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- Open Access
Comparative one-factor-at-a-time, response surface (statistical) and bench-scale bioreactor level optimization of thermoalkaline protease production from a psychrotrophic Pseudomonas putida SKG-1 isolate
https://doi.org/10.1186/1475-2859-10-114
© Singh et al; licensee BioMed Central Ltd. 2011
- Received: 4 October 2011
- Accepted: 28 December 2011
- Published: 28 December 2011
Abstract
Background
Production of alkaline protease from various bacterial strains using statistical methods is customary now-a-days. The present work is first attempt for the production optimization of a solvent stable thermoalkaline protease by a psychrotrophic Pseudomonas putida isolate using conventional, response surface methods, and fermentor level optimization.
Results
The pre-screening medium amended with optimized (w/v) 1.0% glucose, 2.0% gelatin and 0.5% yeast extract, produced 278 U protease ml-1 at 72 h incubation. Enzyme production increased to 431 Uml-1 when Mg2+ (0.01%, w/v) was supplemented. Optimization of physical factors further enhanced protease to 514 Uml-1 at pH 9.0, 25°C and 200 rpm within 60 h. The combined effect of conventionally optimized variables (glucose, yeast extract, MgSO4 and pH), thereafter predicted by response surface methodology yielded 617 U protease ml-1 at glucose 1.25% (w/v), yeast extract 0.5% (w/v), MgSO4 0.01% (w/v) and pH 8.8. Bench-scale bioreactor level optimization resulted in enhanced production of 882 U protease ml-1 at 0.8 vvm aeration and 150 rpm agitation during only 48 h incubation.
Conclusions
The optimization of fermentation variables using conventional, statistical approaches and aeration/agitation at fermentor level resulted in ~13.5 folds increase (882 Uml-1) in protease production compared to un-optimized conditions (65 Uml-1). This is the highest level of thermoalkaline protease reported so far by any psychrotrophic bacterium.
Keywords
- Response Surface Methodology
- Enzyme Production
- Agitation Speed
- Alkaline Protease
- Aeration Rate
Background
Proteases are one of the most exploited industrial groups of enzymes accounting for nearly 60% of the total worldwide sale of enzymes [1]. In order to meet the growing demand of proteases with cost effectivity, scaling-up of the industrial process is imperative. Joo et al. [2] opined that 30-40% cost of industrial enzymes depends on the growth medium. Major nutritional factors affecting protease production are sources of carbon, nitrogen, growth factors and metal ions [1]. Besides, physical factors such as temperature, pH, aeration/agitation and incubation time also significantly affect the protease production [3]. Therefore, optimization of nutritional and physical parameters for maximum enzyme production is of utmost importance for making the industrial process cost-effective and economically viable.
In conventional 'one-variable-at-a-time' approach, the nutritional/cultural factors are optimized by changing one factor at a time, and keeping other variables constant. This approach is simplest to implement, and primarily helps in selection of significant parameters affecting the enzyme yield. However, this method is not only time restrictive, but also ignores the combined interaction(s) among various physical and nutritional parameters [4]. Conversely, the statistical response surface methodology (RSM) is a useful model for simultaneously studying the effect of several factors influencing the process of enzyme production. This also reduces the number of experiments required in growth medium optimization. Use of factorial designs and regression analyses for generating empirical models makes RSM a good statistical tool [5]. To analyze the effect of various factors in better way, a number of statistical approaches with response surface methodology are attempted for the optimization of enzyme production.
Scaling-up of protease production is also governed by aeration, agitation and dissolved oxygen tension (DOT) of fermenting medium. Agitation and aeration processes are important variables in aerobic fermentation, as oxygen availability and its proper distribution in the fermenting medium is regulatory for protease production. At flask level fermentation process, only agitation can be regulated; however, it results in limited aeration, and hence reduced protease yield. This could be due to less availability of dissolved oxygen (DO), which adversely affects the cell growth and enzyme yield [6]. Hence, bioreactors are preferred over flask level microbial fermentation for optimization of aeration, agitation and dissolved oxygen tension. Although, bioreactors provide efficient oxygen distribution in the fermenting broth, sometimes increased shear can reduce the protease yield. So, a proper ratio of agitation and aeration is required for maximization of protease production [7]. Therefore, optimal conditions for expression of high activity must be first determined in laboratory-scale followed by pilot- and industrial-level fermentors [8].
Keeping the above in view, the present study is an attempt to enhance protease production through optimization of nutritional/physical parameters by conventional (one-variable-at-a-time) and statistical approach (RSM) at shake flask level. Further, the effect of agitation, aeration and dissolved oxygen tension (DOT) on production of a psychro-thermo-alkalistable protease from Pseudomonas putida SKG-1 in bench-scale fermentor (under RSM optimized nutritional and cultural parameters) was also envisaged.
Materials and Methods
Microorganism and protease production
Pseudomonas putida SKG-1 (MTCC 10510) was isolated in our laboratory. This solvent and heavy metal tolerant strain is capable of producing solvent-, psychro-, thermo-, alkali-stable protease [9]. The pure culture was maintained over nutrient agar slants (pH 7.0) at 4°C.
For protease production, 250 ml sterile modified GYE (MGYE) broth [10] of pH 9.0 was taken in 500 ml Erlenmeyer flask, inoculated with 2% (v/v) mother culture of 0.8 OD (A660; 1 cm cuvette) containing 2.8 × 108 colony forming units (cfu) ml-1 and incubated at 25°C in shaker incubator (150 rpm). The fermenting broth (5.0 ml) was periodically drawn aseptically, and growth was assessed by turbidity measurement at 660 nm. Each sample was then centrifuged at 12,000 rpm (4°C) for 10 min, and cell-free supernatant was used for protease assay.
Enzyme assay
The protease activity was assayed by casein digestion method of Shimogaki et al. [11] at 40°C and pH 9.5 (sodium carbonate-bicarbonate buffer). One unit of protease activity is defined as the amount of enzyme required to liberate 1 μg of tyrosine min-1.
Optimization of nutritional and physical parameters
In the conventional scaling-up approach, various nutritional and physical parameters were optimized by maintaining all factors at a constant level in the basal medium, except the one under study. Each subsequent factor was examined after taking into account the previously optimized factor(s). Among carbon sources fructose, xylose, lactose, sucrose, soluble starch, maltose, glycerol and carboxymethyl cellulose (CMC) were supplemented individually by replacing glucose in the basal medium. All sugars were autoclaved separately at 10 psi for 20 min, and added at 1.0% (w/v) level. For the effect of different nitrogen sources, peptone plus yeast extract in MGYE broth were replaced individually by gelatin, urea, casein, casamino acid, beef extract, yeast extract, tryptone, sodium nitrate, ammonium nitrate and ammonium sulfate at 1.0% (w/v) level. Combination of each nitrogen source with 0.5% (w/v) yeast extract (YE) was also attempted. After optimization of carbon and nitrogen sources along with their concentrations, varied levels of yeast extract (0.1-0.7% w/v) were also studied to optimize its dose. Metal cations (0.01% w/v) studied to enhance protease yield were: Ca2+ (CaCl2), Cu2+ (CuSO4), Mg2+ (MgSO4.), Mn2+ (MnSO4), Fe2+ (FeCl2), Zn2+ (ZnSO4) and combination of Ca2+ +Mg2+ ions. The dose of best metal ion was optimized by its supplementation at 0.005-0.05% (w/v) concentrations. Initial pH of the medium (7.0-10.0), shaking speed (0-250 rpm), temperature (10-40°C) and time of incubation (0-72 h) were the physical parameters studied for their effect on bacterial growth and protease production.
