyme extract with 2 mL of buffer solutions at pH 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, and 9.0 for 24 hours at 25°C. The buffers used were 0.1 mol/L citrate (pH 3.5), 0.2 mol/L acetate (pH 4.0 to 5.5), 0.2 mol/L phosphate (pH 6.0 to 8.0), and 0.2 mol/L glycine (pH 9 and 10). Enzyme activity in initial and final times was carried out at optimum temperature and pH for enzyme activity, obtained from the results of the assays of Section. The experiments were duplicated.The stability of enzymes obtained through solid-state fermentation was assessed through their extraction from the fermented medium using the following buffer solutions: 0.1 mol/L citrate (pH 3.5), 0.2 mol/L acetate (pH 4.0, 4.5, 5.0, and 5.5), 0.2 mol/L phosphate (pH 6.0, 6.5, 7.0, 7.5, and 8.0), and 0.2 mol/L glycine (pH 9 and 10).
The extracts were kept at 25°C for 24 h and the residual lipolytic activity was determined at optimum temperature and pH for enzyme activity, according to the results obtained from the assays of Section. Results and DiscussionIn the submerged culture fermentation the microorganisms grow in a liquid medium in which the nutrients are dissolved. In solid-state fermentation the microorganisms grow on the surface of a solid matrix in which the nutrients are adsorbed, and the moisture does not exceed the water retention capacity of this matrix ,. These differences between production methods as well as the differences between the microorganisms used in fermentation processes can lead to obtaining lipases with different characteristics. Effect of pH and Temperature on the Optimal Activity of Enzymatic ExtractsThe pH and temperature have great influence on the enzyme activity, being important to define these parameters for the characterization of the enzymes obtained.
After fungal growth in culture media of submerged and solid-state fermentations, enzymatic extracts were obtained as described in Section and used in the assays mentioned in Section. The results of enzymatic activities were presented in Tables and, which also shows the experimental conditions of the experimental designs used to determine optimum temperatures and pH of the enzymes produced through submerged and solid-state fermentations, respectively.The highest lipolytic activities in the solid-state fermentation (Table ) may be due to the characteristics of this type of cultivation when compared with submerged cultivation. In the solid state fermentation, the concentration of the final product is higher and the fungus has the appropriate characteristics, as tolerance to low water activity and production of enzymes through hyphae. Thermal stability of lipases produced in submerged fermentation using Aspergillus flavus (strain O-8): (a) 40°C, 50°C, 70°C, and 80°C; (b) 70°C, 80°C, and 90°C.In order to confirm the data obtained, tests were carried out at 70°C, 80°C, and 90°C for 8 h, and the results (shown in Figure ) confirm the aforementioned behavior, with an initial enzyme inactivation, caused by thermal shock, and later stability.
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Furthermore, the enzymes exhibited greater stability at higher temperatures in the first hour of testing. The lipases were stable at 70°C to 90°C, with mean residual activities of around 72% after the first hour of incubation.Figure shows the thermal destruction kinetics of the enzyme produced through solid-state fermentation between 35°C and 90°C, which follows the pattern of first-order thermal destruction predicted by the Arrhenius model. Table shows the thermal deactivation constants between 35°C and 90°C, obtained from angular coefficients of the curves shown in Figure, as well as the determination coefficients of regression and the half-life of enzymes at each temperature.
