α-Amylase Biosynthesis Research Articles

Improved Production of α-Amylase by Aspergillus terreus in Presence of Oxygen-Vector

n-Dodecane has been investigated as an oxygen-vector for improving α-amylase biosynthesis using the strain Aspergillus terreus. In aerobic microbial cultivation, continuous supply of oxygen is required especially due to its low solubility in the growth medium, in particular at high viscosity, but the limitations of oxygen mass transfer in these systems can be overcome by the addition of water-insoluble compounds which possess a strong affinity for oxygen, namely oxygen-vectors. The use of n-dodecane (as an oxygen-vector) in the fermentation medium of A. terreus can significantly improve the bioprocess performance and enhance α-amylase production. Using 5% n-dodecane at 35 °C, an increase of 1.8–2 times in the enzymatic activity was recorded. In the oxygen-vector’s absence, the highest amount of biomass was obtained at 35 °C, while in the presence of 5% vol. n-dodecane, the amount of fungal biomass increased by approximately 70%, with a shift in optimum temperature to 40 °C, generating also an enzymatic activity increase of 2.30 times. Moreover, the oxygen-vector’s addition in the fermentation broth influenced the fungal morphological development in the form of larger pellets with a more compact structure compared to the system without n-dodecane, with a positive effect on the fermentation performance (higher α-amylase activity production).

Production of α-amylase(s) by Aspergillus flavus, F7 attacking water hyacinth ground preparation (WHGP) under solid state fermentation

Some environmental and nutritional parameters controlling the biosynthesis of α-amylase from Aspergillus flavus, F7 attacking the water hyacinth have been investigated under solid state fermentation conditions for maximum amylase production. The following optima’s were recorded for the highest α-amylase yield; Incubation period 7 days; temperature, 30oC; pH, 5; inoculum size, 3X108 spores/ml; flask volume 100 ml capacity; hyacinth fresh weight 5 g; tap water, 25 ml. Under these conditions, starch showed remarkable stimulatory effect; nitrogen sources and amino acids have no stimulatory effect. Pyridoxal hydrochloride, B6 at a concentration of 200 ppm exhibited a stimulatory effect on biosynthesis of α-amylase.

Bioconversion of agriculture wastes to produce α-amylase from Bacillus velezensis KB 2216: Purification and characterization

Abstract Bacillus velezensis KB 2216 was the most capable amylase producing strain among 26 isolates. The strain was assayed for alpha-amylase production under submerged fermentation. Amylase was purified through ammonium sulfate precipitation and Sephadex gel chromatography. KB 2216 was recognized to produce 4 different forms of amylase. One form of alpha-amylase was noted to dimeric, named Amy2. The latter SDS-PAGE and zymogram analysis revealed that purified Amy2 was an approximately 97 kDa dimer of two 55 kDa and 42 kDa subunits. Amy2 was exhibited maximum activity at pH 5.5 and 55 °C and highest stability at the range of pH 5.0 to pH 5.5 and 0 °C to 4 °C. Initially, production media inoculated by new MS6 seed media was showed the highest production (17.066 U/ml) compared to conventional seed media (11.420 U/ml). Production media was optimized using various agricultural wastes. The highest α-amylase production achieved using 4% moong husk and 2% soybean cake as a major source of carbon and nitrogen respectively and also in the presence of 1.5% fructose and 0.5% NaNO3 as additional nutrients for enhancement of α-amylase biosynthesis (180.06 U/ml). The optimal physical parameters were assessed to the temperature at 35 °C, pH 5.0 and 72 h incubation period.

Optimization, Production, and Partial Purification of Thermostable -Amylase Produced by Marine Bacterium Bacillus sp. NRC12017

α-Amylase biosynthesis was illustrated under stress circumstances of high temperature and high salinity in aerobically cultivated culture of a newly isolated moderately thermophilic bacterium of spore-forming Bacillus sp. NRC12017 in medium containing starch, peptone and yeast extract. Maximum yield (18.41 U/ml) was took place at pH 6.5 with 200 µl inoculum size at 45°C and an incubation time of 3 days. The ideal volume of the fermentation broth was found to be 15 ml in 100 ml Erlenmeyer flask, supplementation of starch at 2.5% and peptone plus yeast extract at 0.7% brought about the greatest production of α-amylase. Two fractions of α-amylase activities, designated FIII and FIV, were refined from culture filtrate utilizing ammonium sulfate (60 and 80%) and showed the main band at 30 KDa. Both fractions had the highest activity at 250 µl starch in a reaction mixture and pH 6.0-7.0. FIII and FIV showed that a temperature of 50 and 55°C and a reaction time of 20 and 30 min were the best available conditions for their activities, respectively, both fractions were stable up to 65°C and the activity was decreased drastically to 3.22 and 3.26 % when heated at 70°C. Concerning pH stability, a broad range of pH stability (5.0-11.0) was obtained by FIII and FIV.