Statistical optimization of factors affecting protease production by RSM
Experimental range and the levels of four independent variables employed in RSM in terms of actual and coded factors
Variables | Levels | ||
---|---|---|---|
-1 | 0 | +1 | |
Glucose (%, w/v) | 0.75 | 1.00 | 1.25 |
Yeast extract (%, w/v) | 0.30 | 0.50 | 0.70 |
MgSO4 (%, w/v) | 0.0075 | 0.0100 | 0.0125 |
pH | 8.80 | 9.00 | 9.20 |
Experimental designs used in RSM studies by using four independent variables with six centre points showing observed and predicted values of protease production
Standard Order | Factor A (Glucose %, w/v) | Factor B (Yeast extract %, w/v) | Factor C (MgSO4 %, w/v ) | Factor D (pH) | Observed response (Uml-1) | Predicted response (Uml-1) |
---|---|---|---|---|---|---|
1 | 0.75 | 0.30 | 0.0100 | 9.00 | 419 | 418.13 |
2 | 1.25 | 0.30 | 0.0100 | 9.00 | 457 | 455.63 |
3 | 0.75 | 0.70 | 0.0100 | 9.00 | 449 | 451.13 |
4 | 1.25 | 0.70 | 0.0100 | 9.00 | 482 | 483.63 |
5 | 1.00 | 0.50 | 0.0075 | 8.80 | 524 | 562.46 |
6 | 1.00 | 0.50 | 0.0125 | 8.80 | 540 | 541.96 |
7 | 1.00 | 0.50 | 0.0075 | 9.20 | 372 | 370.79 |
8 | 1.00 | 0.50 | 0.0125 | 9.20 | 396 | 394.29 |
9 | 0.75 | 0.50 | 0.0100 | 8.80 | 561 | 561.58 |
10 | 1.25 | 0.50 | 0.0100 | 8.80 | 617 | 612.58 |
11 | 0.75 | 0.50 | 0.0100 | 9.20 | 426 | 425.92 |
12 | 1.25 | 0.50 | 0.0100 | 9.20 | 447 | 444.92 |
13 | 1.00 | 0.30 | 0.0075 | 9.20 | 369 | 365.25 |
14 | 1.00 | 0.70 | 0.0075 | 9.00 | 415 | 413.75 |
15 | 1.00 | 0.30 | 0.0125 | 9.00 | 406 | 402.75 |
16 | 1.00 | 0.70 | 0.0125 | 9.00 | 416 | 415.25 |
17 | 0.75 | 0.50 | 0.0075 | 9.00 | 446 | 446.63 |
18 | 1.25 | 0.50 | 0.0075 | 9.00 | 479 | 482.13 |
19 | 0.75 | 0.50 | 0.0125 | 9.00 | 466 | 466.63 |
20 | 1.00 | 0.50 | 0.0125 | 9.00 | 498 | 501.13 |
21 | 1.00 | 0.30 | 0.0125 | 8.80 | 509 | 512.96 |
22 | 1.00 | 0.70 | 0.0100 | 8.80 | 513 | 511.46 |
23 | 1.00 | 0.30 | 0.0100 | 9.20 | 324 | 329.29 |
24 | 1.00 | 0.70 | 0.0100 | 9.20 | 392 | 391.79 |
25 | 1.00 | 0.50 | 0.0100 | 9.00 | 514 | 514 |
26 | 1.00 | 0.50 | 0.0100 | 9.00 | 514 | 514 |
27 | 1.00 | 0.50 | 0.0100 | 9.00 | 514 | 514 |
28 | 1.00 | 0.50 | 0.0100 | 9.00 | 514 | 514 |
29 | 1.00 | 0.50 | 0.0100 | 9.00 | 514 | 514 |
30 | 1.00 | 0.50 | 0.0100 | 9.00 | 514 | 514 |
Where, Y is the predicted response, βo offset term, βn liner coefficient, βnn squared coefficient, βnm interaction coefficient, Xn nth independent variable, Xn2 squared effect and XnXm interaction effects.
ANOVA for Response Surface Quadratic Model
Source | Sum of squares | df | Mean square | F-value | p-value |
---|---|---|---|---|---|
Model | 1.205E+005 | 14 | 8609.99 | 820.46 | < 0.0001 |
A-Glucose | 3675.00 | 1 | 3675.00 | 350.20 | < 0.0001 |
B-Yeast extract | 2790.75 | 1 | 2790.75 | 265.94 | < 0.0001 |
C-MgSO4 | 1140.75 | 1 | 1140.75 | 108.70 | < 0.0001 |
D-pH | 69008.33 | 1 | 69008.33 | 6575.95 | < 0.0001 |
AB | 6.25 | 1 | 6.25 | 0.60 | 0.4531 |
AC | 0.25 | 1 | 0.25 | 0.024 | 0.8795 |
AD | 256.00 | 1 | 256.00 | 24.39 | 0.0002 |
BC | 324.00 | 1 | 324.00 | 30.87 | < 0.0001 |
BD | 1024.00 | 1 | 1024.00 | 97.58 | < 0.0001 |
CD | 16.00 | 1 | 16.00 | 1.52 | 0.2372 |
A2 | 274.05 | 1 | 274.05 | 26.12 | 0.0002 |
B2 | 30325.24 | 1 | 30325.24 | 2889.76 | < 0.0001 |
C2 | 13950.10 | 1 | 13950.10 | 1329.33 | < 0.0001 |
D2 | 555.00 | 1 | 555.00 | 52.89 | < 0.0001 |
Residual | 146.92 | 14 | 10.49 | ||
Lack of fit | 146.92 | 10 | 14.69 | ||
Pure error | 0.000 | 4 | 0.000 | ||
Cor total | 1.205E+005 | 28 | |||
Standard deviation | 3.24 | R-squared | 0.9988 | ||
Mean | 465.38 | Adjusted R-squared | 0.9976 | ||
Coefficient of variation (C.V.%) | 0.70 | Predicted R-squared | 0.9930 | ||
PRESS | 846.24 | Adequate precision | 121.595 |
Bench-scale bioreactor level optimization
Effect of aeration
Fermentation was performed in a stirred tank bioreactor (Bioflo 110, New Brunswick Scientific Co. Inc. Edison, NJ, USA) of 3 liter capacity. The fermentor was equipped with direct drive dual Rushton style impeller, PID temperature and agitation control, probes and controller for pH and DO. For protease production, fermentation was carried out in one liter GGY broth under conventional and RSM optimized nutritional (glucose 1.25%, gelatin 2%, yeast extract 0.5%, Mg2+ 0.01%) and cultural (pH 8.8, 25°C, 200 rpm) conditions. The medium was inoculated (2%, v/v) with the mother culture of strain SKG-1 (0.8 OD, A660; 1 cm cuvette) containing 2.8 × 108 cfu ml-1. The aeration of culture broth was effected at different rates (0-1.5 vvm), and samples (5.0 ml) were drawn periodically at 12 h intervals. The bacterial growth was assessed by turbidity measurement at 660 nm. The sample broth was then centrifuged at 12,000 rpm (4°C) for 10 min, and cell-free supernatant was used to assay the protease activity. Change in DOT during the course of fermentation was also recorded throughout the incubation period.
