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The objectives of this study were to characterize physicochemical properties of two collagens, tilapia skin (TS) and hybrid catfish skin (HS). Angiotensin-converting enzyme (ACE) inhibitory peptide...

The effect of NaCl (0.1 − 3.0 M) on self-assembly of silver carp myosin at 4 °C was investigated in terms of microstructure, conformation, intermolecular interactions, and particle size distribution. During setting at 4 °C, the self-assembly of myosin showed an obvious concentration dependence. At low concentrations ( 2+ -ATPase activity reached a maximum at 0.6 M NaCl. When the concentrations increased to >1.0 M, myosin further assembled into filaments dominated by hydrophobic interactions. Both rod-rod and head-head interactions contributed to the myosin filaments at high concentrations (1.0 − 3.0 M). The solubility and uniformity of the myosin assemblies decreased as the NaCl concentration increased from 1.0 to 3.0 M.

Permeability of angiotensin I-converting enzyme (ACE) inhibitory peptides (KPLLCS, ELFTT, and KPLL) identified from the in vitro gastrointestinal digest of cooked chicken breast was investigated using the Caco-2 cell model system. KPLLCS was originally the most effective ACE inhibitor (IC50 0.37 μM), but it was degraded during permeation through Caco-2 cells. Among these peptides, KPLL showed the highest permeability in intact form, and partially degraded to KP and LL, which still showed ACE inhibitory activity after permeation. ACE inhibitory activity of permeated KPLL was highest of 56%. KPLL and KP possessed a mixed inhibitor characteristic, while LL was a non-competitive inhibitor. Based on molecular docking, K at N-terminus of KPLL is a key structure contributing to ACE inhibition as it can occupy the active site of ACE. Determining the structural stability and degree of peptide transport across epithelial cell is required to confirm their potential as ACE inhibitors.

Nine novel peptides from tilapia hydrolysate were isolated and identified to be EKL, EKP, HKPA, ELSC, ALSC, ASLCH, SLCH, LPGYF, and LEVPGY. In addition, six fragments from in silico gastrointestinal digestion of the identified peptides, including EK, KPA, SC, CH, PGY, and EVPGY, were synthesized. The most effective parent and digested peptides showing ABTS · + scavenging capacity were LPGYF and SC, respectively. All C- and Y-containing peptides were more effective than ascorbic acid (AsA). In contrast, K-containing peptides exhibited less antioxidant activity. All 15 peptides showed potent intracellular reactive oxygen species scavengers in the AAPH-induced HepG2 cell oxidative stress model. In addition, the digested peptides SC, CH, and PGY up-regulated the expression of CAT and SOD1 in HepG2 cells. The peptide PGY was the most effective cellular antioxidant. Thus, tilapia peptides could be potent nutraceutical products to reduce cellular oxidative stress.

Modulation of inflammation-related immune response on THP-1 macrophages of protein hydrolysates derived from tilapia mince, casein and pea protein, were investigated. The protein substrates were hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinase. The degree of hydrolysis (DH) of casein was observed to be the highest throughout the course of hydrolysis. When challenging THP-1 macrophages, tilapia mince hydrolysate (TMH) enhanced innate immunity through induction of IL-1β and COX-2 expression. Anti-inflammatory activity was observed in casein hydrolysate (CH) and pea protein hydrolysate (PPH) by attenuating lipopolysaccharide- (LPS) induced pro-inflammatory gene expression in THP-1 macrophages. CH suppressed IL-1β, IL-6, IL-8, TNF-α and COX-2, while PPH reduced LPS-induced IL-6 and TNF-α responses. In addition, CH and PPH showed stronger suppression of LPS-induced pro-inflammatory gene expression compared with non-hydrolyzed casein and pea protein. These results suggest that TMH, CH and PPH prepared from V. halodenitrificans SK1-3-7 proteinase are potential functional food ingredients with immunomodulatory activity.

The release of encrypted angiotensin converting enzyme (ACE) inhibitory peptides upon simulated in vitro gastrointestinal (GI) digestion of Korat crossbred (KC) and commercial broiler (BR) breast meat varied with cooking methods. Digestas of KC breast meat heated at 70 °C for 0.5 h (H-0.5) showed the highest ACE inhibitory activity, while digestas of KC chicken heated at 121 °C for 1 h showed the lowest inhibitory activity. ACE inhibitory activity varied with the protein digestibility. After a series of chromatographic techniques, we identified KPLLCS, ELFTT, and KPLL, novel potent ACE inhibitor peptides whose sequences were homologous to that of myosin and whose IC50 values were 0.37, 6.35 and 11.98 μM, respectively. ELFTT and KPLL were resistant to plasmin hydrolysis for up to 60 min. Therefore, KC chicken meat heated at H-0.5 could serve as a source of ACE inhibitory peptides upon GI digestion.

