Advanced Materials Research Vol. 584 (2012) pp 440-444 Online available since 2012/Oct/22 at www.scientific.net © (2012) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/AMR.584.440
Strain Improvement of Streptomyces venezuelae for Enhanced Fibrinolytic Enzyme Production Balasubramanian Bhavani1,a, Balakrishnan Naveena1,b and Nagarajan Partha*1,c Department of Chemical Engineering, Alagappa College of Technology, Anna University, Chennai 600 025, India. a
[email protected],
[email protected], c*
[email protected]
Keywords: Fibrinolytic enzymes, Streptomyces venezuelae, Mutagenesis
Abstract: The fibrinolytic enzymes can be used as a potential drug to cure thrombosis diseases. These enzymes can effectively catalyze the degradation of fibrin in blood clot. To develop safe and cheaper fibrinolytic agents, fibrinolytic enzyme was isolated from Streptomyces venezuelae. Strain improvement was employed to increase the production of fibrinolytic enzymes using random mutagenesis (UV and Ethyl methane sulfonate). The mutants obtained were screened based on their fibrinolytic activity and best mutant was selected for further studies. Mutant obtained by Ethyl Methane Sulfonate was able to yield the fibrinolytic activity of 13 FU/mL in growth medium which was higher than wild strain (6 FU/mL). The results indicated that EMS was effective mutagenic agents for strain improvement of Streptomyces venezuelae for enhanced production of fibrinolytic enzyme. The mutant showed improved growth compared to wild strain. The optimal temperature and pH value of this fibrinolytic enzyme were found to be 40 °C and 8.0, respectively. The strain improvement also improves the stability of Streptomyces venezuelae which showed resistance to temperature and pH at higher values. Invitro assays revealed that fibrinolytic enzyme produced by Streptomyces venezuelae could degrade fibrin suggesting that its future application in pharmaceutical industry as thrombolytic agent is highly promising. Introduction: Fibrinolysis is the process in which the blood clot is broken down. Accumulation of fibrin in blood vessels often increases thrombosis, resulting in myocardial infarction and other serious cardiovascular diseases. According to the report by World Health Organization, 17 million people die of cardiovascular disease per year. Fibrinolytic enzymes are considered as most effective drugs in treatment of thrombosis. Based on their mode of action, fibrinolytic (thrombolytic) agents are classified into two types. One is a plasminogen activator, such as tissue-type plasminogen activator (t-PA) and urokinase which activate plasminogen and lead to systemic lysis of fibrin. The other one is a plasmin-like protease that degrades the fibrin clot directly. The fibrinolytic agents available today for clinical use are mostly plasminogen activators, such as tissue-type plasminogen activator, urokinase are widely used but these agents display low specificity to fibrin, expensive and cause undesirable side effects. To overcome this constraint, fibrinolytic enzymes from microorganisms have been explored. Usually the capacity of a wild strain to produce the biologically interesting product is low. Thus, it is essential to improve the strain continuously to make the fermentation process economically successful [1]. Strain improvement is an essential step in the development of bioprocesses. Classical strain improvement based on sequential random mutagenesis and screening is the leading method for the development of industrial microorganisms [2]. Strain improvement techniques are not only used to All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP, www.ttp.net. (ID: 14.139.161.1-23/11/12,06:46:30)
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increase the product yield or specific production yield but also used to increase the strain stability, resistance to phage infection, response to dissolved oxygen tolerance, production of foam and the morphological form of the organism. The present study aims to mutate the Streptomyces venezuelae through random mutagenesis approach using UV irradiation and EMS mutagens. The mutated strains were employed for the production of fibrinolytic enzyme. Experimental procedure: The fibrinolytic activity of Streptomyces venezuelae was screened by blood (fibrin) clot degradation. Streptomyces venezuelae was exposed to UV radiation and EMS for different time intervals (0-10 min) for strain improvement and incubated at 37 °C for 2 days and their survival percentage was calculated. The plates which showed least survival percentage were selected and their fibrinlolytic activities were tested by fibrinolytic assay [3]. The mutant which showed higher fibrinolytic activity was selected for further studies. The growth of mutant and wild strain was studied at 37 °C and pH 7.2. The effect of temperature and pH on biomass production and fibrinolytic activity was studied by incubating strain at different temperature (20 °C – 60 °C) and medium was adjusted to different pH values (4-10). Results and Discussion: Screening of Fibrinolytic Activity. Blood clot (Fibrin) degradation by Streptomyces venezuelae was confirmed by the colonies formed around the blood clot which is shown in Fig.1.A. Hence it was inferred that the organism Streptomyces venezuelae can effectively degrade the blood clot (Fibrin). This indicates that Streptomyces venezuelae can able to produce fibrinolytic enzymes which degrades fibrin.
A
B
Fig. 1 Screening of Fibrinolytic Activity A. Fibrin clot with Streptomyces venezuelae B. Control (Fibrin clot without inoculation of Streptomyces venezuelae) Response analyses of UV and Chemical mutagenesis. The dose–response analysis for the UV and EMS of Streptomyces venezuelae was done and shown in Fig. 2. The increased period of exposure from the UV source and EMS and the strain to be mutated is directly correlated with the mortality rate [4]. Survival capacity of the organism was decreased with an increase in exposure time of UV and EMS and this was due to lethal DNA damage by mutagenesis. The least survival of 1.2 % and 0.2 % were observed at an exposure period of 10 min for UV and EMS respectively. EMS was considered to be effective mutagen than UV irradiation in Streptomyces venezuelae. This is because of the presence of high G- C content in Streptomyces species as reported by Felczak et al., 1999 [5].
