Characterization of an α‐amylase from sorghum (Sorghum bicolor) obtained under optimized conditions

Sorghum malt α‐amylase can compete with bacterial α‐amylase in industrial applications, if sufficiently stable and produced in a large enough quantity. Conditions for maximal α‐amylase production in sorghum malt and the physico‐chemical properties of the α‐amylase so produced are reported in this study. Sorghum grains were steeped in buffers with varying pH (4.0–8.0) for 24 h, at room temperature, and germinated for another 48 h to obtain the green malt. The buffer that induced the highest quantity of α‐amylase was chosen as the optimal pH and served as the medium for further steeping experiments conducted at different temperatures (10, 20, 30, 40, 50 and 60°C). The α‐amylase activity in the extract was determined in order to obtain the optimum temperature for α‐amylase induction at this particular pH. For the purpose of comparison, the α‐amylase produced at the optimum pH and temperature was purified to apparent homogeneity by a combination of ion‐exchange and size‐exclusion chromatography, and further characterized. Eight‐fold higher α‐amylase activity was induced in pH 6.5 buffer at 20°C compared with water, the traditional steeping medium. The K_m and V_max were estimated to be 1.092 ± 0.05 mg mL^?1 and 3516 ± 1.981 units min^?1, respectively. The activation energy of the purified amylase for starch hydrolysis was 6.2 kcal K^?1 mol^?1. Chlorides of calcium and manganese served as good activators, whereas CuSO₄ inhibited the enzyme with a 42% loss in activity at 312 mm salt concentration. Copyright © 2012 The Institute of Brewing & Distilling     

Some Enzyme Properties of Raw Starch Digesting Amylases from Streptomyces sp. No. 4

Streptomyces sp. No. 4 produce two forms of amylase that attack raw cassava starch. Both forms, amylase‐1 and amylase‐2, were purified by starch adsorption, affinity chromatography, and ion exchange chromatography. The molecular weights of amylase‐1 and amylase‐2 as determined by SDS‐PAGE were 56 kD and 77 kD, respectively. Optimal enzyme activities occurred at pH 5.5 and at 50°C for amylase‐1 and at 45°C for amylase‐2. The activation energy of amylase‐1 and amylase‐2 were 67 and 42 kJ/mol, respectively. Hg²⁺ and pCMB inhibited both enzymes, whereas 2‐mercaptoethanol activated only amylase‐2. EDTA inhibited amylase‐1 but activated amylase‐2. The main product of hydrolysis of raw cassava starch by amylase‐1 was maltose, followed by maltotriose, maltotetraose and dextrin. Amylase‐2 cleaved raw cassava starch to produce glucose and maltose as main products. Both amylase‐1 and amylase‐2 are α‐amylases, as shown by the fast disappearance of iodine staining, the corresponding reaction products and the ability of both enzymes to hydrolyze crosslinked blue starch.     

Partial characterization of β‐ d‐xylosidase from wheat malts

Arabinoxylans (AXs) from wheat malts potentially affect beer quality and production. β‐ d ‐Xylosidase is a key enzyme that degrades the main chains of AXs to produce xylose. This study performed a partial characterization of β‐ d ‐xylosidase from wheat malts. The optimal temperature was 70 °C and the enzyme exhibited excellent thermostability, that is, residual activities were 92.6% at 60 °C for 1 h. The enzyme was stable over a pH range of 3.0–6.0 and showed optimum activity at pH 3.5 and 4.5. Kinetic parameters K_m and V_max of wheat malt β‐ d ‐xylosidase against p‐nitrophenyl‐xyloside were 1.74 mmol L⁻¹ and 0.76 m m min⁻¹, respectively. The enzyme activity was severely inhibited by Cu²⁺, moderately inhibited by Mn²⁺, Mg²⁺, Al³⁺, Ca²⁺, Ba²⁺ and Na⁺ and mildly inhibited by Fe³⁺ and Fe²⁺. The partial enzymatic characterization achieved in this study can be used as a theoretical basis for purifying β‐ d ‐xylosidase from wheat malts. Copyright © 2015 The Institute of Brewing & Distilling     

