Raw Starch Degrading, Acidic-Thermostable Glucoamylase from Aspergillus fumigatus CFU-01: Purification and Characterization for Biotechnological Application *For Correspondence (original) (raw)
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African Journal of Biotechnology, 2015
A fourteen day pilot study carried out showed that high glucoamylase activities were obtained on the 4 and 11 th day of fermentation and the enzymes were harvested on the respective days giving the codes GluAgCSV4 and GluAgCSV11. The optimal pH and optimal temperatures for enzyme activities GluAgCSV4 and GluAgCSV11 were in a range of 6 to 7 and 50 to 55, using cassava, guinea corn and tiger nut starch as substrates, respectively. The enzyme activity (GluAgCSV4) was enhanced by Ca 2+ , Mn 2+ , Fe 2+ and Zn 2+. Co 2+ had inhibitory effect on the enzyme while Pb 2+ completely inactivated the enzyme. The enzyme activity (GluAgCSV11) was enhanced by Ca 2+ and Co 2+. Zn 2+ , Fe 2+ Mn 2+ and Pb 2+ completely inactivated the enzyme. The Michaelis constant K M and maximum velocity V max obtained form Lineweaver-Burk plot of initial velocity data at different substrate concentrations were found to be 90.06 mg/ml and 188.67 µmol/min (using cassava starch as substrate), 173.70 mg/ml and 434.78 µmol/min (using guinea corn starch as substrate) and 28.57 mg/ml and 227.27 µmol/min (using tiger nut starch as substrate), respectively for GluAgCSV4. Also, 271.30 mg/ml and 1000 µmol/min (using cassava starch as substrate, 3093 mg/ml and 10000 µmol/min (using guinea corn starch as substrate) 2625 mg/ml and 10000 µmol/min (using tiger nut starch as substrate), respectively, were obtained for GluAgCSV4.
Journal of Bioscience and Biotechnology Discovery, 2023
Cassava starch can be completely hydrolyzed to produce a sweetener that can be used as a substitute for refined sugars and artificial sweeteners in the food and pharmaceutical industries. This work was designed to isolate and identify a good amylase producing fungus from cassava flour and to extract, purify, and partially characterize α-amylase produced. The enzyme was produced through solid-state fermentation followed by 70% ammonium sulphate precipitation and ion-exchange chromatography on Carboxyl-Methyl (CM) Sephadex C25. The physicochemical properties of the purified enzyme were determined. The peak with the highest activity was pooled from the latter chromatographic step and characterized afterward. The enzyme's specific activity rose from 0.11 to 2.1 U/mg having a yield of 15.8% and a purification fold of 19.1. The optimal pH and temperature of the enzyme were 6.0 and 50°C respectively. The enzyme was observed to be thermo-stable at 50°C for 15 to 30 minutes. The kinetics revealed that the Vmax was 1.25 U/min while Km was 0.2 mg/ml. The enzyme's native and sub-unit molecular weights were found to be 22 and 18.5KDa respectively. The results revealed conclusively that the isolated enzyme from Aspergillus tamarii exhibited the properties of glucoamylase.
Journal of The Science of Food and Agriculture, 2001
A medium was developed to obtain the maximum yield of raw starch-digesting amylase from Aspergillus carbonarius (Bainier) Thom IMI 366159 in submerged culture with raw starch as the sole carbon source. The amylase was puri®ed to apparent homogeneity by sucrose concentration and ion exchange chromatography on S-and Q-Sepharose (fast¯ow) columns. SDS-PAGE revealed two migrating protein bands corresponding to relative molecular masses of 31.6 and 32 KDa. The enzyme was optimally active at pH 6.0±7.0 and 40°C, was unin¯uenced across a relatively broad pH range of 3.0±9.0 and retained over 85% activity between 30 and 80°C after 20 min incubation. The enzyme was strongly activated by Co 2 and only slightly by Fe 2 , while Ca 2 , Hg 2 , EDTA and N-bromosuccinamide elicited signi®cant repression of the enzyme activity. The enzyme hydrolysed amylopectin (K m 0.194 mg ml À1 ), glycogen (K m 0.215 mg ml À1 ), pullulan (K m 0.238 mg ml À1 ), amylose (K m 0.256 mg ml À1 ) and raw potato starch (K m 0.260 mg ml À1 ), forming predominantly maltose and relatively smaller amounts of glucose.
Bangladesh Journal of Scientific and Industrial Research, 2017
A Fourteen day experimental study was carried out to determine the day of highest glucoamylase activity using amylopectin from guinea corn starch as the sole carbon source. Two peaks of high activity were observed on the fifth and twelveth days, and were thus mass produced. Specific activities for crude enzymes were found to be 729.45 U/mg and 1046.82 U/mg for day five and twelve harvested enzymes respectively. Ammonium sulphate saturations, 70% and 20%, were found suitable to precipitate proteins with highest glucoamylase activity for day five and twelve harvested enzymes respectively. After ammonium sulphate precipitation and gel filtration, specific activities were found to be 65.98 U/mg and 180.52 U/mg respectively for day five harvested enzyme and 61.51 U/mg and 272.81 U/mg for day twelve harvested enzyme. The pH optimum for day five harvested enzyme were found to be 7.5, 7.5 and 6.0 using tiger nut, cassava and guinea corn starches as substrates respectively, also, the pH optimum for day twelve harvested enzyme were found to be 5.0, 8.5 and 7.0 using tiger nut, cassava and guinea corn starches as substrate, respectively. Optimum temperatures were found to be 50˚C and 45˚C for day five and twelve harvested enzymes, respectively. K m and V max , of day five harvested enzyme were found to be 770.75 mg/ml and 2500 µmol/min, 158.55 mg/ml and 500 µmol/min and 46.23 mg/ml and 454.53 µmol/min using cassava, guinea corn and tiger nut starches as substrate respectively. K m and Vmax of day twelve harvested enzyme were found to be 87.1 mg/ml and 384.61 µmol/min, 29.51 mg/ml and 243.90 µmol/min, and 2364 mg/ml and 2500 µmol/min, using cassava, guinea corn and tiger nut starches as substrate respectively.
