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.     

THE PURIFICATION OF PROTEASE FROM COWSLIP (PRIMULA VERIS) AND ITS USE IN FOOD PROCESSING

Proteases perform very important functions and are used in different objectives as in vitro and in vivo. In recent years, proteases have been used for clinical, pharmaceutical and industrial applications. Most of these proteases are obtained from animal sources. News of diseases passing over from animals to human (severe acute respiratory syndrome [SARS], mad cow) has created suspicion of contamination in animal food and its product. Therefore, a new vegetal protease source for use in the food industry was studied. Ammonium sulfate fractionation and CM‐sephadex ion exchange chromatography were used in the purification of the enzyme. The purified enzyme has an optimum pH of 7.0 and its optimum temperature was 70C. The V_max and K_m values determined by Lineweaver–Burk graphics were 0.44 mg/mL.min and 40 mM, respectively. The purification degree was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE ). The native molecular weight of the enzyme was 46 kDa using a gel filtration chromatograph. SDS‐PAGE results show that the enzyme has two subunits, which have protease activity at 32 and 14 kD. It was investigated whether the purified and characterized protease enzyme would cause to congeal milk or could produce digestion of chicken and cow meat. The results showed that the protease enzyme can be used for industrial production.     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

Purification and characterisation of an acid protease from the Aspergillus hennebergii HX08 and its potential in traditional fermentation

A protease AP3 from Aspergillus hennebergii HX08 was purified by ammonium‐sulphate precipitation, followed by anion‐exchange chromatography and gel filtration. The molecular weight of acid protease AP3 was 33 kDa (SDS‐PAGE and MALDI‐TOF‐MS). The protease AP3 was identified as an acid protease with MALDI‐TOF/TOF tandem MS. Its optimal temperature and pH were 60°C and 4.0, respectively. Its K _max and V _max were 57.92 mg/mL and 32.57 U/mL, respectively. The enzyme was active over a broad pH and temperature range (pH 3.0–5.0 and 30–60°C), and exhibited high activity and stability in 2–12% (v /v) ethanol solvent. Subsequent studies suggest that the enzyme presents a relatively high substrate affinity to wheat protein (98% of total activity). Its application to solid‐state fermentation of wheat flour with Saccharomyces cerevisiae could increase the hydrolysis degree of wheat protein (28.26%) and amino acid nitrogen concentration of fermented grains (34.21%). Additionally, enhanced S. cerevisiae biomass (37.09%) and alcohol concentration (38.29%) were also observed during the process. Volatile compounds analysis of fermented grains by headspace solid‐phase micro‐extraction and GC‐MS revealed more flavour compounds. These results suggest its potential in food and alcohol industries. Copyright © 2017 The Institute of Brewing & Distilling     

PURIFICATION AND CHARACTERIZATION OF BACILLUS SUBTILIS JM-3 PROTEASE FROM ANCHOVY SAUCE

Bacillus subtilis JM‐3 was isolated from anchovy sauce naturally fermented in an underground cellar at 15 ± 3C for 3 years. The activity of the B. subtilis protease was highest in the 40–60% ammonium sulfate fraction. The yield of the purified protease was 5.3%, and its purification ratio was 35.6 folds. The molecular weight of the B. subtilis protease was 17.1 kDa, and its K_m and V_maxvalues were 1.75 μg/mL and 318 μM 1/min, respectively. The optimal temperature for protease activity was 60C, but optimal stability temperature was 30C. The optimal pH for protease activity and stability was 5.5. Therefore, the B. subtilis JM‐3 protease was classified as an acid protease. The relative activities of the B. subtilis JM‐3 protease were 69, 21 and 1.3% at 10, 20 and 30% NaCl concentrations, respectively. The best substrate for the B. subtilis JM‐3 protease was benzyloxycarbonyl‐glycine‐p‐nitrophenyl ester followed by bovine serum albumin. p‐Toluene‐sulfonyl‐L‐lysine chloromethylketone was the strongest inhibitor followed by soybean trypsin inhibitor, but N‐ethylmaleimide did not inhibit this enzyme. The B. subtilis JM‐3 protease was therefore presumed to be a trypsin‐like serine 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.     

