Stability Of Amylase Research Articles

Stability kinetic study for amylase and protease enzymes under food stain removal conditions

A mechanistic model to evaluate the stability of amylase and protease under food stain removal conditions has been developed. The mechanism was determined identifying the significant factors for each enzyme based on response surface methodology. The amylase stability was jeopardized by the deprotonated peracid-based bleach, the interaction pH–temperature, and the protonated peroxide-based bleach promoted by manganese-based bleaching catalyst. The stability of the protease decreased in presence of protonated peracid-based bleach, acid-base equilibria species from peroxide-based bleach as well as temperature and pH. This work integrates the main mechanisms based on non-linear differential equations for each enzyme. In addition, a kinetic dissolution model was included for the enzymatic spherical particles. The models were validated, obtaining a determination coefficient of 0.84 and 0.90 for amylase and protease respectively for the training data set. For the validation set, a determination coefficient of 0.91 and 0.90 were obtained for amylase and protease, respectively.

Treatment of Starch Wastewater by α‐Amylase Immobilized on Silica Infused Magnetite Nanoparticles for Maltose Syrup Preparation

AbstractCasein decorated silicainfused magnetite (Fe3O4‐SiO2) nanoparticles are employed to immobilize commercial α‐amylase, where glutaraldehyde serves as a cross linker. The optimal concentration of variables, such as casein (1.4%w/v), Fe3O4‐SiO2 (106 µl), glutaraldehyde (55µl) and amylase (1 mg/ml) are defined by a Box Behnken design. The binding of casein, glutaraldehyde and enzyme over the nanoparticles are further confirmed structurally by fourier transform infrared spectroscopy (FTIR) and thermal gravimetric analysis (TGA). The loading capacity is 37 µg amylase over 1 µg of silica infused magnetite nanoparticles. The optimum pH for the catalysis of soluble and immobilized amylases is the same, i.e. pH 7. However, the pH range for catalysis is improved upon immobilization. The temperature optimum of soluble and immobilized amylases are 40 and 70 °C, respectively. The amylase stability is improved upon immobilization, as shown by enhanced half‐life and reduced deactivation rate constant. The immobilized amylase is used for 17 consecutive cycles with retention of 52% of the residual activity. The immobilized amylase produces high maltose syrup using the industrial wastewater containing corn starch. The ISO 5377 protocol determines dextrose equivalence values to be 36% and 24% during cycle 1 and cycle 2, respectively. The findings point out its possible commercial use.

Optimization of surfactants formulations to stabilise proteases and amylases

The stability of amylase and protease with linear alkylbenzene sulphonate, ethoxylated fatty alcohol, alkylpolyglucoside, and silica microparticles was analysed. Protease showed first order deactivation finding the highest stability (deactivation energy 62.9 Kcal/mol) with linear alkylbenzene sulfonate/ethoxylated fatty alcohol/silica microparticles (10/10/1.5% w/w). Other surfactant formulations with ethoxylated fatty alcohol (20% w/w), alkylbenzene sulfonate/ethoxylated fatty alcohol (10/10% w/w) or linear alkylbenzene sulfonate/ethoxylated fatty alcohol/alkylpolyglucoside (10/5/5% w/w) also stabilised the protease.Linear alkylbenzene sulfonate deactivated the amylase. Linear alkylbenzene sulfonate/ethoxylated fatty alcohol/ alkylpolyglucoside (10/5/5% w/w) and linear alkylbenzene sulfonate/ethoxylated fatty alcohol/silica microparticles (10/10/1.5% w/w) minimised the amylase destabilisation. These formulations also showed high detergency.Alkylbenzene sulfonate promoted protein unfolding and reduced the enzymatic activity, and the non-ionic surfactants stabilised the enzymes, possibly because they interact harmlessly with the enzymes and avoid interaction with the anionic surfactants at the same binding sites. Optimised surfactant formulations were suggested for detergent formulation with amylase and protease.

