ENZYMATIC CHARACTERISTICS OF TRYPSIN FROM PYLORIC CECA OF SPOTTED MACKEREL (SCOMBER AUSTRALASICUS)

Trypsin was purified from the pyloric ceca of spotted mackerel (Scomber australasicus) by gel filtration on Sephacryl S‐200 and Sephadex G‐50. The purification and yield were 20‐fold and 81%, respectively, as compared to those in the starting crude extract. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular weight of the enzyme was estimated to be 24,000 Da by SDS–PAGE. The trypsin was stable at pH 5–11 for 30 min at 30C, and its maximal activity against N^α‐p‐tosyl‐L‐arginine methyl ester was pH 8.0. Trypsin was heat‐stable up to about 50C for 15 min at pH 8.0. Optimum temperature of the trypsin enzyme was 60C. The enzyme was stabilized by calcium ion. The purified trypsin was strongly inhibited by serine protease inhibitors such as N‐p‐tosyl‐L‐lysine chloromethyl ketone and soybean trypsin inhibitor, suggesting that it is a trypsin‐like serine protease. N‐Terminal amino acid sequence of spotted mackerel trypsin was IVGGYECTAHSQPHQVSLNS.     

Partial purification and characterisation of cysteine protease in wheat germ

BACKGROUND: Proteases have become an essential part of the modern food and feed industry, being incorporated in a large and diversified range of products for human and animal consumption. The objective of this study was to purify and characterise a protease from wheat germ. RESULTS: After purification a single protease of molecular weight 61–63 kDa (determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) was obtained. The purified protease had optimal activity at 50 °C and maintained its activity completely after incubation at 30 °C for 30 min, while over 47% of the activity was lost after incubation at 80 °C for 30 min. The purified protease had optimal activity and maintained maximum stability at pH 5.5, while the activity decreased after incubation for 30 min at other pH values. The protease was inhibited by Mg²⁺, Mn²⁺, Ba²⁺ and iodacetic acid and stimulated by Li⁺, Ca²⁺, Cu²⁺, β‐mercaptoethanol and dithiothreitol, while Zn²⁺, L ‐cysteine and glutathione had no significant effect on its activity. At pH 5.5 the enzyme had a K_m of 0.562 mg mL^?1 with casein as substrate and showed higher affinity to casein than to bovine serum albumin, ovalbumin and gelatin. CONCLUSION: The purified enzyme from wheat germ was identified as a cysteine protease. Copyright © 2011 Society of Chemical Industry     

Purification and Characterization of a Halotolerant Alkaline Serine Protease from Penicillium citrinum YL-1 Isolated from Traditional Chinese Fish Sauce

A halotolerant alkaline serine protease from Penicillium citrinum YL-1 which was isolated from traditional Chinese fish sauce was purified by ammonium sulfate precipitation, dialysis, and DEAE 52-Cellulose column, thereby resulting in a 4.66-fold increase in specific activity (110.68 U/mg). The molecular weight (MW) was estimated to be 32.27 kDa using SDS-PAGE analysis. The protease exhibited optimal activity toward the substrate casein at pH 8.0 at 40°C and was stable at pH 6.0–8.0 and 4–30°C. Activity was inhibited by NaCl and retained at 28.3, 21.4 and 18.1% of the initial activity after incubation for 6 h at 20, 25 and 30% NaCl concentrations, respectively. The enzyme was stimulated by Mn²⁺ and inhibited by K⁺, Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, and Fe³⁺. K_m and V_max of the protease for casein were 1.93 mg/ml and 56.81 μg/(min·ml), respectively. Protease activity was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), which confirmed the serine protease nature of the enzyme. The protease can hydrolyze tilapia protein in the absence or presence of NaCl (5–30%), thus suggesting that this protease is more halotolerant than the protease from other bacteria with high salinity resistance based on the current literature. These properties make the halotolerant alkaline serine protease a suitable candidate enzyme for fish protein hydrolysis during fish sauce fermentation.     

