Indian Journal of Natural Products and Resources Vol. 2(2), June 2011, pp. 170-173
Characterization of extracellular amylase enzyme produced by Aspergillus flavus MV5 isolated from mangrove sediment B Valentin Bhimba*, S Yeswanth and B Edhaya Naveena Department of Biotechnology, Sathyabama University, Rajiv Gandhi Salai, Chennai-600 119, Tamil Nadu, India Received 12 May 2010; Accepted 24 March 2011 Mangroves provide a unique ecological niche to different microbes which play various roles in nutrient recycling as well as various environmental activities. Analysis of microbial biodiversity from these ecosystems will help in isolating and identifying new and potential microorganisms having high specificity for various applications. Mangroves sediment soils are a resource of fungi providing an end number of enzymes that finds their use in industrial processes. This study indicates the extracellular production of amylase by Aspergillus flavus MV5 was confirmed on GYP agar plates. Further maximum enzyme activity was optimized and the DNA of fungal strains were isolated to sequence the ITS region of 5.8s rRNA with an ITS primer. The novelty of the strain was checked by a BLAST analysis for submission to GenBank. Keywords: Amylase, Aspergillus flavus, Extracellular enzyme production, Mangrove sediment. IPC code; Int. cl. (2011.01) A61K 38/43, A23L 1/00, C12N 9/30
Introduction Mangrove areas or mangals are home to a group of fungi. These organisms are vitally important for nutrient cycling in these habitats and are able to synthesis all the necessary enzymes. Amylases based on their way of breaking down starch molecules are classified into α-amylase, β-amylase and amyloglucosidase (AMG). A very important field in which enzymes have proved their worth over the last 15 to 20 years is the starch industry. Use of fungal amylase in the manufacture of specific types of syrup where conventional acid hydrolysis cannot play a good role, dates back to the 1950s. So, in addition to biotechnology and biochemistry, areas of research in fungi have boosted knowledge of enzymes for industries as well as research. In recent years the potential of using microorganisms as biotechnological sources of industrially relevant enzymes have stimulated interest in the exploration of extracellular enzymatic activity in several microorganisms1-5. Amylases are important enzymes employed in the starch processing industries for the hydrolysis of polysaccharides such as starch into simple sugar constituents1. Although amylases can be obtained from several sources, such as plants and animals, the enzymes from microbial sources —————— *Correspondent author, E-mail:
[email protected]
generally meet industrial demand2. Microbial amylases have successfully replaced chemical hydrolysis of starch in starch processing industries. Enzyme activity, we all know is controlled by many factors, including environment, enzyme inhibitors and few more. The present study focus on the extracellular amylase produced by the fungi Aspergillus flavus at different pH and substrate concentration. The results indicate the maximum enzyme production at pH 6.8. So this study can further be extended with different substrates to find optimum conditions under which the fungal amylase is at its best in terms of stability to yield the desired results or products. Materials and Methods Collection of sample
The fungal strains used were isolated from the mangrove sediment soil using Sabourauds Agar with the supplementation of 50% sterile seawater at 28°C in petri plates. Confirmation of amylase producing strain
Amylase producing fungi was assessed by growing on glucose yeast extract peptone (GYP) agar medium (glucose, 1g; yeast extract, 0.1g; peptone, 0.5 g; agar, 16 g; distilled water, 1000ml; pH 6) with 2% soluble starch. After incubation for 5 days, the plates were
BHIMBA et al: EXTRACELLULAR AMYLASE PRODUCED BY ASPERGILLUS FLAVUS FROM MANGROVE SEDIMENT
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flooded with 1% iodine in 2% potassium iodide. The clear zone surrounding the colony reveals the presence of amylase activity.
The genomic DNA was isolated and the ITS region of 5.8s RNA was amplified using primers ITS1 and ITS5 and sequenced using automated sequencer6.
