PURIFICATION AND CHARACTERIZATION OF TWO TOMATO POLYGALACTURONASEISOENZYMES

The properties of tomato polygalacturonases at two ripening stages were investigated. Two isoenzymes, PG I and II, were isolated from underripe fruits with an orange skin color. Fully ripe fruits contained only polygalacturonase II. PG I and II were purified by chromatography on DEAE-Sephadex A-50, Sephacryl S-200 and CM-agarose chromatography. PG I had a M_r of 199,500 as determined by Sephacryl S-300 gel filtration and was 50% inactivated at 66.5°C and pH 4.5 after incubation for 5 min. It had an activation energy (E_a) of 16.8 Kcallmol (70.3 times 10³ Jlmol), V_max of 27.7 units/mg protein and K_m value of 7.5 times 10⁻² mM polygalacturonic acid. PG II had a M_r of 45,700 and was 50% inactivated at 58°C under the same conditions. Both isozymes had a pH optimum of 4.6. PG II had an E_a value of 14.8 Kcallmol (61.9 times 10³ Jlmol), V_max value of 58.8 units/mg protein and K_m value of 3.8 times 10⁻² mM polygalacturonic acid. PGI gave rise to only one band during electrophoresis in polyacrylamide gels, whereas PG II showed one major and one minor band both with PG activity. Gel electrophoresis in the presence of sodium dodecyl sulfate resulted in two major bands (M_r= 47,500 and 41,000) for PG I and only one major band (M_r= 47,500) for PG II. PG I is composed of several subunits, all of which are glycoproteins.     

Chromatographic Isolation and Characterization of a Novel Peroxidase from Large Lima Legumes

ABSTRACT:  A novel peroxidase with antifungal activity was isolated from the large lima bean ( Phaseolus limensis ) seeds. The procedure entailed extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Toyopearl, and gel filtration on Sephadex G-75. The enzyme was adsorbed on Affi-gel blue gel and SP-Toyopearl, and possessed a molecular weight of 34 kDa in SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. There was an almost 110-fold increase in specific activity of the purified peroxidase compared with that of the crude extract. The enzyme exhibited a pI of 8.6 by isoelectric focusing electrophoresis, indicating that it is a basic protein. The optimum pH and the optimum temperature were 5.5 and 30 °C, respectively. The enzyme was stable up to 55 °C. It potently suppressed mycelial growth in Fusarium solani, Mycosphaerella arachidicola, and Pythium aphanidermatum with an IC₅₀ of 76, 103, and 119 μM, respectively.     

PURIFICATION AND PROPERTIES OF GLUCOSE 6-PHOSPHATE DEHYDROGENASE FROM CORIANDER (CORIANDRUM SATIVUM) LEAVES

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP⁺ oxidoreductase, EC 1.1.1.49; G6PD) was purified from coriander (Coriandrum sativum) leaves; the kinetic behavior and some properties of the enzyme were also investigated. The purification was done at 4C and involved two steps: ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 26.4% and had a specific activity of 1.826 U/mg protein. Optimum pH, stable pH, optimum temperature, molecular weight, K_M and V_max values for NADP⁺ and glucose 6-phosphate (G6-P) were also determined.
The overall purification was about 74-fold. SDS-PAGE of the purified enzyme showed a single band. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm.
The molecular mass was estimated to be 74.4 kDa by SDS-PAGE and 73.2 kDa by Sephadex G-200 gel filtration column chromatography. The enzyme had an optimum pH at 8.5 and was stable at pH 8.0 in 0.1 M Tris-HCl buffer. The optimum temperature was at 30C. The K_M values for NADP⁺ and G6-P were 0.026 mM and 0.116 mM, respectively. The V_max values for these substrates were 0.035 EU/mL and 0.038 EU/mL, respectively.     

