Production Of Thermostable Α-amylase Research Articles

High purity glucomannan after ultrasonic-assisted extraction and α-amylase liquefaction of porang (Amorphophallus oncophyllus) flour

Glucomannan extracted from the porang plant (Amorphophallus oncophyllus) is encapsulated by starch impurities. The main objectives of this study were to evaluate the effect of purifying glucomannan using thermostable α-amylase, ultrasonic-assisted extraction (UAE), and the combination of both treatments on glucomannan purity and the physiochemical properties of the porang flour, such as viscosity. This research was conducted through four main steps as follows: thermostable α-amylase production and preparation, liquefaction, extraction, and purification of glucomannan with ethanol, and the glucomannan properties assay. The liquefaction process was carried out at 70°C, at which α-amylase exhibited maximum activity. The combined treatment of UAE and αamylase liquefaction successfully purified glucomannan from porang flour. This treatment significantly reduced starch and ash contents, increased glucomannan content to 89.69%, lowered viscosity to 160 cP, and enhanced solubility to 46.18%. This treatment improved the transparency and lightness of the flour. The pure glucomannan granules obtained by combining UAE and the enzyme treatment showed regular shaped and relatively smooth with more hydroxyl and carbonyl groups than the other treatments. These results provide an alternative method to obtain pure glucomannan by combining UAE and thermostable α -amylase liquefaction.

Optimization of Independent Variables for the Production of Extracellular Alpha Amylase by Bacillus subtilis IMD34 Using Plackett-Burman Design

Bacillus species are widely used for thermostable α-amylase production for various applications such as in the detergent and food industries. This research aimed to study the production of α-amylase by B. subtilis IMD34 in submerged fermentation conditions using Plackett-Burman (PB) design, along with the optimization of certain environmental factors for optimal growth of amylolytic bacteria. Plackett-Burman design was used to evaluate the effect of ten independent variables on α-amylase production. Maximum enzyme activity was recorded in a medium containing (g/l); starch (6.0), peptone (12.0), K2HPO4 (1.0), Na2HPO4 (2.0), NaCl (0.6), Na2SO4 (0.1), MgSO4.7H20 (1.0), FeCl3.6H2O (0.02), CaCl2.2H2O (0.02) and with an agitation speed (200 rpm). From the pareto chart it was observed that the most important independent variables for amylase production were starch, peptone, NaCl, Mg2SO4, CaCl2.2H2O and agitation. Optimization of environmental parameters to assess their effect on the growth of bacteria showed that optimal pH, incubation time and temperature were recorded at 6, 48h and 37˚C. Evaluating independent variables statistically using PB design revealed that some of the medium’s components had a positive impact on α-amylase production by B. subtilis IMD34.

Statistical factorial designs for optimum production of thermostable α-amylase by the degradative bacterium Parageobacillus thermoglucosidasius Pharon1 isolated from Sinai, Egypt

BackgroundThis study aimed to isolate potent thermophilic and amylolytic bacteria from a hot spring of Pharaoh’s bath, Sinai, Egypt, and screen its degradative activity. The amylolytic activity was further optimized using a statistical full factorial design followed by response surface methodology. ResultsA thermophilic bacterium was isolated from the hot spring of Pharaoh’s Bath, Sinai, Egypt. The isolate produced amylase, cellulase, and caseinase and was identified by 16S rRNA gene sequencing as Parageobacillus thermoglucosidasius Pharon1 (MG965879). A growth medium containing 1% soluble starch was found to optimize the amylase production. Dinitrosalycalic acid method (DNS) was used to estimate the amount of reducing sugar produced. Statistical full factorial and response surface designs were employed to optimize physical variables affecting the α-amylase production and determine the significant interactions of the studied variables during the fermentation process. According to the results obtained by the response optimizer, the maximum amylase activity reached 76.07 U/mL/ min at 54.1°C, pH 5.6 after 98.5 h incubation under aerobic conditions. Moreover, the produced enzyme was thermostable and retained most of its activity when exposed to a high temperature of 100°C for 120 min. Maximum enzyme activity was attained when the enzyme was incubated at 70°C for 30 min. ConclusionsThis is the first report of the production of thermostable α-amylase by the potent thermophilic Parageobacillus thermoglucosidasius. The enzyme endured extreme conditions of temperature and pH which are important criteria for commercial and industrial applications.

