Optimum Conditions For Enzyme Production Research Articles

Production, purification, and determination of the biochemical properties of β-glucosidase in Trichoderma koningii via solid substrate fermentation.

The β-glucosidase enzyme was obtained from Trichoderma koningii Oudem. NRRL 54330 under optimal conditions by solid substrate fermentation (SSF) using corn cobs as substrate. The enzyme was purified by two-step procedures, ammonium sulphate precipitation and cefarose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography, followed by biochemical and kinetic characterisation. The β-glucosidase was obtained from T.koningii using ground corn cob as substrate and Na2HPO4, pH 9, as humidification medium. The optimum conditions for enzyme production by SSF were 30 °C and 6days. The purification efficiency of the obtained β-glucosidase was calculated to be 22.56-fold with a yield of 73.51 %. In the determination of β-glucosidase activity, p-nitrophenyl-β-d-glucopyranoside (pNPG) substrate was used, and the optimum pH and temperature values at which β-glucosidase showed high activity were determined to be pH 3.0 and 75 °C. The purity of the enzyme and the presence/number of subunits were checked using two different electrophoretic methods, SDS-PAGE and NATIVE-PAGE electrophoretic methods. The K m and V max values of the purified enzyme were determined to be 0.16 mM and 2000 EU respectively. Itwas also found that d-(+)-glucose and δ-gluconolactone inhibitors exhibited competitive inhibition of β-glucosidase in the presence of pNPG.

Anticancer Efficacy of Purified Extracellular L-asparaginase from Aspergillus niger and Yield Enhancement by Agro-industrial Wastes

Nowadays L-asparaginase enzyme is of great importance medically as an anticancer agent and in the food industry through acrylamide mitigation so this work aimed to screen thirty-one fungal isolates recovered from rhizosphere soil by using the dilution plate method for L-asparaginase production. Twenty-four isolates (77.4%) were L-asparaginase producers. Aspergillus niger and A. quadrilineatus were the highest producers with enzyme activity 9.808±0.18930 and 7.348±0.12328 U/ml, respectively. The optimum conditions for enzyme production were at 30 °C for 72 h, pH 6 at 160 rpm, and 0.1% of KH2PO4 in presence of 2% glucose and 1.5% sucrose as carbon source and 1% L-asparagine by A. niger and A. quadrilineatus, respectively. Ammonium sulfate precipitation, Sphedax G-200, and SDS-PAGE were performed for L- asparaginase purification and molecular weight determination. Purified L-asparaginase molecular weights were approximately 50.36 KDa from A. niger with specific activity 50.4 U/mg and 27.8 KDa from A. quadrilineatus with specific activity 37.4 U/mg. Purified L-asparaginase significantly inhibited the proliferation of HCT-116, HePG-2, and MCF-7cell lines with IC50 concentrations of 28.9, 36.1, and 82.6 µg/ml, respectively and did not exhibit any antibacterial efficacy. Enhancement of L-asparaginase production using agro-industrial wastes was evaluated. Maximum yield (23.548 ± 0.00000 U/ml) was obtained by cultivation of A. niger on a mixture of onion and pomegranate peels powder (50%: 50% w/w) and cultivation of A. quadrilineatus on pomegranate peels only.

Bioconversion of chicken feather wastes by keratinolytic bacteria

Rhodococcus erythropolis, Geobacillus stearothermophilus and two Bacillus species (Bacillus pumilis, and Bacillus licheniformis) were evaluated for protease production using feathers as the sole carbon and nitrogen source. The B. licheniformis produced enzymes optimally at 40°C and pH 10.0, while B. pumilis performed optimally at 37°C and pH 7.0. Furthermore, the optimum conditions for enzyme production by G. stearothermophilus were 55°C and pH 7.0–8.0, while for R. erythropolis they were 37°C and pH 7.0. The maximum proteolytic activities of the protease produced by B. licheniformis, G. stearothermophylus and B. pumilis were 50.41, 9.91 and 35.41 U/ml after 48, 72 and 48h of culture, respectively. With R. erythropolis, the maximum enzyme activity was 33.39 U/ml after 96h of culture. Also, the production of soluble protein showed the same pattern as that of proteolytic protease. When the initial pH value was 10.0, culturing the feathers (30g l−1) for four days at 40°C resulted in the maximum production of soluble proteins (8.28mg/ml) by B. licheniformis. These results suggest that new strategies for waste management have emerged after the introduction of keratinases in sustainable material development, which has industrials applications in green technologies.