Purification of trypsin/chymotrypsin inhibitor from jack fruit seeds

A trypsin/chymotrypsin inhibitor (JSTI) was isolated from jack fruit seeds (Artocarpus integrifolia Hook f) by ammonium sulphate fractionation and chromatography on DEAE–cellulose and Sephadex G-100. During all stages of purification, the ratio of trypsin and chymotrypsin inhibitory activities remained constant. The purified preparation was found to be homogeneous by gel filtration, polyacrylamide gel electrophoresis (PAGE) and ultra-centrifugation. From the sedimentation coefficient, S _20w value of 3·5 ± 0·15 S. the molecular weight of JSTI was calculated to be 30·00 ± 2·50 kamu. The inhibitor showed a molecular weight of 24·55 kamu on a Sephadex G-75 column when eluted with 6 M guanidine hydrochloride, Under non-denaturing conditions, JSTI exhibited anomalous behaviour on a Sephadex G-200 column. On SDS–PAGE, the inhibitor showed two major bands with molecular weights of 26·30 and 15·00 kamu and two minor bands with molecular weights of 19·50 and 12·00 kamu. The carboxyamidomethylated JSTI showed three trypsin inhibitory activity bands on PAGE, suggesting the presence of isoinhibitors.     

TRYPSIN INHIBITORS OF WINGED BEAN TUBERS (PSOPHOCARPUS TETRAGONOLOBUS): PURIFICATION AND PROPERTIES

Two trypsin inhibitors (WBT-TI-I and WBT-TI-II) which differ in their physicochemical properties have been isolated from winged bean tubers, using affinity chromatography and ion-exchange chromatography. WBT-TI-I inhibits trypsin, has a molecular weight of 45,000 (± 1000) and has two subunits of M_r 22,000 as determined by gel filtration, ultracentrifugation and SDS-PAGE studies. WBT-TI-II inhibits trypsin in addition to chymotrypsin and pepsin, and has a molecular weight of 24,000 (± 1000) as estimated by gel-filtration, ultracentrifugation and SDS-PAGE. Although WBT-TI-I and WBT-TI-II were homogeneous by gel filtration, ultracentrifugation and SDS-PAGE, they were electrophoretically heterogeneous on PAGE at pH 8.3. Since WBT-TI-II, when purified by immunoaffinity chromatography, did not show heterogeneity by PAGE at pH 8.3, it was concluded that heterogeneity arose from modification of the native inhibitor on trypsin affinity column. WBT-TI-I and WBT-TI-II are immunologically different. The antibodies of WBT-TI-II cross react with winged bean seed extract.     

Electrophoretic characterisation of low-molecular weight wheat protein of variable solubility

A low mol. wt protein (S protein) which has a high affinity for endogenous polar lipid was isolated from wheat gluten, partially purified and compared electrophoretically with low mol. wt components of several previously studied wheat protein preparations. Mobilities of S protein components in acidic polyacrylamide gel electrophoresis (PAGE) were very close to those of the chloroform/methanol soluble low mol. wt proteins (CM proteins). A fraction of S protein separated by gel filtration chromatography on Sephadex G-50 (S-III protein) was almost identical to CM proteins by two-dimensional electrophoresis. Bands with mobilities similar to those of S-III protein were present in PAGE patterns of gliadin, the low mol. wt fractions IV and V obtained from gliadin by gel filtration chromatography on Sephadex G-200, and the albumin/globulin fraction of flour prepared by the Osborne solubility fractionation. Because of its variable solubility S protein cannot be unambiguously classified according to the Osborne solubility classification of plant proteins.     

SEPARATION OF STEROLS AND TRITERPENE ALCOHOLS FROM UNSAPONIFIABLE FRACTIONS OF THREE PLANT SEED OILS

Preparative HPLC was used to separate sterols and triterpene alcohols from the unsaponifiable matters in plant oils from Camellia weiningensis L., Brassica juncea L. and Microula sikkimensis . The isolated sterols and triterpene alcohols were acetylated and further purified by AgNO₃ impregnated silica gel preparative thin layer chromatography (TLC). The isolated acetyl derivatives of sterols and triterpene alcohols were identified by melting point, specific rotation, infrared and mass spectrometry. The sterols were brassicasterol, campesterol, stigmasterol, β-sitosterol and Θ⁵-avenasterol, Θ⁷-avenasterol, Θ⁷-stigmastenol and α-spinasterol. The triterpene alcohols were cycloartanol, cycloartenol, 24-methylenecycloartanol, cyclobranol, dammaradienol, tirucalla-7,24-dienol, butyrospermol, β-amyrin, germanicol, α-4-taraxasterol and lupeol.     