Partial characterisation of a new barley amylase

A new amylolytic enzyme found in barley (Hordeum vulgare) grain was partially characterised with respect to physicochemical properties and enzymatic activity. The enzyme preparation showed one antigenically homogeneous amylolytic band. Isoelectric focusing resolved the new amylase into two components, one isoelectric at pH 4.5, the other at pH 3.0. During focusing the original activity of the new amylase decreased by 80%. The purified preparation was inactivated by pH-values below 4.5 and above 9.0 and also by temperatures above + 40°C for 1 h. The new amylase splits the 1,4-α-glycosidic linkages, clearly by endo-attack, of starch, amylopectin, amylose and β-limit dextrin optimally at pH 6.5 at +40°C giving K_m-values 8.9 × 10^?3, 4.4 × 10^?3, 6.6 × 10^?3 and 1.7 × 10^?3 g/ml, respectively. The hydrolysis products from β-limit dextrin were 24% glucose and 46% maltose in the total digest. Mercuric chloride, pCMB,^a EDTA^a and TRIS^a have no noticeable effect on the new amylase, indicating that it is stable under conditions where the other amylolytic enzymes are deactivated. The new amylase seems to be a hydrolase acting on o-glycosyl compounds, EC 3.2.1., but could not be identified with any of the amylolytic enzymes of vegetable origin studied previously.     

Inhibition of amylases present in ruminal particle‐associated micro‐organisms

The effect of different substances potentially inhibitory of ruminal amylase activity in sheep was assessed using biochemical and electrophoretic assays. Most amylase activity was detected in the particle‐associated fraction (70%) of the ruminal contents (which was selected for the assays) in comparison with the bacterial (21%) and extracellular (9%) fractions. Salts of divalent ions such as Sn²⁺, Hg²⁺, Cu²⁺ and Zn²⁺ produced 90, 82, 65 and 44% inhibition of amylase activity respectively when assayed at a relative concentration of 5 × 10^?3 mol l^?1. Organic acids such as tannic, formic, ascorbic and benzoic acid produced 79, 48, 43 and 37% inhibition respectively, whereas chelators such as EDTA, EGTA and 1,10‐phenanthroline produced an inhibition ranging from 32 to 37%. Substrate SDS‐PAGE zymograms allowed the identification of different amylase‐active bands in ruminal extracts, showing a wide range of relative molecular masses (from 36 to more than 100 kDa). Such zymograms also confirmed the effect of some inhibitors. The reversibility of the inhibitory effect of some of the assayed substances was assessed. ZnSO₄ was the most persistent inhibitory substance even at low concentrations and, owing to its low toxicity, appears to be an adequate substance to reduce the high in vitro ruminal degradation of starch. Implications for the process of enzymatic digestion of starch are discussed. © 2002 Society of Chemical Industry     

Purification and characterization of a novel glucoamylase from Fusarium solani

Thermostable enzymes are currently being investigated to improve industrial processes of starch saccharification. A novel glucoamylase was purified to electrophoretic homogeneity from the culture supernatant of Fusarium solani on a fast protein liquid chromatographic system (FPLC). The recovery of glucoamylase after gel filtration on FPLC was 31.8% with 26.2-fold increase in specific activity. The enzyme had a molecular mass of 40 kDa by SDS-PAGE and 41 kDa by gel filtration. The glucoamylase exhibited optimum activity at pH 4.5. The K_cat and K_m were 441/min and 1.9 mg/ml, respectively, for soluble starch, specificity constant (K_cat/K_m) was 232. The enzyme was thermally stable at 50 °C and retained 79% activity after 60 min at this temperature. The half-life of the enzyme was 26 min at 60°C. The enzyme was slightly stimulated by Cu²⁺ and Mg²⁺ and strongly inhibited by Hg²⁺, Pb²⁺, Zn²⁺, Ni²⁺ and Fe³⁺.     

