Maximum Α-amylase Activity Research Articles

OPTIMIZATION OF α-AMYLASE SYNTHESIS BY BACILLUS VELEZENSIS USING TAGUCHI EXPERIMENTAL DESIGN

This research employs the Taguchi experimental design to optimize the production of α-amylase, a versatile enzyme with diverse industrial applications, using Bacillus velezensis. Thirteen key parameters, including pH, temperature, agitation, inoculum size, aeration and various carbon and nitrogen sources, were systematically investigated using the Taguchi L27 (313) orthogonal array. Initial screening of six seed media formulations identified SM6 (0.5% peptone, 0.5% yeast extract, 0.1% K2 HPO4 ) as the optimal medium, producing 2.8 U mL-1 of α-amylase. Further optimization under the Taguchi framework yielded a maximum α-amylase activity of 1097.31 U mL-1 and total protein of 1230 mg mL-1 at pH 5, 34°C, 4% moong husk as carbon source, and 2% soybean cake as nitrogen source. Two-factor interaction analysis revealed that a temperature of 34°C with 2% soybean cake, and 4% moong husk with 2% soybean cake, provided the best results. Validation experiments confirmed a 31.2% enhancement in α-amylase production under the optimized conditions. This study highlights the efficacy of the Taguchi design in systematically optimizing complex bioprocesses, paving the way for improved α-amylase synthesis with B. velezensis.

Bread spoilage fungi as creators of α amylase using two types of wheat flour

Fungal spoilage of bread can be a great problem; however, it can be explored as a producer of enzymes. The fungi were isolated from breads, and their activity for α-amylase production was planned. The results identified nine fungi on spoiled breads. Aspergillus fumigatus occurred with 85% frequency, followed by other isolates. Starch yeast (SY), white flour (WF), and black flour (BF) were applied as substrates for α-amylase activity using fungal isolates. The SY was the best, followed by WF and BF for α-amylase activity. Using SY, A. niger showed the greatest potency for α-amylase (7.67 U/mL) unlike Monilia sitophila, which reflected low α-amylase activity (2.69 U/mL). Using WF, A. fumigatus reflected high amylase activity (5.76 U/mL) while A. niger, A. terreus, and Penicillium expansum showed less activity (5.12 U/mL, 4.41 U/mL, and 3.56 U/mL, respectively). The temperature 30 °C and pH 6 were the optimum for α-amylase activity by A. niger, A. fumigatus, and P. chrysogenum, using the three media, but α-amylase activity of A. fumigatus at 40 °C was higher than at 20 °C. At the ninth day of incubation, the maximum α-amylase activity was reported using SY, while at the twelfth day, maximum activity was reported using WF and BF.

Purification and characterization of moderately thermostable raw-starch digesting α-amylase from endophytic Streptomyces mobaraensis DB13 associated with Costus speciosus.

Endophytic actinobacteria are known to produce various enzymes with potential industrial applications. Alpha-amylase is an important class of industrial enzyme with a multi-dimensional utility. The present experiment was designed to characterize a moderately thermostable α-amylase producing endophytic Streptomyces mobaraensis DB13 isolated from Costus speciosus (J. Koenig) Sm. The enzyme was purified using 60% ammonium sulphate precipitation, dialysis, and Sephadex G-100 column chromatography. Based on 12% SDS-PAGE, the molecular weight of the purified α-amylase was estimated to be 55 kDa. The maximum α-amylase activity was achieved at pH 7.0, 50°C and it retained 80% of its activity at both pH 7.0 and 8.0 after incubation for 2 h. The α-mylase activity is strongly enhanced by Ca2+, Mg2+, and inhibited by Ba2+. The activity remains stable in the presence of Tween-80, SDS, PMSF, and Triton X-100; however, β-mercaptoethanol, EDTA, and H2O2 reduced the activity. The kinetic parameters Km and Vmax values for this α-amylase were calculated as 2.53 mM and 29.42 U/mL respectively. The α-amylase had the ability to digest various raw starches at a concentration of 10 mg/mL at pH 7.0, 50°C, where maize and rice are the preferred substrates. The digestion starts after 4 h of incubation, which reaches maximum after 48 h of incubation. These results suggest that S. mobaraensis DB13 is a potential source of moderately thermostable α-amylase enzyme, that effciently hydrolyzes raw starch. It suggesting that this α-amylase is a promising candidate to be use for industrial purposes.