Thermozyme Amylase from Enterobacter sp Extremophiles in Bioethanol Production

Bioethanol is an alternative fuel to replace fossil fuels due to the reduction of fossil fuels. Bioethanol is produced from starch and converted to sugar by thermozyme amylase. Thermozyme amylase produced by thermophile bacteria can be obtained from Pariangan hot springs which have a temperature of 55 °C, pH of 9.2, and have a high level of bacterial diversity. The aim of this study was to obtained isolates of thermozyme amylase-producing bacteria that have the potential for bioethanol production. The research method were bacteria isolation, amylase stability test, temperature and pH optimization, and bioethanol production. The result showed that Enterobacter sp has been isolated from Pariangan hot springs is stable up to 5 hours of incubation, temperature and pH optimum was 85 °C, pH 8.5, and fructose as a carbon source. Thermozyme amylase converts starch into sugar under optimum conditions with a yield of 9.8% bioethanol

World biocatalysis & biocatalyst market analysis to 2024 - market forecast to grow at a CAGR of 15% from 2019 to 2024

The purpose of the study is isolation and application of novel Hot spring bacterial enzymes. It also reports on purification and characterization of thermostable α-amylase from a newly hot spring isolate, Exiguobacterium sp. This thermostable amylase is Ca2+-independent an added improvement in starch saccharification process at a higher temperature because it removes the addition of Ca2+ for improving the stability of amylases. Maximum enzyme activity was obtained at 45 °C at pH 8.5 and stability at concentration of 3.0% NaCl. Thermostable α-amylase from Exiguobacterium sp was purified by 3.9 fold with 54.6% recovery and specific activity was 1083 U/ml. The molecular weight of α-amylase was 54 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Apparent Km and Vmax value was 5.88 mg/ml and 250µmol/min/ml, respectively for the hydrolysis of soluble starch. An initial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the amylase is predominantly turn structure and a detailed structural composition showed alpha helix 10.8%, Beta sheet 27.1%, Turn 29.8% and Randomness 32.3% respectively. The amylase combined with soap-nut extract was able to de stain blood stained cloth within 30 min

Preparation of a Poly(ethylene glycol)‐Based Cross‐Linked Network from a Click Reaction for Enzyme Immobilization

AbstractIn this study, a polyethylene glycol (PEG)‐maleimide film was prepared by a thiol‐ene click reaction. The covalent immobilization of amylase was carried out onto the PEG‐maleimide based photo‐curable network via epoxy and C=O groups without any linker. The epoxy and carbonyl groups on the PEG backbone improved the affinity of the enzyme. The immobilization yield of amylase onto the film was 72.46%. The immobilization capacity was also determined as 313 mg/g. The thermal stability of amylase improved via covalent attachment. Immobilized amylase showed maximum activity at 40 °C and pH 6.5. The immobilized amylase continued to retain 66% of its initial activity after 30 days.

Immobilization and Characterization of Bacillus Thuringiensis HCB6 Amylase in Calcium Alginate Matrix

Free enzyme in solution react with substrates to result in products which cannot be recovered for reuse. These problems can be overcome to a certain extent by the use of enzyme immobilization method. Immobilized enzymes are more robust and more resistant to condition changes. More importantly, the heterogeneous immobilized enzyme systems allow an easy recovery of both enzymes and products, multiple re-uses of enzymes, and continuous operation of enzymatic processes. Entrapment of enzymes in Ca-alginate is one of the simplest methods of immobilization. The aim of this research was to obtain the optimum condition of the making of immobilized amylase beads using a Ca-alginate bead and to determine its characteristics. The optimization of immobilized amylase beads includes variation of sodium alginates and variations of enzyme contact time with CaCl2. The characterization of immobilized amylase includes determination of optimum substrate concentration, optimum pH, and optimum incubation time as well as amylase stability test. Amylase activity was determined by using dinitro salicylic (DNS) method. The results showed that the optimum immobilized amylase obtained at alginate concentrations of 5% (w/v), contact time of 60 minutes and immobilization efficiency of 67.5%. Furthermore, immobilized amylase showed optimum substrate concentration of 1.5-2.5% (w/v), optimum pH of 6, an optimum incubation time of 20 minutes with the activity of 179.8 U/mL. The KM value for free amylase and immobilized amylases were 0.3 mM and 0.12 mM respectively. Vmax value for free amylase and immobilized amylases were 105.3 U/mL and 10.1 U/mL respectively. Immobilized Amylase can be used up to six times with the residual activity of 52.7%.

