Investigation on recovery of alkaline protease from
The membrane separation processes are the most widespread in the field of biotechnology, and they are more easily operated and scaled up in comparison to other bioseparation processes such as chromatography, and electrophoresis. Among the various membrane separation processes, ultrafiltration is one of the processes that functions under pressure gradient which is mostly used for separation and purification of products including enzymes and other proteins [
Proteases are commercially important industrial enzymes accounting 60% of the total enzyme sales with two thirds of the proteases produced are from microorganisms [
Huge amount of municipal wastewater sludge has been generating in Canada. Due to increase in population and other developments sludge management is becoming a crucial environmental concern due to strict regulations on sludge disposal. So, bioconversion of wastewater sludge into value added products is economic and ecofriendly approach. The use of wastewater sludge for the production of alkaline protease with
The aim of the present study was to recover and concentrate the alkaline protease activity from culture filtrate of fermented wastewater sludge using ultrafiltration technique. The efficiency of enzyme was examined in the presence of standard commercial detergents and the enzyme was characterized with respect to the effect of various additives such as stabilizers and inhibitors on the stability at higher temperatures and in alkaline pH.
The wastewater secondary sludge samples collected from municipal wastewater treatment of Communauté Urbaine de Quebec (CUQ, Quebec) were used. The experiments were conducted at a sludge suspended solids concentration of 30 g/L. The sludge was centrifuged in order to obtain higher suspended solids concentration (30 g/L). The sludge characteristics were measured according to standard methods [
Physical and chemical characteristics of secondary sludge.
Characteristics | Concentration |
---|---|
Physical characteristics: | |
Total solids (g/L) | 29 ± 1.2 |
Volatile solids (g/L) | 18 ± 0.6 |
Suspended solids (g/L) | 22 ± 0.9 |
Volatile suspended solids (g/L) | 17 ± 0.7 |
pH | 5.7 ± 0.3 |
Chemical characteristics: | |
Total carbon (%, dry total solids) | 38.12 ± 1.5 |
Total nitrogen (%, dry total solids) | 5.5 ± 0.3 |
Ammonical nitrogen (mg N/kg) | 680 ± 22.1 |
Total phosphorus (mg P/kg) | 12 422 ± 42 |
Orthophosphates (mg P/kg) | 7 780 ± 24 |
Metals (in mg/kg) (dry basis): | |
Al3 | 13 305 ± 41 |
Ca2+ | 16 160 ± 45 |
Cd2+ | 3.5 ± 0.16 |
Cr3+ | 66.5 ± 2 |
Cu2+ | 270 ± 9 |
Fe2+ | 10 365 ± 30 |
Mg2+ | 1874 ± 62 |
Mn2+ | 198 ± 6.9 |
K | 1720 ± 53 |
Pb2+ | 61 ± 2 |
Zn2+ | 477 ± 15 |
A loopful of bacterial growth from a nutrient agar plate was used to inoculate a 500 mL Erlenmeyer flask containing 100 mL of sterilized nutrient broth (composition: 0.3% beef extract and 0.5% peptone) (sterilized at 121°C for 15 min). The flask was incubated in a shaker incubator (New Brunswick) at 35°C with 220 rpm for 12 h. 500 mL Erlenmeyer flasks containing 100 mL of sterilized sludge were then inoculated with 2% (v/v) inoculum from the above flask. The flasks were incubated in the same way for 12 h and these actively growing cells were used as inoculum for fermentor experiments.
A fermenter (Biogénie Inc., Quebec) of 15 L capacity equipped with accessories and automatic control systems for dissolved oxygen, pH, antifoam, impeller speed, aeration rate and temperature and with working volume of 10 L sludge supplemented with 1.5% (w/v) soybean meal and 1.5% (w/v) lactose (sterilized at 121°C for 30 min) was used for production of extracellular alkaline protease. The medium was inoculated with 4.5% (v/v) inoculum. Temperature and pH of the fermentation medium were controlled at 35°C and 7.5, respectively. Dissolved oxygen concentration was maintained above 20% (1.56 mg O2/L) saturation (critical oxygen concentration) by agitating the medium initially at a speed of 200 rpm and finally increased up to 500 rpm and air flow rate was controlled automatically using a computer controlled system. The fermented broth was collected aseptically in HDPE bottles (VWR Canlab, Canada) after 42 h of fermentation and sealed with paraffin and preserved at 4°C for further use.
