Debittering and Hydrolysis of a Tryptic Hydrolysate of β-casein with Purified General and Proline Specific Aminopeptidases from Lactococcus lactis ssp. cremoris AM2

ABSTRACT: In this study, purified β-casein was hydrolysed with trypsin to produce a bitter substrate. The role of 3 aminopeptidases, a general aminopeptidase lysyl- para -nitroanilide hydrolase (KpNA-H), X-prolyl dipeptidyl aminopeptidase (Pep X) and aminopeptidase P (Pep P) each purified from Lactococcus lactis ssp. cremoris AM2, in the hydrolysis and debittering of the tryptic hydrolysate of β-casein, was then studied. The hydrolysates were analyzed for percentage degree of hydrolysis (DH%) and bitterness score. Results indicate that the hydrolysis and debittering potential of the general aminopeptidase (KpNA-H) is limited in the absence of proline specific aminopeptidases. Statistically significant (p < 0.001) reductions in bitterness were obtained following incubation of the tryptic digest of β-casein with specific combinations of the above aminopeptidases.     

Debittering of a Tryptic Digest of Bovine p-casein Using Porcine Kidney General Aminopeptidase and X-Prolydipeptidyl Aminopeptidase from Lactococcus lactis subsp. cremoris AM2

ABSTRACT: The role of leucine aminopeptidase (LAP) from pig kidney cytosol and X-prolyldipeptidyl aminopeptidase (Pep X) from Lactococcus lactis subsp. cremoris AM2 in the hydrolysis and debittering of a tryptic digest of β-casein was studied. Hydrolysis was monitored by quantifying the release of primary amino groups and bitterness by use of a trained sensory panel. Sequential incubation of the bitter tryptic hydrolysate with LAP, Pep X and LAP resulted in higher levels of hydrolysis and significantly (P < 0.001) lower levels of bitterness than incubation with LAP alone. The results demonstrate the central role proline-specific aminopeptidases can play in the hydrolysis and debittering of food protein hydrolysates.     

Constituents isolated from Glehnia littoralis suppress proliferations of human cancer cells and MMP expression in HT1080 cells

Crude extract and solvent-partitioned fraction of Glehnia littoralis were found to possess different anti-proliferative effects against AGS, HT1080 and U937 human cancer cells. The crude extracts and solvent fractions dose-dependently inhibited cell proliferation. Especially, n-hexane and 85% aqueous MeOH fractions exhibited comparatively higher anti-proliferative effects and reduced expressions of Bcl-2, COX-2 and iNOS genes. Systematic separation of all solvent fractions by chromatographic methods led to the isolation of three glucopyranosides, four furanocoumarins and two polyacetylenic alcohols. All the nine compounds were evaluated for their inhibitory effects against both proliferation of human cancer cells and expressions of MMP-2 and -9 in HT1080 cells. Two polyacetylenic alcohols exerted the highest inhibitory activity against both human cancer cell lines, and MMP-2 and -9. These results suggest that G. littoralis may possibly be used as a valuable chemopreventive agent or food supplement for reducing cancer risk.     

Contribution of muscle aminopeptidases to flavor development in dry-cured ham

The activity of muscle aminopeptidases (alanyl, arginyl, leucyl and pyroglutamyl aminopeptidases) have been assayed along the processing of dry-cured ham. The generation of free amino acids resulting from aminopeptidase action on N-terminal of proteins and peptides has been also analyzed. The assayed aminopeptidases, except pyroglutamyl aminopeptidase, showed good stability. Alanyl and arginyl aminopeptidases have optimal neutral pH near the pH in ham and, in addition, their spectrum of activity against terminal amino acids is in coincidence with the observed release of free amino acids in ham. So, both aminopeptidases appear to be the main contributors to the generation of free amino acids during the processing of dry-cured ham.     