Isolation and screening of cellulose and organic matter degrading bacteria from aquaculture ponds for improving water quality in aquaculture

Aquaculture is one of the agroindustrial activities that has the highest growth rate in last decades and provides undeniable benefits for humanity such as providing food, generating jobs as well as contributing to economic development. However, intensive aquaculture also is one of the most criticized activities because of their environmental impacts, in which feed is the main source of waste and is responsible for most of the environmental impacts of aquaculture. Besides high concentration of organic and inorganic wastes from uneaten feed and excreta of aquaculture animals, amount of insoluble fiber (e.g., cellulose, hemicellulose, lignin) in aquaculture water is significant due to the majority of aquaculture feed is plant-based feed and contain a high amount of fiber. The polluted aquaculture water results in not only increase of environmental treatment cost but also outbreaks of aquaculture animal diseases. Improving water quality and reducing the accumulation of pollutants in aquaculture crops are therefore constant concerns. In this study, we isolated and screened cellulose and organic matter degrading bacteria from aquaculture ponds for producing probiotics which can improve aquaculture water quality and feed digestibility of aquaculture animals. From sediment and water samples collected at fish ponds in Phu Vang district, Thua Thien Hue provine, we isolated 215 bacterial strains, of which 25 isolates exhibited at least one of cellulolytic, amylolytic and proteolytic activities. Particularly, the strain Bacillus sp. W12 produced enzymes that are capable of degrading both cellulose and organic matters (cellulase, protease and amylase). The optimum conditions for enzyme production by Bacillus sp. W12 were obtained at pH 7, temperature 30°C, 72 h of incubation and 0.4% of NaCl. Activity of the enzymes reached the highest activity at pH 6-8 and temperature 30 - 40°C. The present study showed the strain Bacillus sp. W12 as a potential probiotic candidate for degrading cellulose and organic substrates in aquaculture water as well as improving feed digestibility of aquaculture animals.

Optimization of Arginine deiminase production from a local higher productive isolate Enterococcus faecium M1

Arginine deiminase (ADI) is an important enzyme in many biological applications and a cancer treatment agent, recently increasing attention is focused to choose a higher productive strain from different bacterial sources. Eighty nine Enterococcus isolates were obtained from 215 samples (20 clinical UTI specimens, 52 human stool specimens, 6 soil and 11 from sewage water), fifty seven isolates were ADI producer. Twenty five isolates were more active in enzyme production, fourteen of them were identified as E. faecium and 11 isolates were E. faecalis. E. faecium isolate M1 obtained from UTI was the most efficient in ADI production, the specific activity of ADI produced from this isolate was 2.64U/mg protein. The effect of culture medium (Mineral salt broth) components on ADI production and other cultural conditions were determined to find the optimum conditions for enzyme production. The maximum ADI production was achieved when the medium was supplemented with 20mM arginine, 1% sucrose, 1% casein, pH 7.5 and incubated at 37ºC for 18 hours. Under these conditions, the specific activity of ADI was 5.1U/mg protein. By concluding, the present study was designed to select ADI higher productive isolate and optimum culture conditions for increasing the production of this enzyme in order to use it as a potent cancer treatment agent after purifying and characterizing it in the future.

Efficient Degradation of Feather by Keratinase ProducingBacillussp.

Keratinase producing microorganisms are being increasingly utilized for degradation and recycling of poultry feather waste. Two native strains BF11 (Bacillus subtilis) and BF21 (Bacillus cereus) degrading keratin completely were characterized. The native strains produced more than 10 KU/mL of enzyme. Strain improvement resulted in isolation of MBF11 and MBF21 from BF11 and BF21 isolates, respectively. Optimization of nutritional and physical parameters of these MBF isolates at laboratory scale increased the overall keratinase activity by 50-fold resulting in a yield of 518–520 KU/mL. Fermentation media designed with starch as carbon source and soya bean meal as nitrogen source supported high levels of enzyme production. The optimum conditions for enzyme production were determined to be pH 8.5 and temperatures of 45–55°C for MBF11 and 37°C for MBF21, respectively. Culture filtrate showed a significant increase in the amounts of cysteine, cystine, methionine, and total free amino acids during the fermentation period. The ratio of organic sulphur concentration was also considerably higher than that of the inorganic sulphate in the culture filtrate suggesting the hydrolysis of disulphide by the isolates.

