Production Of Extracellular Α-amylase Research Articles

Optimization of culture conditions for enhanced production of extracellular α-amylase using solid-state and submerged fermentation from Aspergillus tamarii MTCC5152.

Amylases are one of the main enzymes used in various industries such as food, fermentation, textile, and pharmaceuticals. Microorganisms are the potent sources of amylase enzyme, apart from plant and animal sources. Fungal amylases are more stable than bacterial amylases. The production of extracellular α-amylase from Aspergillus tamarii MTCC5152 using solid-state and submerged fermentation (SSF and SmF) and the various nutritional factors influencing its production were studied. A higher activity of α-amylase (519.40u/g) was attained in a medium having wheat bran (WB) alone as the substrate at an initial moisture content of 70% (v/w) with 2.5% (v/w) of inoculum level (containing 106 spores/ml) after 4 days of incubation at 28°C by SSF. Addition of 1% glucose to WB containing basal medium enhanced α-amylase production (6.49u/ml) after 4 days of incubation by SmF method. Comparative evaluation of enzyme production by SSF and SmF methods produced better results in SSF method.

Production of extracellular α-amylase by single-stage steady-state continuous cultures of Candida wangnamkhiaoensis in an airlift bioreactor.

The kinetics of growth and α-amylase production of a novel Candida wangnamkhiaoensis yeast strain were studied in single-stage steady-state continuous cultures. This was performed in a split-cylinder internal-loop airlift bioreactor, using a variety of carbon sources as fermentation substrates. Results showed that the steady-state yields of cell mass from carbohydrates were practically constant for the range of dilution rates assayed, equaling 0.535 ± 0.030, 0.456 ± 0.033, and 0.491 ± 0.035 g biomass/g carbohydrate, when glucose, maltose, and starch, respectively were used as carbon sources. No α-amylase activity was detected when glucose was used as the carbon source in the influent medium, indicating that α-amylase synthesis of C. wangnamkhiaoensis is catabolically repressed by glucose. Contrastingly, maltose and starch induce synthesis of α-amylase in C. wangnamkhiaoensis, with starch being the best α-amylase inducer. The highest α-amylase volumetric and specific activities (58400 ± 800 U/L and 16900 ± 200 U/g biomass, respectively), and productivities (14000 ± 200 U/L·h and 4050 ± 60 U/g biomass·h, respectively) were achieved at a dilution rate of 0.24 h-1 using starch as the carbon source. In conclusion, single-stage steady-state continuous culture in an airlift bioreactor represents a powerful tool, both for studying the regulatory mechanisms of α-amylase synthesis by C. wangnamkhiaoensis and for α-amylase production. Furthermore, results showed that C. wangnamkhiaoensis represents a potential yeast species for the biotechnological production of α-amylase, which can be used for the saccharification of starch. This offers an attractive renewable resource for the production of biofuels (particularly bioethanol), representing an alternative to fossil fuels with reduced cost of substrates.

Optimization of Growth Conditions to Identify the Superior <i>Bacillus</i> Strain Which Produce High Yield of Thermostable Alpha Amylase

Thermostable α-amylases hold a very important place in commercial industrial applications in Sri Lanka. Therefore, the main aim of this study was to identify superior Bacillus strain and optimize growth conditions that could yield high α-amylase production. Three Bacillus strains, B. amyloliquefaciens ATCC 23350, B. licheniformis ATCC 14580 and B. megaterium ATCC 14581 were used for the study. Shake flask culture experiments were conducted to identify the effect of various fermentation conditions such as growth temperature, incubation period, carbon source, nitrogen source, initial pH and carbon concentration on extracellular α-amylase production. DNSA assay was carried out to determine the enzyme activity. The highest temperature for enzyme activity was reported by B. licheniformis at 85°C, followed by B. amyloliquefaciens at 75°C and B. megaterium at 45°C. Both B. amyloliquefaciens and B. licheniformis were able to give their optimum enzyme production at 37°C, while B. megaterium at 30°C in 150 rpm with initial pH of 7. B. licheniformis and B. amyloliquefaciens gave their optimum yield of the enzyme after 48 h of incubation while B. megaterium gave after 24 h of incubation. Among the carbon sources tested cassava starch was able to give the highest enzyme production. For B. amyloliquefaciens, the highest yield of the enzyme was obtained with 2% of starch, tryptone as a nitrogen source and initial pH of 7. Maximum enzyme production for B. licheniformis was obtained with 1.5% of starch, KNO3 as a nitrogen source and initial pH of 6. For B. megaterium 1% of starch, tryptone and pH 7.5 induced the optimum α-amylase production. According to the results obtained, B. amyloliquefaciens is the highest thermostable alpha amylase producer. However, according to the industrial requirement, B. licheniformis can also be used as an enzyme producer due to its stability in higher temperatures.