The enzyme had higher thermal stability at 35°C and 40°C, which can be observed from the high half-lives ( ), around 6 and 4.3 h, respectively. Above 50°C, the half-life considerably decreased to 29 min between 60°C and 70°C. Figure shows the graph of ln ( ) as a function of absolute temperature (K), used to calculate the energy of thermal deactivation, which was 60.33 kJ/mol for the studied enzyme. Kinetics of thermal destruction of the enzymatic extracts produced by Aspergillus niger in solid-state fermentation: (a) at temperatures of 35°C to 90°C.: enzyme residual activity, (b) linear regression of the thermal deactivation constants obtained at 35°C to 90°C (ln of data) as function of inverse of absolute temperature for calculating the energy of thermal deactivation of the enzyme.Lipase obtained with solid-state fermentation showed higher energy of deactivation than those obtained by Diaz et al. and Lima et al. , which were 30 and 34.2 kJ/mol for the lipases produced by Rhizopus homothallicus and Bacillus megaterium, respectively. However, they had lower deactivation energy than the ones obtained by Maldonado , which were 330, 140, and 182 kJ/mol for the lipases produced by Geotrichum candidum in media containing peptone, hydrolyzed yeast, and macerated clarified corn water, respectively.
The activation energy reflects the dependence of the thermal deactivation constant with respect to temperature , , and the higher the constant, the greater the variation of the thermal deactivation constant with the temperature variation.Razak et al. reported that fungal lipases in general are unstable above 40°C, with moderate stability, contrary to what has been observed in lipases produced by bacteria such as Bacillus and Pseudomonas , which are thermostable above 60 C. However, the lipases produced by Rhizopus sp. Maintained 50% or more of their activity when heated for 60 min between 40 and 55°C and lipases produced by Geotrichum-like R59 showed thermostability, with maximum residual activity after incubation at 60°C for 1 h.Shu et al. reported that the lipases produced by Antrodia cinnamomea had 50% residual activity between 25°C and 40°C. Lipase from Aspergillus niger NCIM 1207 was stable at 40°C for 3 h; however the treatment at 50°C for 1 h caused 52% loss of activity.
showed that the lipases produced by Penicillium corylophilum were completely inactivated after 30 min at 60°C. However, the lipases produced by Rhizopus sp. Had moderate thermostability, with 70% residual activity at temperatures of 40 to 55°C. Ginalska et al.
showed that the lipases produced by Geotrichum sp. Had 100% residual activity after 1 hour of incubation at 60°C, and 50% residual activity at 70°C for 45 min. Furthermore, Sharma et al.
reported that the lipase produced by Bacillus sp. RSJ-1 presented 90 and 70% residual activity after treatment at 50°C for 120 and 240 min, respectively. Lipase obtained from Iftikhar et al. showed that lipases retained 80% of its activity at 25–30°C by wild and 100% of its activity at 20–50°C by mutant strain of R. By further increase in the incubation temperature, the activity of the enzyme was greatly inhibited.Compared with the aforementioned enzymes, lipases obtained in this study through submerged fermentation had higher thermostability and may have applications in industrial processes that require high temperatures. Enzymatic processes that occur at higher temperatures have higher reaction rates.
It may be possible to use thermostable lipases in the synthesis of biopolymers, pharmaceuticals, agrochemicals, cosmetics, biodiesel, and aromas. According to Diaz et al. even for identical lipases produced by different methods of cultivation (submerged and solid-state), there may be thermostability differences caused by the binding of nonprotein compounds derived from the culture medium through noncovalent bonds to the lipases, changing their physical and chemical properties.Enzyme thermostability may be affected by production conditions, such as the producer microorganism, the method of cultivation, and the medium used.
Thermostability is the result of the protein’s amino acid sequence, which provides a more rigid conformation to the enzyme through intramolecular interactions, with the internalization of hydrophobic residues and superficial exposure of hydrophilic residues. Lipase thermostability may also be affected by the presence of compounds such as short-chain alcohols, metals, and ions as Ca +2 and Mg +2 which bind to the surface of enzymes whose binding sites are generally formed by negatively charged groups. According to Iyer and Ananthanarayan thermal stabilization of lipases may be caused by the presence of divalent ions, anions ( ), or cations ( ). And were present in the lipase production culture medium, which, if not consumed by the fungus for growth and synthesis, remain soluble after the separation of cells, and become part of the lipolytic extract. That may explain the thermostability of the produced enzymes. However, if this enzyme extract containing lipases were purified for further use, causing the removal of these ions of the culture medium, the study of the stability of the purified protein would be needed. PH StabilityThe pH stability of enzymatic extracts obtained through submerged and solid-state fermentation was determined according to Section treating these extracts with different buffers for 24 h and after the enzymatic activity was determined using the optimized pH and temperature for the enzymes of each fermentation process (Section ).Figure shows the residual lipolytic activity as a function of pH for the enzymes produced through solid-state and submerged fermentation.