Enterococcus faecium CN-25 was isolated from a traditionally Thai fermented fish roe, som-kai-pla, and was found to possess enterocin A and B genes. Among the 30 microorganisms tested, the produced bacteriocin exhibited antagonistic activity against Listeria monocytogenes TISTR 1327. The bacterium maximally produced bacteriocin at a concentration of 1828 AU/ml at the beginning of the stationary phase (at 18 h) in the inexpensive rice bran medium. The antilisterial activity of the compound was not affected by heating at 60–100 °C for up to 30 min. High antilisterial activity remained at pH 2. Partially purified CN-25 bacteriocin derived from E. faecium CN-25 completely inhibited the growth of L. monocytogenes at the minimum concentration of 2.38 μg/ml. The compound had no effect on the efflux of ATP from L. monocytogenes but triggered the intracellular hydrolysis of ATP, as evidenced by a decrease in the luminescence of total ATP. The bacteriocin also depleted the transmembrane electrical potential (ΔΨ) but had no effect on the transmembrane pH gradient (ΔpH). Therefore, the partially purified CN-25 peptide may be useful for improving the product safety of fermented fish products.

Angiotensin I-converting enzyme (ACE) inhibitory activity of tilapia mince (TM) hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinases with a 48% degree of hydrolysis showed the highest ACE inhibitory activity with an IC 50 of 0.54 mg/mL. After ultrafiltration (UF) and chromatographic separation via anion exchange, cation exchange and size exclusion chromatography, the fraction with the highest ACE inhibitory activity with an IC 50 of 0.15 mg/mL was obtained. The peptides showed uncompetitive inhibition characteristics with an inhibition constant (K i ) of 0.18 mg/mL. The peptides showed high thermostability at 100 and 121 °C, as well as pH stability at a wide pH range of 2–10, and also maintained ACE inhibitory activity after in vitro gastrointestinal digestion. The most potent novel ACE inhibitory peptide identified was MILLLFR with an IC 50 of 0.12 µM. TM hydrolysate prepared by V. halodenitrificans SK1-3-7 proteinases could serve as a source of peptides with ACE inhibitory activity.

Protein hydrolysate from frame, bone and skin (FBSH) of threadfin bream was prepared using Virgibacillus sp. SK33 proteinase and fractionated using sequential ultrafiltration membranes with molecular weight cut-offs (MWCO) of 30, 5 and 1 kDa, respectively. Four fractions, namely FBSH-I (>30 kDa), FBSH-II (5-30 kDa), FBSH-III (1-5 kDa), and FBSH-IV (

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 CaCl 2 and 0.15 M NaCl. The activity increased with CaCl 2 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.

The angiotensin I-converting enzyme (ACE) inhibitory activity of protein hydrolysates from tilapia muscle fractions, namely mince (M), washed mince (WM), and sarcoplasmic protein (SP), were investigated. Each fraction was hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinases for up to 24 h. After 8 h of hydrolysis, the M hydrolysate (48% degree of hydrolysis (DH)) showed the highest ACE inhibitory activity, with an IC50 value of 0.54 mg/ml, while the SP hydrolysate exhibited the lowest DH and ACE inhibition. In vitro gastrointestinal digestion reduced the ACE inhibitory activity of the M hydrolysate but enhanced its transport across Caco-2 cell monolayers. The transported peptides were found to contain 3–4 amino acid residues showing strong ACE inhibition. The novel ACE inhibitory peptide with the highest inhibition was found to be MCS, with an IC50 value of 0.29 μM. Therefore, tilapia mince hydrolyzed by V. halodenitrificans proteinases contained ACE inhibitory peptides that are potentially bioavailable.

Proteinase inhibitory activity of sarcoplasmic protein (SP) extracted from common carp (Cyprinus carpio) muscle and its gel-improving ability were investigated. SPs displayed 89% and 54% inhibitory activity toward trypsin at 40 and 65 °C, respectively. Protein bands with molecular mass of 69, 50, 44, 41, and 35 kDa appeared on trypsin inhibitory activity staining under nonreducing condition when incubated at 40 °C, while 2 protein bands at 54 and 35 kDa were observed at 65 °C. Addition of SP at 0.18 g protein/100 g increased textural properties of threadfin bream surimi gel. However, when SP was added in combination with various CaCl2 concentrations (0.1% to 0.5%) it did not further improve textural properties as compared to the addition of SP alone. Retention of myosin heavy chain of threadfin bream surimi was greater with the addition of SP. These results indicated that the gel-enhancing effect of common carp SP was due to the inhibitory activity toward endogenous trypsin-like proteinases in threadfin bream surimi. Practical Application: Sarcoplasmic protein from common carp muscle could be used as a functional protein ingredient that minimizes muscle proteolysis and improves textural properties of surimi containing trypsin-like endogenous 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 (P0.05). 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.

