Thermostable alpha-amylase production by an extreme thermophile Bacillus thermooleovorans

AIMS: alpha-Amylase production by a newly isolated thermophile, Bacillus thermooleovorans, was studied under different cultivation conditions. METHODS AND RESULTS: The influence of various carbon and nitrogen sources on alpha-amylase production was quantified in batch fermentation in shake flasks. Starch and tryptone were observed to be the ideal carbon and nitrogen sources, respectively. Cultivation of the organism in a chemically defined medium consisting of glucose, riboflavin, cysteine, MgSO4, K2HPO4 and NaCl led to a near twofold increase in the production of alpha-amylase in comparison with that in the complex medium. The increase in enzyme production was achieved using vitamins and amino acids. When the organism was grown in a laboratory fermenter in the optimized complex medium, the noticeable effects were the near abolition of the lag phase, a 2.2-fold increase in enzyme production and a reduction in optimal production time from 12 to 4-5 h. CONCLUSION: Enhancement of amylase production was achieved under various cultivation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus thermooleovorans produces a calcium-independent and thermostable amylase which can find use in starch saccharification.     

Comparison of some properties of free and immobilized alpha-amylase by Aspergillus sclerotiorum in calcium alginate gel beads

Some properties of immobilized alpha-amylase by Aspergillus sclerotiorum within calcium alginate gel beads were investigated and compared with soluble enzyme. Optimum pH and temperature were found to be 5.0 and 40 degrees C, respectively, for both soluble and immobilized enzymes. The immobilized enzyme had a better Km value, but kcat/Km values were the same for both enzymes. Entrapment within calcium alginate gel beads improved, remarkably, the thermal and storage stability of alpha-amylase. The half life values of immobilized enzyme and soluble enzyme at 60 degrees C were 164.2, and 26.2 min, respectively. The midpoint of thermal inactivation (Tm) shifted from 56 degrees C (for soluble enzyme) to 65.4 degrees C for immobilized enzyme. The percentages of soluble starch hydrolysis for soluble and immobilized alpha-amylase were determined to be 97.5 and 92.2% for 60 min, respectively.     

Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15

A thermostable alkaline alpha-amylase producing Bacillus sp. A3-15 was isolated from compost samples. There was a slight variation in amylase synthesis within the pH range 6.0 and 12.0 with an optimum pH of 8.5 (8mm zone diameter in agar medium) on starch agar medium. Analyses of the enzyme for molecular mass and amylolytic activity were carried out by starch SDS-PAGE electrophoresis, which revealed two independent bands (86,000 and 60,500 Da). Enzyme synthesis occurred at temperatures between 25 and 65 degrees C with an optimum of 60 degrees C on petri dishes. The partial purification enzyme showed optimum activity at pH 11.0 and 70 degrees C. The enzyme was highly active (95%) in alkaline range of pH (10.0-11.5), and it was almost completely active up to 100 degrees C with 96% of the original activity remaining after heat treatment at 100 degrees C for 30 min. Enzyme activity was enhanced in the presence of 5mM CaCl2 (130%) and inhibition with 5mM by ZnCl2, NaCl, Na-sulphide, EDTA, PMSF (3mM), Urea (8M) and SDS (1%) was obtained 18%, 20%, 36%, 5%, 10%, 80% and 18%, respectively. The enzyme was stable approximately 70% at pH 10.0-11.0 and 60 degrees C for 24h. So our result showed that the enzyme was both, highly thermostable-alkaline, thermophile and chelator resistant. The A3-15 amylase enzyme may be suitable in liquefaction of starch in high temperature, in detergent and textile industries and in other industrial applications.     

Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus

AIMS: An investigation was carried out on the purification and characterization of an alkaline protease from Bacillus pumilus MK6-5. METHODS AND RESULTS: An alkalophilic Bacillus pumilus MK6-5 was grown in a laboratory fermenter containing 1% reverse osmosis concentrated cheese whey powder, 0.25% corn steep liquor, 1% glucose, 0.5% tryptone, 1% sodium citrate, 0.02% MgSO4.7H2O and 0.65% Na2CO3 at 35 degrees C and pH 9.6, agitation at 250 rev min(-1) and aeration of 1 vvm for 60 h. When the enzyme was purified using ammonium sulphate precipitation, ion exchange and gel filtration chromatographies, a 26.2% recovery of enzyme with 36.6-fold purification was recorded. The purified protease was found to be homogenous by SDS-PAGE with molecular mass estimate of 28 kDa. The enzyme was optimally active at pH 11.5 and temperature of 55-60 degrees C. The Km and kcat values observed with synthetic substrates at 37 degrees C and pH 8.0 were 1.1 mmol l(-1) and 624 s(-1) for Glu-Gly-Ala-Phe-pNA and 3.7 mmol l(-1) and 826 s(-1) for Glu-Ala-Ala-Ala-pNA, respectively. The kinetic data revealed that small aliphatic and aromatic residues were the preferred residues at the P1 position. Inhibition profile exhibited by PMSF suggested the B. pumilus protease to be an alkaline serine protease. CONCLUSIONS: Bacillus pumilus MK6-5 produced a calcium-dependent, thermostable alkaline serine protease. SIGNIFICANCE AND IMPACT OF THE STUDY: The thermostable alkaline protease from Bacillus pumilus MK6-5 will be extremely useful in ultrafiltration membrane cleaning due to its ability to work in broad pH and temperature ranges, and tolerance to detergents, unlike the mesophilic proteases which face these limitations.     

Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme

Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

A novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1: purification and characterization

This study reports the purification and characterization of a novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1. Maximum alpha-amylase activity (53 U mL(-1)) was obtained at 45 degrees C after 44 h of incubation. The enzyme was purified using ammonium sulfate precipitation, ion exchange and gel filtration chromatography, and showed a molecular weight of 56 kDa by SDS-PAGE. This enzyme exhibited maximum activity at pH 5.0, performed stability over a broad range of pH 4.5-11.0, and was optimally active at 40-50 degrees C. The enzyme preparation had a strong digesting ability towards various raw starches and efficiently hydrolyzed raw corn starch at a concentration of 20% and pH 5.0, which were normally used in the starch industries, in a period of 12h. By analyzing its partial amino acid sequences, the enzyme was proposed to be a novel alpha-amylase.     

Co-production of alpha-amylase and beta-galactosidase by Bacillus subtilis in complex organic substrates

Various nutrients belonging to three categories, carbon, organic nitrogen and complex organic sources, were investigated for the first time in terms of their effect on the co-production of extracellular thermostable alpha-amylase and beta-galactosidase by Bacillus subtilis, a bacterium isolated from fresh sheep's milk. Among the organic nitrogen sources tested, tryptone and corn steep liquor favored their production. Substitution of soluble starch by various starchy substrates, such as corn flour, had a positive effect on both enzyme yields. Furthermore, a two-fold higher production of both enzymes was achieved when corn steep liquor or tryptone was used in combination with the different flours. Among the divalent cations examined, calcium ions appeared to be vital for alpha-amylase production. The crude alpha-amylase and beta-galactosidase produced by this B. subtilis strain exhibited maximal activities at 135 degrees C and 65 degrees C, respectively, and were also found to be significantly stable at elevated temperatures.     

Purification and characterization of an extracellular alpha-amylase produced by Lactobacillus manihotivorans LMG 18010(T), an amylolytic lactic acid bacterium

This work presents the purification and characterization of an extracellular alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) produced by a new lactic acid bacterium: Lactobacillus manihotivorans able to produce L(+) lactic acid from starch. The molecular weight was found to be 135 kDa. The temperature and pH optimum were 55 degrees C and 5.5, respectively, and pI was 3.8. The alpha-amylase had good stability at pH range from 5 to 6 and the enzyme was sensitive to temperature, losing activity within 1 h of incubation at 55 degrees C. Higher thermal stability was observed when the enzyme was incubated in presence of soluble starch. K(m) value and activation energy were 3.44 mg/ml and 32.55 kJ/mol, respectively. Amylose was found to be a better substrate than soluble starch and amylopectin. Al(3+), Fe(3+), and Hg(2+) (10 mM) almost completely inhibited the alpha-amylase.     

Enhanced secretion and low temperature stabilization of a hyperthermostable and Ca2+-independent alpha-amylase of Geobacillus thermoleovorans by surfactants

