Effects of limited proteolysis on broad bean polyphenoloxidase

Partially purified broad bean polyphenoloxidase was treated with a variety of proteases. Enzyme activity increased after treatment with chymotrypsin, pepsin, subtilisin, thermolysin, trypsin, and Staphylococcus aureus V₈ protease but this activation was masked when assayed in the presence of sodium dodecylsulfate. The enzyme was resistant to proteolysis when treated with chymotrypsin, elastase, pepsin, thermolysin, and V₈ protease. Proteinase K, subtilisin and trypsin treatment resulted in partial degradation of the enzyme but only trypsin generated an active enzyme form with a slightly smaller molecular weight. Electrophoresis, followed by enzyme staining and or Western blotting, showed that trypsin treatment converted the active 45 kd enzyme into a 43 kd enzyme within 20 min. Treatment with proteinase K, subtilisin and trypsin also produced inactive higher molecular weight forms of purified polyphenoloxidase that were identified by Western blotting. The use of antibodies coupled with Western blotting also identified inactive, active, and latent polyphenoloxidase after protease treatment, and suggests that immunological methods can be used to establish the types and amounts of polyphenoloxidase present in a given tissue.     

Purification of diamine oxidase and its properties in germinated fava bean (Vicia faba L.)

Background: γ‐Aminobutyric acid (GABA) is a non‐protein amino acid with bioactive functions for human health. Diamine oxidase (DAO, EC 1.4.3.6) is one of the key enzymes for GABA formation. In the present study, this enzyme was purified from 5 day germinated fava bean and its properties were investigated in vitro. Results: The molecular mass of the enzyme estimated by Sephadex G‐100 gel filtration was 121 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) displayed a single band at a molecular mass of 52 kDa. The enzyme had optimal activity at 40 °C and retained its activity after being incubated at 30 °C for 30 min. It showed higher activity at pH 6.5 than at other pH values. The enzyme was significantly inhibited by Mg²⁺, Cu²⁺, Fe³⁺, aminoguanidine, ethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraacetic acid disodium salt (EDTA‐Na₂), L ‐cysteine and β‐mercaptoethanol. The K_m value of DAO was 0.23 mmol L^?1 for putrescine and 0.96 mmol L^?1 for spermidine. However, the enzyme did not degrade spermine. Conclusion: DAO from germinated fava bean was purified. The optimal reaction temperature and pH of the enzyme were mild. The enzyme had higher affinity to putrescine than to spermidine and spermine. Copyright © 2011 Society of Chemical Industry     

Evidence of cell-associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation

Abstract: Cell‐associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation were extracted and characterized. Proteinases were effectively released when washed cells were incubated in 0.3 mg/mL lysozyme in 50 mM Tris‐maleate (pH 7) at 37 °C for 2 h. Major cell‐associated proteinases exhibited molecular mass of 17, 32, and 65 kDa, but only a 32‐kDa proteinase showed strong amidolytic activity toward Suc‐Ala‐Ala‐Pro‐Phe‐AMC. Activity of all cell‐associated proteinases was completely inhibited by phenylmethanesulfonyl fluoride, indicating a characteristic of serine proteinase. In addition, a 65‐kDa serine proteinase was also inhibited by ethylenediaminetetraacetic acid, implying a metal‐dependent characteristic. Optimum activity toward a synthetic peptide substrate was at 50 °C and pH 8 and 11. Proteinases with molecular mass of 17 and 32 kDa exhibited caseinolytic activity at 25% NaCl and activity based on a synthetic peptide substrate increased with NaCl concentrations up to 25%, suggesting their role in hydrolyzing proteins at high salt concentrations. This is the first report of liberated cell‐associated proteinases from a moderate halophile, Virgibacillus sp. Practical Application: The cell‐associated proteinases could be extracted from Virgibacillus sp. SK 33 using lysozyme. The extracted enzyme could be applied to hydrolyze food proteins at NaCl content as high as 25%. In addition, this study demonstrated that not only extracellular but also cell‐associated proteinases are key factors contributing to protein‐degrading ability at high salt environment of Virgibacillus sp. SK 33.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF A PROTEINASE INHIBITOR FROM TEPARY BEAN (PHASEOLUS ACUTIFOLIUS) SEEDS

In a qualitative screening of 36 accessions of tepary beans seeds, all the accessions inhibit the activity of bovine trypsin and trypsin-like proteinases from the insect P. truncatus, and the majority of them inhibit α-amylase activity of several important insect pests. A protein proteinase inhibitor was purified from accession L-242-45, using fractional precipitation, gel filtration, ion exchange chromatography and reverse-phase HPLC. The protein showed an apparent molecular weight of 7,100 by PAGE. However, contrary to other inhibitors previously reported, the inhibitory activity was only present in the trimeric form. The protein was characterized as a serine-proteinase inhibitor that recognized trypsin, chymotrypsin and trypsin-like proteinases, but it also recognized aspartic acid proteinases from different insects. It contained no carbohydrate residues and showed a high stability at 96C at low pH.     

