Alpha-amylase Production Research Articles

Development of a solid-state fermentation process for production of bacterial α-Amylase from agro-byproducts and its optimization

Solid-state fermentation (SSF) was carried out using different agro-byproducts as substrate for the production of α-amylase using a bacterial culture of Bacillus licheniformis. Among all the substrates wheat bran supported maximum growth and produced maximum alpha amylase (154.17 IU) under Triton –X as extraction medium. Production optimization was conducted using wheat bran. The maximum alpha amylase production was obtained at pH 6 (170.34 IU) 40°C (168.78 IU), 48 h (155.06 IU) and with 80% initial moisture (171.89 IU). Supplementation with 1% maltose further enhanced enzyme yield (178.46 IU). However, glucose and lactose inhibited the enzyme production. Increase in the enzyme yield (171.68 IU and 172.36 IU) was obtained when fermentation medium was supplemented with 1% soy peptone and (0.15 M) ammonium chloride, respectively. Addition of 0.01 M phosphate concentration also enhanced amylase production. Therefore, from the present study it can be concluded that starch digesting α- amylase produced by Bacillus licheniformis in solid state fermentation could be important in biotechnological application.

Evaluation ofChlorella Vulgarisas a Source of Fermentable Sugar using Amylase ProducingBacillus Cereus

Bacillus cereus that was previously isolated from the soil and screened for alpha-amylase production was used for hydrolysis of Chlorella vulgaris starch content. The Dinitrosalicylic acid method was used to determine the amount of reducing sugar produced. Maximum reducing sugar yield of 2.10 mg/ml was obtained for pre-treated C. vulgaris biomass, 2.06 mg/ml for untreated biomass and 2.56 mg/ml for corn starch (control) after 24 hours incubation. When the production parameters where optimized, maximum reducing sugar yield was obtained at a pH of 6.5, temperature of 35°C, incubation time of 24 hours and 4% inoculums concentration for pretreated C. vulgaris biomass. Also, a 1.3 fold reducing sugar yield was enhanced when compared with non-optimized conditions indicating the usefulness of optimization on biochemical processes. The maximum reducing sugar obtained for hydrolysis of pretreated C. vulgaris biomass is similar to the reducing sugar yield of corn starch. Also, the carbohydrate content of C. vulgaris hydrolysis was 66% which is comparable to the carbohydrate content of corn (about 73%). This indicates the potential of C. vulgaris as an alternative substrate for fermentable sugar production.

Production of Alpha-Amylase from Mixed Actinomyces Spp Cultured at Room Temperature Using Nelson’s Colorimetric Method

Production of Alpha-Amylase from Mixed Actinomyces Spp Cultured at Room Temperature Using Nelson’s Colorimetric Method

English

This study is concerned with the production and optimization of alpha amylase by Bacillus amyloliquefaciens IIB-14 in the presence of additional carbon and nitrogen sources using solid state fermentation. Alpha amylase is of special importance in the starch fermentation industry, as it has significant commercial applications in starch processing, beverages, foods, bread and baking, medicines, textile, paper, pharmaceuticals, sugar and detergent industries. Maltose, glucose, lactose and soluble starch were supplemented as carbon sources. Urea, peptone, yeast extract and nutrient broth as organic nitrogen sources while NH4NO3, (NH4)2SO4, NaNO3 and KNO3 as inorganic nitrogen sources were evaluated. Among them, a marked improvement in enzyme production (70.15 U/mg/min) was obtained with lactose, yeast extract and NH4NO3 at a level of 1.5, 1 and 1%, respectively. The performance of fuzzy-logic system control was found to be highly promising for the improved substrate conversion rate of more than 75%. The process parameters were further identified using Plackett-Burman design. The correlation (1.045E+0025) among variables demonstrated the model terms as highly significant (HS) indicating commercial utility of the culture used (p<0.05). The maximal enzyme activity (84.24 U/mg/min) of crude enzyme was found at pH 7 and temperature 70°C incubated for 30 min.   Key words: Alpha amylase, Bacillus amyloliquefaciens, solid state culture, carbon and nitrogen supplementation, fuzzy-logic control, two-factorial experimental design

Effect of starch containing organic substrates on alpha amylase production in Bacillus strains