AIMS: Selection of suitable surfactants for enhancing and stabilizing alpha-amylase of Geobacillus thermoleovorans. METHODS AND RESULTS: Geobacillus thermoleovorans was cultivated in shake flasks containing 50 ml of starch-yeast extract-tryptone (SYT) medium with/without surfactants. Titres of the enzyme in media were monitored. The enzyme was also preserved at 4 degrees C with/without surfactants and enzyme activities were determined. Among polyethylene glycol (PEGs) of different molecular weights, PEG 8000 (0.5%, w/v) caused a slight increase in the enzyme titre, while Tween-20, Tween-40 and Tween-60 (0.03%, w/v) exerted a significant stimulatory effect on enzyme secretion. In the presence of SDS, Tween-80 and cholic acid (0.03%, w/v), the enzyme production was nearly twofold higher than that in the control. The anionic (SDS, cholic acid) and non-ionic (Tweens) detergents increased the cell membrane permeability, and thus, enhanced alpha-amylase secretion. Furthermore, anionic surfactants exhibited stabilizing effect on the enzyme during preservation at 4 degrees C. CONCLUSIONS: PEG 8000 and the ionic detergents (SDS, cholic acid and Tween-80) were more effective in the solubilization of cell membrane components, and enhancing enzyme yields than the cationic detergents such as CTAB (N,Cetyl-N,N,N-trimethyl ammonium bromide). Further, these surfactants were found to stabilize the enzyme at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The secretion of Ca2+-independent hyperthermostable alpha-amylase was enhanced in the presence of certain anionic and non-ionic detergents in the medium. Furthermore, the surfactants stabilized the enzyme during preservation at 4 degrees C. The use of this enzyme in starch hydrolysis eliminates the addition of Ca2+ in starch liquefaction and its subsequent removal by ion exchange from sugar syrups.     

Purification and characterization of the extracellular alpha-amylase from Clostridium acetobutylicum ATCC 824.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography (mono Q) and gel filtration (Superose 12). The enzyme had an isoelectric point of 4.7 and a molecular weight of 84,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a monomeric protein, the 19-amino-acid N terminus of which displayed 42% homology with the Bacillus subtilis saccharifying alpha-amylase. The amino acid composition of the enzyme showed a high number of acidic and hydrophobic residues and only one cysteine residue per mole. The activity of the alpha-amylase was not stimulated by calcium ions (or other metal ions) or inhibited by EDTA, although the enzyme contained seven calcium atoms per molecule. alpha-Amylase activity on soluble starch was optimal at pH 5.6 and 45 degrees C. The alpha-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a Km of 3.6 g . liter-1 and a Kcat of 122 mol of reducing sugars . s-1 . mol-1. The alpha-amylase showed greater activity with high-molecular-weight substrates than with low-molecular-weight maltooligosaccharides, hydrolyzed glycogen and pullulan slowly, but did not hydrolyze dextran or cyclodextrins. The major end products of maltohexaose degradation were glucose, maltose, and maltotriose; maltotetraose and maltopentaose were formed as intermediate products. Twenty seven percent of the glucoamylase activity generally detected in the culture supernatant of C. acetobutylicum can be attributed to the alpha-amylase.     

Production and characterization of a thermostable alpha-amylase from Nocardiopsis sp. endophyte of yam bean

Thermostable amylolytic enzymes have been currently investigated to improve industrial processes of starch degradation. Studies on production of alpha-amylase by Nocardiopsis sp., an endophytic actinomycete isolated from yam bean (Pachyrhizus erosus L. Urban), showed that higher enzyme levels were obtained at the end of the logarithmic growth phase after incubation for 72 h at pH 8.6. Maximum activity of alpha-amylase was obtained at pH 5.0 and 70 degrees C. The isolated enzyme exhibited thermostable properties as indicated by retention of 100% of residual activity at 70 degrees C, and 50% of residual activity at 90 degrees C for 10 min. Extracellular enzyme from Nocardiopsis sp. was purified by fractional precipitation with ammonium sulphate. After 60% saturation produced 1130 U mg-1 protein and yield was 28% with purification 2.7-fold. The enzyme produced by Nocardiopsis sp. has potential for industrial applications.     

Expression of a bacterial α-amylase gene in transgenic rice seeds

An alpha-amylase gene from Bacillus stearothermophilus under the control of the promoter of a major rice-seed storage protein was introduced into rice. The transgenic line with the highest alpha-amylase activity reached about 15,000 U/g of seeds (one unit is defined as the amount of enzyme that produces 1 mumol of reducing sugar in 1 min at 70 degrees C). The enzyme produced in the seeds had an optimum pH of 5.0-5.5 and optimum temperature of 60-70 degrees C. Without extraction or purification, the power of transgenic rice seeds was able to liquify 100 times its weight of corn powder in 2 h. Thus, the transgenic rice could be used for industrial starch liquefaction.     

A highly thermostable and alkaline amylase from a Bacillus sp. PN5

A highly thermostable alkaline amylase producing Bacillus sp. PN5 was isolated from soil, which yielded 65.23 U mL(-1) of amylase in medium containing (%) 0.6 starch, 0.5 peptone and 0.3 yeast extract at 60 degrees C, pH 7.0 after 60 h of incubation. Maximum amylase activity was at pH 10.0 and 90 degrees C. The enzyme retained 80% activity after 1 h at pH 10.0. It exhibited 65% activity at 105 degrees C and had 100% stability in the temperature range between 80 and 100 degrees C for 1 h. In addition, there was 86.36% stability after 1-h incubation with sodium dodecylsulphate. These properties indicated possible use of this amylase in starch saccharification and detergent formulation.     