Glutaminase or L-glutamine aminohydrolase (EC 3.5.1.2) is an enzyme that catalyzes the formation of glutamic acid and ammonium ion from glutamine. This enzyme functions in cellular metabolism of every organism by supplying nitrogen required for the biosynthesis of a variety of metabolic intermediates, while glutamic acid plays a role in both sensory and nutritional properties of food. So far there have been only a few reports on cloning, expression and characterization of purified glutaminases. Microbial glutaminases are enzymes with emerging potential in both the food and the pharmaceutical industries. In this research a recombinant glutaminase from Bacillus licheniformis (GlsA) was expressed in Escherichia coli, under the control of a ptac promoter. The recombinant enzyme was tagged with decahistidine tag at its C-terminus and could be conveniently purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The enzyme could be induced for efficient expression with IPTG, yielding approximately 26,000 units from 1-l shake flask cultures. The enzyme was stable at 30°C and pH 7.5 for up to 6h, and could be used efficiently to increase glutamic acid content when protein hydrolysates from soy and anchovy were used as substrates. The study demonstrates an efficient expression system for the production and purification of bacterial glutaminase. In addition, its potential application for bioconversion of glutamine to flavor-enhancing glutamic acid has been demonstrated.

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

An extracellular proteinase from Virgibacillus sp. SK33, isolated from 1 month-old fish sauce, was purified to electrophoretic homogeneity, using hydrophobic interaction chromatography and hydroxyapatite with purification fold of 2.5 and 7% yield. The anomalous molecular weight (MW) of 19 kDa was obtained from SDS–PAGE, whereas a MW of 33.7 kDa was determined by MALDI-TOF. Optimum conditions for catalytic activity were 55 °C and pH 7.5. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferentially hydrolysed Suc-Ala-Ala-Pro-Phe-AMC, indicating a serine proteinase with subtilisin-like characteristics. K m and k cat of the purified proteinase were 27 μM and 12 s −1 , respectively. Proteinase activity, toward both synthetic and anchovy substrates, increased with NaCl up to 25%. The proteinase exhibited high stability in both the absence and presence of NaCl up to 25%. Approximately 2.5-fold increase in activity was observed in the presence of divalent cations, including Ca 2+ , Mg 2+ and Sr 2+ at 100 mM. MALDI-TOF MS and LC–ESI-MS/MS analyses, as well as N-terminal sequences, revealed that the purified enzyme did not match microbial proteinases in the database, indicating it to be a novel proteinase.

A NaCl-activated proteinase produced by Virgibacillus sp. SK33 was purified to homogeneity using phenyl-Sepharose and Sephadex G-75 with a yield of 12% and purification of 2.6-fold. A single protein was detected at approximately 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, three subunits with molecular weights of 27,858, 33,918, and 35,368 Da were obtained from MALDI-TOF mass spectra, implying that the enzyme was a heterotrimer. The isoelectric point of the proteinase was 5.4. Optimum catalytic activity was at 55 degrees C and pH 7.5. The enzyme showed serine characteristics as it was completely inhibited by phenylmethanesulfonyl fluoride. The purified proteinase showed broad specificity toward oxidized insulin B including Gln4, Cys7, Glu13, Ala14, Leu15,17, Tyr16,26, Arg22, Phe24,25, and Lys29. Dominant cleavage sites of the enzyme were Tyr16-Leu17 and Phe25-Tyr26, indicating that it preferably hydrolyzed aromatic amino acids located on the P1 site. Among various substrates studied, the enzyme hydrolyzed anchovy protein to the greatest extent at 4 M NaCl. Activity increased with either CaCl2 or NaCl concentration with the maximum 2-fold increase at either 50 mM CaCl2 or 4 M NaCl. The enzyme was also highly stable up to 500 mM CaCl2 or 4 M NaCl. The proteinase showed high stability in various organic solvents (25%, v/v) including dimethylsulfoxide, methanol, acetonitrile, and ethanol. Results of peptide mass fingerprint and de novo peptide sequencing showed that the purified proteinase is a novel proteinase. The proteinase from Virgibacillus sp. SK33 could have a potential application in high ionic strength environments and aqueous-organic solvent systems.

