Comparison of muscle proteinase activity among fish species

The effects of temperature during storage, portion of muscle and growth stage of fish on the activity level of carp muscle acid (cathepsin D, EC 3.4.23.5), neutral and alkaline proteinases were examined. Icing storage, freezer storage and portion of muscle did not affect each proteinase activity (P less than 0.05), but acid proteinase activity was affected by growth stage (P less than 0.05) and the level decreased from small to large fish. The activity levels of three kinds of proteinases were compared among species (P less than 0.05) and the following order was obtained. Acid proteinase, white croaker = rainbow trout = carp greater than sardine = common mackerel, sardine greater than lizardfish, common mackerel = lizardfish. Neutral proteinase, rainbow trout greater than carp = white croaker greater than lizardfish = sardine = common mackerel. Alkaline proteinase, rainbow trout = sardine greater than white croaker = carp = common mackerel greater than lizardfish.     

Purification and Some Properties of Two Aminopeptidases from Sardines

Two fish aminopeptidases designated as aminopeptidases I and II were purified by DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and isoelectric focusing. The final preparations of enzymes I and II were judged nearly homogenous by polyacrylamide gel I, electrophoresis. The molecular weights of enzymes I and II were determined by gel filtration to be 370,000 and 320,000, respectively. The isoelectric points were 4.1 (I) and 4.8 (II), Both enzymes were inhibited by EDTA and activated by Co⁺⁺. Bestatin could inhibit enzyme I but not enzyme II. Enzymes I and II rapidly hydrolyzed not only synthetic substrates containing alanine or leucine but also di-, tri-, and tetra-alanine. Judged from all of these properties, sardine aminopeptidases resemble human alanine aminopeptidase. Enzyme I retained more than 70% of its original activity in 15% NaCl, suggesting the enzyme participates in hydrolyzing fish proteins and peptides during fish sauce production.     

Separation of cathepsin D-like proteinase and acid thiol proteinase of squid mantle muscle

When the proteinases of the squid mantle muscle were extracted in the presence of dithiothreitol (DTT), the acid proteinase activity increased, indicating that the squid mantle muscle contains a considerable amount of the acid thiol proteinase. The crude extract hydrolyzed neither alpha-N-benzoyl-D,L-arginine-p-nitroanilide (BAPA) nor azocasein, thus refuting the presence of cathepsins B and L in the mantle muscle. The cathepsin D-like proteinase and the acid thiol proteinase were separated by Sephadex A-50 column chromatography. Each of the above partially purified proteinases was able to degrade carp actomyosin at pH 2.5 and 5.0, respectively.     

Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce

A gram-positive thermotolerant bacterium, designated strain RKK-04, was isolated from a fermented Thai fish sauce broth as it demonstrated high proteolytic activity. A phylogenetic analysis based on comparisons of 16S rRNA gene sequences showed that strain RKK-04 is Bacillus licheniformis. The proteolytic enzyme, which was purified 80-fold with 18% yield, has a molecular mass of 31 kDa and an isoelectric point higher than 9.3. The optimum pH and temperature of the enzyme activity were found to be 10.0 and 50°C, respectively. The addition of diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride completely inhibited enzymatic activity. These results showed that the enzyme is a subtilisin-like alkaline serine proteinase. On the other hand, the enzyme exhibited unique cleavage sites in oxidized insulin B-chain that differed from those of other subtilisin-like proteases. High enzymatic activity was also retained under high salt conditions (30% NaCl). The myosin heavy chain of fish protein was completely digested by reaction with this enzyme. Thus the halotolerant proteinase from B. licheniformis RKK-04 is a key enzyme for fish sauce fermentation.     