Production of Thermostable ?-Amylase Through Solid State Fermentation (SSF) by Using Thermophilic Anoxybacillus sp.

The production of extracellular α-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1) by a newly isolated thermophilic bacterium Anoxybacillus sp. was studied in solid state fermentation (SSF). Bacterial strain was isolated from a thermal spring of Omer, Afyonkarahisar in Turkey. Agricultural wastes such as banana husk, wheat bran, rice husk, apple bark, orange bark, maize oil cake, lentil bran and pistachio shell were used for α-amylase production as solid substrates. Growth on rice husk gave the highest α-amylase activity. The maximum enzyme activity obtained was 3.628 U/mg of under optimum conditions of an fermentation time of 48 h, an incubation temperature of 60°C, a pH of 6.0, a substrat particle size 1.500 µm, an initial moisture level of 60% and an inoculum level of 40% (v/w).

세포외 고온성 α-아밀라제를 생산하는 Alicyclobacillus acidocaldarius 의 분리 및 효소생산용 최적 배양 조건

A thermophilic Alicyclobacillus acidocaldarius, which produces thermostable α-amylase, was isolated from the hot water effluent of a boiled rice mill near Tirupati, Andhra Pradesh, India. The effect of different culture conditions on the growth and production of extracellular α-amylase by thermophilic A. acidocaldarius was investigated in laboratory scale. The results showed that the optimum conditions for the production of α-amylase are a temperature of 60°C, pH of 6.0, and medium starch concentration of 1.0%, and yeast extract and tryptone of 0.2%. Surfactants, like Tween-20 and SDS, up to 0.02%, were found to increase the bacterial growth and enzymes. Further increase in their concentration resulted in significantly decreased enzyme production.

High level production of extracellular β-galactosidase from Bacillus licheniformis ATCC 12759 in submerged fermentation

Alpha amylase (E.C. 3.2.1.1) of Bacillus licheniformis ATCC 12759 was shown to produce extracellular α-amylase in submerged fermentation.Various nutrients belonging to three categories, carbon, nitrogen and amino acid sources, were investigated in terms of their effect on the production of extracellular α-amylase by Bacillus licheniformis ATCC 12759. Amongst carbon sources sugars, arabinose, sucrose, corn flour and corn starch supported maximum amylase production. Casein, sodium nitrate, ammonium sulphate, ammonium chloride, and ammonium nitrate where the best organic sources and inorganic sources respectively. Among the amino acid sources tested, L-cysteine, L-tryptophane, Lvaline, L-phenylalanine, L-methionine and L-lysine were favored the production respectively. FeSO4, ZnSO4 and CuSO4 inhibited α-amylase production. Maximum α-amylase production (1074.8±35.0 U/mg) was obtained in a medium containing 0.01% L-cysteine in 72 h at 37 o C.

