Brazilian Journal

of Chemical ISSN 0104-6632

Engineering Printed in Brazil
www.abeq.org.br/bjche

Vol. 36, No. 02, pp. 615 - 625, April - June, 2019
dx.doi.org/10.1590/0104-6632.20190362s20180014

ALKALINE PROTEASE PRODUCTION

BY Bacillus licheniformis LBA 46 IN A BENCH
REACTOR: EFFECT OF TEMPERATURE
AND AGITATION
Jessika G. dos S. Aguilar1*, Ruann J. S. de Castro1 and Hélia H. Sato1
1
Universidade de Campinas, Faculdade de Engenharia de Alimentos, Departamento de Ciência de Alimentos, Campinas, SP, Brasil.
E-mail: [email protected] - ORCID: 0000-0003-4435-7565; ORCID: 0000-0003-2649-8567; ORCID: 0000-0002-8170-7088

(Submitted: January 11, 2018 ; Revised: September 17, 2018 ; Accepted: September 21, 2018)

Abstract - The production of protease from Bacillus licheniformis LBA 46 was studied in a 6 L reactor using
the experimental design tool. The higher protease production was obtained in the exponential phase of growth
reaching maximum activity (~3,000 U/mL) after 48 h of fermentation at 30 ºC and 300 rpm in a culture medium
made of agroindustrial by-products. In the thermostability study, the semi-purified enzyme retained about 78% of
the initial activity after 120 min at 50 ºC. The protease was purified 3.33 times by ammonium sulfate precipitation
and DEAE-Sepharose column chromatography and had a molecular mass estimated at 40 kDa by SDS-PAGE.
The purified protease showed optimum activity at 50 and 60 ºC, optimal activity in pH 8.5 and stability in the
range between pH 5-10 after 24 h of incubation at 4 ºC, presenting more than 86% of the initial activity.
Keywords: Bacillus licheniformis; Fermentation; Optimization; Protease; Purification.

INTRODUCTION alkaline proteases were initially marketed for use in

detergents and the market for these industrial enzymes
Industries of food, pharmaceutical, agricultural and expanded substantially during the 1960s. Until
medical have been taking advantage of using Bacillus nowadays they are one of the most widely studied
sp. because of their wide range of physiological groups of enzymes because of their extensive types of
characteristics and ability to produce enzymes and application in several sectors such as detergent, textile,
other metabolites (Gupta et al. 2002; Schallmey et leather and food industries (Benmrad et al. 2016;
al. 2004; Voigt et al., 2004). Bacillus subtilis and Bouacem et al. 2016; Ward et al. 2009). The main
Bacillus licheniformis species are attractive industrial microbial strains used in enzyme production are still
microorganisms recognized as GRAS (generally Bacillus species, used principally to produce alkaline
recognized as safe), which have high growth rates serine proteases and neutral proteases (Schallmey et
leading to shorter fermentation times and posses the al. 2004; Ward 2011; Pant et al. 2015).
ability to secrete extracellular proteins (Ward et al. On an industrial scale, microorganisms are
2009; Ward 2011; Parrado et al. 2014). cultivated in reactors under the best conditions of
Since the advent of enzymology, one of the most production (Moo-Young and Chisti 1994; Gupta et al.
important classes of hydrolytic enzymes, which have 2002). According to Potumarthi et al. (2007), mixing
been extensively studied, is the microbial proteases in the reactor is important during the production
(Furhan and Sharma 2014; Hadjidj et al. 2018). Each of proteases, which is transmitted by aeration and
enzyme has a peculiar characteristic of performance, agitation. The temperature is another important
which makes it suitable for several applications. The function to control in a fermentative process, so

* Corresponding author: Jessika G. dos S. Aguilar - E-mail: [email protected]

616 Jessika G. dos S. Aguilar et al.

there is a necessity of defining a better combination the effects of temperature and agitation for the reactor
of these factors within the fermentation process for fermentation, resulting in a total of 7 tests, which were
maximum efficiency and productivity. Based on carried out in random order. Table 1 shows the coded
this, the aim of this study was to verify the effects and real values of the variables studied.
of modifying temperature and agitation conditions
during the submerged fermentation of B. licheniformis Table 1. Factorial design, coded and real values of the
LBA 46 in a reactor on protease production, using the variables studied (temperature and agitation).
experimental design process.