Intracellular distribution of fish muscle alkaline proteinase

1. Intracellular distribution of a muscle alkaline proteinase was investigated on four kinds of fish. 2. The total activity of the muscle proteinase of carp, Cyprinus carpio, was larger in both myofibrillar (Mf) and microsomal (Mic) fractions than the activity in mitochondrial, lysosomal, and supernatant fractions. The activity found in Mf fraction seemed to due to the Mic enzyme which was not separated from Mf fraction. 3. The relative specific activity was mostly found in Mic fraction in the species tested. 4. The results indicate that the distribution pattern of fish muscle alkaline proteinase is different from those of cathepsin D and acid phosphatase. 5. The Mic fraction hydrolyzed Mf and sarcoplasmic proteins. The rates were 40 and 55%, respectively, of the rate when casein was used as a substrate.     

Hydrolysis of acid-treated protein by a preparation of proteases

Because of less glutaminase activity, soy sauce made with a preparation of proteases from yellow-green Aspergilli contains less glutamic acid than soy sauce made by the traditional shoyu koji method. Thus, an acid treatment was developed to increase this amino acid in enzyme-made shoyu. Amide bonds of glutamine and asparagine in protein molecules were hydrolyzed at 100°C for 30 min with 1.3 N HCl (acid treatment). Using this method, glutamic acid per total nitrogen freed from various proteins by the concerted action of proteinases and peptidases of yellow-green Aspergillus increased to 1.0 to 3.8 times that of control (no acid treatment). An increase of about 31% of glutamic acid per total nitrogen resulted from the acid treatment method in soy sauce made with an enzyme preparation of proteases.     

Proteolytic enzymes in sea urchin eggs: characterization, localization and activity before and after fertilization

The pH versus proteinase activity curve (casein or hemoglobin plus urea substrate) for homogenates of unfertilized Lytechinus eggs reveals two regions of maximum activity: one between pH 3.5 and 4.3, and another of far greater magnitude from pH 8.0 to 11.0. The two classes of proteinases can be separated on a sucrose density gradient. Both the acid and alkaline proteinases in homogenates prepared in isotonic monovalent salt solutions are remarkably stable at pH 7.4 and 0°C. Using synthetic peptide substrates, an enzyme with the specific esterase activity of chymotrypsin was demonstrated; this enzyme accounts for the major part of the proteinase activity at alkaline pH. In addition, an enzyme with specific esterase activity of trypsin was shown to be present, but of low activity. The proteinase activity at acid pH is largely due to an enzyme resembling cathepsin D. The data also suggest the presence of cathepsin B and cathepsin IV (or catheptic carboxypeptidase). When eggs are homogenized in isotonic NaCl plus KCl at pH 7.4, 0.02 M tris buffer at 0°C, all of the alkaline proteinase, and 85–90% of the acid proteinase activity is sedimented at 10,000 g. The presence of any proteinase activity in the supernatant phase represents an artifact of the preparative procedures used. The granules which possess the proteinase activity are contained entirely in the yolk fractions; and the acid proteinase is contained in a population of granules which sediment more readily than those which contain the alkaline proteinase. The acid proteinase resembles the lysosomal acid hydrolases in that it is readily released from the particulates; in contrast, the alkaline proteinase is bound relatively firmly. In contradistinction to reports in the literature, no changes in proteinase activity nor intracellular distribution could be detected following fertilization.     

Novel fibrinolytic enzymes from Virgibacillus halodenitrificans SK1-3-7 isolated from fish sauce fermentation

Virgibacillus sp. SK1-3-7 exhibited the highest fibrinolytic activity among 25 bacterial isolates obtained from fish sauce fermentation. Results of 16S rRNA gene sequence analysis showed 99% homology to Virgibacillus halodenitrificans ATCC 49067. It was, therefore, identified as V. halodenitrificans SK1-3-7. Fibrinolytic enzymes from V. halodenitrificans SK1-3-7 were partially purified using ammonium sulfate fractionation, hydrophobic and ion-exchange chromatographies. The enzymes with molecular weight of 20- and 36-kDa showed fibrinolytic activity on a fibrin zymogram. The enzymes were stable between pH 4 and 10 and below 60 °C. The enzymes were activated by 20 mM CaCl₂ and 0.15 M NaCl. The activity increased with CaCl₂ up to 100 mM and increased with NaCl concentration up to 2 M. In addition, the residual fibrinolytic activity of 61% was found at 4 M NaCl. The enzymes were completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-pNA, suggesting a subtilisin-like serine proteinase. V. halodenitrificans SK1-3-7 enzymes hydrolyzed fibrin to a greater extent than did plasmin. In addition, the enzymes were resistant to pepsin and trypsin digestion. The de novo peptide homology analysis of a 20- and 36-kDa proteinase revealed no matches to bacilli serine proteinases, suggesting that both were novel fibrinolytic enzymes.     