Production of Extracellular α-amylase by Streptomyces albidoflavus

Production of Extracellular α-amylase by Streptomyces albidoflavus

Enhancing α-Amylase and Cellulase in vivo Enzyme Expressions on Sago Pith Residue Using Bacilllus amyloliquefaciens UMAS 1002

The effect of Solid State Fermentation (SSF) parameters on the production of extracellular α-amylase and cellulase (endoglucanases) by Bacillus amyloliquefaciens UMAS 1002 using sago pith residue hampas in shaker flasks was investigated in this study. The appropriate incubation period, temperature, pH, agitation speed, inoculum concentration, hampas concentration and additive substrate effect were optimized for enzyme production. The activity of α-amylase and cellulase was at 14.19 and 13.15 IU mL-1, respectively in optimal culture medium. Maximum yield for both enzymes were achieved by employing 4% w/v hampas in 0.2 M citrate buffer at pH 6 and incubated at 40°C for 6 h with agitation speed of 100 rpm. Inoculum concentrations were found to be optimum at 3% v/v and 4% v/v for α-amylase and cellulase, respectively. Enzyme activity was 2.8 (10.80 IU mL-1) and 3.2 (9.38 IU mL-1) fold higher for α-amylase and cellulase respectively when 1% w/v soluble starch was applied as additive substrates with 0.5% hampas. However in optimal media that consist of 4%w/v of hampas, addition of 1% w/v soluble starch intend to inhibit both enzyme productions. Result revealed that temperature, pH and shaking condition were the most significant factors for the production of α-amylase and cellulase enzyme. Temperature influenced enzyme production by affecting the other parameters including bacterial growth, pH, Dissolved Oxygen (DO) and reducing sugars. Nevertheless, shaking condition could affect DO concentration that in turn affected bacterial growth and enzymes production too.

Solid state fermentation for production of α-amylase by a thermotolerant Bacillus subtilis from hot-spring water

The production of extracellular α-amylase by thermotolerant Bacillus subtilis was studied in solid state fermentation (SSF). The effect of wheat bran (WB) and rice husk (RH) was examined. The appropriate incubation period, moisture level, particle size and inoculum concentration was determined. Maximum yields of 159,520 and 21,760 U g −1 were achieved by employing WB and RH as substrates in 0.1 M phosphate buffer at pH 7 with 30% initial moisture content at 24 and 48 h. Particle size and inoculum concentration were found to be 1000 μm, 20% and 500 μm, 15% for WB and RH, respectively. Enzyme yield was 7.3-fold higher with WB medium compared with RH.

Optimization of α-amylase and glucoamylase production by recombinant strains ofSaccharomyces cerevisiae

Replacement of the regulatory sequence of theBacillus amyloliquefaciens α-amylase gene (AMY1) by the yeast alcohol dehydrogenase gene promoter (ADC1p) resulted in increased levels of extracellular α-amylase production inSaccharomyces cerevisiae. Negative regulation of glucoamylase synthesis by theSTA10-encoded repressor was alleviated by replacing the nativeSTA2 gene promoter fromS. cerevisiae var.diastaticus withADC1p. Enhanced degradation of starch was achieved when the modified versions of theAMY1 andSTA2 genes were introduced jointly intoS. cerevisiae.

Parametric optimization of extracellular α-amylase production by thermophilicBacillus coagulans

The culture parameters required for optimum production of extracellular α-amylase by the thermophilicBacillus coagulans are described. The optimum pH, temperature and incubation period for amylase production were 7, 50°C and 48 h, respectively. Age of inoculum (48 h) and its level, (2%) were critical for maximum amylase yield. The enzyme secretion was high in rice starch and beef extract as compared to other carbon and nitrogen sources tested. The addition of mustard oil cake (1%) and agitation at 1.7 Hz resulted in an enhancement of α-amylase secretion.

Stepwise introduction of regulatory genes stimulating production of .ALPHA.-amylase into Bacillus subtilis: Construction of an .ALPHA.-amylase extrahyper producing strain.

The production of extracellular α-amylase in Bacillus subtilis is probably regulated by many genetic elements, such as amyR, tmrA7, pap, amyB and sacU. Additional genetic elements, C-108 and A-2 for production of the α-amylase were found in D-cycloserine and ampicillin resistant mutants (C108 and A2) of B. subtilis 6160, respectively. Strain C108 increased the production of α-amylase about 5 times and protease about 80 times compared to parental 6160 strain. Strain A2 showed a nearly 6-fold increased α-amylase production.These genetic elements displayed a synergistic effect with other genetic factors in production of extracellular α-amylase when these elements were transferred by DNA mediated transformation. By stepwise introduction of these and other genetic elements into B. subtilis 6160 by transformation and mutation, strains with higher α-amylase producing activity were obtained. The finally obtained strain, T2N26, produced about 1,500-2,000 times more α-amylase than parental 6160 strain.

