Saturated Ammonium Sulfate Research Articles

Isolate and Extract for Milk Clotting Enzymes from the leaves of <i>Moringa Oleifera, Carica Papaya </i>and<i> Mangifera Indica </i>and Use in Cheese Making: The Case of Western Hararage Region, Ethiopia

Milk-clotting activity was screened from leaves of Carica papaya, Mangifera indica and Moringa oleifera in order to use the leaf with the highest milk-clotting activity as a source of the enzyme. The results of the present study showed that milk-clotting activity was only detected in the leaf extracts of Carica papaya and Moringa oleifera and the leaf extract from the leaf of Mangifera indica showed no activity. Ammonium sulfate precipitation was used in this study and the results showed that the highest milk-clotting activity was obtained with 40 % ammonium sulfate saturation. Maximum temperature for the crude extract of Carica papaya and Moringa olifera showed milk clotting activity at 70°C and 65°C respectively and also showed the highest clotting activity at a pH of 7.5 and 7 respectively. There was more than 80% retention of the milk-clotting activity of the crude extract of Carica papaya and Moringa olifera after 1h incubation at 60°C and 55°C respectively and the result also indicated that the crude extract retained more than 80% of its activity between pH 5 to pH 7.5 and pH 5 to pH 6 respectively. Finally the crude extract of Carica papaya and Moringa oleifera has a highest enzymatic activity at a concentration of 70 and 90 grams respectively.

Juglans regia and Ribes nigrum as potential nutraceuticals: Source of thermostable superoxide dismutase enzyme.

In the present study, superoxide dismutase (SOD) extracted from dry fruits; Juglans regia (Walnut; W) and Ribes nigrum (Munakka; M) was partially purified into 0%-40% and 40%-80% fractions based on ammonium sulfate saturation levels. The partially purified fractions (0%-40%) exhibited purification level of 3.09- (W) and 3.22- (M) fold with specific activity 79.32Umg-1 (W) and 125.23Umg-1 (M). SOD from both the sources was found to be thermally stable, that is, 80°C (W) and 70°C (M). Kinetic studies showed Km values to be 3.33mM (W) and 2.86mM (M), whereas the activation energy (Ea ) calculated as 24.52 KJmol-1 (W) and 26.25KJmol-1 (M). Na+ , Mn2+ , and Ba2+ ions acted as potential inhibitors, whereas Fe2+ stimulated SOD from both the sources. Among these metal ions, Na+ exhibited uncompetitive inhibition in both cases; with Ki values of 0.7mM (W) and 0.9mM (M), suggesting the more prominent binding affinity and effectiveness. PRACTICAL APPLICATIONS: Awareness need to be created among people for multifactorial health benefits of nutraceuticals in day-to-day life. Nutritional consumption from fruits, nuts, and vegetables safeguard against various maladies like cardiovascular diseases, diabetes, and cancers. Superoxide dismutase (EC 1.15.1.1) is a standout among the most critical metal-containing enzymes that act as a main line of defense against oxidative stress. Antioxidant-based drugs and formulations have been developed in the recent years and research is emphasized on its impact on oxidative stress levels. In this study, Juglans regia (W) and Ribes nigrum (M) were found to have thermostable SOD enzyme with excellent antioxidant properties. Thermal stability of an enzyme improves its significance making it industry friendly with therapeutically vital products, alongside their utilization as supplement in numerous therapeutic formulations.

Extraction of curcuminoids from Curcuma longa: comparative study between batch extraction and novel three phase partitioning

Curcuminoids, the active components of dried rhizome of Curcuma longa have been extracted using batch extraction and three-phase partitioning (TPP) process. The effect of different processing parameters, namely different solvents, extraction time, ammonium sulfate concentration, slurry to tert-butanol ratio, and solute to aqueous ratio on extraction efficiency of TPP, was studied to attain maximum extraction yield. The highest yield of 58.38 mg/g was achieved at 40±2 °C in 150 min, with saturated ammonium sulfate 30% (w/v), slurry to tert-butanol ratio 1:1 (v/v), and turmeric powder to water ratio 1:40 (w/v) in TPP. However, batch extraction using ethanol as a solvent yielded 52.77 mg/g in 180 min extraction time at 40±2 °C with 1:40 (w/v) turmeric powder to water ratio and 400 rpm agitation speed. In view of reference method, i.e., Soxhlet extraction (100%), TPP showed 65.63% yield in 150 min and batch exhibited 59.92% in 180 min. The turmeric extracts obtained by different methods exhibited excellent antioxidant and anti-inflammatory activities equivalent to their respective reference standards. Hence, TPP extraction process assures a rapid and improved recovery of curcuminoids with excellent therapeutic properties.