Characterization of purified ��-amylase produced by Aspergillus terreus NCFT 4269.10 using pearl millet as substrate

Abstractα-amylase was produced by Aspergillus terreus NCFT 4269.10 using both liquid static surface (LSSF) and solid-state fermentation using pearl millet residues as substrate. The maximum production of α-amylase was noticed at 30°C incubated for 96h. The crude α-amylase was purified to electrophoretic homogeneity and characterized. Characterization of amylase confirmed that the purified α-amylase was found to be most stable at pH 5.0, 60°C temperature, and a substrate concentration of 1.25%. The enzyme was active for 40 min at 70°C with an optimum enzyme–substrate reaction time of 60 min. Amylase was compatible with all detergents tested having highest activity with Surf excel followed by Henko and Ariel. SDS and Tween 20 reduced the activity. Among the metal ions tested, the maximum α-amylase activity was attained in the presence of Ca2+, followed by Mg2+ and Mn2+. The activity of α-amylase was not considerably affected in the presence of ethylenediaminetetraacetic acid and Triton X-100. Amylase activi...

Purification and characterization of thermostable α-amylase from thermophilic Anoxybacillus flavithermus

This study reports on the purification and characterization of thermostable α-amylase (α-1-4 d-glucan glucanohydrolase EC 3.2.1.1) from a newly isolated Anoxybacillus flavithermus. A. flavithermus was used, which was isolated from hot water springs of Ömer, Afyonkarahisar, Turkey. The gram-positive, spore-forming, motile, moderately thermophilic bacteria were found to be a strain of A. flavithermus analysed by 16S rRNA comparison. The optimal conditions for bacterial growth were determined to be at 20thh, 55°C and pH 6.0. Maximum α-amylase activity was obtained at 55°C at pH 7.0 after 24h of incubation. Thermostable α-amylase from A. flavithermus was purified by 70% (NH4)2SO4 and ion-exchange chromatography (5.2-fold; 65.8% yield). The molecular weight of α-amylase was 60kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The α-amylase hydrolyzed soluble starch at 55°C with Km: 0.005mM and Vmax: 3.5μmolmin−1.

Pigment, fatty acid and extracellular enzyme analysis of a fungal strainThelebolus microsporusfrom Larsemann Hills, Antarctica

ABSTRACTA cold-tolerant fungal strainThelebolus microsporuswas investigated for the first time for its pigment and fatty acid production. High-performance liquid chromatography analysis confirmed the presence of carotenoid pigment. Gas chromatography observations showed the presence of major fatty acids: myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), heptadecanoic acid (17:0), linolenic acid (18:3) and linoleic acid (18:2). Of these, linolenic acid, a polyunsaturated fatty acid, was present in substantial quantity, suggesting that it may have a role in adapting the fungus to low Antarctic temperatures by modulating membrane fluidity. The commercial application of linolenic acid is as a food supplement for humans suffering from eczema, cardiovascular disease and diabetic neuropathy. Another fatty acid, linoleic acid, a precursor of 1-octen-3-ol, is the principal aromatic compound in most fungi and has also been documented in this strain. Screening of the fungal culture for extracellular enzyme activity for amylase, protease, lipase, chitinase and cellulase was carried out. The isolate showed maximum α-amylase activity at 20°C, suggesting effective applications as a detergent additive, in textile processing and in the food industry.

