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Enhanced production of thrombinase by Streptomyces venezuelae: Kinetic studies on growth and enzyme production of mutant strain
Bioresource Technology 111 (2012) 417–424
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Enhanced production of thrombinase by Streptomyces venezuelae: Kinetic studies on growth and enzyme production of mutant strain Balakrishnan Naveena a, Kannapan Panchamoorthy Gopinath b, Punniavan Sakthiselvan a, Nagarajan Partha a,⇑ a b
Department of Chemical Engineering, A.C. College of Technology, Anna University Chennai, Chennai 600 025, India Department of Chemical Engineering, Adhiparasakthi Engineering College, Melmaruvathur 603 319, India
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 11 November 2011 Received in revised form 10 February 2012 Accepted 13 February 2012 Available online 21 February 2012
This investigation provides the enhanced production of thrombinase, a fibrinolytic enzyme using mutant Streptomyces venezuelae. Initially the mutagenesis of the marine isolate was done by UV and Ethyl methane sulfonate (EMS) and their mutational efficiencies were compared. The mutants were selected based on their high thrombinase activity and used for further studies. The mutant was found to be more halo and thermo tolerant comparing to wild. The effect of Dissolved oxygen level was also determined and the mutant offered the maximum specific growth rate as 0.2404 (h1). The mutant showed high resistance to higher initial lactose concentration and the inhibition concentration was found to be 155.1 mg/mL. The effect of S0/X0 ratio on specific substrate consumption and production rate were also investigated. Both mutant and wild showed increase in specific substrate consumption and production rate at higher S0/X0 ratio but the mutant showed better values than the wild strain. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Random mutagenesis Thrombinase S0/X0 ratio Growth kinetics Enzyme production kinetics
1. Introduction Thrombolytic agents which include urokinase, streprokinase, and tissue plasminogen activator (t-PA), have been widely applied for the lysis of fibrin clots in the case of ischemia (acute myocardial infarction, pulmonary embolism, ischemic stroke, and arterial thrombosis). Due to their short half life, high expensive and excessive risk of hemorrhagic complication, researches for safe and more economic thrombolytic enzymes have been explored for the past few decades. Based on their mode of action, thrombolytic agents are divided into two types: plasminogen activators and plasmin like proteins (Peng et al., 2005). The former type of thrombolytic agents that includes tissue type plasminogen activator (t-PA), urokinase and streptokinase activates the endogenous fibrinolytic system to generate plasmin which eventually hydrolyzes fibrin (Balaraman and Prabakaran, 2007). While the later type of thrombolytic agents that includes nattokinase, lumbrokinase and chymotrypsin like fibrinolytic enzyme reported by Mander et al. (2011) directly hydrolyzes fibrin. Many thrombolytic enzymes have been so far reported from various sources such as plants (Chung et al., 2010), animals (Zhang et al., 1995; Nakajima et al., 1993) and microbes (Peng et al., 2005). Among all these sources, microbes are most preferred due to their broad biochemical diversity, feasibility of mass culture and ease in genetic manipulation.
⇑ Corresponding author. Tel.: +91 9790445938. E-mail address: [email protected]
(N. Partha).
0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.02.056
Though, numerous fibrinolytic enzymes from various microbes including bacteria, actinomyces, fungi, and algae have reported, only few have been explored from marine microorganisms. More recently, marine microorganisms have been recognized as a productive source of novel secondary metabolites and exhibit salt tolerance (Toledo et al., 2006; Okami, 1986). Hence in this study we attempted to produce thrombinase by Streptomyces venezuelae from marine environment as it is a reservoir of novel natural products. Another major criterion for the production of enzyme is selection of substrate. The enzyme has been so far reported from various substrates such as chick peas, corn starch, and beef extract, however the cost of the substrate must be low for a commercial production. Hence in this study we attempted the enzyme production using dairy and tannery industrial wastes in order to minimize the production cost and reduce environmental pollution generated by those wastes. Several investigations have been proved that use of such industrial wastes minimizes the pollution and also enhances the product yield as they are rich in carbon and nitrogen substrates (Maiorella and Castillo, 1984; Ravindranath et al., 2010). To make an efficient bioprocess of enzymes and to satisfy commercial needs, the source of the microorganism and the substrate are not only enough to consider, some other strategies also to be focused. Among those, one of the most important strategies is strain improvement which mainly increases the production efficiency. Strain improvement concerns about developing or modifying organisms used in the production of commercially important products such as antibiotics and enzymes, with the overall aim of
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reducing production costs. This actually deals the enhancement of the productivity of an organism, but it may also be desirable to manipulate specific characteristics such as the ability to utilize cheaper substrates such as industrial wastes. Many attempts have made inorder to increase the yield of thrombinase production such as selection of the culture medium, formulation of fermentation medium (Seo and Lee, 2004) and optimization of environmental conditions (Chitte and Dey, 2002; Liu et al., 2005b) but only few researches have been reported thrombinase by genetically improved strains. The traditional approach of strain improvement is mutagenesis of a suitable strain followed by random screening of mutants. This approach has been successful and has benefited in recent years from the application of important advances in science and technology. Random mutagenesis and recombinant DNA technology have been adopted to improve the enzyme yield and simplify the downstream processes (Peng et al., 2005). An important such technique, random mutagenesis, is used to induce mutation in organisms using UV, Ethyl methyl sulfonate (EMS) and Ethidium bromide (EtBr) for better distinctiveness (Chandra et al., 2008). In recent years, many attempts were made to improve the strains using physical and chemical mutagenesis. Rajoka et al. (1997) improved the production of xylanase by Cellulomonas biazotea mutated by gamma rays. Savergave et al. (2011) screened high erythritol producing Candida magnolia by UV and chemical mutagenesis. Production of thrombinase by mutants with improved enzyme production potential has not been given much attention. Therefore in this study we gave our attention to enhance the thrombinase production through strain improvement on S. venezuelae. Besides strain improvement, some of the other important facts have also to be considered, they are their tolerance to extreme pH, temperature and salinity. Moreover changes in their kinetic parameters such as maximum specific growth rate, maximum specific consumption rate and maximum production rate before and after strain improvement have also to be concerned. Batch experiments have been usually employed to optimize the operation conditions and to evaluate the kinetic parameters (Vazquez-Rodriguez et al., 1999). However great number of investigations show that the biomass yield is influenced by the ratio of initial substrate concentration, (S0) to the initial biomass concentration, (X0) (Wang et al., 2007). It has been stated that the effect of S0/X0 on batch fermentation influence the change in composition of microbial community i.e., the larger the S0/X0 ratio, the greater the change in the structure of microbial community (Chudoba et al., 1992). However, in previous researches only effects of X0 and S0 on biomass growth and production of fibrinolytic enzyme were determined, the importance of their ratios on consumption rate and production rate were not sufficiently concerned. Hence, in this research attempts were made to evaluate and compare the effect of S0/X0 ratio on specific consumption rate and specific production rate of both wild and mutant strains of Streptomyces venezuelae using tannery and dairy industrial wastes.
2. Methods 2.1. Inoculum preparation Streptomyces venezuelae isolated from marine water sample was maintained in agar slants of Starch casein agar (SCA) containing (component g/L) Starch, 10; K2HPO4, 2; KNO3, 2; Nacl, 2; Casein, 0.3; MgSO47H2O, 0.05; CaCO3, 0.02; FeSO47H2O, 0.01 and agar, 20. The seed culture was prepared using the same media (pH 7) without agar and incubated at 30 °C for 4 days. The inoculum obtained at exponential phase was used for further bioprocess studies.