Production of Schardinger beta-dextrin by soluble and immobilized cyclodextrin glycosyltransferase of an alkalophilic Bacillus sp.

Succinylated cyclodextrin glycosyltransferase (EC 3.2.1.19) of an alkalophilic Bacillus sp. was adsorbed on a vinylpyridine copolymer. The enzyme had about 25% of the activity of soluble enzyme added. No increase of pH or thermal stability of the enzyme was observed by the adsorption, whereas optimum temperature for the enzyme action was shifted from 50 to 55 degrees C. The enzyme converted starch to cyclodextrine without significant loss of activity under the conditions of 4 times reusing of 6 hr conversion by the batch system or 2 weeks continuous reaction by the column system at 55 degrees C and pH 8.0. About 46% of the potato starch solution [15% (w/v)] was converted to cyclodextrins by the enzyme, and 52% was converted by the simultaneous action of the enzyme and alkaline pullulanase of alkalophilic Bacillus sp. (No. 202-1). These values were almost the same as those obtained by the soluble enzyme or enzymes system.     

Purification and characterization of the extracellular alpha-amylase from Streptococcus bovis JB1.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from maltose-grown Streptococcus bovis JB1 was purified to apparent homogeneity by ion-exchange chromatography (Mono Q). The enzyme had an isoelectric point of 4.50 and an apparent molecular mass of 77,000 Da, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was rich in acidic and hydrophobic amino acids. The 15-amino-acid NH2-terminal sequence was 40% homologous with the Bacillus subtilis saccharifying alpha-amylase and 27% homologous with the Clostridium acetobutylicum alpha-amylase. alpha-Amylase activity on soluble starch was optimal at pH 5.0 to 6.0. The enzyme was relatively stable between pH 5.5 and 8.5 and at temperatures below 50 degrees C. When soluble potato starch was used as the substrate, the enzyme had a Km of 0.88 mg.ml-1 and a kcat of 2,510 mumol of reducing sugar.min-1.mg of protein-1. The enzyme exhibited neither pullulanase nor dextranase activity and was 40 to 70% as active on amylopectin as on amylose. The major end products of amylose hydrolysis were maltose, maltotriose, and maltotetraose.     

Characterization of a thermostable Bacillus stearothermophilus alpha-amylase

Liquefying-type Bacillus stearothermophilus alpha-amylase was characterized. The coding gene was cloned in Bacillus subtilis and the enzyme was produced in three different host organisms: B. stearothermophilus, B. subtilis, and Escherichia coli. Properties of the purified enzyme were similar irrespective of the host. Temperature optimum was at 70-80 degrees C and pH optimum at 5.0-6.0. The enzyme was stable for 1 h in the pH range 6.0-7.5 at 80 degrees C. The enzyme was stabilized by Ca2+, Na+, and bovine serum albumin. About 50% of the activity remained after heating at 70 degrees C for 5 days or 45 min at 90 degrees C. Metal ions Cd2+, Cu2+, Hg2+, Pb2+, and Zn2+ were inhibitory, whereas EDTA, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, and Tendamistat were without effect. The enzyme was fully active after treatment in acetone or ethanol at 55 or 70 degrees C, respectively, for 30 min. Sodium dodecyl sulfate (1%) did not affect stability, whereas 6 M urea denatured totally at 70 degrees C. The Km value for soluble starch was 14 mg/ml. Mr is 59,000 and pI 8.8. The only difference between the enzymes produced in different hosts was in signal peptide processing.     

Production of amylase by Aspergillus foetidus on rice flour medium and characterization of the enzyme

Aspergillus foetidus ATCC 10254 was selected from nine starch-utilizing microorganisms for its high amylolytic activity. This mould produced high levels of extracellular alpha-amylase in rice starch medium and degraded the available starch efficiently. Optimal conditions for enzyme production on 2.0% rice medium included 28 degrees C, initial pH of 6.6, and supplementations with 0.02% NaNO2, 0.08% KH2PO4, and 0.08% corn steep liquor. Eleven-fold purification of the enzyme was obtained after ammonium sulphate and ethanol precipitations from spent medium. The molecular weight was estimated at 41 500. Optimum pH and temperature for enzyme activity were 5.0 and 45 degrees C. Michaelis-Menten constants were 1.14 mg/ml on amylopectin, 2.19 mg/ml on soluble starch and 7.65 mg/ml on amylose. Amylose produced substrate inhibition while glucose or maltose did not inhibit the enzyme. This alpha-amylase may be used as a saccharifying enzyme for rice starch. Aspergillus foetidus ATCC 10254 also presents a potential for treatment of starch-containing waste waters.     

Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme. Unlike the P. furiosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions characteristic of that enzyme family. The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration. Both the dimer and monomer retained starch-degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels. The P. furiosus alpha-amylase was a liquefying enzyme with a specific activity of 3,900 U mg-1 at 98 degrees C. It was optimally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 h at 98 degrees C, the P. furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis alpha-amylase (Taka-therm).     

Purification, characterization, and synergistic action of phytate-resistant alpha-amylase and alpha-glucosidase from Geobacillus thermodenitrificans HRO10

The alpha-amylase (1, 4-alpha-d-glucanohydrolase; EC 3.2.1.1) and alpha-glucosidase (alpha-d-glucoside glucohydrolase; EC 3.2.1.20) secreted by Geobacillus thermodenitrificans HRO10 were purified to homogeneity (13.6-fold; 11.5% yield and 25.4-fold; 32.0% yield, respectively) through a series of steps. The molecular weight of alpha-amylase was 58kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The alpha-amylase activity on potato starch was optimal at pH 5.5 and 80 degrees Celsius. In the presence of Ca(2+), the alpha-amylase had residual activity of more than 92% after 1h of incubation at 70 degrees Celsius. The alpha-amylase did not lose any activity in the presence of phytate (a selective alpha-amylase inhibitor) at concentrations as high as 10mM, rather it retained 90% maximal activity after 1h of incubation at 70 degrees Celsius. EGTA and EDTA were strong inhibitory substances of the enzyme. The alpha-amylase hydrolyzed soluble starch at 80 degrees Celsius, with a K(m) of 3.05mgml(-1) and a V(max) of 7.35Uml(-1). The molecular weight of alpha-glucosidase was approximately 45kDa, as determined by SDS-PAGE. The enzyme activity was optimal at pH 6.5-7.5 and 55 degrees Celsius. Phytate did not inhibit G. thermodenitrificans HRO10 alpha-glucosidase activity, whereas pCMB was a potent inhibitor of the enzyme. The alpha-glucosidase exhibited Michaelis-Menten kinetics with maltose at 55 degrees Celsius (K(m): 17mM; V(max): 23micromolmin(-1)mg(-1)). Thin-layer chromatography studies with G. thermodenitrificans HRO10 alpha-amylase and alpha-glucosidase showed an excellent synergistic action and did not reveal any transglycosylation catalyzed reaction by the alpha-glucosidase.     

Statistical optimization of a high maltose-forming,hyperthermostable and Ca2+-independent alpha-amylase production by an extreme thermophile Geobacillus thermoleovorans using response surface methodology

AIM: Statistical optimization for maximum production of a hyperthermostable, Ca2+-independent and high maltose-forming alpha-amylase by Geobacillus thermoleovorans. METHODS AND RESULTS: G. thermoleovorans was cultivated in 250 ml flasks containing 50 ml of chemically defined glucose-arginine medium (g l(-1): glucose 20; arginine 1.2; riboflavin 150 microg ml(-1); MgSO4. 7H2O 0.2; NaCl 1.0; pH 7.0). The medium was inoculated with 5 h-old bacterial inoculum (1.8x10(8) CFU ml(-1)), and incubated in an incubator shaker at 70 degrees C for 12 h at 200 rev min(-1). The fermentation variables optimized by 'one variable at a time' approach were further optimized by response surface methodology (RSM). The statistical model was obtained using central composite design (CCD) with three variables: glucose, riboflavin and inoculum density. An over all 24 and 70% increase in enzyme production was attained in shake flasks and fermenter because of optimization by RSM, respectively. A good coverage of interactions could also be explained by RSM. The end products of the action of alpha-amylase on starch were maltose (62%), maltotriose (31%) and malto-oligosaccharides (7%). CONCLUSIONS: RSM allowed optimization of medium components and cultural parameters for attaining high yields of alpha-amylase, and further, a good coverage of interactions could be explained. The yield of maltose was higher than maltotriose and malto-oligosaccharides in the starch hydrolysate. SIGNIFICANCE AND IMPACT OF THE STUDY: By applying RSM, critical fermentation variables were optimized rapidly. The starch hydrolysate contained a high proportion of maltose, and therefore, the enzyme can find application in starch saccharification process for the manufacture of high maltose syrups. The use of this enzyme in starch saccharification eliminates the addition of Ca2+.