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Fig. 2 Effect of EMS and UV treatment on percent survival of Streptomyces venezuelae Selection of Mutants. The positive mutants were selected on the basis of fibrinolytic enzyme production is shown in Fig. 3. All 7 colonies those obtained through UV mutation showed lower fibrinolytic activity than the wild strain. Among 3 colonies those obtained from EMS mutation, two of them showed higher fibrinolytic activity than the wild strain. Therefore EMS mutated colony which showed higher fibrinolytic activity than the wild strain was selected for further studies. The similar result was reported by Jaivel et al., 2010 [6] for the production of lovastatin using mutant Aspergillus terrus. Fibrinolytic activity (FU/ml)
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Fig. 3 Effect of mutation (UV and EMS) on Fibrinolytic enzyme production
Growth Analyses of Mutant and Wild strain. The growth pattern of the organism was measured on dry weight basis. The growth curve of the wild and mutant strain obtained by their cultivation from 0 to 96 h is shown in Fig. 4. The growth of mutant was faster than the wild strain. The mutant strain showed improved growth pattern compared to the wild strain. The maximum growth was observed at third day (76 h).
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Fig. 4 Growth curve of Wild strain and Mutant strain Effect of Temperature on Biomass and Fibrinolytic activity. The temperature of the environment directly affects the enzyme activity and growth of cells; every species has an ideal temperature for growth that is influenced by its physiology [7]. The effect of temperature on biomass and fibrinolytic activity are shown in Fig.5. It can be seen that maximum biomass production and fibrinolytic activity of wild strains was attained at 40 °C and its biomass production and fibrinolytic activity completely lost above 40 °C as reported by Kim et al [8]. For mutant strains, it can be seen that maximum production and activity was observed at 40 °C. Above 40 °C, biomass production and fibrinolytic activity decreases by increasing temperature but still stable up to 50 °C. This indicates that mutant strain was found to exhibit a good resistance to temperature variations. 12
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Effect of pH on Biomass and Fibrinolytic activity. The fibrinolytic enzymes belong to proteases are generally active at neutral and alkaline pH, with an optimum between pH 8.0 and 10 [9]. The effect of pH on biomass production and fibrinolytic activity of wild and mutant strains is shown in Fig. 7. The stability of the fibrinolytic enzyme activity from the mutant strain toward different pH was much higher than that obtained from the wild. The maximum activity of the fibrinolytic enzyme of wild and mutant strain was at pH 8 and it decreases by increasing the pH value as reported by Raafat et al [10].
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Fig.7 Effect of pH on A. Biomass and B. Fibrinolytic activity Conclusion Mutagenesis using Ethyl methane sulfonate was found to be effective than UV irradiation in case of Streptomyces venezuelae. The mutants obtained were screened based on their fibrinolytic activity and best mutant was selected. The mutant showed improved growth and enzyme yield compared than wild strain. Mutation was found to improve the stability of the Streptomyces venezuelae hence it has been concluded that the enzyme produced by mutant would show extreme tolerance to a broad range of pH and temperature. Moreover use of mutants for large scale bioprocess enhances the yield and also it would favor the utilization of industrial wastes as production medium which is currently under investigation. Refererences [1] K. Sreedevi, J. VenkateswaraRao, N. Lakshmi, M. Fareedullah: J. Microbiol. Biotech. Res. Vol. 1(2) (2011), p.96-100. [2] J. Gong, H. Zheng, Z. Wu, T. Chen, X. Zhao: Biotechnol. Advances Vol. 27 (2009), p. 996– 1005 [3] X.Wei, M. Luo, L. Xu, Y. Zhang, X. Lin, P. Kong, H. Liu: J. Agric. Food Chem. Vol.59 (2011), p.3957–3963. [4] K.P. Gopinath, S. Murugesan, J. Abraham, K. Muthukumar: Bioresource Technol. Vol. 100 (2009), p. 6295–6300 [5]M. Felczak, A. Bebenek, I. Pietrzykowska: Mutagenesis, Vol. 14 (1999), p. 295–300. [6] N. Jaivel, P. Marimuthu: Int. J. of Engg. Sci. and Technol., Vol. 2(7) (2010), p. 2612-2615. [7] M. L. Shuler, F. Kargi: Bioprocess engineering. New Jersey: Prentice-Hall Inc (1992). [8] H.K .Kim, G.T. Kim, D.K. Kim, W.A. Choi, S.H Park, Y.K. Jeong, I.S Kong: J. Fermentation and Bioengg. Vol.84 (1997), p. 307-312. [9] S. H. Kim, N. S. Choi: Biosci., Biotechnol., Biochem. Vol. 64 (2000), p.1722–1725. [10] A.I. Raafata, E. Araby, S. Lotfya: Carbohydrate Polymers Vol. 87 (2011), p. 1369– 1374.
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Strain Improvement of Streptomyces venezuelae for Enhanced Fibrinolytic Enzyme Production 10.4028/www.scientific.net/AMR.584.440