2.2. Induction of mutagenesis The cells were harvested from the culture media by centrifugation at 6000 rpm for 10 min. The cell pellet obtained was washed twice with distilled water and was resuspended in sterile deionised water. Further the colony count was done by Colony counting method and the count was adjusted to 2.36 107 CFU/mL. The UV mutagenesis was carried out by taking 10 mL of the cell suspension in a sterile petridish and exposed to a germicidal lamp for 0–25 min. And for chemical mutation, the cell pellet in deionized water was resuspended in 20 mL of 20 lL ethyl methyl sulfonate (EMS) solution and was shaken thoroughly in an orbital shaker for 30 min. Aliquots were withdrawn at regular intervals of 5 min and serially diluted for viable cell counting by spread plate technique and screening. The % survival was calculated using the following Eq. (1):
S¼
Ni Nd Ni
ð1Þ
where S is the % survival, Ni is the initial viable cell count in CFU/mL and Nd is the viable cell count after mutation in CFU/mL. 2.3. Selection of mutants To select the mutant colonies, 1 mL of the mutated sample was serially diluted to obtain a dilution of 105. From the diluted sample, 0.1 mL was inoculated in a medium containing (component g/ L) Tryptone, 9.47; Meat extract, 4.74; Yeast extract, 0.95; Sodium pyruvate, 9.47; Glycine, 11.36; Lithium chloride, 4.73; thrombin 0.45 U/ml, EDTA 25.0 mL, Bovine fibrinogen, 5.0; Potassium tellurite, 25.0; and was incubated at 37 °C for 24 h. After incubation the broth was centrifugated at 4000 rpm for 5 min. The supernatant was collected for each sample and subjected for fibrin degradation assay. The samples which showed higher enzyme activity compared with the wild were considered as positive mutants and the rest were considered as negative. Further, the mutant sample with highest enzyme activity was isolated and used for further experiments. 2.4. Fermentation media The medium comprises of the following components 2% (w/v) lactose in whey which is derived from the dairy industry waste, 10% (w/v) peptone, 0.7% (w/v) de-limed flesh which is the waste collected from the leather industry, 1.5% (w/v) K2HPO4 and 0.7% (w/v)Triton X100. The medium was autoclaved at 121 °C for 20 min. The experimental design with various fermentation conditions for further analyses is represented in Table 1. 2.5. Evaluation of efficiency of growth and thrombinase production Batch experiments were carried out to analyze the growth and production efficiency of mutated strains at various initial pH, temperature and Sodium chloride (NaCl) concentration. As given in Table 1, these fermentation conditions were varied. The experiments were carried out as triplicates by inoculating 0.1 mL of mutant and wild culture in separate Erlen-meyer flasks containing 1000 mL of sterile fermentation medium and incubated for 4 days with rotary shaking at 200 rpm and their average values were taken in to consideration. Samples were collected at every 3 h interval from the two culture flasks and subjected for centrifugation at 1118g separately. The initial weight of the aluminum foil was taken. The pellet obtained was placed in the foil and kept at 55 °C for 10 min until got dried. The weight of the foil with the dried pellet was measured. From the weight difference the biomass concentration was calculated and the supernatant was used as
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where, l is the specific growth rate, lmax is the maximum specific growth rate, Ks is the half velocity constant (mg/mL) and KI is the substrate inhibition (mg/mL).
Table 1 Experimental design for various analyses. S. No.
Experimental analyses
1
Different set points pH
Temperature, °C
Salinity, % (w/v)
Effect of pH
5–10 (varied)
4
2
Effect of temperature
Optimum
3
Effect of salinity
Optimum
Room temperature37 °C 20–60 °C (varied) Optimum
4
Effect of Dissolved oxygen concentration Determination of assimilable carbon source Effect of S0/X0 on substrate consumption and enzyme activity
Optimum
Optimum
2–10 (varied) Optimum
Optimum
Optimum
Optimum
Optimum
Optimum
Optimum
5 6
2.8. Effect of various S0/X0 on substrate consumption and enzyme activity
4
X ln Xo
2 0 13 X max e: l m ¼ ln exp4exp@ ðk tÞ þ 1A5 Xo ln Xmax
ð3Þ
In Eq. (2), where X0, Xmax, X, t, lm and k denote initial biomass concentration (mg/mL), maximum biomass concentration (mg/ mL) and biomass concentration (mg/mL), incubation time (h), maximum specific growth rate (h1) and lag time (h), respectively. And in Eq. (3), where P, A, Pm, t and k denote Enzyme activity (FU/ mL), Enzyme production potential (FU/mL), specific enzyme production rate (h1), incubation time (h) and lag time (h), respectively. 2.6. Effect of Dissolved oxygen concentration on specific growth rate To determine the effect of Dissolved oxygen (DO) concentration, the growth was analyzed by varying the DO concentration from 2% to 12% (w/v). This was done by using Dissolved oxygen (DO) controller model DO-60 in conjunction with the bioreactor (2 L capacity) to measure and control the oxygen level. The specific growth rates obtained for various DO concentrations were fitted using the following Monod equation.
lm ½DO K DO þ ½DO
ð4Þ
where, l is the specific growth rate, lm is the maximum specific growth rate, KDO is the half velocity constant (mg/mL). 2.7. Determination of assimilable carbon source The assimilable carbon was evaluated by varying the initial concentration of lactose in whey from 0 to 100 mg/mL using the following Haldane’s expression.
l¼
lmax ½S K s þ ½S þ ½KSI
l ¼ lm
S0 =X 0 K S=X þ ðS0 =X 0 Þ S0 =X 0 K S=X þ ðS0 =X 0 Þ
ð6Þ
ð7Þ
where, l is the specific consumption rate, lm is the maximum specific consumption rate, S0/X0 is the ratio of initial substrate concentration to initial biomass concentration, P is the specific production rate, Pmax is the maximum specific thrombinase production rate and K s=x ¼ eðDG0 DG=RTÞ (DG0 and DG are the change of the standard free energy and overall free energy, respectively). The initial lactose concentration (S0) was varied from 20 to 140 mg/mL and the initial biomass concentration (X0) was varied from 0.1 to 1 mg/mL. 2.9. Fibrin degradation assay
ð2Þ
Xo
e:Pm P ¼ A:exp exp ðk t Þ þ 1 A
l¼
The effect of various S0/X0 on substrate consumption and enzyme activity were analyzed by varying S0 at a constant X0 and by varying X0 at a constant S0 using the following equations (Liu et al., 2005a).
P ¼ Pmax
crude enzyme extract. Thus the biomass concentration was determined for all further experiments. Crude extract samples obtained for every 3 h were collected. The enzyme activity for each sample was measured through fibrin degradation assay. The specific growth rate and production rate were calculated from the biomass concentration and enzyme activity obtained at various time intervals for each initial pH, temperature and salt concentration using the following modified Gompertz equations (Gopinath et al., 2011).
419
ð5Þ
The enzyme activity of the crude extract was quantitatively determined by fibrin degradation assay (Wang et al., 2009). Initially 0.4 mL of 0.72% fibrinogen was placed in a test tube with 0.1 mL of 245 mM phosphate buffer (pH 7) and incubated at 37 °C for 5 min. To this 0.1 mL of a 20 U/mL thrombin solution was added and incubated at 37 °C for 10 min. After incubation 0.1 mL of diluted enzyme solution was added, and incubation continued at 37 °C. This solution was again mixed twice at 20 min interval. After 1 h, 0.7 mL of 0.2 M trichloroacetic acid (TCA) was added, mixed and centrifuged at 15,000 rpm for 10 min. Then, 1 mL of the supernatant was collected and the absorbance at 275 nm was measured under UV- Spectrophotometer. According to this assay, 1 unit of enzyme activity is defined as a 0.01-perminute increase in absorbance at 275 nm of the reaction solution. The enzyme activity was expressed in FU/mL (Fibrin degradation unit per mL). 3. Results and discussion 3.1. Mutagenesis analysis The dose response analysis for both UV mutagenesis and chemical mutagenesis of marine Streptomyces venezuelae was done and the % survival values are shown in Table 2. The dosage was expressed in terms of time of exposure to the UV irradiation and varied in the range 2–10 min, to analyze the survival capacity and mutation effect. The marine isolate did not respond well to UV mutagenesis when compared to the chemical mutation by EMS. Since the DNA of the isolate contains more GC basepairs than AT basepairs as it belongs to the group of Actinomyces, mutation by EMS causes GC transversions. Similar trend was found by Baltz (1998), in the case of Streptomyces, where the survival capacity obtained by EMS mutation was higher than UV. At the same time, while comparing with those results the survival capacity of the mutant was severely affected as the dosage of EMS was increased. The transversion mutation in DNA caused by Ethyl methane sulfonate was responsible for the DNA damage. Since the self
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Table 2 Dose response for UV and EMS mutagenesis. S. No.
