Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0–11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (K_m) and catalytic constant (K_cat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s⁻¹, respectively. The catalytic efficiency (K_cat/K_m) was 238 s⁻¹ mM⁻¹.     

Characterisation of a β-N-acetylhexosaminidase from a commercial papaya latex preparation

A β-N-acetylhexosaminidase (β-NAHA) (EC 3.2.1.52) with molecular mass of 64.1 kDa and isoelectric point of 5.5 was purified from a commercial papaya latex preparation. The optimum pH for p-nitrophenyl-N-acetyl-β-d-glucosaminide (pNP-β-GlcNAc) hydrolysis was five; the optimum temperature was 50 °C; the K_m was 0.18 mM, V_max was 37.6 μmol min⁻¹ mg⁻¹ and activation energy (E_a) was 10.3 kcal/mol. The enzyme was thermally stable after holding at 30–45 °C for 40 min, but its activity decreased significantly when the temperature exceeded 50 °C. Heavy metal ions, Ag⁺ and Hg²⁺, at a concentration of 0.25 mM and Zn²⁺ and Cu²⁺, at a concentration of 0.5 mM, significantly inhibited enzyme activity. The β-NAHA had only one active site for binding both pNP-β-GlcNAc and p-nitrophenyl-N-acetyl-β-d-galactosaminide (pNP-β-GalNAc). A prototropic group with pKa value of about five on the enzyme may be involved in substrate binding and transformation, as examined by Dixon–Webb plots.     

An extra-cellular alkaline metallolipase from Bacillus licheniformisMTCC 6824: Purification and biochemical characterization

An extra-cellular lipase produced by Bacillus licheniformis MTCC 6824 was purified to homogeneity by ammonium sulphate fractionation, ethanol/ether precipitation, dialysis, followed by anion-exchange chromatography on Amberlite IRA 410 (Cl⁻ form) and gel exclusion chromatography on Sephadex G 100 using Tris–HCl buffer (pH 8.0). The crude lipase extract had an activity of 41.7 LU/ml of culture medium when the bacterium was cultured for 48 h at 37 °C and pH 8.0 with nutrient broth supplemented with sardine oil as carbon source. The enzyme was purified 208-fold with 8.36% recovery and a specific activity of 520 LU/mg after gel exclusion chromatography. The pure enzyme is a monomeric protein and has an apparent molecular mass of 74.8 kDa. The lipase had a V_max and K_m of 0.64 mM/mg/min and 29 mM, respectively, with 4-nitro phenylpalmitate as a substrate, as calculated from the Lineweaver–Burk plot. The lipase exhibited optimum activity at 45 °C and pH 8.0, respectively. The enzyme had half-lives (T_1/2) of 82 min at 45 °C, and 48 min at 55 °C. The catalytic activity was enhanced by Ca²⁺ (18%) and Mg²⁺ (12%) at 30 mM. The lipase was inhibited by Co²⁺, Cu²⁺, Zn²⁺, Fe² even at low concentration (10 mM). EDTA, at 70 mM concentration, significantly inhibited the activity of lipase. Phenyl methyl sulfonyl fluoride (PMSF, 70 mM) completely inactivated the original lipase. A combination of Ca²⁺ and sorbitol induced a synergistic effect on the activity of lipase with a significantly high residual activity (100%), even after 45 min, as compared to 91.5% when incubated with Ca²⁺ alone. The lipase was found to be hydrolytically resistant toward triacylglycerols with more double bonds.     

A β-galactosidase from chick pea (Cicer arietinum) seeds: Its purification,biochemical properties and industrial applications