CHARACTERISTICS OF TWO TRYPSIN ISOZYMES FROM THE VISCERA OF JAPANESE ANCHOVY (ENGRAULIS JAPONICA)

Two isozymes of trypsin (TR‐I and TR‐II) were purified from the viscera of Japanese anchovy (Engraulis japonica) by gel filtration and anion‐exchange chromatography. Final enzyme preparations were nearly homogeneous in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and the molecular weights of both enzymes were estimated to be 24,000 Da by SDS‐PAGE. The N‐terminal amino acid sequences of the TR‐I, IVGGYECQAHSQPHTVSLNS, and TR‐II, IVGGYECQPYSQPHQVSLDS, were found. Both TR‐I and TR‐II had maximal activities at around pH 8.0 and 60C for hydrolysis of Nα‐p‐tosyl‐L‐arginine methyl ester hydrochloride. The TR‐I and TR‐II were unstable at above 50C and below pH 5.0 and were stabilized by calcium ion.     

Isolation and characterization of pepsin‐solubilized collagen from the integument of sea cucumber (Stichopus vastus)

BACKGROUND: Sea cucumber (Stichopus vastus) is considered an underutilized resource, since only its stomach and intestines are eaten raw as salad in a few countries and the remaining parts, especially the integument rich in collagen, is discarded. Hence a valuable by‐product having potential nutraceutical and pharmaceutical applications is wasted. In the present investigation, pepsin‐solubilized collagen (PSC) from the integument of S. vastus was isolated, purified and characterized. RESULTS: Sodium dodecyl sulfate–polyacrylamide gel electrophoretic analysis showed that the purified collagen was of type I, consisting of three α1 chains of approximately 122 kDa each. The peptide map of PSC digested by V8 protease was different from that of calf skin type I collagen. Fourier transform infrared spectroscopy revealed that the triple helical structure was well preserved in isolated collagen. The denaturation temperature of PSC was 21.23 °C and showed good gel‐forming capability at pH 6.5 and 300 mmol L^?1 NaCl. CONCLUSION: It is inferred that the collagen isolated from S. vastus integument has potential for use as an alternative to land‐based mammalian collagen in food, nutraceuticals and pharmaceutical industries. © 2012 Society of Chemical Industry     

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.     

Antimicrobial Effect of Bacteriocin KU24 Produced by Lactococcus lactis KU24 against Methicillin‐Resistant Staphylococcus aureus

Bacteriocin KU24 produced by Lactococcus lactis KU24 exhibited an inhibitory effect against methicillin‐resistant Staphylococcus aureus (MRSA). Bacteriocin KU24 was inactivated by protease XIV, showing that it has a proteinaceous nature on S. aureus ATCC 33591. Also, bacteriocin KU24 exhibited a strong heat stability (121 °C for 15 min) and pH stability (pH 3 to 9). The mode of inhibition was determined for S. aureus ATCC 33591 by treatment of 0, 250, and 500 AU/mL of bacteriocin KU24. S. aureus ATCC 33591 was inhibited by added bacteriocin KU24, while control was increased. The cell membranes of S. aureus ATCC 33591 were damaged with treatment of 500 AU/mL of bacteriocin KU24. Also, bacteriocin KU24 inhibited the occurrence of mecA gene, the methicillin resistance gene in S. aureus ATCC 33591. Bacteriocin KU24 was purified by C₁₈ Sep‐Pack column, cation exchange chromatography, and gel filtration chromatography, and molecular mass is approximately 6.5 kDa by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. These results demonstrate that bacteriocin KU24 can be used as an alternative antimicrobial agent for the treatment of infection of MRSA in the food industry.     