Production of alkaline amylase
Results and Discussion Currently, two major classes of α-amylases are commercially produced through microbial fermentation. Unlike the bacterial α-amylase that attacks the alpha 1, 4 bonds, the fungal α-amylase attacks the second linkage from the non-reducing terminals (i.e. C4 end) of the straight segment, resulting in two glucose units at a time. Fungal amylase and amyloglucosidase, a type of enzyme mixture amylase be used for the production of corn syrup and conversion of cereal mashes to sugars in brewing. In the present study, primary screening for the enzyme amylase was carried out by the starch hydrolysis method. Among the six isolates, one fungal isolates exhibited higher amylolytic activity in starch agar medium and was selected for further studies. Temperature and pH are most important factors which does influence enzyme activity to a noticeable extent. Among the fungi, most amylase production studies have been done with mesophilic fungi within the temperature range of 25-37°C7,8. In the present studies with regard to temperature stability indicated a general increase in its stability with time (5-50 min). The enzyme retained its activity for 30 to 40 min thus proving to be thermo-stable. It showed the highest stability at the 50th minute and least stability at the 5th minute for all the species (Fig. 1) Amylase production at optimum level has been reported between 50-55°C for the thermophilic fungal cultures such as Talaromyces emersonii, Thermomonospora fusca and Thermomyces lanuginosus9,10. The important factors that determine the growth and morphology of microorganisms is pH as they are sensitive to the concentration of hydrogen ions present in the medium. Earlier studies have revealed that fungi required slightly acidic pH and bacteria required neutral pH for optimum growth. pH is known to affect the synthesis and secretion of α-amylase just like its stability. Aspergillus spp. such as A. oryzae, A. ficuum and A. niger were found to give significant yields of α-amylase at pH=5.0-6.0 in SmF 11-13. This is another clear indication that the enzyme can be employed for a wide range of pH change from slightly acidic to alkaline and vice versa. The maximum enzyme production was observed at pH 6.8 in the
The culture of amylase producing strain was inoculated in 100ml of the production medium(g/l) in flasks (KH2P04-1.4g; NH4NO3-10g; KCl-0.5g; MgS047H20-0.1g; FeS04 ·7H2O-0.01g; Soluble starch 20g; pH to 6.5) that were incubated in a shaker for 72 h at 27°C. Extraction of amylase from fungi
The fungal mycelium was removed from the enzyme production medium by filtering through Whattman number 1 filter paper. The filtrate contains the crude amylase. Enzyme assay for amylase at different pH and substrate concentration and incubation time
In 1 ml of enzyme extract added 1ml of 1% soluble starch in citrate-phosphate buffer (pH 6.5) and incubated in a water bath at 40°C for 30 minutes. Blank consisting of 2ml of the enzyme extract that was boiled for 20 min (boiling inactivates the enzyme) and starch solution was added and treated with the same reagent as the experimental tubes. The reaction was stopped by adding 2 ml of DNS reagent (1.0 g of 3, 5, di nitrosalicyclic acid, 20 ml of NaOH and 30 g of sodium potassium tartarate in 100 ml) and boiled for 5 min at 80°C. After cooling 20 ml of distilled water was added and the absorbance was read at 540 nm. Enzyme activity was checked at different pH (6.2,6.8,7.5,8.0) and substrate concentration (0.2,0.4,0.6,0.8,1,1.2,1.4 ml) and incubation time (5,10,20,30,40,50 min). Glucose standard
10 mg of glucose was dissolved in 10 ml of distilled water and various concentrations were taken for standard graph. The amount of glucose was calculated as mg/ml in the test sample and the standard formula is: Absorbence of the test Absorbence of standard
× Concentration of standard
Molecular characterization and identification of elite fungi by ITS sequencing
The fungi were grown in culture in potato dextrose broth at room temperature in the dark for 48 to 72 h.
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Fig.1 Effect of temperature on amylase production
present study. The crude enzyme extract was allowed to react with different substrate concentrations (0.2 to 1.4) and maximum activity was found with 1% starch as the substrate (Fig. 2). Molecular characterization and identification of elite fungi
The ITS region is now perhaps the most widely sequenced DNA region in fungi. It has typically been most useful for molecular systematics at the species level, and even within species (e.g., to identify geographic races). In the present study the DNA was isolated from the fungal strain and the ITS region
Fig. 2 Effect of substrate concentration on different pH for production of amylase
BHIMBA et al: EXTRACELLULAR AMYLASE PRODUCED BY ASPERGILLUS FLAVUS FROM MANGROVE SEDIMENT
of 5.8s rRNA was amplified using specific primers ITS1 and ITS4 and sequence was determined using automated sequencers. Blast search sequence similarity was found against the existing non redundant nucleotide sequence database thus, identifying the fungi as Aspergillus flavus. The percentage of similarity between the fungi and database suggests it as novel strain and named as Aspergillus flavus MV5 and made publically available in GenBank with an assigned accession number GU815344 (http://www.ncbi.nlm.nih.gov/nuccore/291195943). The 19th century witnessed an enzyme revolution as they proved their role in starch processing, pharmaceutical and feed enzyme industries. More appropriate exploitation of enzymes in the treatment of agro industrial wastes is another domain demanding the role of enzymes. The results furthermore give hope for a pharmaceutical composition wherein bonding amylase with another one auxillary material will be a good option to treat digestive disorders. Conclusion Mangrove ecosystems provide shelter and nurturing sites for many microorganisms and with every marine researcher aware of their role to accomplish an environment friendly technological development, search for diverse endophytic fungi for novel enzymes will sure remain a mainstream area of research. In addition to temperature, pH, effects of substrate concentration and inhibitor concentration on the kinetics of amylase-catalyzed reaction can be studied. Another study following these results could be the comparison of the action of amylase preparations from mangrove fungi with that from human saliva thereby determining the breakdown and delivery of nutrients in the food we eat. Acknowledgement The authors place on record their gratitude to the Department of Biotechnology, Govt. of India for sanctioning this study under the student project scheme for the year 2009-2010 as approved by the Tamil Nadu Sate Council for Science and Technology (TNSCST), an autonomous body under Govt. of Tamil Nadu.
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