A β-galactosidase from Cicer arietinum seeds has been purified to apparent electrophoretic homogeneity using a combination of various fractionation and chromatographic techniques, giving a final specific activity of 220 units mg⁻¹, with approximately 1840 fold purification. Analysis of the protein by SDS–PAGE revealed two subunits with molecular masses of 48 and 38 kDa, respectively. These bands were further confirmed with LC–MS/MS, indicating that Chick pea β-galactosidase (CpGAL) is a heterodimer. Molecular mass was determined to be 85 kDa by Superose-12 FPLC column, which is in agreement with the molecular mass suggested by mass spectroscopy to be 83 kDa. The optimum pH of the enzyme was 2.8 and it hydrolysed o-nitrophenyl β-d galactopyranoside (ONPG) with a K_m value of 1.73 mM at 37 °C. The energy of activation (E_a) calculated in the range of 35 to 60 °C, using Arrhenius equation, was determined to be 11.32 kcal mol⁻¹. The enzyme could also hydrolyse lactose, with an optimum pH of 4.0 at 40 °C. K_m and E_a for lactose hydrolysis was found to be 10 mM and 10.57 kcal mol⁻¹, respectively. The enzyme was found to be comparatively thermostable showing maximum activity at 60 °C for both ONPG and lactose. Galactose was found to be the competitive inhibitor. β-Galactosidase also exhibited glycoproteineous properties when applied on Con-A Sepharose column. The enzyme was localised in germinated seeds with X-gal activity staining and shown to be expressed prominently at grown radical tip and seed coat. Sequence alignment of CpGAL with other known plant β-galactosidase showed high amino acid sequence homology.     

Determination of selenium in garlic (Allium sativum) and onion (Allium cepa) by electro thermal atomic absorption spectrometry

A separation/enrichment procedure has been developed for the determination of selenium in garlic and onion samples by electrothermal atomic absorption spectrometry (ET-AAS) as a slurry sampling after preconcentration with 3,3-diaminobenzidine (DAB) reagent on the activated carbon. The influences of pH, time, amount of carbon and complexing reagent were outlined. The effect of acids used in the digestion of samples was also studied and compared. Selenium level was found to be 0.024 μg g⁻¹ for onion (n = 5; LOD – 0.5 μg L⁻¹; LOQ – 1.7 μg L⁻¹) and 0.015 μg g⁻¹ for garlic (n = 5; LOD – 1.3 μg L⁻¹; LOQ – 3.3 μg L⁻¹). Three different samples of garlic were analyzed by k₀-instrumental neutron activation analysis (k₀-INAA) at the Jozef Stefan Institute (JSI). The data obtained by k₀-INAA show that the content of selenium overlapped the results obtained by ET-AAS.     

Characterization of polyphenoloxidase (PPO) and total phenolic contents in medlar (Mespilus germanica L.) fruit during ripening and over ripening

Characterization of polyphenoloxidase (PPO) enzyme and determination of total phenolic concentrations during fruit ripening and over ripening in medlar (Mespilus germanica L.) were determined. During ripening, PPO substrate specificity, optimum pH and temperature, optimum enzyme and substrate concentrations were determined. Among the five mono- and di-phenolic substrates examined ((p-hydroxyphenyl) propionic acid, l-3,4-dihydroxyphenylalanine, catechol, 4-methylcatechol and tyrosine), 4-methylcatechol was selected as the best substrate for all ripening stages. A range of pH 3.0–9.0 was also tested and the highest enzyme activity was at pH 7.0 throughout ripening. The optimum temperature for each ripening stage was determined by measuring the enzyme activity at various temperatures over the range of 10–70 °C with 10 °C increments. The optimum temperatures were found to be 30, 20 and 30 °C, respectively, for each ripening stage. Optimum enzyme and substrate concentrations were found to be 0.1 mg/ml and 40 mM, respectively. The V_max and K_m value of the reaction were determined during ripening and found to be 476 U/mg protein and 26 mM at 193 DAFB (days after full bloom) – stage 1, 256 U/mg protein and 12 mM at 207 DAFB – stage 2, 222 U/mg protein and 8 mM at 214 DAFB – stage 3. For all ripening stages sodium metabisulfite markedly inhibited PPO activity. For stage 1 of ripening, Cu²⁺, Hg²⁺ and Al³⁺, for stage 2, Cu²⁺ and Hg²⁺, and for stage 3, Cu²⁺, Hg²⁺, Al³⁺ and Ca²⁺ strongly inhibited diphenolase activity. Accordingly, it can be concluded that as medlar fruit ripen there is no significant changes in the optimum values of polyphenoloxidases, although their kinetic parametres change. As the fruit ripening progressed through ripe to over-ripe, in contrary to polyphenoloxidase activity, there was an apparent gradual decrease in total fruit phenolic concentrations, as determined by using the aqueous solvents and water extractions.     

Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin

A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). The effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS–PAGE. Optimum pH and temperature were 9.0 and 65 °C, respectively. The enzyme was stable after incubation for 30 min in a wide pH range (6.0–11.5) and at 55 °C. The kinetic parameters K_m, k_cat and k_cat/K_m were 0.47 ± 0.042 mM, 1.33 s⁻¹ and 2.82 s⁻¹ mM⁻¹, respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe²⁺, Hg²⁺, Zn²⁺, Al³⁺, Pb²⁺, and was highly inhibited by trypsin inhibitors. The trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. The features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries).     

Identification of pepsinogens and pepsins from the stomach of European eel (Anguilla anguilla)

Three pepsinogens (PG-I, PG-II and PG-III) were purified to homogeneity from the stomach of freshwater fish European eel (Anguilla anguilla) by ammonium sulphate fractionation, column chromatography on anion exchange and gel-filtration. Their molecular masses were determined as 36, 37 and 36 kDa, respectively, by SDS–PAGE, which were in agreement with the results obtained by Sephacryl S-200 and Superdex 75 gel-filtration. PG-I, PG-II and PG-III converted into corresponding pepsins with molecular masses of approximately 30 kDa at pH 2.0 and showed maximal activity at pH 3.5, 2.5 and 2.5. Optimal temperatures of these pepsins were 40, 40 and 35 °C, using bovine haemoglobin as substrate. Western blot analysis revealed that anti-sea bream PG-I and PG-II polyclonal antibodies cross-reacted with all three PGs of European eel. On the other hand, anti-sea bream PG-III and PG-IV antibodies cross-reacted with PG-II and PG-III of European eel, while no cross-reaction with PG-I was detected. The kinetic constants of K_m and k_cat of pepsins (P-I, P-II and P-III) for haemoglobin were calculated as 8.8 × 10⁻⁵ M, 23.7 s⁻¹; 9.2 × 10⁻⁵ M, 19.4 s⁻¹ and 7.0 × 10⁻⁵ M, 34.4 s⁻¹, respectively.     

Purification and characterization of sea bream (Sparus latus Houttuyn) pepsinogens and pepsins

Four pepsinogens (PG-I, PG-II, PG-III, and PG-IV) were identified in sea bream (Sparus latus Houttuyn) stomach and were purified to homogeneity by 20–60% ammonium sulfate fractionation and several chromatographies. The molecular weights of the four purified PGs were 36, 32, 32, and 34 kDa, respectively. All the pepsinogens converted into pepsins within a few minutes under pH 2.0. The molecular weights of all the four pepsins were approximately 30 kDa as determined by SDS–PAGE. Optimum pHs of the four enzymes were 3.0–3.5, and optimum temperatures were 45–50 °C. Western blot analysis revealed that anti-PG-II antibody cross-reacted with all the four pepsins while scarcely any immunological cross-reaction existed between anti-PG-I antibody and other pepsins. From the Lineweaver–Burk double reciprocal plots, the K_ms of pepsins (P-I, P-II, P-III, and P-IV) for hemoglobin were calculated as 8.7 × 10⁻⁸ M, 1.0 × 10⁻⁷ M, 8.6 × 10⁻⁸ M, and 7.3 × 10⁻⁸ M, respectively.     

Characterisation of a thermostable family 42 β-galactosidase from Thermotoga maritima

A β-galactosidase gene (TM_0310) of Thermotoga maritima MSB8 was expressed in Escherichia coli. The recombinant β-galactosidase (designated BgalB) was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. BgalB belongs to the glycoside hydrolase family 42. Its molecular mass was estimated to be 78 kDa and 76 kDa by SDS–PAGE and gel filtration, respectively. The enzyme was optimum at pH 5.5, and it was quite stable over the pH range 5.0–11.4 at 70 °C. It was optimally active at 80 °C and was stable up to 75 °C. Besides, BgalB exhibited broad substrate specificity with a preference for p-nitrophenyl-β-galactopyranoside (pNPGal). K_m values of the purified enzyme for pNPGal, o-nitrophenyl-β-galactopyranoside (oNPGal) and pNP-β-fucopyranoside were 2.7 mM, 12.5 mM and 1.4 mM, respectively. These properties make this enzyme an interesting candidate for biotechnological applications. This is the first report of the family 42 β-galactosidases from T. maritima.     

