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With emphasis on thermal behavior in presence of different pH conditions and salts, the kinetic and thermodynamic parameters of purified polygalacturonase (PG) of E. carotovora subsp. carotovora (Ecc) BR1were studied since characterization of an enzyme is significant in the context of burgeoning biotechnological applications. Thermodynamic parameters for polygalacturonic acid hydrolysis by purified PG were as follow: ΔH* = 7.98 kJ/mol, ΔG* = 68.86 kJ/mol, ΔS*= −194.48 J/mol/K, ΔGE−S = −1.04 kJ/mol and ΔGE−T = −8.96 kJ/mol. The turn over number (kcat) was 21/sec. Purified PG was stable in 20-50-C temperature range and was deactivated at 60-C and 70-C. Thermodynamic parameters (ΔH*, ΔG*, ΔS*) for irreversible inactivation of PG at different temperatures (30-60-C) were determined, where effectiveness of various salts and different pH (4-8) individually for thermalstability of PG were characterized. The efficacy of various salts for thermal stability of PG was in the following order: MgCl2 >KCl >BaCl2 >CaCl2 >NaCl. Present work projects kinetics, thermodynamics of substrate hydrolysis as well as thermalstabilization studies of PG from Erwinia species are reported.
Key words: Polygalacturonase, Erwinia carotovora, Thermodynamic, Kinetic, Thermalstabilization.
Pectinases have tremendous potential in the enzyme industries. They are one of the upcoming enzymes of the commercial sectors, especially in the food, textile, waste treatments in the paper and pulp industries . They also aid in maintaining ecological balance by causing decomposition and recycling of waste plant materials .
Almost all of the commercial preparations of pectinases are produced from fungal sources, mainly from Aspergillus niger. The microbial pectinases account for almost 25% of the global food enzyme sales . The application of pectinases in various fields are widening, demanding the discovery of new strains producing pectinases with novel properties. Therefore, to determine the characteristics of pectinase is essential for its efficient and effective applications . Biochemical and thermal-stabilization characterizations will help to establish additional information required to maintain the desired level of enzyme activity over a long period of time and improved stability. These are important parameters taken into account in the selection and design of an enzyme .
Plant pathogenicity and spoilage of fruits and vegetables by rotting are some manifestations of pectinolytic enzymes . Extracellular pectinolytic enzymes produced by E. carotovora subsp. carotovora and other soft rot Erwinia sp. are required in the elicitation of tissue-macerating (soft-rotting) disease in wide variety of plants . Pectin degrading enzymes, particularly endo-polygalacturonase (PG) are among the initial glycanases synthesized and secreted during majority of phytopathogenic microbial infections . There is also strong correlative evidence supporting the involvement of endo-PG in causing soft-rotting or tissue maceration and are therefore considered as virulence determinant of soft rot Erwinia. PG (EC 184.108.40.206) is a member of the pectinase family that acts on α (1,4)- linkages of polygalacturonic acid (PGA) in pectin, a cementing substance in plant cell wall, causing structural degradation .
Bacterial pectinases particularly produced by Bacillus spp. are reported in different publications for varied applications . In the present studies the potential of PG produced by Ecc BR1 is examined, which is important for the development of industrial applications. This report is novel since the kinetic and thermodynamic characterization of PG by Ecc BR1 and its thermal behavior with respect to different pH and salt conditions are explained for the first time.
Materials and Methods
Microorganism and culture conditions
The bacterial strain E. carotovora subsp. carotovora (Ecc) BR1 used in this study was isolated from macerated tissue of Brinjal fruit (Solanum melongena var. esculentum ) and identified on the basis of standard 16s rRNA gene sequencing. Gene sequence has been deposited in NCBI-Genebank (accession no.: FJ187821). The bacterium was cultivated and routinely maintained on Nutrient agar medium containing 0.5% pectin.