This study was conducted to identify and screen indigenous Bacillus strains from different soil samples for alpha amylase activity. Strains positive for the enzyme activity wereBacillus subtilis, Bacillus mycoids, Bacillus licheniformis and Bacillus cereus. These were further studied for amylase production in the presence of starch containing organic substrates such as powdered peels of potato, melon, banana, mango and apple. Solid state fermentations were carried out in mineral salt medium for 24 and 48 h at 37°C. Crude enzyme extract was used for amylase assay to determine the enzyme activity against standard curve of maltose. As a result, banana peel was found to be the best solid organic substrate for the highest alpha amylase production with 331.37, 241.45, 233.12 and 183.17 IU/ml/min in B. subtilis, B. licheniformis, B. cereus and B. mycoids, respectively in 24 h. Melon and potato were next to banana as nutrition, and the level of amylase production in all Bacillus strains with potato peels was comparable to melon peels with slight variations. All strains have shown minimum amylase production with apple or mango peels. A decline in the enzyme activity of the strains was observed after 24 h with all five substrates. Key words: Bacillus, alpha amylase activity, organic substrates, solid state fermentation.

Thermostable alpha-amylase enzyme production from Bacillus laterosporus: Statistical optimization, purification and characterization

Production and characterization of a thermostable alpha-amylase enzyme from Bacillus laterosporus isolate was investigated in this study. Box-Behnken design (BBD) of response surface methodology (RSM) was applied to evaluate the optimal level of four medium components (yeast extract, starch, peptone and NaCl). R2 value of 0.867 showed that model was well fitted with the experimental data and linear effect of starch, yeast extract and peptone were found significant on alpha-amylase production. Optimum values of yeast extract, starch, peptone and NaCl were predicted as 0.58%, 2.44%, 2.34% and 0.11%, respectively, with maximum enzyme activity of 4.838 U/ml. Enzyme purification was carried out by ammonium sulphate precipitation and size exclusion chromatography (SEC). Maximum purification was obtained by SEC with 4.71 fold. Effect of temperature and pH were studied on purified enzyme and the maximum activity was observed at optimal conditions of temperature (60 °C) and pH 7. Presence of Ca2+ ions and EDTA does not affect the enzyme activity, however reduced activity of the enzyme was observed in presence of Mg2+ ions, SDS and β-mercaptoethanol.

Utilization of Agricultural By-Products for Alpha-Amylase Production under Solid State Fermentation by Bacillus Subtilis

The production of alpha-amylase by Bacillus subtilis was investigated under different cultivation conditions on two different solid substrates as the support, i.e. oil palm empty fruit bunch (OPEFB) fibers and rice straw using solid state fermentation (SSF) process. Both solid substrates were first pre-treated using NaOH to remove lignin and silica and to break down the hemicellulose structures. The influences of incubation time, incubation temperature and the additional carbon and nitrogen sources on the production of alpha-amylase by B. subtilis were investigated. After 48 h of incubation, the highest enzyme activities were obtained at 39.9 U/g and 23.5 U/g for rice straw and OPEFB fibers, respectively. The optimum temperature for alpha-amylase production was observed at 45°C for the OPEFB fibers and 55°C for the rice straw. Among the defined carbohydrates, the addition of glucose (0.02 g/g dry substrate) has significantly improved the production of alpha-amylase. Similar effect was observed when yeast extract (0.01 g/g dry substrate) was supplemented as the additional nitrogen source. Rice straw appeared to be the best solid substrate in producing alpha-amylase (276 U/g) in SSF under these optimum cultivation conditions.

Concomitant production of two proteases and alpha-amylase by a novel strain of Bacillus subtilis in a microprocessor controlled bioreactor

We describe the simultaneous production of Bacillus subtilis based proteases and alpha amylase using a computer controlled laboratory scale 7.5 L batch bioreactor. The present strain is the first to be reported that concomitantly produces these two industrially important enzymes. The growth and sporulation of Bacillus subtilis was monitored and maximum production of alkaline protease and alpha amylase was found to coincide with maximum sporulation. Two types of proteases were detected in the fermentation broth; a neutral and an alkaline protease most active in a pH range of 7.0-8.0 and 8.0-10, respectively. Maximum production of proteases was observed at an incubation temperature of 37°C while that of alpha amylase was observed at 40°C. The optimum aeration and agitation levels for protease production were 0.6 L/L/min and 200rpm, respectively, and for alpha amylase were 0.6 L/L/min and 150 rpm. The kinetic parameters Yp/x and qp were also found to be significant at the given fermentation conditions.