High molecular weight heat-stable alkaline proteinase from white croaker and chum salmon muscle: comparison of the activating effects by heating and urea

Effects of heating and urea on the heat-stable alkaline proteinase from white croaker and chum salmon muscle were compared in order to know the regulating mechanism of the proteinase. Chum salmon proteinase required a higher temperature for activity and was more heat-stable than white croaker proteinase. In the presence of 5M urea, the activity was observed to some degree at 37 degrees C only in white croaker proteinase, while both proteinases lost their activities at usual assay temperature around 60 degrees C. These results suggest that the stability of the regulatory and catalytic subunits of the proteinases is somewhat different among fish species.     

Hydrolytic activity of Virgibacillus sp. SK37, a starter culture of fish sauce fermentation, and its cell-bound proteinases

Fish sauce production relies on a natural fermentation process requiring 12-18 months for process completion. Virgibacillus sp. SK37 has been shown to be a potential strain for fish sauce acceleration. However, hydrolytic activity of proteinases bound at cell surface of this strain has not been well elucidated. Addition of 0.2 % CaCl(2) (w/w) in conjunction with starter cultures of Virgibacillus sp. SK 37 increased protein hydrolysis as measured by α-amino group content throughout fermentation (P < 0.05). Cell-bound proteinases from Virgibacillus sp. SK 37 were extracted into a free form by incubating the washed cells in Ca(2+)-free buffer at 37 °C for 2 h. Cell-bound proteinases revealed molecular mass of 19, 20, 22, 32, 34, and 44 kDa based on a synthetic peptide zymogram. The proteinases showed subtilisin-like serine characteristics with the highest activity at 50 °C and pH 8 and 11. Activity of the extracted proteinases increased ~4 times at ≥100 mM CaCl(2). In addition, CaCl(2) enhanced thermal stability of the extracted proteinases. Enzymes showed proteolytic activity in either the absence or presence of 10 and 25 % NaCl toward fish muscle, soy protein isolate, and casein substrates. Cell-bound proteinases were likely to play an important role in protein hydrolysis during fish sauce fermentation.     

Protein turnover in muscle: Inhibition of the calcium activated proteinase by mersalyl without inhibition of the rate of protein degradation

The relative roles of neutral and lysosomal proteinases in degrading intracellular proteins have been examined in rat gastrocnemius muscle. A comparison of the relative activities of the proteinases shows that cathepsin B is 10 times more active in muscle than the calcium activated proteinase. This dramatic difference suggests that, if the calcium activated proteinase is required for protein degradation, it might be rate limiting. $\text{In}\text{,}\text{vivo}$ rates of protein degradation were measured after pulse labeling with [³H]N-ethylmaleimide. The rates were not diminished by intramuscular injection of mersalyl at concentrations that inhibited the calcium activated proteinase by at least 35% throughout the 72 h period of the experiments. On the other hand, the lysosomal proteinase, cathepsin B, increased after mersalyl treatment to 370% by 72 h. Therefore, we conclude that lysosomes are necessary for the degradation of modified proteins in muscle and we question the role of the calcium activated proteinase in this process.     

Studies on an Alkaline Proteinase of Aspergillus sydowi

An alkaline proteinase of Aspergillus sydowi (Bainier et Sartory) Thom et Church has been purified approximately 4.5-fold from a culture filtrate by fractionation with ammonium sulfate, treatment with acrynol and Alumina gel C_γ, and DEAE-Sephadex column chromatography. The purified proteinase obtained as needle crystals was monodisperse in both the ultracentrifuge and the electrophoresis on polyacrylamide gel.

