Papaya Polygalacturonase and its Role in Thermally Injured Ripening Fruit

The polygalacturonase (E.C. 3.2.1.15) (PGase) activity in papayas increased with fruit ripeness. PGase activity was highest in the placenta with the activity decreasing outwardly from the placenta to the exocarp. Extended hot water treatments (46°C for 65 and 90 min) caused delayed softening of the fruit tissue which was correlated to a decrease in PGase activity. The decrease in PGase activity after the heat treatments was more severe in the riper fruits (quarter and half ripe) than the less ripe fruits (colorbreak stage).     

Purification and Characterization of a Pullulan-Hydrolyzing Glucoamylase from Sclerotium rolfsii

The pullulan-hydrolyzing enzyme from the culture filtrates of Sclerotium rolfsii grown on soluble starch as a carbon source has been purified by ultrafiltration (Amicon, PM-10), ion-exchange chromatography (DEAE-Cellulose DE-52) and gel filtration chromatography (Bio-Gel P-150). The enzyme moved as a single band in non-denaturing polyacrylamide gel electrophoresis carried out at pH 2.9 and 7.5. The relative molecular mass of the enzyme was estimated to be 64.000 D by SDS-PAGE and 66.070 D by gel filtration on Bio-Gel P150. The enzyme hydrolyzed pullulan optimally at 50°C between pH 4.0–4.5, whereas, soluble starch was optimally hydrolyzed at a pH of between 4.0–4.5 and at 65°C. The Michaelis constant (Km) for pullulan was 5.13mg·ml⁻¹ (V_max 1.0U · mg⁻¹) and for soluble starch, it was 0.6mg · ml⁻¹ (V_max 8.33 U · mg⁻¹). The enzyme was observed to be a glycoprotein (12–13% carbohydrate by weight) and had a strong affinity for Concanavalin A. The enzyme hydrolyzed α-D-glucans in an exo-manner, which resulted in the release of glucose as the sole product of hydrolysis. Acarbose, a maltotetraose analog, was found to be a potent inhibitor of both pullulan and starch hydrolysis (100% inhibition at 0.06 μM). The enzyme has been characterized as a glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) showing a significant action on pullulan.     

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.     

Ultrafiltration for Recovery and Reuse of Peptidoglutaminase in Protein Deamidation

Ultrafiltration (UF) of B. circulans cell extract using a 100kd hollow-fiber membrane had no effect on peptidoglutaminase (PGase) activity and allowed 34 and 26% of PGases I and II to permeate. UF, with 30kd spiral membrane, slightly increased PGase activity in retentate but no activity was detected in permeate. Retentate was used to sequentially deamidate four batches of a soy protein hydrolysate at 30°C for 2 hr, then PGase was recovered by UF. The 100kd membrane resulted in substantial PGase loss in sequential tests due to permeation and inactivation. A 100% conversion and 97% of PGase were obtained after tests with the 30 kd membrane. Multiple recovery and use of PGase by a suitable membrane is possible.     

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,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.     

Ultrathin-layer isoelectric focusing of partially purified peroxidase from tomato fruit

Partially purified peroxidase from tomato fruits was obtained by gel filtration on Ultrogel AcA 34 of a pH 5·5 extract prepared by homogenising the fruits with 0·4m McIlvaine buffer containing 0·2% Triton-X 100, subsequent centrifugation at 2000 × g and ultrafiltration of the supernatant. This fraction, with a molecular weight of 43 000 daltons, showed an optimum activity of pH 5·3 with guaiacol as the hydrogen donor, a K_m for H₂O₂ of 0·8 mm at optimum guaiacol concentration, a K_m of 9·0 mm for guaiacol at optimum H₂O₂ concentration and competitive cyanide inhibition with a K_i of 1·0 μm. Ultrathin-layer isoelectric focusing in 50 μm polyacrylamide gels resulted in the separation of eight peroxidase isozyme bands, detected mainly in the range pH 2 to 4. Parallel experiments carried out with horseradish peroxidase (Boehringer, Mannheim) exhibited the main enzyme activities in the range pH 7 to 9.     

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.     