Model of the regulation of activity of immobilized enzymes (amylases) in soil

The preservation of activity of extracellular enzymes in soil is presently associated with their immobilization on organic or inorganic carriers. Enzyme immobilization results, however, in a significant decrease in enzymatic activity. In the present work, the mechanism responsible for promotion of the catalytic activity was revealed, as well as the favorable effect of low-molecular alkylhydrozybenzenes of the class of alkylresorcinols, which are common in soil organic matter, on stability of immobilized enzymes (exemplified by amylases) by their post-translational modification. Optimal conditions (enzyme to sorbent ratio, pH optimum, CaCl2 concentration, and sorption time) for amylase sorption on a biological sorbent (yeast cell walls) were determined and decreased activity of the immobilized enzyme compared to its dissolved state was confirmed. Alkylresorcinols (C7AHB) at concentrations of 1.6 to 80 mM were found to cause an increase of amylase activity both in the case of already sorbed enzymes (by 30%) and in the case of a free dissolved enzyme with its subsequent immobilization (by 50–60%). In both cases, the optimal C7AHB concentration was 16 mM. Amylase stability was determined for C7AHB-modified and unmodified enzymes immobilized on the biological sorbent after two cycles of freezing (–20°C) and thawing (4°C). Inverse dependence was revealed between increasing stability of C7AHB-modified enzymes and an increase in their activity, as well as higher stability of immobilized modified amylases than of the dissolved modified enzyme. Investigation of the effect of C7HOB-modification in the preservation of activity in immobilized amylases after four freeze–thaw cycles revealed: (1) better preservation of activity by the modified immobilized enzymes compared to immobilized ones; (2) differences in the dynamics of activity loss within compared pairs, with activity of immobilized amylases decreasing after the second cycle to a lower level (42%) than activity of the modified immobilized enzymes after the fourth cycle (48%). These results demonstrate that in the preservation of activity of extracellular enzymes in soil both stabilization mechanisms are of importance: immobilization on organic carriers and modification of the enzyme conformation by low-molecular compounds with the functions of chemical chaperones.

Optimization of Thermo-Alkali Stable Amylase Production and Biomass Yield from Bacillus sp. Under Submerged Cultivation

The present context was investigated to optimize amylase production and cell biomass of poultry-associated Bacillus sp. using a conventional as well as statistical approach. Box-Behnken design (BBD) matrix at N = 29 was employed to optimize four independent variables, selected from one factor at a time (OFAT) technique, for maximum amylase production and biomass yield. The relative activity of crude amylase obtained from the isolate showed stability at high temperature (60 °C) and alkaline condition (pH 9) up to 4 h of incubation, thereby indicating its alkali-tolerant and thermo-stable property. The BBD resulted in enhanced amylase activity of 145.32 U/mL when the basal medium was slightly acidic (pH 6) and kept at a temperature of 35 °C with the shaking speed of 130 rpm, in addition to being incubated for 24 h. The selected factors, when employed with this statistical optimization approach, showed 1.5-fold and 2-fold enhancements in the amylase production and biomass yield respectively compared to the OFAT method. Analysis of variance (ANOVA) revealed high coefficient of determination (R2) of 0.96 to 0.99 for both the responses at significant level (p < 0.05). Three-dimensional response and 2D contour plot of the quadratic model showed interdependent interaction between the effective variables. Long-term thermo-alkali stability of amylase obtained from Bacillus sp. suggested not only its wide applications in pharmaceutical, food and biotechnological industries, but also suggested a potent replacement of existing amylases on the market.

Thermostable amylase production from hot spring isolate Exiguobacterium sp: A promising agent for natural detergents

The purpose of the study is isolation and application of novel Hot spring bacterial enzymes. It also reports on purification and characterization of thermostable α-amylase from a newly hot spring isolate, Exiguobacterium sp. This thermostable amylase is Ca2+-independent an added improvement in starch saccharification process at a higher temperature because it removes the addition of Ca2+ for improving the stability of amylases. Maximum enzyme activity was obtained at 45°C at pH 8.5 and stability at concentration of 3.0% NaCl. Thermostable α-amylase from Exiguobacterium sp was purified by 3.9 fold with 54.6% recovery and specific activity was 1083U/ml. The molecular weight of α-amylase was 54kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Apparent Km and Vmax value was 5.88mg/ml and 250µmol/min/ml, respectively for the hydrolysis of soluble starch. An initial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the amylase is predominantly turn structure and a detailed structural composition showed alpha helix 10.8%, Beta sheet 27.1%, Turn 29.8% and Randomness 32.3% respectively. The amylase combined with soap-nut extract was able to de stain blood stained cloth within 30min