Production of α-Amylase by Aspergillus terreus NCFT 4269.10 Using Pearl Millet and Its Structural Characterization.

In this investigation, Aspergillus terreus NCFT4269.10 was employed in liquid static surface (LSSF) and solid state (SSF) fermentation to assess the optimal conditions for α-amylase biosynthesis. One-variable-at-a-time approach (quasi-optimum protocol) was primarily used to investigate the effect of each parameter on production of amylase. The maximum amylase production was achieved using pearl millet (PM) as substrate by SSF (19.19 ± 0.9 Ug−1) and also in presence of 1 mM magnesium sulfate, 0.025% (w/v) gibberellic acid, and 30 mg/100 ml (w/v) of vitamin E (~60-fold higher production of amylase) with the initial medium pH of 7.0 and incubation at 30 °C for 96 h. In addition, maltose, gelatin and isoleucine also influenced the α-amylase production. Amylase was purified to homogeneity with molecular mass around 15.3 kDa. The enzyme comprised of a typical secondary structure containing α-helix (12.2%), β-pleated sheet (23.6%), and β-turn (27.4%). Exploitation of PM for α-amylase production with better downstream makes it the unique enzyme for various biotechnological applications.

Inhibitory effect of chemical combinations on seed germination and pre-harvest sprouting in hybrid rice

Pre-harvest sprouting (PHS) is a primary cause of decreases in quality and economic value in rice seeds. This study examined the inhibitory effects of four combinations of maleic hydrazide, eugenol and uniconazole on rice PHS and seed quality. After the four treatments, delayed grain germination was observed compared with the control, mean germination time was prolonged by 17.5 % and germination index was decreased by an average of 18.2 %. Activity of α-amylase was significantly reduced, by 26.7–83.5 %, during grain germination and early seedling growth. We then used qRT-PCR to further analyze the mRNA expression levels of four genes involved in α-amylase biosynthesis. The expression of OsAMY3B and OsAMY3E was significantly down-regulated by the four combinations of chemicals. In field trials, each of the four combinations resulted in a significantly lower sprouting rate and sprouting index compared with the control. In addition, freshly harvested rice seeds showed no significant differences in the mean germination time or germination index between the four combination treatments and the control. After 1 month of storage, there was still no significant difference between the four combination treatments and the control with regard to the mean germination time and germination index. These results suggest that the selected combinations strongly inhibited the seed germination speed and PHS of rice in the field. However, these combinations had no inhibitory effects on the final germination percentage and showed no negative effect on seed quality after short-term storage. The combinations of inhibitors used in this study can be applied to decrease the PHS damage caused by high temperatures and moist weather during the production of hybrid rice.

OPTIMIZATION OF CULTIVATION CONDITIONS FOR α-AMYLASE PRODUCER ACHROMOBACTER SP. 7а

Aim. The investigation of the optimal conditions for α-amylase production by Achromobacter sp. 7а. Methods. For studying the effect of different sources of carbon and nitrogen on the α-amylase biosynthesis the starch and sodium nitrate in a base culture medium were replaced by the various sources of nitrogen and carbohydrates in an amount equivalent to the total carbon content (44.4%) and nitrogen (16.5%) in the basic medium. α-Amylase activity was determined by iodometric method, protein content – by Lowry et al. The screening experiments were carried out to identify the significant mineral nutrients of base medium. Determination of optimal concentrations of carbon and nitrogen sources in the medium was carried out by bifactorial experiment on four levels. Results. It is established that the highest level of enzyme synthesis is observed on the 3 day of deep cultivation. It is shown that mix of malt with starch in the ratio 1:9 and nitrate of sodium are the optimum sources of carbon and nitrogen respectively, for the maximum enzyme producing. The optimum conditions of α-amylase biosynthesis were established: temperature is 28 °C, initial рН value 6.0, the volume of a nutrient medium is 100 ml, number of seed material 15% and intensity of hashing 210 rpm. Conclusions. The α-amylase production was rised fourfold as a result of bifactorial experiment and selection of optimum conditions of cultivation of Achromobacter sp. 7а.