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

The cross-linking of β-lactoglobulin (BLG) was efficiently catalysed by microbial transglutaminase (MTG) but not by fish (threadfin bream) liver transglutaminase (FTG). BLG cross-linking was inhibited by 2 mM 5-(biotinamido) pentylamine (BPNH2) and MTG incorporated BPNH2 into BLG ∼5 times more than was FTG. The glutaminyl sites for the incorporation of BPNH2 into BLG by FTG and MTG were identified using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS analyses showed that MTG and FTG incorporated 4 and 1 residues of BPNH2 per molecule of BLG, respectively. The BPNH2-tagged BLG was digested by trypsin and BPNH2-tagged peptides were selectively purified by avidin-affinity chromatography. Amino acid sequences of BPNH2-tagged peptides were identified by comparing their MALDI-TOF mass spectra with the theoretical mass profiles from the MASCOT database. The BPNH2-modification sites catalysed by MTG were glutamine (Q)13, Q68, Q15 or Q20, Q155 or Q159, whilst FTG only incorporated BPNH2 into BLG at Q68. The different reactivities between FTG and MTG might be due to the different accessibilities of these TGases to the Q residues as well as to differences in substrate specificities.

Sarcoplasmic proteins extracted from threadfin bream (TB, Nemipterus sp.) contained transglutaminase (TGase) activity exhibiting a pH optima at 7.5. Activity increased with CaCl2 and reached the maximum at 5 mmol/L, showing the Ca 2þ -dependent characteristic. TGase activity staining on native-polyacrylamide gel electrophoresis (native-PAGE) showed two fluorescent bands catalyzing incorporation of mono- dansylcadavarine (MDC) into di-methylated casein (DMC). However, both fluorescent bands showed only one major protein band with a molecular weight of about 66 kDa on sodium dodecylsulfate-PAGE (SDS-PAGE), suggesting the presence of isozymes. The TB sarcoplasmic protein (TBSP) concentrated using a 30-kDa ultrafiltration membrane induced cross-linkings of bovine serum albumin when incubated at 25 � C for 6 h. Addition of the concentrated TBSP at 1.6 g protein/100 g along with 0.1 g CaCl 2/100 g resulted in a 1.8-fold increase in the breaking force of the proteinase-laden lizardfish surimi pre

Cell-bound proteinase from Virgibacillus sp. SK37 isolated from the first month of fish sauce fermentation was characterized. The enzyme showed the maximum activity at 65 °C, pH 7.0 and 9.5, using azocasein as a substrate. The enzyme required at least 10 mmol/l Ca 2+ to effectively hydrolyze casein and the extent of casein degradation increased with Ca 2+ concentration. Ethylenediaminetetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF) largely inhibited the activity, indicating a characteristic of Ca 2+ -activated serine proteinase. Among six synthetic substrates tested, the enzyme preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, indicating a subtilisin-like proteinase. Although activity towards actomyosin at 20 g/100 ml NaCl decreased to 63% compared to at 5 g/100 ml, the enzyme showed high stability at 25 g/100 ml NaCl, 30 °C. This was the first study to report biochemical characteristics of cell-bound proteinase from a moderately halophilic bacterium isolated from fish sauce.

Transglutaminase (TGase) from the threadfin bream ([TB]Nemipterus sp.) liver was partially purified using ion exchange, size exclusion and affinity chromatography. Three protein bands with molecular weight (Mw) of 95, 63 and 43 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were observed. Only one distinct fluorescent band appeared on the TGase activity staining of the native-PAGE. When the protein band was eluted and analyzed on the SDS-PAGE, it showed a single band with an Mw of 95 kDa. The enzyme required Ca2+up to 1 mM for full activation. TGase was also activated by 10 mM Sr2+. Dithiothreitol had no effect on activity. TGase activity was unaffected by NaCl up to 0.6 M and reduced to 75% at 1.2 M NaCl. Optimum pH and temperature was 8.5–9.0 and 50C, respectively. TGase activity was markedly inhibited by the sulfhydryl reagents. The enzyme catalyzed the cross-linking of the TB myosin heavy chains. PRACTICAL APPLICATIONS Threadfin bream (TB) liver transglutaminase (TGase) exhibited Ca2+-dependent characteristic similar to muscle TGase. The enzyme catalyzed the cross-linking of myosin, and therefore, could be applied to improve the textural properties of muscle proteins. The enzyme remained its high activity up to 1.2 M NaCl (≈7% w/v), indicating its potential application in foods containing a relatively high NaCl. The biochemical characteristics of the TB liver TGase are critical information leading to more understanding in the nature of the enzyme as well as determining the optimum conditions for food applications. Moreover, the presented purification scheme could be adopted to recover TGase from the TB liver, which is currently a solid waste of the surimi industry.

