Extracellular alkaline protease production by the top five high yielding strains and NCIM strains Identification of the highest yielding strains Table 4. Characters shown by Bacillus sp. Time course of growth and alkaline protease production by Bacillus sp.
K 25 Effect of temperature of incubation Fig. Effect of temperature of incubation on alkaline protease production by Bacillus sp. K 25 Effect of pH Fig. Effect of initial pH of medium on alkaline protease production by Bacillus sp. K 25 Effect of carbon and nitrogen sources Table 7. Effect of different carbon sources on alkaline protease production by Bacillus sp.
K 25 Table 8. Effect of various nitrogen sources on alkaline protease production by Bacillus sp. K 25 Table 9. Effect of using starch and soyabean meal at different concentrations on alkaline protease production by Bacillus sp. K 25 Effect of salts Table Effect of using different concentrations of sodium chloride on alkaline protease production by Bacillus sp.
K 25 Table Effect of different salts on alkaline protease production by Bacillus sp. Effect of different concentrations of calcium chloride on alkaline protease production by Bacillus sp. Effect of age of inoculum on alkaline protease production by Bacillus sp. K 25 Effect of size of inoculum Table Effect of inoculum size on alkaline protease production by Bacillus sp. K 25 Effect of agitation Table Effect of agitating the culture on alkaline protease production Effect of period of incubation Table Effect of incubation period on alkaline protease accumulation in the culture of Bacillus sp.
Alkaline protease production by Bacillus pumilus K by SSF, using different commercially available substrates Effect of size of particles of wheat bran Table Effect of using wheat bran of different particle size on alkaline protease production by Bacillus pumilus K Effect of moisture level, temperature of incubation and incubation period Table A factorial design with 4 possible combinations and 3 central points was used to optimize and evaluate the effects of temperature and agitation for the reactor fermentation, resulting in a total of 7 tests, which were carried out in random order.
Table 1 shows the coded and real values of the variables studied. Thumbnail Table 1 Factorial design, coded and real values of the variables studied temperature and agitation. The microbial growth kinetics of B. Samples of the culture media were collected at different times and inoculated into Petri dishes containing nutrient agar using the pour plate technique. Protease activity, protein and reducing sugar were determined as described below. Protease activity was determined according to the method described by Charney and Tomarelli Charney, J.
A colorimetric method for the determination of the proteolytic activity of duodenal juice. The Journal of Biological Chemistry, , Advantages of an acid protease from Aspergillus oryzae over commercial preparations for production of whey protein hydrolysates with antioxidant activities. Biocatalysis and Agricultural Biotechnology, 3, The reaction mixture contained 0.
The reaction was stopped by adding 0. One protease activity unit was defined as the amount of enzyme which caused an increase of 0. Determination of protein: A modification of the Lowry methods that gives a linear photometric response. Analytical Biochemistry, 48, Use of dinitrosalicylic acid reagent for determination of reducing sugar.
Analytical Chemistry, 31, The value of the temperature coefficient, Q 10 , was determined according to Eq. Enzyme Kinetics, third ed. Academic Press, New York Kinetic parameters for thermal inactivation - To determine the thermal inactivation of the protease, the enzyme was incubated in 0. Samples were collected periodically throughout the incubation period and residual activity was determined at the optimal temperature and pH of protease activity.
The activation energies for denaturation E ad were calculated by plotting ln k d vs. Thermodynamic parameters for thermal inactivation - Thermodynamic parameters of the protease were projected using the Eyring absolute rate expression Eq. All terms were previously described in the equations above. The protease of B.
The precipitate was dissolved in 0. Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature, , The run was performed at V for 30 min. Protein bands were visualized by staining with Coomassie Brilliant Blue R The effect of pH on protease activity was determined by univariate assay using 0.
The effect of pH on protease stability was determined using the same buffers and pH values already mentioned. The residual enzyme activity was then determined. The results were expressed as a percentage and relative activity. The experimental design, matrix and statistical analysis were developed using the Statistica 7. Using mL of culture medium in Erlenmeyer flasks, it was found that temperature variations caused variations in the protease production.
Therefore, the reactor studies were carried out using the reactor in this temperature range. Figure 1 presents the responses obtained in the factorial design for the effect of temperature and agitation during the kinetics of protease production by B. It can be observed that, for the majority of the analyzed times, the central points assays and assay 3 presented values of activity greater than the other tests.
This information indicates that there is no adequate fit for a 1st order model, so there is a necessity of evaluating the curvature. Table 2 presents the estimated regression coefficients for each variable, their interaction and the statistical analysis of each effect for significance assessment.
Thumbnail Table 2 Regression coefficients, standard error, t calc and p -value during the protease production by B. The values for the coefficients of temperature and agitation from 48 h of fermentation were high, negative for temperature and positive for agitation, which means that, when the temperature decreased and the agitation increased, the protease activity was at its highest.
The assay 3 fitted perfectly with these conditions, and it was chosen for the protease production. The pH of the culture medium provides some important information. The initial pH 7 of the culture medium decreased to The pH initially dropped, probably due to acid production from glucose utilization during the growth phase with the increase in the number of microbial cells, but when the enzymatic production was initiated, the pH started to increase Singh et al.
The culture medium used is a complex medium, which presents a variety of proteins and peptides from the yeast extract, dried whey protein and corn steep liquor. According to Chu et al. Enzyme and Microbial Technology, 14, When microbial cells use organic nitrogen amino acids and proteins , the medium becomes more alkaline, resulting in a pH increase, and when ammonium ion is used, the medium turns more acidic, resulting in a pH decrease.