Short Communication: Protein profiles of Giant Mudskipper and its potential use as biomarker candidate for heavy metal contamination in Barito Estuary, Indonesia

Abstract. Hidayaturrahmah, Mabrur, Santoso HB, Sasmita R, Rahmy USA, Badruzsaufari. 2019. Short Communication: Protein profiles of Giant Mudskipper and its potential use as biomarker candidate for heavy metal contamination in Barito Estuary, Indonesia. Biodiversitas 20: 745-753. Giant mudskipper (Periphthalmodon schlosseri, Pallas 1770) is one of important biological diversity that potential as bio-indicator in environment assessment which is heavy metal contamination. These fish are susceptible to ambient environment, that effect to their physiological mechanism including protein synthesis and expression. This research aims to identify protein profiles of giant mudskipper muscle and review its potential application as a biomarker for heavy metal contamination in Barito Estuary of South Kalimantan. Total proteins were isolated from fish muscle using Tris EDTA buffer and then precipitated using Ammonium sulfate salt. The protein concentration was measured using Bradford assay and then separated based on molecular weight using the SDS-PAGE method. The result showed muscle proteins of giant mudskipper could be precipitated on optimum condition at Ammonium sulfate saturation 60-70% which protein concentration 5.106 mg.mL-1. The protein separated into 20 bands for raw and precipitated proteins which molecular weight ranging from 33 into 184 kDa. Further study is needed for protein identification based on amino acids sequence of protein bands for biomarker discovery and validation.

Cross-linked esterase aggregates (CLEAs) using nanoparticles as immobilization matrix

The present study focusses on the enhancement of the catalytic activity and stability of an acetylesterase enzyme isolated from Staphylococcus spp. as Cross-Linked Enzyme Aggregates (CLEAs). The various parameters governing the activity of CLEAs were optimized. The magnetite and graphene oxide nanoparticles were successfully prepared via the chemical co-precipitation and Hummer's method, respectively. These nanoparticles supported the preparation as magnetite nanoparticle-supported cross-Linked Enzyme Aggregates (MGNP-CLEAs) and graphene oxide-supported Cross-Linked Enzyme Aggregates (GO-CLEAs). The activity and stability of these immobilized CLEAs were compared with the free enzyme at various temperature, pH, and organic solvents along with its storage stability and reusability. The immobilized preparations were analyzed by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared spectroscopy (FT-IR) techniques. Acetylesterase precipitated with 60% saturated ammonium sulfate salt (SAS) solution and cross-linked with 100 mM glutaraldehyde for 4 h at 30 °C was found to be optimal to produce CLEAs with highest activity recovery of 99.8%. The optimal pH at 8.0 and temperature at 30 °C remained the same for both the free and immobilized enzyme, respectively. Storage stability significantly improved for the immobilized enzyme as compared to free enzyme. SEM showed type-I aggregate and FT-IR revealed the successful immobilization of the enzyme. MGNP-CLEAs were found to have better activity and stability in comparison to other immobilized preparations.

Rapid identification of triptolide in Tripterygium wilfordii products by gold immunochromatographic assay

Triptolide colloidal gold immunoassay strip was prepared by using colloidal gold labeled monoclonal antibody purified with saturated ammonium sulfate, triptolide succinate-bovine serum albumin and sheep anti-mouse IgG for the first time in our lab. Under optimized conditions, the detection limit of TP strip prepared in this study were determined to be 1 μg/mL and the total analysis time (including sample preparation) was less than 50 min. Sixty-six batches of Tripterygium wilfordii (T. wilfordii) and its adulterations were analyzed. It was found that 28 batches of T. wilfordii were positive as detected by the strips and 38 batches were negative. The result of test strip detection was in consistent with that from the UFLC-ESI-MS/MS method. Gold immunochromatography assay was demonstrated as a rapid, cost-effective, reliable technique for on-site screening of T. wilfordii and its preparations. The prepared colloidal gold immunoassay strip can be conveniently used for rapid identification of triptolide containing products.