1 2 3 4 5 6
UV mutagenesis
EMS mutagenesis
Time of exposure, min
Response, % survival
Time of exposure, min
Response, % survival
0 5 10 15 25 25
100 92 75 53 28 8
0 5 10 15 20 25
100 62 47 29 15 3
DNA repair mechanism fails in certain instances the mutation caused by EMS became permanent. Moreover prolonged incubation causes uncontrolled DNA damage which leads to cell death and hence a suitable incubation limit for good mutagenesis to occur can be determined by selection process. 3.2. Selection of mutants The positive mutants were selected on the basis of thrombinase activity produced by the colonies derived from the plates. The wild strain produced 25 FU/mL and the strains which produced more than this value of thrombinase activity were considered as positive mutants. On the whole seven strains showed more than 30 FU/mL and most of them were showed such a higher activity in 20 min exposure of EMS and beyond this level all showed negative results. None of the strains mutated by UV showed more enzyme activity than the wild. Finally all the seven mutants obtained from EMS, which showed increased thrombinase activity were selected for further studies. 3.3. Effect of initial pH The pH of the industrial wastes keeps on changing and hence when using those wastes as substrate, adaptation of microorganism to various pH makes them more selective to bioprocess of enzymes. To compare the growth rate and production efficiency of wild and mutant strains, the initial pH was varied from 5 to 10. Though both the strains showed highest specific growth rate and specific production rate at pH 7, mutant showed more tolerance even at pH 5 and 10 (Fig. 1), than the wild strain. Hence it was proved that the mutant can able to survive under extreme pH conditions. Similar results were reported by Gopinath et al. (2009b) for the degradation of congo red by Bacillus sp. mutant. 3.4. Effect of temperature The influence of temperature on specific growth rate and specific production rate of the wild and mutant strains was investigated by varying the temperature from 20 to 60 °C. The results obtained demonstrated that the growth and production efficiency was almost same in the temperature range 20–60 °C for mutant strains. From Fig. 2, it was inferred that though the wild strain was found to survive under all tested temperatures, the growth was insignificant at extreme temperatures and hence the specific growth rate and specific production rate were found to be very low. While compared with the wild strain, the mutants were known to exhibit good resistance to the wide range of temperatures (Gopinath et al., 2009b). 3.5. Determination of salt tolerance The industrial wastes such as Delimmed flesh usually have high concentration of salts, thus while using those wastes as substrates,
Fig. 1. Effect of pH on specific growth rate, l (h1) (A) and specific thrombinase production rate, Pm (h1), (B) of wild and mutant Streptomyces venezuelae.
the microorganism used for the production should be highly salt tolerant. Hence adaptation to high salinity is necessary for an effective bioprocess. Thus to compare the salt tolerance of wild and mutant strains, batch experiments were carried out at the initial NaCl concentrations varying from 2% to 10% (w/v) (Fig. 3). Specific growth rate and specific production rate of both wild and mutant strain S. venezuelae were found to increase with increase in salinity as it was isolated from the marine environment. Similar trend was found in degradation of congo red by Pseudomonas sp. mutant (Gopinath et al., 2011). The results showed that the mutant strains provided higher specific growth rate and specific production rate even at high salinity 10% (w/v) compared to wild strain. Hence mutant was found to exhibit high salt tolerance and therefore can be effectively used for production of thrombinase using industrial wastes having high concentrations of salt. Moreover increase in salt concentration, shorten the lag period, increases the velocity of cell growth (Sherman et al., 1922) and could provide microbial products, in particular the enzymes, that could be safer having no or less toxicity or side effects when derived from marine microorganisms particularly sea water which is saline in nature and chemically closer to human blood plasma (Sabu, 2003). 3.6. Growth and enzyme production kinetic analysis at optimum conditions Under the optimized fermentation conditions (pH 7; 30 °C and 6% (w/v) salinity) the time profile of cell growth and thrombinase production was analyzed and modeled using modified gompertz Eqs. (2) and (3), respectively. The experimental data obtained for
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421
Fig. 3. Effect of salinity, % (w/v) on Specific growth rate, l (h1), (A) and Specific thrombinase production rate, Pm (h1), (B) of wild and mutant Streptomyces venezuelae. Fig. 2. Effect of temperature on specific growth rate, l (h1), (A) and specific thrombinase production rate, Pm (h1), (B) of wild and mutant Streptomyces venezuelae.
both wild and mutant strain of S. venezuelae at pH, 7; temperature, 30 °C and salinity, 6% (w/v) were fitted through Matlab 7.0 and represented in Fig. 4. At optimum pH, temperature and salinity, both the biomass concentration and enzyme activity were maximum at 99 h, which infers that the thrombinase production is growth associated as reported by Wei et al. (2011). The specific growth rate and enzyme production rate obtained at optimum conditions were found to be 0.184 l (h1) and 0.167 Pm (h1) for wild and 0.204 l (h1) and 0.193 Pm (h1) for mutant respectively. Though both wild and mutant showed maximum growth and enzyme production at the same time interval, mutant showed higher growth and enzyme activity while compared to wild. 3.7. Monod kinetics for the effect of DO concentration Fig. 5A. shows the specific growth rate variations for different DO concentrations and the obtained kinetic values are represented in the following equation.
l¼
0:1891½DO 0:956 þ ½DO
ð8Þ
l¼
0:2404½DO 0:860 þ ½DO
ð9Þ
The specific growth rate was found to increase with the increase in DO concentration up to certain level and attained steady state. As revealed by Shuler and Kargi (2002) above a critical oxygen concentration the growth rate becomes independent of the DO concentration and below that level, oxygen remains as a rate limiting factor. Based on saturation concentration, specific growth rate varies with DO concentration below the critical level. Hence the extent of growth depends on the DO concentration until it crosses the critical level. 3.8. Evaluation of substrate inhibition kinetics The growth patterns of the isolated seven mutated strains were analyzed and compared with the wild strain. Among the seven mutated strains three showed similar growth pattern with the wild S. venezuelae. The remaining four showed enhanced growth while compared with the wild strain and one strain was selected randomly among these for the evaluation of substrate inhibition kinetics using Haldane model. The corresponding plot for this model is shown in Fig. 5B. The plot infers that the mutant strain show high resistance to higher initial substrate concentration compared to the wild strain and the corresponding kinetic parameters are given in Table 3. The model equation for the substrate inhibition of wild and mutant are as follows:
l¼
0:3726:½S 2
½S 57:56 þ ½S þ 132:3
ð10Þ
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Fig. 4. Growth (A) and Enzyme (B) production kinetics obtained at optimum pH, 7; Temperature, 30 °C and Salinity, 6% (w/v).
l¼
0:5457:½S 2
½S 49:65 þ ½S þ 155:1
ð11Þ
As can be inferred from Eq. (11), inhibitory constant value is 155.1 mg/mL, which was considered as the highest concentration. It was proved that the specific growth rate increases after mutagenesis as reported by Gopinath et al. (2009b) for the degradation of congo red by Bacillus sp. mutant. The analysis of microbial growth at higher concentration of the substrate proves the tolerance of the mutant species in the cases of industrial wastes when used as substrate for the production of thrombinase and this inhibitory analysis is necessary to understand the capability of the organism to resist the higher concentration of substrate as reported by Gopinath et al. (2009a) in biodegradation of Congo red by Bacillus sp.
