Extracellular Amylase Production Research Articles

Production and Optimization of Extracellular α-amylase From Halophilic Bacteria Cytobacillus oceanisediminis Isolated From Wadi-El-Natrun, Egypt

The current study aimed to maximize α-amylase enzyme production by halophilic bacteria Cytobacillus oceanisediminis isolated from soil of Wadi-El-Natrun, Egypt. The results showed that of 21 halophilic bacterial isolates recovered from saline soil samples, eight isolates were high amylase producers. Interestingly, the bacterial isolate AHB6 exhibited the highest extracellular amylase production and was selected for further studies. This isolate was molecularly identified based on the 16srRNA gene sequencing as Cytobacillus oceanisediminis and has the accession number OM759999. The optimum temperature for α-amylase enzyme was 45 °C and exhibited thermostability at 45 °C for 1 h and retained more than 80% of its original activity. The α-amylase enzyme was active in almost all tested pHs and reached its highest activity at pH 9. Also, the optimum culture conditions for α-amylase enzyme production were wheat bran as carbon source, 2 g/l K2HPO4 as potassium source, 6 g/l MgSO4 as magnesium source and 0.5 g/l KNO3 as nitrogen source. These findings suggest the applicability of bacterial isolate Cytobacillus oceanisediminis as a potent producer of α-amylase for industrial purposes. Keywords: Amylase; Optimization; Cytobacillus oceanisediminis; Starch hydrolysis; Thermo-halophilic bacteria.

A Novel Halo-Acid-Alkali-Tolerant and Surfactant Stable Amylase Secreted from Halophile Bacillus siamensis F2 and Its Application in Waste Valorization by Bioethanol Production and Food Industry.

The extracellular amylase production level by the moderate halophile Bacillus siamensis F2 was optimized, and the enzyme was biochemically characterized. The culture parameters for NaCl, carbon, nitrogen, pH, and temperature were optimized for high titers of amylase production. Growing B. siamensis F2 cultures in Great Salt Lake-2 medium with additions of (in g/L) NaCl (100), starch (30), yeast extract (2), KNO3 (2), and MgSO4 (1) at pH 8, 30 °C resulted in the maximum amylase production (4.2 U/ml). The amylase was active across a wide range of salinities (0 to 30% NaCl), pH (5.0-10.0), and temperatures (20-70 °C) and showed good stability with surfactants (sodium dodecyl sulfate (SDS) and Triton X-100); hence, it was identified as halo-acid-alkali-tolerant and surfactant stable. Temperature, pH, and salinity were optimal for amylase activity at 50 °C, pH 7, and 5% NaCl, respectively. It also generates amylase by utilizing agricultural wastes like sugarcane bagasse, sweet potato peel, and rice husk. Based on the performance of B. siamensis F2 using agricultural wastes and synthesizing amylase, the current study attempted to produce bioethanol by coculturing with baker's yeast using sugarcane bagasse and sweet potato peel as a substrate, which yielded 47 and 57 g/L of bioethanol, respectively. Besides bioethanol production, amylase secreted by F2 was also employed for juice clarification for better yield and clarity and for softening dough to produce better-quality buns. This novel amylase may have many potential applications in waste valorization, biorefinery sectors, and food industries.

Production of extracellular amylase contributes to the colonization of Bacillus cereus 0–9 in wheat roots