Effect of agitation at optimized aeration
The growth and protease production was further studied by varying the agitation speed from 100 to 250 rpm at optimized aeration rate. Other experimental conditions remained the same.
Statistical analysis
Each set of experiment was performed thrice, and all values presented here are average of three independent experiments. The standard deviation for each value is ≤ 5%.
Results and Discussion
Protease production
Bacterial growth and extracellular protease production in modified GYE broth (pH 9.0) at 25°C and 150 rpm during 96 h incubation.
Optimization of nutritional and physical parameters
Carbon source
Effect of different carbon and nitrogen sources on protease (Uml-1) production (A) and growth (B) of Pseudomonas putida SKG-1 at pH 9.0, 25°C and 150 rpm during 96 h incubation
Nutritional Parameters (% w/v) | Incubation Time (h) | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Carbon sources | 12 | 24 | 36 | 48 | 60 | 72 | 84 | 96 | |||||||||
A | B | A | B | A | B | A | B | A | B | A | B | A | B | A | B | ||
Glucose | (2.5) | 2 | 1.62 | 5 | 3.10 | 13 | 3.81 | 24 | 4.01 | 33 | 4.71 | 41 | 4.58 | 39 | 4.49 | 36 | 4.35 |
Glucose | (2) | 4 | 1.21 | 7 | 2.41 | 15 | 3.42 | 28 | 4.13 | 42 | 4.51 | 58 | 4.32 | 56 | 4.18 | 53 | 4.12 |
Glucose | (1.5) | 6 | 1.15 | 14 | 2.20 | 20 | 3.13 | 34 | 3.82 | 48 | 4.35 | 67 | 4.17 | 65 | 3.9 | 62 | 3.70 |
Glucose | (1) | 10 | 1.01 | 18 | 1.87 | 26 | 2.57 | 37 | 3.02 | 46 | 3.53 | 65 | 3.43 | 63 | 3.28 | 59 | 3.15 |
Glucose | (0.5) | 8 | 1.01 | 15 | 1.65 | 19 | 2.29 | 28 | 2.78 | 35 | 3.05 | 44 | 2.91 | 42 | 2.80 | 40 | 2.69 |
Glucose | (0.0) | 6 | 0.91 | 13 | 1.65 | 21 | 2.10 | 27 | 1.75 | 26 | 1.57 | 23 | 1.41 | 21 | 1.32 | 19 | 1.18 |
Fructose | (1) | 6 | 0.98 | 11 | 1.61 | 18 | 2.18 | 26 | 2.87 | 38 | 3.37 | 54 | 3.16 | 52 | 3.05 | 50 | 2.73 |
Lactose | (1) | 00 | 0.46 | 4 | 0.78 | 10 | 1.14 | 16 | 1.65 | 21 | 1.83 | 33 | 1.74 | 31 | 1.64 | 29 | 1.58 |
Sucrose | (1) | 4 | 0.62 | 9 | 1.05 | 17 | 2.21 | 28 | 2.96 | 32 | 3.41 | 43 | 3.29 | 41 | 3.10 | 38 | 2.84 |
Maltose | (1) | 5 | 0.78 | 8 | 1.18 | 15 | 2.29 | 26 | 2.85 | 38 | 3.48 | 49 | 3.32 | 48 | 3.22 | 45 | 3.07 |
Soluble starch | (1) | 7 | 0.52 | 19 | 1.12 | 28 | 1.89 | 42 | 2.91 | 54 | 3.12 | 71 | 2.94 | 69 | 2.93 | 66 | 2.88 |
CMC | (1) | 00 | 0.42 | 5 | 0.61 | 9 | 1.11 | 14 | 1.84 | 20 | 2.59 | 27 | 2.41 | 27 | 2.38 | 25 | 2.26 |
Glycerol | (1) | 8 | 0.83 | 14 | 1.03 | 21 | 1.85 | 30 | 2.57 | 34 | 2.91 | 40 | 2.78 | 39 | 2.66 | 34 | 2.40 |
Xylose | (1) | 3 | 0.67 | 8 | 1.05 | 14 | 1.85 | 19 | 2.44 | 23 | 3.06 | 36 | 2.83 | 344 | 2.71 | 31 | 2.62 |
Nitrogen sources | |||||||||||||||||
Cas amino acid | (1) | 3 | 0.75 | 5 | 1.16 | 7 | 1.98 | 13 | 2.68 | 17 | 2.96 | 21 | 2.78 | 20 | 2.71 | 19 | 2.64 |
Ammonium sulfate | (1) | 00 | 0.12 | 00 | 0.31 | 3 | 0.67 | 5 | 0.88 | 8 | 0.76 | 8 | 0.67 | 5 | 0.60 | 4 | 0.48 |
Ammonium nitrate | (1) | 00 | 0.11 | 00 | 0.41 | 00 | 0.61 | 2 | 0.57 | 4 | 0.40 | 4 | 0.38 | 2 | 0.26 | 00 | 0.21 |
Sodium nitrate | (1) | 00 | 0.16 | 00 | 0.46 | 00 | 0.92 | 4 | 1.34 | 7 | 1.28 | 7 | 1.06 | 6 | 0.96 | 4 | 0.73 |
Urea | (1) | 00 | 0.12 | 00 | 0.36 | 00 | 0.71 | 3 | 1.32 | 6 | 1.17 | 6 | 0.95 | 4 | 0.73 | 1 | 0.41 |
Yeast extract | (1) | 3 | 0.94 | 6 | 1.62 | 8 | 2.90 | 11 | 3.28 | 13 | 3.51 | 15 | 3.30 | 14 | 3.18 | 12 | 3.09 |
Beef extract | (1) | 2 | 0.71 | 5 | 1.05 | 8 | 1.87 | 9 | 2.24 | 10 | 2.56 | 12 | 2.43 | 12 | 2.28 | 10 | 2.21 |
Tryptone | (1) | 4 | 0.79 | 6 | 1.24 | 11 | 2.10 | 16 | 2.81 | 19 | 3.15 | 23 | 2.94 | 22 | 2.80 | 21 | 2.71 |
Casein | (1) | 1 | 0.72 | 5 | 1.18 | 9 | 1.92 | 11 | 2.43 | 14 | 2.78 | 18 | 2.60 | 17 | 2.56 | 15 | 2.40 |
Gelatin | (1) | 3 | 0.81 | 8 | 2.05 | 14 | 2.78 | 22 | 3.27 | 38 | 3.40 | 57 | 3.28 | 55 | 3.21 | 51 | 3.18 |
Peptone | (1) | 4 | 0.86 | 7 | 1.47 | 13 | 2.32 | 21 | 2.97 | 24 | 3.28 | 28 | 3.15 | 27 | 3.06 | 25 | 2.84 |
Nitrogen sources with yeast extract (0.5) | |||||||||||||||||
Casamino acid | (1) | 5 | 1.07 | 11 | 2.13 | 24 | 2.88 | 31 | 3.07 | 40 | 3.12 | 51 | 2.86 | 49 | 2.69 | 47 | 2.51 |