Aminopeptidase Activities from Lactobacillus sake in Models of Curing Ingredients and Processing Conditions for Dry Sausage

The effect of curing agents and dry sausage processing parameters on four aminopeptidases (AP 1, AP 2, AP 3, and AP 4) of Lactobacillus sake was investigated. NaCl exerted a strong inhibitory effect against AP 1 but activated the remaining aminopeptidases. NaNO₃ caused inhibition of AP 3 and AP 4. Ascorbic acid inhibited AP 1 and AP 3 while glucose only reduced AP 3 activity. Water activities and processing temperatures affected activity of all aminopeptidases. Acid pH decreased aminopeptidase activity except for AP 4. The combined effects of curing agents and process parameters to simulate different stages during dry sausage processing indicated that AP 2 and AP 4 may remain active throughout the process.     

Characterisation of leucyl aminopeptidase from Solanum tuberosum tuber

Potato juice (a waste product from the starch industry) is a potential source of novel enzymes for food applications. For use in the production and improvement of food protein hydrolysates, commercially available exopeptidases, predominantly aminopeptidases, are recommended. The present study was performed to explore possible biotechnological interest of leucyl aminopeptidase (LAP) activity in the potato tuber. The LAP from potato tuber was purified and characterised. Specific LAP activity was increased 200-fold by purification of the crude extract. The purified enzyme had a pH optimum of 9.0 and temperature optimum of 45 °C. LAP hydrolysed leucine-, alanine- and lysine-p-nitroanilide to a similar degree. The most efficient inhibitor was 1,10-phenanthroline. Almost all divalent cations tested inhibited the enzyme activity, while Co²⁺ stimulated LAP activity by over 100%. The purified LAP had a molecular weight of 90 kDa with an isoelectric point of 5.45. Sodium dodecylsulfate–polyacrylamide gel electrophoresis revealed one band of 48 kDa.     

Comparison of the effects of various attenuation methods on cell permeability and accessibility of intracellular enzymes in Lactococcus lactis strains

Three Lactococcus lactis cheese starter cultures were exposed to various cell attenuation methods including sonication, chemical treatments, heat-shock and freeze-shock. Treated cells were subsequently assessed for changes in cell permeability and accessibility to the intracellular peptidases Pep X and Pep N. Generally, for all three strains, as the duration of sonication increased, the levels of intracellular peptidases in the cell free extract increased. For the other treatments, cell pellets of strains were prepared and treated with isopropyl alcohol (IPA), sodium dodecyl sulphate (SDS), hexadecyltrimethylammonium bromide (CTAB), heated over the range 45–65 °C or freeze-shocked using liquid nitrogen. The effects of various treatments on cell permeability were assessed using flow cytometry after staining with the Live/Dead^® BacLight? reagent kit. Treatment of cell pellets with CTAB generated a unique and highly permeabilized sub-population having the highest levels of accessible intracellular enzyme activity. Treatment with SDS also resulted in highly permeabilized cells and enhanced accessibility to Pep X or Pep N. The findings in this study are significant for the production of permeabilized L. lactis cell fractions having enhanced accessibility to intracellular enzymes.     

Immunogenicity and contraceptive potential of three infertility-relevant zona pellucida 2 epitopes in the marsupial brushtail possum (Trichosurus vulpecula)

