25-08-2017, 09:32 PM
Optimization of a-amylase production from free and immobilized cells of Aspergillus niger
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Introduction
α-amylase enzymes are all α-1,4-glucan 4-
glucanohydrolases (E.C. 3.2.1.1) that are important
enzymes employed in the starch processing industries for
the hydrolysis of polysaccharides such as starch into
simple sugar constituents as reported by Akpan et al.
(1999a, 1999b); Fogarty et al. (1980); Haq et al. (2002).
Starch degrading enzymes like amylase have received a
great deal of attention because of their perceived
technological significance and economic benefits. This
enzyme is also used for the commercial production of
glucose. In storage tissues such as seeds, starch a
polysaccharide of glucose is hydrolyzed for utilization by
the growing seedlings to meet its energy requirement.
Nowadays the new potential of using microorganism as
biotechnological sources of industrially relevant enzymes
has stimulated renewed interest in the exploration of
extra cellular enzymatic activity in several
microorganisms.
Material and Methods
Isolation and identification of Aspergillus niger strain
The Aspergillus culture was isolated from the soil by the serial
dilution method of Clark et al. (1988), one gram soil sample was
dissolved in 100 ml sterilized distilled water. The soil suspension
was diluted up to 10+3 to 10-3 and 0.5ml of diluted suspension was
used and the micro-organism producing the starch digesting α-
amylase screened according to method the described by Bergmann
et al. (1988). Aspergillus niger colonies producing large clear zones
were picked up and purified by streaking on PDA. Identification was
based on cell and colony morphology characterstics as per the
method described by Rasper and Fennel (1965). The young
colonies of Aspergillus niger were aseptically picked up and
transferred to PDA slants and incubated at 27° C for 4-5 days for
maximum growth.
Immobilization of Aspergillus niger cells
Aspergillus niger cells were immobilized and enzyme was isolated
as per the method described by Abraham et al. (1991).
Growth Medium
The medium contained (% w/v): Starch (1%); KH2PO4 (0.2%);
(NH4)2SO4 (0.14%); CaCl2 (0.03%); MgSO4.7H2O (0.03%), Urea
(0.03%), Peptone (0.1%), trace element solution (0.01%), Triton X-
100 (0.02%). Trace element solution contained in 500ml (2.5 g
FeSO4, 1.0 g CoCl2, 1.76 g ZnSO4, 0.98 g MnSO4). The pH of the
medium was adjusted to 4.8 with concentrated HCl, medium was
sterilized by autoclaving at 121°C and 15-17 psi for thirty minutes.
Inoculum and fermentation
A standardized inoculum size of conidia (each ml of cells
suspension contained 2.0 X 106 cells) was transferred from a stock
culture in 250ml flask containing 50 ml of growth medium. The
flasks were incubated for 72 hrs at 28°C ± 2°C on a rotatory shaker
at 150 rpm. On the last day of incubation period (72 hrs), the fungal
mass was separated by centrifugation at 4500 rpm for 10 min.
Effect of Carbon Sources on Extra Cellular α-
Amylase Production
Flasks containing production were media supplemented
with carbon sources (glucose, sucrose, starch,
carboxymethyl cellulose, fructose, sorbitol, xylose,
galactose and dextrin). The influence of these carbon
sources were tested at different concentrations (0.5 to
2.0%). Starch, Sucrose and dextrin and galactose were
good carbon sources for amylase production. Xylose,
fructose and sorbitol could be considered as moderate
source while carboxymethyl cellulose was a poor source
of amylase as it was found that 0.5 % concentration is
good carbon source for amylase production but
carboxymethyl cellulose and glucose at 0.5%
concentration produced low amount of alpha amylase.
Starch was recorded to be the best carbon source for
production of alpha amylase from both free and
immobilized cells of Aspergillus niger (Table 1).
Conclusion
It was concluded in our present study that both nutritional
and cultural condition were required for optimum growth
and production of alpha amylase from free and
immobilized cells of A. niger, for a final breakthrough to
achieving commercially relevant quantities of this enzyme
and will review the current state of knowledge on the
molecular basis of enzyme production by filamentous
fungi in coming future prospects.