BackgroundBacteria usually secrete a variety of extracellular enzymes to degrade extracellular macromolecules to meet their nutritional needs and enhance their environmental adaptability. Bacillus cereus 0–9, a biocontrol bacterial strain isolated from wheat roots, has three genes annotated as encoding amylases in the genome, but their functions are unknown, and whether they are involved in the colonization process of the bacterium remains to be further studied.MethodsMutant gene strains and fluorescently tagged strains were constructed by homologous recombination, and amylase protein was expressed in the prokaryotic Escherichia coli BL21(DE3) expression system. The iodine staining method was used to measure the activity of amylase proteins. We further observed the colonization abilities of the test strains in wheat roots through frozen section technology.ResultsThe results showed that there were three amylase-encoding genes, amyC, amyP and amyS, in the B. cereus 0–9 genome. Among the three amylase encoding genes, only amyS produced extracellular amylase whose secretion was related to signal peptide at position 1–27. The AmyS protein encoded by the amyS gene is an α-amylase. The growth of Rhizoctonia cerealis was inhibited 84.7% by B. cereus 0–9, but the biocontrol ability of the ΔamyS strain decreased to 43.8% and that of ΔamyS/amyS was restored when the amyS gene was complemented. Furthermore, the biocontrol ability of the ΔamySec strain was decreased to 46.8%, almost the same as that of the ΔamyS mutant. Due to the deletion of the amyS gene, the colonization capacities of ΔamyS (RFP) and ΔamySec (RFP) in wheat roots decreased, while that of ΔamyS/amyS (RFP) was restored after the amyS gene was complemented, indicating that the amyS gene influences the colonization of B. cereus 0–9 in wheat roots. In addition, the colonization and biocontrol abilities of the mutant were restored after the addition of sugars, such as glucose and maltose.ConclusionsB. cereus 0–9 encodes three genes annotated as amylases, amyC, amyP and amyS. Only the deletion of the amyS gene with a signal peptide did not produce extracellular amylase. The AmyS protein encoded by the amyS gene is an α-amylase. Our results indicated that the amyS gene is closely related to the colonization abilities of B. cereus 0–9 in wheat roots and the biocontrol abilities of B. cereus 0–9 to fight against R. cerealis. The extracellular amylase produced by B. cereus 0–9 can hydrolyze starch and use glucose, maltose and other nutrients to meet the needs of bacterial growth. Therefore, it is very possible that the secretion and hydrolytic activities of extracellular amylase can promote the colonization of B. cereus 0–9 in wheat roots and play important roles in the prevention and control of plant diseases. Our results contribute to exploring the mechanisms of microbial colonization in plant roots.

Optimization of Amylase Production From Nocardiopsis sp. DW-4 Isolated From Dwarka, Coastal Region of Gujarat

Halophilic actinomycete Nocardiopsis sp. DW-4 was isolated from Dwarka, the coastal region of Gujarat. The isolate was studied for the extracellular amylase production. The DW-4 strain displayed diversity with respect to growth patterns and production of extracellular amylase. ‘One factor variable at time’ method was used to obtain maximum amylase production. The influence of starch, salt and pH was studied on the amylase production and growth of DW-4. The isolate DW-4 could grow and produce amylase at 5-15% salt and starch 1-3% and produced optimum amylase at 5% NaCl and pH 8. Optimization of the amylase production in conjunction with the growth of the organisms is significant.

Production of extracellular amylase by Aspergillus niger under submerged fermentation using jack fruit rag as the carbon source

Production of extracellular amylase by Aspergillus niger was studied under submerged fermentation using jackfruit rag as the carbon source. Different parameters, such as incubation period, pH of the culture broth and level of substrate were changed to optimise the conditions for amylase production. Maximum enzyme production ~ 8400 units/g was obtained in 5 days old cultures, grown at pH, 6.5 and 30°C with substrate level 20 gL -1 . As nitrogen sources NH 4 Cl, KNO 3 casein, peptone and beef extract were tested. Except NH 4 Cl all other sources enhanced the amylase production. Study on the kinetics of extracellular and intracellular amylase production revealed that extracellular amylase production was always higher than that in intracellular. Crude amylase obtained from culture broth was partially purified by ammonium sulphate fractionation followed by DEAE Cellulose chromatography. Partially purified enzyme exhibited optimum pH and incubation temperature at pH 6 and 60 ° C respectively and higher thermal and pH stability at 50-60°C and pH 5-7 respectively and enhanced activity with Ca 2+ . These unique features of the enzyme indicates its suitability for various industrial applications. Shorter incubation period and lower substrate cost offer the potential for inexpensive production of amylase, making the process industrially and economically feasible.

Aspergillus oryzae S2 AmyA amylase expression in Pichia pastoris: production, purification and novel properties.