PURIFICATION AND PROPERTIES OF CYCLODEXTRIN GLUCANOTRANSFERASE FROM AN ALKALOPHILIC BACILLUS sp. 7-12

Cyclodextrin glucanotransferases (EC 2.4.1.19) (CGTase) are industrially important enzymes for production of cyclodextrin (CD) from starch. γ‐CD yield of CGTase from alkalophilic Bacillus species is usually much lower than β‐CD, while from alkalophilic Bacillus sp. 7‐12. γ‐CD yield is close to β‐CD. A CGTase from alkalophilic Bacillus sp. 7‐12 was purified and characterized. When purified by ammonium sulfate fractionation, DEAE‐cellulose column chromatography and Sepharose CL‐6B column chromatography, the enzyme obtained consisted of a single band that did not dissociate into subunits by SDS polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was determined to be 69,000 Da by SDS‐PAGE. The enzyme showed a K_mof 1.24 mg/mL and V_max0.101 µM/min when potato starch was used as substrate. The enzyme was stable below 70C with an optimum activity at 60C, and stable at pH range 6–10 with an optimum pH at 8.5. The enzyme activity was strongly inhibited by Ag⁺, Cu²⁺, Mg²⁺, Al³⁺, Co²⁺, Zn²⁺, Fe²⁺and slightly inhibited by Sn²⁺, Mn²⁺. The ions Ca²⁺and K⁺, EDTA and DTT had no influence on the enzyme activity.     

Starch re‐crystallization kinetics as a function of various cations

Starch retrogradation is the main cause of quality deterioration in starch‐containing foods during storage. The current work investigates the effect of different cations on the retrogradation of corn starch and the potential of reducing starch retrogradation with the aim of preparing products with an extended shelf life. To gelatinize the starch, starch–water suspensions containing various chloride salts (LiCl, NaCl, KCl, MgCl₂, CaCl₂, and NH₄Cl) were heated in a DSC, stored up to 504 h at 8°C, and reheated again. Analysis of gelatinization behavior for each salt type indicates a relationship to the a_W‐value of the starch–water system. The degree of re‐crystallization was calculated using the Avrami equation, and indicates that the starch re‐crystallization rate (k) is significantly (p < 0.01) reduced with the addition of a cation, unlike the reference (starch–water systems without salt). Further, bivalent cations such as Ca²⁺, Mg²⁺ decreased the starch re‐crystallization rate (k) more than univalent cations (Li⁺, NH, Na⁺, and K⁺). This result may be based on the theory that high cations with higher charge densities show greater hydration, and, therefore, lower a_W‐values, than cations with lower charge densities. The results illustrate important results for predicting starch quality change when using sodium replacements.     

Purification and Characterization of a Halotolerant Alkaline Serine Protease from Penicillium citrinum YL-1 Isolated from Traditional Chinese Fish Sauce

A halotolerant alkaline serine protease from Penicillium citrinum YL-1 which was isolated from traditional Chinese fish sauce was purified by ammonium sulfate precipitation, dialysis, and DEAE 52-Cellulose column, thereby resulting in a 4.66-fold increase in specific activity (110.68 U/mg). The molecular weight (MW) was estimated to be 32.27 kDa using SDS-PAGE analysis. The protease exhibited optimal activity toward the substrate casein at pH 8.0 at 40°C and was stable at pH 6.0–8.0 and 4–30°C. Activity was inhibited by NaCl and retained at 28.3, 21.4 and 18.1% of the initial activity after incubation for 6 h at 20, 25 and 30% NaCl concentrations, respectively. The enzyme was stimulated by Mn²⁺ and inhibited by K⁺, Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, and Fe³⁺. K_m and V_max of the protease for casein were 1.93 mg/ml and 56.81 μg/(min·ml), respectively. Protease activity was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), which confirmed the serine protease nature of the enzyme. The protease can hydrolyze tilapia protein in the absence or presence of NaCl (5–30%), thus suggesting that this protease is more halotolerant than the protease from other bacteria with high salinity resistance based on the current literature. These properties make the halotolerant alkaline serine protease a suitable candidate enzyme for fish protein hydrolysis during fish sauce fermentation.     

Purification of diamine oxidase and its properties in germinated fava bean (Vicia faba L.)