Hyperactive α-amylase production by Aspergillus oryzae IFO 30103 in a new bioreactor

To improve the α-amylase production in solid-state fermentation (SSF) condition utilizing a new bioreactor (NB) system. In NB system, 20 g of wheat bran moistened with liquid medium in 1:1 ratio (w/v) was taken on the tray present inside the upper vessel and an additional 80 ml medium was supplemented into the lower vessel. Oxygen uptake rate was improved by supplying compressed air that lifted the liquid medium into the upper vessel and touched the substrate bed. This condition probably facilitated the heat transfer to liquid medium, reduce water loss and catabolite repression. With 1% glucose supplementation, maximum α-amylase activity of 22 317 Ugds(-1) was produced by Aspergillus oryzae IFO 30103 within a very short incubation period (48 h) at 2-cm bed height with air flow rate of 0·1 l min(-1) g(-1) wheat bran at 32°C and initial medium pH of 6. Within a short incubation period, significantly high α-amylase activity was obtained and it is higher than those reported to date at bioreactor scale operating with a fungal strain. The reactor is novel and can overcome some of the major problems associated with SSF process. A. oryzae IFO 30103 is reported as the best fungal source for α-amylase production.

A potential new isolate for the production of a thermostable extracellular - amylase

Two hundred and seventy Bacillus species were isolated from soil samples in Khartoum State and tested for α- amylase production. 20 potential isolates of α- amylase producer were obtained during primary screening. Secondary screening of these isolates yielded a high thermostable alkaline α- amylase producing isolate. Of all the species tested, Bacillus licheniformis gave maximum α-amylase activity of 0.7947 U/mg/ml at pH of 8 in iodine method, and (0.024 U/mg/ml) in 3.5.dinitrosalicylic acid (DNS) method. Characterization of the extra-cellular crude α- amylase was further evaluated for its biochemical properties as an enzyme for industrial use. The production of α- amylase following growth of the microorganism was found to be at optimum temperature and pH of 70°C and 9.0, respectively. Key words: Thermostable α- amylase, alkaline α- amylase, Bacillus licheniformis.

English

The present study is aimed at assessing the ability of Bacillus subtilis to convert starch into reducing sugars. Maximum enzyme activity showed by B. subtilis was 236 Uml-1. B. subtilis showed optimum growth at pH 8 and optimum temperature for the growth of bacterial isolate was found to be 45°C. The optimal pH and temperature of the amylase activity were 7.0 and 37°C, respectively. The enzyme was found to be stable in the pH range of 5 to 10. Maximum α-amylase activity was determined in 1% starch. The enzyme was found to be in two forms with relative molecular mass of 43 and 18 kDa in SDS-polyacrylamide gel electrophoresis. B. subtilis can be exploited for starch conversion biotechnologies. Key words: Starch, α-amylase, Bacillus subtilis.

Alpha-amylase production by toxigenic fungi

This study is concerned with the screening of Alternaria alternata (Fr.) Keissler and Alternaria tenuissima (Kunze ex Pers.) Wilts strains for the biosynthesis of α-amylases. Nine strains of A. alternata and three strains of A. tenuissima were grown on enzyme production medium (EPM) and potato dextrose agar (PDA) using three pH levels (4.5–6.5); then the selected strains, able to produce bigger zones of starch hydrolysis on solid media, were subjected the testing of their amylolytic efficacy in liquid medium. In primary screening, the amylolytic activity of all the strains was tolerant to a wide range of initial culture pH values (4.5–6.5). Of all the cultures tested, A. alternata strains FCBP-100 and FCBP-385, and A. tenuissima strains FCBP-183 and FCBP-252 exhibited the maximum potential in terms of starch hydrolysis at pH 4.5 on EPM, and hence were selected for further studies. In secondary screening, the optimum pH of fermentation medium was adjusted to 4.5 using 0.05 M citrate buffer for the estimation of amylolytic enzyme activities. At 48 h incubation, the maximum α-amylase activity (31.8 units mL−1) was discerned by A. tenuissima strain FCBP-252.