Ammonium sulfate | (1) | 1 | 0.26 | 3 | 0.51 | 4 | 0.96 | 7 | 1.43 | 9 | 2.14 | 14 | 1.85 | 14 | 1.79 | 12 | 1.52 |
Ammonium nitrate | (1) | 00 | 0.14 | 1 | 0.25 | 2 | 0.63 | 6 | 0.94 | 4 | 1.33 | 6 | 1.07 | 5 | 0.96 | 5 | 0.81 |
Sodium nitrate | (1) | 00 | 0.17 | 1 | 0.31 | 3 | 0.76 | 4 | 1.13 | 6 | 1.50 | 9 | 1.36 | 8 | 1.20 | 7 | 1.06 |
Urea | (1) | 00 | 0.12 | 2 | 0.27 | 2 | 0.60 | 5 | 0.99 | 7 | 1.26 | 8 | 1.14 | 8 | 1.07 | 6 | 0.91 |
Peptone | (1) | 10 | 1.01 | 18 | 1.87 | 26 | 2.57 | 37 | 3.02 | 46 | 3.53 | 65 | 3.43 | 63 | 3.28 | 59 | 3.15 |
Beef extract | (1) | 6 | 1.03 | 13 | 1.84 | 19 | 2.35 | 24 | 2.61 | 30 | 2.81 | 34 | 2.65 | 32 | 2.53 | 29 | 2.48 |
Tryptone | (1) | 5 | 1.11 | 15 | 1.73 | 26 | 2.42 | 32 | 2.95 | 39 | 3.20 | 53 | 3.16 | 51 | 2.98 | 50 | 2.79 |
Casein | (1) | 4 | 0.92 | 7 | 1.46 | 12 | 1.99 | 19 | 2.71 | 24 | 3.06 | 30 | 2.81 | 28 | 2.65 | 26 | 2.50 |
Gelatin | (0.5) | 7 | 0.63 | 18 | 1.30 | 31 | 2.43 | 52 | 3.05 | 88 | 3.26 | 106 | 3.19 | 103 | 3.10 | 95 | 3.09 |
Gelatin | (1) | 11 | 1.11 | 27 | 2.05 | 49 | 2.95 | 81 | 3.45 | 139 | 3.68 | 171 | 3.58 | 168 | 3.44 | 164 | 3.26 |
Gelatin | (1.5) | 18 | 1.14 | 45 | 2.12 | 78 | 3.33 | 121 | 4.41 | 183 | 4.56 | 210 | 4.49 | 203 | 4.38 | 191 | 4.24 |
Gelatin | (2) | 29 | 1.23 | 84 | 2.62 | 115 | 3.84 | 182 | 4.71 | 239 | 4.96 | 278 | 4.87 | 269 | 4.80 | 251 | 4.71 |
Gelatin | (2.5) | 27 | 1.20 | 76 | 2.64 | 112 | 3.99 | 171 | 4.92 | 230 | 5.20 | 264 | 5.14 | 256 | 5.00 | 239 | 4.82 |
Gelatin | (3) | 9 | 0.97 | 19 | 1.86 | 42 | 2.53 | 74 | 3.54 | 128 | 3.96 | 154 | 3.75 | 149 | 3.60 | 144 | 3.48 |
Nitrogen source
Effect of yeast extract, metal ions and physical factors on protease (Uml-1) production (A) and bacterial growth (B) at pH 9.0, 25°C and 150 rpm during 96 h incubation
Yeast extract and metal ions (%, w/v) | Incubation Time (h) | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Yeast extract | 12 | 24 | 36 | 48 | 60 | 72 | 84 | 96 | |||||||||
A | B | A | B | A | B | A | B | A | B | A | B | A | B | A | B | ||
Yeast extract | (0.0) | 9 | 0.96 | 17 | 2.41 | 28 | 3.21 | 41 | 3.52 | 66 | 3.74 | 89 | 3.66 | 84 | 3.60 | 81 | 3.51 |
Yeast extract | (0.1) | 14 | 1.08 | 31 | 2.40 | 52 | 3.40 | 76 | 3.75 | 93 | 3.84 | 110 | 3.72 | 108 | 3.65 | 105 | 3.61 |
Yeast extract | (0.2) | 17 | 1.16 | 54 | 2.47 | 67 | 3.46 | 103 | 3.82 | 154 | 3.95 | 178 | 3.81 | 170 | 3.74 | 159 | 3.69 |
Yeast extract | (0.3) | 20 | 1.18 | 65 | 2.49 | 92 | 3.67 | 152 | 4.21 | 203 | 4.38 | 219 | 4.20 | 213 | 4.14 | 208 | 4.07 |
Yeast extract | (0.4) | 28 | 1.21 | 79 | 2.53 | 109 | 3.79 | 167 | 4.63 | 227 | 4.77 | 255 | 4.64 | 246 | 4.58 | 239 | 4.50 |
Yeast extract | (0.5) | 29 | 1.23 | 84 | 2.62 | 115 | 3.84 | 182 | 4.71 | 239 | 4.96 | 278 | 4.87 | 269 | 4.80 | 251 | 4.71 |
Yeast extract | (0.6) | 26 | 1.30 | 67 | 2.97 | 105 | 4.11 | 178 | 4.91 | 231 | 5.17 | 269 | 5.04 | 257 | 4.97 | 248 | 4.91 |
Yeast extract | (0.7) | 21 | 1.39 | 48 | 3.16 | 92 | 4.50 | 163 | 5.20 | 227 | 5.35 | 246 | 5.26 | 241 | 5.18 | 237 | 5.03 |
Metal ions | |||||||||||||||||
No metal | 29 | 1.23 | 84 | 2.62 | 115 | 3.84 | 182 | 4.71 | 239 | 4.96 | 278 | 4.87 | 269 | 4.80 | 251 | 4.71 | |
ZnSO4 | (0.01) | 6 | 1.34 | 17 | 3.62 | 36 | 4.93 | 67 | 5.82 | 92 | 6.05 | 103 | 5.87 | 100 | 5.54 | 94 | 5.17 |
CuSO4 | (0.01) | 5 | 0.51 | 12 | 1.32 | 41 | 2.15 | 98 | 2.62 | 159 | 2.94 | 186 | 2.77 | 182 | 2.61 | 174 | 2.54 |
FeCl2 | (0.01) | 8 | 0.62 | 31 | 1.40 | 74 | 1.95 | 154 | 2.17 | 186 | 2.31 | 210 | 2.27 | 207 | 2.08 | 201 | 1.83 |
MnSO4 | (0.01) | 4 | 0.82 | 15 | 1.43 | 34 | 3.05 | 78 | 3.67 | 133 | 4.15 | 147 | 3.94 | 145 | 3.80 | 141 | 3.64 |
CaCl2 | (0.01) | 33 | 1.16 | 86 | 3.08 | 143 | 4.60 | 196 | 5.23 | 289 | 5.82 | 342 | 5.53 | 336 | 5.38 | 327 | 4.94 |
MgSO4 + CaCl2 | (0.005 + 0.005) | 36 | 1.19 | 92 | 2.97 | 158 | 4.82 | 209 | 5.34 | 318 | 5.95 | 377 | 5.76 | 372 | 5.60 | 364 | 5.23 |
MgSO4 | (0.005) | 36 | 1.23 | 77 | 2.86 | 159 | 3.90 | 216 | 5.69 | 297 | 5.91 | 354 | 5.57 | 349 | 5.21 | 342 | 5.19 |