The optimum pH and temperature for the activity were 8.0 and 40°C, respectively. Fifty per cent of the activity was lost at 45°C within ten minutes and 95% at 50°C. At 5°C, the enzyme was highly stable at the range of pH 6 to 9. None of metallic salts tested promoted the activity, but Zn⁺⁺, Ni⁺⁺ and Hg⁺⁺ were found to be inhibitory. Sulfhydryl reagent, reducing and oxidizing reagents tested except iodine had no effect on the activity, but potato inhibitor, DFP and NBS caused a marked inhibition.

The alkaline proteinase from Aspergillus sydowi was markedly protected from inactivation by the presence of Ca⁺⁺ in the enzyme solution. The protective effect of Ca⁺⁺ was influenced remarkably by the pH values of the enzyme solution, i.e., optimum concentrations of Ca⁺⁺ for the protective effect at pH 7.1, 7.5 and 7.8 were 10^?2, 10^?3 and 10^?4 M, respectively. Conversely, at higher pH values such as 9.0, Ca⁺⁺ accelerated the rate of inactivation. There was a parallelism between the loss in activity and the increase in ninhydrin-positive material in the enzyme solution.

The proteinase acted on various denaturated proteins, but not on native proteins. In digestion of casein by the proteinase, 92% of nitrogen was turned into soluble form in 0.2 m trichloroacetic acid solution, with 14~17% of peptide bonds being hydrolyzed. Casein hydrolyzed with the Asp. sydowi proteinase was further hydrolyzed by Pen. chrysogenum, B. subtilis or St. griseus proteinases, which further increased the free amino residues in the reaction mixtures. On the contrary, the Asp. sydowi proteinase reacted only slightly on casein hydrolyzed by the above-mentioned proteinases.     

Gas Chromatographic Determination of Optical Isomers of α-Phenylethylamine and α-Phenyl-β-(p-tolyl)-ethylamine on Optically Active Stationary Phases

The nondialyzable melanoidins prepared from glucose-butylamine (I) and xylose–butylamine (II) reaction systems, freeze-dried powder obtained from the dialyzable fraction of the glucose–butylamine reaction system (III) and N-butyl-glucosylamine (IV) were pyrolyzed at 350°C for 0.5-2 hr and the volatile pyrolysate was investigated. To trap the volatile compounds, Tenax GC trapping and cold trapping methods were used. Identification of these volatiles was made by gas chromatography-mass spectrometry, using a glass capillary column. The volatile components in the pyrolysates of I, II, III and IV were qualitatively similar to each other. The major volatile components in the pyrolysates of I, II, III and IV were identified as two furans, 1-butanol, two 1-butylpyrroles, 1-butylpyrrolidine, 1-butylaziridine and two N-butylamides. The results are discussed in relation to those obtained from previously investigated sugar-amino acid melanoidins.     

Volatile Components Formed by Thermal Degradation of Nondialyzable Melanoidins Prepared from Sugar–Amino Acid Reaction Systems

The nondialyzable melanoidins prepared from glucose-glycine (I), xylose-glycine (II) and glucose-β-alanine (III) reaction systems were pyrolyzed at 350°C for 0.5 ~ 2hr and the volatile pyrolyzates were investigated. To trap the volatile compounds, cold trapping and Tenax GC trapping methods were used. Identification of these compounds was made by gas chromatographymass spectrometry using a glass capillary column. The volatile components of I and II were qualitatively similar to each other. Major components of volatile pyrolyzates of I and II were identified as methyl acetate, five furans, toluene, two pyrroles, two pyridines and acetic acid. The major volatile components of III were methyl acetate, five furans, two 2-cyclopentenones, pyrrole, two pyridines and acetic acid.     

