Partial Purification and Characterization of Extracellular Fructofuranosidase with Transfructosylating Activity from <Emphasis Type="Italic">Candida</Emphasis> sp.

The present work was carried out with the aim to investigate some properties of an extracellular fructofuranosidase enzyme, with high transfructosylating activity, from Candida sp. LEB-I3 (Laboratory of Bioprocess Engineering, Unicamp, Brazil). The enzyme was produced through fermentation, and after cell separation from the fermented medium, the enzyme was concentrated by ethanol precipitation and than purified by anion exchange chromatography. The enzyme exhibited both fructofuranosidase (FA) and fructosyltransferase (FTA) activities on a low and high sucrose concentration. With sucrose as the substrate, the data fitted the Michaellis–Menten model for FA, showing rather a substrate inhibitory shape for fructosyltransferase activity. The K _m and v _max values were shown to be 13.4 g L⁻¹ and 21.0 μmol mL⁻¹ min⁻¹ and 25.5 g L⁻¹ and 52.5 μmol mL⁻¹ min⁻¹ for FA and FTA activities, respectively. FTA presented an inhibitory factor K _i of 729.8 g L⁻¹. The optimum conditions for FA activity were found to be pH 3.25–3.5 and temperatures around 69 °C, while for FTA, the optimum condition were 65 °C (±2 °C) and pH 4.00 (±0.25). Both activities were very stable at temperatures below 60 °C, while for FA, the best stability occurred at pH 5.0 and for FTA at pH  4.5–5.0. Despite the strong fructofuranosidase activity, causing hydrolysis of the fructooligosaccharides (FOS), the high transfructosilating activity allows a high FOS production from sucrose (44%).     

Production and Application of Xylanase from Penicillium sp. Utilizing Coffee By-products

The lignocellulosic coffee by-products such as coffee pulp, coffee cherry husk, silver skin, and spent coffee were evaluated for their efficacy as a sole carbon sources for the production of xylanase in solid-state fermentation using Penicillium sp. CFR 303. Among the residues, coffee cherry husk was observed to produce maximum xylanase activity of 9,475 U/g. The process parameters such as moisture (50%), pH (5.0), temperature (30 °C), particle size (1.5 mm), inoculum size (20%), fermentation time (5 days), carbon source (xylose), and nitrogen source (peptone) were optimized and the enzyme activity was in the range of 19,560–20,388 U/g. The enzyme production was further improved to 23,494 U/g with steam as a pre-treatment. The extracellular xylanase from the fungal source was purified to homogeneity from culture supernatant by ammonium sulfate fractionation, DE32-cellulose with a recovery yield of 25.5%. It appeared as a single band on SDS-PAGE gel with a molecular mass of approximately 27 kDa. It had optimum parameters of 50 °C temperature, pH 5.0, K _m 5.6 mg/mL, and V _max 925 μmol mg⁻¹ min⁻¹ with brichwood xylan as a substrate. The crude enzyme hydrolysed lignocellulosic substrate as well as industrial pulp. Production of xylanase utilizing coffee by-products constitutes a renewable resource and is reported for the first time.     

Properties of thermostable extracellular FOS-producing fructofuranosidase from <Emphasis Type="Italic">Cryptococcus</Emphasis> sp.

The present work was devoted to investigations concerning the fructooligosaccharide producing activity of Cryptococcus sp. LEB-V2 (Laboratory of Bioprocess Engineering, Unicamp, Brazil) and its extracellular fructofuranosidase. After cell separation, the enzyme was purified by ethanol precipitation and anion exchange chromatography. The enzyme showed both fructofuranosidase (FA) and fructosyl transferase (FTA) activity. With sucrose as substrate, the data failed to fit the Michaelis–Menten behaviour, showing a substrate inhibitory model. The K _m, K _i and v _max values were shown to be 64 mM, 3 M and 159.6 μmol mL⁻¹ min⁻¹ for FA and 131 mM, 1.6 M and 377.8 μmol mL⁻¹ min⁻¹ for FTA, respectively. The optimum pH and temperature were found to be around 4.0 and 65 °C, while the best stability was achieved at pH 4.5 and temperatures below 60 °C, for both the FA and FTA. Despite the strong FA activity, the high transfructosylating activity allowed for good FOS production from sucrose (35% yield).     

