Bacillus licheniformis strain SHG10 was used in this study as PGase producer. This bacterium was previously isolated from Egyptian soil and identified as B. licheniformis SHG10 strain (unpublished data). Its 16S rDNA nucleotide sequence was submitted in the GenBank at NCBI [National Center for Biotechnology Information] under the accession number [GenBank: JN853580]. Moreover, this bacterial strain was deposited in the DSMZ [Leibniz Institute-German Collection of Microorganisms and Cell Cultures] under the accession number [DSM 28096].
Different pectin-containing materials were co-utilized in this study as a sole carbon source for the growth of the producer bacterial strain and as an inducer for PGase as well. Pectin-containing materials used in this study included synthetic citrus pectin, wheat bran, orange peel waste, lemon peel waste, banana peel waste, artichokes peel waste and pomegranate peel waste. The last five pectin–containing materials were collected from different sites (e.g.; local Egyptian markets, domestic effluents and agricultural fields). The collected pectin–containing materials were washed with distilled water then were allowed to dry at 60°C for five hrs. After that, the dried pectin–containing materials were prepared in the form of small cut pieces preparation before their incorporation into the fermentation broth. Synthetic citrus pectin was purchased from Sigma –Aldrich Co. While, wheat bran was obtained from flour mills companies in Alexandria, Egypt.
Peptone yeast broth (Bernhardt et al. 1978) was used to activate the bacterial producer strain. PA medium is peptone yeast broth with 1.5% agar agar. Polygalacturonase core production medium of (Soares et al. 1999) with slight modifications was used in this study. This modified medium was used during the initial steps of the optimization process. The modified PGase core production medium contained the following components in % (w/v): 0.5 g pectin, 0.14 g (NH4)2SO4, 0.6 g K2HPO4, 0.2 g KH2PO4, 0.01 g MgSO4 and 0.3 g yeast extract unless otherwise stated.
A fine touch of B. licheniformis SHG10 preserved on PA slant was streaked on PA agar and was incubated at 37°C for overnight. One colony was picked to inoculate 20 ml of PY medium in 100 ml Erlenmeyer flask. The inoculated broth was incubated at 37°C with agitation speed of 200 rpm for 4 hrs until the culture OD at 420 nm reached 0.5. Then this growing culture (seed broth) was used to inoculate the fermentation broth (production medium). The inoculum size of the seed broth used to inoculate the fermentation broth was 2%(v/v) unless otherwise stated.
The PGase activity was assayed by estimating the amount of reducing sugars released under assay conditions. Determination of the amount of released reducing sugars as galacturonic acid was carried out as reported previously (Miller 1959) using 2-hydroxy-3,5-dinitrobenzoic acid [(DNSA), Shanghai Orgpharma Chemical Co., Ltd., China]. Succinctly, the reaction mixture contained 0.5 mL of 0.5% citrus pectin (Sigma-Aldrich Co.) as a substrate (dissolved in 50 mM Tris-HCl, pH7.6) and 0.5 mL crude enzyme (fermentation broth). This mixture was incubated at 37°C for 20 min. After that, the enzymatic reaction was stopped by addition of 1 mL of DNSA followed by boiling for 10 min. Then the final volume was completed to 4 mL by distilled water and the developed color was measured at 540 nm. Control reactions were prepared as mentioned above except that DNSA was added prior addition of the crude enzyme. A standard curve with α-galacturonic acid (Sigma-Aldrich Co.) was established. One unit (arbitrary unit) of enzyme activity was defined as the amount of enzyme that releases one μg of α-galacturonic acid per min from citrus pectin as a substrate in 50 mM Tris-HCl, pH 7.6 at 37°C.
The protein content of the crude enzyme solution was performed as reported previously using Folin -Lowry reagent (Lowry et al. 1951). A standard curve using bovine serum albumin was established.
Pectic oligosaccharides determination
Pectic oligosaccharides were determined as reported previously by the method of Miller (1959). Briefly, 0.5 mL of the fermentation broth was added to 1 mL of DNSA. Then, the reaction mixture was boiled for 10 min. After that, the absorbance of the developed color was measured at 540 nm against blank (the same as the reaction except that water was added instead of fermentation broth).