Production of novel antimicrobial protein from Bacillus licheniformis strain JS and its application against antibiotic-resistant pathogens

Bacillus licheniformis strain JS has been shown to produce antimicrobial protein (AMP) using wheat bran as carbon and nitrogen source. Monitoring of AMP production revealed maximum after 72 h and extracted optimally at 70% saturation of ammonium sulphate. The extracted protein was further purified by ion exchange column chromatography which was eluted at 0.3 M NaCl concentration. The 16 kDa purified antimicrobial protein shows more activity against Gram-positive bacteria Bacillus cereus as compared to other bacteria. Further, combinatorial effect of AMP with antibiotics showed increased efficiency of Kanamycin, Neomycin and Streptomycin. So, the AMP extracted from B. licheniformis strain JS could be promising to increase the efficiency of present antibiotics against antibiotic-resistant pathogens. Thus, this study could be useful to design new therapeutic strategies to treat infectious diseases.

Aktivitas Antibakteri dan Antioksidan Peptida Susu Kambing Hasil Hidrolisis dengan Protease Lactobacillus plantarum S31

ABSTRACT Research on the hydrolysis of milk with various protease obtained from the digestive tract, plants and animals has been widely carried out, but the hydrolysis process of goat milk with protease enzymes from Lactobacillus plantarum S31 isolated from bekasam is still widely unknown. This study aims to determine the antibacterial and antioxidant activity of peptides hydrolyzed by protease enzymes from L. plantarum S31. Isolation and purification of extracellular proteases was carried out with 80% ammonium sulfate saturation, dialysis and G50 Sephadex matrix. The results of hydrolysis showed that fraction 11 has the highest antioxidant activity. Goat milk with pH 5 has a greater inhibitory activity of about 29%, which is 5% compared to goat milk pH 7. This fraction also has a quite good antibacterial activity of Entero Phatogenic Escherichia coli (EPEC K1.1) bacteria, Staphylococcus aureus and Listeria monocytogenes. Keywords: goat milk, hydrolysis, antibacterial, antioxidant, Lactobacillus plantarum

Purification and recovery of laccase produced by submerged cultures of Trametes versicolor by three-phase partitioning as a simple and highly efficient technique

Abstract In this work, three-phase partitioning (TPP) was used for the purification of laccase from liquid cultures of Trametes versicolor. For determining the optimal conditions of TPP process, parameters such as initial pH (6.5, 7.0, 7.5, 8.0), ammonium sulphate saturation (20%-80%) and the water phase to tert-butanol ratio (1:0.5, 1:1, 1:2), were analyzed. The best conditions with 73% recovery and 24-fold purification was obtained with the use of 50% saturation with ammonium sulphate, water phase to tert-butanol ratio of 1:1 and initial pH 7.0. The molecular mass of the purified laccase secreted by analyzed strain T. versicolor was found for ≈ 66 kDa. The results showed that TPP is an efficient method for the fractionation and purification of laccase obtained from liquid cultures of T. versicolor and it allows for obtaining the relatively pure enzyme without the use of time-consuming and costly chromatographic methods.

Introducing a cost-effective method for purification of bioactive flagellin from several flagellated gram-negative bacteria

The objective of this study was to introduce a simple and cheap method for purification of flagellin. So, flagellin proteins of Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), Citrobacter freundii (C. freundii) and Salmonella typhi (S. typhi) were purified by a modified simple method. Bacterial cultures were precipitated by centrifugation. Precipitates were washed twice and flagellin proteins were detached by shaking vigorously (in PBS pH = 2), and then flagellin proteins were precipitated by ammonium sulfate saturation. Evaluation of purification efficiency and concentration were examined by SDS-PAGE and Bradford assay. Polyclonal antibodies were produced against S. typhimurium FliC and cross-reactivity of anti-S. typhimurium was assessed against other flagellins. Bioactivity of flagellins was evaluated by cell proliferation and IL-8 protein expression assay in HEK293 cells, and also, IL-6 and TNF-α genes expression in chicken cells. Results showed a single band for flagellin proteins of all bacteria on %10 SDS-PAGE, which concentration ranged from 150 to 400 μg/mL. All flagellin proteins increased cell proliferation, and IL-8 levels were increased after treatment by flagellins and levels of IL-6 and TNF-α were increased after treatment with S. typhimurium FliC. All flagellin proteins showed cross-reactivity with antibodies. Findings showed that application of our method, not only reduced time and cost, but also, the purified flagellin proteins had acceptable bioactivity.