The Influences of Steeping Duration and Temperature on the α- and β-Amylase Activities of Six Thai Rice Malt Cultivars (Oryza sativa L. Indica)

A preliminary study of malting conditions for six Thai rice cultivars was conducted. Three non-glutinous rice cultivars (KDML105, PT60, and WR) and three glutinous rice cultivars (SPT, RD6, and KND) were selected. The steeping durations (24, 48, and 72 h) and temperatures (20, 25 and 30°C) were investigated for their effect on α- and β-amylase, the key enzymes for malt quality evaluation. During steeping, the production of both enzymes was lower than at the germination process. The longer the steeping duration, the lower the maximum β-amylase activity obtained. The contradictory effect was observed for α-amylase activity, near the end of the germination time. Additionally, temperature influenced the water absorption content as well as the amylolytic enzyme activity. Particularly at 30°C, the maximum β-amylase activity (6.7 unit/mg protein) was found in KND malt steeped for 24 h, and maximum α-amylase activity (20 unit/mg protein) was found in PT60 malt steeped for 72 h. The amount of enzyme production depended on the variety rather than the amylose content in the rice. The optimal condition for malting rice regarding β-amylase activity and α-amylase activity was analyzed at 30°C, with steeping for 24 h and germination for 4–5 days.

A novel raw starch digesting α-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 α-amylase from a newly isolated Bacillus sp. YX-1. Maximum α-amylase activity (53 U mL −1) was obtained at 45 °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 °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 12 h. By analyzing its partial amino acid sequences, the enzyme was proposed to be a novel α-amylase.

Production of bacterial α-amylase by B. amyloliquefaciens under solid substrate fermentation

Production of α-amylase by Bacillus amyloliquefaciens, under solid substrate fermentation (SSF) was investigated in shaken-culture. The maximum α-amylase activity was obtained under the following optimized conditions: corn gluten meal (CGM) 30 g/l, yeast extract (YE) 10 g/l, agitation rate 150 rpm and fermentation temperature 33 °C. The results showed that α-amylase production in a medium with CGM was five times higher than that in the medium contained starch and other components. The temperature of fermentation was found to be most crucial factor in α-amylase production.

Enhanced production of mouse α-amylase by feeding combined nitrogen and carbon sources in fed-batch culture of recombinant Pichia pastoris

The optimal culture conditions for attaining high levels of expression of mouse α-amylase in the methylotrophic yeast Pichia pastoris were investigated. Cell growth and enzyme production were found to be optimal at a temperature of 28 °C and a pH of 6.0. Yeast extract and peptone were the most appropriate nitrogen sources for the expression of α-amylase. When a mixture of 20 g/l peptone and 20 g/l yeast extract was used as the nitrogen source, α-amylase activity reached 300 U/ml. The expression of α-amylase in a fed-batch culture using methanol as the sole carbon source reached 720 U/ml at 3 days of culture. However, when a methanol-to-glycerol mixture was used at ratio of 1:0.5 at a feed rate of 3 g/h, the maximum α-amylase activity was 1.14 kU/ml. Under the optimal culture conditions, when 40 g/l of a mixture of yeast extract and peptone (1:1) was added to the culture at 3 and 5 days the final α-amylase activity increased to 2.4 kU/ml, which was 5.8- and 2.1-fold higher than the activity in the batch culture and in the fed-batch culture with feeding a mixture of methanol and glycerol at a ratio of 1:0.5, respectively. This study demonstrates that an addition of the appropriate amount of an organic nitrogen source to the fed-batch culture is effective to improve high level of expression of mouse α-amylase in the methylotrophic yeast Pichia pastoris.

Production and purification of α-amylase from hydrogen producing Enterobacter cloacae IIT-BT 08

Enterobacter cloacae IIT-BT08 was found to produce both α-amylase and hydrogen in a batch system using soluble starch as substrate. Incubation time, temperature, pH and substrate concentration for the maximum α-amylase activity (130 U/ml) were 8 h, 37 °C, 6.00 and 10 g/l of soluble potato starch respectively. However, the optimum temperature and pH for the crude α-amylase activity were 60 °C and 4 respectively. The maximum rate of hydrogen production was observed at 10th h of fermentation and corresponding hydrogen yield was 7.6 mmol H2/g soluble potato starch.