Purification and partial characterization of β-glucanase produced by <Emphasis Type="Italic">Trichoderma viride</Emphasis> TP09 isolated from sewage of beer-making

As an initial investigation to improve the insoluble yeast β-1, 3-glucan solubility, a novel β-glucanase from Trichoderma viride TP09 was purified in the culture supernatant and partially characterized. By 70% saturation ammonium sulfate and chromatography on DEAE-Sepharose CL-6B column, β-glucanase was purified 28.7-fold, with recovery of 45.2% of the initial activity. The molecular weight of this enzyme was estimated to be 54.6 KD by SDS-PAGE. The optimum pH and the optimum temperature for the enzyme were 5.0 and 50 °C, respectively. The enzyme showed high stability within the range of pH 3.0–5.0 and thermostability between 30 and 70 °C. The enzyme activity was inhibited by Fe³⁺, Mg²⁺, Mn²⁺, Cu²⁺, and stimulated by Zn²⁺, Ca²⁺, Fe²⁺. Substrate specificity studies revealed the enzyme to be a β-1, 3–1, 4-glucanase. The β-glucanase showed preference for β-1, 3 linkage and β-1, 4 linkage, but had no activity on α-1, 4 and α-1, 6 linkage. The above results indicated that the enzyme extracted from T. viride TP09 of the beer-making sewage could be used as a potential predominant tool to enhance solubility of the insoluble yeast β-1, 3-glucan. These findings may lead to an enhanced solubility and expedite the progress of application in immunotherapy.     

Sensitive Determination of Decoquinate in Milk by High-Performance Liquid Chromatographic Coupled to Laser-Induced Fluorescence Detection

A new and sensitive method based on high performance liquid chromatography with laser-induced fluorescence detection has been developed for the determination of decoquinate in milk. Laser source was obtained with a He–Cd laser using a continuous excitation wavelength at 325 nm. Decoquinate exhibits moderate fluorescence, but it is increased using Ca(NO₃)₂ in the mobile phase. The chromatographic separation was performed on a Luna C₁₈ 5-mm reversed phase column, which solves the broadening of peaks and peak tailing compared with other columns tested. The mobile phase, delivered at 1 ml min⁻¹, consisted of methanol–calcium nitrate (0.025 M)–acetonitrile (83/13/4 v/v/v). Decoquinate was successfully cleaned up from milk by solid-phase extraction using C₁₈ cartridges. The method was found to be linear between 0.16 and 16.33 ng ml⁻¹. The results of recovery studies were found to be satisfactory; an average recovery rate of 88.7% was obtained. The LOQ of decoquinate in milk was 0.16 ng ml⁻¹. The intraday relative standard deviation (RSD) was 4%, and interday assay gave an RSD of 4.4%.     

Antioxidant peptides isolated from sea cucumber Stichopus Japonicus

The sea cucumber Stichopus japonicus protein was hydrolyzed by pepsin, trypsin, papain, acid protease and neutral protease, respectively, to get five kinds of peptide fractions: pepsin peptides (PP), trypsin peptides (TP), acid protease peptides (AP), neutral protease peptides (NP) and papain peptide (PAP). Antioxidative activities of all peptide fractions were evaluated by hydroxyl radical– (·OH) and Superoxide anion (O₂ ^{· −} )–scavenging activity. Trypsin peptide (TP) exhibited the highest antioxidative activity compared to other peptide fractions. In considering scavenging effects on hydroxyl radicals (·OH) and Superoxide anions (O₂ ^{· −} ), TP was employed for isolation, purification and identification of antioxidant peptide. To purify and characterize antioxidative peptide, two steps gel filtration, one-step ion-exchange column chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) were used. The purified antioxidative peptide TP2b-1 was a novel peptide and was sequenced as GPEPTGPTGAPQWLR, in which the low molecular weight and some amino acid constituents played important role in the radical-scavenging effects according reports. The IC₅₀ values of TP2b-1 were 138.9 μM on ·OH and 353.9 μM on O₂ ^{· −} .     