Job Strain and Shift Work Influences on Biomarkers and Subclinical Heart Disease Indicators: A Pilot Study

This pilot study is one of the first to examine the impact of job strain and shift work on both the autonomic nervous system (ANS) and the hypothalamic-pituitary-adrenal (HPA) axis using two salivary stress biomarkers and two subclinical heart disease indicators. This study also tested the feasibility of a rigorous biological sampling protocol in a busy workplace setting. Paramedics (n = 21) self-collected five salivary samples over 1 rest and 2 workdays. Samples were analyzed for α-amylase and cortisol diurnal slopes and daily production. Heart rate variability (HRV) was logged over 2 workdays with the Polar RS800 Heart Rate monitors. Endothelial functioning was measured using fingertip peripheral arterial tonometry. Job strain was ascertained using a paramedic-specific survey. The effects of job strain and shift work were examined by comparing paramedic types (dispatchers vs. ambulance attendants) and shift types (daytime vs. rotating day/night). Over 90% of all expected samples were collected and fell within expected normal ranges. Workday samples were significantly different from rest day samples. Dispatchers reported higher job strain than ambulance paramedics and exhibited reduced daily alpha-amylase production, elevated daily cortisol production, and reduced endothelial function. In comparison with daytime-only workers, rotating shift workers reported higher job strain, exhibited flatter α-amylase and cortisol diurnal slopes, reduced daily α-amylase production, elevated daily cortisol production, and reduced HRV and endothelial functioning. Despite non-statistically significant differences between group comparisons, the consistency of the overall trend in subjective and objective measures suggests that exposure to work stressors may lead to dysregulation in neuroendocrine activity and, over the long-term, to early signs of heart disease. Results suggest that further study is warranted in this population. Power calculations based on effect sizes in the shift type comparison suggest a study size of n = 250 may result in significant differences at p = 0.05. High compliance among paramedics to complete the intensive protocol suggests this study will be feasible in a larger population.

English

  In this article, we report the optimization of bacterial extracellular alpha amylase production from Bacillus amyloliquefaciens IIB-14, a locally isolated bacterial culture, using response surface methodology (RSM). Different agricultural by-products were evaluated for enzyme activity under solid state fermentation (SSF). Of those, wheat bran (10 g) was found to be optimal when moistened with 0.02 M phosphate buffer at a level of 1:1.25. A marked improvement in enzyme productivity (52.06 U/mg/min) was observed at 72 h after the cultivation at 40°C (pH 7.2). The pre-grown bacterial inoculum (24 h old) was used at a level of 20%, v/v. The combined effect of cultural and nutritional variables on bacterial amylase was further investigated using RSM. The procedure limited the number of actual experiments performed while allowing for possible interactions between the optimal components including temperature, pH and inoculum. No previous work has used statistical analysis in determining the interactions among these parameters for alpha amylase production. The p-value of the coefficient for quadratic effect was 0.002, suggesting that this was the principal experimental variable having maximal effect on enzyme productivity. The results are economically significant (p ≤ 0.05) and marked the commercial utility of the isolated bacterial culture.   Key words: Agricultural by-products, alpha amylase, Bacillus spp., fermentation, response surface methodology, solid substrate fermentation.

Bioseparation of alpha-amylase by forming insoluble complexes with polyacrylate from a culture of Aspergillus oryzae grown in agricultural wastes