Studies on α-amylase production byBacillus licheniformisMIR-61

An acid α-amylase hyperproducing strain, designated as MIR-61, was isolated in a screening procedure from South American soil samples. MIR-61, a 60°C thermoresistant strain, was identified using 98 biochemical and morphological tests and characterized as Bacillus licheniformis by numerical taxonomy. Batch cultures of B. licheniformis MIR-61 showed extracellular α-amylase and α-glucosidase activities during the exponential growth phase. The production of α-amylase was studied at free and constant pH values at 37 and 45°C. Maximum α-amylase activity (4,767 kU/dm3 in a liquid medium) was detected at 45°C at a constant pH (7.0) in the late exponential phase. The α-amylase production by B. licheniformis MIR-61 is 10 to 300 times higher than the enzyme production reported in strains of the same species. Optimum α-amylase activity was found at 50 to 67°C in an acid pH range from 5.5 to 6.0. These properties would allow its use in starch industry processes.

Increased production of α-amylase from Thermomyces lanuginosus by the addition of Tween 80

The thermophilic fungus Thermomyces lanuginosus was cultivated in shake flasks for up to 120 h with low molecular weight dextran as carbon source supplemented with either Tween 80 or Triton X-100. Addition of Tween 80 to the growth medium gave a 2.7-fold increase in maximum α-amylase activity as compared with controls. Triton X-100 did not affect α-amylase production. In the presence of Tween 80, the α-amylase secretion was stimulated from the third day of cultivation whereas the general protein secretion was increased after only 24 h of cultivation. The amount of biomass produced increased slightly by high concentrations of Tween 80 but decreased by the addition of Triton X-100. Maximum total extracellular protein increased more than three-fold with increasing concentrations of Tween 80 in the medium. Neither the hyphal growth unit length (G) nor the hyphal diameter were affected by Tween 80. Tween 80 did not change the degree of glycosylation of the α-amylase. The addition of Tween 80 appears to give a general stimulation of protein secretion.

Selective growth of a mutant in continuous culture of Bacillus caldolyticus for production of ?-amylase

In a continuous culture of Bacillus caldolyticus strain SP, which requires maltose as an inducer for production of α-amylase in batch culture, a predominant mutant strain M1 which produced high amounts of α-amylase in the absence of maltose in batch culture, developed. The change of cell population from strain SP to strain M1 in maltose-casitone medium was linear with time in the transient state after the change from batch to continuous culture at a dilution rate of 0.17 h-1, and was completed in about 11 generations of bacterial growth. The dilution rate effect of continuous culture on α-amylase activity was almost the same with both strains SP and M1. The maximum α-amylase activity of 380 units/ml was observed at an intermediate dilution rate that was 11.5 times higher than α-amylase activity at the end of a batch culture using the same medium. It was deduced that the enhancement of α-amylase production in continuous culture was attributed partly to the predominant growth of a mutant strain with higher α-amylase productivity.

Comparative Studies on the Production of Extracellular α-Amylase by Three Mesophilic <i>Bacillus</i> Isolates

In the present study three mesophilic Bacillus isolates were analyzed for their α-amylase activity in shakeflask cultures. The organisms were capable to produce hydrolysis zone around their colonies on starch agar medium. The effect of various fermentation conditions on α-amylase production was investigated, and in every case it was found that B. subtilis was the best producer of the enzyme, which was followed by the newly isolated Bacillus sp. and B. amyloliquefaciens. The synthesis of extracellular α-amylase by the bacteria was repressed by the presence of readily metabolizable carbon source like glucose in the culture medium. Maximum α-amylase activity by the Bacillus isolates was obtained at 37°C with an initial medium pH 7.0 under agitation at 160-180 rpm for 72 h of growth. Keywords: Bacillus species, α-Amylase, Enzyme production, Shake-flask cultureDOI: http://dx.doi.org/10.3329/bjm.v24i2.1257 Bangladesh J Microbiol, Volume 24, Number 2, December 2007, pp 129-132