Extracellular Protease from Pseudomonas marinoglutinosa: Some Properties and Its Action on Fish Actomyosin

A bacterium isolated from Indian mackerel (Rastrelliger kanagurta) and identified as Pseudomonas marinoglutinosa, was found to produce appreciable amounts of extracellular protease when grown in nutrient medium. This enzyme which degraded several proteins, was found to be most active against mackerel myofibrillar proteins. The optimum temperature and pH range for enzyme activity were 50°C and 7–8 respectively. Treatment of mackerel actomyosin with the protease at 0–2°C for 4 days resulted in degradation of the protein as assessed by release of tyrosine, loss in Mg⁺⁺-dependent ATPase activity and changes in SDS-polyacrylamide gel electrophoretic patterns.     

Purification and properties of cysteine protease from rhizomes of Curcuma longa (Linn.)

BACKGROUND: Turmeric rhizome (Curcuma domestica Linn.) contains proteases and has proteolytic activity. Curcumin from turmeric rhizomes has been used for healing manu ailments, including cancer have been used for healing many ailments, including cancer. The purpose of this study was to purify turmeric protease and to research their biochemical characteristics. RESULTS: Cysteine protease from C. domestica has been purified to homogeneity using acetone precipitation followed by preparatory native polyacrylamide gel electrophoresis (PAGE). This protocol resulted in six fold purification with 28% final recovery. The purified turmeric protease showed a prominent single peak and band on high‐performance liquid chromatography and sodium dodecyl sulfate–PAGE, respectively, and an estimated molecular weight of 43 KDa, and exhibited optimal activity between 37 and 60 °C. The protease activity of the turmeric protease was significantly inhibited by iodoacetic acid. The turmeric protease had higher alanine and glutamate content and cleaved synthetic peptides N‐Cbz‐Ile‐Pro and N‐Cbz‐Phe‐Leu in a time‐dependent manner. Peptide mass fingerprint using matrix‐assisted laser desorption/ionization–time of flight mass spectroscopy revealed peptide matches to proteasome subunit alpha type 3 of Oryza sativa ssp. japonica (Rice). The turmeric protease showed antifungal activity at 10 µg mL^?1 towards pathogens Pythium aphanidermatum, Trichoderma viride and Fusarium sp. CONCLUSION: Cysteine addition significantly activated turmeric protease. The protease inhibition test suggested that turmeric protease belonged to the cysteine type. The biochemical characteristics of turmeric protease described in this paper can provide useful information for potential end uses of turmeric protease for pharmaceutical industry applications such as therapeutics. Copyright © 2009 Society of Chemical Industry     

Comparative study on proteolysis of two species of bigeye snapper,Priacanthus macracanthus and Priacanthus tayenus

Proteolytic activity in muscle from two species of bigeye snapper (Priacanthus macracanthus and Priacanthus tayenus) was studied. Autolysis of mince and washed mince at 50 and 60 °C was compared. Higher degradation of myosin heavy chain was observed in both mince and washed mince from P macracanthus than in those from P tayenus, especially when the incubation time was increased. Autolysis of washed mince from both species was inhibited by soybean trypsin inhibitor, suggesting that myofibril‐associated proteases were serine proteases. When sarcoplasmic proteolytic activity in P macracanthus muscle was studied, two activity peaks with an optimum temperature of 60 °C were observed at pH 6.5 and 8.5. The activities of both peaks were mostly inhibited by soybean trypsin inhibitor, suggesting that the major protease was a serine protease. Major sarcoplasmic proteolytic activity in P macracanthus muscle was found at M_r 62 000 on sodium dodecyl sulphate substrate gel. For P tayenus sarcoplasmic proteolytic activity, two activity peaks with an optimum temperature of 60 °C were found at pH 5.0 and 8.5. The pH 5.0 peak activity was effectively inhibited by pepstatin A, while the pH 8.5 peak activity was inhibited by several inhibitors. The results indicated that various sarcoplasmic proteases were present in P tayenus muscle. The two species contained different sarcoplasmic proteases in terms of composition and activity level. P macracanthus muscle generally had higher sarcoplasmic proteolytic activities than P tayenus muscle. Copyright © 2003 Society of Chemical Industry     