3.9. Effect of various S0/X0 ratio on specific substrate consumption rate The influence of S0/X0 on substrate consumption rate is illustrated in Fig. 6A and B. Fig. 6A shows that the specific consumption rate of both wild and mutant strain S. venezuelae were increased with the increasing S0/X0 ratio at a constant X0, this was due to higher initial energy level as because the cells were provided with higher quantity of substrate. However at constant lactose concentration (S0), the specific consumption rate was found to decrease with decrease in S0/X0 ratio (Fig. 6B), as because of the increase in cell density in short period which resulted in devoid of
Fig. 5. Effect of Dissolved oxygen concentration on specific growth rate, l (h1) (A) and the effect of mutagenesis on substrate inhibition kinetics of the marine isolate, Streptomyces venezuelae (B).
substrate. In addition it was reported that if the S0/X0 ratio is low, the cell growth will be repressed by the low availability of carbon source (Soto et al., 2002). Moreover while compared with wild strain, mutant showed improved specific consumption rate and the corresponding kinetic parameters obtained were shown in Table 4. The maximum substrate consumption rate (lm) for mutant was found to be slightly higher than the wild S. venezuelae at constant X0, which proves the efficient utilization of the carbon substrate by the mutant cells and substrate conversion in to product.
3.10. Effect of S0/X0 ratio on specific production rate of thrombinase The specific production rate of thrombinase got influenced with the change in S0/X0 ratio which is shown in Fig. 6C and D and the kinetic parameters obtained are given in Table 3. Both wild and mutant strain showed increase in specific production rate of the enzyme with increase in S0/X0 ratio and this was due to improved enzyme activity. Moreover increase in enzyme activity was the result of higher substrate consumption and hence the production rate of thrombinase was found to be analogous to substrate consumption rate. Though both the strains showed same profile in both the cases i.e., at constant X0 and S0, mutant was proved to be more efficient than wild. It was also reported that both growth and enzyme production contribute to substrate consumption by Mohammad et al. (2007). As the enzyme production is growth dependent, the production rate might have increased with
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B. Naveena et al. / Bioresource Technology 111 (2012) 417–424 Table 3 Substrate inhibition parameters for wild and mutant strain of Streptomyces venezuelae. S. No.
Strain
Maximum specific growth rate, lmax (h1)
Half velocity constant, Ks (mg/mL)
Inhibition constant, KI (mg/mL)
1 2
Wild Mutant
0.3726 0.5457
57.74 49.65
132.3 155.1
Alexander, (1984), that is if the initial cell density is much greater than the number of new microorganisms, i.e., X0 >> S0, the microbial growth during bioprocess becomes insignificant on a proportional basis. Hence it was proved that specific production rate of thrombinase not only depends on specific growth rate but also on the specific consumption rate. 4. Conclusion
increasing specific growth rate. At a constant X0, the cells were supplied with more energy due to higher initial substrate concentration and which in turn increases the production of thrombinase thus there was a rise in specific production rate. However at a constant S0, while varying the initial biomass concentration (X0), the specific production rate was found to decrease since the substrate consumption was low (Fig. 6B). As inferred above, this might be due to cell population density which lowered the specific growth rate. This is evident with the inference of Simkins and
Strain improvement by random mutagenesis using Ethyl methane sulfonate was found to be effective than UV irradiation. It was found that the mutant strain is highly thermotolerant, halophilic than wild and can even grow at extreme pH conditions. S. venezuelae mutant was considered as highly resistant to higher initial concentrations of lactose as the KI value was too high. The importance of S0/X0 ratios on substrate consumption rate and specific thrombinase production rate of both strains was also interpreted. Mutant showed enhanced specific substrate consumption and production rate at higher S0/X0 ratios. Hence, proved that for an
Fig. 6. Influence of initial S0/X0 ratio in substrate consumption rate, l (h1) (A- at constant initial concentration of biomass, (X0); (B)- at constant initial concentration of Lactose, (S0)) and specific production rate, P (h1) ((C)- at constant initial concentration of biomass, (X0); (D)- at constant initial concentration of Lactose, (S0)) of both mutant and wild Streptomyces venezuelae.
Table 4 Kinetic parameters obtained for the effect of S0/X0 ratios. Strain
Wild Mutant
Substrate consumption rate at constant X0
Substrate consumption rate at constant S0
Thrombinase production rate at constant X0
Thrombinase production rate at constant S0
Maximum specific consumption rate, lm (h1)
Ks/x, (mg/mL)
Maximum specific consumption rate, lm (h1)
Ks/x, (mg/mL)
Maximum specific production rate, Pmax (h1)
Ks/x, (mg/mL)
Maximum specific production rate, Pmax (h1)
Ks/x, (mg/mL)
0.697 0.730
156.2 152.4
0.349 0.376
66.08 60.21
0.391 0.406
196.8 171.6
0.266 0.293
81.28 78.48
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efficient thrombinase production, the S0/X0 ratio should also considered.
References Balaraman, K., Prabakaran, G., 2007. Production & purification of a fibrinolytic enzyme (thrombinase) from Bacillus sphaericus. Indian J. Med. Res. 126, 459–464. Baltz, R.H., 1998. New genetic methods to improve secondary metabolite production in Streptomyces. J. Ind. Mirobiol. Biot. 20, 360–363. Chandra, M., Kalra, A., Sangwan, N.S., Gaurav, S.S., Darokar, M.P., Sangwan, R.S., 2008. Development of mutant of Trichoderma citrinoviride for enhanced production of cellulases. Bioresour. Technol. 100, 1659–1662. Chitte, R.R., Dey, S., 2002. Production of a fibrinolytic enzyme by thermophilic Streptomyces species. World J. Microbiol. Biotechnol. 18, 289–294. Chudoba, P., Capdeville, B., Chudoba, J., 1992. Explanation of biological meaning of the S0/X0 ratio in batch culture. Water Sci. Technol. 26, 743–751. Chung, D.M., Choi, N.S., Maeng, P.J., Chun, H.K., Kim, S.H., 2010. Purification and characterization of a novel fibrinolytic enzyme from Chive (Allium tuberosum). Food Sci. Biotechnol. 19, 697–702. Gopinath, K.P., Asan Meera Sahib, H., Muthukumar, K., Velan, M., 2009a. Improved biodegradation of Congored by using Bacillus sp. Bioresour. Technol. 100, 670– 675. Gopinath, K.P., Murugesan, S., Abraham, J., Muthukumar, K., 2009b. Bacillus sp. mutant for improved biodegradation of Congo red: random mutagenesis approach. Bioresour. Technol. 100, 6295–6300. Gopinath, K.P., Kathiravan, M.N., Srinivasan, R., Sankaranarayanan, S., 2011. Evaluation and elimination of inhibitory effects of salts and heavy metal ions on biodegradation of Congo red by Pseudomonas sp. mutant. Bioresour. Technol. 102, 3687–3693. Liu, Y., Liu, Q.S., Tay, J.H., 2005a. Initial conditions-dependent growth kinetics in microbial batch culture. Process Biochem. 40, 155–160. Liu, J.G., Xing, J.M., Chang, T.S., Ma, Z.Y., Liu, H.Z., 2005b. Optimization of nutritional conditions for nattokinase production by Bacillus natto NLSSE using statistical experimental methods. Process Biochem. 40, 2757–2762. Mander, P., Cho, S.S., Simkhada, J.R., Choi, Y.H., Yoo, J.C., 2011. A low molecular weight chymotrypsin-like novel fibrinolytic enzyme from Streptomyces sp. CS624. Process Biochem. 46, 1449–1455. Maiorella, B.L., Castillo, F.J., 1984. Ethanol, biomass, and enzyme production for whey waste abatement. Process Biochem. 8, 157–161. Mohammad, F., EI-Tayeb, O., Aboulwafa, M., 2007. Optimization of the industrial production of bacterial a amylase in Egypt. V. Analysis of kinetic data for enzyme production by two strains of Bacillus amyloliquefaciens. Afr. J. Biotechnol. 7, 4537–4543. Nakajima, N., Mihara, H., Sumi, H., 1993. Characterization of potent fibrinolytic enzymes in earthworm, Lumbricus rubellus. Biosci. Biotechnol. Biochem. 57, 1726–1730.