Characterization of digestive enzymes from de-oiled mackerel (Scomber japonicus) muscle obtained by supercritical carbon dioxide and n-hexane extraction as a comparative study

The oil in mackerel muscle was extracted using an environmental friendly solvent, supercritical carbon dioxide (SC-CO2) at a semi-batch flow extraction process and an n-hexane. The SC-CO2 was carried out at temperature 45°C and pressures ranging from 15 to 25MPa. The flow rate of CO2 (27g/min) was constant at the entire extraction period of 2h. The highest oil extracted residues after SC-CO2 extraction was used for activity measurement of digestive enzymes. Four digestive enzymes were found in water soluble extracts after n-hexane and SC-CO2 treated samples. Amylase, lipase and trypsin activities were higher in water soluble extracts after SC-CO2 treated samples except protease. Among the four digestive enzymes, the activity of amylase was highest and the value was 44.57uM/min/mg of protein. The water soluble extracts of SC-CO2 and n-hexane treated mackerel samples showed same alkaline optimum pH and pH stability for each of the digestive enzymes. Optimum temperature of amylase, lipase, protease and trypsin was 40, 50, 60 and 30°C, respectively of both extracts. More than 80% temperature stability of amylase, lipase, protease and trypsin were retained at mentioned optimum temperature in water soluble extracts of both treated samples. Based on protein patterns, prominent protein band showed in water soluble extracts after SC-CO2 treated samples indicates no denaturation of protein than untreated and n-hexane.

Microencapsulation of Purified Amylase Enzyme from Pitaya (Hylocereus polyrhizus) Peel in Arabic Gum-Chitosan using Freeze Drying

Amylase is one of the most important enzymes in the world due to its wide application in various industries and biotechnological processes. In this study, amylase enzyme from Hylocereus polyrhizus was encapsulated for the first time in an Arabic gum-chitosan matrix using freeze drying. The encapsulated amylase retained complete biocatalytic activity and exhibited a shift in the optimum temperature and considerable increase in the pH and temperature stabilities compared to the free enzyme. Encapsulation of the enzyme protected the activity in the presence of ionic and non-ionic surfactants and oxidizing agents (H2O2) and enhanced the shelf life. The storage stability of amylase is found to markedly increase after immobilization and the freeze dried amylase exhibited maximum encapsulation efficiency value (96.2%) after the encapsulation process. Therefore, the present study demonstrated that the encapsulation of the enzyme in a coating agent using freeze drying is an efficient method to keep the enzyme active and stable until required in industry.

Enhanced production and characterization of a solvent stable amylase from solvent tolerant Bacillus tequilensis RG-01: thermostable and surfactant resistant.

Ten bacterial strains isolated from the soil samples in the presence of cyclohexane were screened for amylase production. Among them, culture RG-01 was adjudged as the best amylase producer and was identified as Bacillus tequilensis from MTCC, Chandigarh. The isolate showed maximum amylase production (8100 U/mL) in the presence of starch, peptone, and Ca2+ ions at 55°C pH 7.0 within 24 h of incubation. The enzyme was stable in the presence of n-dodecane, isooctane, n-decane, xylene, toluene, n-hexane, n-butanol, and cyclohexane, respectively. The presence of benzene, methanol, and ethanol marginally reduced the amylase stability, respectively. The enzyme was showed it 100% activity at 55°C and pH 7.0 with 119% and 127% stability at 55°C and pH 7.0, respectively. The enzyme was also stable in the presence of SDS, Tween-40, Tween-60, and Tween-80 (1%) and was found stimulatory effect, respectively. Only Triton-X-100 showed a moderate inhibitory effect (5%) on amylase activity. This isolate (Bacillus tequilensis RG-01) may be useful in several industrial applications owing to its thermotolerant and organic solvents and surfactants resistance characteristics.

