EFFECT OF SUBSTRATE SIZE ON THE ACTIVITY OF TOMATO POLYGALACTURONASE

SUMMARY— Three polygalacturonic acid preparations with widely differing molecular weight distributions were obtained by controlled enzymatic hydrolysis of pectic acid. A study of the action of tomato polygalacturonase on the polygalacturonic acids and pectic acid revealed that the activity h dependent on the molecular size of the substrate. Pectic acid was hydrolyzed optimally at pH 5, with little activity below pH 4. Decreasing the molecular weight of the substrate resulted in a progressive shift of the pH optimum to the acid side. For the smallest substrate, the activity extended to below pH 2. Monovalent cations enhanced the activity at low pH, and this effect was also dependent on the molecular weight of the substrate. Below pH 4, pectic acid inhibited 70% of the hydrolysis of low molecular weight substrates by tomato polygalacturonase. The incomplete inhibition is attributed to the presence of a polygalacturonase isoenzyme which is not inhibited by high molecular weight polygalacturonic acids.     

PURIFICATION AND CHARACTERIZATION OF INVERTASE FROM DATES (PHOENIX DACTYLIFERA L., VAR. ZAHDI)

Zahdi dates (Phoenix dactylifera) contain invertase at all development stages; the highest specific activity is present in the late yellow stage. The enzyme was purified to homogeneity, as determined by disc gel electrophoresis and isoelectric focusing, by a combination of techniques including ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sepharose 4B and Sephadex G-150 columns. A complex of invertase with a high molecular weight pectic substance of the date could not be dissociated by ammonium sulfate or DEAE-cellulose chromatography but the complex was dissociated by gel filtration on a Sepharose 4B column at pH 4.0 and ionic strength of 0.5 M. The enzyme contained 8.2% carbohydrate covalently linked probably via an amide linkage to aspartic acid. Molecular weight determination by exclusion gel chromatography and sedimentation equilibrium gave values of 130,000 and 97,100 ± 1,300, respectively. The enzyme is probably composed of two identical subunits as shown by SDS polyacrylamide gel electrophoresis. Amino acid analyses showed the enzyme to be low in sulfur-containing amino acids. Date invertase is an acid β-fructofuranosidase with a pH optimum between 3–4 and with a K_m and k_cat for sucrose of 6mM and 49 sec^-1, respectively. Activation energies for denaturation of enzyme and conversion of substrate to product were determined to be 48.7 and 17.6 kcal/mole, respectively. Chemical modification indicated that sulfhydryl groups are probably not essential for activity while carboxyl groups may be involved in the active site of the enzyme.     

PURIFICATION AND CHARACTERIZATION OF A MALATE DEHYDROGENASE FROM PHASEOLUS MUNGO

A malate dehydrogenase (MDH) was identified and isolated from the seeds of the mung bean (Phaseolus mungo). The procedure entailed extraction, ammonium sulfate precipitation, ion exchange chromatography on CM‐Sephadex and high performance liquid chromatography on POROS HS‐20. The purified protein exhibited a molecular mass of 38 kDa in SDS‐polyacrylamide gel electrophoresis under both nonreduced and reduced conditions. The pI was 9.7 by isoelectric focusing. The specific activity of the MDH was estimated to be 199 U/mg. The enzyme expressed its optimum activity at pH 7.2, 35C, and showed stable activity below 40C. The Km for oxaloacetate was 112 µM. The partial N‐terminal amino acid sequence data analysis of the first 20 amino acids of the mung bean MDH revealed 95 and 80% homology with two reported MDH from soya bean (Glycine max) and potato (Solanum tuberosum), respectively.     

IRRADIATION OF INDUSTRIAL ENZYME PREPARATIONS II. CHARACTERIZATION OF FUNGAL PECTINASE BY THIN-LAYER ISOELECTRIC FOCUSING AND GEL FILTRATION

Industrial dry fungal pectinase from A. niger was irradiated with doses (up to 1 Mrad) of ⁶⁰ Co-γrays effective in reducing microbial contamination. The pectinase was characterized by thin-layer isoelectric focusing and gel filtration in order to detect possible radiation-induced structural alterations. Thin-layer isoelectric focusing revealed at least fifteen multiple forms with pectin-depolymerizing activity, with isoelectric points in the range pH 4.5–7. Heterogeneity of pectinesterase was also demonstrated, the main band occurring around pH 4. By thin-layer gel filtration the molecular weight of the pectin-depolymerase was estimated as being about 36,000, and that of pectinesterase as about 33,000. Radiation-induced changes of the charge properties or molecular size of the irradiated pectinase preparation were not observed. The feasibility of using ionizing radiation for the reduction of microbial contamination of industrial enzyme preparations looks promising.     