MATERIALS AND METHODS

Fermentation
Microorganism and culture medium
The microorganism used was a strain of B.
licheniformis LBA 46 from the culture collection
of the Laboratory of Food Biochemistry, School of
Food Engineering, UNICAMP, Brazil. The culture Kinetics of microbial growth and protease production
medium used was proposed by Contesini (2014), with The microbial growth kinetics of B. licheniformis
modifications, containing agroindustrial by-products LBA 46 and the protease production were carried out
as carbon and nitrogen sources (32 g/L of sugar cane in a 6 L reactor containing 3 L composed of 32 g/L
molasses (Fio de Ouro®); 6 g/L of corn steep liquor of sugar cane molasses (Fios de Ouro®); 6 g/L of corn
(Corn Products®); 2 g/L of yeast extract (Prodex-Lac steep liquor (Corn Products®); 2 g/L of yeast extract
SD®) and 20 of g/L dried whey (Alibra®), adjusted to (Prodex-Lac SD®) and 20 g/L dried whey (Alibra®),
adjusted to pH 7, at 300 rpm, 30 ºC and 0.8 vvm.
pH 7).
Samples of the culture media were collected at different
times and inoculated into Petri dishes containing
Inoculum preparation
nutrient agar using the pour plate technique. Petri
The microorganism was grown in nutrient agar
dishes were incubated at 30 ºC for 24 h. Microbial
(1 g/L of meat extract; 2 g/L of yeast extract; 5 g/L
growth was expressed as colony forming units (CFU)/
of peptone; 5 g/L of sodium chloride and 15 g/L of mL. Protease activity, protein and reducing sugar were
agar, pH 7) slants and incubated at 30 ºC for 18-24 h. determined as described below.
After growth, a bacterial cell suspension was prepared
by adjusting the absorbance at 620 ηm to 0.49-0.51. Protease activity determination
Erlenmeyer flasks containing the cell suspension and Protease activity was determined according to the
culture medium were incubated at 30 ºC and 200 rpm method described by Charney and Tomarelli (1947) and
for 36-40 h. modified by Castro and Sato (2014), using azocasein
as the substrate. The reaction mixture contained 0.5
Submerged fermentation in a reactor mL of 0.5% azocasein in 0.05 M sodium phosphate
The fermentation of B. licheniformis LBA 46 was buffer, pH 7, and 0.5 mL of the enzymatic extract
performed in a New Brunswich Bioflo II reactor with which were incubated for 40 min at 60 ºC. The reaction
a capacity for 6 L and a working volume of 3 L. The was stopped by adding 0.5 mL of 10% trichloroacetic
inoculum represented 10% of the culture medium acid (TCA). The reaction mixture was centrifuged at
and was prepared as described above. Foaming 17,000 x g for 15 min at 15 ºC. An aliquot of 1 mL of
was controlled during fermentation using the anti- the supernatant obtained was neutralized with 1 mL
foam DC*FG-10 (Dow Corning®), which dripped of 5 M KOH. One protease activity unit was defined
automatically when the foam level reached the sensor. as the amount of enzyme which caused an increase of
The air flow rate was maintained at 0.8 vvm. The pH 0.01 in absorbance at 428 ηm.
value was monitored using a calibrated potentiometer.
The total fermentation time was 72 h, and samples Protein and reducing sugar determination
(15 mL) were collected every 12 h and centrifuged at Protein quantification was carried out by Lowry’s
11,000 x g for 15 min at 5 ºC. The cell-free supernatant method with some modifications (Hartree, 1972).
was used as the enzyme extract for determination of The calculations were based on a standard curve of
protease activity. bovine serum albumin (BSA) and were expressed
in mg/mL. The reducing sugars were quantified
Optimization of temperature and agitation with dinitrosalicylic acid, DNS (Miller, 1959). The
A factorial design with 4 possible combinations calculations were based on a standard glucose curve
and 3 central points was used to optimize and evaluate and were expressed in mg/mL.