Background: γ‐Aminobutyric acid (GABA) is a non‐protein amino acid with bioactive functions for human health. Diamine oxidase (DAO, EC 1.4.3.6) is one of the key enzymes for GABA formation. In the present study, this enzyme was purified from 5 day germinated fava bean and its properties were investigated in vitro. Results: The molecular mass of the enzyme estimated by Sephadex G‐100 gel filtration was 121 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) displayed a single band at a molecular mass of 52 kDa. The enzyme had optimal activity at 40 °C and retained its activity after being incubated at 30 °C for 30 min. It showed higher activity at pH 6.5 than at other pH values. The enzyme was significantly inhibited by Mg²⁺, Cu²⁺, Fe³⁺, aminoguanidine, ethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraacetic acid disodium salt (EDTA‐Na₂), L ‐cysteine and β‐mercaptoethanol. The K_m value of DAO was 0.23 mmol L^?1 for putrescine and 0.96 mmol L^?1 for spermidine. However, the enzyme did not degrade spermine. Conclusion: DAO from germinated fava bean was purified. The optimal reaction temperature and pH of the enzyme were mild. The enzyme had higher affinity to putrescine than to spermidine and spermine. Copyright © 2011 Society of Chemical Industry     

Purification,properties and heat inactivation of pectin methylesterase from apple (cv Golden Delicious)

Pectin methylesterase from apple (cv Golden Delicious) was extracted and purified by affinity chromatography on a CNBr‐Sepharose®‐PMEI column. A single pectin methylesterase peak was observed. Isoelectric points were higher than 9. Kinetic parameters of the enzyme were determined as K_m = 0.098 mg ml⁻¹ and V_max = 3.86 µmol min⁻¹ ml⁻¹ of enzyme. The optimum pH of the enzyme was above 7.5 and its optimum temperature was 63 °C. The purified PME required the presence of NaCl for optimum activity, and the sodium chloride optimum concentration increased with decreasing pH (from 0.13 M at pH 7 to 0.75 M at pH 4). The heat stability of purified PME was investigated without and with glycerol (50%), and thermal resistance parameters (D and Z values) were calculated showing that glycerol improved the heat resistance of apple PME. © 2000 Society of Chemical Industry     

Polyphenol oxidase properties,anti-urease,and anti-acetylcholinesterase activity of Diospyros lotus L. (Plum Persimmon)

Diospyros lotus fruit polyphenol oxidase was purified using affinity chromatography, resulting in a 15-fold enrichment in specific activity. The purified enzyme, having 16.5 kDa molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibited the highest activity toward 4-methylcatechol. Maximum diphenolase activity was reached at pH 7.0 and 60°C in the presence of 4-methylcatechol. K_m and V_max values were calculated as 3.8 mM and 1250 U/mg protein, respectively. Ascorbic acid was a promising inhibitor with an IC₅₀ value of 0.121 µM. The activity of the purified enzyme was stimulated by Fe²⁺, Sr²⁺, Zn²⁺, and K⁺ and deeply inhibited by Hg²⁺, at 1 mM final concentration. Aqueous extract of Diospyros lotus L. fruit showed strong substantial urease and acetylcholinesterase inhibition, with IC₅₀ values of 1.55 ± 0.05 and 16.75 ± 0.11 mg/mL, respectively.     

Phytosterols in banana (Musa spp.) flower inhibit α‐glucosidase and α‐amylase hydrolysations and glycation reaction

Three phytosterols were isolated from Musa spp. flowers for evaluating their capabilities in inhibiting glucosidase and amylase activities and glycation of protein and sugar. The three phytosterols were identified as β‐sitosterol (PS1), 31‐norcyclolaudenone (PS2) and (24R)‐4α, 14α, 4‐trimethyl‐5α‐cholesta‐8, 25(27)‐dien‐3β‐ol (PS3). IC₅₀ values (the concentration of inhibiting 50% of enzyme activity) of PS1, PS2 and PS3 against α‐glucosidase were 283.67, 11.33 and 43.10 μg mL^?1, respectively. For inhibition of α‐amylase, the IC₅₀ values of PS1, PS2 and PS3 were 52.55, 76.25 and 532.02 μg mL^?1, respectively. PS1 was an uncompetitive inhibitor against α‐amylase with K_m at 5.51 μg mL^?1, while PS2 and PS3 exhibited a mixed‐type inhibition with K_m at 52.36 and 2.49 μg mL^?1, respectively. PS1 and PS2 also significantly inhibited the formation of advanced glycation end products (AGEs) in a BSA–fructose model. The results suggest that banana flower could possess the capability in prevention of the diseases associated with abnormal blood sugar and AGEs levels, such as diabetes.     

