Production of Extra Cellular α-amylase using Bacillus megaterium isolated from White Mangrove (Avicennia marina)
Bacillus megaterium isolated from leaves of Avicennia marina (Forssk.) was screened for their ability to produce α-amylase, studied in submerged fermentation by using an Adlof-Kuhner orbital shaker. The levels of amylase production detected in culture supernatants varied greatly with the type of carbon source used such as starch, lactose and glucose. Effect of different nitrogen sources revealed that peptone increase the enzyme yield. The enzyme activity increased between 1.5 and 3 g L-1 of yeast extract concentration and optimal concentration of peptone for the production of amylase was detected as 6 g L-1. The maximum enzyme activity was obtained under optimum conditions of an incubation period of 72 h, an incubation temperature of 35°C and pH of 6.5.
November 02, 2010; Accepted: January 11, 2011;
Published: February 07, 2011
Enzymes are among the most important products acquired for human needs in the
areas of industrial, environmental and food biotechnology through microbial
sources. Alpha amylase is a hydrolytic enzyme and in recent years, interest
in its microbial production has increased dramatically due to its wide spread
use in food, textile, baking and detergent industries (Asgher
et al., 2007). α-amylase has been derived from several fungi,
yeast, bacteria and actinomycetes; however enzymes from fungi and bacteria sources
have dominated applications in industrial sectors (Pandey
et al., 2000). The major advantage of using microorganisms for production
of amylases is an economical bulk production capacity and microbes are also
easy to obtain enzymes of desired characteristics. The production of amylases
by fermentation has been thoroughly investigated and its affected by a
variety of physiochemical factors. Spectrum of applications of α-amylase
has widened in many sectors such as clinical, medicinal and analytical chemistry.
Besides their use in starch saccharification, they also find applications in
baking, brewing, detergent, textile, paper and distilling industry (Ramachandran
et al., 1978). Bacillus megaterium has been ideal for studies
of cell structure, protein localization, sporulation, and membranes (McCool
and Cannon, 2001).
Mangroves are known to stabilise coastal sediments through their aboveground
aerial root complex. Mangroves inhabit intertidal zones with high salinity (Liang
et al., 2008) and can tolerate a wide range of salinities under natural
conditions. The mangroves adaptations to landward depression and seaward
depression localized topographic differences were important in view of changes
in intertidal hydrology, the latter being linked to changes in topography. The
grey mangrove Avicennia marina has the ability to adapt its pneumatophores
to micro-topographical irregularities in the otherwise regularly sloping intertidal
zone (Dahdouh-Guebas et al., 2007). Avicennia
marina (Forssk.) Vierh. is the most common species planted for mangrove
restoration and is highly salt tolerant (Khan and Aziz,
The present investigation reported the optimization of physio-chemical factors such as pH, temperature, carbon and nitrogen sources for the production of amylase using Bacillus megaterium first time isolated from leaves of white mangrove Avicennia marina under submerged fermentation.
MATERIALS AND METHODS
Isolation and screening of micro organism: The rod shaped, mainly
aerobic spore-forming Gram positive bacteria Bacillus megaterium was isolated
from the aerial part of leaves of white mangrove Avicennia marina in the Vellar
estuary of Southeast coast of Tamil Nadu (11°29 N Lat, 79°46
E Long) India during the month of August 2010. The primary screening of the
strain was done by starch agar plate method (Shaw et
al., 1999). The selection of thermophilic bacteria was done by growing
them on a medium containing 2% Bactotryptone, 1% Bacto yeast extract, 1% NaCl
and 2% agar at pH 7.0. The screening of bacteria capable of producing starch
digesting enzymes was done by growing them on medium containing 1% soluble starch,
0.2% yeast extract, 0.5% peptone, 0.05% MgSO4, 0.05% NaCl, 0.015%
CaCl2 and 2% agar at pH 7.0. The plates were stained with Grams
iodine solution (2% 2I and 0.2% potassium iodide) and largest halo-forming zone
was considered as the most promising strain. Later microbiology of the isolated
strain was determine according to the methods describe in Barges manual of systematic
bacteriology (Seneath et al., 1986) and strain maintained
on nutrient agar slant at 4°C for further studies. All the analytical chemicals
and media components were purchased from Hi-media (Mumbai) and Sigma chemicals
Enzyme production medium: The composition of production medium used was soluble starch 50 g, yeast extract 0.5 g, KH2PO4 10 g, (NH2)2SO4 10.5 g, MgSO4 0.3 g, CaCl2 0.5 g, FeSO4 0.013 g, MnSO4 0.7H2O,0.004 g, ZnSO4 .2H2O 0.004 g, CaCl2 0.0067 g and 1000 mL of distilled water. The pH was adjusted to 6.8 and the media were sterilized in an autoclave for 15 min at 121°C. The media were inoculated with a loop full of growing culture of Bacillus megaterrium and then incubated at 32°C in an orbital shaker set at 100 rpm for 24 h. The media were centrifuged at 8000 g, for 10 min at 4°C to obtain cell free filtrate.