A synthetic cDNA-AmyA gene was cloned and successfully expressed in Pichia pastoris as a His-tagged enzyme under the methanol inducible AOX1 promoter. High level of extracellular amylase production of 72 U/mL was obtained after a 72h induction by methanol. As expected, the recombinant strain produced only the AmyA isoform since the host is a protease deficient strain. Besides, the purified r-AmyA showed a molecular mass of 54kDa, the same pH optimum equal to 5.6 but a higher thermoactivity of 60°C against 50°C for the native enzyme. Unlike AmyA which maintained 50% of its activity after a 10-min incubation at 60°C, r-AmyA reached 45min. The higher thermoactivity and thermostability could be related to the N-glycosylation. The r-AmyA activity was enhanced by 46% and 45% respectively in the presence of 4mM Fe2+ and Mg2+ ions. This enzyme was more efficient in bread-making since such ions were reported to have a positive impact on the nutriment quality and the rheological characteristics of the wheat flour dough. The thermoactivity/thermostability as well as the iron and magnesium activations could also be ascribed to the presence of an additional C-terminal loop containing the His tag.

Fermentative production of extracellular amylase from novel amylase producer, Tuber maculatum mycelium, and its characterization

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22 ± 2 °C) under static conditions which resulted in 1.41 U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50 °C. The enzyme showed good thermostability at 50 °C by retaining 98% of the maximal activity after 100 min of incubation. The amylase activity was marginally enhanced in presence of Cu2+ and Na+ and slightly reduced by K+, Ca2+, Fe2+, Mg2+, Co2+, Zn2+, and Mn2+ ions at 1 mM concentration.

Media optimization for extracellular amylase production by Pseudomonas balearica vitps19 using response surface methodology

In this study, we optimized the process for enhancing amylase production from Pseudomonas balearica VITPS19 isolated from agricultural lands in Kolathur, India. Process optimization for enhancing amylase production from the isolate was carried out by Response Surface Methodology (RSM) with optimized chemical and physical sources using Design expert v.7.0. A central composite design was used to evaluate the interaction between parameters. Interaction between four factors–maltose (C-source), malt extract (Nsource), pH, and CaCl2 was studied. The factors pH and CaCl2 concentration were found to affect amylase production. Validation of the experiment showed a nearly twofold increase in alpha amylase production. Amylase production was thus optimized and increased yield was achieved.

Enhanced Production of α-Amylase from <I>Bacillus licheniformis</I> SKB 4

The effect of some inductive factors including incubation temperature, aeration, inorganic phosphate source, supplementation of amino acids and vitamins on production of extracellular amylase by Bacillus licheniformis SKB 4 was optimized in a submerged fermentation. The maximum production of enzyme was obtained in highly aerobic condition at 42°C, pH 6.5 and 24 h incubation period. 1%, v/v inoculumn concentration exerts best result for growth and enzyme production. Several chemical factors such as inorganic phosphate (KH 2 PO 4 ; 0.3%), amino acids (0.5 mg %; glutamic acid, cysteine, isoleucine), vitamins (biotin, 50μg/ml; pyridoxine, 50μg/ml; riboflavin, 20μg/ml), metallic salt MgSO 4 (1 mM) and surfactant Tween 80 (0.075% (v/v) have positive effect on amylase production. Among the raw food grains used for amylase production, soya bean seed favoured enzyme production and rate production is much higher (2.5 fold) than the normal culture medium, whereas cereals and other pulses have no effect on enzyme production within normal incubation time. Use of raw food grain as culture medium and optimization of certain factors are the most important part for production of amylase in industrial sector.

Mode of Association, Enzyme Producing Ability and Identification of Autochthonous Bacteria in the Gastrointestinal Tract of Two Indian Air-Breathing Fish, Murrel (Channa punctatus) and Stinging Catfish (Heteropneustes fossilis)

The mode of association of epithelium-associated bacteria in the gastrointestinal (GI) tract of two Indian air-breathing fish species, the murrel, Channa punctatus and the stinging catfish, Heteropneustes fossilis was demonstrated through scanning and transmission electron microscopy (SEM and TEM). The SEM examination revealed substantial numbers of rod shaped bacterial cells associated with the microvillus brush borders of enterocytes in proximal (PI) and distal regions (DI) of the GI tract of both the fish species. The TEM investigation indicated endocytosis and translocation of bacteria in the microvilli. The isolated bacterial strains (two each from the PI and DI of murrel and stinging catfish) were quantitatively evaluated for their extracellular amylase, cellulase and protease production. All the bacterial strains exhibited high cellulolytic activity than that of amylolytic and proteolytic enzymes. Only two strains, CPF1 and CPF2, isolated from the PI of murrel exhibited high proteolytic activity. Maximum amylase activity was exhibited by the strain, HFH5, isolated from the DI of stinging catfish. Totally six most promising enzyme-producing autochthonous bacterial strains were identified based on partial 16S rRNA gene sequence analytical results. All the strains showed close (92–99 %) similarity to Bacillus licheniformis.

