Screening of Amylase and Cellulase Enzymes from Marine Actinomycetes
K. Viswanathan*, L. Jeyanthi Rebecca
Department of Industrial Biotechnology, Bharath Institute of Higher Education and Research (BIHER),
Chennai, Tamilnadu, India
Actinomycetes have an important role in the Industrial enzymes, Antibiotics, vitamins and organic acids. We have isolated 114 actinomycetes strains from Chennai coastal region by using starch casein agar plate method. Out of these 114 actinomycetes strains, 13 actinomycetes strains showed amylase activity by starch agar plate method and 12 strains showed cellulase activity by cellulase agar plate method, which was indicated by clear zone of dark blue color formation for amylase activity and dark yellow color formation for cellulase activity. Based on the maximum amylase and cellulase producing strains MB23 and BN08 was found to be a potential strains for further investigation.
Actinomycetes are gram positive bacteria, showing a filamentous growth like fungi. Actinomycetes are group of microorganism which is been used industrially for preparation antibiotics and enzymes. The microbial enzyme has been extensively used in Industries for clean, eco-friendly and effective biotechnology process in various fields. It is been recently identified that marine microorganisms has a rich source for isolation of Industrial enzymes. In the production of antibiotics such as streptomycin, gentamicin, fifamycin, erythromycin and enzymes like cellulose, amylase and lipase from the metabolites of Actinomycetes.
Marine microbial organisms produce many enzymes and many organic complexes; it is present in coastal sediments and soil. Enzymes like Protease, cellulases, amylase, gelatinase, pectinases and ureases are obtained from the coastal sediments. Actinomycetes produce different enzymes and it appears to be a rich source for the production of industrially enzymes.
In starch industries and syrup preparation from oligosaccharide and monosaccharide, enzyme amylase plays a significant role. Amylase enzyme needed for resizing the cloth materials. Cellulase enzyme system which completely controls the enzymatic hydrolysis process comprises three classes of soluble extracellular enzymes. Cellulase enzymes system actively involved in color extraction from juices, detergents influencing the color brightness and softening, biostoning of jeans, pretreatment of industrial wastes and biomass and improve nutritional quality of forage. Different sources were examined for actinomycetes strains[7-9].
The present work focuses on the isolation of amylase and cellulose producing actinomycetes strains from Chennai coastal region for the application of industrial purpose.
MATERIALS AND METHODS:
The different marine soil samples were collected from Chennai coastal region, Tamilnadu.
Isolation of actinomycetes:
Actinomycetes strains were isolated by starch casein agar plate method. The subculture was stored at 4˚C for further investigation.
Screening of Amylase enzyme from Actinomycetes strains:
Actinomycetes strains were tested for amylase activity by starch agar plate method containing: Peptone 5g/l, Yeast extract 1.5g/l, Beef extract 1.5g/l, Nacl 5g/l, Soluble starch 10g/l and agar 20g/l. The media was sterilized and plated on sterile petriplates. The test actinomycetes strains are then streaked on the respective substrate starch agar medium and the plates are incubated at a room temperature for 48hrs. After that gram’s iodine solution was spread on the plate and left for 5 min. The actinomycetes strain secretes amylase, which produce zone of clearance or decolonization against the blue color back ground. The subculture was stored at 4˚C for further investigation.
Screening of Cellulase enzyme from Actinomycetes strains:
Actinomycetes strains were tested for Cellulose activity by Cellulose agar plate method containing: NaNo3 2g/l, K2HPO4 1g/l, Mgso4 0.05g/l, Kcl 0.05g/l, FeSO4 0.01g/l, Carboxy methyl cellulose 10g/l and agar 20g/l. The media was sterilized and plated on sterile petriplates. The test actinomycetes strains are then streaked on the respective substrate Cellulose agar medium and the plates are incubated at a room temperature for 48hrs. After incubation, flood the plates with 0.3% Congo red solution for 10 min followed with de-staining solution for 10 min. Cellulase production is visualized by a translucent zone around the stains. The subculture was stored at 4˚C for further investigation.
Assay of amylase and Cellulase enzymes:
Amylase and cellulose were identified by using Spectrophotometer assay method [11, 12]. The activity of amylase and cellulose were assayed by incubating 0.5 ml of crude enzyme with 0.5 ml of starch soluble (1%, w/v) and 0.5 ml CMC (1%, w/v) prepared in 0.1 M sodium phosphate buffer at pH 7.0. After incubation at 37˚C for 60 min the reaction was stopped by the addition of 2 ml Dinitrosalicylic (DNS) reagent and measurement was done for absorbance of amylase and Cellulase activity at 550 nm in spectrophotometer and expressed in units.
RESULTS AND DISCUSSION:
Isolation of marine actinomycetes:
In the present study, 114 actinomycetes strains were isolated from marine soil samples at Chennai costal region. Amylase and Cellulase enzyme producing actinomycetes strains were streaked on the starch agar plate method.