Amylase Production on Solid State Fermentation by Bacillus Spp

Twenty bacterial isolates were isolated from different sources. Preliminary screening for amylotic bacteria was performed on starch solid media. Out of them; 11 isolates showed positive results when flooded with iodine solution. The amylotic activity index was 93, 95 and 99. Identification of all isolates revealed that they belonged to the genus Bacillus. Out of all, three strains of Bacillus designated as B1, B2, and B11 were chosen for further study; according to their saccharification activity measured with the DNS method. Optimum conditions for enzyme production were measured, using wheat bran solid state fermentation (SSF) method. The optimum conditions for the three strains were generally found to be 55%- 75% moisture contents ; not less than 48 hrs incubation time (72 hours max. ); maximum enzymes production was attained at incubation temperature of 30℃ - 50℃; although the amylase retained more than 89% of its activity at temperature ranging from20℃ -70℃ , and optimum pH of 3.5.Qualitative analysis revealed that glucose and maltose was produced by Bacillus strains, B11 enzymes from hydrolysis of soluble starch.

Extraction and purification of L-Asparaginase II from local isolate of Proteus vulgaris

Forty one isolates of genus Proteus were collected from 140 clinical specimens such as urine, stool, wound, burn, and ear swabs from patients of both sex. These isolates were identified to three Proteus spp. P. mirabilis, P. vulgaris and P. penneri .The ability of these bacteria to produce L-asparaginase II by using semi quantitative and quantitative methods was determined. P. vulgaris Pv.U.92 was distinguished for high level of L-asparaginase II production with specific activity 1.97 U/mg. Optimum conditions for enzyme production were determined; D medium with 0.3% of L-asparagine at pH 7.5 with temperature degree 35°C for incubation. Ultrasonication was used to destroy the P. vulgaris Pv.U.92 cells then ASNase II was extracted and purified throughout several purification steps including precipitation with (NH4)2SO4(60-80%), DEAE-cellulose ion exchanger chromatography followed by Sephacryl S-300 filtration. The specific activity was 155.6 U/ mg and the purification fold was 27.3 with 10.4% yield.

Extraction and purification of L-Asparaginase II from local isolate of Proteus vulgaris

Forty one isolates of genus Proteus were collected from 140 clinical specimens such as urine, stool, wound, burn, and ear swabs from patients of both sex. These isolates were identified to three Proteus spp. P. mirabilis, P. vulgaris and P. penneri .The ability of these bacteria to produce L-asparaginase II by using semi quantitative and quantitative methods was determined. P. vulgaris Pv.U.92 was distinguished for high level of L-asparaginase II production with specific activity 1.97 U/mg. Optimum conditions for enzyme production were determined; D medium with 0.3% of L-asparagine at pH 7.5 with temperature degree 35°C for incubation. Ultrasonication was used to destroy the P. vulgaris Pv.U.92 cells then ASNase II was extracted and purified throughout several purification steps including precipitation with (NH4)2SO4(60-80%), DEAE-cellulose ion exchanger chromatography followed by Sephacryl S-300 filtration. The specific activity was 155.6 U/ mg and the purification fold was 27.3 with 10.4% yield.

Production of α-amylase by Aspergillus oryzae from cassava bagasse and wastewater sludge under solid-state fermentation

The potential of cassava bagasse (from cassava starch production) and biological sludge (biosludge, from an activated sludge treatment system) was investigated for α-amylase production using Aspergillus oryzae TISTR 3605 under solid-state fermentation and the optimum conditions for enzyme production were determined. The optimum conditions for α-amylase production were 48 h of incubation, using a ratio of cassava bagasse and biosludge of 3:2 (w/w) at a moisture content of 60%, with 1 mL inoculum and distilled water as the moisturizing agent. The influence of supplementation with carbon sources (glucose and starch) suggested that only glucose increased α-amylase production and then slightly. However, supplementing with nitrogen sources (peptone and ammonium sulfate) had no positive effect on amylase production. The results suggested that cassava bagasse and wastewater sludge had potential to be used as carbon and nutrient sources, respectively, for microorganisms to produce α-amylase. However, further study is needed to improve the α-amylase yield. © 2011 American Institute of Chemical Engineers Environ Prog, 2012

Purification and Characterization of Nitrate Reductase (NAR) from Pseudomonas sp. SH7 Isolate

Sixty five soil samples and fifteen water samples were collected from different places in which previously explosions were occurred in Iraq. Seven isolates showed ability to utilize 0.1mM trinitrotoluene (TNT) and/or 0.2mM glycerol trinitrate (GTN) as a sole carbon and nitrogen source and one of these isolates showed the highest nitrate reduction which was classified and coded as Pseudomonas sp. SH7. The highest nitrate reductase activity extracted by sonication while optimum conditions for enzyme production in minimal media pH 7 containing 0.25mM GTN at 35oC for 3 days under aerobic condition. Nitrate reductase was purified by 40-60% ammonium sulphate, ion exchange and gel filtration. Nitrate reductase molecular weight determined by SDS-PAGE was 115 kD. The characterization of purified enzyme activity and stability was higher at a pH between 6.5-7.5 and. Maximum activity was at 35oC and stable at 30-40oC for 15 min., while for heat sensitivity 100% activity observed at 45oC for 20 min. Treatment with 200 µM azide and 500 µM cyanide inhibited the activity by 76 and 91% respectively.