Virgibacillus sp. SK33 newly isolated from one-month-old Thai fish sauce was studied for its proteinase production. Neopeptone broth (NEO), halobacterium broth (HL) and HL without either yeast extract (HL-Y), peptone (HL-P) or casamino acid (HL-C) were found to be suitable for proteinase production, whereas fish broth (FB) and skim milk salts broth (SKS) appeared to suppress proteinase production. Moreover, yeast extract, peptone and casamino acid equally stimulated proteinase production. The optimal enzyme production for Virgibacillus sp. SK33 was at 5% NaCl, 40 °C. Maximum proteinase production was achieved at 36 h and maximum cell growth was obtained at 72 h in the modified HL supplemented with only yeast extract (Ym). Extracellular proteinase of Virgibacillus sp. SK33 exhibited optimum activity at 50 °C and pH 8, 10 and 11. Crude proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF), indicating the presence of serine alkaline proteinase. The enzymes preferentially cleaved Suc-Ala-Ala-Pro-Phe-AMC, a substrate for subtilisin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) activity staining showed molecular weights of 56, 46, 42, 32, 25 and 19 kDa. All proteinases exhibited caseinolytic activity at 25% NaCl. Proteolytic activity towards synthetic substrate increased about 4 times in the presence of 25% NaCl, indicating the characteristic of NaCl-activated proteinase. In addition, crude proteinase from Virgibacillus sp. SK33 showed higher proteolytic activity towards anchovy than did commercial proteinases at 25% NaCl, demonstrating its potential for protein hydrolysis at high salt content.

The effect of CaCl 2 on aggregation and conformational changes of tropical tilapia ( Oreochromis niloticus ) actomyosin incubated at 4 and 40 °C was investigated. Aggregation of tilapia actomyosin incubated at 40 °C for 30 min increased with addition of 10–100 mM CaCl 2 . Formation of higher molecular weight protein (HMP) at 40 °C was enhanced by addition of >10 mM Ca 2+ ion, but suppressed by 2 mM N-ethylmaleimide (NEM) and 1 mM phenylmethnesulfonyl fluoride (PMSF), suggesting the involvement of endogenous transglutaminase (TGase). Moreover, addition of 10–100 mM CaCl 2 destabilized actomyosin as evident by an increase in aniline naphthalenesulfonate surface hydrophobicity (S 0 -ANS) and loss of α-helical structure at 40 °C. However, CaCl 2 only increased S 0 -ANS of actomyosin incubated at 4 °C without disturbing its secondary structure. Both e-(γ-glutamyl)lysine isopeptide bonds and hydrophobic interactions appeared to be involved in HMP aggregates formed at 40 °C. Breaking force and deformation of actomyosin gels incubated at 40 °C for 30 min increased with added CaCl 2 level and reached the maximum at 100 mM CaCl 2 , corresponding to an increased intensity of HMP observed on 5% SDS-PAGE. Ca 2+ improved gelation during setting at 40 °C by not only activating endogenous TGase but also promoting hydrophobic interactions among unfolded actomyosin. Setting was also induced to a lesser extent at 4 °C in the presence of >10 mM CaCl 2 .

BACKGROUND: Tilapia (Oreochromis niloticus) sarcoplasmic proteins contain substantial transglutaminase (TGase) activity. The enzyme catalyzes the protein cross-linking reaction, resulting in a more elastic gel. The objective was to investigate the gel-enhancing effect of sarcoplasmic proteins from tilapia as related to TGase activity. RESULTS: Total TGase activity of sarcoplasmic proteins concentrate (SpC) increased about 3.6-fold after ultrafiltration using 30 kDa membrane, but specific activity remained unchanged, indicating minimal TGase purification by ultrafiltration. Addition of 1 mg mL−1 SpC containing 40 units TGase activity induced cross-linking of tilapia actomyosin, and the extent of cross-linking increased with added level of SpC. Myosin heavy chain (MHC) and troponin were preferably cross-linked by tilapia SpC, while actin and tropomyosin were not affected. Higher retention of MHC was observed concomitantly with greater content of cross-linked protein when SpC was added to lizardfish surimi. Lizardfish surimi with 10 g kg−1 SpC added and pre-incubated at 37 °C for 1 h exhibited 91.6% and 26.7% increase in breaking force and deformation, respectively, when compared to the control. CONCLUSIONS: Residual TGase activity in SpC played an important role in catalyzing the protein cross-linking and enhancing actomyosin gelation. SpC could be a potential ingredient for improving textural properties of fish protein gel. Copyright © 2007 Society of Chemical Industry