Production and purification of PG
A liquid medium containing (g/l): peptic digest of animal tissue, 5.0, sodium chloride, 5.0, beef extract, 1.5, yeast extract, 1.5, pectin, 5.0 with pH adjusted to 6.5 was sterilized by autoclaving at 121°C for 15min. One percent inoculum of Ecc BR1 (1-108 CFU/ml) was added to 500ml of liquid media in 2 liter Erlenmeyer flask and incubated for 24h at 30°C on rotary shaker maintained at 180rpm. The culture was harvested at the end of the growth phase, centrifuged at 15,880 -g for 15min at 4°C and the supernatant was precipitated with acetone (1:1 v/v). After storing it for 4h, the precipitate was centrifuged, dissolved in 50 mM sodium acetate buffer (pH 5.0) and dialyzed against 5 mM sodium acetate buffer (pH 5.0). Then it was chromatographed on a CM-cellulose column (1.5 cm - 12 cm) at a flow rate of 0.5 ml/min by linear gradient elution technique with same buffer of increasing ionic strength from 5 to 500 mM .
Molecular mass determinations and activity staining assay
The SDS-PAGE was performed using 12% polyacrylamide gel according to the method of Laemmli . Protein Molecular weight markers (Banglore GenieTM, India; PMWH range: 29-205KDa, carbonic anhydrase, 29 kDa; ovalbumin, 43 kDa; bovine serum albumin, 66 kDa; myosin-Rabit muscule, 205 kDa) were run alongside the samples. After electrophoresis the gel was stained by silver salts . Activity staining assay for polygalacturonase was performed using polygalacturonic acid as a substrate incorporated in 12% polyacrylamide gel and after electrophoresis incubated overnight at 30°C in 0.05M sodium acetate buffer (pH-5.0) contained in 0.2M NaCl. After incubation gel was stained with 0.01% (w/v) Toluidine blue-O .
Polygalacturonase activity was determined by measuring reducing sugar released as a result of hydrolysis of polygalacturonic acid using a dinitrosalicylic acid (DNS) reagent . The reaction mixture containing of 200 μl 0.5% (w/v) polygalacturonic acid (PGA) in 50 mM sodium acetate buffer (pH-5.0) and 100 μl of appropriately diluted enzyme solution was incubated at 40°C for 20 min. Absorbance of the reaction products was monitored at 540nm. The reducing sugars formed were quantified using D-galacturonic acid as a standard. One Unit of enzyme activity was defined as the amount of enzyme required to release 1μmole of D- galacturonic acid per min at 40°C at pH-5.0.
Effect of pH and temperature on activity
The effect of pH on PG was determined by measuring activity at 40°C for 20 min, using different buffers: 50mM phthalate buffer (pH 2.6-3.0), in 50mM Na-acetate buffer (pH 4.0, 5.0, 5.4), 50mM phosphate buffer (pH 6.0,7.0) and 50mM glycine-NaOH buffer (pH 8.0, 9.0, 10.0). Optimum temperature and activation energy (Ea) were determined by incubating appropriate amount of the enzyme with 0.5% PGA at various temperatures ranging from 20° to 70°C in 50mM Na-acetate buffer (pH 5.0) for 20 min. Ea was calculated by using Arrhenius plot . Ea was calculated from the slope of a linear plot of 1000/T versus ln[PG activity], Ea= -slope-R, where R (gas constant) = 8.314 J/K/mol. The effect of temperature on the rate of reaction was expressed in terms of temperature quotient (Q10), which is the factor by which the rate increases due to a raise in the temperature by 10°C. Q10 was calculated by rearranging the equation :
Where, E = Ea = activation energy.
Estimation of kinetic parameters
The Michaelis constants (Km and Vmax values) were determined by measuring the activity reaction rates (under the conditions given earlier) at substrate concentrations ranging from 0.01 to 0.5% (w/v) PGA with fixed amount of enzyme in 50mM Na-acetate buffer pH 5.0 at 40°C for 20 min. The Km and Vmax values were obtained from the Lineweaver-Burk plot (1/[S] versus 1/V), and catalytic efficiency i.e. the ratio Vmax/Km, and kinetic constants (kcat, kcat/Km) were determined . To determine substrate specificity, 0.5% (w/v) solution of pectic substrates viz. PGA and pectin with a 28%, 35% and 95% degree of esterification (DE) were used. Specificity in terms of specific activity was measured by PG assay.
Estimation of thermodynamic parameters for polygalacturonic acid hydrolysis
The thermodynamic parameters for substrate hydrolysis were calculated by rearranging the Eyring's absolute rate equation derived from the transition state theory [21, 4]:
Where kb is the Boltzmann's constant (R/N) = 1.38-10−23 J/K, R the gas constant = 8.314 J/K/mol, N the Avogadro's number = 6.02-1023/mol, T the absolute temperature (K), h the Planck's constant = 6.626-10−34 J s, ΔH* the enthalpy of activation and ΔS* is the entropy of activation:
Where activation energy Ea of enzyme for substrate hydrolysis was determined using an Arrhenius model is described in Section 2.5.
