CHARACTERIZATION OF AN 'ENDOPOLYGALACTURONASE' OF THE YEAST KLUYVEROMYCES MARXIANUS

Analytic isoelectric focusing showed that the ‘endopolygalacturonase’ from Kluyveromyces marxianus consists of 21 multiple enzyme components. Neither changes in cultivation conditions, enzyme substrate or reaction conditions resulted in any quantitative or qualitative differences in the enzyme patterns. Two of these multiple enzyme forms, the main band, (IP = pH 5.8) which accounted for approximately 95% of the total activity, and an ‘acid-band’ (IP = pH 2.7), were purified by means of preparative isoelectric focusing and their molecular weights and amino acid compositions were determined. The molecular weight of the main band was established as 76,000 daltons (2 subunits of 47,900 and 28,100), The molecular weight of the ‘acid-band’ was 32,200. Both amino acid analyses and molecular weight determinations suggested that the proteins are chemically and physically different. The pH optimum was 4 for the pure enzyme on different pectin substrates, e.g., high molecular weight pectic acid, low molecular weight pectic acid and highly methylated pectins B and C. The temperature optimum obtained for pure enzyme with high or low molecular-weight pectic acid as substrate was 40° C. V_max and Km-value determination at different pH values with low molecular weight pectic acid as a substrate was used to identify the catalytically active groups at the active site. They were tentatively identified as an unprotonated α-carboxyl group and a protonated carboxyl group of aspartic acid.     

Purification and properties of a new,commercial, thermostable Bacillus stearothermophilus α‐amylase

A rapid purification has been developed for Bacillus stearothermophilus α‐amylase starting with the commercial enzyme product. The two‐step procedure, using hydrophobic interaction chromatography and ion exchange, results in a 6.8‐fold increase in specific activity with an 86% recovery of starting activity. The molecular weight of the enzyme was 58,000 when measured by SDS‐PAGE The enzyme preparation consisted of three isozymes with pl values of 8.1, 8.0, and 7.8–7.7. The enzyme mixture had a broad pH optimum of pH 4.0–7.0 at 40° C and a narrower optimum of pH 5.2–6.5 at 90° C. The temperature optimum was 80° C when measured by the starch‐iodine assay; hydrolysis of maltodextrin indicated a constant, maximum rate between 70° C and 100° C. Calcium was shown to increase the half life of the enzyme at 90° C approximately 10‐fold; addition of 10% maltodextrin and calcium increased the half life of the enzyme 80‐fold.     

Heat inactivation and kinetics of polyphenoloxidase from palmito (Euterpe edulis)

Polyphenoloxidase (PPO, EC 1.14.18.1)was extracted from palmito (Euterpe edulis Mart) using 0.1 M phosphate buffer, pH 7.5. Partial purification of the enzyme was achieved by a combination of (NH₄)₂SO₄precipitation (35–90% saturation) and Sephadex G-25 and DEAE-cellulose chromatography. The purified preparation gave five protein bands on polyacrylamide gel electrophoresis, three of them with PPO activity. The K_mvalues for chlorogenic acid, caffeic acid, catechol, 4-methylcatechol and catechin were 0.57, 0.59, 1.1, 2.0 and 6.25 mM , respectively. PPO has a molecular weight of 51 000 Da, maximum activity at pH 5.6 with chlorogenic acid as substrate, and was stable between pH 5.0 and 8.0. The enzyme was heat stable at 50–60°C and inactivated at 75°C. The heat stability of palmito PPO was found to be pH dependent; at 50°C and pH 4.0 the enzyme was fully inactivated after 30 min. The pH/activity studies showed two groups with pK values c 4.6 and 6.7 involved in PPO catalysis.     

Purification and properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.     