Okami, Y., 1986. Marine microorganisms as a source of bioactive agents. Microb. Ecol. 12, 65–78. Peng, Y., Yang, X., Zhang, Y., 2005. Microbial fibrinolytic enzymes: an overview of source, production, properties and thrombolytic activity in vivo. Appl. Microbiol. Biotechnol. 69, 126–132. Rajoka, M.I., Bashir, A., Malik, K.A., 1997. Mutagenesis of Cellulomonas biazotea for enhanced production of xylanases. Bioresour. Technol. 62, 99–108. Ravindranath, E., Chitra, K., Shamshath Begum, S., Navaneetha Gopalakrishnan, A., 2010. Effect of recirculation rate on anaerobic treatment of fleshing using UASB reactor with recovery of energy. J. Sci. Ind. Res. 69, 790–793. Savergave, L.S., Gadre, R.V., Vaidya, B.K., Narayanan, K., 2011. Strain improvement and statistical media optimization for enhanced erythritol production with minimal byproducts from Candida magnoliae mutant R23. Biochem. Eng. J. 55, 92–100. Sabu, A., 2003. Sources, properties and applications of microbial therapeutic enzymes. Indian. J. Biotechnol. 2, 334–341. Seo, J.H., Lee, S.P., 2004. Production of fibrinolytic enzyme from soybean grits fermented by Bacillus firmus NA-1. J. Med. Food. 7, 442–449. Sherman, J.M., Holm, G.E., Albus, W.R., 1922. Salt effects in bacterial growth III. Salt effects in relation to the lag period and velocity of growth. J. Bacteriol. 7, 583– 588. Shuler, M.L., Kargi, F., 2002. Bioprocess Engineering: Basic Concepts, second ed. Prentice-Hall India, New Delhi. Simkins, S., Alexander, M., 1984. Models for mineralization kinetics with variables of substrate concentration and population density. Appl. Environ. Microbiol. 47, 1299–1306. Soto, O., Sanchez, O., Aspe, E., Roeckel, M., 2002. Denitrification kinetics of simulated fish processing wastewater at different ratios of nitrate to biomass. Biotechnol. Lett. 24, 1173–1176. Toledo, G., Green, W., Gonzalez, R.A., Christoffersen, L., Podar, M., Chang, H.W., Hemscheidt, T., Trapido-Rosenthal, H.G., Short, J.M., Bidigare, R.R., Mathur, E.J., 2006. High throughput cultivation for isolation of novel marine microorganisms. Oceanography 19, 120–125. Vazquez-Rodriguez, G., Palluy, F., Goma, G., Rols, J.L., 1999. Procedures in ready biodegradability testing: effects of the inoculation and the monitored parameter. Environ. Technol. 20, 301–308. Wang, J., Fang, F., Yu, H.Q., 2007. Substrate consumption and biomass growth of Ralstonia eutropha at various S0/X0 levels in batch cultures. Bioresour. Technol. 98, 2599–2604. Wang, S.L., Chen, H.J., Liang, T.W., Lin, Y.D., 2009. A novel nattokinase produced by Pseudomonas sp. TKU015 using shrimp shells as substrate. Process Biochem. 44, 70–76. Wei, X., Luo, M., Xu, L., Zhang, Y., Lin, X., Kong, P., Liu, H., 2011. Production of fibrinolytic enzyme from Bacillus amyloliquefaciens by fermentation of chickpeas, with the evaluation of the anticoagulant and antioxidant properties of chickpeas. J. Agric. Food Chem. 59, 3957–3963. Zhang, Y., Wisner, A., Xiong, Y., Bon, C., 1995. A novel plasminogen activator from snake venom. J. Biol. Chem. 270, 10246–10255.
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Enhanced production of thrombinase by Streptomyces venezuelae: Kinetic studies on growth and enzyme production of mutant strain Balakrishnan Naveena a, Kannapan Panchamoorthy Gopinath b, Punniavan Sakthiselvan a, Nagarajan Partha a,⇑ a b
Department of Chemical Engineering, A.C. College of Technology, Anna University Chennai, Chennai 600 025, India Department of Chemical Engineering, Adhiparasakthi Engineering College, Melmaruvathur 603 319, India
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 11 November 2011 Received in revised form 10 February 2012 Accepted 13 February 2012 Available online 21 February 2012
This investigation provides the enhanced production of thrombinase, a fibrinolytic enzyme using mutant Streptomyces venezuelae. Initially the mutagenesis of the marine isolate was done by UV and Ethyl methane sulfonate (EMS) and their mutational efficiencies were compared. The mutants were selected based on their high thrombinase activity and used for further studies. The mutant was found to be more halo and thermo tolerant comparing to wild. The effect of Dissolved oxygen level was also determined and the mutant offered the maximum specific growth rate as 0.2404 (h1). The mutant showed high resistance to higher initial lactose concentration and the inhibition concentration was found to be 155.1 mg/mL. The effect of S0/X0 ratio on specific substrate consumption and production rate were also investigated. Both mutant and wild showed increase in specific substrate consumption and production rate at higher S0/X0 ratio but the mutant showed better values than the wild strain. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Random mutagenesis Thrombinase S0/X0 ratio Growth kinetics Enzyme production kinetics
1. Introduction Thrombolytic agents which include urokinase, streprokinase, and tissue plasminogen activator (t-PA), have been widely applied for the lysis of fibrin clots in the case of ischemia (acute myocardial infarction, pulmonary embolism, ischemic stroke, and arterial thrombosis). Due to their short half life, high expensive and excessive risk of hemorrhagic complication, researches for safe and more economic thrombolytic enzymes have been explored for the past few decades. Based on their mode of action, thrombolytic agents are divided into two types: plasminogen activators and plasmin like proteins (Peng et al., 2005). The former type of thrombolytic agents that includes tissue type plasminogen activator (t-PA), urokinase and streptokinase activates the endogenous fibrinolytic system to generate plasmin which eventually hydrolyzes fibrin (Balaraman and Prabakaran, 2007). While the later type of thrombolytic agents that includes nattokinase, lumbrokinase and chymotrypsin like fibrinolytic enzyme reported by Mander et al. (2011) directly hydrolyzes fibrin. Many thrombolytic enzymes have been so far reported from various sources such as plants (Chung et al., 2010), animals (Zhang et al., 1995; Nakajima et al., 1993) and microbes (Peng et al., 2005). Among all these sources, microbes are most preferred due to their broad biochemical diversity, feasibility of mass culture and ease in genetic manipulation.
⇑ Corresponding author. Tel.: +91 9790445938. E-mail address: [email protected]
(N. Partha).