PURIFICATION AND CHARACTERIZATION OF α‐GALACTOSIDASE FROM PEANUTS1

Alpha‐galactosidase was characterized in two peanuts market types, Runner and Spanish. The enzyme was purified 54 fold using ammonium sulfate precipitation, anion exchange chromatography and Size Exclusion High‐performance Liquid Chromatography. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis indicated that the enzyme has a molecular weight of 30, 000 Da, and isoelectric focusing showed a pI of 5.2. The optimum temperature and pH were 50C and 6.0, respectively. The enzyme had a K_m of 0.221 mM when p‐nitrophenyl α‐D‐galactopyranoside (PNPG) was used as a substrate and 80.8 mM when raffmose was a substrate. Raffmose and galactose were found to be competitive inhibitors when PNPG was the substrate: K_i values were 25.4 and 189, respectively. The enzyme was very sensitive to Hg⁺⁺, Ag⁺⁺ and to a lesser extent to Cu⁺⁺. However, ethylcne diamine tetraacetic acid did not have an effect indicating no requirement for cations. The two peanut types tested showed identical enzyme activities.     

On lipoxygenase and enzymes which decompose linoleic acid hydroperoxides in rye (author's transl)]

An enzyme fraction from rye containing lipoxygenase activity was investigated. The molecular weight of lipoxygenase was found to be about 102000. Two bands groups with isoelectric points between 5.1-5.5 and 5.8-6.4 were obtained by isoelectric focusing. Three isoenzymes could be separated by ion exchange chromatography. Lipoxygenase has optimum activity at pH 7.3-7.5 and predominantly forms 13-hydroperoxy-9-cis, 11-trans-octadecadienoic acid (13-LHPO). In rye the 13-LHPO is converted to alpha-ketols by a high molecular protein fraction. This isomerase converts the LHPO formed by rye lipoxygenase predominantly to 12,13-ketohydroxy acids. The Michaelis Constant of isomerase is 3-5 X 10(-5), using LHPO as substrate. At low protein concentrations the reaction velocity of LHPO-conversion increases linearly with protein concentration.     

PURIFICATION AND SOME CHARACTERISTICS OF PECTATE LYASE FROM PSEUDOMONAS FLUORESCENS GK-5

Pseudomonas fluorescens GK-5 produced up to 35 U per ml of a single extracellular pectate lyase in a gluconate-yeast extract medium. The enzyme was purified by chromatography on CM Bio-Gel A at pH 7.0, and on crosslinked pectate, first at pH 7.0 and then at pH 9.0. The pure enzyme (specific activity 956 U per mg protein) has a molecular weight of 42300 and an isoelectric point of 10.3. Its amino acid composition was determined. The pectate lyase is an endo enzyme, requiring calcium ions for its activity. The enzyme is maximally active at pH 9.4 and ionic strength 0.01. It has a K_m value of 0.10 mg polygalacturonic acid per ml and a V_max of 1.3 millimoles of unsaturated products released per min per mg of enzyme protein. The organism produces soft rot in potato. The enzyme macerates potato tissue optimally at about pH 8. Cell-bound pectate lyase was also found. Molecular and kinetic properties of the cell-bound enzyme are identical to those of the extracellular enzyme.     

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 Characterization of Bacillus acidopullulyticus Pullulanase for Enzymatic Starch Modification

The pullulanase (EC 3.2.1.41) of Bacillus acidopullulyticus was purified using anion exchange chromatography and preparative isoelectric focusing. The purified preparation migrated as a single protein band upon SDS gel electrophoresis and its molecular weight was estimated to be 102,000. Two main activity bands having pl-values 5.0 and 5.2 were detected by analytical isoelectric focusing. The enzyme was purified 4-fold with the yield 38% by this two-step purification procedure. The purified pullulanase showed maximal activity at 50°C and pH 5 and was slightly activated by Ca₂₊. It was stable at 50°C but totally lost its activity at 60ºC in one hour. This purified pullulanase efficiently hydrolyzed the α-1,6-glucosidic bonds of pullulan and gelatinized starch.     

Low molecular weight serine protease from the viscera of sardinelle (Sardinella aurita) with collagenolytic activity: Purification and characterisation

A new low molecular weight (LMW) serine-protease from sardinelle (Sardinella aurita) viscera was purified using ammonium sulphate precipitation and Sephadex G-100 gel filtration, with a 3.82-fold increase in specific activity. The molecular weight of the enzyme was estimated to be 14.2 kDa by SDS-PAGE. The optimum pH and temperature for the enzyme activity were around pH 8.0 and 60 °C, respectively. The purified protease was strongly inhibited by phenylmethylsulphonyl fluoride, a serine-protease inhibitor, and soybean trypsin inhibitor. The N-terminal amino acid sequence of the first 10 amino acids of the purified protease was APVQPCVVVI. This sequence showed low homology with several peptidases, suggesting that the enzyme is a new protease. Interestingly, the protease was found to cleave collagen type I and hydrolyze succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin. Our findings indicate that the S. aurita protease is a new LMW enzyme with collagenolytic activity.     

