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.     

INACTIVATION OF MALT PEPTIDASES DURING MASHING*

Malt contains at least eight different peptidases: three carboxypeptidases (pH optima 4·8, 5·2, 5·7), three aminopeptidases active on aminoacyl-β-naphthylamides (pH optima in hydrolysis of peptides at pH 5·8-6 5), and two (amino)peptidases acting on Leu-Tyr and Ala-Gly at higher pH (pH optima 8·6, 7·8). We have studied the progressive inactivation of these peptidases during mashing with a temperature programme from 45° to 70° C (pH 5·8 → 5·7). All peptidases were stable at 45° C. The five aminopeptidases were inactivated at different rates during a 30-min incubation at 55° C. The carboxypeptidases retained 70–100% of their activities at this temperature, and one of them had 40% residual activity after 60 min at 70° C. Liberation of amino acids continued at a considerable rate during the incubation at 70° C, probably catalysed by the heat-stable carboxypeptidase. Malt carboxypeptidases are therefore more heat-stable in mashing conditions than the aminopeptidases. This property, in combination with their high activities and suitable pH optima, makes them the most important enzymes in the production of free amino acids in mashing.     

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%.     

Production and Application of a Maltogenic Amylase by a Strain of Thermomonospora viridis TF-35

An extracellular and thermostable maltogenic amylase-producing moderate thermophile (Thermomonospora viridis TF-35), which grew well at 28–60°C, with optima at 45°C and pH 7, was isolated from soil. Maximal enzyme production was attained after aerobical cultivation for 32 h at 42°C with a medium (pH 7.3) composed of 2% (w/v) soluble starch, 2% gelatin hydrolyzate, 0.1% K₂HPO₄ and 0.02% MgSO₄ · 7H₂O. The partially purified enzyme, which was most active at 60°C and pH 6.0 and stabilized with Ca²⁺, converted about 65, 80, 75, 75, 65 and 60% of maltotriose, maltotetraose, maltopentaose, amylose, amylopectin and glycogen into maltose as a major product under the conditions used, respectively. Glucose and small amounts of maltooligosaccharides were also formed concomitantly as by-products. The molar ratio of maltose to glucose from maltotriose were larger than 1 during all stages of the hydrolysis. About 70 and 76% of 25% (w/v) potato starch liquefites having a 3.5 DE value were converted into maltose by the enzyme in the absence and presence of pullulanase during the saccharification, respectively. About 90 and 94% of the starch liquefites were also converted into maltose with relatively low contents of maltooligosaccharides by the cooperative 2 step reaction with the enzyme after obtaining starch hydrolyzates containing about 85 and 90% maltose by the simultaneous actions of soybean ß-amylase and debranching enzymes.     

Factors Influencing the Production and Activity of a Streptococcus thermophilus Lipase

The cells from 32 strains were disrupted by pressure. All the strains except two showed lipase activity. Optimum temperature for lipase production was 44°C. Production of lipase was inhibited by addition of butter oil (29%), milk (41%) and casein (12%) and stimulated by soybean oil (39%), cream (27%) and corn oil (21%) to the medium. The enzyme was stable when stored at 37°C and 45°C for 1 wk. The enzyme was partially inactivated by pasteurization treatment (65°C/30 min). Optimum temperature and pH for lipase activity was 45°C and 9.0, respectively. The enzyme was relatively more active toward tributryin than toward natural lipids.     

Induction,purification and characterization of malolactic enzyme from Oenococcus oeni SD-2a

The aim of this study was to purify a malolactic enzyme (MLE) from Oenococcus oeni (O. oeni) strain and determine its properties in detail. O. oeni SD-2a was cultivated in the ATB broth supplemented with 7 g/L l-malic acid for harvesting the cells. After harvest, the cells were washed and disrupted for purification of MLE. MLE was purified from the supernatant of the disrupted cells through protamine sulfate precipitation, anion exchange chromatography and gel filtration chromatography. The purified MLE was identified using mass spectrometry. The MLE was purified by 43-fold with a yield of 0.42 % and possessed a specific activity of 419.2 U/mg. The purified enzyme with a nominal molecular mass of 59 kDa and a theoretical pI of 4.76 exhibited a maximum enzyme activity at 35 °C and pH 6.0, which retained over 50 % of its initial activity in the presence of 14 % (v/v) ethanol. Mn²⁺ was proven to be the most effective divalent cation to promote enzyme activity. Under the conditions of temperature 30 °C and pH 6.0, the K _m and V _max of MLE on l-malic acid were 12.5 × 10^?3 M and 43.86 μmol/(min × mg), respectively. Moreover, the purified enzyme exhibited a higher stability with 0.1 M NaCl in addition and had a half-life of 30 days at 4 °C.     