Evaluation of brewer's spent grain hydrolysate as a substrate for production of thermostable α-amylase by Bacillus stearothermophilus

In the present study, BSG was hydrolysed using cellulolytic enzymes and used as a growth medium supplement for cultivation of the thermophilic bacterium, Bacillus stearothermophilus in the production of α-amylase. A central composite design involving five parameters and four levels viz. starch, peptone, KCl, and MgSO4 along with BSG hydrolysate was used to derive the optimal media composition. The fermentation was conducted using shake flasks for 36 h at a temperature of 50 °C and pH 7.0 at 220 rpm. Optimization trials revealed that maximal amylase production (198.09 U/ml) occurred with a medium composition of starch (0.2% w/v), peptone (0.2% w/v), KCl·4 H2O (0.02% w/v), MgSO4·7 H2O (0.01% w/v) and hydrolysate (0.22% v/v). A 1.3-fold increase in amylase activity was obtained following novel media composition. All the factors considered in the study were found to be significant. The enzyme was purified by three step purification strategy, characterised and tested for anti-biofilm activity.

Scale-up thermostable α-amylase production in lab-scale fermenter using rice husk as an elicitor by Bacillus licheniformis-AZ2 isolated from Qinarje Hot Spring (Ardebil Prov. of Iran)

Background and purpose: Amylases are commercially important enzymes with various biotechnological, clinical and medical applications. This study aimed at scaling up α-amylase production elicited by rice husk in stirred-fermenter using Bacillus lichneniformis-AZ2 isolated from Qinarje Hot Spring. Materials and methods: Effect of temperature, aeration rate and agitation speed on bacterial growth and ɑ-amylase production were investigated under batch fermentation process in a 3-Lit stirred-fermenter. OFAT method was followed to select optimum level of each parameter. Other factors were set upon the results of previous experiments carried out in shake-flask scale. Results: Maximum α-amylase production of 17.66 ± 0.87 U/mL (2.1 folds more than shake-flask cultures) was achieved in stirred-fermenter with optimized agitation speed of 100 rpm and 1 vvm aeration rate at 37oC after 60 h of incubation. This time was shorter than the corresponding fermentation time obtained from shake-flask experiments by half. A comparison of kinetic parameters of fermentation in stirred-fermenter and shake-flask cultures revealed that B. licheniformis-AZ2 was more active to synthesize ɑ-amylase in fermenter. In shaken cultures Qx, Qp, Yp/x, µmax, qp and td, were 0.27 (g/L/h), 228.6 (U/L/h), 13.64 (U/g), 0.055 (h-1), 0.76 (U/g/h) and 12.48 h, whereas in stirred-fermenter the above values were 0.40 (g/L/h), 723.1 (U/L/h), 45.17 (U/g), 0.120 (h-1), 5.42 (U/g/h) and 5.78 h, respectively. Conclusions: SmF in stirred-fermenter is a potential strategy for ɑ-amylase production. Although for commercialization further studies are needed in pilot-scale. Rice husk as a low-cost agro-waste is preferable to use as the carbon and energy sources, which provides a great ɑ-amylase elicitation.

Production of thermostable extracellular α-amylase by a moderate thermophilic Bacillus licheniformis isolated from Qinarje Hot Spring (Ardebil prov. of Iran)

Background and Purpose: Amylases are most important industrial enzymes that account for about 30% of the world’s food, feed, fermentation, textile, detergent and cellulosic industries. This study aimed at optimum production of thermostable α-amylase via moderate thermophilic bacterium (Bacillus licheniformis) which was recently isolated from Qinarje Hot spring.Material and Methods: Initially, ability of bacterium for amylase activity was determined by starch hydrolysis test using Gram’s iodine staining. Then bacterial growth pattern and amylase production curves in basal production medium were graphically determined at different time intervals. Finally, effect of different temperature, pH, carbon source, nitrogen source, minerals and inoculum size were studied on bacterial growth and amylase production using turbidimetric and DNS method, respectively.Results: Optimum enzyme production achieved after 84 hours of inoculation from cultures growing at 40 ˚C and pH 9.0 in a medium containing 0.03% (w/v) of CaCl2, compared to the basal medium, results showed that the best enzyme production happened with inoculum size of 4% (v/v). The addition of 1% (w/v) rice husk (as a Carbon source) enhanced enzyme productivity up to 160% and substitution of the peptone and yeast extract with 1% (w/v) of tryptone (as a Nitrogen source) increased the α-amylase production up to 160%.Conclusion: Our findings show that B. licheniformis-AZ2 strain has an ability to produce the thermostable α-amylase which is suitable in starch processing and food industries. To be commercialized, further investigation is required for enhancement of the enzyme production.Keywords: Bacillus licheniformis; Optimization; Basal medium; Agricultural by-products.