Fermented broth was centrifuged at 9000x g for 30 min according to the procedure of Brar et al
The equipment used for ultrafiltration was of tangential flow filtration type (PREP/SCALE-TFF, Cartridges Millipore) with recirculation. The fluid was tangentially pumped along the surface of the membrane. Pressure was applied to force a portion of the fluid through the membrane to the permeate side. The supernatant from centrifuge was fed into the ultrafiltration equipment by a pump (Casy Load, Master Flex, Millipore). The supernatant was brought to room temperature (25°C) in order to conduct ultrafiltration study. The process consisted of feeding aseptically a volume (1 L) of the supernatant from the centrifugation step referred to as “feed” through the membrane in order to concentrate the active components to a concentrated volume referred to as “retentate” which was 18% of the volume of the supernatant [
After each ultrafiltration operation, liquid in the membrane was completely drained. Taking into account the type of medium used in this study (biological environment), it was recommended to use an alkaline solution (0.1 N NaOH). The alkaline solution was passed through the membrane until the membrane was clean. Later, the membrane was removed and inverted to facilitate complete washing. Resistance of the membrane can be determined by “normalized permeability weight” (NWP)
Membranes with molecular weight cut-off (MWCO) of 10 kDa and 100 kDa were used in the present study (Millipore, prep/scale spiral wound TFF-1). The membrane was made up of regenerated cellulose and was of the type spiral wound TFF-1 module PLCC with a surface area of 0.1 m2. Supernatant was passed first through 100 kDa membrane to eliminate all other sludge impurities and final permeate was collected as enzyme source to carry out optimization studies using membrane of 10 kDa. Concentrated enzyme was used for characterization purpose. Characteristics of the membranes were presented in Table
Characteristics of the membrane.
Description of membrane | Characteristics |
---|---|
Type | Prep/scale spiral wound TFF-1 |
Filter type | Ultrafiltration |
Length, cm (in.) | 23.4 (9.2) |
Diameter, cm (in.) | 5.8 (2.3) |
Minimum working volume (mL) | 150 |
pH range | 2.0–13.0 |
Configuration | Spiral wound cartridge |
Filtration area (m2) | 0.23 |
Operation temperature (°C) | 4–50 |
Filter material | Regenerated cellulose |
Maximum Intel pressure, bar (psi) | 0–5.5 (0–80) |
Recirculation rate (L/min) | 1.0–6.0 |
Molecular weight cut off (MWCO), kDa | 10 |
Transmembrane pressure and flux of the feed are important parameters to be controlled in ultrafiltration. For the optimization, the experiment was carried out for various values of TMP (70–110 kPa) and feed fluxes (455–2500 L/h/m2). In a typical ultrafiltration process, lower permeate flow results in higher solute concentration in the retentate. Samples were withdrawn to determine the suspended solids, total solids, soluble protein, and protease activity in the retentate and permeate.
The total solids of the samples (supernatant, retentate, and permeate) were measured by drying 30 mL volume at 105°C [
The modified method of Kunitz [
The activity of protease was measured at different pH values in the absence and presence of 10 mm CaCl2. The pH was adjusted using different buffers; acetate buffer (pH 5), phosphate buffer (pH 6-7), borate buffer (pH 8-9), bicarbonate buffer (pH 10), and Robinson and stokes buffer (pH 11-12). Reaction mixtures were incubated at 37°C and the activity of the enzyme was measured.
Stability of the enzyme was determined by incubating the reaction mixtures at various pH values using different relevant buffers (pH 5–12) for 2 h at 37°C. The residual activity after incubation was determined under standard assay conditions. Residual activities are obtained at respective pH values assuming the activity of enzyme before the incubation is 100%.
Optimum temperature was determined by activity assay on casein at pH 10 from 30°C–90°C in the absence and presence of 10 mm CaCl2 and relative protease activities were assayed at standard assay conditions using casein as substrate.