脱脂羊脑蛋白水解多肽的分离纯化及抗氧化活性

为制备羊脑蛋白抗氧化肽,本实验对脱脂羊脑蛋白含量及氨基酸组成进行了分析;采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）对枯草芽孢杆菌中性蛋白酶不同酶解时间的酶解液分子质量进行了分析;采用交联葡聚糖凝胶Sephadex G-25和Sephadex G-15对羊脑酶解产物进行了逐级分离纯化,以羟自由基（·OH）和亚硝酸根离子清除能力为指标对分离组分进行抗氧活性评价,并对纯化后的组分抗氧化活性进行了测定。结果表明,脱脂羊脑粉中蛋白含量为60.55%,在测定的17 种氨基酸中,谷氨酸和天冬氨酸这两种酸性氨基酸含量最高,且含有7 种必需氨基酸;羊脑蛋白经酶解后,分子质量集中在10 kD以下;经Sephadex G-25纯化后,得到了6 个组分,其中组分F4的抗氧化活性最强,组分F4经SephadexG-15纯化后,得到3 个组分,其中组分F4-2的抗氧化活性最强,组分F4-2对1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基、·OH、超氧阴离子自由基（O2－·）、亚硝酸根离子的半数抑制率IC50分别为1.64、2.47、7.98、5.14 mg/mL。     

Interaction of Low Molecular Weight Phenolics with Proteins (BSA)

Mixtures of bovine serum albumin (BSA) and several low molecular weight phenolics were incubated and fractionated using G-50 Sephadex chromatography. Fractions corresponding to the protein and the possible phenolic-protein complexes and fractions corresponding to the free phenolics were collected, and their phenolic content was determined. Among the selected commercial phenolic standards tested ( p -coumaric acid, p -hydroxybenzoic acid, protocatechuic acid, caffeic acid and (+)-catechin), the strongest BSA-binding affinity was demonstrated by 3,4-dihydroxy benzoic and cinnamic acids (protocatechuic acid and caffeic acid), whereas p -hydroxybenzoic acid did not interact with BSA. The methodology was also applied to a phenolic extract obtained from lentils containing p -coumaric acid, a p -coumaric acid derivative, (+)-catechin and procyanidins B₃ [(+)-catechin-(4αÕ8)-(+)-catechin] and B₁ [(-)-epicatechin-(4αÕ8)-(+)-catechin]. Interactions observed among the lentil phenolics and BSA were comparable to those observed among the commercial phenolic standards and BSA.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF WHITE KIDNEY BEAN (PHASEOLUS VULGARIS)β-AMYLASE INHIBITORS FROM TWO EXPERIMENTAL CULTIVARS

β-Amylase inhibitors WKB 858B and WKB 858B were purified to homogeneity from different cultivars of white kidney beans by extraction from the ground beans and by sequential heat treatment, ethanol fractionation, DEAE-cellulose chromatography, Sephadex G-75 gel chromatography and CM-cellulose chromatography. The inhibitors were homogeneous by 7.5% polyacrylamide gel electrophoresis; no isoinhibitors were found. Inhibitors WKB 858A and WKB 858B had isoelectric points of 5.0 and 4.65, respectively, and molecular weights of 42,000 and 20,000, respectively, by FPLC Superose 12 gel filtration chromatography. Inhibitor WKB 858A had molecular weights of 40,000 and 38,000 by Sephadex G-75 gel filtration chromatography and by native gel electrophoresis, respectively. Inhibitor WKB 858A contained 11.0% carbohydrate, N-linked to asparagine residues, with a composition of 1 fucose, 1 xylose, 4 galactose, 8 N-acetylglycosamine and 13 mannose residues per mol of inhibitor. Amino acid analysis of Inhibitor WKB 858A gave a high content of Asx, Glx, Ser, Thr and Val (combined total of 60% molar ratio) and low content of sulfur amino acids (0.8% molar ratio of Met and no 1/2 cystine). No-SH groups were found. The amino acid composition was similar to that of eight other a-amylase inhibitors from beans. Inhibitor WKB 858A formed a 1:1 stoichiometric complex with porcine pancreatic a-amylase with a Ki of 1.0 × 10^?11 M at pH 5.4 and 30C; it had no trypsin inhibitory activity. At pH 6.90 and 30C, the rate of complex formation between Inhibitor WKB 858A and porcine pancreatic β-amylase was 2.76 times faster at 1.385 vs 0.035 ionic strength (with Na₂SO₄), indicating hydrophobic bonds are most important in complex formation.     