Lipases produced through submerged fermentation by Aspergillus flavus were stable at pH ranging from 3.5 to 6.5 for 24 h, with residual activities greater than 80%. At pH 7 to 10 there was a reduction in the stability of enzymes with residual activity of around 50%. PH stability of lipases produced (●) by Aspergillus flavus (strain O-8) through submerged fermentation and (■) by Aspergillus niger (strain O-4) through solid-state fermentation.Lipase produced through solid-state fermentation by Aspergillus niger had greater stability at pH greater than 7.0, with residual activity greater than 60%. In acidic pH (4 to 6), the stability of the enzyme after 24 h was around 50%.
It was found that the enzyme showed optimal activity at acidic pH (6.0), while the highest stability was observed with alkaline pH.This behavior is similar to that reported by Mhetras et al. , who reported that lipases produced by Aspergillus niger NCIM 1207 were stable when pH was alkaline (pH 8 to 11) despite having had optimum activity at an acidic pH. Sharma et al. reported that the lipases produced by Bacillus sp. RSJ-1 had 84 and 82% residual activity, respectively, after 2 h at pH 8 and 9.
The lipases produced by Candida sp. Were stable at pH ranging from 7.5 to 8.5 for 15 min. ConclusionLipase produced by the Aspergillus flavus (strain O-8) through submerged fermentation had maximum activities at 37°C and pH 7.2. The thermal stability was 72% after 1 h of exposure to temperatures of 70 to 90°C and pH stability greater than 80% in acidic pH, which are desirable traits for industrial application. On the other hand, lipases produced through solid-state fermentation with Aspergillus niger (O-4) had optimum temperature and pH around 35°C and pH 6.0 and stability at room temperature (63.6% and 26.8% of residual activity after 1 h of exposure to 50 and 60°C, resp.), lower than that observed with enzymes obtained through submerged fermentation. The pH stability was higher in alkaline pH, with residual activity greater than 60% after 24 h of exposure.
Conflict of InterestsThe authors declare that there is no conflict of interests regarding the publication of this paper. AcknowledgmentsThe authors thank CNPq for the financial support. They are very grateful to the researchers Fabio Rodrigo Duarte and Siu Mui Tsai, from Cena/USP, who carried out the identification of fungi used in this work.
. 1.6k Downloads.AbstractFifty-five bacterial strains isolated from soil were screened for efficient poly-3-hydroxybutyrate (P3HB) biosynthesis from xylose. Three strains were also evaluated for the utilization of bagasse hydrolysate after different detoxification steps. The results showed that activated charcoal treatment is pivotal to the production of a hydrolysate easy to assimilate.
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Burkholderia cepacia IPT 048 and B. Sacchari IPT 101 were selected for bioreactor studies, in which higher polymer contents and yields from the carbon source were observed with bagasse hydrolysate, compared with the use of analytical grade carbon sources. Polymer contents and yields, respectively, reached 62% and 0.39 g g −1 with strain IPT 101 and 53% and 0.29 g g −1 with strain IPT 048. A higher polymer content and yield from the carbon source was observed under P limitation, compared with N limitation, for strain IPT 101. IPT 048 showed similar performances in the presence of either growth-limiting nutrient.
In high-cell-density cultures using xylose plus glucose under P limitation, both strains reached about 60 g l −1 dry biomass, containing 60% P3HB. Polymer productivity and yield from this carbon source reached 0.47 g l −1 h −1 and 0.22 g g −1, respectively.