Improving production of hyperthermostable and high maltose-forming
a-amylase by an extreme thermophile Geobacillus thermoleovorans
using response surface methodology and its applications
J.L. Uma Maheswar Rao, T. Satyanarayana
*
Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India
Received 17 October 2005; received in revised form 22 December 2005; accepted 24 December 2005
Available online 10 February 2006
Abstract
By cultivating Geobacillus thermoleovorans in shake flasks containing cane molasses medium at 70 °C, the fermentation variables were
optimized by ‘one variable at a time’ approach followed by response surface methodology (RSM). The statistical model was obtained by
central composite design (CCD) using three variables (cane-molasses, urea and inoculum density). An overall 1.6- and 2.1-fold increase
in enzyme production was achieved in the optimized medium in shake flasks and fermenter, respectively. The a-amylase titre increased
significantly in cane-molasses medium (60 U ml
À1
) as compared to that in the synthetic medium (26 U ml
À1
). Thus the cost of enzyme
produced in cane molasses medium (€0.823 per million U) was much lower than that produced in the synthetic starch–yeast extract-tryp-
tone medium (€18.52 per million U). The shelf life of bread was improved by supplementing dough with a-amylase, and thus, the enzyme
was found to be useful in preventing the staling of bread. Reducing sugars liberated from 20% and 30% raw pearl millet starch were
fermented to ethanol; ethanol production levels attained were 35.40 and 28.0 g l
À1
, respectively.
Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: Ca
2+
-independent a-amylase; Response surface methodology; Cane molasses; Antistaling; Ethanol
1. Introduction
a-Amylase (EC 3.2.1.1) is one of the enzymes of world-
wide interest in food, pharmaceutical and fermentation
industries. This enzyme is used in the conversion of starch
into different sugar syrups. Industrial applications gener-
ally require amylases with a very specific hydrolysis pro-
file. A need for more thermostable a-amylases devoid of
requirement for Ca
2+
for their activity/stability in starch
saccharification has been emphasized (Antranikian,
1992; Malhotra et al., 2000), since the added Ca
2+
must
be removed from the product streams by using ion-
exchangers.
The conventional practice of single factor optimization
by maintaining other factors at an unspecified constant
level does not depict the combined effect of all the factors
involved. The method requires a large number of experi-
ments to determine optimum levels, which is tedious and
time consuming. Optimizing all the effecting parameters
can eliminate these limitations of a single factor optimiza-
tion process collectively by statistical experimental design
using Response Surface Methodology (RSM). It is well
documented that extracellular amylase production by
microbes is greatly influenced by media components, espe-
cially carbon and nitrogen sources, minerals and physical
factors such as pH, temperature, agitation, dissolved
oxygen and inoculum density (Babu and Satyanarayana,
1993; Dey et al., 2001; Gigras et al., 2002). Statistical opti-
mization not only allows quick screening of a large exper-
imental domain, but also reflects the role of each of the
components. Application of RSM has gained attention of
0960-8524/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2005.12.022
*
Corresponding author. Tel.: +91 11 2112008; fax: +91 11 26885270.
E-mail addresses: tsnarayana@vsnl.net, tsnarayana@gmail.com (T.
Satyanarayana).
Bioresource Technology 98 (2007) 345–352