Capsicum annuum proteinase inhibitor ingestion negatively impacts the growth of sorghum pest Chilo partellus and promotes differential protease expression

BackgroundChilo partellus is an important insect pest infesting sorghum and maize. The larvae internalize in the stem, rendering difficulties in pest management. We investigated the effects of Capsicum annuum proteinase inhibitors (CanPIs) on C. partellus larvae by in-vitro and in-vivo experiments.MethodsRecombinant CanPI-7 (with four-Inhibitory Repeat Domains, IRDs), -22 (two-IRDs) and insect proteinase activities were estimated by proteinase assays, dot blot assays and in gel activity assays. Feeding bioassays of lab reared C. partellus with CanPI-7 and -22 were performed. C. partellus proteinase gene expression was done by RT-PCR. In-silico structure prediction of proteinases and CanPI IRDs was carried out, their validation and molecular docking was done for estimating the interaction strength.ResultsLarval proteinases of C. partellus showed higher activity at alkaline pH and expressed few proteinase isoforms. Both CanPIs showed strong inhibition of C. partellus larval proteinases. Feeding bioassays of C. partellus with CanPIs revealed a dose dependent retardation of larval growth, reduction of pupal mass and fecundity, while larval and pupal periods increased significantly. Ingestion of CanPIs resulted in differential up-regulation of C. partellus proteinase isoforms, which were sensitive to CanPI-7 but were insensitive to CanPI-22. In-silico interaction studies indicated the strong interaction of IRD-9 (of CanPI-22) with Chilo proteinases tested.ConclusionsOf the two PIs tested, CanPI-7 prevents induction of inhibitor insensitive proteinases in C. partellus so it can be explored for developing C. partellus tolerance in sorghum.General significanceIngestion of CanPIs, effectively retards C. partellus growth; while differentially regulating the proteinases.     

Identification of novel halotolerant bacillopeptidase F-like proteinases from a moderately halophilic bacterium, Virgibacillus sp. SK37

Aims: Virgibacillus sp. SK37 isolated from Thai fish sauce produced numerous NaCl‐activated subtilisin‐like proteinases. Our objectives were to purify, characterize and identify these extracellular proteinases. Methods and Results: Three major subtilisin‐like enzymes including 19, 34 and 44 kDa were partially purified and showed maximum activity at pH 8, 55–60°C, 25–30% NaCl and 70–100 mmol l^?1 CaCl₂. Enzymes showed stability at 0–30% NaCl and <20 mmol l^?1 CaCl₂ and were completely inhibited by phenylmethanesulphonyl fluoride but not by ethylenediaminetetraacetic acid. The isoelectric points of 19‐, 34‐ and 44‐kDa proteinases were at 3·6, 5·2 and 3·8, respectively, based on 2D electrophoresis. Peptide mass fingerprint and de novo peptide homology analysis of tryptic peptides using MALDI‐TOF and LC–MS/MS, respectively, suggested that all three enzymes were novel and homologous to bacillopeptidase F. Conclusions: The three major proteinases are a member of bacillopeptidase F‐like enzymes exhibiting thermophilic and halotolerant characteristics with high stability at 30% NaCl. Significance and Impact of the Study: This is the first report on bacillopeptidase F‐like proteinases in genus Virgibacillus with a distinct halotolerant feature. They showed potential to be a processing aid for food and biotechnological applications, particularly in high salt condition.     

Nicotianamine chelates both FeIII and FeII. Implications for metal transport in plants

Nicotianamine (NA) occurs in all plants and chelates metal cations, including Fe^II, but reportedly not Fe^III. However, a comparison of the Fe^II and Zn^II affinity constants of NA and various Fe^III-chelating aminocarboxylates suggested that NA should chelate Fe^III. High-voltage electrophoresis of the FeNA complex formed in the presence of Fe^III showed that the complex had a net charge of 0, consistent with the hexadentate chelation of Fe^III. Measurement of the affinity constant for Fe^III yielded a value of 10^20.6, which is greater than that for the association of NA with Fe^II (10^12.8). However, capillary electrophoresis showed that in the presence of Fe^II and Fe^III, NA preferentially chelates Fe^II, indicating that the Fe^IINA complex is kinetically stable under aerobic conditions. Furthermore, Fe complexes of NA are relatively poor Fenton reagents, as measured by their ability to mediate H₂O₂-dependent oxidation of deoxyribose. This suggests that NA will have an important role in scavenging Fe and protecting the cell from oxidative damage. The pH dependence of metal ion chelation by NA and a typical phytosiderophore, 2′-deoxymugineic acid, indicated that although both have the ability to chelate Fe, when both are present, 2′-deoxymugineic acid dominates the chelation process at acidic pH values, whereas NA dominates at alkaline pH values. The consequences for the role of NA in the long-distance transport of metals in the xylem and phloem are discussed.     