Purification and characterization of cathepsin B from the gut of the sea cucumber (<Emphasis Type="Italic">Stichopus japonicas</Emphasis>)

Cathepsin B from the gut of sea cucumber (Stichopus japonicas) was purified 81-fold with a 3% recovery by ammonium sulfate fractionation and a series chromatography on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-Gel 3000 SWxl. The purified protein appeared as a single band on Native-PAGE but showed 2 bands of 23 and 26 kDa on SDS-PAGE. The optimum activity was found at pH 5.5 and 45°C. The enzyme was stable at pH 4.5–6.0 and the thermal stability was up to 50oC. The enzyme was strongly inhibited by E-64, iodoacetic acid, and antipain, demonstrating it is a cysteine protease containing sulfhydryl groups. Cu²⁺, Ni²⁺, and Zn²⁺ could strongly inhibit the enzyme activity. The amino acid sequences of the purified enzyme were acquired by mass spectrometer, which did not show any homology with previously described cathepsins, suggesting it may be a novel member.     

Use of Metals and Anion Species with Chemometrics Tools for Classification of Unprocessed and Processed Coconut Waters

Coconut water is a natural isotonic, nutritive, and low-caloric drink. Preservation process is necessary to increase its shelf life outside the fruit and to improve commercialization. However, the influence of the conservation processes, antioxidant addition, maturation time, and soil where coconut is cultivated on the chemical composition of coconut water has had few arguments and studies. For these reasons, an evaluation of coconut waters (unprocessed and processed) was carried out using Ca, Cu, Fe, K, Mg, Mn, Na, Zn, chloride, sulfate, phosphate, malate, and ascorbate concentrations and chemometric tools. The quantitative determinations were performed by electrothermal atomic absorption spectrometry, inductively coupled plasma optical emission spectrometry, and capillary electrophoresis. The results showed that Ca, K, and Zn concentrations did not present significant alterations between the samples. The ranges of Cu, Fe, Mg, Mn, PO₄³⁻, and SO₄²⁻ concentrations were as follows: Cu (3.1–120 μg L⁻¹), Fe (60–330 μg L⁻¹), Mg (48–123 mg L⁻¹), Mn (0.4–4.0 mg L⁻¹), PO₄³⁻ (55–212 mg L⁻¹), and SO₄²⁻ (19–136 mg L⁻¹). The principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to differentiate unprocessed and processed samples. Multivariated analysis (PCA and HCA) were compared through one-way analysis of variance with Tukey–Kramer multiple comparisons test, and p values less than 0.05 were considered to be significant.     

Evaluation of Nano Zinc (ZnO) for Surface Enhancement of ATR–FTIR Spectra of Butter and Spread