Precipitation of insoluble complexes between alpha-amylase from Aspergillus oryzae and polyacrylic acid was studied by us in a previous work as a strategy of protein concentration and purification. Here we studied the effect of these polyelectrolytes on the stability of the enzyme at the pH of higher interaction (3.00), as well as the stability of the precipitates formed after applied the above methodology. The polymers showed a stabilizing effect on the activity of alpha-amylase at pH 3.00, given the kinetic constants achieved when adjusting the experimental data to a process of first order inactivation: 2.20e−2±2e−3 for alpha-amylase alone, 7.7e−3±3e−4 and 8.2e−3±2e−4 for polyacrylic acid 240,000 and 100,000, respectively.Also, this stabilizing effect is intensified when complexes are in the solid precipitated form before redissolve, given the lower kinetic constants of inactivation obtained: 1.7e−3±2e−4 and 2.6e−3±2e−4 for polyacrylic acid 240,000 and 100,000, respectively.In a subsequent step, a strain of A. oryzae was used as enzymatic source. In order to stimulate alpha-amylase production and secretion by the microorganism, we used a minimum culture medium with wheat processing residues as substrate. This has economic and environmental benefits, because of the recycling of agricultural wastes. This culture showed a maximum productivity at the fourth day of incubation.When the technique of concentration and purification of alpha-amylase was implemented from this culture we obtained purification factors around two and recoveries around 70%. These results demonstrate that this methodology is suitable for the concentration and purification of alpha-amylase from A. oryzae. Besides, it is inexpensive since it uses agricultural materials without commercial value.

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.

Thermostable \alpha-amylase from moderately halophilic Halomonas sp. AAD21

The moderately halophilic Halomonas sp. strain AAD21, which produces extracellular thermostable \alpha-amylase, was isolated from the Çamaltı Saltern area located in İzmir, Turkey. NaCl, carbon, and nitrogen sources in the growth medium were optimized to enhance \alpha-amylase yield. The highest enzyme yield was measured in the presence of 20% NaCl with peptone as the nitrogen and starch as the carbon sources in the fermentation broth. This microorganism was also found to utilize waste potato peel as a carbon source for \alpha-amylase production. Concentrations of carbon and nitrogen sources were optimized using a statistical approach, and \alpha-amylase activity increased from 4.07 U mL^-^1 min^-^1 to 26.25 U mL^-^1 min^-^1. Maximum \alpha-amylase production was achieved at the end of 48 h of growth in the presence of 20% NaCl, 4.12% starch, 1.0% peptone, 0.2% KCl, 2% MgSO_{4}.7H_{2}O, and 0.03% trisodium citrate pentahydrate. The optimum pH and temperature of the \alpha-amylase were found to be 7.0 and 50 °C, respectively. The \alpha-amylase synthesized by Halomonas sp. AAD21 was also thermostable. Crude \alpha-amylase did not lose its original activity after 2 h of incubation at 50 °C and 60 °C, and it retained 70% of its original activity after 120 min of incubation at 90 °C.

Concomitant production of two proteases and alpha-amylase by a novel strain of Bacillus subtilis in a microprocessor controlled bioreactor

We describe the simultaneous production of Bacillus subtilis based proteases and alpha amylase using a computer controlled laboratory scale 7.5 L batch bioreactor. The present strain is the first to be reported that concomitantly produces these two industrially important enzymes. The growth and sporulation of Bacillus subtilis was monitored and maximum production of alkaline protease and alpha amylase was found to coincide with maximum sporulation. Two types of proteases were detected in the fermentation broth; a neutral and an alkaline protease most active in a pH range of 7.0–8.0 and 8.0–10, respectively. Maximum production of proteases was observed at an incubation temperature of 37°C while that of alpha amylase was observed at 40°C. The optimum aeration and agitation levels for protease production were 0.6 L/L/min and 200rpm, respectively, and for alpha amylase were 0.6 L/L/min and 150 rpm. The kinetic parameters Yp/x and qp were also found to be significant at the given fermentation conditions.

Production, purification and characterization of two α-amylase isoforms from a newly isolated Aspergillus Oryzae strain S2

A new fungal strain that was isolated from old sweet soy sauce was identified, based on subsequent microscopic studies and analyses of rRNA18S gene sequence, intergenic region rRNA 18S-23S, and aflatoxins production tests, as an Aspergillus oryzae strain. The latter was noted to produce two extracellular α-amylases, namely AmyA and AmyB. The monitoring of alpha-amylase production in the presence and absence of various protease inhibitors indicated that AmyB could be formed from the proteolysis of AmyA. The enzymes were purified to homogeneity through fractional acetone precipitation, size exclusion, and anion exchange chromatography. The molecular masses estimated for AmyA and AmyB by SDS-PAGE were 50 and 42 kDa, respectively. The NH 2-terminal of the purified proteins showed the same amino acid sequences. Further biochemical characterization assays revealed that both enzymes attained maximal activity at pH 5.6 and 50 °C. They were activated and stabilized by Ca 2+ and were noted to produce maltose and maltotriose as major starch hydrolysis end products. Overall, the findings of the present study indicate that both AmyA and AmyB exhibit a number of promising properties that make them potential strong candidates for application as additives in the bread making industry.