The thermostability of enzyme was measured by incubating the enzyme preparation at different temperatures ranging from 30°C–90°C for 180 min in the absence and presence of 10 mm CaCl2. The residual activity after incubation was determined under the standard assay conditions. Residual activities are obtained at respective temperatures assuming the activity of the enzyme before the incubation is 100%.
The effect of various protease inhibitors (5 mm) such as serine inhibitors (phenylmethyl sulphonyl fluoride [PMSF] and diisopropyl fluorophosphate [DFP]), cysteine-inhibitors (p-chloromercuric benzoate [
To study the effect of various metal ions (Ca2+, K+, Fe2+, Zn2+, Hg2+, Mg2+, Mn2+, Cu2+, Co2+, Na+) on enzyme activity, metal salt solutions were prepared in a concentration of 10 mm and 1 mL of metal solution was mixed with 5 mL of enzyme and was incubated for 2 h. Enzyme activities were measured at standard assay conditions. The activity is expressed in terms of relative activity assuming that the activity of the enzyme in the absence of metal salts just before the initiation of the treatment is 100%.
The effect of various protein substrates such as casein, BSA, egg albumin, and gelatin were determined under assay conditions by mixing 1 mL of the enzyme and 5 mL of assay buffer containing the protein substrates (1.2% w/v). After incubation at 60°C for 10 min, the reaction was stopped by adding 10% TCA (w/v). The undigested protein was removed by filtration (whatman filter paper, 934-AH) and the absorbance of the filtrate was measured at 275 nm. The protease activity towards casein was taken as a control.
Protease enzyme stability with commercial detergents was studied in the presence of 10 mm CaCl2. The detergents used were Merit Selection (Metrobrands, Montreal, Quebec), La Parisienne (Lavo Inc., Montreal, Quebec), Arctic Power (Phoenix brands Canada), Bio-vert (Savons Prolav Inc., Canada) and Sunlight (the Sun products of Canada corporation, Ontario). The detergent solutions (0.7% w/v) were prepared in distilled water and incubated with the partially purified enzyme (2 mL recovered enzyme and 1 mL detergent of 0.7%) up to 3 h at 60°C. At every 30 min interval, the protease activity was estimated under standard assay conditions. The control was maintained without any detergent and enzyme activity was taken as 100%.
Application of protease enzyme (2 mL recovered enzyme) as detergent additive in removing blood stains was studied on white square cotton cloth pieces measuring 4 × 4 cm prestained with goat blood according to the method of Adinarayana [ Flask with distilled water (100 mL) + stained cloth. Flask with distilled water (100 mL) + stained cloth + 1 mL of respective detergent solution at 7 mg/mL. Flask with distilled water (100 mL) + stained cloth + 1 mL of respective detergent solution (7 mg/mL) + 2 mL of enzyme solution (114 IU/mL).
The above flasks were incubated at 60°C for 15 min. After incubation, cloth pieces were taken out, rinsed with water, and dried. Visual examination of various pieces exhibited the effect of enzyme in removal of stains. Untreated cloth pieces stained with blood were taken as control.
The TMP was given by [
Maximum values of protease activity, soluble protein, total solids, and suspended solids at optimum TMP and feed flux.
Parameters | Supernatant | Retentate | |
Before UF | TMP (90 kPa) | Feed flux (714 L/h/m2) | |
Protease (IU/mL) | 15 ± 0.72 | 69 ± 3.4 | 67.3 ± 3.3 |
Soluble protein (mg/mL) | 1.7 ± 0.08 | 7.8 ± 0.39 | 7.6 ± 0.37 |
Total solids (g/L) | 12 ± 0.6 | 55.2 ± 2.75 (9.9 g/180 mL | 54 ± 2.7 (9.72 g/180 mL) |
Suspended solids (g/L) | 0.6 ± 0.03 | 2.7 ± 0.13 (0.48 g/180 mL) | 2.52 ± 0.12 (0.45 g/180 mL |
Protease activity and protein concentration in the retentate at different transmembrane pressures.
Total solids (TS) and suspended solids (SS) concentration in the retentate at different transmembrane pressures.
Profiles of protease activity, soluble protein, total solids, and suspended solids versus different feed fluxes at TMP of 90 kPa were presented in Figures
Protease activity and protein concentration in the retentate at different feed flux rates.