ISOLATION AND PARTIAL CHARACTERIZATION OF A NOVEL BACTERIOCIN PRODUCED BY LACTOCOCCUS LACTIS SSP. LACTIS MC38

This work presents the isolation and partial characterization of a new lactococcal bacteriocin produced by Lactococcus lactis ssp. lactis MC38. The bacteriocin demonstrated broad spectrum of inhibition activity against both pathogenic and food spoilage organisms, and various lactic acid bacteria. This antimicrobial substance appeared to be proteinaceous because its activity was completely inactivated by proteinase K and α‐chymotrypsin. It was heat and pH stable. The apparent molecular mass of the purified bacteriocin, determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, was 8.0 kDa. The amino acid composition of the studied bacteriocin was found to be quite different from known lactococcal bacteriocins. The calculation of the number of amino acid residues in the bacteriocin molecule revealed that it contained 62 amino acids.     

Chymotrypsin from the hepatopancreas of cuttlefish (Sepia officinalis) with high activity in the hydrolysis of long chain peptide substrates: Purification and biochemical characterisation

Chymotrypsin from the hepatopancreas of cuttlefish (Sepia officinalis) was purified to homogeneity, with a 120-fold increase in specific activity and 23% recovery. The molecular weight of the purified chymotrypsin was estimated to be 28 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The optimum pH and temperature for the chymotrypsin activity were pH 8.5 and 55 °C, respectively, using succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide (SAAPFpNA) as a substrate. The enzyme was extremely stable in the pH range of 7.0–10.0 and highly stable up to 50 °C after 1 h incubation. This proteinase was strongly inhibited by chymostatin, soybean trypsin inhibitor, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, but was not inhibited by tosyl-l-phenylalanine chloromethyl ketone, N-carbobenzoxy-phenylalanine chloromethyl ketone or N_α-tosyl-l-lysine chloromethyl ketone. The enzyme hydrolysed long chymotrypsin peptide substrates SAAPFpNA, SAAPLpNA and ZAALpNA and did not hydrolyse short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SAAPFpNA, with k_cat 18 s^?1 and K_m 22 μM. However, the enzyme had a lower K_m for SAAPLpNA, 54 μM.The N-terminal amino acid sequence of the first 20 amino acids of the purified chymotrypsin was IVGGQEATIGEYPWQAALQV.     

Proteinase inhibitory activity of sarcoplasmic proteins from threadfin bream (Nemipterus spp.)

BACKGROUND: Thailand is the second largest surimi producer in the world and 50% of surimi is produced from threadfin bream. During surimi processing, sarcoplasmic proteins are removed through water washing and discarded in the waste stream. This study was aimed at investigating the proteinase inhibitory activity of sarcoplasmic proteins. RESULTS: Sarcoplasmic proteins from threadfin bream (TBSP) exhibited inhibitory activity toward trypsin but did not inhibit papain and chymotrypsin. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis under non‐reducing condition stained by trypsin inhibitory activity revealed three protein bands of molecular mass of 95, 41 and 37 kDa. Inhibitory activity of TBSP reached a maximum when subjected to 45 °C and completely disappeared at 60 °C. The breaking force and deformation of lizardfish surimi gel with added TBSP and pre‐incubated at 37° for 20 min increased with additional levels of TBSP (P < 0.05). Trichloroacetic acid–oligopeptide content of lizardfish surimi gel with added TBSP decreased with the addition of 4 g kg^?1 TBSP (P < 0.05). Retention of myosin heavy chain (MHC) increased when TBSP concentration was increased. TBSP effectively protected MHC from proteolysis at 37 °C to a similar extent as egg white powder, but efficacy of TBSP was not observed at 65 °C. CONCLUSION: TBSP could be applied to reduce proteolytic degradation of lizardfish surimi or other surimi associated with trypsin‐like proteinase, rendering an improvement in surimi gelation set at 37–40 °C. Copyright © 2009 Society of Chemical Industry     

Purification and characterisation of cathepsin D from the digestive gland of the pelagic squid Todarodes sagittatus