Production of thermostable, Ca+2-independent, maltose producing α-amylase by Streptomyces sp. MSC702 (MTCC 10772) in submerged fermentation using agro-residues as sole carbon source

The purpose of this study was to determine the influence of growth conditions and medium composition on the thermostable, Ca+2-independent, maltose producing α-amylase production in submerged fermentation (SmF) by Streptomyces sp. MSC702—a strain newly isolated from mushroom compost. Production of α-amylase enzyme by a Streptomyces strain was detected on semi-synthetic media containing rice bran and wheat bran in a 1:2 ratio as the sole carbon source. The effect on α-amylase production of different medium ingredients and additives, such as NaCl, additional carbon sources, nitrogen sources, metal salts, nucleotides, phytohormones, surfactants and other chemicals, was examined. NaCl played no notable role in the biosynthesis of α-amylase, with the strain being able to grow at 14% (w/v) NaCl with ~76% α-amylase activity. Among the different cations tested, Hg2+ showed no inhibitory effect on α-amylase production. The best combination of physical parameters for the maximum production of α-amylase was a temperature of 50°C, pH 7.0 and an incubation period of 48 h. The best combination of chemical parameters with rice bran 1.0% (w/v) and wheat bran 2.0% (w/v) was dipotassium hydrogen phosphate 0.1% (w/v), magnesium sulfate 0.1% (w/v) with d-inositol (as additional carbon source) 1.0% (w/v), peptone (as nitrogen source) 0.1% (w/v), manganese sulphate (as additional metal ion) 0.05% (w/v) and Tween-80 (as surfactant) 1.0% (v/v). With the application of these culture conditions, α-amylase production increased by ~1.42 fold.

The Efficiency of Temperature-Shift Strategy to Improve the Production of α-Amylase by Bacillus sp. in a Solid-State Fermentation System

The performance of temperature-shift strategies on the production of α-amylase by Bacillus sp. in solid-state fermentation systems was investigated. Switching the temperature from an initial set point (37 °C, 40 °C, or 45 °C) to 28 °C increased the production of α-amylase significantly in all treatments, with different rates. The effects of having a constant incubation temperature and performing the temperature-shift operation from higher to lower temperatures (37 °C, 40 °C, or 45 °C) to 32 °C and vice versa were also studied. The results showed that the α-amylase biosynthesis could successfully be improved by shifting the incubation temperature from 40 °C for the initial 20 h of fermentation to 32 °C after a span of 20–48 h. An enhancement of 29.5% in the production of α-amylase after 48 h was recorded compared to the treatment that was shifted from 37 °C to 32 °C. This finding is particularly attractive for large-scale industrial fermentation from an economic point of view.

A comparative study of bioassays based on enzyme biosynthesis in Escherichia coli and Bacillus subtilis exposed to heavy metals and organic pesticides

Two enzyme assays, namely β-galactosidase in Escherichia coli (E.C.3.2.1.23) and α-amylase in Bacillus subtilis (E.C.3.2.1.1), were compared following exposure to various concentrations of heavy metals and organic pesticides. It was shown that α-amylase biosynthesis in the Gram-positive bacteria was more sensitive to heavy metals, particularly to mercury as low as 0.1 ppm, compared to β-galactosidase in the Gram-negative bacteria. The results of organic pesticide treatment were more clear-cut, when comparing both assays. Alpha-amylase test system can detect the inhibition by the organometallic fungicides Maneb and Mancozeb at as low as 0.1 ppm, while no inhibition with β-galactosidase system up to as high as 100 ppm. The effect of organic insecticide endosulfan on α-amylase was also seen at very low concentrations (0.048 ppm). The electrophoretic analysis also revealed the inhibition of enzyme biosynthesis at the tested toxicant concentrations.