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 Alkaline Protease Production by Bacillus licheniformis LBA 46 in a Bench Reactor: Effect of Temperature and Agitation 617

Determination of kinetic and thermodynamic 2.303

parameters of semi-purified protease D= (5)
kd
Activation energy and temperature coefficient (Q10)
To determine the activation energy (Ea),
measurements of protease activity were performed Thermodynamic parameters for thermal
with incubation at different temperatures, 30-80 ºC. Ea inactivation - Thermodynamic parameters of the
was calculated from the slope of the plot of 1000/T vs. protease were projected using the Eyring absolute rate
ln (protease activity), Ea = - slope x R. expression (Eq. 6).
The value of the temperature coefficient, Q10, was
 −∆H   ∆S 
determined according to Eq. 1 (Dixon and Webb, k T    

1979). This measure is used to relate the reaction rate k d =  b  e e

RT   R 
(6)
 h 
with a 10 ºC increase in the reaction temperature.

 E × 10  where kb is the Boltzmann constant (1.38 x 10-23 J/K); T

Q10 antilog ε  a 2 
= (1) is the absolute temperature (K); h is the Planck constant
 RT  (6.63 x 10-34 J.s); ΔH is the enthalpy of activation (kJ/
where R is the gas constant (8.314 J/Kmol) and T is the mol) and ΔS is the entropy of activation (J/mol K).
absolute temperature (K). The enthalpy of activation, ΔH, was calculated
using Eq. 7. The activation free energy, ΔG was
Determination of Km and Vmax calculated using Eq. 8 and the activation entropy, ΔS
Kinetic parameters (Michaelis Mentem constants, was determined according to Eq. 9. All terms were
Km and maximum velocity, Vmax) were determined at previously described in the equations above.
the optimal temperature and pH of protease activity
using different concentrations of azocasein as substrate ∆H = E ad − RT (7)
(1-10 mg/mL).
k h
Determination of kinetic and thermodynamic ∆G =−RT ln  d  (8)
parameters for thermal inactivation  kbT 
Kinetic parameters for thermal inactivation - To
determine the thermal inactivation of the protease, the ( ∆H − ∆G )
enzyme was incubated in 0.05 M sodium phosphate ∆S = (9)
T
buffer, pH 7, at temperatures of 50-70 ºC for 120 min
in the absence of substrate. Samples were collected Purification and characterization of purified protease
periodically throughout the incubation period and The protease of B. licheniformis LBA 46 strain
residual activity was determined at the optimal was produced using the optimized conditions of
temperature and pH of protease activity. temperature and agitation. The supernatant was
The value of the deactivation constant (kd) expressed separated by centrifugation and fractionated with 80%
as an exponential decay and found by plotting ln (A/ ammonium sulfate. The precipitate was dissolved in
A0) vs. time was measured according to Eq. 2. 0.05 M phosphate buffer, pH 7, and dialyzed against
distilled water at 5 ºC and freeze-dried. The freeze-
A = A 0 e− kd t (2)
dried protease was applied to a 20 mL DEAE-Sepharose
where A and A0 is the protease activity at a determined ion exchange column (HiPrep™ DEAE FF 16/10, GE,
time t and at an initial time, respectively. Little Chalfont, UK) equilibrated with 0.05 M sodium
The activation energies for denaturation (Ead) were phosphate buffer, pH 7, and the proteins were eluted
calculated by plotting ln (kd) vs. 1/RT as described (5 mL/min) with a linear 0 to 1 M sodium chloride
in Eq. 3. The time when the residual activity reaches gradient (Äkta Purifier, GE, Little Chalfont, UK).
50% (apparent half-life) was estimated by Eq. 4. The Fractions containing protease activity were pooled and
D-value, which is defined as the time required for analyzed by SDS-PAGE (Vertical Slab Mini-Protean
a 90% reduction in the initial enzyme activity at a Electrophoresis System, Bio-Rad Laboratories,
specific temperature, was calculated as shown in Eq. 5. Hercules, CA, USA) as described by Laemmli (1970).
The run was performed at 110 V for 30 min. The
 − E ad 
  molecular weight of the enzyme was estimated using
k d = Ae RT 
(3) molecular mass markers (Thermo Fisher Scientific
Ruler™ Unstained Protein Ladder) ranging from 10
ln ( 0.5 ) to 200 kDa. Protein bands were visualized by staining
t1/ 2 = (4)
kd with Coomassie Brilliant Blue R-250.