0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.02.056
Though, numerous fibrinolytic enzymes from various microbes including bacteria, actinomyces, fungi, and algae have reported, only few have been explored from marine microorganisms. More recently, marine microorganisms have been recognized as a productive source of novel secondary metabolites and exhibit salt tolerance (Toledo et al., 2006; Okami, 1986). Hence in this study we attempted to produce thrombinase by Streptomyces venezuelae from marine environment as it is a reservoir of novel natural products. Another major criterion for the production of enzyme is selection of substrate. The enzyme has been so far reported from various substrates such as chick peas, corn starch, and beef extract, however the cost of the substrate must be low for a commercial production. Hence in this study we attempted the enzyme production using dairy and tannery industrial wastes in order to minimize the production cost and reduce environmental pollution generated by those wastes. Several investigations have been proved that use of such industrial wastes minimizes the pollution and also enhances the product yield as they are rich in carbon and nitrogen substrates (Maiorella and Castillo, 1984; Ravindranath et al., 2010). To make an efficient bioprocess of enzymes and to satisfy commercial needs, the source of the microorganism and the substrate are not only enough to consider, some other strategies also to be focused. Among those, one of the most important strategies is strain improvement which mainly increases the production efficiency. Strain improvement concerns about developing or modifying organisms used in the production of commercially important products such as antibiotics and enzymes, with the overall aim of
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reducing production costs. This actually deals the enhancement of the productivity of an organism, but it may also be desirable to manipulate specific characteristics such as the ability to utilize cheaper substrates such as industrial wastes. Many attempts have made inorder to increase the yield of thrombinase production such as selection of the culture medium, formulation of fermentation medium (Seo and Lee, 2004) and optimization of environmental conditions (Chitte and Dey, 2002; Liu et al., 2005b) but only few researches have been reported thrombinase by genetically improved strains. The traditional approach of strain improvement is mutagenesis of a suitable strain followed by random screening of mutants. This approach has been successful and has benefited in recent years from the application of important advances in science and technology. Random mutagenesis and recombinant DNA technology have been adopted to improve the enzyme yield and simplify the downstream processes (Peng et al., 2005). An important such technique, random mutagenesis, is used to induce mutation in organisms using UV, Ethyl methyl sulfonate (EMS) and Ethidium bromide (EtBr) for better distinctiveness (Chandra et al., 2008). In recent years, many attempts were made to improve the strains using physical and chemical mutagenesis. Rajoka et al. (1997) improved the production of xylanase by Cellulomonas biazotea mutated by gamma rays. Savergave et al. (2011) screened high erythritol producing Candida magnolia by UV and chemical mutagenesis. Production of thrombinase by mutants with improved enzyme production potential has not been given much attention. Therefore in this study we gave our attention to enhance the thrombinase production through strain improvement on S. venezuelae. Besides strain improvement, some of the other important facts have also to be considered, they are their tolerance to extreme pH, temperature and salinity. Moreover changes in their kinetic parameters such as maximum specific growth rate, maximum specific consumption rate and maximum production rate before and after strain improvement have also to be concerned. Batch experiments have been usually employed to optimize the operation conditions and to evaluate the kinetic parameters (Vazquez-Rodriguez et al., 1999). However great number of investigations show that the biomass yield is influenced by the ratio of initial substrate concentration, (S0) to the initial biomass concentration, (X0) (Wang et al., 2007). It has been stated that the effect of S0/X0 on batch fermentation influence the change in composition of microbial community i.e., the larger the S0/X0 ratio, the greater the change in the structure of microbial community (Chudoba et al., 1992). However, in previous researches only effects of X0 and S0 on biomass growth and production of fibrinolytic enzyme were determined, the importance of their ratios on consumption rate and production rate were not sufficiently concerned. Hence, in this research attempts were made to evaluate and compare the effect of S0/X0 ratio on specific consumption rate and specific production rate of both wild and mutant strains of Streptomyces venezuelae using tannery and dairy industrial wastes.
2. Methods 2.1. Inoculum preparation Streptomyces venezuelae isolated from marine water sample was maintained in agar slants of Starch casein agar (SCA) containing (component g/L) Starch, 10; K2HPO4, 2; KNO3, 2; Nacl, 2; Casein, 0.3; MgSO47H2O, 0.05; CaCO3, 0.02; FeSO47H2O, 0.01 and agar, 20. The seed culture was prepared using the same media (pH 7) without agar and incubated at 30 °C for 4 days. The inoculum obtained at exponential phase was used for further bioprocess studies.
2.2. Induction of mutagenesis The cells were harvested from the culture media by centrifugation at 6000 rpm for 10 min. The cell pellet obtained was washed twice with distilled water and was resuspended in sterile deionised water. Further the colony count was done by Colony counting method and the count was adjusted to 2.36 107 CFU/mL. The UV mutagenesis was carried out by taking 10 mL of the cell suspension in a sterile petridish and exposed to a germicidal lamp for 0–25 min. And for chemical mutation, the cell pellet in deionized water was resuspended in 20 mL of 20 lL ethyl methyl sulfonate (EMS) solution and was shaken thoroughly in an orbital shaker for 30 min. Aliquots were withdrawn at regular intervals of 5 min and serially diluted for viable cell counting by spread plate technique and screening. The % survival was calculated using the following Eq. (1):
S¼
Ni Nd Ni
ð1Þ
where S is the % survival, Ni is the initial viable cell count in CFU/mL and Nd is the viable cell count after mutation in CFU/mL. 2.3. Selection of mutants To select the mutant colonies, 1 mL of the mutated sample was serially diluted to obtain a dilution of 105. From the diluted sample, 0.1 mL was inoculated in a medium containing (component g/ L) Tryptone, 9.47; Meat extract, 4.74; Yeast extract, 0.95; Sodium pyruvate, 9.47; Glycine, 11.36; Lithium chloride, 4.73; thrombin 0.45 U/ml, EDTA 25.0 mL, Bovine fibrinogen, 5.0; Potassium tellurite, 25.0; and was incubated at 37 °C for 24 h. After incubation the broth was centrifugated at 4000 rpm for 5 min. The supernatant was collected for each sample and subjected for fibrin degradation assay. The samples which showed higher enzyme activity compared with the wild were considered as positive mutants and the rest were considered as negative. Further, the mutant sample with highest enzyme activity was isolated and used for further experiments. 2.4. Fermentation media The medium comprises of the following components 2% (w/v) lactose in whey which is derived from the dairy industry waste, 10% (w/v) peptone, 0.7% (w/v) de-limed flesh which is the waste collected from the leather industry, 1.5% (w/v) K2HPO4 and 0.7% (w/v)Triton X100. The medium was autoclaved at 121 °C for 20 min. The experimental design with various fermentation conditions for further analyses is represented in Table 1. 2.5. Evaluation of efficiency of growth and thrombinase production Batch experiments were carried out to analyze the growth and production efficiency of mutated strains at various initial pH, temperature and Sodium chloride (NaCl) concentration. As given in Table 1, these fermentation conditions were varied. The experiments were carried out as triplicates by inoculating 0.1 mL of mutant and wild culture in separate Erlen-meyer flasks containing 1000 mL of sterile fermentation medium and incubated for 4 days with rotary shaking at 200 rpm and their average values were taken in to consideration. Samples were collected at every 3 h interval from the two culture flasks and subjected for centrifugation at 1118g separately. The initial weight of the aluminum foil was taken. The pellet obtained was placed in the foil and kept at 55 °C for 10 min until got dried. The weight of the foil with the dried pellet was measured. From the weight difference the biomass concentration was calculated and the supernatant was used as
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where, l is the specific growth rate, lmax is the maximum specific growth rate, Ks is the half velocity constant (mg/mL) and KI is the substrate inhibition (mg/mL).
Table 1 Experimental design for various analyses. S. No.
Experimental analyses
1
Different set points pH
Temperature, °C
Salinity, % (w/v)
Effect of pH
5–10 (varied)
4
2
Effect of temperature
Optimum
3
Effect of salinity
Optimum
Room temperature37 °C 20–60 °C (varied) Optimum
4
Effect of Dissolved oxygen concentration Determination of assimilable carbon source Effect of S0/X0 on substrate consumption and enzyme activity
Optimum
Optimum
2–10 (varied) Optimum
Optimum
Optimum
Optimum
Optimum
Optimum
Optimum
5 6
2.8. Effect of various S0/X0 on substrate consumption and enzyme activity
4
X ln Xo
2 0 13 X max e: l m ¼ ln exp4exp@ ðk tÞ þ 1A5 Xo ln Xmax
ð3Þ
In Eq. (2), where X0, Xmax, X, t, lm and k denote initial biomass concentration (mg/mL), maximum biomass concentration (mg/ mL) and biomass concentration (mg/mL), incubation time (h), maximum specific growth rate (h1) and lag time (h), respectively. And in Eq. (3), where P, A, Pm, t and k denote Enzyme activity (FU/ mL), Enzyme production potential (FU/mL), specific enzyme production rate (h1), incubation time (h) and lag time (h), respectively. 2.6. Effect of Dissolved oxygen concentration on specific growth rate To determine the effect of Dissolved oxygen (DO) concentration, the growth was analyzed by varying the DO concentration from 2% to 12% (w/v). This was done by using Dissolved oxygen (DO) controller model DO-60 in conjunction with the bioreactor (2 L capacity) to measure and control the oxygen level. The specific growth rates obtained for various DO concentrations were fitted using the following Monod equation.