PURIFICATION AND PHYSICOCHEMICAL PROPERTIES OF AN EXTRA CELLULAR AMYLASE FROM A STRAIN OF BACILLUS AMYLOLIQUEFACIENS ISOLATED FROM DRY ONION POWDER

An extracellular α-amylase from Bacillus amyloliquefaciens, isolated from dry onion powder, has been purified to homogeneity by ammonium sulfate fractionation, adsorption on starch, column chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100 column. The enzyme consisted of one polypeptide chain with a molecular weight of 60,000. The isoelectric point was pH 5.2, the pH optimum 5.5 and the temperature optimum ranging from 50°-70°C. Prolonged digestion by trypsin did not affect the catalytic properties of the enzyme. The K_m for starch was 6.9 mg/ml. The enzyme was quite stable at 50°C, but lost about 85% of its activity at 60° after 30 min (pH 6.0).     

Purification and Properties of a Thermostable Inulinase (β-D-Fructan Fructohydrolase) from Bacillus stearothermophilus KP1289

A thermophilic soil isolate, Bacillus stearothermophilus KP1289, that grew from 41 °C to 69 °C, produced extracellular inulinases in the presence of inulin. One (inulinase II) of these enzymes was purified to homogeneity. The molecular weight (M_r) and the isoelectric point of the enzyme were estimated as 54,000 and 5.0, respectively. The enzyme was active between 30 and 75 °C and at pH 4.5—8.6 with an optimum at 60 °C and pH 6.1. At 69 °C and pH 7.0 the half-life of the enzyme was 10 min. The enzyme released fructose exo-wise from the non-reducing end of inulin (M_r = 4,5000). The Michaelis constant, catalytic center activity, and specificity constant for inulin at 60 °C and pH 5.0 were 80 mM (360 mg/mL), 460 s^—1, and 5.8 s^—1 mM^—1, respectively. The ratio of specificity constants for inulin, sucrose, and raffinose was 1:0.50:0.16. The enzyme was classified as a thermophilic thermostable β-D -fructan fructohydrolase (EC 3.2.1.80).     

Purification and Characterization of a Novel Protopectin-Solubilizing Enzyme from Aspergillus niger Z-25

A mutant, Aspergillus niger Z-25 derived from A. niger XZ-131 with N⁺ supplementation, produced a protopectin-solubilizing enzyme (protopectinase). The enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation and gel chromatographies on DEAE-Sephadex A-50 and Sephadex G-100 columns. The molecular weight of the protopectinase was estimated at 68.4 KD by SDS-polyacrylamide gel electrophoresis. The enzyme was stable from pH 3.0 to 7.0 and up to 60°C. The optimum pH and temperature for enzyme activity was 4.0 and 50°C, respectively. The purified enzyme had 268-U/mL PPase (protopectinase) activity on citrus protopectin, and also showed 100-U/mL PGase (polygalacturonase) activity, which catalyzed the hydrolysis of polygalacturonic acid. The Km value for protopectin was 0.215 mg/mL, while the Km value for polygalacturonic acid was 39.09 mg/ml. The PPase/PGase q-value was 2.68, more than 0.5. Therefore the enzyme was considered a novel pectinase and classified as protopectinase.     

Pectin lyase and polygalacturonase of Aspergillus niger pathogenic for yam tubers (Diascorea rotundata)

Polygalacturonase and pectin lyase of Aspergillus niger partially purified by ethanol, ammonium sulphate precipitation, DEAE-cellulose and Sephadex G-150 column chromatography were characterized. Polygalacturonase gave optimum activity at pH 4–5, and at 35°C. It was stable at pH 3–7 and at 20–50°C. The molecular weight was 38020. For pectin lyase optimum activity occurred at pH 5 and 45°C. The enzyme was stable at pH 3–4 and at 40–50°C. The molecular weight was 30 900. Yam tissue was optimally macerated at pH 4–5 by the enzymes. At pH 4.5, potassium sorbate (0.6 mg/ml), benzoic acid (0.8 mg/ml) and sodium benzoate (1.0 mg/ml) caused complete inhibition of polygalacturonase activity. With pectin lyase, this effect was achieved with potassium sorbate and benzoic acid each at 0.9 mg/ml, but not with sodium benzoate.     