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618 Jessika G. dos S. Aguilar et al.

Determination of the optimum pH of activity and program (Statsoft®/Dell, USA), Tukey’s test and
stability of purified protease Pearson correlation were carried out in Minitab 16.1.1
The effect of pH on protease activity was determined (Minitab Inc., USA). All the analyses were carried out
by univariate assay using 0.1 M acetate buffer (pH in triplicate and evaluated considering a p-value lower
4-5), 0.1 M sodium phosphate buffer (pH 6-8), 0.1 M than 10% (p ≤ 0.10).
Tris-HCl buffer (pH 9), 0.1 M carbonate-bicarbonate
buffer (pH 10) and 0.1 M NaOH-bicarbonate buffer RESULTS AND DISCUSSION
(pH 11).
The effect of pH on protease stability was Experimental design for the kinetics of protease
determined using the same buffers and pH values production in a reactor
already mentioned. The enzyme solutions were Preliminary tests were performed to verify the
incubated at different pH values for 24 h at 4 ºC in effects of temperature (in the range of 30-37 ºC) on
the absence of substrate. The residual enzyme activity extracellular protease production by B. licheniformis
was then determined. The results were expressed as a LBA 46. Using 100 mL of culture medium in
percentage and relative activity. Erlenmeyer flasks, it was found that temperature
variations caused variations in the protease production.
Determination of the optimum temperature of activity Therefore, the reactor studies were carried out using
and stability of purified protease the reactor in this temperature range. Figure 1 presents
The protease activity was tested at different the responses obtained in the factorial design for the
temperatures (between 30 ºC and 80 ºC, pH 7). Relative effect of temperature and agitation during the kinetics
activities were determined by defining the maximum of protease production by B. licheniformis LBA 46 in
enzyme activity, at a specific temperature, as 100%. a reactor for 72 h of fermentation.
The thermal stability of the enzyme was evaluated The estimated enzymatic activity for the kinetics
by preincubation at various temperatures (between 30 at 72 h (Figure 1A) showed that the values remained
ºC and 80 ºC, pH 7) for 1 h with subsequent cooling, high (> 1000 U/mL) in 71.43% of the assays analyzed.
and the residual enzymatic activity was determined. Under the conditions evaluated, the microorganism
produced protease in the range from 30-37 ºC and 200-
Statistical analysis 300 rpm.
The experimental design, matrix and statistical It can be observed that, for the majority of the
analysis were developed using the Statistica 7.0 analyzed times, the central points (assays 5-7) and

Figure 1. Kinetics of the protease production (A), pH values (B), reducing sugars (C) and protein (D) measured
during fermentation of B. licheniformis LBA 46 in a bench reactor during 72 h of fermentation.

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 Alkaline Protease Production by Bacillus licheniformis LBA 46 in a Bench Reactor: Effect of Temperature and Agitation 619