Screening of Amylase enzyme:
Amylase screening was carried out by starch agar plate method. 13 actinomycetes strains showed amylase enzyme production which was identified by formation of clear zone against dark blue plates respectively (Figure 1).
Figure: 1 Amylase enzyme producing actinomycetes strains
Strains BN17 and MB33 Strains MB13, MB44 and MB51 Strains BN09 and MB23
Screening of Cellulase enzyme:
Cellulase screening was carried out by starch agar plate method. 12 actinomycetes strains showed cellulase enzyme production which was identified by formation of clear zone against dark brown plates respectively (Figure 2).
Figure: 2 Cellulase Enzyme producing actinomycetes strains
BN08, BN37 and BN01 MB15, MB40, MB52 and MB10 BN21 NK06
Amylase and Cellulase activity:
Enzyme activity was tested by spectrophotometric assay, 13 actinomycetes strains produced amylase activity and 12 actinomycetes strains produced cellulase activity. From the above results, the actinomycetes strain MB23 and BN08 showed maximum amylase and cellulase activity (Figure 3 and Figure 4).
Figure: 3 Amylase Enzyme Activity
Figure: 3 Cellulase Enzyme Activity
Amylase, cellulase and protease are the industrially important enzymes having wider applications in industries like leather, pharmaceutical, laundry, fermentation, food, textile, paper and waste processing. The target organism and enzymes should be tolerable for production process at different variables for effective industrial enzymes production. In this study most of the actinomycetes strains showed more number of enzyme activities. In the present study the isolates showed a potential to produce a wide range of enzymes from the natural selection of microorganisms in order to survive in the competing environment. Earlier studies have also proved that marine bacteria including marine actinomycetes are exhibiting diverse pattern in secreting extracellular enzymes[14-15]. In the present work, 114 actinomycetes isolates were isolated from different soil samples in Chennai coastal regions and they are identified by colony morphology and Gram staining. Most of researcher’s in earlier investigations have also reported that actinomycetes from soil and water bodies possess high number of enzymatically active actinomycetes. Amylase activity in actinomycetes and other microorganism has been reported[16-23] and Cellulase activity in microorganism has been reported [24-29]. Amylase and cellulase enzymes are important enzymes in the food and pharmaceutical industries. In current study the actinomycetes strain MB23 and BN08 is selected for enzyme production. The enzyme activities from the actinomycetes are differing from soil to soil and isolate to isolate depending upon the growth and physical condition of the isolates.
The present study highlighted that the potential amylase and cellulase activity from actinomycetes strains (MB23 and BN08) which can be used for the production of industrial products. Further investigations are required to make the production of amylase and cellulase enzyme from the marine actinomycetes strains.
CONFLICT OF INTEREST STATEMENT:
We declare that we have no conflict of interest.
The authors are grateful to Bharath Institute of Higher Education and Research for their encouraging support and our special thanks to Armats Biotek Training and Research Institute for providing us with the laboratory facilities required for this research work.
1. Adinarayana K, Ellaiah P. Response surface optimization of the critical medium component for the production of alkaline protease by a newly isolated Bacillus sp. Indian J.Pharm.Sci. Vo.l5, 2002: 272-278.
2. Gulve R.M, Deshmukh A.M. Antimicrobial activity of the marine Actinomycetes. Inter. Multidiscipl. Res. J. vol.2 (3); 2012: 16-22.
3. Suneetha V.V, Laksmi V.V. Actinomycetes as a source of soil enzymes. Enzyme. Microbial. Tech. vol. 6, 2004: 31-36.
4. Tonkova A. Microbial starch converting enzymes of the amylase family. In: Ray, C.R. and O.P. words. Mirobial biotech. Horticult. vol.1, 2006: 421- 472.
5. Zhou L, Yeung K, Yuen C. Combined cellulase and wrinkle free treatment on cotton fabric. J. Dong. Hua. Univ. vol.18, 2001: 11–15.
6. Klemm D, Heublein B, Fink H.P, Bohn A. Cellulose: fascinating biopolymer and sustainable raw material. Angew Chemical International Education. Vol.44, 2005: 3358–3393.
7. Souvik Paul, Kavitha S, Vimala R. isolation of Actinomycetes and Optimization of Process Parameters for Antimicrobial Activity. Research J. Pharm. and Tech 2016, 9(11): 1913-1921.
8. Vishnu K, Vaze. Biodegradation of Pesticide Cypermethrin by Phosphatase and Esterase Enzymes Produced by Actinomycetes. Research J. Pharm. and Tech. 2017, 10(2): 445-448.
9. Sujatha J, Suriya P, Rajeshkumar S. Biosynthesis and Characterization of silver Nanoparticles by Actinomycetes isolated from Agriculture field and its application on Antimicrobial activity. Research J. Pharm. and Tech. 2017, 10(6): 1963-1968.