Polyphenoloxidase activity from strawberry fruit (Fragaria ananassa,Duch., cv Selva): characterisation and partial purification

In this work, polyphenoloxidase (PPO) from Selva strawberry fruit (Fragaria × ananassa, Duch) was extracted, characterised and partially purified. The activity of PPO was analysed in crude extracts obtained from either fresh fruits or acetone powder. The presence of NaCl and Triton X‐100 in the extraction buffer caused a marked increase in enzyme extractability. The enzyme showed an apparent K_m value of 11.2 mM with pyrocatechol as substrate. The maximum enzyme activity was observed at 50 °C and pH 5.3–6.0 without SDS and pH 7.2 in the presence of SDS. The presence of SDS increased PPO activity at pH 7.2 but diminished it at pH 6.0. The enzyme showed high thermal stability and maintained activities equal to or greater than 50% of its maximum activity in the 2.6–9.3 pH range. One polyphenoloxidase isoenzyme was detected in crude extracts of all ripening stages, showing an isoelectric point of 7.3. The specific activity of PPO decreased continuously through fruit ripening. Maximum specific activities were found at the ‘small green’ and ‘large green’ ripening stages. A total enzyme extract was partially purified by means of (NH₄)₂SO₄ precipitation and cationic exchange chromatography in an FPLC system. The purification grade achieved was near 25. The partially purified enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 135 ± 4 kDa for the native protein. © 2000 Society of Chemical Industry     

Lipase from Vicia faba minor

Lipase was partially purified from small faba beans by ethanol precipitation and Sephadex gel filtration and characterised by disc gel electrophoresis, isoelectric focusing and molecular weight determination in the presence of sodium dodecyl sulphate. Kinetic studies demonstrated that the properties of the enzyme conformed generally to those of lipases from other sources. The isoelectric point was pH 4·8, and electrophoresis at pH 9·3 revealed one lipase band in the R_f0·25–0·31 region. The molecular weight was 210,000 ± 20,000. The possible importance of lipase is discussed with respect to the degradation of small faba bean lipids and to chemical changes occurring during storage of processed faba beans.     

Inhibition of Raw Starch Digestion by One Glucoamylase Preparation from Black Aspergillus at High Enzyme Concentration

Raw starch digestion by glucoamylase I (Ab. G-I) preparation from black Aspergillus was inhibited significantly at relatively high concentration of the enzyme. The properties of this enzyme were studied together with those of another glucoamylase I (Nor. G-I), also from black Aspergillus. The two glucoamylases do not differ so much in their physico-chemical properties such as molecular weight, pH and thermal stability, pH and temperature optimum, substrate specificity, debranching activity, isoelectric point etc. The adsorption rate of both enzymes on raw starch increased by the increase of enzyme concentration. The raw starch digestion rate by adsorbed Ab. G-I, however, was decreased with the increase of concentration of enzyme whereas the same was increased in case of Nor. G-I. The inhibitory effect was weaker at 60°C or above.     

THERMAL AND HIGH-PRESSURE STABILITY OF PURIFIED PECTIN METHYLESTERASE FROM PLUMS (PRUNUS DOMESTICA)

Pectin methylesterase (PME) from greengage plums (Prunus domestica) has been extracted and purified using affinity chromatography. Only one band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis was obtained, with an estimated molecular weight of 31 kDa. On isoelectric focusing electrophoresis, two bands with neutral isoelectric points (6.8 and 7.0) were detected. The optimal pH and temperature for plum PME activity were 7.5 and 65C, respectively. A study of purified plum PME thermostability was performed at pH 7.5 and 4.0, indicating a higher thermostability at pH 7.5 than at pH 4.0. A biphasic inactivation behavior was observed for thermal treatments (54–70C), whereas its pressure inactivation could be described by a first‐order kinetic model in a pressure range of 650–800 MPa at 25C. Purified plum PME was found to be relatively stable to thermal and pressure (≤600 MPa) treatments, compared to PME from other fruits.     