The effect of nano zinc (ZnO) particles in surface enhancement of attenuated total reflection–Fourier transform mid-infrared spectroscopy (ATR–FTIR) has been studied in butter and spread. Due to the health implications associated with consumption of trans fats, the studies also included the determination of band corresponding to trans fats of butter/spread in the nano-zinc-treated samples. The FTIR spectra of nano-zinc-treated butter showed enhancement of bands related to (―C―O, ―CH₂―) at wave number 1,238 cm⁻¹ and (O―C―C) band of esters at wave number 1,100 cm⁻¹. Shifting in wave number to 1,150 cm⁻¹ and reduction in its peak intensity was observed with the band corresponding to 1,162 cm⁻¹ (―C―O stretch). Reduction in peak intensity of the bands at about 2,915 and 2,850 cm⁻¹ (C―H groups) was also observed. In the case of spread, nano zinc reduced the peak intensities of FTIR bands at 2,915 cm⁻¹, 2,850 cm⁻¹ (―C―H― (CH₂) stretches), 1,746 cm⁻¹ (―C═O ester), 1,465 cm⁻¹ (―C―H (CH₂, CH₃)), 1,375 cm⁻¹ (―C―H (CH₃)) and 1,156 cm⁻¹ (C―O, ―CH₂). Trans fats band corresponding to ═C―H stretch (trans bonds, 966 cm⁻¹), was observed in pure butter and spread. Trans fatty acids in butter and spread were quantified with the aid of calibration/validation standards using trielaidin and triolein and by using partial least squares regression analysis. However, the band at 966 cm⁻¹ was hindered in Zn-treated butter and reduced in Zn-treated spread.     

Purification and characterization of Aspergillus oryzae LK-101 salt-tolerant acid protease isolated from soybean paste

A novel salt-tolerant acid protease was produced from Aspergillus oryzae LK-101 (AOLK-101). The AOLK-101 protease was purified to homogeneity by ammonium sulfate precipitation, DEAE-Sephadex A-50 and Sephadex G-100 chromatographies in order. The specific activity and the purification ratio of the purified protease were 2,301 unit/mg and 11.6 fold, respectively, with 25 kDa of molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrpphoresis (SDS-PAGE). Its optimal pH and temperature were pH 6.5 and 50°C, respectively. This protease was relatively stable at pH 4.5–7.5, below 40°C, and up to 10% salt concentration. The protease was moderately inhibited by Ag²⁺ and Zn²⁺, and strongly by ethylenediamide tetraacetic acid (EDTA) and phenylmethysulfonyl fluoride (PMSF), but activated by Cu²⁺ and Mn²⁺. Therefore, the AOLK-101 protease was a serine protease based on the influence of metal ions and inhibitors. K _m , V _max , k _cat , and k _cat /K _m values of AOLK-101 protease for hammastein milk casein were 1.04 mg/mL, 124.84 unit/L, 163.5/sec, and 3.9×10⁶/m·sec, respectively.     

Sorption Isotherms of Barnyard Millet Grain and Kernel

The moisture sorption isotherms of grain and kernel of barnyard millet (Echinochloa frumentacea) were determined at 20, 30, 40, and 50 °C. A gravimetric static method was used under 0.112–0.964 water activity (a _w) range for the determination of sorption isotherms. The models were compared using the coefficient of determination (r ²), reduced chi-square (χ ²) values, and on the basis of residual plots. In grain, modified Chung–Pfost (r ² > 0.99; χ ² < 0.7) and modified Oswin (r ² > 0.99; χ ² < 0.55) models were found suitable for predicting the M _e –a _w relationship for adsorption and desorption, respectively. Modified Henderson model was found to give the best fit (r ² > 0.99 and χ ² < 0.55) for describing the adsorption and desorption of the kernel. The isosteric heat, calculated using Clausius–Clapeyron equation, was varied between 46.76 and 61.71 kJ g⁻¹ mol⁻¹ at moisture levels 7–21% (d.b.) for grain and 47.11–63.52 kJ g⁻¹ mol⁻¹ at moisture level between 4% and 20% (d.b.) for kernel. The monolayer moisture content values ranged from 4.3% to 6% d.b. in the case of adsorption of barnyard millet grain and 5.2–6.6% d.b. in the case of desorption at the temperature ranges of 50–20 °C. The monolayer moisture values of barnyard millet kernel ranged from 4.4% to 6.67% d.b. in adsorption and 4.6% to 7.3% d.b. in desorption in the temperature ranges of 50–20 °C.     