Partial Purification and Biochemical Characterization of ?-Glucosidase from Corn by Three-Phase Partitioning

α-Glucosidase was first purified from corn by three-phase partitioning(TPP) and then characterized. Ammonium sulfate and t-butanol were used in order to obtain three phases. Effect of different process parameters such as; ammonium sulfate concentration, enzyme to t-butanol ratio and pH required for efficient purification of the corn α-glucosidase was studied to get highest purification fold and activity recovery. Optimum purification parameters of the TPP system were determined as 50% (w/v) ammonium sulfate saturation with 1.0:0.75 (v/v) ratio of crude extract: t-butanol at pH 4.5. Under optimized conditions α-glucosidase was purified with 3.8 purification fold and 60% activity recovery. The molecular weight of α-glucosidase was determined approximately as 29 kDa by using sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS–PAGE). Characterization studies showed that, optimum pH and temperature of α-glucosidase were pH 4.5 and 55°C, respectively. The purified enzyme was found to be very stable at a temperature range of 25-60°C and a pH range of 3.5-5.5. Kinetic constants (KM and Vmax) were calculated from Lineweaver-Burk plot as 0.64 mM and 0.45 U, respectively. With 20 mM of CaCl2 concentration α-glucosidase activity was significantly increased to 130%. TPP is a useful strategy to concentrate and purify α-glucosidase for its applications.

Inhibitory activity of salivary glycoproteins on phytohemagglutins (PHA): Possible molecules to enhance nutritional quality of red kidney beans

Food allergy caused by red kidney bean (Phaseolus vulgaris L.) is of serious health concern and is mainly due to its phytohemagglutinins (PHA) content. PHA can enter the circulation after oral uptake and cause IgE mediated allergy. However, studies describing enhancement of nutritional quality of red kidney beans by targeting PHA are not reported. This study was carried out to identify, PHA-inhibitory molecules present in saliva secretions. Results describe that PHA can be effectively inhibited by salivary glycoproteins. Fractionation of salivary proteins by ammonium sulphate precipitation revealed that, PHA-inhibitory proteins can be specifically precipitated at 30-60% of ammonium sulphate saturation. Gel filtration chromatography and lectin-blot analysis of 30-60% ammonium sulphate fraction suggest that only high molecular weight glycoproteins can act as potent inhibitors of PHA. In conclusion, human saliva secretions contain inhibitory glycoproteins which can be used to inhibit PHA effectively. If these glycoproteins are purified to homogeneity, can be used as potent food supplements in order to neutralize allergic potential of PHA, thus increasing the nutritional value of red kidney beans.

Extraction, partial purification and characterization of alkaline protease from rainbow trout (Oncorhynchus Mykiss) viscera

In this study, crude alkaline proteases were recovered from rainbow trout (Oncorhynchus mykiss) viscera and partially purified by use of different saturation of ammonium sulfate. The enzyme exhibited highest yield, purity and activity when precipitated at a saturation of 40–60% compared to other ranges of saturation. Molecular weight for extracted protease was between 8-24 kDa. The protease had caseinolytic activity over a wide range of temperatures (30-55 °C) and pH (4-12). Soybean trypsin inhibitor and trypsin-chymotrypsin inhibitor strongly inhibited the enzyme activity but it was stable in the presence of surfactant, oxidizing reagents and organic solvents. The proteases had serine protease activity but no collagenase activity was detected. The current study showed that partially purified protease from the digestive tract of rainbow trout could be applicable in food and detergent industry because of its good activity over a wide temperature and pH range and its good thermal stability.

Partial Purification and Characterization of Polyphenol Oxidase from the Wild Edible Mushroom Lepiota Procera Using Three-Phase Partitioning

Abstract Polyphenol oxidase (PPO) from the wild edible mushroom Lepiota procera is partially purified and biochemically characterized using three-phase partitioning (TPP), which is an easily applied and effective method. This method includes ammonium sulfate saturation at different concentrations, t-butanol addition (1:1; 1:5), and adjustment of pH. Optimum purification parameters with 20% ammonium sulfate saturation and 1:1 t-butanol conditions led to the highest activity at bottom phase with 8.4 fold purification. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the molecular weight of the purified enzyme to be 35 kDa. The partially purified PPO presented a high level of activity with L-DOPA (Michaelis-Menten constant, Km – 0.12 mM), followed by caffeic acid (0.27 mM) and 4-methylcatechol (0.46 mM) and it is classified as a catecholase type of PPO. The enzyme had peak activity at a temperature of 40 °C and a pH value of 7.0. These results demonstrate a new enzyme source and easy purification method, useful for various industrial applications.