assay 3 presented values of activity greater than the 2,661.17 U/mL after 48, 60 and 72 h of fermentation,
other tests. This information indicates that there is no corresponding to protease productivity values equals to
adequate fit for a 1st order model, so there is a necessity 51, 46.8 and 36.7 U/mL.h, respectively. The values for
of evaluating the curvature. According to the p-value the coefficients of temperature and agitation from 48
obtained (p ≤ 0.10), the analysis of curvature was h of fermentation were high, negative for temperature
significant. Table 2 presents the estimated regression and positive for agitation, which means that, when the
coefficients for each variable, their interaction and temperature decreased and the agitation increased, the
the statistical analysis of each effect for significance protease activity was at its highest. The assay 3 fitted
assessment. perfectly with these conditions, and it was chosen for
For protease activity, the temperature, the agitation the protease production.
and the interaction between them showed effects Figure 1 (B, C, D) shows the pH values, reducing
on the factorial design, and the calculated p-value sugars and protein measured during fermentation by B.
confirmed the presence of all significant effects after licheniformis LBA 46 in a reactor. The pH of the culture
48 h of fermentation with 90% of confidence (Table medium provides some important information. The
2). The maximum protease activity was reached initial pH (7) of the culture medium decreased to 6-6.8
under the conditions of assay 3 (30 ºC and 300 rpm), after 12 h and then increased to reach 6.8-8 after 72 h
which presented activities of 2,448.83, 2,627.33 and in the most assays. The pH initially dropped, probably
Table 2. Regression coefficients, standard error, tcalc due to acid production from glucose utilization during
and p-value during the protease production by B. the growth phase with the increase in the number of
licheniformis LBA 46 in a bench reactor during 72 h microbial cells, but when the enzymatic production
of fermentation. was initiated, the pH started to increase (Singh et al.,
2004). 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. (1992), the acidification
or alkalinization of the medium during the microbial
growth reflects the substrate consumption. 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
1C, the consumption of sugars had a similar profile
for the 7 assays. Protein content of the culture medium
increased in all assays, reaching about 3.5-5 mg/mL
after 72 h of fermentation (Figure 1D), representing
the increase in the protease production.
Dey et al. (2016) evaluated the improvement of
protease production by B. licheniformis NCIM-2042
in a 2.2 L bioreactor containing 30.8 g/L starch, 78.89
g/L soybean meal, 0.5 g/L MgSO4, and 5.3 g/L NaCl,
pH 7.4. The effect of aeration (1, 2 and 3 vvm) and
agitation (150-210 rpm) were tested and the maximum
protease production, 382.46 U/mL, was achieved
using 180 rpm and 2 vvm, after 84 h of incubation at
37 ºC. On the contrary, in this study a higher protease
production (>2400 U/mL) was obtained after 48 h
*tcalc calculated with 3 degrees of freedom. of fermentation using lower temperature and lower
R² > 0.96 for all responses. agitation than those used by Dey et al. (2016).

Brazilian Journal of Chemical Engineering, Vol. 36, No. 02, pp. 615 - 625, April - June, 2019
620 Jessika G. dos S. Aguilar et al.