Benzyloxybenzaldehydethiosemicarbazone: Extractive Spectrophotometric Reagent for the Determination of Cu(II) in Food and Water Samples

Benzyloxybenzaldehydethiosemicarbazone (BBTSC) was prepared and developed a new method for the simple, highly selective, and extractive spectrophotometric determination of copper(II) with BBTSC at wavelength 370 nm. The metal ion formed a bluish green colored complex with BBTSC in acetate buffer of pH 5.0, which was easily extractable into n-butanol with 1:1(metal/ligand) composition. The method obeys Beer’s law in the range of 0.5–5.2 ppm. The molar absorptivity and Sandell’s sensitivity were found to be 1.5 × 10⁴ l mol⁻¹ cm⁻¹ and 0.00412 g cm⁻², respectively. The correlation coefficient of the Cu(II)–BBTSC complex was 0.998, which indicated an excellent linearity between the two variables. The repeatability of the method was checked by finding the relative standard deviation (RSD; n = 10), which was 0.377% and its detection limit 0.0204 μg ml⁻¹. The interfering effect of various cations and anions were also studied. The proposed method was successfully applied to the determination of copper(II) in food and water samples. Comparing the results with those obtained using an atomic absorption spectrophotometer tested the validity of the method.     

Supercritical CO<Subscript>2</Subscript> extraction of <Emphasis Type="Italic">Alkanna</Emphasis> species and investigating functional characteristics of alkannin-enriched yoghurt during storage

Alkannin is a potent pharmaceutical substance with a wide spectrum of biological activities. In the scope of this study, supercritical CO₂ extraction and sonication with hexane were applied to various Alkanna species, which were then subjected to hydrolysis. Total alkannins were quantified by HPLC/DAD and incorporated into yoghurt. Viscosities, pH values and microbial analyses were reported at 7 days of intervals for 21 days of storage. A. tinctoria possessed the highest amounts of alkannins and total phenols (686.3 mg GAE/g extract). The results revealed no significant changes in pH values (4.1–4.0), viable counts of Streptococcus salivarius ssp. thermophilus (80–150 × 10⁶ cfu g⁻¹) and slightly lower viscosities of enriched yoghurts (8,250–6,750 cPs) compared with the control (4.15–4.0; 110–105 × 10⁶ cfu g⁻¹; 12,600–11,310 cPs) during storage. However, viable counts of Lactobacillus delbrueckii ssp. bulgaricus of enriched yoghurts (87 × 10³ cfu g⁻¹) were much better than the control (191 × 10³ cfu g⁻¹), indicating a significant decrease in post acidification and generation of bitter peptides. Among the species investigated, A. tinctoria is the most promising source, obtained at higher yields via supercritical fluid extraction technology as a green alternative to solvent extraction and thus can be utilized at industrial scale in order to develop yoghurt products with improved health benefits.     

Electrochemical Determination of Ascorbigen in Sauerkrauts

Ascorbigen (ABG), 2-C-(indol-3-yl)methyl-α-l-xylo-hex-3-ulofuranosono-1,4-lactone, was synthesised and its structure and purity was confirmed by means of ¹H NMR and ¹³ C NMR spectra and HPLC-PDA, respectively. The electrochemical behaviour of ABG was studied by cyclic voltammetry (CV) method for pH within the range of 3.0–7.0 and further characterisation was performed by differential pulse voltammetry (DPV) at pH 5.0. Voltammetric studies of ABG at glassy carbon electrode showed one irreversible oxidation peak (centred at E _p = 0.950 V for pH 5.0). The anodic peak current potential related to the irreversible oxidation of ABG was shifted towards more positive potentials with decreasing pH values. The linear response between concentration of ABG within the range of 0.08–0.75 mM and recorded current was observed. The DPV method was shown to be more sensitive when compared to the CV method. ABG showed a significant reducing activity provided by CV, whilst the antioxidant activity of ABG against O₂^−• was negligible. Electrochemical behaviour of ABG standard was applied successfully for the quantification of natural ABG in sauerkrauts by DPV and HPLC-CoulArray methods. Based on the developed analytical methods, ABG content increased during natural fermentation of cabbage at 0.5% and 1.5% NaCl levels, and results were comparable with those found in the literature.     