Preparation of lactose‐free milk by using salt‐fractionated almond (Amygdalus communis) β‐galactosidase

β‐galactosidase was isolated from almond (Amygdalus communis) extract by ammonium sulfate precipitation. Almond proteins precipitated by using ammonium sulfate and then dialysed exhibited 5.3‐fold purification of β‐galactosidase, and the yield of enzyme preparation was 96.5%. The partially purified β‐galactosidase exhibited pH and temperature optima at pH 5.5 and 50 °C, respectively. The enzyme was significantly stable against heat, pH, calcium and magnesium ions and D ‐galactose. The almond β‐galactosidase preparation exhibited over 89% activity even after 2 months storage at 4 °C. Hydrolysis of lactose in milk and whey was performed in a stirred batch process by using this enzyme preparation. These observations indicated that the hydrolysis of lactose increased continuously with time. The enzyme could hydrolyse 94% of lactose in buffer solution and whey whereas 90% of lactose hydrolysis was achieved in milk. The main aim of the present study was to prepare lactose‐free milk, which must be free from contamination, and the process should be inexpensive. Copyright © 2007 Society of Chemical Industry     

Characterisation of cellulose-hydrolysing enzymes from the fungus Bipolaris sorokiniana

Cellulose-hydrolysing enzymes from the phytopathogenic fungus Bipolaris sorokiniana were partially purified and characterised. The enzyme production was variable according to the carbon source. β-Glucosidase and cellobiohydrolase activities were higher by growing the fungus on cellulose than on other carbon sources. Carboxymethyl cellulase production was stimulated by other carbohydrates, mainly lactose. Partial enzyme purification was carried out by liquid chromatography on Sepharose CL4B. The purification was about 17-fold, with a yield of 41% as judged by assay with p-nitrophenyl-β-D -glucopyranoside as substrate. The optimum pH and temperature were 5.0 and 55–60 °C respectively. The enzyme was stable at 28 and 37 °C but lost about 50% of its initial activity after 120 min at 55 °C. Saccharification of cellulosic materials such as crystalline cellulose, filter paper and wheat straws was carried out using the partially purified enzyme, resulting in the production of reducing sugars. © 1999 Society of Chemical Industry     

Properties of Trypsin from the Pyloric Ceca of Atlantic Cod (Gadus morhua)

Trypsin (EC 3.4.21.4) was isolated from the pyloric ceca of Atlantic cod and purified to homogeneity by affinity chromatography. The enzyme catalyzed the hydrolysis of benzoyl arginine p-nitroanilide (BAPA, pH 8.2 and 25°C) such that V_max was 250 BAPA units per micromole trypsin and K_m was 1.48 mM. For the hydrolysis of tosyl arginine methyl ester (TAME, pH 8.1 and 25°C), V_max was 18.2 × 10³ TAME units/micromole trypsin, and K_m 0.22 mM. The pH and temperature optima with BAPA substrate were 7.5 and 40°C, respectively. Atlantic cod trypsin was most active and stable at alkaline pH. The enzyme was heat labile, losing more than 50% of its activity after incubation at 50°C for 30 min. Amino acid analysis of Atlantic cod trypsin revealed that the enzyme was rich in residues such as serine, glycine, glutamate and aspartate, but poor in basic amino acid residues compared to trypsins from warm blooded animals.     

Optimisation of the Mashing Procedure for 100% Malted Proso Millet (Panicum miliaceum L.) as a Raw Material for Gluten‐free Beverages and Beers

Proso millet is a gluten‐free cereal and is therefore considered a suitable raw material for the manufacturing of foods and beverages for people suffering from celiac disease. The objective of this study was to develop an optimal mashing procedure for 100% proso millet malt with a specific emphasis on high amylolytic activity. Therefore, the influence of temperature and pH on the amylolytic enzyme activity during mashing was investigated. Size exclusion chromatography was used to extract different amylolytic enzyme fractions from proso millet malt. These enzymes were added into a pH‐adjusted, cold water extract of proso millet malt and an isothermal mashing procedure was applied. The temperatures and pH optima for amylolytic enzyme activities were determined. The α‐amylase enzyme showed highest activity at a temperature of 60°C and at pH 5.0, whereas the β‐amylase activity was optimum at 40°C and pH 5.3. The limit dextrinase enzyme reached maximum activity at 50°C and pH 5.3. In the subsequent mashing regimen, the mash was separated and 40% was held for 10 min at 68°C to achieve gelatinisation. The next step in the mashing procedure was the mixture of the part mashes. The combined mash was then subjected to an infusion mashing regimen, taking the temperature optima of the various amylolytic enzymes into account. It was possible to obtain full saccharification of the wort with this mashing regimen. The analytical data obtained with the optimised proso millet mash were comparable to barley wort, which served as a control.     