Production of fibrinolytic protease from various fungal isolates and species 2.Determination of optimum conditions for enzyme production from Pleurotus ostreatus

The optimum conditions for production of fibrinolytic protease from an edible mushroom Pleurotus ostreatus grown on the solid medium , Sus medium, composed of Sus wastes (produced from extracted medicinal plant Glycyrrhiza glabra) were determined. Addition of 5% of Soya bean seeds meal in Sus medium recorded a maximum fibrinolytic protease activity resulting in 7.7 units / ml. The optimum moisture content of Sus medium supplemented with 5% Soya bean seeds meal was 60% resulting in 7.2 units / ml.Pleurotus ostreatus produced a maximum fibrinolytic protease activity when the spawn rate,pH of medium and incubation temperature were 2,6 and 30°C, respectively. The maximum fibrinolytic protease activity was 7.6 units / ml when incubation period of Pleurotus ostreatus at the end of 3rd week (vegetative or mycelium stage), then lowered to 6.2 and 4.4 units/ml in the end of 4th week (reproduction or fruit bodies stage) and 5th week (after harvesting of fruit bodies), respectively. Although the minimum fibrinolytic protease activity was recorded in the end of 4th and 5th weeks, production of fibrinolytic protease regard to a byproduct after harvesting of fruit bodies.

Lipase Production by Solid-State Fermentation: Cultivation Conditions and Operation of Tray and Packed-Bed Bioreactors

The production of lipase by Penicillium simplicissimum in solid-state fermentation was studied using babassu cake as the basal medium. Tray-type and packed-bed bioreactors were employed. In the former, the influence of temperature; content of the medium, and medium supplementation with olive oil, sugarcane molasses, corn steep liquor, and yeast hydrolysate was studied. For all combinations of supplements, a temperature of 30 degrees C, a moisture content of 70%, and a concentration of carbon source of 6.25% (m/m, dry basis) provided optimum conditions for lipase production. When used as single supplements olive oil and molasses also were able to provide high lipase activities (20 U/g). Using packed-bed bioreactors and molasses-supplemented medium, optimum conditions for enzyme production were air superficial velocities above 55 cm/min and temperatures below 28 degrees C. The lower temperature optimum found for these reactors is probably related to radial heat gradient formation inside the packed bed. Maximum lipase activities obtained in these bioreactors (26.4 U/g) were 30% higher than in tray-type reactors.

Biosynthesis of tannase and hydrolysis of tannins to gallic acid by Aspergillus awamori — optimisation of process parameters

Tannin acyl hydrolase (tannase) is an industrially important enzyme produced by a large number of fungi. Tannic acid concentration, agitation speed and pH during the fermentation were identified as important process parameters effecting cell growth and enzyme synthesis by Aspergillus awamori. These parameters were optimised in a laboratory bioreactor by response surface methodology using Box and Behnken factorial design to determine the optimum conditions for enzyme production and gallic acid accumulation. Under optimum process conditions for enzyme synthesis, the fermentation run lasted 60 h with an initial tannic acid concentration of 35.0 g l −1, yielding biomass concentration of 7.13 g l −1 containing 771 IU of intracellular tannase per gram dry cell weight and 19 g l −1 of gallic acid. However, maximum gallic acid accumulation (40.3 g l −1) was obtained in 24 h with an initial substrate concentration of 45 g l −1.

Some factors affecting endo‐β‐1,4‐glucanase production by two Bacillus strains isolated from Zimbabwean hot springs

AbstractTwo Bacillus strains capable of producing endo‐β‐1,4‐glucanase enzyme were isolated from Zimbabwean hot springs. Strain HR68 produced the enzyme when cultured using both carboxymethylcellulose (CMC) and simple sugars as carbon source while strain CH43 produced the enzyme on CMC only. The optimum conditions for enzyme production were an initial pH of 6 and a temperature of 50 °C for strain HR68 while for strain CH43 the optimum conditions for enzyme production were an initial pH of 5 and a temperature of 50 °C. A fall in enzyme production was observed after about 4 to 8 h of growth for both isolates. The fall in production coincided with a rise in proteolytic activity in cultures of both isolates. Strain HR68 produced endo‐β‐1,4‐glucanase when grown in continuous culture using glucose as the carbon source.