The free energy of substrate binding and transition state formation was calculated using the following derivations:
where Ka = 1/Km :
Thermal stability assay
Thermal inactivation was determined by incubating enzyme at 20, 30, 40, 50, 60 and 70°C temperatures. Aliquots were withdrawn at 10 min intervals, cooled on ice for 1 h and assayed for PG activity.
Effect of pH or salts on thermal stability
A pH range from 4.0 to 8.0 was selected for thermal stability. The pH of enzyme solutions were adjusted with acetic acid (1M) or NaOH (1M) followed by the thermal stability assay. Selection of salts concentration was CaCl2 (1 mM), NaCl (0.1M), MgSO4·7H2O (5 mM), KCl (0.15 M), BaCl2 (0.5 mM) on the basis of earlier observations by Nasuno and Starr . The stability of purified enzymes with each salt was studied separately; by incubating each salt with the enzyme at 4°C for 10 min. The remaining unbound salts were removed by dialysis for 1h followed by thermal stability assay.
Estimation of deactivation rate constant
The residual activity was determined by comparing enzyme activity after heating with that of freshly prepared unheated enzyme. The deactivation rate of these enzymes was calculated by first order expression :
The Kd (deactivation rate constant or first order rate constant) values were calculated from a plot of time (t) versus ln[Et/E0] at a particular temperature.
The half-life of an enzyme is defined as the time required by the enzyme to loose half of its initial activity, which is given by:
Estimation of thermodynamic parameters for thermal deactivation
Thermodynamic parameters were calculated by rearranging the Eyring absolute rate equation . ΔH* and ΔS* values were calculated from the slope and intercept of a 1/T versus ln[Kd/T] plot, respectively. So that,
Free energy change were calculated by using the following relationship
Energy of deactivation was estimated using the Arrhenius equation:
Energy (E) involved in this deactivation process was calculated from the slope of a linear plot of 1/T versus ln[Kd] , E= -slope-R, where R (gas constant) = 8.314 J/K/mol. Thermalstability of enzyme in the presence of different salts and pH was determined by heating the enzyme in the presence of the salt or in specific pH in sealed tubes for different times at 30, 40, 50 and 60°C. Enzyme activity was measured before and after incubation period at respective temperature to demonstrate residual activity under each condition. All experiments were conducted in triplicate and results shown are mean values.
Results and discussion
Purification and characterization of PG
The purification of PG (Mw- 43 kDa) produced from locally (Gujarat, India) isolated Ecc BR1 strain grown under submerged condition is summarized in Table 1 and its purification to apparent homogeneity was completed in two steps, checked on SDS-PAGE stained by silver salts and activtity staining for polygalacturonase (Figure 1). The overall purification was achieved upto 20.6-fold with 6.1% recovery (Table 1).
Optimum PG activity was observed in range of pH 4.0-6.0, maximum at pH 5.0 and 40°C temperature (Figure 2A,B). The activation energy (Ea) for substrate hydrolysis of purified PG was 10.6 kJ/mol, a value lower than reported for PG from other sources . The overall Q10 (temperature quotient) of purified PG was 1.03 between 20-70°C.
Determination of kinetic parameters of purified PG
Purified PG showed typical Michaelis-Mentan profile. Km and Vmax values of PG determined through Lineweaver-Burk plot for the hydrolysis of polygalcturonic acid at 40°C were 0.67 mg/ml and 2.381 μM/ml/min respectively. The apparent value of Km is in proximity with previously reported values for other PG (0.5 mg/ml) from Rhizopus sp. , (0.49 mg/ml) from Burkholderia cepacia , (0.8 mg/ml) from Sclerotinia sclerotiorum . The estimated Km value was 7.0 and 1.2 times lower than Saccharomyces cerevisiae and S. sclerotiorum polygalacturonase respectively. This indicates that the current polygalacturonase has a higher affinity for PGA than above two microorganisms . The turn over number Kcat was 21/sec and the apparent second-order rate constant (Kcat/Km) was 31.34 (Table 2). PG of Ecc BR1 has specific activity of 6.84 U/mg determined from kinetic parameters. The catalytic efficiency value (Vmax/Km) of purified PG from Ecc BR1 was 3.55 μmoles/min/mg substrate (Table 2), which is useful for measuring efficiency of enzyme in commercial applications . The purified PG was capable of hydrolyzing different variety of pectic substrates, with different DE percentage. It show high substrate specificity towards polygalacturonic acid (PGA) (3.1 U/mg protein) and 35% DE pectin (3.01 U/mg protein).While lower substrate specificity towards 28% DE pectin (1.0 U/mg protein) and 95% DE pectin (0.4 U/mg protein) was observed. Thus concomitant with increase in DE of pectic substrates PG activity was found to be decrease.