α-Amylases in raw and fermented African oil bean seeds (Pentaclethra macrophylla benth)

The !-amylases of raw and fermented seeds of African oil bean (Pentaclethra macrophylla Benth) were isolated, partially purified (tenfold) and subjected to different thermal and pH conditions. The specific activities of the purified enzyme from raw and fermented seeds were 0.037 ml^-1 min^-1 and 0.88 ml^-1 min^-1 respectively. The !-amylase from fermented seeds was more thermostable with optimum activity at 70 °C, compared to the optimum temperature of 60 °C obtained for the raw seed enzyme. The assayed !-amylases were stable over a wide range of pH (3.0-7.0), with optimum activity found at pH 6.0 and pH 5.0 for raw and fermented seeds respectively. The results show that the !-amylase of Pentaclethra macrophylla complements the microbial amylases in the mainly bacterial fermentation of African oil bean seeds to 'ugba', a highly nutritious food condiment. While the !-amylase in the raw seeds was basically the plant enzyme, that isolated from the fermented seeds was a combination of plant and microbial !-amylases.     

Characterization of Polyphenoloxidase from Ravat 51 and Niagara Grapes

Polyphenoloxidase (PPO) was isolated from two varieties of grapes grown in the northeastern United States and its characteristics were studied. The temperature and pH optima for both enzymes were 25°C and 5.5, respectively. The thermal inactivation of PPO followed first-order kinetics; with the Niagara enzyme being more heat stable than Ravat PPO. The substrate specificity of the grape PPO clearly showed high affinity toward the o-diphenolic compounds, with a high affinity toward caffeic acid. Inhibition studies indicated that L-cysteine and sodium diethyldithiocarbamate were the most potent.     

Biochemical studies of cocoa bean polyphenol oxidase

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purified from cocoa beans. The properties of the enzyme were studied. The Michaelis constant K_m for catechol was 1 × 10^?2 M . The pH optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at pH 6.8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3-(3,4-dihydroxy phenyl)-DL -alanine, 3-hydroxytyramine hydrochloride and 4-methyl catechol but showed no activity towards tyrosine, p-cresol, and 4-hydroxy-phenol. A rapid deactivation of the enzyme was observed when catechol of concentration > 40 mM was used as substrate. The enzyme activity was inhibited by ascorbic acid, L -cysteine, sodium bisulphite and thiourea.     

Purification of a lipase from the hepatopancreas of oil sardine (Sardinella longiceps linnaeus) and its characteristics and properties

Lipase has been purified from the hepatopancreas of oil sardine (Sardinella longiceps) by defatting, water extraction, ammonium sulphate fractionation and chromatography on DEAE Sephadex and Sephadex G-100. The preparation was homogeneous on polyacrylamide disc gel electrophoresis and on gel filtration through Sephacryl S-200. The enzyme showed a molecular weight of 54000±57000 with 6.1% of carbohydrate. The pH and temperature optima of purified sardine lipase were 8 and 37°C respectively. Sardine lipase remained stable up to 45°C (15 min) and in the pH range 5 to 9.5. The K_m values obtained for the substrates tributyrin and triacetin were 4 × 10^?2 and 30 × 10^?2, respectively. The effect of halogens and various metal ions on sardine lipase activity, substrate specificity, amino acid and carbohydrate composition are also reported.     

Purification and characterisation of thermo-alkaline pectinase enzyme from Hylocereus polyrhizus

The thermo-alkaline pectinase enzyme from Hylocereus polyrhizus was purified 232.3-fold with a 73.3 % recovery through ammonium sulphate precipitation, gel filtration, and ion exchange chromatography. Ion exchange chromatography combined with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme was monomeric with a molecular weight of 34.2 kDa. The pectinase exhibited broad specificity towards polygalacturonic acid, arabinan, oat spelt xylan, and pNP-α-glucopyranoside. The optimum pH and temperature were 8.0 and 75 °C, respectively. This enzyme was stable over a wide pH range (3.0–11.0) and at relatively high temperature (85 °C for 1 h). The K_m and V_max values of pectinase towards polygalacturonic acid were 2.7 mg/ml and 34.30 U/mg proteins, respectively. In addition, the enzyme activity was inhibited by Ni²⁺, Al³⁺, and Fe²⁺ and was increased in the presence of Ca²⁺ and Mg²⁺ by 120 and 112 %, respectively. The purified pectinase demonstrated robust stability in response to surfactants and oxidising agents. EDTA, which is a powerful chelating agent, did not exert any significant effect on the enzyme stability. Thus, enzymes with these unique properties may be widely used in different types of industries and biotechnological applications.     