In a previous study, three infertility-relevant epitopes of possum ZP2 (Pep12 (amino acids 111-125), Pep31 (amino acids 301-315), and Pep44 (amino acids 431-445)) were identified using sera from possums (Trichosurus vulpecula) immunized with recombinant possum zona pellucida 2 (ZP2) constructs, and a synthetic peptide library of possum ZP2 protein. In this study, the three peptides were conjugated to keyhole limpet hemocyanin and 300 mug of each conjugated peptide were administered subcutaneously to female possums (n = 20 per peptide) in complete Freund's adjuvant. Immunogen doses were repeated 3 and 6 weeks later using incomplete Freund's adjuvant. Control animals were immunized with either phosphate-buffered saline only (n = 10) or 300 mug keyhole limpet hemocyanin (n = 10), administered with the same adjuvants. Serum antibodies from animals immunized against these three epitopes bound to the corresponding possum ZP2 peptides, recombinant possum ZP2 protein constructs, and native zona. Possum fertility was assessed following superovulation and artificial insemination. Peptides Pep12 and Pep31 had no significant effects on fertility parameters (P > 0.05). However, animals immunized with Pep44 had lower egg fertilization rates (immunized 19.5% versus control 60.5%, P < 0.05) and produced significantly fewer embryos than control animals (immunized 0.5 embryos versus control 2.4 embryos, P < 0.05). The number of Pep44-immunized females that produced embryos was reduced by 64%. Identification and characterization of possum infertility-relevant epitopes on possum ZP2 protein will assist development of safe, humane, and possum-specific immunocontraceptive vaccines for controlling the introduced possums in New Zealand.     

Application of High Pressure for Selective Activity Regulation of Starter Cultures Aminopeptidases Involved in Ripening of Brined Cheeses

The combined effect of high pressure processing and temperature on aminopeptidases activity of lactic acid bacteria used as starter cultures in brined cheese manufacturing, in order to find the optimum process conditions for acceleration of the significant long-duration cheese ripening step, was investigated.The effect of high hydrostatic pressure (HP) (100–450 MPa) combined with temperature (20–40 °C) on the activity of five aminopeptidases (PepN, PepX, PepY, PepC, and PepA) of Streptococcus thermophilus ACA-DC 0022 and Lactococcus lactis ACA-DC 0049, used as the starter culture for white Greek brine cheese (feta) production, was studied. S. thermophilus aminopeptidases PepN, PepX, PepA, and PepC were activated at pressures up to 200 MPa, and all studied temperatures (20–40 °C), while for L. lactis, PepN, X, and Y were activated at pressures up to 300 MPa and temperatures up to 30 °C and PepA at the same temperature range but milder pressures (up to 200 MPa). For L. lactis, PepC an increase in activity was observed at all studied pressures but only at 20 °C. A multi-parameter equation was used for predicting the activation of all aminopeptidases in the pressure and temperature domain. Overall, processing at 200 MPa and 20 °C may be selected as the optimum conditions for maximum activation of all aminopeptidases of both S. thermophilus ACA-DC 0022 and L. lactis ACA-DC 0049. A 20-min treatment at these conditions leads to an average threefold increase in activity which could lead to better and faster maturation of white cheese.     

Enzymatic hydrolysis of hard‐to‐cook bean (Phaseolus vulgaris L.) protein concentrates and its effects on biological and functional properties

Inadequate postharvest handling and storage under high temperature and relative humidity conditions produce the hard‐to‐cook (HTC) defect in beans. However, these can be raw material to produce hydrolysates with functional activities. Angiotensin I‐converting enzyme (ACE) inhibitory and antioxidant capacities were determined for extensively hydrolysed proteins of HTC bean produced with sequential systems Alcalase‐Flavourzyme (AF) and pepsin–pancreatin (Pep‐Pan) at 90 min ACE inhibition expressed as IC₅₀ values were 4.5 and 6.5 mg protein per mL with AF and Pep‐Pan, respectively. Antioxidant activity as Trolox equivalent antioxidant capacity (TEAC) was 8.1 mm  mg^?1 sample with AF and 6.4 mm  mg^?1 sample with Pep‐Pan. The peptides released from the protein during hydrolysis were responsible for the observed ACE inhibition and antioxidant activities. Nitrogen solubility, emulsifying capacity, emulsion stability, foaming capacity and foam stability were measured for limited hydrolysis produced with Flavourzyme and pancreatin at 15 min. The hydrolysates exhibited better functional properties than the protein concentrate.     