Transformation of Bacillus subtilis in alpha-amylase productivity by deoxyribonucleic acid from B. subtilis var. amylosacchariticus.

Deoxyribonucleic acid (DNA) of Bacillus subtilis var. amylosacchariticus showed almost the same ability as B. subtilis Marburg to induce transfer of several genetic markers in DNA-mediated transformation. DNA-DNA hybridization data also showed an intimate relationship between the two strains. Genetic elements involved in the production of extracellular alpha-amylase (EC 3.2.1.1.) in B. subtilis var. amylosacchariticus were studied by using DNA-mediated transformation. Two Marburg derivatives, NA20(amyR2) and NA20-22(amyR1), produced about 50 and 10 U of alpha-amylase per mg of cells, respectively, whereas B. subtilis var. amylosacchariticus produced as much as 150 U of the enzyme per mg of cells. When B. subtilis var. amylosacchariticus was crossed with strain NA20-22 as recipient, transformants that acquired high alpha-amylase productivity (about 50 U/mg of cells) were obtained. Genetic analysis revealed that a regulator gene (amyR) for alpha-amylase synthesis was found in B. subtilis var. amylosacchariticus, as in the case of B. natto 1212 (amyR2) and B. subtilis Marburg (amyR1). The allele was designated amyR3; it is phenotypically indistinguishable from amyR2, but is readily distinguishable from amyR1. The presence of amyR3 was not sufficient for an organism to render production of an exceptional amount of alpha-amylase. Extra-high alpha-amylase producers could be obtained by crossing B. subtilis var. amylosacchariticus as donor with strain NA20 as recipient. The transformants produced the same or even greater amounts of the enzyme than the donor strain. Results suggest the presence of another gene that is involved in the production of the exceptional amount of alpha-amylase.

Membrane mutation related to the production of extracellular α-amylase and protease in [formula omitted][formula omitted

Mutants which had a genetic character to increase the production of both α-amylase and protease simultaneously, were isolated from a transformable strain of Bacillus subtilis Marburg by NTG treatment. This mutation seems to have occurred at a single gene of the bacterial chromosome and was not linked to aro 116 which was closely linked to the α-amylase gene. When this mutation and an α-amylase regulator gene ( amyR h) coexisted in one strain, their synergistic effect on extracellular α-amylase production ws observed. The introduction of this mutation resulted in a loss of competence for the transformation. The SDS disc gel electrophoretic profiles of the membrane proteins from the original strain, the mutants and transformants with this mutation showed a remarkable difference in one component.

Production and Partial Characterization of Extracellular α-Amylase by <i>Trichoderma viride</i>

The production of extracellular α-amylase by the mesophilic fungus Trichoderma viride was optimized in shake-flask cultivation. Overall, the data imply that α-amylase from fungus was induced by a variety of starchy substrate but maximum enzyme production was stimulated by corn starch at a concentration of 4.0%. Enzyme synthesis was affected by nitrogen sources, and maximal activity was attained with inorganic than organic nitrogen sources. Effect of different salts in the culture medium was evaluated and it was found that Mg2+, Fe2+, K+ and, to some extent, Ca2+ and Na+ play an important role for optimum production of α-amylase by the fungus. Maximum enzyme production was obtained after 3 days of incubation in a fermentation medium with initial pH 5.0 at 30° under continuous agitation at 180 rpm. These properties make the enzyme suitable for industrial uses. Key words: Trichoderma viride, α-Amylase, Enzyme production optimization, Shake-flask cultureDOI: http://dx.doi.org/10.3329/bjm.v25i2.4870 Bangladesh J Microbiol, Volume 25, Number 2, December 2008, pp 99-103