PURIFICATION AND SOME PHYSICAL AND CHEMICAL PROPERTIES OF RED KIDNEY BEAN (PHASEOLUS VULGARIS) α-AMYLASE INHIBITOR

Red kidney bean contains more amylase inhibitor than do California white bean and cowpea while garbanzo bean and Westan and Westley lima beans do not contain inhibitor. Red kidney bean amylase inhibitor was purified to homogeneity by selective heat treatment (60°C) of a water extract at pH 4. 0, fractionation with ethanol and successive chromatography on DEAE- and CM-cellulose chromatography. The inhibitor has an apparent molecular weight of 49,000 by Sephadex gel filtration and contains 8. 6% carbohydrate probably covalently linked via an amide linkage to asparagine. The inhibitor probably contains four subunits perhaps of three different types. The inhibitor is high in aspartic acid, glutamic acid, serine, threonine and valine, low in cysteine/cystine and does not contain proline. Stable 1:1 complex formation between inhibitor and porcine pancreatic α-amylase was demonstrated by gel filtration on Sephadex G-100. The inhibitor has activity against porcine pancreatic α-amylase, human salivary α-amylase, and Tenebrio molitor (yellow corn meal worm) larval midgut α-amylase but is inactive against Bacillus subtilis α-amylase, Aspergillus oryzae α-amylase, barley α-amylase and red kidney bean α-amylase.     

Optimising the free radical scavenging activity of shrimp protein hydrolysate produced with alcalase using response surface methodology

Response surface methodology (RSM) was used to optimise the hydrolysis parameters of Acetes chinensis by Alcalase 2.4L in order to obtain a hydrolysate with potent radical scavenging activity. The parameters were temperature, pH and enzyme concentration/substrate concentration (E/S) ratio with degree of hydrolysis (DH) being the response. The results showed that the optimum condition was: temperature at 57 °C, pH at 8.0, E/S ratio at 2.6AU 100 g⁻¹ shrimp, hydrolysis time 3 h. The DH was 26.32%, the hydroxyl radical scavenging activity was up to 88.12% and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was 35.61%. The gel column filtration chromatography by a Sephadex G-25 column yielded five fractions. The molecular weight of the most potent free radical scavenging activity fraction ranged from 915 to 207 Da, its IC₅₀ for hydroxyl radical was 0.03 mg mL⁻¹, and IC₅₀ for DPPH radical was 8.86 mg mL⁻¹.     

花生抗真菌蛋白的纯化及活性鉴定

利用亲和色谱Affi-gel和葡聚糖凝胶色谱SephadexG-75从花生种子中分离出一种抗真菌蛋白。试验结果表明:此种纯化的抗真菌蛋白对植物致病菌苹果轮纹病菌(Physalosporapiricola)、棉花枯萎病菌(Fusariumoxysporum)、瓜果腐霉病菌(PythiumapHanidermatum)的生长具有明显抑制作用。通过SDS-聚丙烯酰胺凝胶电泳鉴定达到电泳纯,分子质量为34.4kDa。还原和非还原状态下的此抗真菌蛋白均显示单一区带,说明该抗真菌蛋白为单倍体蛋白。     