MgSO4 | (0.01) | 41 | 1.20 | 106 | 3.10 | 197 | 4.73 | 304 | 6.07 | 386 | 6.18 | 431 | 6.10 | 428 | 5.83 | 417 | 5.26 |
MgSO4 | (0.02) | 38 | 1.80 | 81 | 4.13 | 175 | 5.20 | 286 | 6.22 | 364 | 6.38 | 378 | 6.15 | 376 | 6.03 | 371 | 5.86 |
MgSO4 | (0.03) | 35 | 1.62 | 62 | 3.57 | 139 | 5.14 | 246 | 6.11 | 327 | 6.15 | 356 | 5.96 | 352 | 5.81 | 346 | 5.66 |
MgSO4 | (0.04) | 34 | 1.45 | 66 | 3.40 | 122 | 4.93 | 212 | 5.88 | 305 | 5.07 | 321 | 5.82 | 314 | 5.73 | 306 | 5.43 |
MgSO4 | (0.05) | 27 | 1.40 | 59 | 2.89 | 108 | 4.76 | 176 | 5.32 | 264 | 5.73 | 309 | 5.24 | 301 | 5.10 | 296 | 5.08 |
Effect of physical parameters on bacterial growth and protease production in optimized medium containing (gl -1 distilled water): glucose, 10.0; gelatin, 20.0; yeast extract, 5.0 and MgSO 4 .7H 2 O, 0.1 | |||||||||||||||||
Incubation Time (h) | |||||||||||||||||
pH | 12 | 24 | 36 | 48 | 60 | 72 | 84 | 96 | |||||||||
A | B | A | B | A | B | A | B | A | B | A | B | A | B | A | B | ||
7.0 | 00 | 0.63 | 00 | 1.73 | 00 | 2.95 | 00 | 4.38 | 00 | 4.92 | 00 | 4.78 | 00 | 4.64 | 00 | 4.51 | |
7.5 | 00 | 0.69 | 00 | 1.83 | 7 | 3.06 | 19 | 4.57 | 32 | 5.10 | 38 | 4.85 | 35 | 4.69 | 31 | 4.32 | |
8.0 | 12 | 0.93 | 21 | 2.40 | 34 | 3.32 | 48 | 4.87 | 64 | 5.30 | 87 | 5.14 | 81 | 5.02 | 78 | 4.85 | |
8.5 | 30 | 1.18 | 76 | 3.04 | 165 | 4.52 | 213 | 5.77 | 294 | 6.08 | 311 | 5.84 | 308 | 5.61 | 304 | 5.22 | |
9.0 | 41 | 1.20 | 106 | 3.10 | 197 | 4.73 | 304 | 6.07 | 386 | 6.18 | 431 | 6.10 | 428 | 5.83 | 417 | 5.26 | |
9.5 | 5 | 1.08 | 17 | 3.06 | 48 | 4.26 | 92 | 5.17 | 135 | 5.50 | 146 | 5.34 | 143 | 5.21 | 138 | 5.02 | |
10.0 | 00 | 0.25 | 00 | 0.34 | 00 | 0.21 | 00 | 0.19 | 00 | 0.16 | 00 | 0.13 | 00 | 0.12 | 00 | 0.09 | |
Agitation speed (rpm) | |||||||||||||||||
50 | 14 | 0.62 | 26 | 1.44 | 41 | 2.58 | 58 | 3.06 | 81 | 3.30 | 97 | 3.16 | 95 | 3.08 | 93 | 3.01 | |
100 | 27 | 0.93 | 93 | 2.17 | 139 | 3.86 | 187 | 4.23 | 249 | 4.95 | 283 | 4.70 | 279 | 4.40 | 273 | 4.19 | |
150 | 41 | 1.20 | 106 | 3.10 | 197 | 4.73 | 304 | 6.07 | 386 | 6.18 | 431 | 6.10 | 428 | 5.83 | 417 | 5.26 | |
200 | 53 | 1.46 | 127 | 3.81 | 261 | 5.48 | 375 | 6.32 | 514 | 6.21 | 509 | 6.03 | 501 | 5.64 | 492 | 5.17 | |
250 | 46 | 1.30 | 113 | 4.05 | 254 | 5.72 | 368 | 6.29 | 491 | 6.14 | 483 | 5.80 | 476 | 5.64 | 468 | 5.37 | |
Temperature (°C) | |||||||||||||||||
10 | 4 | 0.42 | 10 | 0.79 | 18 | 1.13 | 27 | 1.84 | 32 | 2.26 | 46 | 2.81 | 53 | 2.51 | 51 | 2.42 | |
15 | 13 | 0.61 | 32 | 1.06 | 59 | 1.92 | 104 | 2.68 | 167 | 3.96 | 219 | 3.74 | 217 | 3.58 | 216 | 3.42 | |
20 | 24 | 0.92 | 45 | 1.75 | 112 | 3.87 | 287 | 5.11 | 423 | 4.83 | 421 | 4.70 | 418 | 4.47 | 413 | 4.26 | |
25 | 53 | 1.46 | 127 | 3.81 | 261 | 5.48 | 375 | 6.32 | 514 | 6.21 | 509 | 6.03 | 501 | 5.64 | 492 | 5.17 | |
30 | 31 | 1.14 | 84 | 2.59 | 196 | 4.05 | 308 | 5.40 | 435 | 5.27 | 431 | 5.13 | 427 | 4.86 | 421 | 4.33 | |
35 | 18 | 0.85 | 31 | 1.92 | 58 | 2.73 | 87 | 3.42 | 132 | 3.25 | 132 | 3.14 | 129 | 3.01 | 126 | 2.90 | |
40 | 2 | 0.80 | 3 | 1.43 | 7 | 1.84 | 10 | 2.17 | 12 | 1.94 | 11 | 1.72 | 8 | 1.54 | 6 | 1.24 |
Metal ions
Table 5 reveals that Mg2+ and Ca2+ ions individually and in combination enhanced the protease production. However, maximum enzyme (431 Uml-1) was produced with Mg2+ ions alone, and was therefore, selected for further studies. Other metal ions (Cu2+, Fe2+, Zn2+, Mn2+) reduced protease production (Table 5). Among different levels of Mg2+ (0.005-0.05% w/v) employed, 0.01% (w/v) was most effective. Any deviation in Mg2+ concentration from optimum 0.01% (w/v) adversely affected protease yield (Table 5). Our results are in accordance with the findings of other researchers, who also reported Mg2+ as the best metal ion supplement for protease production and bacterial growth [18]. Rahman et al. [17] also reported enhanced protease production in the presence of Mg2+ by P. aeruginosa strain K. The stimulating effect of CaCl2 was reported by Mabrouk et al. [19]. They attributed this effect to the stabilizing nature of CaCl2 on alkaline protease.