Purification and characterisation of exo- and endo-inulinase from Aspergillus ficuum JNSP5-06

Three exoinulinases (Exo-I, Exo-II, and Exo-III) and two endoinulinases (Endo-I and Endo-II) were purified from the culture broth of Aspergillus ficuum JNSP5-06 by ammonium sulphate precipitation, DEAE-cellulose column chromatography, Sepharose CL-6B column chromatography and preparative electrophoresis. The molecular weights of Exo-I, Exo-II, Exo-III, Endo-I, and Endo-II were determined to be 70 kDa, 40 kDa, 46 kDa, 34 kDa, and 31 kDa, respectively. Using inulin as the substrate, their K_m values were 43.1 mg/ml, 31.5 mg/ml, 25.3 mg/ml, 14.8 mg/ml, and 25.6 mg/ml, respectively. These five inulinases were stable below 50 °C with optimum activity at 45 °C, and were stable at a pH range of 4–8 with an optimum pH at 4.5 for exoinulinase and at 5.0 for endoinulinase. The inulinase activity was completely inhibited by Ag⁺ and strongly inhibited by Fe²⁺ and Al³⁺, whereas K⁺, Ca²⁺, Li²⁺, EDTA and urea had no significant influence on the inulinase activity.     

Helvellisin,a novel alkaline protease from the wild ascomycete mushroom Helvella lacunosa

A 33.5-kDa serine protease designated as helvellisin was isolated from dried fruiting bodies of the wild ascomycete mushroom Helvella lacunosa. It was purified by using a procedure which entailed ion exchange chromatography on DEAE-cellulose, CM-Sepharose, Q-Sepharose, and FPLC-gel filtration on Superdex 75. The protease was characterized by unique N-terminal amino acid sequence, thermostability and pH stability. The protease exhibited a pH optimum of 11.0 and a temperature optimum of 65 °C, with about 40% activity remaining at 87 °C and pH 5 and 13. Helvellisin demonstrated a protease activity of 14 600 U/mg toward casein. The K_m of the purified protease for casein was 3.81 mg/ml at pH 11.0 and 37 °C. The V_max was 5.35 × 10^− 2 mg ml^− 1 min^− 1. It was adversely affected by phenylmethylsulfonyl fluoride, suggesting that it is serine protease. The activity of the protease was enhanced by Mg²⁺, Fe²⁺ and Mn²⁺, but was curtailed by Cu²⁺, Hg²⁺ and Fe³⁺. It was devoid of antifungal and ribonuclease activities.     

Determination of minor carbohydrates in carrot (Daucus carota L.) by GC–MS

Minor carbohydrates present in carrot (Daucus carota L.) have been studied by GC–MS analysis of their trimethylsilyl derivatives because of their remarkable role in a variety of biological functions. Scyllo-inositol and sedoheptulose (d-altro-2-heptulose), identified for the first time in this paper were present in all the carrots analysed in concentrations ranging 1.5–5.8 and 1.4–24.6 mg g⁻¹ dried weight, respectively. Other minor carbohydrates detected in carrot were myo- inositol (2.2–9.8 mg g⁻¹) and mannitol (traces-1.3 mg g⁻¹). Whereas small amounts (close to 2 mg g⁻¹) of scyllo-inositol were experimentally determined in other vegetables from the Apiaceae family (parsley, coriander and fennel), sedoheptulose was only detected at trace levels.     

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5 kDa according to SDS–PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40 °C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe²⁺ > Hg²⁺ > Mn²⁺ > K⁺ > Mg²⁺ > Li⁺ > Cu²⁺). Trypsin activity decreased continuously as NaCl concentration increased (0–30%). K_m and k_cat values were 0.13 mM and 1.46 s⁻¹, respectively. Results suggest the enzyme have a potential application where room processing temperatures (25–35 °C) or high salt (30%) concentration are needed, such as in fish sauce production.     