A proteinase from the digestive gland of squid (Todarodes sagittatus) was purified from a neutral extract by ammonium sulphate fractionation, S-Sepharose chromatography and gel filtration on Sephadex G-75. Inhibition by pepstatin showed that the enzyme is an aspartic proteinase. The enzyme is fairly stable in the pH range 2.5-7, and optimum pH for haemoglobin digestion is 3.7. Combined results from gel filtration and sodium dodecyl sulphate electrophoresis indicate a molecular weight of 38 kDa, and that the enzyme consists of two different subunits with molecular weights approximately 10 and 28 kDa. Analytical electrofocus-ing showed an enzyme pi of 8.5. As judged from the physical properties and the amino acid composition, it is concluded that the enzyme is a cathepsin D. This enzyme probably counts for the major part of proteinase activity in the digestive gland.     

Preparation and characterization of the N and C monoferric lobes of buffalo lactoferrin produced by proteolysis using proteinase K

The two glycosylated N- and C-terminal lobes of buffalo lactoferrin have been produced by limited proteolysis using proteinase K. Lactoferrin is a single chain glycoprotein of molecular mass 80 kDa with two iron-binding sites and two structural lobes connected by a short peptide. Purified samples of lactoferrin, isolated from buffalo colostrum, were subjected to hydrolysis using trypsin, chymotrypsin, pepsin, subtilisin and proteinase K. The first three proteinases produced two major fragments of approximately 35 and 23 kDa together with small molecular mass peptides. Trypsin and chymotrypsin partly digested lactoferrin, while pepsin converted all the intact lactoferrin into fragments. Subtilisin hydrolysis produced fragments of 40 and 26 kDa together with low molecular mass peptides. However, SDS-PAGE of the proteinase K hydrolysis product gave a clear band at 40 kDa together with a band indicating a substantial quantity of low molecular mass peptides (< 14.4 kDa). Upon ion-exchange chromatography this product gave two major fractions, which were further purified by gel filtration and identified as the C and N lobes from their N-terminal sequences. Thus, the 40 kDa band in SDS-PAGE of the proteinase K hydrolysis product contained two fragments of equal molecular mass. On further hydrolysis with proteinase K, the N lobe was completely hydrolysed into low molecular mass peptides, while only a small fraction of the C lobe was converted into small products. This suggested that an inhibitory fragment was present in the C lobe that was released on hydrolysis to small fragments and prevented complete digestion of the C lobe by high-affinity binding to the active site of proteinase K. This fragment was isolated from the lactoferrin-proteinase K complex and its sequence determined to be Val-Ala-Gln-Gly-Gly-Ala-Ala-Gly-Leu-Ala. Circular dichroism studies indicated a high alpha-helical content in the native lactoferrin while comparatively lower helical structures were present in the N and C lobes. In addition, the iron saturations of the N and C lobes appeared to be lower than that of the native protein.     

Albacore tuna (Thunnus alalunga) spleen trypsin partitioning in an aqueous two-phase system and its hydrolytic pattern on Pacific white shrimp (Litopenaeus vannamei) shells

Partitioning behaviors of trypsin from the spleen of albacore tuna (Thunnus alalunga) in an aqueous two-phase system (ATPS) were investigated. Partitioning behaviors of proteins were influenced by polyethylene glycol (PEG) molecular mass and concentration, types and concentration of salts, NaCl concentration, and temperature. Trypsin was preferentially partitioned into the PEG-rich top phase. The best ATPS conditions for trypsin partitioning from albacore tuna spleen were 15% PEG 4000–15% NaH₂PO₄ at 40°C without the addition of NaCl, which increased the purity by 5.54-fold with the recovered activity of 71.92%. Based on SDS-PAGE, the enzyme after ATPS separation was near homogeneity and the result of SDS-substrate gel electrophoresis revealed that the band intensity of enzyme in ATPS fraction increased, indicating the enhanced specific activity of splenic extract. The study further investigated the effect of fractionated trypsin on the hydrolysis of Pacific white shrimp (Litopenaeus vannamei) shells. Electrophoretic study revealed that trypsin after ATPS separation was a potential enzyme for extraction of carotenoprotein from Pacific white shrimp shell waste. Therefore, ATPS was an effective method for purification and recovery of trypsin from the spleen of albacore tuna and it could be used as an alternative cheap proteinase for the extraction of carotenoprotein from shrimp shells.     