Winged bean lipoxygenase—Part 2: Physicochemical properties

Winged bean lipoxygenase (linoleate: oxygen oxidoreductase EC 1.13.11.12) isoenzymes FI and FII were isolated and purified according to the method of Truong et al. (1980).FI and FII were both highly specific for linoleic acid. They exhibited optimal activity at pH 6·0 and 5·8, respectively at 30°C. An activation energy of 4·5 kcal mol^?1 was calculated for this lipoxygenase within the temperature range of 30–50°C.At 0·075% Tween 20, FI and FII had K_m values for linoleic acid of 0·44 and 0·37 × 10^?3M, respectively, compared to 0·4 × 10^?3M for the crude enzyme. Maximal activity was obtained at 1·6 × 10^?3M. Higher levels of Tween 20 inhibited the lipoxygenase activity.Both isoenzymes had identical average molecular weight of 80 000 daltons by gel filtration and SDS gel electrophoresis.FI and FII isoenzymes were strongly inhibited by Hg⁺⁺, Mn⁺⁺, Mg⁺⁺ and Fe⁺⁺⁺ and activated by Zn⁺⁺, Co⁺⁺ and Fe⁺⁺. A difference in the degree of inhibition or activation was observed between FI and FII response. Ca⁺⁺ inhibited both FI and FII but the former was more sensitive to Ca⁺⁺. KCN also inhibited the two isoenzymes.Among the antioxidants tested, butylated hydroxytoluene and butylated hydroxyanisole most effectively inhibited both FI and FII at only 10^?6M. Sulphydryl reagents such as iodoacetamide and dithiothreitol have little effect on the lipoxygenase isoenzyme activity.The lipoxygenase isoenzymes were more stable at neutral pH. The enzyme in the crude extract and especially in situ was more stable to heat treatment.     

The detection of aflatoxin B1 in peanut kernels,peanut butter and maize using a monoclonal antibody based enzyme immunoassay

A simple procedure was validated for the routine immunochemical analysis of AFB₁ in peanut kernels, peanut butter and maize. The specificity, affinity and sensitivity of the monoclonal antibody (McAb) employed was such that minimal sample preparation was required. The enzyme immunoassay (EIA) had a sensitivity for standard AFB₁ of 0·2 ng ml⁻¹ with a working range up to 30 ng ml⁻¹ and was not significantly affected by matrix interference of samples. Essential protocol features were (1) blending substrate with methanol: water; (2) filtering blended sample; and (3) analysis of filtrate by EIA after dilution with buffer. Average recoveries of AFB₁ spiked samples at levels of 6–400 ppb were 90–112·5%. Using laboratory prepared samples contaminated with Aspergillus flavus there was high positive correlation (r = 0·97) when EIA results were compared with thin layer chromatography (TLC) techniques.     

脱酰胺对热诱导小麦面筋蛋白构象及凝胶性质的影响

利用蛋白质谷氨酰胺酶(PGase)对小麦面筋蛋白进行处理,研究PGase脱酰胺及热诱导对小麦面筋蛋白构象及其凝胶性质的影响。结果表明,PGase脱酰胺能够在一定程度上抑制小麦面筋蛋白的热诱导聚集行为,提高小麦面筋蛋白热诱导凝胶的强度和保水性。当PGase添加量为3 U/g时,其凝胶强度和保水性均达到最大值,分别为903. 27 N和78. 70%。研究还表明,PGase处理后小麦面筋蛋白与水相互作用增加,从而提高了脱酰胺小麦面筋蛋白热诱导凝胶中非可冻结水含量。扫描电镜结果显示PGase脱酰胺结合热诱导处理的小麦面筋蛋白形成了更加有序的凝胶网络结构。     

ISOLATION,PURIFICATION AND ELECTROPHORETIC PROPERTIES OF AN α-AMYLASE FROM MALTED BARLEY

An α-amylase component from malted barley was isolated and purified using aqueous extraction at pH 8·0, heat treatment of the extract at 70°C, specific precipitation with glycogen and ion exchange chromatography on carboxymethyl (CM) and diethylaminoethyl (DEAE) cellulose. The enzyme preparation was shown to be pure by disc electrophoresis at pH 8·9 and iso-electricfocusing on polyacrylamide gel in a pH 4–8 gradient.     

Membrane Reactor for Enzymic Deamidation of Food Proteins

A soy protein hydrolysate was deamidated with peptidoglutaminase (PGase) retained within a 30kd spiral membrane. Reactor was operated at 30°C and 6.5 L/m²/hr flux in recycle mode to 60% conversion, then in diafiltration mode for 2 hr. Time course, predicted by a Michaelis-Menten equation integrated for mixed zero- and first-order kinetics with the corrections for the ultrafiltration (UF) interactions, matched that measured experimentally. The equation could be used to predict the potential activity and performance of PGase in UF reactors to achieve control of reactions for the optimal enzymic process. At the end of the run, 96% conversion and 99% of PGase were obtained. This method has the potential of being scaled-up for the production of enzyme-free deamidated proteins.     

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%.     

Proximate composition and chemical assay of the methionine,lysine and tryptophan concentrations of some forest tree seeds

The proximate compositions of the seeds of twenty-five species of tree were determined and methionine, lysine and tryptophan were colorimetrically estimated. The lowest crude protein value (8·8%) was obtained in Afzelia bella (calyx) while Parkia clappertoniana had the highest value (33·2%). Extractable lipids ranged from 1·0% in Cassia nodosa to 47·8% in Khaya senegalensis. The highest value of crude fibre (26%) was obtained in Tectona grandis while Cassia siemea had the highest ash value (9·7%). Nitrogen-free extract ranged from 21·6% for Jatropha curcas to 85·4% for Afzelia bella (calyx). The highest values of $\text{4·19}\text{g}\text{16}\text{g}$ N for methionine, $\text{8·70}\text{g}\text{16}\text{g}$ N for lysine and $\text{3·10}\text{g}\text{16}\text{g}$ N for tryptophan were obtained in Tectona grandis, Afzelia pachyloba and Khaya ivorensis, respectively.     

Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope

Saccharomyces cerevisiae cell envelope polyphosphatase was isolated in highly active and stable form by extraction from cells with zwittergent TM-314 followed by chromatography of the extract on phosphocellulose and QAE-Sephadex in the presence of 5 mM -MgCl₂, 0·5 mM -EDTA and 0·1% Triton X-100. The enzyme possessed a specific activity of 220 U/mg and after 30 days retained 87% of its activity at ?20°C. Polyphosphatase molecular mass was determined to be about 40 kDa by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme hydrolysed polyphosphates with various chain lengths (n = 3–208), had low activity for GTP and did not split pyrophosphate, ATP and p-nitrophenylphosphate. On polyphosphates with chain lengths n = 3, 9 and 208, K_m values were 1·7 × 10^?4, 1·5 × 10^?5 and 8·8 × 10^?7 M respectively. Polyphosphatase was most active and stable at pH 6·0–8·0. The enzyme showed maximal activity at 50°C. The time of half inactivation of polyphosphatase at 40, 45 and 50°C was 45, 10 and 3 min, respectively. In the absence of divalent cations and also with Ca²⁺ or Cu²⁺, the enzyme showed practically no activity. The ability of divalent cations to activate polyphosphatase was reduced in the following order: Co²⁺ > Mg²⁺ > Mn²⁺ > Fe²⁺ > Zn²⁺. Polyphosphatase was completely inhibited by 1 mM -ammonium molybdate and 50 μM -Zn²⁺ or Cu²⁺ (in the presence of Mg²⁺).     

Degradation of myofibrils and formation of premer omyosin by a neutral protease produced by Pseudomonas fragi

Rabbit muscle myofibrils were treated with a neutral protease (200: 1-protein: enzyme) isolated from Pseudomonas fragi (P. fragi) at 0° C in 100 mm KCl, 5·0 mm CaCl₂, 0·2 mm DTT (dithiothreitol) and 20 mm TRIS. HCl (pH 7·50) for 0–60 min. SDS-polyacrylamide disc gel electrophoresis showed extensive fragmentation of the myofibrillar proteins. Three of the major products correspond in molecular weight to heavy meromyosin (HMM), light meromyosin (LMM) and the recently discovered premeromyosin (PMM). Results demonstrated that the neutral protease produced by P. fragi is capable of hydrolysing myosin and probably plays an important role in microbial breakdown of the muscle proteins during meat spoilage.     

Thermal inactivation kinetics parameters of browning enzymes in starfruit (Averrhoa carambola L.) juice

This study aimed to evaluate the thermal inactivation kinetics of polyphenol oxidase (PPO) and peroxidase (POD) in starfruit juice. It followed the Malaysia Food Regulations 1985 and CODEX STAN 247-2005. Glucose, fructose and sucrose were the main sugars in starfruit juice. The total soluble solids, pH, titratable acidity, and total phenolics content of the starfruit juice produced were 8.13 ± 0.25 °Brix, 3.80 ± 0.05, 0.43% ± 0.02% malic acid, and 93.67 ± 4.96 mg GAEL⁻¹, respectively. Thermal inactivation kinetics of PPO and POD followed the first-order kinetic model. The decimal reduction time at 83.6 °C (D_83.6) of PPO and POD was 198.48 and 98.4 s, respectively, while the thermal resistance constant (z value) of PPO and POD was 12.8 and 5.4 °C, respectively. In conclusion, PPO might be a suitable signal for thermal processing on starfruit juice since it has higher heat resistance than POD.     

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).     

Chemical evaluation of the effect of roasting on the nutritive value of maize (Zea mays, Linn.)

This study reports on the effects of roasting on the chemical composition of maize (Zea mays). Proximate analysis showed no significant difference (p > 0·05) between raw and roasted maize in ether extract, crude protein, crude fibre, ash and carbohydrate content; except moisture content (p < 0·05), which showed a 42·3% decrease. Elemental composition analysis showed decreases of potassium (13·8%) and calcium (41·1%). Significant differences (p < 0·05) were observed for vitamins B₁, B₂ and C contents with 26·8%, 32·4% and 35·1% destruction, respectively. Amino acid analysis showed losses for lysine (26·7%), iso-leucine (20·8%) and leucine (23·4%).There was significant (p < 0·05) variation in phytic acid, oxalic acid, tannin and hydrocyanic acid with reductions of 15·4%, 6·02%, 51·3% and 34·6% respectively.The effect of these changes on the nutritive value of roasted maize is discussed.