Total solids (TS) and suspended solids (SS) concentration in the retentate at different feed flux rates.
pH is a determining factor in the expression of an enzyme activity as it alters the ionization state of the amino acid or ionization of substrate. The ionization state of enzymes is undoubtedly one of the most crucial parameters that control substrate binding, catalytic enzyme action, and three-dimensional structure of enzyme molecule. Effect of pH on enzyme activity (permeate of 100 kDa) and stability in the presence and absence of 10 mm CaCl2 are presented in Figure
Effect of pH on the activity and stability of alkaline protease in the absence and presence of 10 mM CaCl2 .
Temperature profiles on enzyme activity in the presence and absence of 10 mm CaCl2 are shown in Figure
Effect of temperature profiles on activity of enzyme in the absence and presence of 10 mM CaCl2 .
Temperature profile on enzyme stability was presented in Figure
Effect of temperature on stability of alkaline protease enzyme (ultrafiltrated samples) in the presence of 10 mM CaCl2.
Inhibition studies primarily give an insight of the nature of an enzyme, its cofactor requirements and the nature of the active center [
Effect of enzyme inhibitor and chelator on protease activity.
Inhibitor/chelator (5 mM) | Relative enzyme activity (%) |
---|---|
Control | 100 |
78 ± 3.8 | |
91 ± 4.3 | |
Phenylmethyl sulphonyl fluoride (PMSF) | 0 |
Diisopropyl fluorophosphate (DFP) | 10 ± 0.5 |
Iodoacetate | 96 ± 4.4 |
Ethelene diamine tetra acetic acid (EDTA) | 110 ± 5.6 |
Dithiotheritol | 100 ± 4.8 |
Effect of various metal ions with 5 mm concentration on the enzyme activity was tested and results were presented in Table
Effect of metal ions on alkaline protease activity.
Metal ions (5 mm) | Residual alkaline protease activity (%) |
---|---|
Control | 100 |
Ca2+ | 125 ± 6.1 |
K+ | 6 ± 0.26 |
Fe2+ | 66 ± 3.1 |
Zn2+ | 98 ± 4.7 |
Hg2+ | 23 ± 1.1 |
Mg2+ | 99 ± 4.8 |
Mn2+ | 114 ± 5.5 |
Cu2+ | 95 ± 4.6 |
Co2 + | 91 ± 4.4 |
Na+ | 96 ± 4.7 |
Effect of some native proteins as substrates on enzyme was studied and results were presented in Table
Activity of alkaline protease against different natural proteins.
Protein (2 mg/mL) | Relative enzyme activity (%) |
---|---|
Casein | 100 |
Gelatin | 3 ± 0.14 |
BSA | 54 ± 2.6 |
Egg albumin | 35 ± 1.7 |
Protease used for detergent additive is expected to be stable in the presence of various commercial detergents. The protease from
Compatibility of alkaline protease activity from
Relative enzyme activity (%) | ||||||
Time (h) | Control | Sunlight | La Parisienne | Merit selection | Arctic power | Bio-vert |
0.0 | 100 | 100 | 100 | 100 | 100 | 100 |
0.5 | 97 | 95 | 90 | 89 | 91 | 88 |
1.0 | 93 | 91 | 82 | 80 | 83 | 81 |
1.5 | 90 | 88 | 71 | 69 | 72 | 70 |
2.0 | 84 | 81 | 65 | 61 | 64 | 64 |
2.5 | 80 | 75 | 57 | 54 | 60 | 59 |
3.0 | 74 | 62 | 52 | 48 | 55 | 50 |
Protease produced by
Washing test of alkaline protease from
In this study, enzyme was concentrated and characterized to apply as additive in detergents as detergent industry needs concentrated enzyme in order to have higher efficiency. The recovery of alkaline protease using ultrafiltration process with an optimum transmembrane pressure of 90 kPa and feed flux of 714 L/h/m2 showed a recovery of 83% of the protease activity. The protease from
The authors are sincerely thankful to Natural Sciences and Engineering Research Council of Canada (Grants nos. A4984, Canada Research Chair) for financial support. The views and opinions expressed in this paper are those of authors and should not be construed as opinions of the U.S. Environmental Protection Agency.