Enzyme assay: Amylase assay was made by using a reaction mixture 4 mL
consisted of 1 of enzyme solution and 2 mL of soluble starch in phosphate buffer,
pH 6.8 ( Wood and Bhat, 1988). The mixture was incubated
for 10 min at 32°C. Level of reducing sugars was determined by Dinitrosalicylate
method (Miller, 1959) and is expressed in units (one
unit is the amount of enzyme which releases 1 μmole glucose.
Optimization of culture conditions: The factors such as temperature,
pH, source of carbon and nitrogen affecting production of amylase were optimized
by varying parameters one at a time. The experiments were conducted in 200 mL
of Erlenmeyer flask containing production medium. The optimum pH was determined
by adjusting pH of the media at a range from 4.5-7.0. The media was incubated
at 26-36°C for 24-120 h to check preferred temperature and incubation time
for enzyme production. Similarly, the effect of various carbon sources viz.
(glucose, galactose, maltose, lactose and sucrose 1%) and nitrogen sources namely
peptone, beef extract, yeast extract and casein each at 0.5% were tested. Each
experiment was carried out in triplicate and their values were averaged.
RESULTS AND DISCUSSION
Isolation and screening of amylase producing microorganism: The production
of extra cellular α-amylases by submerged fermentation has been thoroughly
investigated and it is affected by a variety of physiochemical factors. Many
bacteria produce extra cellular amylase during the fermentation of starch. The
organism was isolated from the leaves of a white mangrove species by serial
dilution and screened by zone hydrolysis method and later identified as Bacillus
megaterium (Table 1) according to Bergeys manual.
The attached bacterial variations may be related to atmospheric and leaf temperature
and probable inhibiting substance released by the plant leaves. The isolation
of heterotrophic marine bacteria from the leaves of Avicennia marina
had also been made by Mahasneh (2002). B. megateium
was also capable of Polyhydroxybutarate accumulation up to 65% of the cell dry
weight when grown in a synthetic medium based on sucrose and ammonium feed (Sabra
and Abou Zaid, 2008). After 72 h of incubation, every 1 h interval, the
culture filtrate was analyzed for amylase activity. In addition Bacillus
megaterium was isolated and identified from the cassava dumpsites and used
for amylase production (Oyeleke and Oduwole, 2009).
Effect of pH and temperature: The pH of the growth medium plays an important
role in terms of inducing enzyme production. Similarly our study reported the
amylase production varied at different range of pH. The best pH for growth was
6.5 where as the optimum temperature was 32°C and strain was capable to
produce amylase by hydrolyzing starch. The activity of amylase was about 35%
higher at pH 6.5 than pH 5.0 (Fig. 1) and 30% higher at 35°C
than 40°C (Fig. 2). Some of the researchers previously
reported the optimum pH of amylase production from Bacillus sp. at 7
(Vidyalakshmi et al., 2009; Alamri,
2010) and 7.5-8.5 (Amoozegar et al., 2003).
Hence that, pH for the amylase production from Bacillus sp., ranges between
6.5-8.5. Although the optimum temperature for α-amylase production by Bacillus
megaterium and Bacillus sp. exhibited at 60°C (Takasaki,
1989) and 70°C (Asgher et al., 2007).