Determination of thermodynamic parameters of purified PG for polygalacturonic acid hydrolysis
There is paucity of information regarding thermodynamic parameters of PG of other microbes for substrate hydrolysis. The enthalpy of activation (ΔH*), Gibbs free energy (ΔG*) and entropy of activation (ΔS*) for polygalacturonic acid hydrolysis by purified PG of Ecc BR1 were calculated shown in Table 2.
The low enthalpy value of purified PG showed that the formation of transition state or activated complex between enzyme-substrate was very efficient. Moreover, as explained earlier by Muhammad et al. , low ΔG* value suggested that the conversion of its transition complex (E-S) into products was more spontaneous. The low entropy value indicates the transition complex of PG had less disorder. The feasibility and extent of chemical reaction is best determined by measuring change in ΔG* for substrate hydrolysis, i.e. the conversion of E-S complex into product .The free energy for activation of substrate binding (ΔG*E-S) and the free energy for the formation of activation complex (ΔG*E-T) of the purified PG again confirmed that the enzyme has high affinity towards polygalacturonic acid for hydrolysis and its spontaneous conversion into D-galacturonic acid (Table 2).
Thermalstabilty studies of purified PG
Thermalstability represents the capability of enzyme molecule to resist thermal unfolding in absence of substrate, while thermophilicity is the ability of an enzyme to work at elevated temperatures in the presence of substrate. The stability of enzymes in this case is always judged by residual activity .
The kinetics of thermal inactivation of the PG from the Ecc BR1 was measured. The enzyme was incubated at 20-70°C. Pseudo first order plot was applied to determine the extent of thermal inactivation (Figure 3). The PG was found to be stable at 20, 30, 40 and 50°C with half life (t1/2) of 426, 192.5, 126 and 99 min respectively. During further increase in temperature, inactivation rate increased. At 60 and 70°C inactivation rate increased drastically with decrease in half life of 36 and 10 min respectively.
Thermal inactivation of enzymes occurs in two steps  i.e. N ↔ U* → I, Where N is native enzyme, U* the unfolded inactive enzyme, which could be reversibly refolded upon cooling and I is the inactivated enzyme formed after prolonged exposure to heat and therefore cannot be recovered upon cooling.
The inactivation curves of purified PG of Ecc BR1 were not linear in the range of temperatures studied (Figure 3). Rather, a biphasic nature was evident, implying that inactivation of PG was first-order process commonly observed for enzyme inactivation . Purified PG being studied in this work was found as thermostable as the PG from commercial preparations Pectinase CCM .
Thermal deactivation of purified PG at varying pH
The extent of deactivation of an enzyme is measured by the deactivation rate. The deactivation rate is proportional to active enzyme concentration. Kd or the deactivation rate constant is considered as the proportionality constant . Deactivation rate and half-life were studied at 30, 40, 50, and 60°C with different pH-3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 conditions (Figure 4 A,C). pH is one of the main factor affecting tertiary and quaternary structure of protein and enzyme. In many cases the rate of deactivation depends on the pH of the enzyme solution . A pH range from 4.0 to 8.0 was selected since the PG was found to have optimum activity at pH 5.0. As shown in Figure 4A,C at 40 °C (the temperature optima) the PG was stable at all the pH conditions (pH 4.0-8.0). At 60°C ( higher than optimum temperature) stability of PG increased with the increasing pH to alkalinity, indeed the order of stability in various pH conditions was following: pH 8 ≥ pH 6 > pH 7 > pH 5 > pH 4 (Figure 4A,C). At higher pH the folding of PG may relax due to changes in the balance of electrostatic and hydrogen bond which result in increased stability [14, 30].