10. Viswanathan K, Jeyanthi Rebecca L. Antimicrobial Activity of Marine Actinomycetes against Human Pathogenic Bacteria. J. Pharm. Sci. and Res. Vol.9 (11), 2017: 2086-2088.
11. Bernfeld P, Amylase α and β methods. Enzymology. Vol.1, 1985: 149-158.
12. Ghose TK. Measurement of Cellulase activities. Pure and applied chemistry. vol.52, 1987: 257-268.
13. Arjit Das E.B, Mahdi K, Prashanthi S, Sandeep and Sourav B. Enzymatic screening and random amplified polymorphic DNA finger printing of soil Streptomycetes isolated from Wayanad District in Kerala, India. J. Bio. Sci. Vol.12, 2012: 43-50.
14. Ramesh S, Mathivanan N. Screening of marine actinomycetes isolated from the Bay of Bengal, India for antimicrobial activity and industrial enzymes. World J. Microbiol. Biotechnol. Vol. 25, 2009: 2103-2111.
15. Jayaprakashvel M, Ramesh S, Sownthararajan K, Mathivanan N. Marine bacterial population in seawater of Bay of Bengal: Their adoptive characteristics and enzyme production. J. Biotechnol, 2008: 136S-S527.
16. Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R. Advances in microbial amylases. Biotechnol Appl Biochem. Vol.31 (2), 2000: 135–152.
17. Chakraborty S, Raut G, Khopade A, Mahadik K, Kokare C. Study on calcium ion independent amylase from haloalkaliphilic marine Streptomyces strain A3. Ind J Biotech. Vol.11, 2012: 427–437.
18. Rajagopalan G, Krishnan C. Alpha-amylase production from catabolite derepressed Bacillus subtilis KCC103 utilizing sugarcane bagasse hydrolysate. Biores Technol, Vo.99, 2008: 3044–3050.
19. Reddy NS, Nimmagadda A, Sambasiva Rao KRS. An overview of the microbial α-amylase family. Afr. J. Biotechnol. Vol.2, 2003: 645–648.
20. Syed DG, Agasar D, Pandey A. Production and partial purification of amylase from a novel isolate Streptomyces gulbargensis. J Ind Microbiol Biotech. Vol.36 (2), 2009: 189–194.
21. Ashwini K, Shanmugamsampath Kuma. Partial-purification of Alpha-Amylase from Marine Streptomyces gancidicus-ASD-KT852565. Research J. Pharm. and Tech. 2016, 9(6):731-734.
22. Shamalee Nitin Narappanawar, Shruti Sunil Ranade, Swarnita Kandpal, Veena S, K.V. Bhaskara Rao. Isolation of α-Amylase Producing Bacteria from Naini lake and Assessment of their Anti-Oxidant and anti-Fungal Properties. Research J. Pharm. and Tech. 2016, 9(3):245-250.
23. Subbaiya R, Priyanka S, Suresh D, Masilamani Selvam M, Balachandar R, Chozhavendhan S.. Application of Response Surface Methodology in Process Parameter Optimization of Media for Production of Amylase. Research J. Pharm. and Tech 2018, 11(12): 5273-5281.
24. Jang HD and Chenks. Production and characterisation of thermostable cellulase from Streptomyces transformant T3-World. J. Microbiol. Biotechnol. Vol.19, 2003: 263-268.
25. Yassien MAM, Jiman-Fatani AAM, Asfour HZ. Production, purification and characterization of cellulase from Streptomyces sp. Afri Journal of Microbio. Vol.4, 2014: 348-354.
26. Arunachalam R, Wesley EG, George J, Annadurai G. Novel approaches for Identification of streptomyces nobortoensis TBGH-V20 with cellulase production. Curr. Res, Bacteriol, Vol.3 (1), 2010: 15- 26.
27. Da Vinha FNM, Gravina-Oliveira MP, Franco MN, Macrae A, Da Silva Bon EP, Nascimento HZ. Cellulase production by Streptomyces viridobrunneus SCPE- 09 using lignocellulosic biomass as inducer substrate. Appl Biochem Biotech. Vol.164 (3), 2011: 256–67.
28. Bharathi V, Jannathul Firdous, Resni Mona, Noorzaid Muhamad. Efficacy of Cellulose Degrading Bacteria from Soil in Production of Cellulase from Corn Waste. Research J. Pharm. and Tech 2018, 11(9): 4024-4028.
29. Utharalakshmi N, Ganesh Kumar A, Narendrakumar G. Optimization of Cellulase Producing Aspergillus flavus SB4 by Solid State Fermentation using Response Surface Methodology (RSM)-CCD. Research J. Pharm. and Tech 2015, 8(4): 349-354.
Accepted on 20.05.2019 © RJPT All right reserved
Research J. Pharm. and Tech 2019; 12(8): 3787-3790.