News & Notes: Specificity of a Neutral Zn-Dependent Proteinase from Thermoactinomyces sacchari Toward the Oxidized Insulin B Chain

The proteolytic specificity of the neutral Zn-dependent proteinase from Thermoactinomyces sacchari was determined by analysis of the peptides obtained after incubation with the oxidized insulin B chain as a substrate. The enzyme is an endopeptidase with broad specificity. In total, 12 peptide bonds in the B chain of insulin were hydrolyzed. The major requirement is that a hydrophobic residue such as Leu, Val, or Phe should participate with the α-amino group in the bond to be cleaved. However, hydrolysis of bonds at the N-terminal side of His, Thr, and Gly was also observed. The peptide bond Leu 15–Tyr 16 in the oxidized insulin B chain, which is the major cleavage site for the alkaline microbial proteinases, is resistant to the attacks of the enzyme from Thermoactinomyces sacchari and other neutral proteinases. The proteolytic activity of the Zn-dependent proteinase from T. sacchari is different from those of other metalloendopeptidases from microorganisms. Received: 10 November 1999 / Accepted: 15 December 1999     

Purification and characterization of serine proteinase from a halophilic bacterium, Filobacillus sp. RF2-5

In order to find a unique proteinase, proteinase-producing bacteria were screened from fish sauce in Thailand. An isolated moderately halophilic bacterium was classified and named Filobacillus sp. RF2-5. The molecular weight of the purified enzyme was estimated to be 49 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10-11 under 10% NaCl, and was highly stable in the presence of about 25% NaCl. The activity was strongly inhibited by phenylmethane sulfonyl fluoride (PMSF), chymostatin, and alpha-microbial alkaline proteinase inhibitor (MAPI). Proteinase activity was activated about 2-fold and 2.5-fold by the addition of 5% and 15-25% NaCl respectively using Suc-Ala-Ala-Phe-pNA as a substrate. The N-terminal 15 amino acid sequence of the purified enzyme showed about 67% identity to that of serine proteinase from Bacillus subtilis 168 and Bacillus subtilis (natto). The proteinase was found to prefer Phe, Met, and Thr at the P1 position, and Ile at the P2 position of peptide substrates, respectively. This is the first serine proteinase with a moderately thermophilic, NaCl-stable, and NaCl-activatable, and that has a unique substrate specificity at the P2 position of substrates from moderately halophilic bacteria, Filobacillus sp.     

Production and Further Characterization of an Alkaline Elastase Produced by Alkalophilic Bacillus Strain Ya-B

The characteristics of the obligate alkalophilic Bacillus sp. strain Ya-B, which produces alkaline elastase extracellularly, were examined. This strain grew at pH 7.0 only in the presence of 1% or more NaCl. Its fatty acid distribution pattern was similar to that of other Bacillus species in which iso-C₁₅ and anteiso-C₁₅ were the most abundant fatty acids. About 120 mg of enzyme was recovered from 1 liter of culture broth in a medium (pH 10.1) containing mainly glucose, soymeal, and glycerol. The antiserum against this enzyme did not recognize microbial proteinases, such as subtilisins, but reacted with proteinase C, which was purified from commercial pronase. Chemical modification studies revealed that certain histidine and tyrosine residues might be involved in the enzyme activity. This enzyme underwent a partial unfolding at pHs higher than 12.0, as indicated by the circular dichroism study.