Statistical Optimization of Thermostable α-Amylase Production by a Newly Isolated Rhizopus oryzae Strain FSIS4 Using Decommissioned Dates

A new Rhizopus oryzae strain FSIS4 isolated from wheat seed cultivated in Algerian Sahara was evaluated for the production of a thermostable α-amylase enzyme. Medium components were optimized by a statistical approach for the optimal production of α-amylase using an agro-industrial residue (decommissioned dates). The Plackett–Burman design was performed to screen the fermentation variables and response surface methodology for optimizing the significant factors affecting the production. The optimum levels for maximum production of α-amylase (approx 10,151 IU) were determined. A 1.8-fold increase of production was obtained in comparison to the non optimized medium. Partial characterization of the enzyme showed optimal temperature and pH at 60 °C and 5.0, respectively. The enzyme was found to be stable at 80 and 90 °C, with a half-life of 165 and 105 min, respectively. According to the large literature, this is the first report on the production of α-amylase by the thermophilic R. oryzae strain FSIS4. The thermo and pH stability of the α-amylase suggest its wide application in the food and pharmaceutical industries.

Bioprocess exploration for thermostable α-amylase production of a deep-sea thermophile Geobacillus sp. in high-temperature bioreactor

ABSTRACTGeobacillus sp. 4j, a deep-sea high-salt thermophile, was found to produce thermostable α-amylase. In this work, culture medium and conditions were first optimized to enhance the production of thermostable α-amylase by statistical methodologies. The resulting extracellular production was increased by five times and reached 6.40 U/ml. Then, a high-temperature batch culture of the thermophile in a 15 l in-house-designed bioreactor was studied. The results showed that a relatively high dissolved oxygen (600 rpm and 15 l/min) and culture temperature of 60°C facilitated both cell growth and α-amylase production. Thus, an efficient fermentation process was established with initial medium of pH 6.0, culture temperature of 60°C, and dissolved oxygen above 20%. It gave an α-amylase production of 79 U/ml and productivity of 19804 U/l·hr, which were 10.8 and 208 times higher than those in shake flask, respectively. This work is useful for deep-sea high-salt thermophile culture, where efforts are lacking presently.

Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei

ObjectivesIn an attempt α-amylase gene from Pyrococcus woesei was amplified and cloned into a pTYB2 vector to generate the recombinant plasmid pTY- α-amylase. MethodsEscherichia coli BL21 used as a host and protein expression was applied using IPTG. SDS-PAGE assay demonstrated the 100 kDa protein. Amylolytic activity of proteins produced by transformed E. coli cells was detected by zymography, and the rate of active α-amylase with and without the intein tag in both soluble conditions and as inclusion bodies solubilized by 4M urea were measured. ResultsAmylolytic activity of ∼185,000 U/L of bacterial culture was observed from the soluble form of the protein using this system. ConclusionThese results indicate that this expression system was appropriate for the production of thermostable α-amylase.

