Bioresource Technology 96 (2005) 1201–1204

Short Communication

Pearl millet, a source of alpha amylase production byBacillus licheniformis

Ikram-ul-Haq *, Hamad Ashraf, Qadeer M.A., Javed Iqbal

Biotechnology Research Laboratory, Department of Botany, G.C. University Lahore, Pakistan

Received 13 February 2003; received in revised form 22 September 2004; accepted 22 September 2004

Available online 1 January 2005

Abstract

The present study is concerned with the selection of economically available agricultural starchy substrate for the production of

alpha amylase by Bacillus licheniformis. Different agricultural starchy substrates such as soluble starch, hordium, pearl millet, rice,

corn, gram and wheat starch were tested for the production of alpha amylase by parental and its mutant derivatives. The production

of alpha amylase was 10-folds better by the mutant strain B. licheniformis GCUCM-30 than the parental strain when pearl millet

starch at 1.5% level and nutrient broth concentrations at the level of 0.25% was supplemented to the fermentation medium.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Pearl millet; Bacillus licheniformis

1. Introduction

Alpha amylase, an extracellular, enzyme degraded a,

1-4 linkage of starch and extensively used in starch lique-faction, paper, food, pharmaceutical and sugar industries

(Nigam and Sing, 1995). Highly active alpha amylase is

required to meet the demand of above-mentioned indus-

tries. Mutant strains of Bacillus have better ability for the

production of alpha amylase (Zhao and Quirong, 1994;

Allan et al., 1997). The mutant strains of Bacillus can be

derived by mutagenesis and extensive screening (Bin

et al., 1999; Ashraf et al., 2001).The fermentable carbonsources such as glucose, starch, lactose or fructose that

are obtained after the processing of agricultural products.

Therefore, they became very expensive for commercial

production of alpha amylase. These expensive products

can be replaced in the fermentation medium with the eco-

nomically available agricultural by-products (Ghosh and

Chandra, 1984). The flours of different grains such as

wheat, barley and rice can be used in the fermentation

0960-8524/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2004.09.012

* Corresponding author.

E-mail address: ikrhaq@yahoo.com (I. Haq).

medium to increase the productivity of alpha amylase

by the bacterium (Pratima and Umender, 1989; Mamo

and Gessesse, 1999). We have recently reported that the

alpha amylase of B. licheniformis GCBU-8 shows greatpromise for the production of alpha amylase using an

economical medium (Haq et al., 2003). Because of the im-

mense potential of the enzyme for industrial applications,

further studies were needed to optimize carbon and nitro-

gen sources for enhanced production of alpha amylase. A

study has been under taken to optimize agricultural

starches as carbon source and nutrient broth as nitrogen

source for the biosynthesis of alpha amylase. The resultsof comparative study of Bacillus strains for the selection

of suitable low cost agricultural non-processed starch

for the production of alpha amylase is discussed.

2. Methods

2.1. Organism

The parental B. licheniformis GCB-36 and its mutant

derivatives of GCUB-8, GCCM-23, GCUCM-30 were

1202 I. Haq et al. / Bioresource Technology 96 (2005) 1201–1204

obtained from Biotechnology Laboratory, Department

of Botany, G.C. University, Lahore (Ashraf et al.,

2001). The strains were maintained on the nutrient

starch agar slopes.

2.2. Inoculum preparation

Fifty ml of inoculum medium containing (%w/v)

nutrient broth 1.0, soluble starch 1.0, NaCl 0.5, CaCl20.2 in 100ml of phosphate buffer (pH7.5) was trans-

ferred to each of 250ml cotton plugged conical flask.

The flasks were sterilized in an autoclave at 15 lb/in.2

pressure (121 �C) for 15min. After cooling the medium,

a loopful of bacteria was aseptically transferred to eachflask. The flasks were then rotated in the rotary shaking

incubator (200rpm) at 40 �C for 24h.