Partial Purification and Characterization of an Acid Phosphatase from Papaya

A phosphatase in papaya was extracted, partially purified, and characterized. With p-nitrophenyl phosphate as substrate, the enzyme had a pH optimum of 6.0, which categorized it as an acid phosphatase, a temperature optimum of 37°C, and a Km of 1.0 mM. Heat inactivation of papaya acid phosphatase was biphasic, and the kinetics of both phases were first order reactions. D values at 60°, 65°, 70°C for the heat resistant phase were 21.0, 11.7, and 4.0 min, respectively. For the heat labile and heat resistant isozymes of papaya acid phosphatase, the activation energies, E_a, for thermal inactivation were 60.0 Kcal/mole and 37.8 Kcal/mole, respectively. The apparent molecular weight of the enzyme as determined by gel filtration was 120,000 daltons.     

Acid phosphatase in the tubers of Dioscorea species and its purification from the white yam (D. rotundata poir)

Acid phosphatase activity was determined in 15 cultivars from four species of yam. A 12-fold purification of the enzyme from Dioscorea rotundata (cv. chikakwondo) gave a homogeneous preparation as demonstrated by polyacrylamide gel electrophoresis. This enzyme preparation has an apparent molecular weight of 115 000 ±2000 and an optimum activity at a pH of 5·20 and a temperature of 50°C. The K_m of the enzyme is 3·81 mM with disodium p-nitrophenylphosphate (p-NNP) as a substrate. The energy of activation, heat of activation, energy of inactivation and heat of inactivation are 7·0, 6·4, 4·41 and 4·34 kcal M^?1, respectively. Although it has very little activity with most organic phosphoric acid esters, it is significantly inhibited by Ca²⁺, Hg²⁺ and EDTA and activated by Mg²⁺. The enzyme has a half-life of 50,17 or 13 days, respectively, when stored at 6-8°C, 0°C or room temperature (29±2°C).     

Purification and Characterization of Extremely Thermostable Exo-oligo-1,6-glucosidase from a Caldoactive Bacillus sp. KP 1228

A p-nitrophenyl-α-D -glycopyranoside-hydrolyzing oligo-1,6-glucosidase (dextrin 6-α-glucanohydrolase, EC 3.2.1.10) of a caldoactive Bacillus sp. KP 1228 capable of growing at 51–82°C was purified to homogeneity. The molecular weight was estimated as 140,000. The enzyme consisted of two identical subunits each comprising a threonine residue at the NH₂-terminus. The enzyme was most active at 85°C and pH 5.1, and stable for 10 min up to 85°C at pH 6.8. The enzyme had no antigenic determinant common to oligo-1,6-glucosidases from Bacillus cereus ATCC 7064 (mesophile), Bacillus coagulans ATCC 7050 (facultative thermophile) and Bacillus thermoglucosidasius KP 1006 (DSM 2542) (obligate thermophile). A strong correlation between the increase in proline content and the rise in thermostability of these 4 proteins was observed.     

Purification and Some Properties of Glutaminase fromActinomucor taiwanensis,Starter of Sufu

Glutaminase of Actinomucor taiwanensis was purified approximately 96-fold with a yield of 18%, by sequential fractionation with ammonium sul-phate, anion exchange with DEAE-Sepharose CL-6B and gel filtration with Sephacryl S-200. The pH and temperature optima of purified glutaminase were 8·0 and 45°C, respectively. Glutaminase was stable at a temperature up to 35°C and at pH values of 6·0–8·0. The molecular weight was 80000 as determined from SDS-PAGE. The enzyme activity was markedly inhibited by HgCl₂. In the presence of 100 g litre⁻¹ NaCl, the enzyme activity was inhibited 50%.     

Enzymatic Properties of β‐1,3‐Glucanase from Streptomyces sp Mo.