Moreover, the highest α-amylase activity produced by Bacillus megaterium
was achieved in our investigation at the temperature of 35°C and pH of 6.5.
||Biochemical characterization of isolated bacteria isolated
from auxiliary buds of white mangrove
|+: Positive; -: Negative
||Effect of pH of anylase production
||Effect of various range of temperature on anylase production
||Anylase production on various incubation period
Effect of time course on amylase production: When the culture was incubated
at 96 h, the maximum activity detected was 156 U mL-1. There was
a two fold increase in activity at 96 h incubation as compared to 24 h (Fig.
3). Extension beyond the optimum time course was generally accompanied by
a decrease in the growth rate and enzyme productivity, which gradually declined
after 96 h of incubation (Alamri, 2010). The short incubation
period for Bacillus sp. compared with other bacteria and fungi offers
the potential for inexpensive enzyme production (Bernfeld,
Effect of carbon sources: A number of carbon sources (1% w/v) were tested
in order to determine their effect on growth and α-amylase production.
Among the effect of various carbon sources maltose was the best to enhance the
amylase activity of 156 U mL-1 which was 7% higher than other carbon
sources are shown in Fig. 4. The similar observation had been
reported by Thomas et al. (1980). Growth and
enzyme production did not alter when potato and corn starch were used as carbon
sources. The same findings were reported by Oliveira et
al. (2007). They mentioned that the induction of α-amylase requires
substrates having α-1, 4 glucoside bond, including starch and maltose.
The biosynthesis of α-amylase in most species of the genus Bacillus
was represed by readily metabolizable substrates, especially glucose, by a mechanism
of catabolite repression (Lin et al., 1998).
||Anylase production on different carbon sources. 1: Glucose,
2: Galactose, 3: Lactose, 4: Maltose, 5: Sucrose, 6: Xylose
||Anylase production on different nitrogen sources. 1: Yeast
extract, 2: Neat extract, 3: Beef extract, 4: Peptone, 5: Casein
In addition Bacillus licheniforms, starch used as carbon source for
amylase production (Adeyanju et al., 2007). Besides,
Sarikaya and Gurgun (2000) reported that the highest
α-amylase yield obtained by the addition of Na-citrate and sucrose for
the strains of Bacillus subtilis and Bacillus amyloliquefaciens.
Among different carbon sources disaccharides are more suitable for amylase production
compared to polysaccharides.
Effect of nitrogen sources: Among the nitrogen sources, peptone was ideal to increase the enzyme activity of 140 U mL-1 which was about 10% higher than casein, meat and yeast (Fig. 5). The enzyme was susceptible to reagents that react with thiol groups and had an exo-action on starch yielding maltose with a p-anomeric configuration. It is concluded that the principal starch-hydrolysing enzyme from B. megaterium is a 1, 4-α-glucan maltohydrolase similar in its properties to other Bacillus, plant, and amylases.
The present study revealed white mangrove (Avicennia marina) leaves also a one of the habitation for microbial population. The production of amylase from Bacillus megaterium showed significant application for industrial enzyme production.
The authors are gratefully acknowledge to the Prof. Dr. T. Balasubramanian Dean and Prof. K. Kathiresan (source of Mangrore plant) Faculty of Marine Sciences, Annamalai University, Parangipettai, Tamil Nadu, India for providing all support during the study period.
Adeyanju, M.M., F.K. Agboola, B.O. Omafuvbe, O.H. Oyefuga and O.O. Adebawo, 2007. A thermostable extracellular alpha amylase from Bacillus licheniformis isolated from cassava steep water. Biotechnology, 6: 473-480.
Direct Link |
Alamri, S.A., 2010. Isolation phylogeny and characterization of new α- amylase producing thermophilic Bacillus sp. from the Jazan Region, Saudi Arabia. Int. J. Biotechnol. Biochem., 6: 537-547.
Direct Link |
Amoozegar, M.A., F. Malekzadeh and K.A. Malik, 2003. Production of amylase by newly isolated moderate halophile, Halobacillus sp. strain MA-2. J. Microbiol. Methods, 52: 353-359.
Asgher, M., M.J. Asad, S.U. Rehman and R.L. Legge, 2007. A thermostable alpha amylase from a moderately thermophilic Bacillus subtilis strain for starch processing. J. Food Eng., 79: 950-955.