lm ½DO K DO þ ½DO
ð4Þ
where, l is the specific growth rate, lm is the maximum specific growth rate, KDO is the half velocity constant (mg/mL). 2.7. Determination of assimilable carbon source The assimilable carbon was evaluated by varying the initial concentration of lactose in whey from 0 to 100 mg/mL using the following Haldane’s expression.
l¼
lmax ½S K s þ ½S þ ½KSI
l ¼ lm
S0 =X 0 K S=X þ ðS0 =X 0 Þ S0 =X 0 K S=X þ ðS0 =X 0 Þ
ð6Þ
ð7Þ
where, l is the specific consumption rate, lm is the maximum specific consumption rate, S0/X0 is the ratio of initial substrate concentration to initial biomass concentration, P is the specific production rate, Pmax is the maximum specific thrombinase production rate and K s=x ¼ eðDG0 DG=RTÞ (DG0 and DG are the change of the standard free energy and overall free energy, respectively). The initial lactose concentration (S0) was varied from 20 to 140 mg/mL and the initial biomass concentration (X0) was varied from 0.1 to 1 mg/mL. 2.9. Fibrin degradation assay
ð2Þ
Xo
e:Pm P ¼ A:exp exp ðk t Þ þ 1 A
l¼
The effect of various S0/X0 on substrate consumption and enzyme activity were analyzed by varying S0 at a constant X0 and by varying X0 at a constant S0 using the following equations (Liu et al., 2005a).
P ¼ Pmax
crude enzyme extract. Thus the biomass concentration was determined for all further experiments. Crude extract samples obtained for every 3 h were collected. The enzyme activity for each sample was measured through fibrin degradation assay. The specific growth rate and production rate were calculated from the biomass concentration and enzyme activity obtained at various time intervals for each initial pH, temperature and salt concentration using the following modified Gompertz equations (Gopinath et al., 2011).
419
ð5Þ
The enzyme activity of the crude extract was quantitatively determined by fibrin degradation assay (Wang et al., 2009). Initially 0.4 mL of 0.72% fibrinogen was placed in a test tube with 0.1 mL of 245 mM phosphate buffer (pH 7) and incubated at 37 °C for 5 min. To this 0.1 mL of a 20 U/mL thrombin solution was added and incubated at 37 °C for 10 min. After incubation 0.1 mL of diluted enzyme solution was added, and incubation continued at 37 °C. This solution was again mixed twice at 20 min interval. After 1 h, 0.7 mL of 0.2 M trichloroacetic acid (TCA) was added, mixed and centrifuged at 15,000 rpm for 10 min. Then, 1 mL of the supernatant was collected and the absorbance at 275 nm was measured under UV- Spectrophotometer. According to this assay, 1 unit of enzyme activity is defined as a 0.01-perminute increase in absorbance at 275 nm of the reaction solution. The enzyme activity was expressed in FU/mL (Fibrin degradation unit per mL). 3. Results and discussion 3.1. Mutagenesis analysis The dose response analysis for both UV mutagenesis and chemical mutagenesis of marine Streptomyces venezuelae was done and the % survival values are shown in Table 2. The dosage was expressed in terms of time of exposure to the UV irradiation and varied in the range 2–10 min, to analyze the survival capacity and mutation effect. The marine isolate did not respond well to UV mutagenesis when compared to the chemical mutation by EMS. Since the DNA of the isolate contains more GC basepairs than AT basepairs as it belongs to the group of Actinomyces, mutation by EMS causes GC transversions. Similar trend was found by Baltz (1998), in the case of Streptomyces, where the survival capacity obtained by EMS mutation was higher than UV. At the same time, while comparing with those results the survival capacity of the mutant was severely affected as the dosage of EMS was increased. The transversion mutation in DNA caused by Ethyl methane sulfonate was responsible for the DNA damage. Since the self
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Table 2 Dose response for UV and EMS mutagenesis. S. No.
1 2 3 4 5 6
UV mutagenesis
EMS mutagenesis
Time of exposure, min
Response, % survival
Time of exposure, min
Response, % survival
0 5 10 15 25 25
100 92 75 53 28 8
0 5 10 15 20 25
100 62 47 29 15 3
DNA repair mechanism fails in certain instances the mutation caused by EMS became permanent. Moreover prolonged incubation causes uncontrolled DNA damage which leads to cell death and hence a suitable incubation limit for good mutagenesis to occur can be determined by selection process. 3.2. Selection of mutants The positive mutants were selected on the basis of thrombinase activity produced by the colonies derived from the plates. The wild strain produced 25 FU/mL and the strains which produced more than this value of thrombinase activity were considered as positive mutants. On the whole seven strains showed more than 30 FU/mL and most of them were showed such a higher activity in 20 min exposure of EMS and beyond this level all showed negative results. None of the strains mutated by UV showed more enzyme activity than the wild. Finally all the seven mutants obtained from EMS, which showed increased thrombinase activity were selected for further studies. 3.3. Effect of initial pH The pH of the industrial wastes keeps on changing and hence when using those wastes as substrate, adaptation of microorganism to various pH makes them more selective to bioprocess of enzymes. To compare the growth rate and production efficiency of wild and mutant strains, the initial pH was varied from 5 to 10. Though both the strains showed highest specific growth rate and specific production rate at pH 7, mutant showed more tolerance even at pH 5 and 10 (Fig. 1), than the wild strain. Hence it was proved that the mutant can able to survive under extreme pH conditions. Similar results were reported by Gopinath et al. (2009b) for the degradation of congo red by Bacillus sp. mutant. 3.4. Effect of temperature The influence of temperature on specific growth rate and specific production rate of the wild and mutant strains was investigated by varying the temperature from 20 to 60 °C. The results obtained demonstrated that the growth and production efficiency was almost same in the temperature range 20–60 °C for mutant strains. From Fig. 2, it was inferred that though the wild strain was found to survive under all tested temperatures, the growth was insignificant at extreme temperatures and hence the specific growth rate and specific production rate were found to be very low. While compared with the wild strain, the mutants were known to exhibit good resistance to the wide range of temperatures (Gopinath et al., 2009b). 3.5. Determination of salt tolerance The industrial wastes such as Delimmed flesh usually have high concentration of salts, thus while using those wastes as substrates,
Fig. 1. Effect of pH on specific growth rate, l (h1) (A) and specific thrombinase production rate, Pm (h1), (B) of wild and mutant Streptomyces venezuelae.
the microorganism used for the production should be highly salt tolerant. Hence adaptation to high salinity is necessary for an effective bioprocess. Thus to compare the salt tolerance of wild and mutant strains, batch experiments were carried out at the initial NaCl concentrations varying from 2% to 10% (w/v) (Fig. 3). Specific growth rate and specific production rate of both wild and mutant strain S. venezuelae were found to increase with increase in salinity as it was isolated from the marine environment. Similar trend was found in degradation of congo red by Pseudomonas sp. mutant (Gopinath et al., 2011). The results showed that the mutant strains provided higher specific growth rate and specific production rate even at high salinity 10% (w/v) compared to wild strain. Hence mutant was found to exhibit high salt tolerance and therefore can be effectively used for production of thrombinase using industrial wastes having high concentrations of salt. Moreover increase in salt concentration, shorten the lag period, increases the velocity of cell growth (Sherman et al., 1922) and could provide microbial products, in particular the enzymes, that could be safer having no or less toxicity or side effects when derived from marine microorganisms particularly sea water which is saline in nature and chemically closer to human blood plasma (Sabu, 2003). 3.6. Growth and enzyme production kinetic analysis at optimum conditions Under the optimized fermentation conditions (pH 7; 30 °C and 6% (w/v) salinity) the time profile of cell growth and thrombinase production was analyzed and modeled using modified gompertz Eqs. (2) and (3), respectively. The experimental data obtained for
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421
Fig. 3. Effect of salinity, % (w/v) on Specific growth rate, l (h1), (A) and Specific thrombinase production rate, Pm (h1), (B) of wild and mutant Streptomyces venezuelae. Fig. 2. Effect of temperature on specific growth rate, l (h1), (A) and specific thrombinase production rate, Pm (h1), (B) of wild and mutant Streptomyces venezuelae.