Optimization of the Medium for the Production of Extracellular Amylase by the Pseudomonas stutzeri ISL B5 Isolated from Municipal Solid Waste.

The management of municipal solid waste is one of the major problems of the present world. The use of microbial enzymes for sustainable management of the solid waste is the need of the time. In the present study, we have isolated a potent amylase producing strain (ISL B5) from municipal solid waste. The strain was identified as Pseudomonas stutzeri (P. stutzeri) both biochemically and by 16S rDNA sequencing. The optimization studies revealed that the strain ISL B5 exhibited maximum activity in the liquid media containing 2% starch (2.77 U/ml), 0.8% peptone (2.77 U/ml), and 0.001% Ca2+ ion (2.49 U/ml) under the pH 7.5 (2.59 U/ml), temperature 40°C (2.63 U/ml), and 25 h of incubation period (2.49 U/ml). The highest activity of crude enzyme has also been optimized at the pH 8 (2.49 U/ml).

Molecular Identification of Newly Isolated Bacillus Strains from Poultry Farm and Optimization of Process Parameters for Enhanced Production of Extracellular Amylase Using OFAT Method

Molecular Identification of Newly Isolated Bacillus Strains from Poultry Farm and Optimization of Process Parameters for Enhanced Production of Extracellular Amylase Using OFAT Method

Production and Characterization of α-Amylase from an Extremely Halophilic Archaeon, Haloferax sp. HA10.

Haloarchaea are found at very high concentrations in salt-conditioned environments, hence produce enzymes which are able to catalyze reactions under harsh conditions, typical of many industrial processes. In the present study, culture conditions for extracellular amylase production from Haloarchaea isolated from a solar saltern were optimized and the purified enzyme was characterized. Haloferax sp. HA10 showed maximum amylase production at 3 M NaCl, 37 °C, pH=7 and 1% starch content. Purified α-amylase was a calcium-dependent enzyme with an estimated molecular mass of about 66 kDa and many industrially useful properties. It was found to be stable in a broad range of pH (from 5 to 9) and NaCl concentrations (from 0.5 to 3.0 M), retaining 48% activity even at 4 M. The optimal temperature for Haloferax sp. HA10 amylase activity was 55 °C (99% activity), and 57% activity was retained at 80 °C, which dropped to 44% with the increase of temperature to 90 or 100 °C. It was able to sustain various surfactants and detergents. To the best of our knowledge the detergent-stable α-amylases from halophilic archaeon have not been reported yet.

Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase.

A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37°C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50°C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time.

Expression and Characterization of Geobacillus stearothermophilus SR74 Recombinant α-Amylase in Pichia pastoris.

Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL−1 at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg−1. The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0–8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t 1/2) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification.

Production and partial characterization of extracellular amylase enzyme from Bacillus amyloliquefaciens P-001

Amylases are one of the most important enzymes in present-day biotechnology. The present study was concerned with the production and partial characterization of extracellular amylase from Bacillus amyloliquefaciens P-001. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Enzyme production was induced by a variety of starchy substrate but corn flour was found to be a suitable natural source for maximum production. Tryptone and ammonium nitrate (0.2%) as nitrogen sources gave higher yield compared to other nitrogen sources. Maximum enzyme production was obtained after 48 hrs of incubation in a fermentation medium with initial pH 9.0 at 42°C under continuous agitation at 150 rpm. The size of inoculum was also optimized which was found to be 1% (v/v). Enzyme production was 2.43 times higher after optimizing the production conditions as compared to the basal media. Studies on crude amylase revealed that optimum pH, temperature and reaction time of enzyme activity was 6.5, 60°C and 40 minutes respectively. About 73% of the activity retained after heating the crude enzyme solution at 50°C for 30 min. The enzyme was activated by Ca2+ (relative activity 146.25%). It was strongly inhibited by Mn2+, Zn2+ and Cu2+, but less affected by Mg2+ and Fe2+.