Gel-forming ability of small scale mud carp (Cirrhiana microlepis) mince and washed mince was investigated with respect to their proteinase and transglutaminase (TGase) activities. Proteinases in sarcoplasmic fluid showed the optimum activity at 65 °C and pH 9, whereas autolytic activity was maximum at 70 °C and pH 10, indicating the presence of heat-stable alkaline proteinases. When mince was washed with three volumes of water twice, TGase and proteinases were mainly removed in the first washing cycle, resulting in a decreased autolytic activity of washed mince. Breaking force of the single washed mince gels was greater than the twice washed mince and the unwashed mince gels (p

Trypsin-like proteinases exhibited the highest activity in viscera and muscle of Indian anchovy (Stolephorus spp.). The molecular weights (MWs) of proteinases in viscera were estimated to be 31, 35, 44, 49 and 57 kDa by activity staining in the presence of 4 mol L−1 NaCl. The MW of proteinase extracted from muscle was 56 kDa. This reflects that only one proteinase was extracted from muscle whereas five proteinases were extracted from viscera. Trypsin-like, chymotrypsin-like and cathepsin L-like proteinases were found in commercial fish sauce samples throughout 12 months of fermentation, suggesting that these proteinases were stable at high salt concentrations (250–300 g kg−1 NaCl) and under acidic conditions (pH 5.2–5.8). In contrast, leucine aminopeptidase activity was detected only in the first month. The MWs of proteinases found in fish sauce after various fermentation periods were estimated to be 37, 47 and 53 kDa, which coincided with the MWs of proteinases found in Indian anchovy. Copyright © 2006 Society of Chemical Industry

The changes in anthocyanins, phenolic compounds, and antioxidant activities of Thai purple rice cooked by various cooking devices, including an electric rice cooker, an autoclave and a microwave oven, were investigated. In raw rice, cyanidin-3-glucoside (cy-3-glu) and peonidin-3-glucoside (pn-3-glu) are predominant anthocyanins, whereas protocatechuic acid (PCA) and vanillic acid (VA) are major free phenolic acids, and ferulic acid (FA) and VA are major bound phenolic acids. The microwave cooking method resulted in a marked loss of phenolics, anthocyanins and antioxidant activities (p

Transglutaminase (TGase) from Tropical tilapia (Oreochromis niloticus) was purified to electrophoretic homogeneity using successive chromatographies of DEAE-Sephacel, Sephacryl S-4 HR and HiTrap Heparin with a yield and purification-fold of 12.9% and 69.8, respectively. The molecular weight (MW) of the purified tilapia TGase was estimated to be 85 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point (pI) of tilapia TGase was 6.53. Optimal temperature and optimal pH of tilapia TGase were 37–50 °C and 7.5, respectively. Optimal concentrations of CaCl2 and dithiothreitol (DTT) were at 1.25 and 5 mM, respectively. The activity of TGase towards monodansylcadaverine (MDC) decreased as the NaCl concentration increased. Chelating agents, ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA), inhibited TGase activity. Tilapia TGase was strongly inactivated by ρ-chloromercuribenzoic acid (PCMB), N-ethylmaleimide (NEM), iodoacetamide (IAA), Cu2+, and Zn2+, suggesting a thiol group at the active site.

The effect of Ca2+ on physicochemical and conformational changes of threadfin bream (TB) myosin and actin during setting at 25 and 40°C was investigated. Ca2+ ion at 10 to 100 mM induced the unfolding of myosin and actin as evident by an increase of surface hydrophobicity (So ANS) at 40 °C. Total SH groups also decreased with an increased Ca2+ concentration, suggesting that Ca2+ promoted the formation of disulfide bonds during setting at 40 °C. Both hydrophobic interactions and disulfide linkages were involved in formation of myosin aggregates at 40 °C and were enhanced by addition of 10 to 100 mM Ca2+. Myosin Ca-ATPase activity decreased when Ca2+ was greater than 50 mM, indicating conformational changes of myosin head. Circular dichroism spectra demonstrated that Ca2+ reduced the α-helical content of myosin and actin incubated at either 25 or 40 °C. Ca2+ induced conformational changes of TB myosin and actin incubated at 40 °C to a greater extent than at 25 °C.