Endotoxin and biogenic amine levels in Atlantic mackerel (Scomber scombrus), sardine (Sardina pilchardus) and Mediterranean hake (Merluccius merluccius) stored at 22 °C

Whole Atlantic mackerel (Scomber scombrus), sardine (Sardina pilchardus) and Mediterranean hake (Merluccius merluccius) from the Croatian Adriatic were stored at 22 °C and changes in histamine, putrescine, tyramine and cadaverine levels were monitored in relation to bacterial endotoxin. After 12 h, histamine levels in sardine were above the legal limit of 50 mg kg^?1, set by the US Food and Drug Administration, and an increase in putrescine content preceded the increase in histamine. After 24 h, histamine contents in mackerel and sardine reached 1090 ± 101 and 577 ± 275 mg kg^?1, respectively, which exceeded the toxic threshold of 500 mg kg^?1. At the same time, the putrescine content was also high in both fish (353–420 mg kg^?1). The time-course of endotoxin production was similar in all fish species stored at 22 °C. A high correlation was found between endotoxin and histamine, and between endotoxin and putrescine in mackerel and sardine. On the other hand, high endotoxin levels in hake, after 24 h, were associated with the low histamine and putrescine content (40–60 mg kg^?1).     

Characterization of a novel protease from Bacillus cereus and evaluation of an eco‐friendly hydrolysis of a brewery byproduct

Proteases and proteolytic enzymes constitute one of the most important groups of enzymes and are attracting worldwide attention in attempts to exploit their physiological and biotechnological applications. In this study, partial purifications and biochemical and antimicrobial characterizations of a protease from Bacillus cereus spp., originally isolated from fermented cabbage, were carried out. The crude extract obtained after purification, involving ammonium sulphate precipitation and dialysis, was designated as a partially purified protease (PPP). The obtained PPP had a specific activity of 0.395–2.539 U/g at 32 °C, with maximum activities for the fractions precipitated at 60 and 80% ammonium sulphate. The PPP activity ranged between 20 and 55 °C, with an optimum temperature at 40 °C. At 60 °C, the PPP retained more than 30% of its activity. The optimum pH for the PPP was achieved at pH 9, indicating the alkaline source of the enzyme. Protease production was specifically dependent on the calcium concentration in the culture medium. Also the robustness of the protease on brewer's spent grain hydrolysis was demonstrated. This suggests a potential eco‐friendly application of the enzyme. Finally, it was found that the PPP inhibited the growth of Escherichia coli O157:H7. This novel property of the PPP liberated by the B. cereus spp. could provide important future benefits to industry. Copyright © 2015 The Institute of Brewing & Distilling     

The effect of the high pressure homogenisation on the activity and stability of a commercial neutral protease from Bacillus subtilis

The activity of a commercial neutral protease from Bacillus subtilis after high pressure homogenisation (HPH) was investigated. The enzyme was processed up to 2000 bar, and the residual activity was measured from 20 to 70 °C during refrigerated storage. Moreover, the effect of HPH at high temperatures was evaluated. No improvement in the activities at 55–70 °C were observed after HPH, while an increase of approximately 30% in the 20 °C‐activity was reached after 2000 bar processing. Thus, HPH shifted the optimum temperature from 55 to 20 °C. The high temperature homogenisation caused no changes in 55 °C‐activity, but reduced 20 °C‐activity three times. It suggests that HPH modifies the protease configuration, changing enzyme performance (maximum activity condition), as the efficacy of lock‐and‐key mechanism is strictly dependent on enzyme spatial structure. The changes can be permanent or not, depending on homogenisation pressure, inlet temperature and enzyme storage conditions. Therefore, the HPH is a promising method to change protease characteristics.     

Wheat-bug damage in New Zealand wheats: Some properties of a glutenin hydrolysing enzyme in bug-damaged wheat

Some properties of a glutenin hydrolysing enzyme present in bug (Nysius huttoni) damaged wheat (Triticum aestivum) were examined using a modified SDS sedimentation test reported previously. The enzyme appears to be a water-soluble alkaline protease with an activity optimum at pH 9.0. It is relatively heat stable, but the temperature optimum for activity is quite low (35–40°C). The enzyme is not inhibited by EDTA or N-ethylmaleimide, but is inhibited by the metal ions Co²⁺, Mn²⁺ and Fe²⁺.     

Quality Improvement of Mackerel Surimi with NADPH-Sulfite Reductase from Escherichia coli

The application of NADPH‐sulfite reductase, produced from Escherichia coli, on mackerel surimi was investigated. The activity of this reductase was very stable. There was about 70%, 80%, and 90% activity left even after 8–wk storage at 25 °C and 12–wk storage at 4 or ‐30 °C, respectively. Increase in mackerel‐surimi gel properties containing reductase was consistent with the amounts of reactive sulfhydryl groups detected. SDS‐PAGE indicated that NADPH‐sulfite reductase could reconstitute myosin heavy‐chain and increase soluble actomyosin. From the data obtained, E. coli NADPH‐sulfite reductase effectively improved the gel properties of mackerel surimi.     