Initial pH
The organism was able to grow in the selected pH range (7.0-10.0), but protease production was restricted to pH range 7.5-9.5 only. At pH 9.0, maximum protease (431 Uml-1) was produced under optimized nutritional conditions. Any deviation in pH from optimum 9.0 adversely affected the bacterial growth and enzyme production. At pH 7.0 and 10.0, protease production was not detected (Table 5). The alkaline pH optimum reveals alkaliphilic nature of strain SKG-1. Joshi et al. [20] also reported maximum protease yield at pH 9.0 by B. cereus isolate. Bacillus sp. strain APP1 was able to grow well in pH range 5.0-12.0, but produced protease maximally at pH 9.0 [21]. Other researchers have reported maximum alkaline protease production at pH 7.0-7.5 also [3, 13, 22]
Agitation
Shaking of cultures significantly affected protease production, which was maximum (514 Uml-1) at 200 rpm. Any change in agitation speed decreased enzyme production, and produced 97, 283, 431 and 49U protease ml-1 at 50, 100, 150 and 250 rpm, respectively (Table 5). Presumably at 200 rpm agitation, the aeration of culture broth increased optimally, which enhanced the supply of dissolved oxygen and uptake of nutrients to the bacterial cells. The decreased enzyme production at > 200 rpm was perhaps due to denaturation of proteases caused by the mechanical damage.
Shaking of aerobic bacterial culture is one of the most decisive factors for growth and protease production, as agitation maintains proper oxygen supply and mixing of growing cells. Oxygen transfer into bacterial cells in aerobic fermentation process strongly affects growth and enzyme production by affecting the metabolic pathways and fluxes [23]. Several other researchers also reported maximum protease production at an agitation speed of 200 rpm [22]. Gupta and Khare [3] reported maximum protease production by P. aeruginosa PseA at 250 rpm.
Temperature
In any bioprocess, specific temperature requirement and its regulation is one of the most critical parameters. Strain SKG-1 was able to grow and produce protease in complete temperature range (10°- 40°C) of study with maximum production at optimum 25°C. Temperature higher or lower than optimum, reduced the bacterial growth, thereby steady decrease in enzyme production (Table 5). The order of enzyme production at other temperatures was (Uml-1): 10°C (53) < 15°C (219) < 20°C (423) < 25°C (514) > 30°C (435) > 35°C (132) > 40°C (12).
The ability of strain SKG-1 to grow at wide temperature range of 10°- 40°C with optimum at 25°C revealed its psychrotrophic nature. Only few psychrotrophic bacterial isolates have been reported for thermostable alkaline protease production. Jackman et al. [24] reported heat stable protease production at 25°C from psychrotrophic pseudomonads. A psychrotrophic Exiguobacterium sp. SKPB5 produced alkaline protease at 30°C with maximum protease activity at 50°C [25].
Incubation time
Protease production and growth response of P. putida SKG-1 in finally optimized medium containing (gl -1 distilled water): glucose, 10.0; gelatin, 20.0; yeast extract, 5.0 and MgSO 4 , 0.1 at initial pH 9.0, 25°C and 200 rpm during 72 h growth.
Statistical optimization of factors affecting protease production by RSM
Table 2 shows predicted responses of Box-Behnken design on the basis of above polynomial equation. This regression equation was assessed statistically for analysis of variance (ANOVA), and the results are predicted in Table 3. ANOVA of regression model demonstrated the determination coefficient (R2) 0.9988, which means 99.88% variability in the response could be explained by this model. The R2 value is always between 0 and 1.0. The model is stronger and predicts better response when R2 value is closer to 1.0 [5]. The value of the adjusted determination coefficient (adjusted R2) is 0.9976. This higher value of adjusted R2 indicates greater significance of the model. A very low value of coefficient of variation (C.V., 0.70%) indicates better precision and reliability of the experiments executed. The adequate precision value of 121.595 measures signal to noise ratio, and a ratio > 4.0 is desirable. In this case, higher ratio indicates an adequate signal, and also proves that model can be used to navigate the design space.
The F- value of 820.46 in Table 3 implies that the model is significant. There is only 0.01% chance that a "model F- value" so large could occur due to noise. ANOVA analysis also indicated that the model term linear glucose (P < 0.0001), yeast extract (P < 0.0001), MgSO4 (P < 0.0001), pH (P < 0.0001), quadratic glucose A2 (P < 0.0002), yeast extract B2 (P < 0.0001), MgSO4 C2 (P < 0.0001), pH D2 (P < 0.0001) and four interaction terms were significant. Smaller the P-value, more significant is the corresponding coefficient. The P < 0.0500 indicates that model terms are significant.
Response-surface curve of alkaline protease production by Pseudomonas putida SKG-1 showing mutual interactions between (a) glucose and yeast extract, (b) glucose and MgSO 4 , (c) glucose and pH, (d) MgSO 4 and yeast extract, (e) pH and yeast extract, (f) pH and MgSO 4 . Other variables, except for two in each figure, were maintained at zero level in coded units.
Figure 3a depicts the production of alkaline protease with respect to glucose versus yeast extract. From the interaction response of glucose with yeast extract, protease yields increased with increasing glucose and yeast extract concentration up to 1.25% and 0.5%, respectively. The enzyme activity decreased at > 0.5% yeast extract concentration. However, the response curve did not show curvature, rather it was flattened. This suggested a demand for higher concentration of glucose. Figure 3b represents the interaction effect of glucose and MgSO4 on production of alkaline protease. With an increase in glucose (0.75-1.25%, w/v) and MgSO4 (0.0075-0.01%, w/v) concentration, the protease yield increased. Thereafter, an increase in MgSO4 concentration up to 0.0125% (w/v) resulted in decreased protease production. The optimal values for glucose and MgSO4 (w/v) were 1.25 and 0.01%, respectively.
Figure 3c reveals that maximum protease was produced at higher level of glucose (1.25%) and slightly lower alkaline pH (8.8) in the design range. This accorded a run number of 10, which is considered as the optimal condition of test variables. Table 2 shows that maximum protease of 617 Uml-1 were produced at pH 8.8 (-1 in coded unit) and glucose at 1.25% (+1 in coded unit). Figure 3d depicts the interaction of two variables, viz., MgSO4 and yeast extract on protease production. The protease production increased with increasing concentration of MgSO4 (0.0075-0.01%, w/v) and yeast extract (0.3-0.5%, w/v). Further increase in their concentrations resulted in decreased alkaline protease production.
Figure 3e illustrates the interaction effect of pH (8.8-9.2) and yeast extract (0.3-0.7%) on alkaline protease production. Maximum enzyme units were produced with 0.5% yeast extract and pH 8.8. Further increase in pH and any deviation in yeast extract concentration from optimal, decreased the enzyme production. The effect of pH and MgSO4 is shown in Figure 3f. The response curve analysis indicated that protease production decreased with increase in pH from 8.8 to 9.2, and increased with enhanced concentration of MgSO4 up to 0.01% (w/v). Further increase in MgSO4 concentration above 0.01% caused decreased enzyme yield.