Distribution of microbial flora,intracellular enzymes and compositional indices throughout a 12 kg Cheddar cheese block during ripening

Two studies were carried out to investigate the distribution of microbial populations, intracellular enzymes and compositional indices within 12 kg Cheddar cheese blocks. In Study A, at day 21, starter and non-starter lactic acid bacteria viability in the top sub-sections were lower than middle sub-sections, but at day 98 this trend was reversed. Lactate dehydrogenase (LDH) activity in cheese juice was significantly higher in the middle sub-sections of the cheese block, but for Pep X activity there was no apparent trend. In Study B, at day 1 and day 14 of ripening, pH values were greater in the top sub-sections compared with the middle sub-sections. Over ripening, percentage salt-in-moisture was significantly greater in middle sub-sections of the cheese block. A paired comparison of pooled data indicated significantly higher LDH activity in the middle sub-sections, with Pep X activity significantly higher in the top sub-sections of the cheese block. No differences were found in the numbers of either live or dead cells in cheese juice expressed from top or middle sub-sections of cheese blocks.     

Kinetic Study of the Combined Effect of High Hydrostatic Pressure and Temperature on the Activity of Lactobacillus delbrueckii ssp. bulgaricus Aminopeptidases

ABSTRACT:  The effect of high hydrostatic pressure (HHP) (100 to 700 MPa) combined with temperature (20 to 40 °C) on the activity of 5 aminopeptidases (PepN, PepX, PepY, PepC, and PepA) of Lactobacillus delbrueckii ssp. bulgaricus ACA-DC 0105, used as starter culture for feta cheese production, was studied. Aminopeptidases PepN, PepX, and PepA were activated at pressures up to 200 MPa, at temperatures up to 40 °C. PepY and PepC appeared to be more sensitive to pressure and temperature treatment leading to inactivation for pressures above 100 and 200 MPa, respectively, combined with temperature above 30 °C. A multi-parameter equation was used for predicting the activation of PepN, PepX, and PepA aminopeptidases in the pressure and temperature domain. Overall, processing at 200 MPa and 20 °C may be selected as the optimum conditions for maximum activation of 4 out of 5 aminopeptidases of L. delbrueckii ssp. bulgaricus. A 20-min treatment at these conditions leads to an average 3-fold increase in activity, which could lead to better and faster maturation of white cheese.     

Membrane‐disruptive property of a novel antimicrobial peptide from anchovy (Engraulis japonicus) hydrolysate

A novel antimicrobial peptide named Pep39 has been isolated from anchovy hydrolysate using ÄKTA protein liquid chromatography and reverse‐phase high‐performance liquid chromatography. Its amino acid sequence was RLFRHAFKAVLRL with a molecular weight of 1626.92. Pep39 demonstrated antimicrobial ability against Escherichia coli ATCC 25922, Salmonella typhi ATCC 50013, Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 9372, with minimal inhibitory concentration (MIC) ranging from 4 to 32 μg mL^?1. No cytotoxicity to mouse red blood cells was observed when its concentration was lower than 30 μg mL^?1. Pep39 induced the influx of fluorescent probe 8‐Anilino‐1‐naphthalenesulfonic acid and the outflow of β‐galactosidase by increasing E. coli outer and inner membrane permeabilities, respectively. Flow cytometric analysis also demonstrated that Pep39 disrupted E. coli cell membrane. These results suggested that the antimicrobial mechanism of Pep39 involved cytoplasmic membrane damage.     

Schizosaccharomyces pombe Pep12p is required for vacuolar protein transport and vacuolar homotypic fusion

In eukaryotic cells, SNARE proteins are essential for intracellular vesicle trafficking. Several SNARE proteins are required for vacuolar protein transport and vacuolar biogenesis in Saccharomyces cerevisiae. Previously we demonstrated that one of the fission yeast SNARE proteins, Pep12p, is not required for vacuolar fusion process in Schizosaccharomyces pombe. We have re-examined the function of S. pombe Pep12p using the newly created pep12(+) deletion strain. Deletion of the fission yeast pep12(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y-and various ion- and drug-sensitivities. GFP-Pep12 fusion protein is mostly localized at the vacuolar membrane and the prevacuolar compartment. The S. pombe pep12Δ mutation phenocopies that of vps33Δ, suggesting that both Pep12p and Vps33p act at the same membrane fusion step in S. pombe, and both mutations cause vacuolar deficiency.     