薤白多糖的分离纯化及组成分析

采用DEAE-纤维素离子交换柱层析和SephadexG-150凝胶柱层析,对薤白粗多糖(PAM)进行了分离纯化;利用醋酸纤维素薄膜电泳和凝胶过滤法鉴定精制薤白多糖纯度;硅胶薄层层析分析精制多糖的单糖组成。结果表明:DEAE-纤维素柱分别用水、0.1mol.L-1NaCl、0.5mol.L-1Na0H洗脱分得三种级分,即水洗级分(PAM-I)、盐洗级分(PAM-II)和碱洗级分(PAM-III);这三种级分再经过SephadexG-150柱进一步分离纯化,得三个主要级分PAM-Ib、PAM-IIa及PAM-III’;经醋酸纤维素薄膜电泳和凝胶过滤法鉴定,它们为均一的多糖;硅胶薄层层析分析显示,PAM-Ib主要由半乳糖和葡萄糖组成,PAM-IIa主要由半乳糖、葡萄糖、果糖、木糖和鼠李糖组成,PAM-III’主要由半乳糖、葡萄糖和木糖组成。     

Some characteristics of polyphenol oxidase purified from edible yam (Dioscorea cayenensis-rotundata cv. Longbô) cultivated in Côte d'Ivoire

Polyphenol oxidase (PPO_Y) of edible yam ( Dioscorea cayenensis-rotundata cv. Longbô ) cultivated in Côte d'Ivoire was purified to homogeneity by a procedure that included ion exchange chromatography, ammonium sulphate fractionation, gel filtration and hydrophobic chromatography using dopamine as a substrate. The relative molecular weight of the PPO_Y was estimated to be 94.75 ± 0.63 and 151.56 ± 2.75 kDa by SDS–PAGE and gel filtration on Sephacryl S100 HR, respectively, indicating this PPO_Y as a multimer. Maximal PPO_Y activity occurred at 30 °C and pH 6.6. The enzyme was stable at 25 °C and its pH stability was in the range of 6.0–7.0. The values V _max/ K _m showed that the enzyme had the greatest reactivity towards dopamine among the substrates used. PPO_Y showed no activity toward the monophenols and was completely inhibited by beta-mercaptoethanol, sodium thiosulphate, l -ascorbic acid, sodium bisulphite and l -cysteine. Data generated by this study might help to better prevent edible yam browning.     

Purification and Characterization of an α-L-Rhamnosidase from Aspergillus terreus of Interest in Winemaking

ABSTRACT: An enzyme with α-L-rhamnosidase activity was purified to homogeneity from a culture filtrate of Aspergillus terreus after growth in a medium containing L-rhamnose as the sole carbon source. The biosynthesis of this enzyme was repressed by glucose. The enzyme had a molecular mass of 96 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 4.6 as determined by analytical isoelectric focusing. The pH and temperature optima for the enzyme were found to be 4.0 and 44 °C, respectively. Using p-nitrophenyl-α-L-rhamnopyranoside as a substrate, the enzyme exhibited Michaelis-Menten kinetics with K_M and V_max values of 0.17 mM and 84 U/mg, respectively. The enzyme was inhibited competitively by L-rhamnose (K₁ 2.5 mM). Divalent cations such as Ca²⁺ Mg²⁺ Zn²⁺ and Co²⁺ stimulated the a-L-rhamnosidase activity, whereas this was inhibited by Hg2+ and Cd2+. Ethanol (12% v/v) and glucose (21% w/v) decreased enzyme activity by approximately 20%, while this was not affected by SO₂.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF α-GALACTOSIDASE FROM ASPERGILLUS ORYZAE

A crude extract of α-galactosidase obtained by fermenting Aspergillus oryzae on wheat bran was purified 35 fold by ethanol precipitation, gel filtration and ion-exchange chromatography. The final preparation was free of protease activity but contained invertase activity. The molecular weight of the enzyme was estimated as 64,000 daltons. The pH and temperature optima were 4.0 and 60°C, respectively. The enzyme was stable over the pH range 3–7.5 and at temperatures up to 55°C (pH 4.0). The K_m values for p-nitrophenyl α-Dgalactopyranoside (PNPG) and raffinose were 4.0 × 10⁻⁴M and 1 × 10⁻²M, respectively. Divalent cations were not required for activity. More than 80% of the oligosaccharides in soy milk were hydrolyzed after 3 h at 50°C using 0.113 PNPG units/mL milk.     