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.     

Effect of High Hydrostatic Pressure (HHP) Treatment on Physicochemical Properties of Horse Mackerel (Trachurus trachurus)

The basic objective of this study was to determine the effect of high hydrostatic pressure (HHP; 220, 250 and 330 MPa), holding time (5 and 10 min) and temperature (7, 15 and 25 °C) on some quality parameters of horse mackerel such as colour changes, thiobarbituric acid (TBA-i) and trimethylamine nitrogen (TMA-N), free amino acid content. HHP increased L ^* values of horse mackerel. The a ^* and b ^* of treated horse mackerel did not change significantly after HHP applications. After, HHP, TBA-i and TMA values of all HHP-treated horse mackerel samples remained unchanged than those of untreated samples. The results obtained from this study showed that the quality of high pressure treated horse mackerel is best preserved at 250 MPa, 7–15 °C for 5 min, 220 MPa, 15–25 °C for 5 min, 250 MPa, 15 °C for 10 min and 330 MPa, 25 °C for 10 min.     

Effect of lipid oxidation and frozen storage on muscle proteins of Atlantic mackerel (Scomber scombrus)

The effect of storage on the lipids and proteins in Atlantic mackerel stored for up to 24 months at ?20 and ?30 °C was studied. Traditional methods including the peroxide value, thiobarbituric acid‐reactive substances (TBARS) and a reverse phase HPLC method were used to determine the primary and secondary lipid oxidation products. All tests showed an increase in lipid oxidation products with storage time and at a higher storage temperature of ?20 °C compared with samples stored at ?30 °C. Antioxidants had a significant effect (P < 0.01) on the inhibition of lipid oxidation, as shown by the reduction in peroxide value and hydroxides, and malondialdehyde formation. Similarly, deterioration of protein structure and functionality in mackerel stored for 3, 6, 12 and 24 months was greater at ?20 than ?30 °C. ATPase activity in the myosin extract of Atlantic mackerel showed a significant decrease (P < 0.01) with progressive frozen storage. Protein solubility in high salt concentration (0.6 M NaCl) decreased (P < 0.01) during storage at both ?20 and ?30 °C but was greater at ?20 °C. Interestingly, antioxidants BHT, vitamin C and vitamin E protected the proteins against complete loss of ATPase activity and protein solubility to a significant level (P < 0.01) for up to 1 year at ?20 °C compared with samples stored without antioxidants. This study confirms the deleterious effect of lipid oxidation products on protein structure and function in frozen fatty fish. © 2002 Society of Chemical Industry     

PROPERTIES OF AN ALKALINE PROTEASE FROM THE SKELETAL MUSCLE OF ATLANTIC CROAKER1

An alkaline protease was partially purified from the skeletal muscle of Atlantic croaker. The protease is a cytoplasmic enzyme and heat stable. The enzyme preparation was shown to degrade fish actomyosin in vitro between 50–60°C. The enzyme is a sulfhydryl protease and does not require Ca⁺⁺ ions for its activity. Preparations of the enzyme do not hydrolyze TAME, BTEE or denatured hemoglobin. Column chromatographic analyses suggest an apparent molecular weight of 80,000 ± 4,000 and the isoelectric point is 6.0 ± 0.2.     

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.     

Biochemical Characterization of an Extracellular Heat‐Stable Protease from Serratia liquefaciens Isolated from Raw Milk

The protease Ser2 secreted by the psychrotrophic strain Serratia liquefaciens L53, a highly proteolytic strain isolated from Brazilian raw milk was purified and characterized. Using azocasein as substrate, Ser2 exhibited activity in a wide range of pH (5 to 10) and temperature (4 to 60 °C). The optimal activity was detected at pH 8.0 and at a temperature of 37 °C. This protease, still active at 4, 7, and 10 °C, was strongly inhibited by chelating agents and by dithiothreitol, a reducing agent. These results confirmed that Ser2 belongs to the peptidase family M10 and requires Ca²⁺, Zn²⁺, and disulfide bridges for stability. This protease is able to hydrolyze three kinds of casein in the preferential order of κ→ β→ α‐casein. Highly heat‐stable in skimmed, semi‐skimmed, and whole milk at 140°C with D‐values of 2.8, 3.9, and 4.5 min, respectively, Ser2 showed a residual activity between 87 and 100 percent after heat‐treatment of 65 °C for 30 min, 72 °C for 20 s, and 140 °C for 4 s that are commonly used in dairy industries. As the protease AprX that is mainly secreted by Pseudomonas genus, Ser2 could be one of the main causes of UHT milk destabilization during storage.     