Isolation and production of thermostable α-amylase from thermophilic Anoxybacillus sp. KP1 from Diyadin hot spring in Ağri, Turkey

A novel amylolytic enzyme producing thermophilic bacterial strain KP1 from the Diyadin hot spring water in Agri, Turkey, was isolated in the present study. Phylogenetic analysis based on the partial 16S rRNA gene, biochemical and physiological tests revealed that the strain KP1 belongs to the genus Anoxybacillus. The pH and temperature optima for the α-amylase production by Anoxybacillus sp. KP1 were 8.0 and 50°C, respectively, where the maximum growth was obtained at the 28th hour of incubation and the highest α-amylase activity was obtained at the 40th hour of incubation (8979.6 U/mL). The optimum pH and temperature for the enzyme activity were 8.0 and 60°C, respectively. The maximum α-amylase production was secreted in the presence of 2% (w/v) soluble starch (10837.7 U/mL). Among the various organic and inorganic nitrogen sources tested, while keeping the beef extract concentration constant, casamino acid (14310.6 U/mL), urea (14126 U/mL), and tryptone (13217.2 U/mL) at a concentration of 2% gave the maximum α-amylase production. The enzyme activity was enhanced in the presence of 1.5 mM Mn2+ (123%), whereas it was strongly inhibited 1.5 mM by Hg2+. Inhibition by 89% was obtained also with sodium dodecyl sulphate (1%). The enzyme was found to be relatively stable at a range of pH and temperature.

Effect of partial pressure of CO2 on the production of thermostable α-amylase and neutral protease by Bacillus caldolyticus

Controlling the concentration of dissolved oxygen is a standard feature in aerobic fermentation processes but the measurement of dissolved CO2 concentrations is often neglected in spite of its influence on the cellular metabolism. In this work room air and room air supplemented with 5% and 10% carbon dioxide were used for aeration during the cultivation of the thermophilic microorganism Bacillus caldolyticus (DSM 405) on starch to produce alpha-amylase (E.C. 3.2.1.1) and neutral protease (E.C. 3.4.24.27/28). The increased CO2 concentrations resulted in a 22% raise in activity of secreted alpha-amylase and a 43% raise in protease activity when compared with aeration with un-supplemented room air. There was no effect on the final biomass concentration. Furthermore, the lag-phase of fermentation was reduced by 30%, further increasing the productivity of alpha-amylase production. Determinations of dissolved CO2 in the culture broth were conducted both in situ with a probe as well as using exhaust gas analysis and both the methods of quantification showed good qualitative congruence.

Isolation of a thermophilic Anoxybacillus flavithermus sp. nov. and production of thermostable α-amylase under solid-state fermentation (SSF)

A new bacteria was isolated from hot-spring water of Gazligol, Afyonkarahisar in Turkey. Based on morphological and biochemical tests and 16S rRNA gene sequence analysis, the isolate belonged to the Anoxybacillus flavithermus species which has 99% similarity with the bacterium DNA. The production of α-amylase by thermophilic Anoxybacillus flavithermus was investigated under solid-state fermentation by using some agricultural waste as substrates. Solid substrates such as rice husk, banana husk, millet, water melon husk, lentil bran, wheat bran and maize oil cake were studied for enzyme production. Of these, rice husk was proved as the best substrate for α-amylase production (1,271 U/mg). The maximum α-amylase production was observed as 1,803 U/mg at 72 h, 1,000 μm particle size, 70% initial moisture content (w/v), and 40% inoculum level (v/w). Among the various nitrogen sources tested, 1% peptone (3,170 U/mg) was found to be the best nitrogen source for α-amylase production. As additional carbon sources, 1% starch (2,364 U/mg) enhanced α-amylase production. The optimum temperature for the activity of α-amylase was found to be 70°C. The enzyme was optimally active at pH 6.0 and stable in the pH range of 6.0–8.0.

Improved thermostable α-amylase activity of Bacillus amyloliquefaciens by low-energy ion implantation

Thermostable α-amylase is of great importance in the starch fermentation industry; it is extensively used in the manufacture of beverages, baby foods, medicines, and pharmaceuticals. Bacillus amyloliquefaciens produces thermostable α-amylase; however, production of thermostable α-amylase is limited. Ion-beam implantation is an effective method for mutation breeding in microbes. We conducted ion-beam implantation experiments using two different ions, Ar(+) and N(+), to determine the survival rate of and dose effect on a high α-amylase activity strain of B. amyloliquefaciens that had been isolated from soil samples. N(+) implantation resulted in a higher survival rate than Ar(+) implantation. The optimum implantation dose was 2.08 × 10(15) ions/cm(2). Under this implantation condition, we obtained a thermally and genetically stable mutant α-amylase strain (RL-1) with high enzyme activity for degrading α-amylase. Compared to the parental strain (RL), the RL-1 strain had a 57.1% increase in α-amylase activity. We conclude that ion implantation in B. amyloliquefaciens can produce strains with increased production of thermostable α-amylase.