Glucoamylases (GAs) from a wild and a deoxy-D-glucose-resistant mutant of a locally isolated Aspergillus niger were purified to apparent homogeneity. The subunit molecular mass estimated by SDS-PAGE was 93 kDa for both strains, while the molecular masses determined by MALDI-TOF for wild and mutant GAs were 72.876 and 72.063 kDa, respectively. The monomeric nature of the enzymes was confirmed through activity staining. Significant improvement was observed in the kinetic properties of the mutant GA relative to the wild type enzyme. Kinetic constants of starch hydrolysis for A. niger parent and mutant GAs calculated on the basis of molecular masses determined through MALDI-TOF were as follows: k cat = 343 and 727 s À1 , K m = 0.25 and 0.16 mg mL À1 , k cat /K m (specificity constant) = 1374 and 4510 mg mL À1 s À1 , respectively. Thermodynamic parameters for soluble starch hydrolysis also suggested that mutant GA was more efficient compared to the parent enzyme.
Purification and properties of two forms of glucoamylase fromAspergillus niger
Folia Microbiologica, 1996
A. niger produced c~-glucosidase, a-amylase and two forms of glucoamylase when grown in a liquid medium containing raw tapioca starch as the carbon source. The glucoamylascs, which formed the dominant components of amylolytic activity manifested by the organism, were purified to homogeneity by ammonium sulfate precipitation, ion-exchange and two cycles of gel filtration chromatography. The purified enzymes, designated GA1 and GA2, a raw starch digesting glucoamylase, were found to have molar masses of 74 and 96 kDa and isoelectric points of 3.8 and 3.95, respectively. The enzymes were found to have pH optimum of 4.2 and 4.5 for GA1 and GA2, respectively, and were both stable in a pH range of 3.5-9.0. Both enzymes were thermophilic in nature with temperature optimum of 60 and 65 ~ respectively, and were stable for 1 h at temperatures of up to 60 ~ The kinetic parameters K m and Vshowed that with both enzymes the branched substrates, starch and amylopectin, were more efficiently hydrolyzed compared to amylose. GA2, the more active of the two glucoamylases produced, was approximately six to thirteen times more active towards raw starches compared to GAl.
Hydrolysis of raw tuber starches by amylase of Aspergillus niger AM07 isolated from the soil
African Journal of …, 2005
Eight Aspergillus niger strains which produced strong starch degrading amylase were isolated from the soil using a medium containing Remazol Brilliant Blue (RBB) starch as substrate. Amylase production was detected by the disappearance of the blue colour around the colony. Among the isolates, A. niger AM07 produced the largest clear zone (7.0mm) on Remazol Brilliant Blue (RBB) agar plate and also gave the highest amylase yield (806 U/ml) in solid-state fermentation process, hence it was selected for further studies. The crude amylase preparation of A. niger AM07 had temperature and pH optima activities at 60 o C and 4.0 respectively. The optimum substrate concentration was 3 %. The action of the crude amylase of A. niger on raw tuber starches of yam, cassava, sweet potato and cocoyam were studied in comparison with the well known maize starch which is a cereal starch. The crude amylase was able to hydrolyze all the raw starches tested. Hydrolysis was significantly (p<0.05) dependent on starch source and length of incubation. At 72-h incubation time, raw cassava starch gave the highest yield of 200.1 mg/g with a conversion efficiency of 198.91% while raw maize starch gave a yield of 109.6 mg/g with 108.95 % conversion efficiency. Raw cocoyam starch was more resistant to hydrolysis and incubation of cocoyam starch beyond 24 h, resulted in decreased yield of reducing sugars. Thin layer chromatography showed glucose as the main sugar produced with low level of maltose.
Starch-starke - STARCH, 1980
Aus den Versuchsergebnissen geht folgendes hervor : Die Einrichtungen zur Kartoffelstarkegewinnung sollten eine moglichst groDe Leistungskapazitat haben. Diese Kapazitat sol1 max. ausgenutzt werden, weil dies zur Senkung des Energieaufwandes bei Erhaltung der entsprechenden Qualitat des Endproduktes fuhrt. Heutzutage verwendete Einrichtungen verbrauchen zu vie1 Wasser und verursachen deshalb neben den allzu groDen Energieverlusten Schwierigkeiten bezuglich der Reinigung. Den Hydrozyklonen im Auswasch-und Raffinationsverfahren sollte im Hinblick auf ihre universelle Verwendbarkeit und den verhaltnismaBig niedrigen Energieverbrauch eine groDere Bedeutung zugemessen werden. Die Untersuchungsergebnisse erlauben grundsatzlich h d erungen im Auswasch-und RaffinationsprozeD, die bei Berucksichtigung durch die Untersuchung der ermittelten optimalen Parameter eine neue technische Anordnung der untersuchten Einrichtungen in der Kartoffelstarkeproduktion gestatten.