both wild and mutant strain of S. venezuelae at pH, 7; temperature, 30 °C and salinity, 6% (w/v) were fitted through Matlab 7.0 and represented in Fig. 4. At optimum pH, temperature and salinity, both the biomass concentration and enzyme activity were maximum at 99 h, which infers that the thrombinase production is growth associated as reported by Wei et al. (2011). The specific growth rate and enzyme production rate obtained at optimum conditions were found to be 0.184 l (h1) and 0.167 Pm (h1) for wild and 0.204 l (h1) and 0.193 Pm (h1) for mutant respectively. Though both wild and mutant showed maximum growth and enzyme production at the same time interval, mutant showed higher growth and enzyme activity while compared to wild. 3.7. Monod kinetics for the effect of DO concentration Fig. 5A. shows the specific growth rate variations for different DO concentrations and the obtained kinetic values are represented in the following equation.
l¼
0:1891½DO 0:956 þ ½DO
ð8Þ
l¼
0:2404½DO 0:860 þ ½DO
ð9Þ
The specific growth rate was found to increase with the increase in DO concentration up to certain level and attained steady state. As revealed by Shuler and Kargi (2002) above a critical oxygen concentration the growth rate becomes independent of the DO concentration and below that level, oxygen remains as a rate limiting factor. Based on saturation concentration, specific growth rate varies with DO concentration below the critical level. Hence the extent of growth depends on the DO concentration until it crosses the critical level. 3.8. Evaluation of substrate inhibition kinetics The growth patterns of the isolated seven mutated strains were analyzed and compared with the wild strain. Among the seven mutated strains three showed similar growth pattern with the wild S. venezuelae. The remaining four showed enhanced growth while compared with the wild strain and one strain was selected randomly among these for the evaluation of substrate inhibition kinetics using Haldane model. The corresponding plot for this model is shown in Fig. 5B. The plot infers that the mutant strain show high resistance to higher initial substrate concentration compared to the wild strain and the corresponding kinetic parameters are given in Table 3. The model equation for the substrate inhibition of wild and mutant are as follows:
l¼
0:3726:½S 2
½S 57:56 þ ½S þ 132:3
ð10Þ
422
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Fig. 4. Growth (A) and Enzyme (B) production kinetics obtained at optimum pH, 7; Temperature, 30 °C and Salinity, 6% (w/v).
l¼
0:5457:½S 2
½S 49:65 þ ½S þ 155:1
ð11Þ
As can be inferred from Eq. (11), inhibitory constant value is 155.1 mg/mL, which was considered as the highest concentration. It was proved that the specific growth rate increases after mutagenesis as reported by Gopinath et al. (2009b) for the degradation of congo red by Bacillus sp. mutant. The analysis of microbial growth at higher concentration of the substrate proves the tolerance of the mutant species in the cases of industrial wastes when used as substrate for the production of thrombinase and this inhibitory analysis is necessary to understand the capability of the organism to resist the higher concentration of substrate as reported by Gopinath et al. (2009a) in biodegradation of Congo red by Bacillus sp.
3.9. Effect of various S0/X0 ratio on specific substrate consumption rate The influence of S0/X0 on substrate consumption rate is illustrated in Fig. 6A and B. Fig. 6A shows that the specific consumption rate of both wild and mutant strain S. venezuelae were increased with the increasing S0/X0 ratio at a constant X0, this was due to higher initial energy level as because the cells were provided with higher quantity of substrate. However at constant lactose concentration (S0), the specific consumption rate was found to decrease with decrease in S0/X0 ratio (Fig. 6B), as because of the increase in cell density in short period which resulted in devoid of
Fig. 5. Effect of Dissolved oxygen concentration on specific growth rate, l (h1) (A) and the effect of mutagenesis on substrate inhibition kinetics of the marine isolate, Streptomyces venezuelae (B).
substrate. In addition it was reported that if the S0/X0 ratio is low, the cell growth will be repressed by the low availability of carbon source (Soto et al., 2002). Moreover while compared with wild strain, mutant showed improved specific consumption rate and the corresponding kinetic parameters obtained were shown in Table 4. The maximum substrate consumption rate (lm) for mutant was found to be slightly higher than the wild S. venezuelae at constant X0, which proves the efficient utilization of the carbon substrate by the mutant cells and substrate conversion in to product.
3.10. Effect of S0/X0 ratio on specific production rate of thrombinase The specific production rate of thrombinase got influenced with the change in S0/X0 ratio which is shown in Fig. 6C and D and the kinetic parameters obtained are given in Table 3. Both wild and mutant strain showed increase in specific production rate of the enzyme with increase in S0/X0 ratio and this was due to improved enzyme activity. Moreover increase in enzyme activity was the result of higher substrate consumption and hence the production rate of thrombinase was found to be analogous to substrate consumption rate. Though both the strains showed same profile in both the cases i.e., at constant X0 and S0, mutant was proved to be more efficient than wild. It was also reported that both growth and enzyme production contribute to substrate consumption by Mohammad et al. (2007). As the enzyme production is growth dependent, the production rate might have increased with
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B. Naveena et al. / Bioresource Technology 111 (2012) 417–424 Table 3 Substrate inhibition parameters for wild and mutant strain of Streptomyces venezuelae. S. No.
Strain
Maximum specific growth rate, lmax (h1)
Half velocity constant, Ks (mg/mL)
Inhibition constant, KI (mg/mL)
1 2
Wild Mutant
0.3726 0.5457
57.74 49.65
132.3 155.1
Alexander, (1984), that is if the initial cell density is much greater than the number of new microorganisms, i.e., X0 >> S0, the microbial growth during bioprocess becomes insignificant on a proportional basis. Hence it was proved that specific production rate of thrombinase not only depends on specific growth rate but also on the specific consumption rate. 4. Conclusion
increasing specific growth rate. At a constant X0, the cells were supplied with more energy due to higher initial substrate concentration and which in turn increases the production of thrombinase thus there was a rise in specific production rate. However at a constant S0, while varying the initial biomass concentration (X0), the specific production rate was found to decrease since the substrate consumption was low (Fig. 6B). As inferred above, this might be due to cell population density which lowered the specific growth rate. This is evident with the inference of Simkins and
Strain improvement by random mutagenesis using Ethyl methane sulfonate was found to be effective than UV irradiation. It was found that the mutant strain is highly thermotolerant, halophilic than wild and can even grow at extreme pH conditions. S. venezuelae mutant was considered as highly resistant to higher initial concentrations of lactose as the KI value was too high. The importance of S0/X0 ratios on substrate consumption rate and specific thrombinase production rate of both strains was also interpreted. Mutant showed enhanced specific substrate consumption and production rate at higher S0/X0 ratios. Hence, proved that for an
Fig. 6. Influence of initial S0/X0 ratio in substrate consumption rate, l (h1) (A- at constant initial concentration of biomass, (X0); (B)- at constant initial concentration of Lactose, (S0)) and specific production rate, P (h1) ((C)- at constant initial concentration of biomass, (X0); (D)- at constant initial concentration of Lactose, (S0)) of both mutant and wild Streptomyces venezuelae.
Table 4 Kinetic parameters obtained for the effect of S0/X0 ratios. Strain
Wild Mutant
Substrate consumption rate at constant X0
Substrate consumption rate at constant S0
Thrombinase production rate at constant X0
Thrombinase production rate at constant S0
Maximum specific consumption rate, lm (h1)
Ks/x, (mg/mL)
Maximum specific consumption rate, lm (h1)
Ks/x, (mg/mL)
Maximum specific production rate, Pmax (h1)
Ks/x, (mg/mL)
Maximum specific production rate, Pmax (h1)
Ks/x, (mg/mL)
0.697 0.730
156.2 152.4
0.349 0.376
66.08 60.21
0.391 0.406
196.8 171.6
0.266 0.293
81.28 78.48
424
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efficient thrombinase production, the S0/X0 ratio should also considered.
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