Application of decolourized and partially purified polygalacturonase and α-amylase in apple juice clarification

Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi) peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103) using wheat bran by solid state fermentation (SSF) process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL) and 0.4% α-amylase (899 U/mL), maximum clarity (%T660nm = 97.0%) of juice was attained after 2 h of incubation at 50 ºC in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property.

Partial purification and some properties of -amylase from Bacillus subtilis KIBGE-HAS.

An extracellular -amylase from Bacillus subtilis KIBGE-HAS was partially purified by ultrafiltration and ammonium sulphate precipitation with 19.2-fold purification and specific activity of 4195 U/mg. The enzyme showed relatively high thermostability and retained 62% of its activity when kept at 70°C for 15 min. -Amylase was highly stable at -18°C and loss of activity was very low during stability study. Metal ions like Mn2+, Ca2+, Co2+, K+, Mg2+, and Fe3+ activated the enzyme, while Hg2+ Ba2+, Cu2+, Na+ and Al3+ strongly inhibited the activity. The α-amylase was highly stable in various surfactants and detergents. In the presence of surfactants such as SDS and Triton X-100 the enzyme activity was found 2.9 and 1.8-fold higher respectively than control. The non-ionic detergents (Tween 20 and Tween 80) exhibited slightly inhibitory effect on the enzyme activity.

Purification and characterization of two alpha-amylases from wheat

Using chromatography on DEAE-cellulose, four forms, A1, A2, A3 and A4 of germinated wheat Sakha 8 alpha-amylases were demonstrated. alpha-Amylases A2 and A3 were purified to homogencity by Sephacryl S-200 column and the homogeneity proved by polyacrylamide gel electrophoresis. The molecular masses of alpha-amylases A2 and A3 were 29,000 and 31,000, respectively. The Km values of alpha-amylases A2 and A3 were 7.8 and 5.0 mg starch/ml, respectively. The affinity between substrate and the two enzymes decreased in the order of potato starch > glycogen > starch > corn starch for alpha-amylase A2 and glycogen > potato starch > starch > corn starch for alpha-amylase A3. The two enzymes were found to have the same pH and temperature optima of 7.5 and 50°C, and were heat stable up to 50°C. The effect of metal ions showed that Ca[+2], Co[+2] and Mg[+2] caused activating effects on alpha-amylases A2 and A3, while Cd[+2], Fe[+2], Cu[+2], Ni[+2] and Hg[+2] caused partial or strong inhibition effects. The three metal chelators, EDTA, citric and oxalic acids, have the same pattern of inhibition toward alpha-amylases A2 and A3, where 1 mM of these chelators caused 50% inhbiition of amylolytic activity and most enzyme activity was lost at 20 mM. This information will have a bearing on their potential use in the food industries

alpha-Amylase from germinated barley hordeum vulgare [cv. Giza 125] seeds

The activity of alpha-amylase during germination of barley [cv. Giza 125], increased from day 0 to day 10, where it exhibited its highest level, followed by a gradual decrease in activity till day 16. alpha-Amylase A2 was purified to apparent homogeneity from 10-day-old germinated barley [cv. Giza 125] by two steps of purification via DEAE-Sepharose ion exchange and Sephacryl S-200 gel permeation chromatographies with a recovery of 24% and 12 fold purification. This amylase, having a molecular weight of 26 kDa was found to be monomeric. The pH and temperature optima of a-amylase A2 was found to be 5 and 50°C, respectively. alpha-Amylase was stable at temperatures up to 50°C for 30 min incubation. The substrate specificity study showed that the enzyme act on the substrate which had alpha-1,4-and alpha-l,6-linkages as glycogen. K[m] and V[max] values were 0.87% starch and 2.85 micro mol reducing sugar liberated/min, respectively. Ca[2+], Li[+], Ba[2+] and Zn[2+] were found to have activating effect and Ni[2+] and Hg[2+] completely inactivated the activity. EDTA, sodium citrate and sodium oxalate had different inhibition effects on the enzyme activity except sodium oxalate at 1 mM which had activation effect. alpha-Nature and endo-action of this amylase was identified by its ability to reduce the viscosity of starch solution

Characterization of a thermostable alpha-amylase from a thermophilic Streptomyces megasporus strain SD12.

An extracellular alpha-amylase (1,4-alpha D-glucan glucan hydrolase; EC 3.2.1.1) was isolated from the cell free broth of Streptomyces megasporus SD12 grown in glucose, soluble starch and raw starch. The enzyme was purified 55-fold with a specific activity of 847.33 U mg-1 of protein and with a yield of 36% activity. The apparent molecular mass of the enzyme was 97 kDa, as estimated by SDS-PAGE. The pI of the enzyme was 5.4 and it was stable at a pH range of 5.5 to 8.5 with an optimum pH 6. The enzyme was stable upto 85 degrees C with a half life of 60 min. With soluble starch as substrate the enzyme exhibited a K(m) and kcat value of 4.4 mg ml-1 and 2335 U min-1 mg-1 of protein respectively. The major end products of starch hydrolysis were maltotriose and maltose depending on the incubation period. The production of the enzyme with agricultural wastes as substrates was 643 to 804 U min-1 mg-1 of protein in submerged fermentation whereas solid state fermentation could produce only 206 U min-1 mg-1 of protein.

Obtención de *-amilasa bacteriana: influencia del shock térmico y de los nutrientes del medio / Isolation of baceterial *-amylase: effect of heat shock and media nutrients

Se estudió la producción de alfa-amilasa empleando una cepa de Bacillus subtilis NRRL 3411. Las variables estudiadas incluyeron shock término, concentración y naturaleza de la fuente de carbono, nitrógeno y factores. Además se realizaron experiencias sobre estabilización de los caldos enzimáticos libres de células. Se determinó que utilizando esporos sometidos a un shock térmico de 100-C de 10 min, en un medio con lactosa, caseína, extracto de levadura y minerales, se alcanzaron valores de 140 unidades de alfa-amilasa en 72 h de proceso. También se demostró que el agregado de 20% de glicerol y 1% de benzoato de sodio aseguran caldos estabilizados a 20-C, por un período de 30 días

Effect of glycosylation of bacterial amylase on stability and active site conformation.

With a view to understand the changes in the conformation of bacterial amylase, the enzyme preparation was conjugated to dextran. Glycosylation of purified bacterial amylase resulted in increased stability against heat, proteolytic enzymes and denaturing agents. Several group specific inhibitors exhibited dose-dependent inhibition and the extent of inhibition was same for native as well as for the glycosylated enzyme. The pH optima of native and glycosylated enzyme remained the same indicating that the ionization at the active site is not greatly influenced as a result of glycosylation. Although the native as well as the glycosylated enzyme bind to the substrate with the same affinity, the rate of reaction differed greatly at 90 and 100 degrees C. At 70 degrees C, the rate of reaction was similar for the conjugated as well as the unconjugated amylase. Thermostability at different temperatures clearly showed that the glycosylated enzyme had greater stability compared to the native enzyme. The divalent cation binding site in the amylase also appears to be unaltered upon glycosylation since EDTA inhibited both enzymes to the same extent and addition of calcium ion restored the activity to almost the same level. These studies showed that conjugating the amylase enzyme with a bulky molecule like dextran does not affect the conformation at the active site.