Streptomyces sp Mo endo‐β‐1,3‐glucanase was found to have hydrolyzing activity toward curdlan and released laminarioligosaccharides selectively. The molecular weight was estimated to be 36000 Da and its N‐terminal amino acid sequence was VTPPDISVTN. The optimal pH was 6 and the enzyme was found to be stable from pH 5 to 8. The optimal temperature was 60 °C and the activity was stable below 50 °C. The enzyme hydrolyzed selectively curdlan containing only β‐1,3 linkages. The enzyme had 89% relative activity toward Laminaria digitata laminarin, which contains a small amount of β‐1,6 linkages compared with curdlan, while Eisenia bicyclis laminarin with a higher amount of β‐1,6‐linkages, was not hydrolyzed. Mo enzyme adsorbed completely on curdlan powder. The enzymatic hydrolysis of curdlan powder resulted in the accumulation of laminaribiose (yield 81.7%). Trisaccharide was inevitably released from the hydrolysis of laminarioligosaccharides with 5 to 7 degrees of polymerization (DP). Although the enzyme cleaved off disaccharide (DP 2) from tetrasaccharide (DP 4), the reaction rate was lower than those of DP 5 to 7. The results indicated that the active site of Mo endo‐β‐1,3‐glucanase can efficiently recognize glucosyl residue chain of greater than DP 5 and hydrolyzes the β‐1,3 linkage between the 3rd and 4th glucosyl residue.     

Purification and Properties of Aminopeptidase I from Penicillium caseicolum

An extracellular aminopeptidase from Penicillium caseicolum was purified from the ammonium sulfate fraction using chromatography on DEAE-Sephacel and chromatofocusing on PBE-94 gel. The purified enzyme exhibited homogeneity upon electrophoresis. The enzyme was most active at pH 7.5 and was Stable within pH range of 6.0 to 7.5. The enzyme was most active at 40°C and was stable up to 50°C. The activity was inhibited by p-chloromercuribenzoic acid and was considerably inhibited by metal chelating reagents. It was highly activated by cobalt and slightly activated by zinc and manganese. The molecular weight of the enzyme was 120,000 by gel filtration on Sephadex G-200. The Michaelis constant for arginine-2-naphthylamide was estimated to be 1.0 × 10⁻⁴ M. Characterization studies indicated that the enzyme was capable of cleaving an aminoterminal leucine and phenylalanine residues. The enzyme showed a wide range of specificity against dipeptides and oligopeptides.     

Purification and enzymatic identification of an acid stable and thermostable α‐amylase from Rhizopus microsporus

Rhizopus microsporus, recently isolated from a solid culture of Heng‐Shui Lao‐Bai‐Gan (HSLBG, a famous distilled liquor in Northern China) was found to produce a novel extracellular acid stable and thermostable α‐amylase. This fungal α‐amylase was purified using ammonium precipitation, Sephadex G‐25 desalination and DEAE‐52 cellulose chromatography. Its molecular weight was estimated to be 75 kDa by SDS–PAGE. The optimum pH and temperature of this enzyme was pH 5.0 and 70°C respectively. Thermostability and kinetic analysis through the Arrhenius and Michaelis–Menten equations revealed that this enzyme showed an exceptional activity at low pH and high temperature. A combination of this thermostability and acid stability could be a valuable trait for the efficient hydrolysis of amylose to glucose in large‐scale biotechnology applications. Copyright © 2012 The Institute of Brewing & Distilling     

Isolation and Characterization of Pectin Methylesterase from Apple Fruit

Two forms of the enzyme pectin methylesterase are evidenced in the apple (Malus communis). They differ both in their charge and molecular weight. The two enzymes were separated by DEAE-cellulose chromatography. Their molecular weights, determined by gel-filtration, were 55000 and 28000 daltons. The heavier form has been purified at homogeneity and subjected to investigations regarding its activity as a function of the pH and temperature, and determination of its kinetic parameters. The enzyme has a Km value of 1.05 mg/mL for citrus pectin and an optimum activity in the pH range between 6.5-7.5. The enzyme was stable up to 40°C. Incubation for 1 min at 90°C leads to its complete inactivation.     