Isoelectric fractionation and some properties of a protease from soyabean seeds

A protease, capable of hydrolysing benzoyl DL -arginine p-nitroanilide(BAPA), and L-amino acid β-naphthylamide derivatives, was purified, by isoelectric focusing in the region pH 3–6, from dormant and 6-day germinated soyabean seeds. The enzyme was focused at pH 4·80. The K_m value using BAPA as substrate was found to be 5·03 × 10⁻⁴M . Maximum activity of the enzyme towards BAPA was obtained in the pH 8·2–8–5 region. Slight activation was observed in the presence of 0·05 M concentration of Ca²⁺ and Mg²⁺ ions. The protease lacked caseinolytic activity, and was not inhibited by Kunitz soyabean trypsin inhibitor.     

Characterization of a β-Glucosidase from an Edible Mushroom,Lycoperdon Pyriforme

A β-glucosidase from Lycoperdon pyriforme, a wild edible mushroom, was characterized biochemically. The enzyme showed a maximum activity at pH 4.0 and 50°C when p-nitrophenyl-β-D-glucoside was used as a substrate. K_m and V_max values were calculated as 0.81 mM and 1.62 U/mg protein, respectively. The enzyme activity was conserved about 85% over a broad range of pH (3.0–9.0) at 4°C after 24 h incubation. The activity was fully retained after 60 min incubation at 20–40°C. Na⁺, Li⁺, Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, Ca²⁺, and Cu²⁺ did not affect the enzyme activity and 0.25% sodium dodecylsulfate inhibited the enzyme activity approximately 76%. Ethylenediamine tetra-acetic acid, phenylmethanesulfonylfluoride, and dithiothreitol showed no or a little negative effect on the enzyme activity. The resistance of the enzyme to some metal ions, chemicals, and ethanol along with the pH stability, can make it attractive for future applications in industry.     

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.     

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

Characterization and Cheese-Making Properties of Rennet-Like Enzyme Produced by a Local Algerian Isolate of Aspergillus niger

An ochratoxin free extracellular acid protease was produced by solid state cultivation of Aspergillus niger FFB1. The purified enzyme (48.7 kDa) showed an optimal milk clotting activity at pH 5.5 and 45°C in the presence of 0.01 M CaCl₂. The enzyme was stable at least 24 h at 35°C in the pH range of 5.5–7.0. Thermal denaturation started above 45°C. Fresh cheese manufactured with reconstituted cow milk and the purified enzyme showed similar basic characteristics (pH 4.5, acid taste, white color) as marketed cheeses obtained with calf rennet. This emphasizes the value of exploiting local biological resources for value added food processing in developing countries.     

SEMICONTINUOUS ETHANOL FERMENTATION WITH SHOCHU DISTILLERY WASTE*

An effective utilization system using distillery waste discharged from Japanese traditional shochu factory was developed. Mugi (barley) shochu distillery waste discharged from a novel vacuum distillation procedure (35–40°C) contained a large number of viable yeast (7 × 10⁶ cells/ml), glucoamylase activity (19.7 units/ml), acid protease activity (940 units/ml), and neutral protease activity (420 units/ml). Ethanol fermentation was achieved with a mash composed of glucose as the sola carbon source and mugi shochu distillery waste. After ethanol fermentation was completed the fermented broth was again distilled at 35–40°C in vacuo and the non volatile residue used in the next ethanol fermentation. In this way, semicontinuous ethanol fermentation system of more than 10 cycles was developed. Even in the distillate of the mash of the 8th fermentation cycle, 7.9% of ethanol, 33.0 ppm of ethyl acetate, 28.5 ppm of isobutyl alcohol, and other aromatic compounds were present. A semicontinuous ethanol fermentation system has been developed for shochu distillery waste which conventionally is treated as wastewater.