Characterization and Cytotoxic Activity of Cytosine Deaminase Enzyme Purified from Locally Isolated Escherichia coli

This research was aimed to the purification and characterization of cytosine deaminase as a medically important enzyme from locally isolated Escherichia coli; then studying its cytotoxic anticancer effects against colon cancer cell line. Cytosine deaminase was subjected to three purification steps including precipitation with 90% ammonium sulfate saturation, ion exchange chromatography on DEAE-cellulose column, and gel filtration chromatography throughout Sephadex G-200 column. Specific activity of the purified enzyme was increased up to 9 U/mg with 12.85 folds of purification and 30.85% enzyme recovery. Characterization study of purified enzyme revealed that the molecular weight of cytosine deaminase produced by E. coli was about 48 KDa, the highest enzyme activity at pH 8.5, and is most stable at pH 7.5 - 9, the enzyme also showed a full activity at a range of temperatures between 45-60 0C. Enzyme activity was strongly inhibited in the presence of mercuric chloride and copper sulphate, when added individually at a constant concentration. However, calcium chloride, manganese chloride and ferric chloride caused a little increase in enzyme activity while sodium azide had no effect on enzyme activity. Upon cytotoxic effect study through micro-cultured tetrazolium assay (MTT) against Caco-2 cell line. Purified cytosine deaminase was found to inhibit the growth of Caco-2 cancer cell line with an IC50 of 242.5 ?g/ml in a comparison to an IC50 of 1864 ?g/ml for crude enzyme. Besides, the enzyme didn’t show significant effect on WRL normal cell line.

Autoantibodies against dsDNA measured with nonradioactive Farr assay-an alternative for routine laboratories.

Autoantibodies against dsDNA are utilized for the diagnosis and prognosis of SLE as they are highly specific and correlate with disease activity/renal involvement. However, different detection methods are used in routine diagnostic laboratories. Farr radioimmunoassay (Farr-RIA) has been designated as the preferred method, since it provides very specific and at the same time quantitative results, enabling follow-up of level variations over time. Using intercalating fluorescent dsDNA dye would enable all the benefits of Farr-RIA without the radioactive material and organic solvents. To develop a modified fluorescent Farr method (Farr-FIA) and compare it to the classical Farr-RIA in regard to laboratory parameters, as well as clinical utility. Assays were tested on sera of 70 SLE patients, 78 other autoimmune patients, and 145 healthy blood donors. DNA for Farr-FIA was isolated from healthy donor, for Farr-RIA, 14C-labeled dsDNA from E. coli was used and mixed with sera in borate-buffered saline, followed by precipitation with saturated ammonium sulfate solution and centrifugation. The supernatant (S) was separated from the precipitate (P), and content of dsDNA was measured with PicoGreen (Invitrogen) in Farr-FIA or radioactive isotope in scintillation solution in Farr-RIA. The results were calculated as a ratio (P-S)/(P+S). Farr-FIA has a diagnostic sensitivity of 53% and diagnostic specificity of 100% (ROC AUC 0.781). Good correlation and agreement were shown between Farr-RIA and Farr-FIA. Also, there is good correlation between Farr-FIA and SLEDAI, comparable to that of Farr-RIA. Farr-FIA differs from Farr-RIA in the changed detection system yielding comparable results and thus could represent a nonradioactive replacement for Farr-RIA.

Production and Partial Purification of Tannase from Serratia Marcescens Isolated from Different Sources

Tannase has different benefits in food, chemical and pharmaceutical fields. Seventeen Serratia marcescens isolates were collected from septicemia, wound infections and hospital environment(babies incubators).These isolates were identified by biochemical tests and Vitek 2 system that contained Vitek GNI card then conformed by16S rRNA gene products(amplified size 179 bp) for genotypic detection. After that, they screened for higher tannase production and Serratia marcescens b9 was a better producer of tannase with a larger diameter of a dark green zone. The tannase activity was increased to 63U/ml when this isolate was cultivated under the optimal conditions which consisted of using nutrient broth supplemented with ber leaves at pH value 5.5 and a temperature equals to 37°C for 72 hours. In the partial purification of tannase, ammonium sulfate was more efficient than organic solvents, since it was found that 70% saturation of ammonium sulfate led to precipitate of tannase with tannase activity of 80U/ml. In contrast, 30% of ethanol, acetone, and isopropanol led to precipitate of tannase with different levels of activity ranged between 45-47U/ml. Consequently, ber leaves have a potential as an effective and much cheaper (economical) substrate for tannase production in comparison with traditionally used substrates like tannic acid.