Characterization of an acidophilic and thermostable α‐amylase from Alicyclobacillus sendaiensis NUST

This paper describes the characterization of an acidophilic and thermostable α‐amylase from Alicyclobacillus sendaiensis NUST. The MW of this enzyme was estimated to be 56 kDa by SDS–PAGE. The enzyme was stable over a range of pH from 2.5 to 5.5 with an optimum around 3.5. Maximum activity of the α‐amylase was observed at pH 3.5 and 85°C in the presence of soluble starch as substrate. The enzyme activity was decreased by Mg²⁺, Cu²⁺, Zn²⁺, Al³⁺, K⁺, Li⁺, Ag⁺, urea, EDTA, trichloroacetic acid and Tween 60 and inhibited by Hg²⁺, Ce²⁺ and SDS, whereas the activity was increased by Mn²⁺, DTT, and β‐mercaptoethanol. Ca²⁺and Fe²⁺ did not affect the enzyme activity.     

Optimisation of porous starch preparation by ultrasonic pretreatment followed by enzymatic hydrolysis

Porous starch granules were formed by the partial hydrolysis of starch using amylase. Fungal amylase and corn starch were chosen as the original amylase and substance from different sources, respectively. Ultrasonic technique as an assistant of enzymatic hydrolysis was used to pretreat raw starch. Scanning electron microscopy was used to examine the structure of native and treated starch, revealing the size of the pores in each occasion. The extent of enzymatic hydrolysis (y%) was markedly enhanced by statistical optimisation of enzymatic conditions. A significant influence of amount, temperature and pH of enzyme has been noted with Plackett–Burman design. It was then revealed with response surface methodology (RSM) that 503.26 U (g substance)^?1 amount of enzyme, temperature of 55 °C and pH of 5.1 were optimum. This optimisation strategy led to the enhancement of y% from 53.4% to 61.38%.     

Purification and some properties of a cysteine proteinase from sorghum malt variety SK5912

A cysteine proteinase from sorghum malt variety SK5912 was purified by a combination of 4 M sucrose fractionation, ion‐exchange chromatography on Q‐ and S‐Sepharose (fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 8.4‐fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2057.1 U mg^?1 protein. SDS—PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 55 and 62 kDa, respectively. The enzyme was optimally active at pH 6.0 and 50 °C, not influenced across a relatively broad pH range of 5.0–8.0 and retained over 60% activity at 70 °C after 30‐min incubation. It was highly significantly (P < 0.001) inhibited by Hg²⁺, appreciably (P < 0.01) inhibited by Ag⁺, Ba²⁺ and Pb²⁺ but highly significantly (P < 0.001) activated by Co²⁺, Mn²⁺ and Sr²⁺. The proteinase was equally highly significantly (P < 0.001) inhibited by both iodoacetate and p‐chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: K_m = 0.33 mg ml^?1; V_max = 0.08 µmol ml^?1 min^?1. Copyright © 2004 Society of Chemical Industry     

Three Citrus flavonoids retard the digestion of starch and its working mechanisms

In this work, the effects of three Citrus flavonoids (naringin, neohesperidin and hesperidin) on α‐amylase catalysed starch digestion were investigated. An inhibitory kinetics experiment demonstrated that they noncompetitively inhibit the activity of α‐amylase with Ki values of, respectively, 25.21 μm , 40.26 μm , 40.69 μm . Surface plasmon resonance assay showed three Citrus flavonoids can bind with starch with K_D values of, respectively, 3.98 ± 0.20 mm , 4.92 ± 0.15 mm and 5.07 ± 0.17 mm . Scanning electron microscopy indicated they can delay the retrogradation of starch. The results of 1, 1‐diphenyl‐2‐picryl hydrazyl radical assay indicate that the antioxidant activities of three Citrus flavonoids are significantly decreased by interacting with starch. These results illustrated three Citrus flavonoids that retard the digestion of starch not only through interaction with α‐amylase, but also through interaction with starch.     