2.3. Fermentation and biomass estimation

Fifty milliliters of the fermentation medium contain-

ing (%w/v) wheat bran 1.25, nutrient broth 1.0, soluble

starch 1.0, lactose 0.5, NaCl 0.5, CaCl2 0.2 in 100mlof phosphate buffer (pH7.5) was transferred to 250ml

of cotton plugged conical flask. The flasks were auto-

claved (15 lb/in.2 pressure, 121 �C, 15min) and then

cooled at room temperature. Each flask was then inocu-

lated with one ml of inoculum and then rotated in the

rotary incubator shaker (X.X2. C, Sanyo Gallenkamp

UK) at 40 �C for 48h. After 48h the fermented broth

was centrifuged (4000rpm) for 15min and substrate freesupernatant was used for the estimation of alpha amy-

lase. The biomass was estimated turbidimetrically with

a spectrophotometer (Model CECIL CE7200 Aquaris

UK) at 650nm. All the experiments were performed par-

allel in triplicates.

2.4. Enzyme assay

The enzyme was assayed according to the method of

Rick and Stegbauer (1974). The enzyme solution at

Table 1

Effect of starch substrates on the production of alpha amylase by B. lichenif

Starchy substrates Parental GCBU-8

Biomass

(g/l)

Enzyme

activity

(IU/ml/min)

Biomass

(g/l)

Enzyme

activity

(IU/ml/

Soluble starch 19 ± 0.12a 100 ± 4a 8 ± 0.02c 150 ± 3

Hordium 16 ± 0.15b 77 ± 3b 10 ± 0.01b 255 ± 2

Pearl millet 16 ± 0.02b 62 ± 2bc 13 ± 0.02a 397 ± 2

Rice 9 ± 0.18cd 32 ± 1d 13 ± 0.03a 291 ± 1

Corn 0 ± 0.14cd 54 ± 3c 10 ± 0.03b 238 ± 4

Gram 10 ± 0.05c 56 ± 1c 8 ± 0.04c 231 ± 5

Wheat 8 ± 0.02d 22 ± 2de 7 ± 0.3d 158 ± 6

Each value is an average of three parallel replicate. ± indicated standard devi

differ significantly at p < 0.05. Medium composition (%w/v) wheat bran 1.25

pH7.5 was incubated at 60 �C using 1% soluble starch

solution. The reducing sugars were measured by adding

3,5-dinitro salicylic acid reagent, boiling for 5min,

cooling and measuring the OD at 540nm in the spectro-

photometer against maltose as standard. One unit of en-

zyme activity is that amount of enzyme which 10minliberates reducing group from 1% Lintner�s soluble

starch. The enzyme activity was converted into IU/ml/

min according to Ashraf et al. (2001).

2.5. Parametric study and statistical analysis

Different agricultural starches such as hordium, pearl

millet, rice, corn, gram and wheat starch were comparedwith the soluble starch for the production of alpha amy-

lase. the pearl millet starch at the level of 0.5–3.0% and

nutrient broth (Peptone 3% and beef extract 2%) 0.0–

2.5% were tested to optimize their supplementation as

carbon and nitrogen sources respectively. Treatment ef-

fects were compared by the method of Snedecor and

Cochrane (1980). Post Hoc Multiple Comparison tests

were applied under one way ANOVA. Significance hasbeen presented in the form of probability (p < 0.05)

values.

3. Results and discussion

Different agricultural raw starches such as pearl mil-

let, rice, gram, hordium, corn and wheat starches wastested for the production of alpha amylase (Table 1).

The starches were added to the fermentation medium

at 1% level. The production of enzyme by the parental

strain was higher in the presence of soluble starch. How-

ever, its mutant derivatives gave optimum production of

alpha amylase in the presence of pearl millet starch. It

might be due to pearl millet contain adequate amount

of nutrients (Carbohydrates 67.1%, Protein 11.6%, Min-erals 2.7%) for the growth of microorganism as well as

for the production of alpha amylase. The productivity

ormis GCB-36 and its mutant derivatives

GCCM-23 GCUCM-30

min)

Biomass

(g/l)

Enzyme

activity

(IU/ml/min)

Biomass

(g/l)

Enzyme

activity

(IU/ml/min)

d 15 ± 0.1c 460 ± 3b 19 ± 0.3b 710 ± 5b

bc 17 ± 0.2a 462 ± 2b 18 ± 0.5c 666 ± 4c

a 16 ± 0.19 b 500 ± 5a 20 ± 0.1a 936 ± 2a

.7b 15 ± 0.14c 350 ± 3c 17 ± 0.3d 633 ± 5c

c 14 ± 0.11d 200 ± 1d 17 ± 0.17d 630 ± 3c

c 10 ± 0.18e 180 ± 3e 17 ± 0.15d 430 ± 4d

d 10 ± 0.2e 178 ± 2e 16 ± 0.21e 400 ± 3d

ation from mean value. The values in columns denoted by superscripts

, nutrient broth 1.0, lactose 0.5, incubation period 48h.