一株嗜热菌木糖异构酶的纯化与性质研究

研究了1株嗜热菌(Anoxybacillus flavithermus)所产木糖异构酶的分离纯化以及酶学性质。结果表明,经硫酸铵沉淀、Sephadex G-75凝胶过滤、纤维素DE-52弱阴离子交换柱和Q Sepharose Fast Flow强阴离子交换层析得到的木糖异构酶,分子量约为181 ku,由4个相同分子量的亚基组成。酶反应的最适温度为80℃,最适为pH为7.0且最适pH范围宽泛,pH6.0~11.0酶反应活性能保持80%左右。该酶热稳定性及耐碱性能良好,70℃保温1 h后酶活仍能保持近80%左右;pH5.0~8.0保温1 h后酶活仍能保持近80%以上,甚至pH12.0保温1 h后酶活性仍能保持40%左右。Mn2+和Co2+对酶活性有明显促进作用,Zn2+、Cu2+以及Al3+对酶活性有一定程度的抑制。     

Characteristics and Concentrations of α-Amylase Inhibitor in Phaseolus vulgaris Biotypes

Flours from six legume species (lentil, lima bean, field pea, chickpea, fababean, mung bean) were devoid of α-amylase inhibitory activity but five biotypes of Phaseolus vulgaris showed activities of 27 to 40 units/mg protein. Air classification of the pin milled flours yielded fine fractions which contained 37% (black bean) to 61% (navy bean) protein and 38 to 66 units of inhibitor activity/mg protein, respectively. The coarse starch fractions were proportionately depleted in protein content and inhibitor activity. The partially purified amylase inhibitor from navy bean was active towards porcine pancreatic α amylase. The optimum pH for inhibition was 5.6 - 5.7, with the inhibitor being most stable at pH 6.9 after 4 h incubation at 37°C. The inhibition was faster at 37°C than at 25°C. Although retaining most of its inhibitory power between 37°C–60°C, the navy bean inhibitor showed a complete loss of activity after 20min at 90°C.     

The linamarase of Leuconostoc mesenteroides: Production,isolation and some properties

Leuconostoc mesenteroides was found to produce highly active linamarase when linamarin was incorporated in its growth medium. The enzyme was isolated from the bacterium and partially purified using diethylaminoethyl (DEAE) cellulose. Its activity was measured spectrophotometrically using linamarin extract. This yielded 62.2 mg CN^? g^?1 of linamarin. A study of some of its properties showed it was active in the temperature range of -10 to +45°C, with an optimum at 29°2°C. Activity was observed over a wide pH range, 4.0–8.0, with optimum at 6.0–6.5. Its pH of stability was 5.5–7.5, while above pH 8.0 there was a rapid loss of activity. Incubating the enzyme at 50°C led to loss of over 90% of its activity within 18 min. The optimal substrate concentration was 0.15–0.20 ml^?1. Whereas above 0.25 mg ml^?1 there was no observable increase in activity, loss of activity became more pronounced below 0.10 mg ml^?1 of substrate.     

Purification and characterization of a phytate-degrading enzyme from germinated oat (Avena sativa)

A phytate-degrading enzyme (myo-inositol hexakisphosphate phosphohydrolase) has been purified about 5,400-fold from germinated oat seedlings to apparent homogeneity. The molecular mass of the native monomeric enzyme was estimated to be about 67 kDa. Optimal pH for degradation of phytate was 5.0 and the optimal temperature 38 °C. Kinetic parameters for the hydrolysis of Na-phytate are K_M 30 µM and k_cat 356 s⁻¹ at 35 °C and pH 5.0. The oat phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyses phytate in a stepwise manner. The first hydrolysis product was identified as D /L -l(1,2,3,4,5) P₅. © 1999 Society of Chemical Industry     

Characterization of Pullulanase and α-Amylase Activities of a Thermus sp. AMD33

Thermostable Thermus sp. AMD 33 pullulanases (I and II) capable of cleaving α-1,6-links in pullulan as well as α-1,4-glucosidic linkages in amylose were purified to electrophoretically homogeneous states. Relative molecular masses and pI values were determined as 135,000 (I and II) by SDS-PAGE and 4.2 (I) and 4.3 (II) by isoelectric focusing, respectively. The pullulanase and α-amylase activities of the purified enzyme II responded similarly to temperature and pH, with optima at 70°C and pH 5.5–6.0. Both activities were activated by Ca²⁺ and inhibited by Hg²⁺, Fe³⁺, NBS, DBS, SDS and urea to almost the same extent. Both activities were also inhibited competitively by CDs. Enzyme II catalyzed the hydrolysis of α-1,6-glucosidic linkages in maltosyl- and maltotriosyl-α-CD as well as that of α-1,4-bonds in amylose and related linear malto-oligosaccarides larger than maltotriose, but exhibited no action on panose, isopanose or glucosyl α-CD.