Bernfeld, P., 1955. Enzymes of starch degradation and synthesis. Adv. Enzymol., 12: 379-428.
Dahdouh-Guebas, F., J.G. Cairo, R. de Bondt and N. Koedam, 2007. Pneumatophore height and density in relation to micro-topography in the grey mangrove Avicennia marina. Belg. J. Botany, 140: 213-221.
Direct Link |
Khan, M.A. and I. Aziz, 2001. Salinity tolerance in some mangrove species from Pakistan. Wetlands Ecol. Manage., 9: 219-223.
Direct Link |
Liang, S., R. Zhou, S. Dong and S. Shi, 2008. Adaptation to salinity in mangroves: Implication on the evolution of salt tolerance. Chin. Sci. Bull., 53: 1708-1715.
Lin, L.L., C.C. Chyau and W.H. Hsu, 1998. Production and properties of a raw starch degrading amylase from the thermophilic and alkaliphilic Bacillus sp. TS- 23. Biotechnol. Applied Biochem., 28: 61-68.
Direct Link |
Mahasneh, A.M., 2002. Heterotrophic marie bacteria attached to leaves of Avicennia marina L. along the qatari coast (Arabia Gulf). J. Biol. Sci., 2: 740-743.
McCool, G.J. and M.C. Cannon, 2001. Pha C and Pha R are required for polyhydroxy alkanoic acid synthase activity in Bacillus megaterium. J. Bacteriol., 183: 4235-4243.
Miller, G.L., 1959. Use of dinitrosalicylic acid regent for determination of reducing sugar. Anal. Chem., 31: 426-428.
Oliveira, A., L. Oliveira, J. Andrade and A. Junior, 2007. Rhizobial amylase production using various starchy substances as carbon substrates. Braz. J. Microbiol., 38: 208-216.
Direct Link |
Oyeleke, S.B. and A.A. Oduwole, 2009. Production of amylase by bacteria isolated from a cassava waste dumpsite in Minna, Niger State, Nigeria. Afr. J. Microbiol. Res., 3: 143-146.
Direct Link |
Pandey, A., P. Nigam, C.R. Soccol, V.T. Soccol, D. Singh and R. Mohan, 2000. Advances in microbial analyses. Biotechnol. Applied Biochem., 31: 135-152.
Ramachandran, N., K.R. Sreekantiah and V.S. Murthy, 1978. Studies on the thermophilic amylolytic enzymes of a strain of Aspergillus niger. Starch Starke, 8: 272-275.
Sabra, W. and D.M. Abou-Zeid, 2008. Improving feeding strategies for maximizing polyhdroxybutyrate yield by Bacillus megaterium. Res. J. Microbiol., 3: 308-318.
CrossRef | Direct Link |
Sarikaya, E. and V. Gurgun, 2000. Increase of the α-amylase yield by some Bacillus strains. Turk. J. Biol., 24: 299-308.
Direct Link |
Seneath, P.H.A., N.S. Mair, M.E. Sharpe and J.G. Holt, 1986. Bergey's Manual of Systematic Bacteriology. 9th Edn., Williams and Wilkins, Baltimore.
Shaw, J.F., F.P. Lans, S.C. Chen and H.C. Chen, 1999. Purification and properties of an extra cellular α amylase from thermos sp. Bot. Bull. Acad. Sin., 36: 195-299.
Takasaki, Y., 1989. Novel maltose producing amylase from Bacillus megaterium G-2. Agric. Biol. Chem., 53: 341-347.
Thomas, M., F.G. Priest and J.R. Stark, 1980. Characterization of an extra cellular p-amylase from Bacillus megaterium sensu stricto. J. Gen. Microbiol., 118: 67-72.
Direct Link |
Vidyalakshmi, R., R. Paranthaman and J. Indhumathi, 2009. Amylase production on submerged fermentation by Bacillus spp. World J. Chem., 4: 89-91.
Wood, T.M. and K.M. Bhat, 1988. Methods for Measuring Cellulases Activites. In: Methods Enzymology Series Biomass Part A. Cellulose and Hemicellulose, Aselson, J.N. and M. Simon (Eds.). Acadmic Press, New York, pp: 87-117.