Purification and Characterization of an Endoprotease from Melon Fruit

An endoprotease was purified from melon fruit (Cucumis melo L.) by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography using t-butyloxycarbonyl-Ala-Ala-Pro-Leu p-nitroanilide as a substrate. The molecular weight was estimated as 26,000 and isoelectric point pH 9.5. It preferentially hydrolyzed peptide bonds of the carboxyl terminal sides of Leu, Ala, His, Gin, and Am. Activity was strongly inhibited by diisopropyl phosphofluoridate, indicating the serine protease nature of the enzyme. The migration distance on electrophoresis, molecular weight and substrate specificity differed from cucumisin, a known protease from melon. This unusual protease may have potential for special food treatment applications.     

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.     

Proteolysis characteristics of Actinomucor elegans and Rhizopus oligosporus extracellular proteases under acidic conditions

Enzymatic hydrolysis of soybean protein isolate by the extracellular proteases from Actinomucor elegans and Rhizopus oligosporus at pH 3.0, 3.5, 5.0, 5.5 and 6.0 was investigated. The activity of the A. elegans protease is lower than that of R. oligosporus, but both proteases exhibit considerable degradation of soybean protein at pH 5.5 and 6.0. The water‐soluble protein content and the degree of hydrolysis of the hydrolysates are increased significantly, and bitterness values are very low. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) reveals that these proteases have different cutting sites on peptide polymers. At pH 5.5, there is a lower content of total free amino acids (39.20 mg per 100 mL; 62.68% hydrophobic amino acids) in the R. oligosporus protease hydrolysate. In conclusion, treatment with R. oligosporus protease at pH 5.5 achieves efficient degradation of soybean protein, suggesting a promising industrial process for making bitterless protein hydrolysates.     

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     

ENZYMATIC PROPERTIES OF A PROTEASE FROM THE HEPATOPANCREAS OF SHRIMP, PENAEVS OMENTAIM

The protease purified from hepatopancreas of shrimp, Penaeus orientals, had high proteolytic activity in the pH range of 7.0 to 9.5. Temperature optimum for hydrolysis of casein was 70C. The protease was stable at neutral and alkaline pH and unstable at acidic pH. The apparent Michaelis‐Menten constant (K_m) and the turnover number (V_max) of the protease on hydrolysis of N‐CBZ‐L‐tyrosine p‐nitrophenyl ester (CBZ‐Tyr‐NE) and N‐CBZ‐L‐phenylalanine p‐nitrophenyl ester (CBZ‐Phe‐NE) ‐were similar, however, those for N‐CBZ‐L‐cysteine p‐nitropher.yl ester (CBZ‐Cys‐NE) were different. K_m and V_max for hydrolysis of casein by the protease were determined to be 0.31% and 5.21s^‐1, respectively. The N‐terminal sequence of the protease showed higher homology with the collagenase of crab and trypsins from Crustacea. Myosin heavy chain (MHC) was the primary substrate during proteolysis with the protease. Actin/tropomyosin were degraded progressively during 2 h incubation but to a lesser extent than MHC.     

Purification and characteristics of serine protease from the head of pacific white shrimp

Serine protease from the head of Pacific white shrimp was purified by the following techniques: ammonium sulfate fractionation, Q-Sepharose HP ion exchange chromatography, and Sephadex G-100 gel filtration. The molecular weight was estimated as 32.8 kDa using SDSPAGE. The optimum pH and temperature of the enzyme for the hydrolysis of casein were determined to be 10.0 and 40°C. It was stable at pH range from 8.0 to 11.0 and had good thermal stability. Pb²⁺, Ca²⁺, Mg²⁺, Cu²⁺, and Mn²⁺ could active the enzyme certainly when Zn²⁺ and Hg²⁺ strongly inhibited the activity. The enzyme was inhibited by the general serine protease inhibitor (PMSF) and the specific trypsin inhibitors (TLCK, SBTI). The modification of various amino acid modifiers for the purified enzyme determined that the enzyme active center included tryptophan, histidine, and serine, moreover, arginine had a certain relationship with the enzyme activity.