MACERATION ACTIVITY OF AN ENDOPOLYGALACTURONASE FROM CANDIDA MACEDONIENSIS

The yeast Candida macedoniensis produces constitutively an extracellular pectinolytic enzyme with a high maceration activity; the culture filtrate is free from foreign enzyme activity. The enzyme formation is optimal under strictly anaerobic conditions with N₂ gassing. The culture medium for optimal enzyme recovery is a nutrient solution containing 1% yeast extract, 2% peptone and 10% sucrose, at pH 3, 28°C. Characterization of the enzyme showed it to be an endopolygalacturonase. The pH and temperature optima of the enzyme differ for pectic acid cleavage and maceration activity, these being 4.5 and 50—53°C and 2.5 and 40°C, respectively. The enzyme activities could not be separated from one another by protein chemical methods (analytical and preparative isoelectric focussing). Dialysis of the enzyme-containing culture filtrate did not decrease the enzyme activity. The endopolygalacturonase from Candida macedoniensis leads to the release of plant cells from the tissue, without destroying the cells by lysing the cell walls. After two hours incubation of the substrate (carrot slices), the tissue mass consisted of cell clumps of up to 15 cells.     

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     

Trypsin-like Enzyme from Sand Crab (Portunus pelagicus): Purification and characterization

Studies with synthetic substrates and specific inhibitors indicated that a protcinase from the hepatopancreas of the sand crab (Portunus pelngicus) was a trypsin-like serine proteinase. The molecular weight of the enzyme estimated by gel filtration and mass spectrometry was ～ 25,000, whereas SDS-PAGE indicated a molecular weight of 34,800. The optimum temperature for hydrolysis of azocasein was 60°C, while inactivation of 50% enzymic activity occurred at 68°C. The enzyme, optimally active at pH 8.0 towards p-tosyl-L-arginine methyl ester and unstable at acid pH, was high in acidic amino acid residues. Under some conditions ihe knzyme readily autodigested. Our results can help understand and avoid problems of meat softening during storage of seafood products.     

Purification,crystallisation and characterisation of carboxypeptidase from wheat bran

A carboxypeptidase was purified and crystallised from wheat bran. Disc gel electrophoresis at pH 4·0 and ultracentrifugal analysis revealed that the enzyme was essentially homogeneous. The sedimentation constant and isoelectric point were determined to be 6·3 S and 6·0, respectively. The molecular weight of the enzyme was estimated to be 118,000 by a gel filtration method. The enzyme liberated carboxyl terminal amino acid residues from a wide range of N-substituted dipeptides and tripeptides which contain l-proline. It had a pH optimum at pH 4·0 for Z-Glu-Tyr (Z-benzyloxycarbonyl). The K_m and k_cat values for Z-Glu-Tyr at pH 4·0 and 30°C were 0·19 mm and 20 s^?1, respectively. The enzyme hydrolysed Z-Gly-Pro-Leu-Gly-Pro and bradykinin sequentially at pH 4·0 from their carboxyl terminal amino acid residues. The enzyme activity was completely inhibited by DFP.     

Molecular weight and amino acid composition of glycinin subunits

Glycinin, the major soyabean globulin, is composed of subunits having molecular weights of about 22,300, and 37,200. On the basis of amino acid analysis, the six submits of glycinin isolated by isoelectric focusing are all different. The ‘acidic’ subunits have higher content of glutamic acid and proline, whereas the ‘basic’ subunits are higher in the hydrophobic amino acids leucine, tyrosine, phenylalanine, valine and alanine.     

PURIFICATION AND CHARACTERIZATION OF THREE POLYPHENOL OXIDASE ISOZYMES FROM LOBSTER (HOMARUS AMERICANUS)

ABSTRACT Three isozymes of polyphenol oxidase (PPO I, PPO II and PPO III) were purified from lobster (Homarus americanus) by ion-exchange chromatography and preparative isoelectric focusing using a Rotofor cell. The purified isozymes migrated as single protein bands in polyacrylamide gels with R_f values corresponding to molecular weights of 32,180, 35,480 and 39,300, respectively. The pI values of the PPO isozymes were 3.89, 4.26 and 4.54, respectively. PPO I was most active at pH 6.5 and most stable from pH 6.0 to 7.0; PPO II was most active within the pH range 6.0 to 7.0, and most stable within the pH range 4.0 to 9.0; while PPO III was most active at pH 7.0 and most stable in the pH range 6.0 to 8.0. The temperature optimum for the PPO-dihydroxyphenylalanine oxidation reaction was 35C with PPO I and 45C with PPO II or III. Lobster PPO I lost about 30% of its initial activity after 30 min incubation at 45C, while PPO II and II retained virtually all their activity after the same heat treatment. The catalytic specificities of the PPO isozymes were relatively higher with dihydroxyphenylalanine as substrate than with chlorogenic acid.