The above optimized results (Table 2 standard order 10) concerning four variables were finally verified by again performing the batch shake flask experiment. The maximum experimental alkaline protease production of 617 Uml-1 was very close to 612 Uml-1 predicted by Box-Behnken design with 1.25% glucose, 0.5% yeast extract, 0.01% MgSO4 and pH 8.8. Thus, under optimized conditions, the protease yield increased from 514 units in conventional optimization trial to 617 Uml-1 using RSM at 60 h incubation. This proved that response surface methodology is a slightly better optimization approach as compared to conventional "one-variable-at-a-time" method in terms of improved protease yield in less time, resource and expenditure. Our findings are in agreement with the results of other researchers who have also reported RSM a better approach for enhanced protease production [4, 5, 16]. Reddy et al. [27] reported a 2.3 folds increase in alkaline protease production using Plackett-Burman and Response surface methodology by Bacillus sp. RKY3. Anbu et al. [28] obtained a good correlation coefficient of 0.9996 using Box-Behnken design and alkaline protease production of 112.90 Uml-1 by Shewanella oneidensis MR-1 strain through response surface optimization.
Bench-scale bioreactor optimization
Effect of aeration
Effect of different aeration rates (0-1.5 vvm) at constant agitation speed (200 rpm) on protease production.
Effect of different aeration rates (0-1.5 vvm) at constant agitation speed (200 rpm) on bacterial growth and change in DOT profile.
Under RSM optimized conditions, protease production was maximum at 60 h incubation, while it was highest in bioreactor trial at 48 h, thereby led to significant time saving of 12 h. Optimization of aeration rate at fixed agitation speed of 200 rpm indicated that oxygen supply to bacterial cell mass is a critical parameter for enzyme production under aerobic fermentation process. This could be achieved by optimizing the agitation speed and maintaining proper aeration; however, an appropriate balance of agitation and aeration is imperative to avoid any mechanical damage to bacterial cells which can reduce the enzyme yield. From the results of aeration optimization, it can be inferred that a direct correlation existed between bacterial growth and protease production. The reduction in DOT followed a similar pattern as experienced in previous experiment. It reduced rapidly during exponential growth phase, and rose again on commencement of stationary phase onwards (Figure 5). Rao et al. [29] reported highest protease production of 238.77 Uml-1 by a Beauveria bassiana isolate on 6th day of fermentation in a 5 litre stirred tank bioreactor at 150 rpm and 0.6 vvm aeration. Maximum protease of 340 Uml-1 by Bacillus licheniformis NCIM-2042 was reported at aeration and agitation rates of 3 vvm and 200 rpm, respectively [7].
Effect of agitation at optimized aeration
Effect of different agitation speeds (100-250 rpm) at constant aeration rate (0.8 vvm) on protease production.
Effect of different agitation speeds (100-250 rpm) at constant aeration rate (0.8 vvm) on bacterial growth and change in DOT profile.
Pattern of bacterial growth, enzyme production and DOT change in GGY broth at 150 rpm and 0.8 vvm aeration at pH 9.0 and 25°C.
It is evident from the findings that an optimum agitation of fermenting broth is necessary for proper oxygen supply to bacterial cells. However, above the optimum speed it may damage the cells or change the cell morphology, which leads to reduced protease yield. Maximum protease production at 300 rpm has also been reported by Potumarthi et al. [7] in a stirred tank bioreactor. Any deviation in agitation speed to 200 or 400 rpm reduced the protease yield. They concluded that mixing is important for maximal protease production by optimizing both agitation and aeration for better oxygen mass transfer rate. This results in better product formation through avoiding/minimizing any mechanical damage to the bacterial cells. Many other researchers also reported the optimum agitation speed range of 150-300 rpm for protease production from different isolates [30–32]. It may, therefore, be inferred that a proper ratio of agitation and aeration is mandatory for appropriate oxygen transfer to bacterial cell mass, and also to minimize the shearing effect for maximum growth and protease production.
Conclusions
The optimization of alkaline protease production from Pseudomonas putida is being reported by conventional as well as statistical response surface methodologies. A 7.9 folds (from 65 to 514 Uml-1) increase in protease production was evident with optimized nutritional (glucose 1%, gelatin 2%, yeast extract 0.5%, Mg2+ 0.01%) and cultural (pH 9.0, 25°C, 200 rpm) conditions during early stationary phase at 60 h fermentation employing conventional method of optimization. Whereas, the response surface methodology enhanced the protease production to 9.5 folds (617 Uml-1) by further optimizing the glucose concentration to 1.25% (w/v) and pH to 8.8. The optimization of process parameters by RSM proved it a time/resource saving and efficient method. Although, it provided better insight of interactions among the parameters that affect enzyme production, development of a better statistical tool is a constant endeavor. Further optimization of agitation (150 rpm) and aeration (0.8 vvm) rates at bench-scale bioreactor level enhanced the enzyme production by ~43% (882 Uml-1) at 48 h fermentation. It not only enhanced the protease yield, but also led to a significant time saving of 12 h. Furthermore, this is the first report on such a high yield of solvent and psychro-thermo-alkali-stable protease from a solvent tolerant psychrotrophic bacterial strain.
Declarations
Acknowledgements
The senior author Santosh K Singh is thankful to University Grants Commission, Government of India, New Delhi, for providing research fellowship under the major research project [Project no. 37-521/2009 (SR)]. The assistance provided by Government of Uttar Pradesh and Department of Science and Technology, Government of India, respectively under the schemes of Center of Excellence and DST-FIST, is duly acknowledged. The help in RSM studies rendered by Dr. Sunil Kumar Tyagi, Department of Statistics, Narendra Dev University of Agriculture and Technology, Faizabad, India, is gratefully acknowledged.