Transglutaminase(TGase) activity of threadfin bream mince was 99.6 units/g of dry weight. After washing and screw-pressed dewatering, 44% residual activity was retained. Covalent cross-linking of myosin heavy chain (MHC) was observed at both 25 and 40°C and supported by increased gel strength. When pre-incubation at 40°C was prolonged to 4 h, breaking force and MHC decreased due to endogenous proteinase(s). TGase activity towards MHC and synthetic substrates was effectively inhibited by iodoacetic acid (IAA). Autolytic activity and degradation of MHC was inhibited by phenylmethanesulfonyl fluoride (PMSF). Addition of 0.2% Ca(2+) significantly improved breaking force and increased MHC cross-linking of surimi gels pre-incubated at 40°C for 2 h. Keywords: transglutaminase, myosin heavy chain, cross-linking, threadfin bream.

The NaCl-activated and detergent-stable proteinases from Virgibacillus halodenitrificans SK1-3-7 isolated from fish sauce fermentation were purified and characterized. The enzymes with molecular masses of 20 and 36 kDa showed caseinolytic activity on a zymogram. Optimum azocaseinolytic activity was at 60 °C and pH 9. The proteolytic activity increased in the presence of 10 mM CaCl2 and 0.5 M NaCl and showed high stability at 0–2 M NaCl. The enzymes were stable at pH 4–10 and 10–50 °C. The enzymes preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-pNA and were completely inhibited by phenylmethanesulfonyl fluoride (PMSF), showing subtilisin-like characteristics. Activity and stability remained high in the presence of H2O2 and various surfactants. The enzymes exhibited high stability (>95 %) in various organic solvents (DMSO, butanol, ethanol, 2-propanol, and acetonitrile) at concentration of 50 %. The V. halodenitrificans SK1-3–7 proteinases showed potential as a biocatalyst in aqueous-organic solvent systems and as an additive in laundry detergent.

Proteolytic degradation of tropical tilapia surimi was biochemically and rheologically characterized to identify a group of proteinase(s) responsible for its textural degradation. Proteolysis of tilapia surimi occurred as the temperature increased and attained the highest activity at 65 °C. Smaller proteins with molecular weight of 116-97 kDa were noted as a result of myosin heavy chain (MHC) degradation. MHC completely disappeared when incu- bated at 65 °C for 4 h. Proteolysis was significantly inhibited by soybean trypsin inhibitor (SB) and leupeptin (LE). Storage modulus (G) of surimi gels mixed with either SB or LE was higher than other inhibitors indicating that serine type proteinase(s) were involved in proteolysis of tropical tilapia surimi.

Pacific whiting surimi paste was ohmically heated to investigate degradation of myosin heavy chain (MHC) caused by endogenous proteinase over a range of 40–85°C and 0.5–35 min. Degradation was best described with an apparent reaction order of 1.4. Changes of degradation rate increased with temperature and reached a maximum at 57°C. Then, rate of MHC degradation decreased with higher temperature and reached a minimum at 75°C. Ea values of activation and inactivation zone were 142.3 and 83.1 kJ/mol, respectively. Generally, failure shear stress and shear strain increased linearly with MHC content. Proteolytic degradation of actin exhibited the same trend as that of MHC but at a slower rate. The synergistic effect of actin in the gelation of whiting surimi was predominant at ≥75°C.

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.

Fish liver transglutaminase (FTG), a Ca(2+)-dependent enzyme, exhibits different characteristics from the Ca(2+)-independent microbial transglutaminase (MTG), leading to potential differences in their substrate specificity and reactivity. The ability of these enzymes to catalyze isopeptide bond formation by incorporating 5-(biotinamido)pentylamine (BPNH2) into peptides derived by tryptic digestion of threadfin bream (TB)-myosin was investigated to identify reaction sites and substrate specificity using a peptidomic strategy. BPNH2 was incorporated into TB-myosin peptides to a greater extent by MTG than FTG. Peptides derived from TB-myosin heavy chain (MHC) shared highest similarity to amberjack-MHC on the basis of a Mascot database search. Amino acid sequences and modification sites of BPNH2-tagged peptides were identified by tandem mass spectrometry based on the amberjack-MHC sequence. The BPNH2 modification sites catalyzed by both TGases were at the myosin rod. Most of the BPNH2 peptides contained charged amino acids (E, R, K) at the glutaminylamide site of reactive glutamine (Q*). The alpha-acrylamide site of Q* contained E, F, or L on peptides catalyzed by both enzymes, I, Q, or A on peptides catalyzed only by FTG, and V on a peptide catalyzed only by MTG. These results demonstrate the different structural requirements for glutaminyl substrates between these two enzymes.