Table 2

Effect of different concentrations of pearl millet starch for the production of alpha amylase by B. licheniformis GCB-36 and its mutant derivatives

Concentrations of

pearl millet starch (%)

Parental GCBU-8 GCCM-23 GCUCM-30

Biomass

(g/l)

Enzyme

activity

(U/ml/min)

Biomass

(g/l)

Enzyme

activity

(U/ml/min)

Biomass

(g/l)

Enzyme

activity

(U/ml/min)

Biomass

(g/l)

Enzyme

activity

(U/ml/min)

0.50 14 ± 0.04a 66 ± 4a 11 ± 0.02c 340 ± 2b 14 ± 0.19c 400 ± 5c 18 ± 0.09d 752 ± 5c

1.00 14 ± 0.08a 62 ± 4ab 13 ± 0.03a 397 ± 7a 15 ± 0.3b 450 ± 7b 20 ± 0.10b 833 ± 4b

1.50 14 ± 0.15a 62 ± 2ab 12 ± 0.02b 280 ± 5c 16 ± 0.14a 507 ± 2a 21 ± 0.14a 936 ± 2a

2.00 13 ± 0.23b 55 ± 3b 13 ± 0.01a 264 ± 6c 16 ± 0.33a 500 ± 3a 19 ± 0.08c 910 ± 5a

2.5 12 ± 0.15c 50 ± 1c 8 ± 0.013d 201 ± 7d 14 ± 0.18c 440 ± 6b 18 ± 0.09d 840 ± 5b

3.0 10 ± 0.09d 34 ± 0.5d 7 ± 0.2d 160 ± 5e 13 ± 0.12d 350 ± 6d 16 ± 0.2e 699 ± 3d

Each value is an average of three parallel replicate. ± indicated standard deviation from mean value. The values in columns denoted by superscripts

differ significantly at p < 0.05. Medium composition (%w/v) wheat bran 1.25, nutrient broth 1.0, lactose 0.5, incubation period 48h.

I. Haq et al. / Bioresource Technology 96 (2005) 1201–1204 1203

of enzyme, as well as biomass formation was greatly

inhibited in the presence of wheat starch, possibly due

to an inhibitor substance for the production of alpha

amylase (Ghosh and Chandra, 1984). The other starchessuch as hordium, rice, corn and gram gave insignificant

production of alpha amylase. The production of enzyme

following growth of the organism was higher by mutant

strain of B. licheniformis GCUCM-30 and varies signi-

ficantly than the other strains of B. licheniformis.

Based on our results, the parental and its mutant

derivatives were compared for the production of alpha

amylase in the presence of pearl millet (Table 2). Thepearl millet starch was added to the fermentation med-

ium at 0.5–3.0% levels. Maximum enzyme activity by

mutant strain GCBU-8 was achieved only when 1.0%

starch was added to the fermentation medium. How-

ever, the other mutant derivatives gave optimum pro-

duction of alpha amylase when 1.5% pearl millet

starch was supplemented in the fermentation medium.

As the amount of the starch was further increased, thegrowth of the organism and the production of alpha

amylase were significantly inhibited. Although, the mu-

tant GCUCM-30 required a little more starch for its

growth and productivity, the remarkable increase in en-

zyme production was obtained by using pearl millet

starch at 1.5% concentration. Moreover, it was noticed

that approximately 50% of the starch remained insolu-

Table 3

Effect of different concentrations of nutrient broth for the production of alp

Concentrations of

nutrient broth (%w/v)

Parental GCBU-8

Biomass

(g/l)

Enzyme

activity

(IU/ml/min)

Biomass

(g/l)