Characteristics of an aqueous two-phase system for α-amylase production

The characteristics of an aqueous two-phase system for the overproduction of extracellular enzyme through α-amylase fermentation by Bacillus amyloliquefaciens were investigated. With higher molecular weight of polyethylene glycol (PEG) or lower molecular weight of dextran, the partition coefficient of α-amylase was increased. α-Amylase biosynthesis was increased when PEG 6000 was included in the medium compared to the medium without PEG. Phosphate addition to the PEG-dextran system improved the partition coefficient of α-amylase, but deactivated α-amylase severely. By using sodium sulfate instead of phosphate, α-amylase deactivation was negligible, and high partitioning of the enzyme in the top phase was obtained.

Phosphate effects in the fermentation of ?-amylase by Bacillus amyloliquefaciens

The effects of phosphate on α-amylase fermentation byBacillus amyloliquefaciens were investigated. It was observed through batch culture that optimal phosphate level which maximizes α-amylase biosynthesis exists. High concentration of phosphate level promotes maltose uptake and growth of the microorganism, while high maltose uptake rate in the microorganism at the same time represses the enzyme biosynthesis presumably due to catabolite repression inside the microorganism. In continuous cultivation, a steady state of α-amylase biosynthesis was obtained by maintaining phosphate level at a certain level. In fed-batch culture, by intermittant feeding of phosphate as well as maltose, higher activity of α-amylase in the broth was obtained compared to the result from single nutrient feeding.

Penicillin Induction of Gibberellin and α-Amylase Biosynthesis in Rice Endosperm

Penicillin induces the synthesis of cr-amylase in embryoless rice endosperm and enhances the gibberellin-induced response. Penicillin induction of cr-amylase can be prevented by inhibitors of nucleic acid and protein synthesis, CCC and 2,4-DNP. A characteristic gibbereliin-like activity in the extracts from penicillin-treated endosperms becomes detectable after 12 h from the addition of penicillin. This gibbereliin-like activity is located on paper chromatograms at the RT typical for GA3 and its formation is blocked by CCC, an inhibitor of G A biogenesis. Glucose has no effect on the biosynthesis of either gibberellin or cr amylase induced by penicillin. The time-course study of the levels of different constituents shows that penicillin probably induces RNA and DNA synthesis in the first place, which results in gibberellin biosynthesis, which in turn stimulates the synthesis of cr-amylase. The possible mode of action of penicillin in higher plants is discussed.

INDUCED BIOSYNTHESIS OF ALPHA-AMYLASE BY GROWING CULTURES OF BACILLUS STEAROTHERMOPHILUS.

Welker, N. E. (Western Reserve University, Cleveland, Ohio), and L. Leon Campbell. Induced biosynthesis of alpha-amylase by growing cultures of Bacillus stearothermophilus. J. Bacteriol. 86:1196-1201. 1963.-The maximal differential rate (K) of alpha-amylase synthesis was usually two to three times that of the sucrose control culture, over an inducer concentration range of 5 x 10(-4) to 1 x 10(-3)m. With maltotetraose, higher concentrations decreased the K value, whereas higher concentrations of maltose were needed to obtain maximal K values. Glucose, in concentrations from 10(-5) to 10(-2)m, had no effect on the differential rate of enzyme synthesis. Cultures growing on maltotriose, maltotetraose, maltopentaose, and maltohexaose exhibited the same growth rate (k) and differential rate of alpha-amylase synthesis over a concentration range of 2.92 x 10(-4) to 1.46 x 10(-2)m. Growth of cultures in various concentrations of pure maltose revealed that with concentrations of maltose ranging from 2.92 x 10(-3) to 1.46 x 10(-2)m the K value for alpha-amylase production increased 18-fold. The amount of maltose utilized, during the growth period, at each concentration of maltose, was constant. Diauxic type growth was observed when maltose was used in addition to another carbon source (i.e., glucose, glycerol, fructose, or sucrose). Maltose was not utilized until the other carbon source had been metabolized. Phenyl-, methyl-, and ethyl-alpha-d-glucoside and methyl-beta-d-maltoside were good inducers of alpha-amylase and would not serve as a carbon source in a chemically defined medium supplemented with 0.1% casein hydrolysate. These compounds were therefore gratuitous inducers of alpha-amylase. Isomaltose, panose, butyl-alpha-d-glucoside, and methyl-alpha-d-maltotetraoside were not effective as inducers of alpha-amylase. Fructose had an inhibitory effect on constitutive (41%) and inducible (55%) alpha-amylase formation; glucose had no effect.