Chuprom et al. (2016) studied the enhancement of

halophilic protease production by Halobacterium sp.
strain LBU50301 using statistical design response to
optimize the medium composition. Using 18.62 g/L
gelatin, 9.13 g/L MgSO4.7H2O, 27.95% (w/v) NaCl,
pH 7.88 as culture medium the protease production
increased 13-fold from 17.80 U/mL in Erlenmeyer
flasks to 231.33 U/mL in a laboratory fermenter.
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. (2016) also observed Figure 2. Fermentation of B. licheniformis LBA 46 in
that the optimization tool is useful for increasing the a bench reactor at temperature of 30 °C and agitation
enzymatic production, as well as the use of a reactor. of 300 rpm: microbial growth, protease production,
However, the values of enzyme activity found in this reducing sugar and protein content.
study were higher than those mentioned above. The The highest activity of protease from B. subtilis UO-
protease of B. licheniformis LBA 46 was produced 01 was reached after 15 h of fermentation (10 U/mL),
in greater quantity (>2400 U/mL) when produced in when the microbial cells entered the post-exponential
a reactor with optimized conditions of temperature phase. This may occur due to the need for nutrients
and agitation (30 ºC and 300 rpm). The strain of B. for microbial survival or due to the need for renewal
licheniformis LBA 46, in the conditions studied, was a of cell proteins at a lower growth rate. The enzyme
better protease producer than the strain investigated by production was associated with microbial growth
Chuprom et al. (2016). (Blanco et al., 2016). Dias et al. (2008) observed
Besides the activity values reported by Dey et al. maximum proteolytic activity of the enzyme produced
(2016) and Chuprom et al. (2016) being lower than by B. subtilis ATCC 6633 (839.8 U/mg) and Bacillus
those found in this study, the culture medium used by sp. UFLA 817 (975.9 U/mg) after 24 h of fermentation,
them was synthetic, unlike the culture medium used coinciding with the end of the exponential phase of
in this work, which was composed of agro-industrial microbial growth. The protease production by Bacillus
low-cost by-products. cereus VITSN04 also had its maximum activity (200
U/mL) associated with the exponential growth phase
Kinetics of microbial growth and protease (Sundararajan et al., 2011).
production The protease from Bacillus megaterium was
In the fermentation of B. licheniformis LBA 46 produced in accordance with the bacterial growth. The
in a 6 L reactor in the best conditions of temperature maximum protease activity (7 U/mL) was achieved
(30 ºC) and agitation (300 rpm), according to assay during the stationary phase after 15 h of fermentation
3, the protease was produced in the exponential phase (Uttatree et al., 2017). Rao e Narasu (2007) observed
of growth reaching maximum activity (~3,000 U/ that the maximum activity (215 U/mL) of the protease
mL) after 48 h of fermentation. The reducing sugar produced by Bacillus firmus 7728 was reached in the
content in the culture medium decreased to 17.9 mg/ stationary phase after 48 h of growth.
mL and 16.2 mg/mL after 36 and 48 h of fermentation, The results presented in this study showed a high
respectively. Cell growth decreased after 48 h of production of protease and high productivity, with
fermentation (Figure 2). values higher than those found in the mentioned
The production of proteases by Bacillus species literature, regardless of the microbial growth phase or
is controlled by a number of complex mechanisms the strain used in the production.
that occur during the transition between exponential
and stationary phases. The production of enzymes Determination of the kinetic and thermodynamic
is related to the growth phase of the microorganism parameters of semi-purified protease
(Strauch and Hock, 1993). According to Strauch and Activation energy and Q10 value
Hock (1993), Jisha et al. (2013) and Contesini (2014), Figure 3 presents the protease activity at different
proteases from Bacillus sp. are mainly produced temperatures (30-80 ºC). It can be observed that the
during the stationary phase of microbial growth. The semi-purified protease from B. licheniformis LBA
extracellular enzyme production pattern depends on 46 showed high activity between 55-65 ºC, with the
the Bacillus strains (Jisha et al., 2013). optimum value at 60 and 65 ºC.

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 Alkaline Protease Production by Bacillus licheniformis LBA 46 in a Bench Reactor: Effect of Temperature and Agitation 621