Improved yield and stability of amylase by multipoint covalent binding on polyglutaraldehyde activated chitosan beads: Activation of denatured enzyme molecules by calcium ions

In this study raw starch digesting amylase (RSDA) from Aspergillus carbonarius (Bainier) Thom IMI 366159 was stabilized by covalent binding on polyglutaraldehyde (PG), glutaraldehyde (G) activated chitosan beads or post immobilization cross linking of enzyme adsorbed on chitosan. Presence of Ca2+ ions (0.5–1.5mM) activated the PG and G derivatives but repressed the crosslinked enzyme. Optimum pH for cross linked derivative increased by 2units but was unaltered for PG and G derivatives. Immobilized amylase exhibited improved thermal and storage stability. Immobilized derivatives had no loss of activity after 1month storage and retained above 90% activity after 10 batch reactions of 60min each. Immobilization successfully stabilized RSDA and immobilized enzyme from A. carbonarius can be applied in numerous industries for cheap, cost effective and environmentally friendly starch hydrolytic processes to simple sugars.

Structure‐based replacement of methionine residues at the catalytic domains with serine significantly improves the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica

The alkaline amylase requires high resistance towards chemical oxidation for use in the detergent and textile industries. This work aims to improve the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica by site-directed mutagenesis based on the enzyme structure model. Five mutants were created by individually replacing methionine at positions 145, 214, 229, 247, and 317 in the amino acid sequence of alkaline amylase with oxidative-resistant serine. The pH stability of the mutant enzymes was almost the same as that of the wild-type (WT) enzyme (pH 7.0-11.0). The stable temperature range of the mutant enzymes M145S and M247S decreased from <50 °C of the WT to <40 °C, while the thermal stability of the other three mutant enzymes (M214S, M229S, and M317S) was almost the same as that of the WT enzyme. The catalytic efficiency (k(cat)/K(m)) of all the mutant enzymes decreased when compared to WT enzyme. The mutant enzymes showed increased activity in the presence of surfactants Tween-60 and sodium dodecyl sulfate. When incubated with 500 mM H(2)O(2) at 35 °C for 5 h, the WT enzyme retained only 13.3% of its original activity, while the mutant enzymes M145S, M214S, M229S, M247S, and M317S retained 55.6, 70.2, 54.2, 62.5, and 46.4% of the original activities, respectively. The results indicated that the substitution of methionine residues at the catalytic domains with oxidative-resistant serine can significantly improve the oxidative stability of alkaline amylase. This work provides an effective strategy to improve the oxidative stability of amylase, and the high oxidation resistance of the mutant enzymes shows their potential applications in the detergent and textile industries.

Mathematical modeling of maize starch liquefaction catalyzed by α-amylases from Bacillus licheniformis: effect of calcium, pH and temperature

The first step of starch hydrolysis, i.e. liquefaction has been studied in this work. Two commercial α-amylases from Bacilllus licheniformis, known as Termamyl and Liquozyme have been used for this purpose. Using starch as the substrate, kinetics of both enzymes has been determined at optimal pH and temperature (pH 7, T = 80 °C) and at 65 °C and pH 5.5. Michaelis-Menten model with uncompetitive product inhibition was used to describe enzyme kinetics. Mathematical models were developed and validated in the repetitive batch and fed-batch reactor. Enzyme inactivation was described by the two-step inactivation model. All experiments were performed with and without calcium ions. The activities of both tested amylases are approximately one hundred times higher at 80 °C than at 65 °C. Lower inactivation rates of enzymes were noticed in the experiments performed at 65 °C without the addition of calcium than in the experiments at 80 °C. Calcium ions in the reaction medium significantly enhance amylase stability at 80 °C and pH 7. At other process conditions (65 °C and pH 5.5) a weaker calcium stabilizing effect was detected.

Characterization of the activity and stability of amylase from saliva and detergent: Laboratory practicals for studying the activity and stability of amylase from saliva and various commercial detergents

This article presents two integrated laboratory exercises intended to show students the role of α-amylases (AAMYs) in saliva and detergents. These laboratory practicals are based on the determination of the enzymatic activity of amylase from saliva and different detergents using the Phadebas test (quantitative) and the Lugol test (qualitative) under different conditions (e.g. variations in temperature and alkalinity). This work also proposes the study of enzyme stability in the presence of several surfactants and oxidizing agents using the same technical approach. The proposed laboratory exercises promote the understanding of the physiological function of this enzyme and the biotechnological applications of AAMYs in the detergent industry. The exercises also promote the understanding that the enzymatic stability and performance are dependent on the organism of origin, and if necessary, these properties could be modified by genetic engineering. In addition, this article reinforces the development of laboratory skills, problem-solving capabilities, and the ability to write a laboratory report. The exercises are proposed primarily as an undergraduate project for advanced students in the biochemical and biotechnological sciences. These laboratory practicals are complementary to the previously published BAMBED article (Biochemistry and Molecular Biology Education Vol. 39, No. 4, pp. 280-290, 2011) on detergent proteases.