PURIFICATION OF THE ANTI-LISTERIAL BACTERIOCINLIKE INHIBITORY SUBSTANCE PRODUCED BY ENTEROCOCCUS FAECIUM 108

Extracellular antimicrobial substances produced by certain enterococci inhibit Listeria monocytogenes. Enterococcus faecium 108, a competitive food isolate, produced a heat-stable and protease-sensitive anti -L. monocytogenes bacteriocin-like substance (Ef108) in Listeria selective enrichment broth and other media. Ef108 activity was purified to homogeneity by a four-step procedure including (NH₄)₂SO₄ fractionation, chromatography on anion exchange QSepharose column, and two Superose 12 gel filtration columns. In activities represented by two peaks (Ef108A and Ef108B), 90% of the crude activity was due to peak B. Ef108A is believed to be a variable aggregate of the active moiety in Ef108B. All Listeria spp. and five L. monocytogenes serotypes were inhibited by Ef108B in several media including Listeria enrichment medium. The Ef108 activity may be due to a bacteriocin-like inhibitory substance produced by E. faecium 108 that may suppress the growth and predominance of Listeria monocytogenes during selective enrichment .     

Production of hard cheese from caprine milk by the use of two types of probiotic cultures as adjuncts

Hard cheeses (Kefalotyri-like) were manufactured from caprine milk with yoghurt as a starter (A), and with its partial replacement with the probiotic adjuncts Lactobacillus rhamnosus LC 705 (B) and/or Lactobacillus paracasei ssp. paracasei DC 412 (C). Both adjuncts retarded the growth of enterococci, and the environment in cheese B did not favour the recovery of lactic acid bacteria (LAB) on Rogosa agar. However, better recovery of the LAB population on M17 agar from cheeses B and C made with adjuncts was recorded early in ripening, and this was accompanied by a greater decrease in pH. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein demonstrated that cheese C, made with Lb. paracasei ssp. paracasei as adjunct, is a better vehicle for delivery of live probiotic cells (10 ⁷   cfu/g) to the gastrointestinal tract than cheese B, made with Lb. rhamnosus ; the latter did not belong to the predominant microflora of one out of the two B cheeses. Urea-PAGE electrophoresis results indicated that adjunct lactobacilli enhanced the degradation of both α _S -casein (α _S -CN) and β-casein (β-CN). In the fresh cheese, hydrolysis of α _S -CN was more rapid than β-CN, and the free amino acid content of B and C was higher than in A. Lipolysis products were also higher in B and C than in A as ripening progressed, and the organoleptic characteristics of these cheeses resulted in higher scores, in the order C > B > A. Thus, making Kefalotyri-like cheese from caprine milk with probiotic lactobacilli, particularly Lb. paracasei ssp. paracasei, as adjunct can be considered an effective way of producing a cheese with a large number of probiotic cells.     

ISOLATION AND SOME PROPERTIES OF THE THERMOSTABLE β-GALACTOSIDASE OF PYROCOCCUS WOESEI EXPRESSED IN ESCHERICHIA COLI

The enzyme with β-galactosidase activity from E. coli BL21(DE3) transformant containing the gene encoding enzyme from Pyrococcus woesei (DSM 3773) was isolated using cell extraction in 0.01 M phosphate buffer (pH 7.2), protein thermopredpitation at 85C, precipitation at acetone/extract ratio of 1:1 (v/v) and gel filtration on Sephadex G-200. The increase in the enzyme specific activity was determined using ONPG as substrate. The activity increased from 2.9 × 10³ U/mg protein to 37 × 10³ U/mg. Thermoprecipitation removed 78% of E. coli protein and retained 92% of the cell extract activity. The acetone precipitation and gel filtration applied in the next purification steps led to homogeneous enzyme with specific activity of 37,700 U/mg protein. The isolated enzyme had a half-life of 23 h and 9 h during incubation at 85C and 100C, respectively, in 0.1 M citrate-phosphate buffer (pH 5.4).