Production ofHalothermotolerant α-Amylase from aModerately Halophilic Bacterium, NesterenkoniaStrain F.

Production of extracellular amylase was demonstrated under conditions of high salinity in aerobically cultivated culture of a newly isolated moderately halophilic Gram-positive coccus, designated strain F in basal medium containing peptone from meat, yeast extract, NaCl (7% w/v) and starch. Biochemical and physiological characterization along with 16S rRNA sequence analysis placed F in the genus Nesterenkonia. The enzyme production was synchronized bacterial growth and reached a maximum level during the early-stationary phase in the basal medium. Maximum yield production was observed when the soluble starch, yeast extract and NaCl (1%, 0.75% and 10% w/v, respectively) were used in the fermentation medium with pH 8.0, inoculated with 7% (v/v) pre-culture medium and incubated in 30°C for 72 h with aeration of 250 rpm (299.25 U/ml). The potential of different carbohydrates in amylase production was in the order: starch> maltose>dextrin>fructose>lactose=glucose>sodium acetate>sodium citrate. The production yield in the presence of different salts was as follows: NaCl> KCl> NaNO3> Na2SO4. Maximum activity for enzyme was assayed in 3.0 M NaCl, pH: 5-7, and 40°C. α-amylase hydrolyzed starch, dextrin and glycogen to form maltose, maltotriose and maltotetraose as major products, and has no effect on amylose and pullulan as substrates. These results suggest that the amylase secreted by Nesterenkonia sp. strain F is industrially important from the perspective of its tolerance to a broad temperature range and its high tolerance to a wide range of salt concentrations (0–3 M NaCl).

Optimization of conditions for extracellular amylase production from Listeria denitrificans

The foodborne Listeria denitrificans was isolated from degraded rice and studied as an amylase producer. This microorganism showed maximum amylase production at temperature 27 ºC and medium pH 7.0, and on 3rd day during incubation period. Maximum amylase production by the isolate was found in medium containing 3.0 % starch as carbon source and 0.50 % yeast extract as nitrogen source. DOI: http://dx.doi.org/10.3329/cujbs.v4i1.13388 The Chittagong Univ. J. B. Sci.,Vol. 4(1&2):73-81, 2009

Amylase production by moderately halophilic Bacillus cereus in solid state fermentation

The production of extracellular amylase by the moderately halophilic bacterium Bacillus cereus was studied in solid state fermentation (SSF). Solid substrates such as rice bran, wheat bran, sugarcane bagasse, black gram husk and green gram husk were studied for enzyme production. Growth on sugarcane bagasse gave the highest amylase activity. The time course for amylase production inferred that 48 h was the optimum duration for higher amylase production. The suitable pH, temperature and inoculum level observed for higher amylase production was pH 7.0, 40.0°C and 2.0%, respectively. The amylase production ofB. cereus was high in maltose (carbon source), yeast extract (nitrogen source), Tween 80 (surfactant) and calcium chloride (metal ion) added medium when compared to other respective sources tested. The halotolerancy of B. cereus for amylase production was 3% sodium chloride. The sugarcane bagasse can be utilized for the cheap production of amylase from Bacillus cereus. It is less expensive in comparison to synthetic media and is readily available for production purposes. Key words: Bacillus cereus, amylase, solid state fermentation (SSF), sugarcane bagasse.

Production and immobilization of a novel thermoalkalophilic extracellular amylase from bacilli isolate

A Thermoalkalophilic amylase was produced from an environmental bacterial isolate. The enzyme was then immobilized through its amino groups onto the epoxy rings of magnetic poly glycidyl methacrylate [m-poly (GMA)] beads. The free enzyme was active within a large pH range, between 7 and 12 and displayed the optimum activity at 95°C and pH 10. The immobilization appeared to increase the stability of the enzyme as its bound form showed optimum activity at 105°C and pH 11.0. Kinetic studies demonstrated that immobilized enzyme had higher Km and lower Vmax values. The activity of the free and bound enzyme was determined, at 37°C and pH 10.0 and pH 11.0, respectively, in the presence of various organic solvents and detergents (5%, v/v). Results obtained indicated that detergents, sodium dodecyl sulfate (SDS) and TritonX-100, caused six fold increase and that various organic solvents also increased the activity of the amylase.