Protease and Amylase Stability in the Presence of Chelators Used in Laundry Detergent Applications: Correlation Between Chelator Properties and Enzyme Stability in Liquid Detergents

AbstractChelators are a common ingredient in most laundry detergents. They have a number of different functions such as reducing water hardness, assisting in keeping particulate soil in suspension and the removal of certain stains, thus complementing the action of the anionic surfactants. Another important group of components in a modern liquid detergent is enzymes, mainly proteases and amylases. As the most commonly used enzymes within the detergent industry are dependent on bound calcium ions to maintain conformational stability and function, the presence of both chelators and enzymes in a liquid detergent presents a challenge. The three commonly used Ca2+ chelators: citrate, DTPA (diethylene triamine pentaacetic acid) and HEDP (1‐hydroxyethane‐1,1‐diyl)bis(phosphonic acid), were studied with regard to their impact on protease and amylase stability in buffer and in a model liquid detergent. Enzyme stability was characterized by differential scanning calorimetry (DSC) and activity studies, and correlated to the chelator‐Ca2+ interaction properties. The results show that a chelator's ability to reduce water hardness and its Ca2+ affinity are in reality two separate aspects in the context of their use in liquid detergents. In the presence of DTPA, stoichiometric surplus of free Ca2+ is required to maintain sufficient amylase and protease stability. In the presence of the weaker chelators, HEDP and citrate, the total Ca2+ concentration is more important to protein stability than stoichiometric balancing between chelator and Ca2+. Thus, for these chelators their total concentration only has a minor impact on the Ca2+ concentration required to maintain or improve enzyme storage stability. The results underline the importance of Ca2+ in liquid detergent formulations, and suggest how proper balancing of chelators and Ca2+ can be used to improve overall enzyme stability.

Biochemical characterization of digestive amylase of wheat bug, Eurygaster maura (Hemiptera: Scutelleridae)

Biochemical characterization of
-amylase in the midgut and salivary glands of Eurygaster maura was conducted. Results showed that
-amylase activities were present in the salivary glands and gut. The activity of
-amylase in the midgut and in the salivary glands was 0.098 and 0.057 U/ml, respectively. The pH of salivary glands and the gut was determined to be in the range of 5- 5.5 (for the salivary glands) and in the range of 6-6.5 (for the gut), using staining indicator. The optimum pH and temperature for salivary glands and midgut amylase activity was 6-7 and 35-40oC, respectively. The stability of amylase was highest in the acidic pH (4-5). Ethylenediamine tetraacetic acid (EDTA), urea, sodium dodecyl sulfate (SDS) and Mg 2+ inhibited the enzyme activity but, NaCl and KCl enhanced enzyme activity. Based on linear regression analysis of reciprocal starch concentration versus reciprocal amylase activity K m and V max were 0.11% and 0.04 mM maltose/min for midgut amylase and 0.298% and 0.071 mM maltose/min for salivary amylase, respectively. Sodium dodecyl sulfate electrophoresis (SDS-PAGE) showed that both midgut and salivary glands contain isozymes.

Heat activation and stability of amylases from Bacillus species

Leitch and Collier sporulating Bacillus medium was used to isolate some strains of Bacillus species from soil, wastewater and food sources in Ibadan, Oyo State, Nigeria, by heat activation method. Heat treatment at 80oC allowed the growth of sporulating Bacillus species, in the culture sample source without other bacteria forms. The amylolytic Bacillus species isolated during the study were identified as Bacillus macerans, Bacillus coagulans Bacillus licheniformis, Bacillus circulans, Bacillus megaterium, Bacillus polymyxa and Bacillus subtilis. Heat treatment at 70oC denatured the
-amylase component of the amylase source while -amylase retained its potency at this temperature. Calcium cations (Ca2+) enhance the enzyme production than Na+ which was less effective. Physiological studies show that an optimum temperature of 40oC was suitable for the enzyme activity while temperature above 60oC reduced its activity unless positive measures are taken to stabilize it with relevant cations like Ca2+.