Purification and some properties of a cysteine proteinase from sorghum malt variety SK5912

A cysteine proteinase from sorghum malt variety SK5912 was purified by a combination of 4 M sucrose fractionation, ion‐exchange chromatography on Q‐ and S‐Sepharose (fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 8.4‐fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2057.1 U mg^?1 protein. SDS—PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 55 and 62 kDa, respectively. The enzyme was optimally active at pH 6.0 and 50 °C, not influenced across a relatively broad pH range of 5.0–8.0 and retained over 60% activity at 70 °C after 30‐min incubation. It was highly significantly (P < 0.001) inhibited by Hg²⁺, appreciably (P < 0.01) inhibited by Ag⁺, Ba²⁺ and Pb²⁺ but highly significantly (P < 0.001) activated by Co²⁺, Mn²⁺ and Sr²⁺. The proteinase was equally highly significantly (P < 0.001) inhibited by both iodoacetate and p‐chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: K_m = 0.33 mg ml^?1; V_max = 0.08 µmol ml^?1 min^?1. Copyright © 2004 Society of Chemical Industry     

Trypsin-like Enzyme from Sand Crab (Portunus pelagicus): Purification and characterization

Studies with synthetic substrates and specific inhibitors indicated that a protcinase from the hepatopancreas of the sand crab (Portunus pelngicus) was a trypsin-like serine proteinase. The molecular weight of the enzyme estimated by gel filtration and mass spectrometry was ～ 25,000, whereas SDS-PAGE indicated a molecular weight of 34,800. The optimum temperature for hydrolysis of azocasein was 60°C, while inactivation of 50% enzymic activity occurred at 68°C. The enzyme, optimally active at pH 8.0 towards p-tosyl-L-arginine methyl ester and unstable at acid pH, was high in acidic amino acid residues. Under some conditions ihe knzyme readily autodigested. Our results can help understand and avoid problems of meat softening during storage of seafood products.     

HYPOTENSIVE ACTIVITY OF MUSCLE PROTEIN AND GLUTEN HYDROLYSATES OBTAINED BY PROTEASE TREATMENT

Protein hydrolysates obtained by treatment with papain, trypsin, chymotrypsin and actinase all exhibited inhibitory activity (IC50: 3.4–41.8 mg%) toward angiotensin‐converting enzyme (ACE) (EC 3.4.15.1). In particular, the protein hydrolysate obtained by treatment with papain showed the highest inhibitory activity (3.7–5.3 mg%). The ACE inhibitory activity of the gluten hydrolysate obtained with actinase was mainly due to peptides of less than 500 Da in molecular mass. On the other hand, the ACE inhibitory activity of the myofibrillar protein hydrolysate obtained with papain was due to peptides of both less and more than 500 Da in molecular mass. The blood pressure of spontaneously hypertensive rats (SHR) administered the myofibrillar protein hydrolysate was significantly reduced at 2 h after administration. The blood pressure of SHR was also reduced at 2 h after administration of the gluten hydrolysate, and this effect continued until 6 h. These hydrolysates may potentially be useful as antihypertensive food materials.     

Characterization of a β-glucosidase isolated from an alpeorujo strain of Candida adriatica

A β-glucosidase-producing strain, Candida adriatica CECT13142, was isolated from olive oil wastes (alpeorujo) and identified by PCR/restriction fragment length polymorphism of the rDNA internal transcribed spacer and sequence analysis of the D1/D2 region of the 26S rRNA gene techniques. The enzyme was purified by sequential chromatography on DEAE-cellulose and Sephadex G-100. The relative molecular mass of the enzyme was estimated to be 50 kDa by SDS-PAGE. The hydrolytic activity of the β-glucosidase had an optimum pH of 8.2 and an optimum temperature of 40°C. The enzyme displayed high substrate specificity and high catalytic efficiency (K_m 0.85 mM, V_max 12.5 U/g of cells) for p-nitrophenyl-β-D-glucopyranoside. Although β-glucosidases have been purified and characterized from several other organisms, the C. adriatica β-glucosidase is able to have optimal activity at alkaline pH.     

RECENTLY IDENTIFIED ENZYMES OF BREVIBACTERIUM LINENS ATCC 9174 - A REVIEW

An extracellular proteinase and an aminopeptidase and an intracellular aminopeptidase and esterase were isolated from Brevibacterium linens ATCC 9174. The proteinase was a serine enzyme with relatively broad specificity onα_s1-andβ-caseins. The extracellular aminopeptidase was a metallo enzyme but the intracellular aminopeptidase was a thiol enzyme with rather narrow specificity for small side chains, e.g., Gly and Ala. The organism possessed two intracellular esterases, one of which was purified to homogeneity. This enzyme was a tetramer of MW 200 kDa, was strongly inhibited by thiol blocking agents and showed a high specificity for short chain fatty acids with no lipolytic activity on tributyrin or milk fat.     