The enzyme showed, with good correlation

(R2 = 0.91), Michaelis-Menten-type kinetics with Km
= 1.60 mg/mL and a high Vmax = 2 x 106 U/g. A similar
Km value (1.92 mg/mL) was reported by Souza et al.
(2015) for acid protease from A. foetidus utilizing
azocasein as substrate. A lower Km value (0.44 mg/
mL) was related for serine protease from Aspergillus
niger (Castro et al., 2014) also using azocasein as
substrate. Using casein as substrate, Abdel-Naby
(2017) determined a higher Km value (3.7 mg/mL)
than the one found in this study for alkaline protease
from B. stearothermophilus. It can be seen that the Km
value depends on the type of substrate evaluated and
the enzyme-producing microorganism.
Figure 3. Effect of temperature on the activity of semi-
purified protease from B. licheniformis LBA 46. Thermal inactivation of semi-purified protease
The thermostability of semi-purified protease
For the range of temperature analyzed (30-80 ºC) from B. licheniformis LBA 46 was studied in the
a linear variation could be observed with the increase range of 50-70 ºC. The enzyme showed higher
in temperature, which suggests that the protease has stability at a temperature of 50 ºC, retaining above
a single conformation at the temperature of transition 80% of the initial activity after 120 min. The protease
(Castro et al. 2014). was rapidly inactivated at 70 ºC in the absence of
The Ea value (47.96 kJ/mol) was high, positive substrate, losing 84% of the initial activity after 30
and showed good correlation (R2 = 0.95), within min of incubation.
the temperature range studied. Abdel-Naby (2017) The half-life of an enzyme is defined as the
described a lower Ea of 17.31 kJ/mol for alkaline amount of time required at a given temperature,
protease from Bacillus stearothermophilus. Souza capable of reducing its initial activity by half.
et al. (2015) also found Ea (19.03 kJ/mol) for acid According to Table 3, the semi-purified protease of
protease from Aspergillus foetidus lower than that B. licheniformis LBA 46 has high thermal resistance,
described in this study. It can be observed that the requiring 693.15 min to reduce half of its activity at
proteases produced by different microorganisms
50 ºC and that value fell as the temperature increased,
have different Ea values. As related in this study, an
reaching 23.90 min at 70 ºC. The D-value, which is
alkaline protease from Nacordiopsis alba showed a
the time required for a 90% reduction in the initial
high Ea value of 36.80 kJ/mol (Gohel and Singh,
enzyme activity was also reduced with increasing
2012). The high reported values mean that to make
temperature, ranging from 2,302.60 to 79.40 min
the activated enzyme-substrate complex, more
energy is required. between 50 and 70 ºC. In relation to the inactivation
The Q10 value is a kinetic parameter used to rate constants (Kd), the values increased with an
determine whether the catalytic reactions are controlled increase in temperature, ranging from 1.0 x 10-3
by temperature or other factors. According to Elias et to 29 x 10-3 min-1. The energy required for thermal
al. (2014), the enzymatic reactions have values of Q10 inactivation (144.50 kJ/mol) was calculated using
ranging from 1 to 2. Values outside this range can be an Arrenius plot. Abdel-Naby (2017) determined a
interpreted as indicative of the involvement of factors similar value for Ea (105.5 kJ/mol) by studying an
other than temperature in the control of the reaction alkaline protease from B. stearothermophilus, which
rate. The Q10 value was determined to be in the range means that both enzymes require a similar amount
of 1.59-1.87, representing the rate of reaction which of energy to be inactivated.
was affected only by a temperature increase.
Table 3. Thermodynamic and kinetic parameters for
thermal inactivation of semi-purified protease from B.
Kinetic parameters, Km and Vmax
licheniformis LBA 46.
The kinetic parameters were calculated according
to a double reciprocal Lineweaver-Burk plot. The Km
value indicates the protease-substrate affinity and a
low Km value indicates higher affinity of the enzyme
for the substrate. The Vmax value could be defined as
the maximum value of initial velocity when all actives
sites are occupied by the substrate.

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622 Jessika G. dos S. Aguilar et al.