Authors’ Affiliations
References
- Adinarayana K, Ellaiah P, Siva Prasad D: Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. AAPS PharmSciTech. 2003, 4: 56-View ArticleGoogle Scholar
- Joo HS, Kumar CG, Park GC, Paik SR, Chang CS: Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties. J Appl Microbiol. 2003, 95: 267-272. 10.1046/j.1365-2672.2003.01982.x.View ArticleGoogle Scholar
- Gupta A, Khare SK: Enhanced production and characterization of a solvent stable protease from solvent tolerant Pseudomonas aeruginosa PseA. Enz Microbial Technol. 2007, 42: 11-16. 10.1016/j.enzmictec.2007.07.019.View ArticleGoogle Scholar
- Vishwanatha KS, Rao AGA, Singh SA: Acid protease production by solid-state fermentation using Aspergillus oryzae MTCC 5341: optimization of process parameters. J Ind Microbiol Biotechnol. 2010, 37: 129-138. 10.1007/s10295-009-0654-4.View ArticleGoogle Scholar
- Doddapaneni KK, Tatineni R, Potumarthi R, Mangamoori LN: Optimization of media constituents through response surface methodology for improved production of alkaline proteases by Serratia rubidaea. J Chem Technol Biotechnol. 2007, 82: 721-729. 10.1002/jctb.1714.View ArticleGoogle Scholar
- Calik P, Calik G, Ozdamar TH: Oxygen transfer effects in serine alkaline protease fermentation by Bacillus licheniformis: Use of citric acid as the carbon source. Enz Microbial Technol. 1998, 23: 451-461. 10.1016/S0141-0229(98)00069-6.View ArticleGoogle Scholar
- Potumarthi R, Ch S, Jetty A: Alkaline protease production by submerged fermentation in stirred tank reactor using Bacillus licheniformis NCIM-2042: Effect of aeration and agitation regimes. Biochem Eng J. 2007, 34: 185-192. 10.1016/j.bej.2006.12.003.View ArticleGoogle Scholar
- Hameed A, Keshavarz T, Evans CS: Effect of dissolved oxygen tension and pH on the production of extracellular protease from a new isolate of Bacillus subtilis K2, for use in leather processing. J Chem Technol Biotechnol. 1999, 74: 5-8. 10.1002/(SICI)1097-4660(199901)74:1<5::AID-JCTB979>3.0.CO;2-T.View ArticleGoogle Scholar
- Singh SK, Singh SK, Tripathi VR, Khare SK, Garg SK: A novel psychrotrophic, solvent tolerant Pseudomonas putida SKG-1 and solvent stability of its psychro-thermoalkalistable protease. Process Biochem. 2011, 46: 1430-1435. 10.1016/j.procbio.2011.03.012.View ArticleGoogle Scholar
- Singh SK, Tripathi VR, Jain RK, Vikram S, Garg SK: An antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease. Microb Cell Fact. 2010, 9: 59-10.1186/1475-2859-9-59.View ArticleGoogle Scholar
- Shimogaki H, Takeuchi K, Nishino T, Odera M, Kudo T, Ohba K, Iwama M, Irie M: Purification and properties of a novel surface active agent and alkaline resistant protease from Bacillus spp. Y. Agric Biol Chem. 1991, 55: 2251-2258. 10.1271/bbb1961.55.2251.View ArticleGoogle Scholar
- Shafee N, Aris SN, Rahman RNZRA, Basri M, Salleh AB: Optimization of environmental and nutritional conditions for the production of alkaline protease by newly isolated bacterium Bacillus cereus strain 146. J Appl Sci Res. 2005, 1: 1-8.Google Scholar
- Rahman RNZRA, Basri M, Salleh AB: Thermostable alkaline protease from Bacillus stearothermophilus F1; Nutritional factors affecting protease production. Ann Microbiol. 2003, 53: 199-210.Google Scholar
- Nadeem M, Qazi JI, Baig S, Syed Q: Effect of medium composition on commercially important alkaline protease production by Bacillus licheniformis N-2. Food Technol Biotechnol. 2008, 46: 388-394.Google Scholar
- Kumar CG, Takagi H: Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnol Adv. 1999, 17: 561-594. 10.1016/S0734-9750(99)00027-0.View ArticleGoogle Scholar
- Puri S, Beg QK, Gupta R: Optimization of alkaline protease production from Bacillus sp. by response surface methodology. Curr Microbiol. 2002, 44: 286-290. 10.1007/s00284-001-0006-8.View ArticleGoogle Scholar
- Rahman RNZRA, Geok LP, Basri M, Salleh AB: An organic solvent-tolerant protease from Pseudomonas aeruginosa strain K: Nutritional factors affecting protease production. Enz Microbial Technol. 2005, 36: 749-757. 10.1016/j.enzmictec.2004.12.022.View ArticleGoogle Scholar
- Thumar J, Singh SP: Secretion of an alkaline protease from a salt-tolerant and alkaliphilic, Streptomyces clavuligerus strain MIT-1. Braz J Microbiol. 2007, 38: 766-772. 10.1590/S1517-83822007000400033.View ArticleGoogle Scholar
- Mabrouk SS, Hashem AM, El-Shayeb NMA, Ismail MS, Abdel-Fattah AF: Optimization of alkaline protease productivity by Bacillus licheniformis ATCC 21415. Biores Technol. 1999, 69: 155-159. 10.1016/S0960-8524(98)00165-5.View ArticleGoogle Scholar
- Joshi GK, Kumar S, Sharma V: Production of moderately halotolerant, SDS stable alkaline protease from Bacillus cereus MTCC 6840 isolated from lake Nainital, Uttaranchal state, India. Braz J Microbiol. 2007, 38: 773-779. 10.1590/S1517-83822007000400034.View ArticleGoogle Scholar
- Chu W-H: Optimization of extracellular alkaline protease production from species of Bacillus. J Ind Microbiol Biotechnol. 2007, 34: 241-245. 10.1007/s10295-006-0192-2.View ArticleGoogle Scholar
- Abusham RA, Rahman RNZRA, Salleh AB, Basri M: Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand. Microb Cell Fact. 2009, 8: 20-10.1186/1475-2859-8-20.View ArticleGoogle Scholar
- Calik P, Calik G, Ozdamar TH: Oxygen-transfer strategy and its regulation effects in serine alkaline protease production by Bacillus licheniformis. Biotechnol Bioeng. 2000, 69: 301-311. 10.1002/1097-0290(20000805)69:3<301::AID-BIT8>3.0.CO;2-4.View ArticleGoogle Scholar
- Jackman DM, Bartlett FM, Patel TR: Heat-stable proteases from psychrotrophic pseudomonads: comparison of immunological properties. Appl Environ Microbiol. 1983, 46: 6-12.Google Scholar
- Kasana RC, Yadav SK: Isolation of a psychrotrophic Exiguobacterium sp. SKPB5 (MTCC 7803) and characterization of its alkaline protease. Curr Microbiol. 2007, 54: 224-229. 10.1007/s00284-006-0402-1.View ArticleGoogle Scholar
- Zeng R, Zhang R, Zhao J, Lin N: Cold-active serine alkaline protease from the psychrophilic bacterium Pseudomonas strain DY-A: enzyme purification and characterization. Extremophiles. 2003, 7: 335-337. 10.1007/s00792-003-0323-x.View ArticleGoogle Scholar
- Reddy LVA, Wee Y, Yun J, Ryu H: Optimization of alkaline protease production by batch culture of Bacillus sp. RKY3 through Plackett-Burman and response surface methodological approaches. Biores Technol. 2008, 99: 2242-2249. 10.1016/j.biortech.2007.05.006.View ArticleGoogle Scholar
- Anbu P, Annadurai G, Lee J-F, Hur B-K: Optimization of alkaline protease production from Shewanella oneidensis MR-1 by response surface methodology. J Chem Technol Biotechnol. 2009, 84: 54-62. 10.1002/jctb.2004.View ArticleGoogle Scholar
- Rao YK, Lu S, Liu B, Tzeng Y: Enhanced production of an extracellular protease from Beauveria bassiana by optimization of cultivation processes. Biochem Eng J. 2006, 28: 57-66. 10.1016/j.bej.2005.09.005.View ArticleGoogle Scholar
- Banerjee CU, Sani RK, Azmi W, Soni R: Thermostable alkaline protease from Bacillus brevis and its characterization as a laundry detergent additive. Process Biochem. 1999, 35: 213-219. 10.1016/S0032-9592(99)00053-9.View ArticleGoogle Scholar
- Joo HS, Kumar CG, Park GC, Kim KT, Paik SR, Chang CS: Optimization of the production of an extra cellular alkaline protease from Bacillus horikoshii. Process Biochem. 2002, 38: 155-159. 10.1016/S0032-9592(02)00061-4.View ArticleGoogle Scholar
- Kanekar PP, Nilegaonkar SS, Sarnaik SS, Kelkar AS: Optimization of protease activity of alkaliphilic bacteria isolated from an alkaline lake in India. Biores Technol. 2002, 85: 87-93. 10.1016/S0960-8524(02)00018-4.View ArticleGoogle Scholar
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