Effect of an elevated carbon dioxide partial pressure on the production of thermostable α-amylase and protease from Bacillus caldolyticus

Effect of an elevated carbon dioxide partial pressure on the production of thermostable α-amylase and protease from Bacillus caldolyticus

Production of thermostable α-amylase using food industry wastes

Production of thermostable α-amylase using food industry wastes

Potato peel as a solid state substrate for thermostable α-amylase production by thermophilic Bacillus isolates

Potato peel was found to be a superior substrate for solid state fermentation, compared to wheat bran, for the production of α-amylase by two thermophilic isolates of Bacillus licheniformis and Bacillus subtilis. Under optimal conditions, B. licheniformis produced 270 units/ml and 175 units/ml of α-amylase on potato peel and wheat bran, respectively, while the corresponding values for B. subtilis were 600 units/ml and 265 units/ml. The enzyme from B.␣licheniformis was optimally active at 90 °C and pH 9.0, while that from B. subtilis at 60 °C and pH 7.0. The nature of the experimental data permitted excellent polynomial fits, on the basis of which, two master equations, corresponding to the isolated strains, were derived for estimation of enzyme activity for any set of values of temperature, particle size, moisture, and incubation time within the indicated ranges.

Thermostable α-amylase and α-galactosidase production from the thermophilic and aerobic Bacillus sp.JF strain

Extracellular thermostable α-amylase (EC 3.2.1.1) and intercellular α-galactosidase (EC 3.2.1.22) from one fermentation of a thermophilic and aerobic Bacillus sp.JF 2 strain were studied and modelled. The cell growth of the thermophilic Bacillus sp.JF 2 strain could be expressed by the Monod model, the μ max was 0.084/h, the activation energy for cell growth and death was E g=7.42 and E d=−18.8 cal/g mol K. The thermostable α-amylase production was expressed by a model combining growth-associated and non-growth-associated models. Intercellular α-galactosidase production was expressed by the growth-associated model; and the relationship equation between the substrate, cell growth, and two enzyme production was also defined, and the all constants were defined.

Immobilization of Bacillus licheniformis cells, producers of thermostable ?-amylase, on polymer membranes

Cells of Bacillus licheniformis 44MB82-G immobilized on different polymer membranes were used for production of thermostable α-amylase. The α-amylase yields of the membrane-immobilized cells were affected by the reactive chemical groups of the carriers and the spacer size. Formaldehyde-activated polysulphone membranes (PS-FA) were the most suitable for effective immobilization. The highest amylase yield (62% increase of the control) and operational stability (97% residual activity after 480 h repeated batch cultivation) were obtained with this system. This was confirmed by scanning electron micrographs. An additional increase of α-amylase production by PS-FA-membrane immobilized cells was achieved in a fluidized-bed reactor.

Thermostable α-amylase production by immobilized Bacillus licheniformis cells in agar gel and on acrylonitrile/acrylamide membranes

Immobilized cells of Bacillus licheniformis 44MB82-G were used for the production of thermostable alpha-amylase. The immobilization was carried out by entrapment in agar gel or by binding to formaldehyde-activated acrylonitrile/acrylamide membranes. The alpha-amylase production after 144 h of cultivation of membrane immobilized cells was 40% higher in comparison with the free cells. The respective value for the agar-entrapped cells was 22%. Similar trends were observed in the repeated batch fermentations performed with the immobilized cells. The scanning electron micrographs (SEM) of the immobilized cells gave additional information about their binding to the respective carriers.

Continuous Production of Thermostable α-Amylase by Immobilized Bacillus Cells in a Fluidized-Bed Reactor

The exponential cells ofBacillus cereus immobilized in calcium alginate and spun into microcylindrical particles were used in a fluidized-bed reactor for continuous synthesis of thermostable α-amylase. The reactor was operated over a period of 30 d with a dilution rate of 0.33 h-1, producing 1000–1200 U/mL of enzyme. The productivity of the reactor was in the range of 330–396 kU/h. A 20-fold increase in the productivity with respect to batch fermentation with free cells was attained.