Optimizing the production of PGase from B. licheniformis SHG10
Optimizing the production of PGase from the producer strain was accomplished through a three successive step plan; one variable at a time approach (OVAT), Plackett-Burman design and Box- Behnken design.
OVAT was employed in this study in order to screen different independent variables that would either stimulate or inhibit the production of PGase enzyme. This approach is based on changing one variable at a time without studying the interaction among the tested variables. The effect of different agro-industrial wastes (e.g., orange, lemon, banana, artichoke, pomegranate peel wastes and wheat bran) and some synthetic carbon sources (e.g., citrus pectin, tryptone, peptone, beef extract, glucose, maltose, sucrose, xylose, fructose and glycerol) on PGase productivity by B. licheniformis was assessed. In addition, the effect of different salts such as NaNO3, KNO3, NH4Cl, CaCl2, MgSO4 and FeSO4 was studied as well. The exact concentrations of the aforementioned tested substances were displayed in Tables 1 and 2.
Plackett – Burman Design (PBD)
Identifying the significant main key determinants (physicochemical independent parameters) in a bioprocess along with studying the linear effect of these tested variables is achieved by applying a powerful statistical approach namely called Plackett–Burman design that developed by two statisticians Plackett and Burman (1946). In this approach evaluating the linear effect of N independent variables on the dependent variable (output of a bioprocess) is tested in a N + 1 experiment. Normally, each independent variable is studied in two levels -1 and +1; low coded level and high coded level, respectively. The design matrix was generated by a statistical software package Minitab version 15. Here, twenty experimental runs (trials) had been conducted. The following polynomial equation from the first order (Equation 3) was put in order to evaluate the linear effect imposed by the ten tested independent variables on the level of PGase enzyme:
Where Y is the level of PGase activity, β
is the model intercept, X
are the tested independent variables (orange peel waste, NaNO3, MgSO4, CaCl2, FeSO4, KNO3, pH, inoculum size, incubation temperature and incubation time, respectively) and β
are the coefficient of the ten tested independent variables. The experimental runs were conducted according to the PBD matrix in 250 ml Erlenmeyer flasks with working volume of 25 ml. All experimental runs were conducted at agitation speed of 150 rpm.
Three key determinants (orange peel waste percent, pH of the production medium and incubation temperature) identified through PBD had significant effects on the level of PGase. In order to determine the optimal level of each key determinant ((independent variable) along with the maximal level of PGase (dependent variable), a response surface methodology approach was applied here. Box- Behnken design, developed by Box and Behnken (1960), was employed in this study. Fifteen experimental runs (trials) had been conducted. The following polynomial equation from the second order (Equation 4) was set in order to estimate the effect of all possible forms of interactions imposed by the above mentioned three independent variables on the level of PGase enzyme:
Where Y is the level of PGase activity, β
is the model intercept, X
are the tested independent variables (orange peel waste percent, pH of the production medium, and incubation temperature, respectively), β
are linear coefficients, (β
) are quadratic coefficients and (β
) are cross interaction coefficients. For statistical calculations, each independent variable X was coded as Xi according to the Equation 5.
is dimensional coded value of the independent variable, xi is the real value of this variable at this coded value, x
is the real value of this variable at the center point (zero level) and Δxi is the step change value. The experimental runs were conducted according to the Box –Behnken matrix in 250 ml Erlenmeyer flasks with a working volume of 25 ml. All experimental runs were conducted at agitation speed of 150 rpm.
Statistical, canonical analyses and contour plots
RSM package (R Development Core Team 2009), available from the Comprehensive R Archive Network at http://CRAN.R-project.org/package=rsm, was used in this study to carry out multiple regression, canonical analyses and graphing of three dimensional contour surface plots.
Effect of different pH and temperature on the activity of PGase
Two buffers were used in this study to cover a wide range of pH, 50 mM sodium acetate buffer, pH 3-6 and 50 mM phosphate buffer, pH 7-11. Five different temperatures (37°C, 40°C, 50°C, 60°C and 70°C) were used to test the optimal activity of PGase.