Alpha Amylase Production by Aspergillus Oryzae Using Solid State Fermentation

Alpha Amylase Production by Aspergillus Oryzae Using Solid State Fermentation

Effects of Sodium Octanoate, Acylated Ghrelin, and Desacylated Ghrelin on the Growth of Genetically Engineered Escherichia Coli

Effects of Sodium Octanoate, Acylated Ghrelin, and Desacylated Ghrelin on the Growth of Genetically EngineeredEscherichia ColiAcylated ghrelin is a 28-amino acid peptide hormone bearing a fatty acid group based on octanoic acid (caprylic acid) at the serine which is located at position 3 and at the N-terminus. If this fatty acid is cleaved from acylated ghrelin, the remaining peptide is referred to as desacylated ghrelin. Free fatty acids (FFAs) can kill or inhibit the growth of bacteria. The purpose of this study was to test this ability using acylated ghrelin, desacylated ghrelin, and sodium octanoate (caprylic acid) as carbon sources for the genetically engineeredEscherichia colistrains MK79 and MK57. For this experimental work, minimal medium was modified by replacing glucose with equal concentrations of acylated ghrelin, desacylated ghrelin, or sodium octanoate. Bacterial optical density, viability, alpha-amylase production, plasmid stability and pH of the growth medium were measured during these experiments. The media that allowed most growth, based on viable cell counts and the OD600of MK79, was minimal medium, followed by the medium containing desacylated ghrelin or acylated ghrelin, and finally the medium containing sodium octanoate. The same order was observed for MK57. Neither of the strains lost plasmids during the entire course of each experiment. There was also little change in the pH of any of the media used for both strains. These results suggest that sodium octanoate, acylated ghrelin, and desacylated ghrelin, when compared with minimal medium, inhibitEscherichia coligrowth. Proliferation was lowest when sodium octanoate was used as the carbon source, followed by acylated ghrelin and desacylated ghrelin. Therefore, the acylated ghrelin found previously in human saliva might help to inhibit pathogenic microorganisms, and acylated ghrelin levels below a critical concentration in saliva could result in an increased risk of oral infection.

Optimized production, purification and characterization of alpha amylase by Brevibacterium linens DSM 20158, using bio-statistical approach

Various cultivation parameters were optimized using a statistical approach to improve the alpha amylase yield by Brevibacterium linens DSM 20158. The Plackett-Burman design was used to screen the fermentation variables followed by the optimization of five significant parameters by response surface central composite design (CCD). The optimum levels for maximum production of enzyme were moisture level (82.5%), inoculum size (4.30%), wheat bran (14.2 g), temperature (33.5°C) and (NH4)2SO4 (0.4 g). Alpha amylase yield was found to be twofold higher than that obtained in the unoptimized reference medium. The closeness of optimized values to experimental values proved the validity of the statistical model. The enzyme was purified 7.88-fold with a 16.80% yield, and it appeared homogeneous on SDS PAGE with a molecular weight of 58 kDa.

Response Surface Methodology for the Optimization of α-Amylase Production by Bacillus subtilis ZJF-1A5

The aim of this work was to optimize the process parameters through the statistical approach for the production of alpha amylase by Bacillus subtilis ZJF-1A5 in submerged fermentation (SMF). The process parameters influencing the enzyme production were identified by using response surface methodology. Among the variables screened, the temperature and time were most significant and showed a positive interaction. The optimum levels were: temperature 35.8 °C, pH 5.03 and time 54h and under these conditions α-amylase yield was up to 191.15 U/mL.

Effect on Alpha-Amylase Production by Employing Polyethylene Glycol at Different Concentrations in Medium

Effect on Alpha-Amylase Production by Employing Polyethylene Glycol at Different Concentrations in Medium