The mechanisms involved in ochratoxin A elimination by Yarrowia lipolytica Y‐2

AbstractBiological control of mycotoxin in cereals, fruits and vegetables have emerged as a promising method. In a previous study, Yarrowia lipolytica Y‐2 isolated by our research team showed biocontrol effect on the post‐harvest decay of grapes and ochratoxin A (OTA) elimination in polytoma medium. The aim of this study was to elucidate the possible mechanisms of OTA elimination by Y. lipolytica Y‐2. The results indicated that OTA elimination by Y. lipolytica Y‐2 was attributed to the degradation action of intracellular enzymes but not extracellular enzymes. A degradation product was identified as ochratoxin alpha (OTα) by liquid chromatography‐tandem mass spectrometry. The intracellular enzymes precipitated with 65% saturation of ammonium sulphate degrade OTA the most quickly and 97.2% OTA was degraded within 4 h. Analysis of this fraction showed that two proteins of carboxypeptidase were expressed in Y. lipolytica Y‐2 but not in Y. lipolytica Polh without the ability to degrade OTA. The results of the protein identification combined with product identification indicated that OTA was degraded to OTα by Y. lipolytica Y‐2 through the hydrolysis activity of carboxypeptidases. Additionally, many proteins of Y. lipolytica Y‐2 involved in stress response and reactive O2 species elimination also played essential role in OTA degradation.

Isolation and partial characterization of serum immunoglobuline in two species of sturgeons, Siberian sturgeon (Acipenser baerii) and Stellate sturgeon (Acipenser stellatus)

The immunoglobulins (Igs) were isolated and purified respectively from serum of Siberian sturgeon (Acipenser baerii) and Stellate sturgeon (Acipenser stellatus) by using 50% saturated ammonium sulfate precipitation. Analysis of the purified Ig of the two species of fishes on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions revealed two bands, heavy chains and light chains respectively. The molecular weight of the heavy chains of two fishes was about 70 kD and the molecular weight of the light chains was 27kD for Siberian sturgeon and 27.5 kD for Stellate sturgeon. The results suggested that the isolated and purified serum Immunoglobuline in two species of sturgeons, Siberian sturgeon (A. baerii) and Stellate sturgeon (A. stellatus) can be used to produce polyclonal and monoclonal antibodies against sturgeon Ig for developing diagnostic tests against a wide variety of pathogens.

Agarose Hydrogel Beads: An Effective Approach to Improve the Catalytic Activity, Stability and Reusability of Fungal Amyloglucosidase of GH15 Family

This study deals with the immobilization of amyloglucosidase within agarose using method of entrapment. Enzyme was produced from Aspergillus fumigatus KIBGE-IB33 and then partially purified using 40% ammonium sulphate saturation. Using 40 gl−1 concentration of agarose and adjusting 3.0 mm size of hydrogels, maximum entrapment yield (78%) was obtained. The kinetic behavior was slightly changed after immobilization as reaction time and reaction temperature increases from 5.0 min (soluble) to 10.0 min (immobilized) and 60 °C (soluble) to 65 °C (immobilized), respectively while, pH optima remained same (pH 5.0). Substrate saturation kinetics revealed that Km was increased from 1.47 to 4.215 mg ml−1 while, the value of Vmax decreased from 947 to 611 kU mg−1 for soluble and entrapped amyloglucosidase, respectively. The stability profile of amyloglucosidase also significantly improved after entrapment in agarose hydrogels at 50, 60 and 70 °C for 120 min with retention of 77, 59 and 25% residual activity, respectively. Furthermore, the t1/2 of soluble and immobilized amyloglucosidase at 60 °C was 167 and 375 min respectively. Due to increase in reusability for various subsequent cycles of entrapped amyloglucosidase, about 8.73 mg ml−1 increase in glucose production was observed as compared to soluble enzyme.