Natural actomyosin (NAM) from Pacific whiting (PW) showed thermal transition temperatures by circular dichroism at 31.8 and 43.1°C, which were lower than those of threadfin bream (TB) NAM, 35.0 and 49.3°C. Endothermic transitions of PW-NAM by differential scanning calorimetry were at 31.8, 42.1 and 75.3°C, compared to 36.1, 50.9 and 78.4°C for TB-NAM. Based on surface hydrophobicity, α-helical content, and solubility, PW-NAM unfolded to a greater extent than did TB-NAM when incubated at 25°C for 4h and 40°C for 2h, suggesting its lower thermal stability. Transglutaminase generally catalyzed more extensive cross-linking of PW-myosin heavy chain (MHC) than TB-MHC, and the MHC cross-linking mediated by microbial transglutaminase (MTG) was greater than by fish transglutaminase (FTG). Textural properties of PW-NAM gels increased approximately 3.6-6.1-fold and 1.3-1.5-fold in the presence of MTG and FTG, respectively.

Virgibacillus sp. SK37 exhibited high extracellular proteolytic activity in skim milk broth containing 10% NaCl. Optimum conditions of the crude proteinase were at pH 8.0 and 65 degrees C. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, suggesting the serine proteinase with a subtilisin-like characteristic. Proteolytic activity increased with NaCl concentration up to 20%. Ca(2+) activated the enzyme activity but reduced enzyme stability at 65 degrees C. Several proteinases with dominant molecular mass (MW) of 81, 67, 63, 50, 38, and 18 kDa were detected on native-polyacrylamide gel electrophoresis (native-PAGE) activity staining in the absence and presence of 25% NaCl. These results demonstrated that Virgibacillus sp. SK37 produced salt-activated extracellular proteinases. Virgibacillus sp. SK37 could be a promising strain for starter culture development used in fish sauce fermentation.

Surface hydrophobicity, reactive and total sulfhydryl groups, circular dichroism, and storage modulus (G') revealed that unfolding of actomyosin began at 35°C, while aggregation took place at 45°C. Optimum temperature of crude transglutaminase (TGase) was found at 37 and 55°C and pH 7-7.5. TGase activity was reduced from 99 unit/g dry weight in mince to 47 unit/g dry weight after screw pressing. Gels incubated at 25°C for 4 h and 40°C for 3 h showed an increased force at failure. Degradation of myosin heavy chain (MHC) was evident when incubation time at 40°C was extended. Iodoacetic acid (IAA) was found to be the most effective TGase inhibitor. Autolytic degradation of threadfin bream was highest at 60°C. Salt had no effect on autolytic activity. Force at failure and deformation of surimi gels decreased as incubation time at 60°C was prolonged, corresponding to a decrease of MHC.

Background Microbial proteases are one of the most commercially valuable enzymes, of which the largest market share has been taken by subtilases or alkaline proteases of the Bacillus species. Despite a large amount of information on microbial proteases, a search for novel proteases with unique properties is still of interest for both basic and applied aspects of this highly complex class of enzymes. Oxidant stable proteases (OSPs) have been shown to have a wide application in the detergent and bleaching industries and recently have become one of the most attractive enzymes in various biotechnological applications. Results A gene encoding a novel member of the subtilase superfamily was isolated from Virgibacillus sp. SK37, a protease-producing bacterium isolated from Thai fish sauce fermentation. The gene was cloned by an activity-based screening of a genomic DNA expression library on Luria-Bertani (LB) agar plates containing 1 mM IPTG and 3% skim milk. Of the 100,000 clones screened, all six isolated positive clones comprised one overlapping open reading frame of 45% identity to the aprX gene from Bacillus species. This gene, designated aprX-sk37 was cloned into pET21d(+) and over-expressed in E. coli BL21(DE3). The enzyme product, designated AprX-SK37, was purified by an immobilized metal ion affinity chromatography to apparent homogeneity and characterized. The AprX-SK37 enzyme showed optimal catalytic conditions at pH 9.5 and 55°C, based on the azocasein assay containing 5 mM CaCl2. Maximum catalytic activity was found at 1 M NaCl with residual activity of 30% at 3 M NaCl. Thermal stability of the enzyme was also enhanced by 1 M NaCl. The enzyme was absolutely calcium-dependent, with optimal concentration of CaCl2 at 15 mM. Inhibitory effects by phenylmethanesulfonyl fluoride and ethylenediaminetetraacetic acid indicated that this enzyme is a metal-dependent serine protease. The enzyme activity was sensitive towards reducing agents, urea, and SDS, but relatively stable up to 5% of H2O2. Phylogenetic analysis suggested that AprX-SK37 belongs to a novel family of the subtilase superfamily. We propose the name of this new family as alkaline serine protease-X (AprX). Conclusions The stability towards H2O2 and moderately halo- and thermo-tolerant properties of the AprX-SK37 enzyme are attractive for various biotechnological applications.