Purification and characterization of a novel fibrinolytic enzyme from chive (Allium tuberosum)

A novel Allium tuberosum fibrinolytic enzyme (ATFE) was purified from the leaf of chive by ion exchange chromatography followed by gel filtration. The molecular mass and iso-electric point (pI) of ATFE were 90 kDa and 4.0 by using 1- or 2-D fibrin zymography, respectively. ATFE was optimally active at pH 4.0 and 40°C. ATFE had a high degrading activity for the Aα-chain of human fibrinogen and hydrolyzed the Bβ-chain slowly, but did not affect the γ-chain, indicating that it is a α-fibrinogenase. The proteolytic activity of ATFE was inhibited completely by phenylmethylsulfonyl fluoride (PMSF), indicating that this enzyme belongs to the serine protease class. ATFE was also inhibited by the 1 mM of Cu²⁺. ATFE exhibited high specificity for Meo-Suc-Arg-Pro-Tyr-pNA (S-2586), a synthetic chromogenic substrate for chymotrypsin. The first 20 amino acid residues of the N-terminal sequence of ATFE were determined as TTKSWNFIGFDETSKXTTYE, which is 60% identical with subtilisin-like serine protease from Narcissus pseudonarcissus.     

Further characterisation of protease inhibitors from pigeon pea (cajanus cajan (l) millsp) seeds

Cajanus trypsin inhibitor (CTI) and Cajanus trypsin-chymotrypsin inhibitor (CTCI) previously purified from cv TAT-10 were further characterised. The modification of the inhibitors revealed the presence of lysine at the trypsin reactive site in both CTI and CTCI. Modification of tyrosine at the reactive site of CTCI did not abolish chymotrypsin inhibition suggesting the presence of leucine or phenylalanine as reported in other chymotrypsin inhibitors. CTCI did not contain tryptophan. Dissociation constants for the inhibitors with bovine trypsin were in the region of 0.69 nmol (CTCI) and 0.029 nmol (CTI). Although the protease inhibitors lost their inhibitory activity on exposure to 2-mercaptoethanol they remained attached to the enzyme. The inhibitors were not very effective against the protease from Helicoverpa armigera which is a serious field pest of Cajanus. Dissociation constants for the inhibitors with the larval enzyme were in the region of 100 nmol.     

Purification and Characterization of β-Glucosidase from Aspergillus niger

Aspergillus niger, an isolate of soil contaminated with effluents from cotton ginning mill was grown in Czapek-Dox medium containing sawdust, Triton-X 100 and urea for production of an extracellular β-glucosidase. β-Glucosidase enzyme was purified (86-fold) from culture filtrate of A. niger by employing ammonium sulphate precipitation and gel filtration on sephadex G-75. The molecular mass of the purified enzyme was estimated to be 95 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The enzyme had an optimal activity on p-nitrophenyl β-D-glucopyranoside at 50°C and pH 5.0. The K_m and V_max of the enzyme on p-nitrophenyl β-D-glucopyranoside at 50°C and pH 5 were 8.0 mM and 166 µmol/min/mg of protein, respectively. The enzyme could hydrolyze cellobiose and lactose but not sucrose. Heavy metals like Hg²⁺, Al³⁺, and Ag⁺ inhibited the activity, whereas Zn²⁺ and detergents such as Triton-X 100 and Tween-80 increased the activity at 0.01%. The enzyme activity increased in the presence of methanol and ethanol.     

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.     

DOUBLE-HEADED TRYPSIN/CHYMOTRYPSIN INHIBITORS FROM HORSE GRAM (DOLICHOS BIFLORUS): PURIFICATION, MOLECULAR AND KINETIC PROPERTIES

Four proteinase inhibitors were purified to homogeneity from horse gram (Dolichos biflorus). These inhibitors are double-headed and inhibit trypsin and chymotrypsin simultaneously and independently. Dissociation constants range between 0.87 and 4.6 × 10^?7 M. Each of the four isoinhibitors possesses a crucial lysine residue at the trypsin reactive site. These inhibitors have molecular masses of 8.5 kDa and isoelectric points of 4.6 to 5.6. They exist mainly as dimers under physiological conditions. Amino acid analysis revealed high levels of half-cystine, serine, aspartate and proline but low levels of methionine and aromatic amino acids. Amino-terminal sequence analysis revealed that each of the four isoinhibitors have a conserved core sequence but are divergent at the N-terminal end. These inhibitors belong to the Bowman-Birk (BBI) family of proteinase inhibitors as reflected by their inhibitory properties, amino acid composition and homology to other BBIs.