Purification and characterisation of trypsins from the pyloric caeca of mandarin fish (Siniperca chuatsi)

Two trypsins of anionic form (trypsin A) and cationic form (trypsin B) from the pyloric caeca of mandarin fish (Siniperca chuatsi) were highly purified by a series of chromatographies, including DEAE-Sephacel, Sephacryl S-200 HR, Q-Sepharose or SP-Sepharose. Purified trypsins revealed a single band on native-PAGE. The molecular weights of trypsin A and B were 21 kDa and 21.5 kDa, respectively, as estimated by SDS–PAGE, both under reducing and non-reducing conditions. Zymography analysis showed that both trypsins were active in degrading casein. Trypsin A and B exhibited maximal activity at 35 °C and 40 °C, respectively, and shared the same optimal pH of 8.5, using Boc-Phe-Ser-Arg-MCA as substrate. The two trypsins were stable up to 45 °C and in the pH range from 4.5 to 11.0. Trypsin inhibitors are effective on these two enzymes and their susceptibilities were similar. Both trypsins were activated by metal ions such as Ca²⁺ and Mg²⁺ and inactivated by Fe²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Al³⁺, Ba²⁺ and Co²⁺ to different degrees. Apparent K_m values of trypsin A and B were 2.18 μM and 1.88 μM, and K_cat values were 81.6 S⁻¹ and 111.3 S⁻¹ for Boc-Phe-Ser-Arg-MCA, respectively. Immunoblotting analysis using anti-common carp trypsin A positively cross-reacted with the two enzymes, suggesting their similarity. The N-terminal amino acid sequence of trypsin B was determined as IVGGYECEAH, which is highly homologous with trypsins from other species of fish.     

Expression of recombinant Thermomonospora fusca xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it

The mature peptide of Thermomonospora fusca xylanase A (TfxA) was successfully expressed in Pichia pastoris under the control of AOX1 promoter. The activity of recombinant T. fusca xylanase A (reTfxA) in culture supernatant was 117.3 ± 2.4 U/mg, which is 3 times higher than that of the native TfxA. The optimal temperature and pH for reTfxA were 60 °C and 6.0, respectively. When treated at 70 °C and pH 6.0 for 2 min, the residual activity of the reTfxA was 70%. The reTfxA was very stable over a wide pH range (5.0–9.0). After incubation over pH 5.0–9.0 at 25 °C for 1 h, all the residual activity of reTfxA was over 80%. The K_m and k_cat values for reTfxA were 2.45 mg/ml and 139 s⁻¹, respectively. HPLC analysis revealed that xylobiose (X2) was the main hydrolysis product released from birchwood xylan and wheat bran insoluble xylan by reTfxA. Hydrolysis results of xylooligosaccharides showed that reTfxA was an endo-acting xylanase and xylobiose, xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolysed. This is the first report on the expression of reTfxA in yeast and on the determining and quantifying of the hydrolysis products released from xylans and xylooligosaccharides by reTfxA.     

Characterisation of a thermostable family 42 β-galactosidase (BgalC) family from Thermotoga maritima showing efficient lactose hydrolysis

A β-galactosidase gene (TM_1195) of Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant β-galactosidase (BgalC), belonging to glycosyl hydrolase (GH) family 42, was purified to homogeneity with 23.4-fold purification and a recovery of 36.6%. Its molecular mass was estimated to be 78 kDa by SDS–PAGE. BgalC exhibited maximum activity at an optimal pH of 5.5 and an optimum temperature of 80 °C. The enzyme displayed important properties, such as stability over a broad pH range of 5.0–9.0 and thermostability up to 75 °C. K_m values of BgalC for p-nitrophenyl-β-galactopyranoside (pNPGal), o-nitrophenyl-β-galactopyranoside (oNPGal) and lactose were 1.21, 7.31 and 6.5 mM, respectively. BgalC was efficient in complete removal of lactose from milk. BgalC is significantly one of the few β-galactosidases from family 42 displaying significant hydrolysis of lactose. These properties make BgalC an ideal candidate for commercial use, in the production of lactose-free milk.     