Enzyme

(IU/ml/m

0.00 07 ± 0.07e 20 ± 2e 9 ± 0.07d 180 ± 3e

0.50 10 ± 0.12d 66 ± 3d 11 ± 0.07c 434 ± 2a

1.00 18 ± 0.13a 70 ± 2c 12 ± 0.05b 388 ± 1b

1.50 17 ± 0.17b 88 ± 4b 12 ± 0.04b 360 ± 3c

2.00 17 ± 0.14b 100 ± 1a 13 ± 0.01a 340 ± 4d

2.5 16 ± 0.12c 77 ± 3c 13 ± 0.01a 362 ± 4c

Each value is an average of three parallel replicate. ± indicated standard devi

differ significantly at p < 0.05. Medium composition (%w/v) wheat bran 1.25,

ble in the crude form in which it was used. The carbon

sources affected not only the mode of amylase formation

but also the velocity with which the carbohydrates are

metabolized. Easily metabolizable carbohydrates mayresult in the better growth of the bacteria along with

reduction in the enzyme formation (Rama and Sriva-

stav, 1995). However, in the complex and crude source

of starch, organism grew very slowly with significant

secretion of alpha amylase in the fermentation medium.

Thus, the pearl millet starch was selected as the inducer

of alpha amylase.

The amount of nitrogen source can play a very criticalrole in the production of alpha amylase (Young et al.,

1995). The effect of different concentrations of nutrient

broth was investigated for the production of alpha amy-

lase by parental and mutant strains of B. licheniformis

(Table 3). The nutrient broth was added to the medium

at 0.25–1.25% levels. The parental strain gave maximum

production of alpha amylase in the presence of 1.0%

nutrient broth. However, their mutant derivatives gaveoptimal enzyme activity in the presence of 0.5% nutrient

broth but B. licheniformisGCUCM-30 gave optimal pro-

duction of alpha amylase in the presence of 0.25% of

nutrient broth. The productivity of the enzyme was

highly insignificant, as the nutrient broth was not supple-

mented in the fermentation medium. Therefore, this mu-

tant required a low quantity of nutrient broth for the

ha amylase by B. licheniformis GCB-36 and its mutant derivatives

GCCM-23 GCUCM-30

activity

in)

Biomass

(g/l)

Enzyme activity

(IU/ml/min)

Biomass

(g/l)

Enzyme

activity

(IU/ml/min)

9 ± 0.1e 130 ± 5d 12 ± 0.3e 452 ± 5d

17 ± 0.09a 550 ± 4a 22 ± 0.4a 960 ± 4a

17 ± 0.2a 515 ± 7ab 21 ± 0.2b 936 ± 2ab

16 ± 0.4b 480 ± 3b 20 ± 0.1c 877 ± 5b

15 ± 0.3c 420 ± 2bc 19 ± 0.1d 840 ± 5bc

14 ± 0.5d 390 ± 6c 19 ± 0.5d 800 ± 3c

ation from mean value. The values in columns denoted by superscripts

nutrient broth 1.0, lactose 0.5, pearl millet 1.5, incubation period 48h.

1204 I. Haq et al. / Bioresource Technology 96 (2005) 1201–1204

productivity of enzyme, possibly due to a low initial

requirement of easily available nitrogen source for its

initial growth, followed by an attack on the complex

sources such as pearl millet starch for its growth as well

as for the production of alpha amylase. The production

of the enzyme by the mutant strain GCUCM-30 washighly significant and varied significantly (p < 0.05) com-

pared to the other mutant derivatives in the presence of

nutrient broth at the level of 0.25%. As the amount of

the nutrient broth was increased, the productivity was

significantly decreased, possibly due to the fact that the

higher concentration of nitrogen source having an ad-

verse effect on the growth of microorganism as well as

on the production of alpha amylase (Hewitt and Solomo-nos, 1996; Pedreson and Nielson, 2000).

4. Conclusion

Based on this study it appears that complex carbohy-

drate sources such as pearl millet starch can serve as

basal and standardized medium for obtaining highyields of alpha amylase from B. licheniformis. With the

addition of pear millet starch, the amount of nutrient

broth was decreased from 1.0% level to 0.25% level by

the mutant derivatives. As compared to soluble starch,

the pearl millet starch showed significant benefit in en-

zyme production in consideration of the low cost of this

starch.

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