Purification and properties of two intracellular aminopeptidases produced by Brevibacterium linens SR3

Two aminopeptidases, designated as aminopeptidase I (API) and aminopeptidase II (APII), were isolated from the disrupted cells of Brevibacterium linens SR3 and purified to homogeneity by a combination of Phenyl-Sepharose, DEAE-Sepharose, Q-Sepharose and Sepharose CL-6B chromatography. Optimal temperature for enzymatic activity was 45°C for API and 37°C for APII, and the pH optimum was found to be 8.5 for API and 8.0 for APII. Divalent metals ions like Co²⁺ and Ni²⁺ increased enzymatic activity while Ca²⁺, Cu²⁺ and Fe²⁺ had an inhibitory effect. The enzymes were strongly inhibited by EDTA, 1,10-Phenantroline and cysteine, indicating that both aminopeptidases were metalloenzymes. The K_m values of API and APII for l-leucine-p-nitroanilide were 1.0 and 0.25 mm, respectively. Native molecular masses of aminopeptidases I and II were 80 and 220 kDa after gel filtration chromatography while molecular masses of 14 and 18 kDa, were seen, after SDS-polyacrylamide gel electrophoresis.     

Development of a strain for efficient degradation of polychlorinated biphenyls by patchwork assembly of degradation pathways

Rhodococcus jostii RHA1 accumulates chlorobenzoates (CBA) during the degradation of polychlorinated biphenyls (PCBs). CBA degradation is considered one of the rate-limiting steps in the complete degradation of PCBs. To reduce the accumulation of CBAs, the upper pathway enzyme genes for PCB degradation of RHA1 were introduced into a CBA-degrading bacterium, Burkholderia sp. NK8. The resulting recombinant strain exhibited no biphenyl 2,3-dioxygenase (BphA) activity encoded by bphAaAbAcAd genes, which encode the large and small subunits of the terminal oxygenase component and the ferredoxin and reductase subunits responsible for electron transfer from NADH to the large subunit. The remaining enzyme genes involved in the transformation of biphenyl to benzoate, bphB2C1D1, which encode dehydrogenase, ring-cleavage dioxygenase and hydrolase, conferred activities to NK8. To obtain the BphA activity of RHA1 in NK8, sets of BphA genes were constructed by combining the bphAaAbAcAd genes of RHA1 and bphA3A4 of Pseudomonas pseudoalcaligenes KF707, encoding the ferredoxin and reductase subunits. Hybrid derivatives of BphA containing the KF707 bphA3 conferred BphA activity to NK8, and a derivative containing the RHA1 bphAaAb and KF707 bphA3A4 genes exhibited the highest BphA activity. A plasmid containing the RHA1 bphAaAb and KF707 bphA3A4 genes plus the RHA1 bphB2C1D1 genes was constructed and introduced into NK8. The resulting recombinant strain efficiently degraded 2-, 3- and 4-chlorobiphenyls with an apparent reduction in CBA accumulation in comparison to the recombinant mutant strain, which had an insertion in the cbeA gene to inactivate CBA dioxygenase.     