However, the half lives (t1/2 ) of PG in different pH conditions did not increase uniformly as pH increased to alkaline condition; only exception was at 60°C where there was steady increase in t1/2 viz 18, 24, 46, 40 and 46 min with increasing pH in the range of 4.0-8.0 respectively. Here at temperature 30°C the t1/2 of purified PG was optimum (166 min) at pH 5.0 and lowest (57min) at pH 4.0. At temperature 40°C the t1/2 values were relatively similar to those in the range of pH 4.0-7.0 respectively. Only at pH 8.0 the half life of PG was found to be greater (125 min) than at pH range of 4.0-7.0 (Figure 4C). The increase in half-life of purified PG in pH range 4.0-8.0 at temperature range 30-60°C is similar to other reports of half life for partially purified PG-I and II from A. niger  and chitinase of Pantoea dispersa .
Thermal deactivation of purified PG in presence of different salts
Deactivation rate and half lives of purified PG were studied at temperature range 30- 60°C in presence of different salts. The deactivation rate increased with increasing temperature, in other words the half life decreased with increasing temperature. The addition of 0.1M NaCl, 0.15M KCl, 1mM CaCl2, 5mM MgCl2 and 0.5mM BaCl2 individualy to purified PG reduced deactivation rates and increased half life at all temperatures studied (Figure 4B,D).
The effectiveness of various salts was in the following order: MgCl2 >KCl >BaCl2 >CaCl2 >NaCl. This indicated that the effect of salt on stability of enzymes was related to the type of salt and its concentration. Stabilization of PG by salt may be due to reduction in unfavourable electrostatic repulsion which led to reduced unfavorable electrostatic free energy. The stability of PG was found to be optimum with MgCl2 where the lower Kd values of PG were obtained at temperature range of 30-60°C (Figure 4B). Overall the salts used in this study were found to increase the stability of PG. Also, the level of salt required to enhance the stability of PG was in the range generally found in soils. Half lives of PG with different salts were found to increase as compared to PG without any salts, especially with MgCl2 where t1/2 were higher at 30-60°C temperature range (Figure 4D). These half lives are far greater than reported for PG-I, II from A. niger .
Entropy change during thermal deactivation of purified PG in presence of salts and pH
In addition to the deactivation study an investigation of other thermodynamic parameters (ΔG*, ΔH*, ΔS* and deactivation energy) is necessary to understand the behaviour of molecules in different physiological conditions and complex process of deactivation to certain extent . Negative entropy is generally found in biocatalytic system due to compaction of enzyme molecule . Accordingly, the transition state of the PG was ordered as revealed by negative ΔS* value which was comparatively lower for its thermal deactivation (Table 3). The change in entropy has been calculated by transition state theory according to Equation no.-(12). Enzyme is rendered more thermostable by stabilizing native form, by putting non-covalent bond including hydrogen bond, salt bridge and hydrophobic interaction or by decreasing the entropy of unfolding . Similar kind of observations were noted on addition of each salt and at every pH for PG of Ecc BR1 (Table 3).
In presence of MgCl2, ΔS* was lower (-126.59 J/mol K) as compared to other salts, while there was marginal difference with ΔS* (-133.04 J/mol K) of PG in absence of salt. The entropy change for thermal inactivation of PG in presence of salts had following order : CaCl2 > NaCl > BaCl2 > KCl > MgCl2 (Table 3). The increased ΔS* indicates an increase in the number of protein molecules in transition activated state .
The PG at pH 4 had lowest value of ΔS* (-136.38 J/mol K) compared to other pH conditions. The entropy of PG in presence of various pH showed following order : pH 4 < pH 7 < pH 5 < pH 6 < pH 8 (Table-3). The magnitude of the entropic term of the enzymatic reaction decreases as the enzyme stability increases . In all cases of different pH entropy changes were found to be negative (Table 3). This observation is consistent with the fact that there must be a compaction of the reacting enzyme molecule, but such changes could arise from the formation of charged particles and the associated gain and ordering of solvent molecules also .