Purification and characterization of hydroperoxide lyase from amaranth tricolor (Amaranthus mangostanus L.) leaves

Hydroperoxide lyase (HPL) was extracted from amaranth tricolor leaves using Triton X-100, and purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography and hydroxyapatite chromatography. The purified HPL preparation consisted of a single band and spot with a molecular mass of about 55 kDa as shown in SDS–PAGE and 2-D PAGE, respectively; the isoelectric point was found to be about 5.4. The maximum activity of the enzyme was observed at pH 6.0 and 25 °C, respectively. The HPL showed higher activity against 13-hydroperoxy-linolenic acid compared to 13-hydroperoxy-linoleic acid. K _m value for 13-hydroperoxy-linolenic acid was 62.7 μM, and the corresponding V _max was 178.5 μM min⁻¹. The activity of HPL was significantly inhibited by nordihydroguaiaretic acid, HgCl₂ and 2(E)-hexenal but not by EDTA and hexanal.     

Comparison Between Systems for Synthesis of Fructooligosaccharides from Sucrose Using Free Inulinase from Kluyveromyces marxianus NRRL Y-7571

This work is focused on the synthesis of the fructooligosaccharides (FOS) from sucrose using free inulinase from Kluyveromyces marxianus NRRL Y-7571 in aqueous and aqueous–organic systems. The most significant variables for the aqueous–organic system were identified using a fractional factorial design. The evaluated variables were the temperature, pH, sucrose concentration, inulinase activity, aqueous/organic ratio, and the polyethylene glycol concentration. The use of sequential experimental design methodology was shown to be very useful in the optimization of the FOS synthesis by inulinase either in aqueous or aqueous–organic systems. For the aqueous–organic system, the maximum Y _FOS reached was 16.7 ± 1.1 wt.% with the following operational conditions: temperature of 40 °C, enzyme activity of 4 U mL⁻¹, organic solvent/total system ratio of 25/100, pH of 6.0, and sucrose concentration of 55%. In the aqueous system, the maximum conversion obtained was 12.8 ± 1.0 wt.% under the following conditions: 40 °C, pH 5.0, 55% sucrose, and inulinase activity 4 U mL⁻¹.     

Purification and properties of a heat-stable enzyme of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Rm12 from raw milk

Pseudomonas fluorescens Rm12 is a kind of Psychrotrophic bacteria growing in cold raw milk. It produced an extracellular heat resistant protease with an estimated molecular weight of 45 kDa by size exclusion chromatography and SDS-PAGE under both reducing and non-reducing conditions. The enzyme, designated Ht13, was purified to electrophoretic homogeneity from the culture supernatant by sequentially using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography and size exclusion chromatography. The specific activity of the enzyme increased 115.5-folds. The optimum pH value and temperature of Ht13 were 7.5 and 40 °C, respectively. Based on its biochemical characteristics, Ht13 can be included in the group of metalloproteases, which was inhibited by 1, 10-phenanthroline and EDTA but not by pepstatin A, chymostatin, STI, E-64, BBI, PMSF and pAPMSF. Mn²⁺ has positive effect on activity and can increased the heat resistance capability, while Ca²⁺ had a negligible effect. For the hydrolysis of azocasein, the Km was 0.012 mg mL⁻¹. The enzyme showed typical heat-stable behavior. After treatment of 160 °C 20 s, the residual activity was 9%. The half-life of the enzyme at 160 °C in buffer with Mn²⁺ was approximately 12 s. Among several main milk proteins, Ht13 can cleave α_s-casein, β-casein and κ-casein. The sequence of 1st–16th amino acids of N-terminal was MSKVKDKAIVSAAQAS, which was same as those proteases excreted from some other P. fluorescens. However, their molecular weights, the activation ion and amino acid composition were different, suggesting Ht13 from P. fluorescens Rm12 is a novel protease.     