Consumption of sugars and protein synthesis were consistent with cell growth. The sugars were consumed and decreased with the advance of the fermentation, the expected behavior, because the sugars are fermented by the microorganisms during their growth to supply their metabolic needs. According to Figure 1 C, the consumption of sugars had a similar profile for the 7 assays.
Protein content of the culture medium increased in all assays, reaching about 3. Dey et al. Studies on the effect of agitation and aeration for the improved protease production by Bacillus licheniformis NCIM Materials Today: Proceedings, 3, The effect of aeration 1, 2 and 3 vvm and agitation rpm were tested and the maximum protease production, Chuprom et al.
Approach toward enhancement of halophilic protease production by Halobacterium sp. Biotechnology Reports, 10, Using According to the authors, the production of proteases obtained in the reactor was higher than that obtained in the fermentation of the Erlenmeyer flasks, since the reactor systems provide more precise control of parameters such as pH, aeration and stirring speed.
As in this work, Chuprom et al. However, the values of enzyme activity found in this study were higher than those mentioned above. The strain of B. Besides the activity values reported by Dey et al. In the fermentation of B. The reducing sugar content in the culture medium decreased to Cell growth decreased after 48 h of fermentation Figure 2. Thumbnail Figure 2 Fermentation of B. The production of proteases by Bacillus species is controlled by a number of complex mechanisms that occur during the transition between exponential and stationary phases.
The production of enzymes is related to the growth phase of the microorganism Strauch and Hock, Strauch, M. Transition-state regulators: Sentinels of Bacillus subtilis post-exponential gene expression. Molecular Microbiology, 7, According to Strauch and Hock , Jisha et al. Versatility of microbial proteases. Advances in Enzyme Research, 1, The extracellular enzyme production pattern depends on the Bacillus strains Jisha et al.
The highest activity of protease from B. This may occur due to the need for nutrients for microbial survival or due to the need for renewal of cell proteins at a lower growth rate. The enzyme production was associated with microbial growth Blanco et al.
Simultaneous production of amylases and proteases by Bacillus subtilis in brewery wastes. Brazilian Journal of Microbiology, 47, Dias et al. Alkaline protease from Bacillus sp. World Journal of Microbiology and Biotechnology, 24, UFLA Journal of Bioscience and Bioengineering, , The protease from Bacillus megaterium was produced in accordance with the bacterial growth.
A novel metal-tolerant, solvent and surfactant stable protease from a new strain of Bacillus megaterium. Biocatalysis and Agricultural Biotechnology , 12, Rao e Narasu Rao, K. Alkaline protease from Bacillus firmus African Journal of Biotechnology, 6, The results presented in this study showed a high production of protease and high productivity, with values higher than those found in the mentioned literature, regardless of the microbial growth phase or the strain used in the production.
It can be observed that the semi - purified protease from B. Thumbnail Figure 3 Effect of temperature on the activity of semi-purified protease from B. A new approach for proteases production by Aspergillus niger based on the kinetic and thermodynamic parameters of the enzymes obtained. Biocatalysis and Agricultural Biotechnology , 4, The E a value Abdel-Naby Abdel-Naby, M. Catalytic, kinetic and thermodynamic properties of stabilized Bacillus stearothermophilus alkaline protease.
International Journal of Biological Macromolecules, 96, Souza et al. Kinetic and thermodynamic studies of a novel acid protease from Aspergillus foetidus. International Journal of Biological Macromolecules , 81, It can be observed that the proteases produced by different microorganisms have different E a values.
As related in this study, an alkaline protease from Nacordiopsis alba showed a high E a value of Purification strategies, characteristics and thermodynamic analysis of a highly thermostable alkaline protease from a salt-tolerant alkaliphilic actinomycete, Nocardiopsis alba OK Journal of Chromatography.
The high reported values mean that to make the activated enzyme-substrate complex, more energy is required. The Q 10 value is a kinetic parameter used to determine whether the catalytic reactions are controlled by temperature or other factors. According to Elias et al. The universality of enzymatic rate-temperature dependency. Trends in Biochemical Sciences, 39, Values outside this range can be interpreted as indicative of the involvement of factors other than temperature in the control of the reaction rate.
The Q 10 value was determined to be in the range of 1. The kinetic parameters were calculated according to a double reciprocal Lineweaver-Burk plot. The K m value indicates the protease-substrate affinity and a low K m value indicates higher affinity of the enzyme for the substrate.
The V max value could be defined as the maximum value of initial velocity when all actives sites are occupied by the substrate. A similar K m value 1. A lower K m value 0. It can be seen that the K m value depends on the type of substrate evaluated and the enzyme-producing microorganism.
The thermostability of semi - purified protease from B. The half-life of an enzyme is defined as the amount of time required at a given temperature, capable of reducing its initial activity by half. According to Table 3 , the semi-purified protease of B.
In relation to the inactivation rate constants K d , the values increased with an increase in temperature, ranging from 1. The energy required for thermal inactivation Thumbnail Table 3 Thermodynamic and kinetic parameters for thermal inactivation of semi-purified protease from B.
According to Batista et al. Extraction, partial purification and characterization of polyphenol oxidase from Solanum lycocarpum fruits. Journal of Molecular Catalysis B: Enzymatic, , Kinetic and thermodynamic properties of novel glucoamylase from Humicola sp. Enzyme and Microbial Technology , 41, The parameters of kinetic inactivation are important since they serve to define and model the use of enzymes in certain industrial applications.