Enthalpy and activation energy change of PG deactivation in presence of salt and pH
Enthalpy change (ΔH*) and deactivation energy (E) of PG were estimated within a temperature range of 30-60°C in the presence of varying salts and pH. It has been reported that enthalpy change of enzymes should be in range of 20-150 kJ/mol during deactivation . Enthalpy value was calculated from Equation-(11). Enthalpy increased in presence of salts with following order: MgCl2 < BaCl2 < KCl < NaCl < CaCl2 (Table 3). Enthalpy increase in varying pH conditions was random but it was within range of 20-150 kJ/mol. ΔH* of PG deactivation was within this range in the presence of each salt and at every pH demonstrating major involvement of enthalpic stabilization of the protein structure in temperature adaptation. The decrease of ΔH* as enzyme stability increased mainly reflects the decrease in cooperation of inactivation and unfolding. For instance, heat-labile enzyme denatures in a shorter temperature range, leading to the sharp slope of the Arrhenius plots and subsequently to high activation energy Ea and ΔH*. Such high cooperativity probably originates from the lower number of interactions required to disrupt the active conformation .
Enthalpies of PG were moderately lower with all salts tested at temperature range of 30-60°C. ΔH* of purified PG show low values in presence of MgCl2 and BaCl2 compared to other salts, indicating that salts allow thermalstabilization at the enzyme. Enthalpy of PG at pH range (pH 5.0-8.0) tested has less variations. The ΔH* value was lower (41.512 kJ/mol) at pH 4.0 as compared to other remaining pH conditions (Table 3).
The deactivation energy (E) was calculated from the slope of a linear plot of 1/T versus ln[Kd] using Equation-(15) and shown in Table 3. It is observed that the deactivation energy of 170 kJ/mol was maximum at pH 8.0 for PG and started increasing at acidic pH range. The deactivation energy increases at alkaline and neutral pH suggesting that at these pH conditions enzyme requires more amount of energy to deactivate. Deactivation energy (E) increased in every condition with the order : KCl < NaCl < CaCl2< BaCl2< MgCl2. This indicates that divalent cationic salts allow enzyme to require more energy for thermal deactivation as compared to remaining monovalent cationic salts.
Free energy change of PG deactivation in presence of salt and pH
Gibbs free energy (ΔG*) measures the spontanaeity of a reaction. In turn, this thermodynamic parameter measures a combination of changes in heat, and entropy that occurs during a reaction. The increase in ΔS* implies an increase in the number of protein molecules in transition activated state which, in turn, gives lower value of ΔG* . The value of Gibbs free energy (ΔG*) (calculated from Equation-13) are shown in Table 4 for PG. For PG enzymes, ΔG* increased with increasing temperature. However, this was comparitively higher to previously reported partially purified PG from A. niger . Iyer and Ananthanarayan  proposed that kinetic analysis of enzyme inactivation mechanisms is of prime importance allowing for better control over biocatalyst use. In addition, the resistance of enzymes to thermal denaturation is due to the intrinsic contribution of the polypeptide chain (i.e. hydrophobic interaction, hydrogen bonding and ionic stabilization).
To assess the contribution of various salt ions to thermal stabilization of PG of Ecc BR1, the effect of different salts at its optimum activation concentration on themalstability of enzymes was studied at 30-60ËšC. Free energy of PG marginaly varies with each salt indicating that thermal stability of the PG was dependent on interaction with salt to certain extent. ΔG* decreased in the following order: MgCl2 >BaCl2 > CaCl2 ≥ NaCl > KCl with increase in temprature. Similar kind of results were also obtained in varying pH condition in ΔG* with the following order: pH 7 > pH 5 > pH 6 > pH 8 > pH 4. Decrease in ΔG* was not uniform as pH increased to alkaline (Table 4). Monovalent cations showed lower ΔG* compared to divalent cation used in this study. It has been reported that the mechanism by which salt affect enzyme stability may be explained by the effect of salt on water structure and hence on the strength of hydrophobic interaction .
In conclusion, purified PG of Ecc BR1 is catalytically efficient enzyme for polygalacturonic acid hydrolysis. Its ability to hydrolyze different types of pectic substrates, as well as kinetic and thermodynamic parameters makes the enzyme versatile and efficient for industrial applications. The stabilty of this enzyme in the presence of different salts and pH may be useful atribute to utilize when applied as alternative commercial preparation. Enzyme deactivation is equally important to an industrial process however scant attention is given to such studies. These studies enhance the knowledge of mechanism of PG which may lead to establishment of inhibition and interaction studies at molecular level. The PG of Ecc BR1could be a potential candidate for specific applications in food, waste treatment centres, textile and paper industries. It should not be ignored that the feasibility of obtaining PG with novel industrial potential produced by new microbial strain is higher, as in this case, not previously reported for this purpose.