Growth kinetics and predictive model of Aeromonas hydrophila in a broth-based system

This study developed a predictive model of Aermonas hydrophila in tryptic soy broth for any combination of temperatures (5 to 40°C), pH (6 to 8), and NaCl (0 to 5%) using a response surface model. A. hydrophila tended to grow within a pH range of 6.0 to 8.0 and could not tolerate NaCl up to 5.0%. The interaction of pH and NaCl did not affect the specific growth rates (SGR). The primary model to obtain the SGR showed a good fit (R²≥0.980). A secondary model was obtained by non-linear regression analysis and calculated as: SGR= 0.4577+0.0529X₁−0.1641X₂−0.1493X₃−0.0016X₁X₂−0.0001X₁X₃+0.0115X₂X₃0.0006X₁ ²+0.0114X₂ ²+0.0150X₃ ² (X₁=temperature, X₂=pH, X₃=NaCl). The appropriateness of the polynomial model was verified by the mean square error (0.0023), bias factor (0.922), accuracy factor (1.343), and coefficient of determination (0.937). The newly secondary model of SGR for A. hydrophila could be incorporated into the tertiary model to predict the growth of A. hydrophila.     

Properties of <Emphasis Type="Italic">Aspergillus subolivaceus</Emphasis> free and immobilized dextranase

Aspergillus subolivaceus dextranase is immobilized on several carriers by entrapment and covalent binding with cross-linking. Dextranase immobilized on BSA with a cross-linking agent shows the highest activity and considerable immobilization yield (66.7%). The optimum pH of the immobilized enzyme is shifted to pH 6.0 as compared with the free enzyme (pH 5.5). The optimum temperature of the reaction is resulted at 60 °C for both free and immobilized enzyme. Thermal and pH stability are significantly improved by the immobilization process. The calculated K _m of the immobilized dextranase (14.24 mg mL⁻¹) is higher than that of the free dextranase (11.47 mg mL⁻¹), while V _max of the immobilized enzyme (2.80 U μg protein⁻¹) is lower than that of the free dextranase (11.75 U μg protein⁻¹). The immobilized enzyme was able to retain 76% of the initial catalytic activity after 5.0 cycles.     

Purification and biochemical properties of pepsins from the stomach of skipjack tuna (Katsuwonus pelamis)

Pepsins 1 and 2 from the stomach of skipjack tuna (Katsuwonus pelamis) were purified to homogeneity by using a series of chromatographic purification involving DEAE-cellulose, Sephadex G-50 and Sephadex G-75 with increase in purity of 246-fold and 213-fold, respectively. Molecular weights of pepsins 1 and 2 were estimated by SDS–PAGE to be 33.9 and 33.7 kDa, respectively. The N-terminal amino acid sequences of the first 20 amino acids of both isoenzymes were YQDGTEPMTNDADLSYYGVI. The optimal pH and temperature for pepsin 1 were 2.5 and 50 °C, respectively, while pepsin 2 showed optimal activity at pH 2.0 and 45 °C. The activity of two pepsins was stable in the pH range of 2–5 and at temperatures up to 50 °C. The activity of purified pepsins was strongly inhibited by pepstatin A in a dose-dependent manner. SDS and cysteine showed inhibitory effects toward both pepsins. Activity of pepsin 2 was slightly activated by NaCl, but NaCl had no effect on pepsin 1. Pepsins 1 and 2 had high affinity and hydrolytic activity toward hemoglobin with K _m of 54 and 71 μM, respectively. k _cat of pepsins 1 and 2 were 38.1 and 44.3 s⁻¹, respectively. Both pepsins effectively hydrolyzed bovine serum albumin, egg white, natural actomyosin from brownstripe red snapper muscle and acid-solubilized collagen from arabesque greenling skin. Nevertheless, the hydrolytic activity was slightly less than that of pepsin from porcine stomach.