Isolation and Purification of Ascorbate Oxidase from Acremonium sp. HI-25.

A screening test was undertaken to isolate microorganisms that produced ascorbate oxidase. The enzyme activity was found in a culture filtrate of a fungal strain (HI-25), newly isolated from a soil sample. Based on the morphological characteristics, this isolate was identified as Acremonium sp. From the examinations of cultural conditions, optimum conditions for enzyme production were found; strain HI-25 was aerobically cultured by a jar fermenter at 25°C in a medium containing 5% glycerol, 2% defatted soybeans, 0.1% monosodium L-glutamate, 0.1% KH2PO4, 0.02% MgSO4 ·7H2O, and 0.01% KCl, pH 6.0. After cultivation, an ascorbate oxidase was purified from the culture filtrate by an ammonium sulfate fractionation, column chromatographies on DEAE-cellulose and Butyl-Toyopearl, and gel filtration twice on Sephadex G-100. The purification was 850-fold with an activity yield of 8.8%. The purified enzyme gave a single band on SDS polyacrylamide gel electrophoresis, and had a molecular weight of 80,000 by SDS polyacrylamide gel electrophoresis and 76,000 by native gel filtration. This enzyme was most active at pH 4.0, 45°C, and was most stable between pH 6.0-10.0 and at temperatures below 60°C.

Dextran .ALPHA.-1,2 debranching enzyme from Flavobacterium sp. M-73: Its production and purification.

A new type of dextranase, dextran α-1,2 debranching enzyme, was detected in the culture fluid of a gram negative bacterium. This dextranase producing microorganism had characteristics of the genus Flavobacterium, and was tentatively named M-73. The optimum conditions for enzyme production were studied.The debranching enzyme was concentrated from the culture supernatant, treated at 55°C to inactivate the concomitant dextranase activity, and purified by column chromatography on DEAE-cellulose. Further purification was performed by biospecific adsorption chromatography using Sephadex G-150, from which the enzyme was eluted with a solution of clinical dextran, and by Polyacrylamide preparative gel electrophoresis. The purified enzyme was electrophoretically homogeneous and had a molecular weight of approximately 125,000. About a 2,700-fold increase in specific activity was achieved (66 units per milligram of protein) with a yield of 14.2%.

An .ALPHA.-1,3-glucanase from Streptomyces sp. KI-8 production and purification.

An α-1, 3-glucanase was detected in the culture supernatant of a micro-organism, which was isolated from soil on agar medium containing α-1, 3-glucan as sole carbon source. The isolated strain was characterized as a strain of Streptomyces, tentatively named KI-8. This enzyme required α-1, 3-glucosidic linkage as an inducer. The optimum conditions for enzyme production were studied. The enzyme was purified by (NH4)2SO4 precipitation, column chromatography on DEAE-cellulose and P (phospho)-cellulose. To eliminate the concomitant β-1, 3-glucanase activity, partially purified enzyme preparation was passed through a column packed with pachyman. Final purification was accomplished by the adsorption chromatography using Sephadex G-150 from which the α-1, 3-glucanase was eluted with a solution of α-1, 3-linked gluco-oligo-saccharides. The purified enzyme was electrophoretically homogeneous and had a molecular weight of approximately 78, 000 by SDS-polyacrylamide gel electrophoresis.

The properties and large-scale production of L-asparaginase from citrobacter.

An intracellular L-asparaginase with antitumour activity was purified from a strain of Citrobacter. The optimum conditions for enzyme production by fermentation on scales up to 2700 l were investigated. Highest enzyme yield was obtained in corn-steep liquor medium (9-2%, W/V) at 37 degrees C. Oxygen limitation was not necessary for high enzyme yield. A total recovery of 4-3% from nucleic-acid-free extract and a 180-fold increase in specific activity were obtained after purificaiton. The specific activity of the purified preparation was 45 i.u./mg protein. The enzyme hydrolysed D-asparagine and L-glutamine at 7 and 5%, respectively, of its activity toward L-asparagine, but L-glutaminase activity could be demonstrated only at substrate concentrations above 5 mM. The Km values for L-asparagine and D-asparagine were 2-6 X 10(-5) and 1-4 X 10(-4) respectively. The anti-lymphoma activity of the enzyme was demonstrated with Gardner lymphosarcoma and was found only slightly less potent that Crasnitin, the most active asparaginase so far tested in this system.