Purification and Characterization of an Oligo-1,6-glucosidase from the Caldoactive Thermophile Bacillus caldotenax

An oligo-1,6-glucosidase (dextrin 6-α-D -glucanhydrolase, EC 3.2.1.10) of the caldoactive thermophile Bacillus caldotenax KP 1213 (YT-G, DSM406) was purified to homogeneity. Its relative molecular weight, Stokes radius, sedimentation coefficient at 20°C in water, molar absorption coefficient at 280nm and pH 6.8, and isoelectric point were estimated to be 64,000, 3.3nm, 4.8S, 122,000M ⁻¹cm⁻¹, and 4.9, respectively. The enzyme N-terminal sequence of twenty residues was determined to be Met¹-Glu-Trp-Ala-Trp-Lys-Glu-Ala-Val-Val-Tyr-Gln-Ile-Tyr-Pro-Arg-Met-Phe-Tyr²⁰. The enzyme shared its antigenic groups in part with the homologue from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile). The B. caldotenax enzyme was most active at 70°C and at pH 6.0–6.8. The best substrate for the enzyme was isomaltotriose of naturally-occurring oligo- and polysaccharides tested. The enzyme half-life at pH 6.8 was 10min at 75°C. The enzyme (Pro, 5.93mol%) fairly matched with a positive relationship between the thermostability of its six bacillary counterparts and their proline mol% contents. This relationship has been found to hold for sixteen bacterial enzymes from other four different groups (α-glucosidases, pullulanases and 3-isopropylmalate dehydrogenases).     

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 and characterisation of carrot (Daucus carota L) pectinesterase

A pectinesterase isoform with an alkaline isoelectric point of over 8.66 was detected in crude extracts of carrot. The enzyme was purified by ion exchange and molecular exclusion chromatography. The molecular weight of the isoform was 25 kDa, determined in native conditions by filtration through Sephadex G‐75 SF. The enzyme showed a high affinity for its substrate, with K_m and V_max values of 0.031 mg ml^?1 and 6.77 units respectively for apple pectin. The pectinesterase activity exhibited an optimum around pH 7.4 and was activated by metallic ions, with optimum activities at NaCl concentrations between 130 and 330 mM and at CaCl₂ concentrations between 15 and 50 mM . The enzyme was activated most by Ca²⁺ and exhibited a greater tolerance of high concentrations of Na⁺. Comparison of its heat stability with other pectinesterases of vegetable origin indicated that the purified isoform was very thermolabile, being rendered inactive by heating for 5 min at 70 °C. The enzyme was inhibited by high concentrations of polygalacturonic acid and competitively inhibited by D ‐galacturonic acid, with a K_i value of 1 mM . Copyright © 2003 Society of Chemical Industry     

Some properties of polyphenol oxidase from lily

A study of crude polyphenol oxidase (PPO) from lily bulbs was carried out to provide information useful for guiding food processing operations. Optimum pH for the enzyme activity in the presence of catechol, were 4.0 and 7.0 at room temperature(approximately 20 °C) and the enzyme was stable in the pH range from 5.0 to 6.5 at 4 °C for 10 h. Its optimum temperature was 40 °C and the heat inactivation of the enzyme followed first‐order kinetics. Lily PPO possessed a diphenolase activity toward catechol, catechin and gallic acid; catechin was the best substrate for the enzyme considering the V_max/K_m ratio. The most effective enzyme inhibitor was sodium sulphite, although ascorbic acid, l ‐cysteine and thiourea were also effective inhibitors at high concentration. But NaCl and citric acid were poor inhibitors of the enzyme. Data generated by this study might help to better prevent lily bulbs browning.