Mate (Ilex paraguariensis St. Hilaire) saponins induce caspase-3-dependent apoptosis in human colon cancer cells in vitro

Saponins are naturally occurring metabolites associated with several health benefits. The objective was to quantify and purify saponins from mate dry leaves, and to assess their anti inflammatory and apoptotic mechanisms in human colon cancer cells in vitro. Matesaponins were extracted with methanol from dry leaves, partially purified and quantified. Leaves contained 10–15 mg/g dry weight total saponins, predominantly matesaponins 1 and 2. HPLC and LC/ESI-MS-MS identified saponins in six preparative chromatographic fractions (A, B, C, D, E, and F). Major matesaponins were identified as 1 [M–H]⁻ = 911 and 2 [M–H]⁻ = 1057, with trace amounts of 3 [M–H]⁻ = 1073, 4 [M–H]⁻ = 1219, and 5 [M–H]⁻ = 1383. Fractions D, E, and F significantly inhibited iNOS (IC₃₅ = 36.3, 29.5, 43.7 μM), PGE₂ (IC₃₅ = 23.1, 22.3, 11.7 μM) and COX-2 (IC₃₅ = 45.7, 32.4, 17.0 μM). Fraction F reduced nuclear translocation of nuclear factor-κB subunits p50 (49.8%) and p65 (49.0%) and induced apoptosis through suppression of Bcl-2 and increased Bax protein expressions and activated caspase-3 activity. Saponins in leaves of mate prevent inflammation and colon cancer in vitro.     

Purification and characterization of olive (Olea europaea L.) peroxidase – Evidence for the occurrence of a pectin binding peroxidase

Peroxidase from olive fruit (Olea europaea L., cv Douro) in a black ripening stage was purified to electrophoretic homogeneity, resulting in four cationic and four anionic fractions. The anionic fractions accounted for 92% of recovered activity and showed molecular masses of 18–20 kDa. The anionic fraction PODa4, the predominant fraction that comprised about 70% of total recovered activity, showed an isoelectric point of 4.4 and optimum pH and temperature of, respectively, 7.0 and 34.7 °C, and apparent K_m values of 41.0 and 0.53 mM, for phenol and H₂O₂, respectively. From the activity-temperature profile, the denaturation temperature and the changes in enthalpy and heat capacity for unfolding of PODa4 were estimated as being, respectively, 36.5 °C, 411.2 and −13.6 kJ mol⁻¹ K⁻¹. The activation energy for phenol oxidation by PODa4 was 99.1 kJ mol⁻¹, corresponding to a calculated temperature coefficient (Q₁₀) of 4. The arabinose (39 mol%) and galacturonic acid (38 mol%) content of the carbohydrate moiety indicated the existence of pectic material in the purified PODa4 fraction. Co-migration of the carbohydrate with the protein band in the isoelectric focusing electrophoresis, points to PODa4 fraction as being a pectin type binding peroxidase.     

Immobilization of naringinase from Aspergillus niger CECT 2088 in poly(vinyl alcohol) cryogels for the debittering of juices

Naringinase, induced from Aspergillus niger CECT 2088 cultures, was immobilized into a polymeric matrix consisting of poly(vinyl alcohol) (PVA) hydrogel, cryostructured in liquid nitrogen, to obtain biocatalytically active beads. The effects of matrix concentration, enzyme load and pH on immobilization efficiency were studied. Between 95% and 108% of the added naringinase was actively entrapped in PVA cryogel, depending on the conditions of immobilization used. The optimal conditions were: 8% (w/v) PVA at pH 7 and 1.6–3.7 U ml⁻¹ of enzyme load. The pH/activity profiles revealed no change in terms of shape or optimum pH (4.5) upon immobilization of naringinase. However, the optimum temperature was shifted from 60 °C to 70 °C and the activation energy of reaction, E_a, was decreased from 8.09 kJ mol⁻¹ to 6.36 kJ mol⁻¹ by immobilization. The entrapped naringinase could be reused through six cycles (runs of 24 h at 20 °C), retaining 36% efficacy for the hydrolysis of naringin in simulated juice.