Figen Ertan

Optimization of α‐Amylase Immobilization in Calcium Alginate Beads

Abstract α‐Amylase enzyme was produced by Aspergillus sclerotiorum under SSF conditions, and immobilized in calcium alginate beads. Effects of immobilization conditions, such as alginate concentration, CaCl2 concentration, amount of loading enzyme, bead size, and amount of beads, on enzymatic activity were investigated. Optimum alginate and CaCl2 concentration were found to be 3% (w/v). Using a loading enzyme concentration of 140 U mL−1, and bead (diameter 3 mm) amount of 0.5 g, maximum enzyme activity was observed. Beads prepared at optimum immobilization conditions were suitable for up to 7 repeated uses, losing only 35% of their initial activity. Among the various starches tested, the highest enzyme activity (96.2%) was determined in soluble potato starch hydrolysis for 120 min at 40°C.

Production and Properties of α‐Amylase from Penicillium chrysogenum and its Application in Starch Hydrolysis

Abstract Fungi were screened for their ability to produce α‐amylase by a plate culture method. Penicillium chrysogenum showed high enzymatic activity. α‐Amylase production by P. chrysogenum cultivated in liquid media containing maltose (2%) reached its maximum at 6–8 days, at 30°C, with a level of 155 U ml−1. Some general properties of the enzyme were investigated. The optimum reaction pH and temperature were 5.0 and 30–40°C, respectively. The enzyme was stable at a pH range from 5.0–6.0 and at 30°C for 20 min and the enzymes 92.1% activitys was retained at 40°C for 20 min without substrate. Hydrolysis products of the enzyme were maltose, unidefined oligosaccharides, and a trace amount of glucose. α‐Amylase of P. chrysogenum hydrolysed starches from different sources. The best hydrolysis was determined (98.69%) in soluble starch for 15 minute at 30°C.

Some Properties of Free and Immobilized α‐Amylase from Penicillium griseofulvum by Solid State Fermentation

Abstract α‐Amylase was produced from Penicillium griseofulvum by an SSF technique. α‐Amylase was immobilized on Celite by an adsorption method. Various parameters, such as effect of pH and temperature, substrate concentration, operational and storage stability, ability to hydrolyze starch and products of hydrolysis were investigated; these findings were compared with the free enzyme. The activity yield of immobilization was 87.6%. The optimum pH and temperature for both enzymes were 5.5°C and 40°C, respectively. The thermal, and the operational and storage stabilities of immobilized enzyme were better than that of the free enzyme. Km and Vmax were calculated from Lineweaver–Burk plots for both enzymes. Km values were 9.1 mg mL−1 for free enzyme, and 7.1 mg mL−1 for immobilized enzyme. The Vmax of the immobilized enzyme was approximately 40% smaller than that of the free enzyme. The hydrolysis ability of the free and immobilized enzyme were determined as 99.3% and 97.9%, respectively. Hydrolysis products of the α‐amylase from P. griseofulvum were maltose, unidentified oligosaccharides, and glucose.

Solid state fermentation for the production of α-amylase from Penicillium chrysogenum using mixed agricultural by-products as substrate

Production of α-amylase from local isolate, Penicillium chrysogenum, under solid-state fermentation (SSF) was carried out in this study. Different agricultural by-products, such as wheat bran (WB), sunflower oil meal (SOM), and sugar beet oil cake (SBOC), were used as individual substrate for the enzyme production. WB showed the highest enzyme activity (750 U/gds). Combination of WB, SOM, and SBOC (1:3:1 w/w/w) resulted in a higher enzyme yield (845 U/gds) in comparison with the use of the individual substrate. This combination was used as mixed solid substrate for the production of α-amylase from P. chrysogenum by SSF. Fermentation conditions were optimized. Maximum enzyme yield (891 U/gds) was obtained when SSF was carried out using WB + SOM + SBOC (1:3:1 w/w/w), having initial moisture of 75%, inoculum level of 20%, incubation period of 7 days at 30°C. Galactose (1% w/w), urea and peptone (1% w/w), as additives, caused increase in the enzyme activity.

THE PRODUCTION OF A NEW FUNGAL α-AMYLASE DEGRADED THE RAW STARCH BY MEANS OF SOLID-STATE FERMENTATION

In this study, it was intended to produce a new fungal amylase by solid-state fermentation and purification and also to determine some of its biochemical properties. It was found that Penicillium brevicompactum had the best enzyme activity according to screening methods with amylase degrading raw starch, and P. brevicompactum was selected as the amylase source. Wheat bran, rice husks, and sunflower oil meal were tested to determine the best solid substrate. Wheat bran was determined as the best of these. The fermentation conditions were optimized for the production of amylase. The optimum fermentation conditions were found to be an initial moisture level for the solid substrate of 55%, moistening agent of 0.1 M sodium phosphate buffer (pH 5.0), incubation period of 7 d, inoculum concentration of 2.5 mL, and incubation temperature at 30°C. Penicillium brevicompactum α-amylase was purified 45.98 times by the starch affinity method. The K m and V max values of α-amylase for soluble starch were 5.71 mg/mL and 666.6 U/mL, respectively. This amylase showed maximum activity at between 30 and 50°C and at pH 5.0. Initial enzyme activity was kept at 100% after incubation at 30°C for 45 min. Enzyme was stable in the pH range of 4.0–5.0. This enzyme was activated by Mn2+, Cu2+, and Na+ ions, and was inhibited by Mg2+, K+, Fe3+, and ethylenediamine tetraacetic acid (EDTA). The molecular mass of P. brevicompactum α-amylase was found to be 32.5 kD by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis.

Comparison of Some Properties of Free and Immobilized α‐Amylase by Aspergillus sclerotiorum in Calcium Alginate Gel Beads

Abstract Some properties of immobilized α‐amylase by Aspergillus sclerotiorum within calcium alginate gel beads were investigated and compared with soluble enzyme. Optimum pH and temperature were found to be 5.0 and 40°C, respectively, for both soluble and immobilized enzymes. The immobilized enzyme had a better Km value, but kcat/Km values were the same for both enzymes. Entrapment within calcium alginate gel beads improved, remarkably, the thermal and storage stability of α‐amylase. The half life values of immobilized enzyme and soluble enzyme at 60°C were 164.2, and 26.2 min, respectively. The midpoint of thermal inactivation (Tm) shifted from 56°C (for soluble enzyme) to 65.4°C for immobilized enzyme. The percentages of soluble starch hydrolysis for soluble and immobilized α‐amylase were determined to be 97.5 and 92.2% for 60 min, respectively.

Determination of the effects of initial glucose on the production of α-amylase from Penicillium sp. under solid-state and submerged fermentation.

The effects of catabolite repression of initial glucose on the synthesis of α-amylase from Penicillium chrysogenum and Penicillium griseofulvum were investigated under solid-state fermentation (SSF) and submerged fermentation (SmF) systems. The results obtained from either fermentation were compared with each other. In the SmF system, initial glucose concentration above 10 mg/mL completely repressed the production of α-amylase from P. chrysogenum and P. griseofulvum. However, the repression in the SSF system was not complete, even when the glucose level was raised to 160 mg/g.