Matrix metalloproteinases (MMPs) inhibitory effects of an octameric oligopeptide isolated from abalone Haliotis discus hannai

Abalone (Haliotis discus hannai) is a marine gastropod, and an important fishery and food industrial resource that is massively maricultured in Asia, Africa, Australia and America. However, its health benefits have rarely been studied for nutraceutical and pharmaceutical application. In this study, the purified abalone oligopeptide (AOP) with anti-matrix metalloproteinases (anti-MMPs) effects was isolated from the digests of abalone intestine using recycle HPLC with a JAI W253 column and an OHpak SB-803 HQ column. The AOP was identified as Ala-Glu-Leu-Pro-Ser-Leu-Pro-Gly (MW = 782.4 Da) with a de novo peptide sequencing technique using a tandem mass spectrometer. The AOP exhibited a specific inhibitory effect against MMP-2/-9 activity and attenuated protein expression of p50 and p65 in the human fibrosarcoma (HT1080) cells, dose-dependently. The results presented illustrate that the AOP could inhibit MMP-2/-9 expression in HT1080 cells via the nuclear factor-kappaB (NF-κB)-mediated pathway. This data suggest that the AOP from H. discus hannai intestine may possess therapeutic and preventive potential for the treatment of MMPs-related disorders such as angiogenesis and cardiovascular diseases.     

Comparison of Proteolytic Enzyme Levels in Chicken,Pig, Lamb and Rabbit Muscle at Point of Slaughter: Role in Meat Tenderisation post mortem

In order to develop a clearer understanding of the biochemical mechanism underlying the process of meat tenderisation, a comparison was made of the levels of a comprehensive range of protein catabolising proteolytic enzymes in muscle from animal species known to differ markedly in rates of meat tenderisation, ie chicken (breast and thigh muscles), pig, lamb and rabbit ( longissimus dorsi muscles). Proteolytic enzyme activities were determined at point of slaughter: (i) for the following protease types using optimized specific fluorimetric assays: alanyl-, arginyl-, leucyl-, and pyroglutamyl aminopeptidases, dipeptidyl aminopeptidases III and IV, tripeptidyl aminopeptidase, proline endopeptidase, calpain and macropain (cytoplasmic proteases); dipeptidyl aminopeptidases I and II, cathepsins B, D H and L (lysosomal proteases)); (ii) using tissue homoge-nate time course assays to measure rates of structural protein degradation (via analytical electrophoresis) by endogenous cytoplasmic or lysosomal proteases. Although chicken (the most rapidly tenderising) muscles contained the highest levels of activity for most proteases measured, there was no general relationship between muscle proteolytic capacity and previously published meat tenderisation rates for the other species investigated. It would therefore appear that known differences in the rate of tenderisation in different species cannot be rationalised solely in terms of species specific differences in muscle proteolytic capacity and, whilst the latter is of importance in the tenderisation process, other potential factors should be taken into consideration.     

Biogenic Polyamines Affect Activity of Aminopeptidase B and Alanyl Aminopeptidase from Porcine Skeletal Muscle

The effect of biogenic polyamines (agmatine, cadaverine, putrescine, spermidine, and spermine) on the activity of pork muscle aminopeptidase and alanyl aminopeptidase was investigated. Agmatine (5 mM) showed a powerful inhibitory effect for both enzymes (more than 50% inhibition). Cadaverine and less intensely putrescine showed similar inhibitory effects for both enzymes. All assayed polyamines inhibited activity of both aminopeptidases except spermine and spermidine which did not affect aminopeptidase B. Thus, one of these inhibitors could be used to avoid the interference of alanyl aminopeptidase when assaying aminopeptidase B in muscle extracts.     

Survey of Rabbit Skeletal Muscle Peptidases Active at Neutral pH Regions

A survey of oligopeptide hydrolases active at neutral pH regions responsible for increased free ammo acids on aging of high ultimate pH muscles were performed examining chromatographic behavior and substrate specificity of rabbit skeletal muscle extract. The DEAE-cellulose chromatography of muscle extract revealed five major activity peaks respectively ascribable to a dipeptidase, an aminotripeptidase, an ammopeptidase, leucine aminopeptidase and the 160,000 dalton-aminopeptidase purified previously by us. Since the 160,000 dahon-ammopeptidase showed a broader substrate specificity than the two other aminopeptidases, it is most likely that this enzyme shares the largest part in the tetrapeptide hydrolysis in rabbit skeletal muscle at neutral pH regions.