Purification and characterisation of β‐mannanase from Lactobacillus plantarum (M24) and its applications in some fruit juices

β‐Mannanase was purified 2619.05‐fold from the Lactobacillus plantarum (M24) bacterium by ammonium sulphate precipitation and ion exchange chromatography (DEAE‐Sephadex). The purified enzyme gave two protein bands at a level of approximately 36.4 and 55.3 kDa in the SDS‐PAGE. The purified mannanase enzyme has shown its maximum activity at 50 °C and pH 8, and it has been also determined that the enzyme was stable at 5–11 pH range and over 50 °C. The V_max and K_m values have been identified as 82 mg mannan mL^?1 and 0.178 mm , respectively. The effects of some metal ions such as Fe²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, Cu²⁺ and Zn²⁺ on the mannanase enzyme have been also investigated, and it has been determined that all metal ions had significant effects on the activation of the mannanase enzyme. In addition, the effectiveness of the purified mannanase enzyme on the clarification of some fruit juices such as orange, apricot, grape and apple has been investigated. During the clarification processes, the enzyme was more effective than crude extracts on the clarification of the peach juice with a ratio of 223.1% at most.     

Characterization of a β-Glucosidase from an Edible Mushroom,Lycoperdon Pyriforme

A β-glucosidase from Lycoperdon pyriforme, a wild edible mushroom, was characterized biochemically. The enzyme showed a maximum activity at pH 4.0 and 50°C when p-nitrophenyl-β-D-glucoside was used as a substrate. K_m and V_max values were calculated as 0.81 mM and 1.62 U/mg protein, respectively. The enzyme activity was conserved about 85% over a broad range of pH (3.0–9.0) at 4°C after 24 h incubation. The activity was fully retained after 60 min incubation at 20–40°C. Na⁺, Li⁺, Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, Ca²⁺, and Cu²⁺ did not affect the enzyme activity and 0.25% sodium dodecylsulfate inhibited the enzyme activity approximately 76%. Ethylenediamine tetra-acetic acid, phenylmethanesulfonylfluoride, and dithiothreitol showed no or a little negative effect on the enzyme activity. The resistance of the enzyme to some metal ions, chemicals, and ethanol along with the pH stability, can make it attractive for future applications in industry.     

Isolation,purification and properties of the peroxidase from the hull of Glycine max var HH2

Soybean hull peroxidase (EC 1.11.1.7), an acidic peroxidase isolated from soybean (Glycine max var HH2) hulls was purified to electrophoretic homogeneity by a combination of ammonium sulphate fractionation, DEAE‐Sephadex A‐50 chromatography, concanavalin A‐Sepharose 4B affinity chromatography and Bio‐Gel P‐60 gel filtration. The specific activity of purified peroxidase was about 57‐fold higher than that of crude extract. The yield was about 16.4%. The molecular weight of the enzyme was estimated to be 38 000 by SDS‐polyacrylamide gel electrophoresis. The peroxidase was a glycoprotein containing about 18.7% carbohydrate, approximately one‐quarter of which was shown to be glucosamine residues. It was found to have an isoelectric point of 3.9. The enzyme was most active at pH 4.6 and 45°C, and was stable in the pH range 2.5–11.5. The enzyme could tolerate heating for 10 min at 75°C without being inactivated, and at 85°C, it took 40 min to inactivate the enzyme 50%, confirming that the peroxidase was a novel thermostable enzyme. Fe ²⁺, Fe³⁺, Sn²⁺, CN ⁻ and N₃⁻ inhibited enzyme activity, while Hg²⁺, Ag⁺, Pb ²⁺, Cr³⁺, EDTA and SDS were not significantly inhibitory. © 1999 Society of Chemical Industry