Thermal inactivation of enzymes is accompanied

by the breakdown of many non-covalent bonds, which
represents an increase in the value of ΔH. According
to Batista et al. (2014), high ΔH values are linked to
high thermal stability of the enzyme. The opening or
unfolding of the enzyme caused by heating increases
its disordered state, which can be measured by the
value of ΔS. An enzymatic reaction can also be
evaluated by measuring the change in ΔG value during
the conversion of an enzyme-substrate complex into a
product (Riaz et al., 2007). A low ΔG value suggests
that this conversion is more spontaneous; however,
high ΔG values indicate high enzyme stability (Batista
et al., 2014).
The values of ΔH, ΔS and ΔG practically did not Figure 4. SDS-PAGE of purified protease from B.
vary within the temperatures analyzed (Table 4), in licheniformis LBA 46. (A) molecular mass markers
this case, these temperatures were not capable of and (B) purified protease.
causing visible changes in the enzymatic behavior, molecular weight (equal to that found in this study)
which remained constant within the range of of the protease at 40 kDa by SDS-PAGE. Other
temperature evaluated. The parameters of kinetic
works have studied different types of proteases
inactivation are important since they serve to define
from Bacillus sp. purification using various methods
and model the use of enzymes in certain industrial
applications. with varied molecular weights. Annamalai et al.
(2013) purified a 33 kDa protease from Bacillus
Table 4. Thermodynamic parameters for thermal alveayuensis CAS 5 using DEAE-cellulose and
inactivation of semi-purified protease from B. Sephadex G-50 columns. Jellouli et al. (2011)
licheniformis LBA 46. purified a 30 kDa protease from B. licheniformis
MP1 using Sephadex G-100 and Mono Q-Sepharose
columns. Lakshmi et al. (2018) purified a protease
from Bacillus cereus strain S8 using ion exchange
followed by gel filtration chromatography. The
estimated molecular weight was 21.8 kDa. As in this
work, several studies in the literature also described
proteases with low molecular weight from Bacillus
Purification of protease sp.: 15 kDa (Adinarayana et al. 2003), 17.10 kDa
The protease from B. licheniformis LBA 46 was (Kim and Kim 2005), 20.10 kDa (Rai et al. 2009),
purified 3.33 fold using 80% ammonium sulfate 30 kDa (Hadjidj et al., 2018).
precipitation and using DEAE-Sepharose column The purified protease of B. licheniformis LBA 46
chromatography. The purified protease showed a presented high activity (> 80%) in the range of pH
specific activity of 628.96 U/mg (Table 5). 6.5-9, optimal activity at pH 8.5 and low activity at
pH 4.0 (15%). The purified protease was stable in
Biochemical characterization of purified protease the range of pH 5-10 after 24 h at 4 ºC, retaining
The molecular weight of purified protease from more than 86% of the initial activity (Figure 5A).
B. licheniformis LBA 46 was estimated as 40 kDa by The purified protease presented optimum activity at
SDS-PAGE (Figure 4). 50 and 60 ºCat pH 7.0. The enzyme was stable at 40
Jalkute et al. (2017) purified the protease from ºC for 1 h in pH 7 and retained 85% of the initial
Bacillus safensis CK about 7-fold by DEAE- activity after 1 h of treatment at 50 ºC, pH 7 (Figure
cellulose column chromatography and estimated the 5B).
Table 5. Summary purification of protease from B. licheniformis LBA 46.

Brazilian Journal of Chemical Engineering

 Alkaline Protease Production by Bacillus licheniformis LBA 46 in a Bench Reactor: Effect of Temperature and Agitation 623

Figure 5. Optimum and stability pH (A) and temperature (B) of purified protease from B. licheniformis LBA 46.
CONCLUSIONS Bacillus alveayuensis CAS 5 using marine wastes.
Food and Bioproducts Processing, 92, 335-342
A protease from B. licheniformis LBA 46 was (2013). https://doi.org/10.1016/j.fbp.2013.08.009
produced using agroindustrial by-products as Batista, K.A., Batista, G.L.A., Alves, G.L.,
sources of carbon and nitrogen in a reactor. The Fernandes, K.F. Extraction, partial purification
highest protease activity was obtained after 48 h of and characterization of polyphenol oxidase from
fermentation at 30 ºC and 300 rpm. The semi-purified Solanum lycocarpum fruits. Journal of Molecular
protease showed high catalytic activity (~1,500,000 Catalysis B: Enzymatic, 102, 211-217 (2014).
U/g) with an optimum at 60 and 65 ºC. The purified https://doi.org/10.1016/j.molcatb.2014.02.017
protease presented optimum activity at 50 and 60 ºC Benmrad, M.O., Moujehed, E., Ben Elhoul, M., Zaraî
and was stable in the range of pH 5-10 after 24 h at 4 Jaouadi, N., Mechri, S., Rekik, H., Kourdali, S.,
ºC. This protease presented interesting characteristics El Hattab, M., Badis, A., Sayadi, S., Bejar, S.,
for potential industrial application. Jaouadi, B. A novel organic solvent- and detergent-
stable serine alkaline protease from Trametes
ACKNOWLEDGMENTS cingulata strain CTM10101. International Journal
of Biological Macromolecules, 91, 961-972 (2016).
The authors are grateful to Alibra® and Corn https://doi.org/10.1016/j.ijbiomac.2016.06.025
Products Brasil® for kindly providing the dried whey Blanco, A.S., Durive, O.P., Pérez, S.B., Montez, Z.D.,
and corn steep liquor, respectively. Guerra, N.P. Simultaneous production of amylases
and proteases by Bacillus subtilis in brewery wastes.
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