Enzymatic debittering of food protein hydrolysates.

Protein hydrolysates have a range of applications in the food and allied healthcare sectors. Bitterness is a negative attribute associated with most food protein hydrolysates. The development of biotechnological solutions for hydrolysate debittering is ongoing. Specific enzymatic debittering strategies have focused on the application of proline specific exo- and endopeptidases given the contribution of proline residues to peptide/hydrolysate bitterness. Hydrolysate manufacturing conditions may also play an important role in bitterness development. Practical solutions to hydrolysate debittering are likely to involve judicious choice of enzymatic processing conditions in conjunction with the use of peptidase activities having targeted hydrolytic specificity.

Cheddar cheese cooking temperature induces differential lactococcal cell permeabilization and autolytic responses as detected by flow cytometry: implications for intracellular enzyme accessibility.

AIMS: To determine the influence of cheese cooking temperature on autolysis and permeabilization of two lactococcal starter strains in broth and in Cheddar cheese juice during ripening. METHODS AND RESULTS: Flow cytometry (FCM) was used to identify and enumerate intact and permeabilized cells in broth and in Cheddar cheese juice. Levels of intracellular enzyme activities were quantified concurrently. Permeabilized cell numbers increased for both strains in broth following a temperature shift from 32 to 38 degrees C and was accompanied by an increase in the level of accessible intracellular enzyme activities. The relative proportions of intact and permeabilized cell populations, as detected by FCM in cheese juice, changed during 42-day ripening. Permeabilized cell populations increased during ripening for both strains; however, an increase in accessible intracellular enzyme activity was observed only for the highly autolytic strain Lactococcus lactis AM2. CONCLUSIONS: Differences in the autolytic and permeabilization response induced by cooking temperature in two lactococcal strains affects intracellular enzyme accessibility in Cheddar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the importance of the autolytic and permeabilization properties of lactic acid bacteria starter strains and their impact on cheese ripening.

Dipeptidyl aminopeptidase IV and aminopeptidase P, two proline specific enzymes from the cytoplasm of guinea-pig brain: their role in metabolism of peptides containing consecutive prolines.

In this study the majority of dipeptidyl aminopeptidase IV and aminopeptidase P activities of guinea-pig brain are reported to reside in the cytoplasm. Both activities were purified and soluble dipeptidyl aminopeptidase IV was found to have a relative molecular mass of 194000 and to be comprised of two equal subunits of relative molecular mass 93000 while native soluble aminopeptidase P had a relative molecular mass of 140000. Both activities require proline or alanine in the penultimate position from the N-terminus. Dipeptidyl aminopeptidase IV removed the N-terminal dipeptide whereas aminopeptidase P removed only the N-terminal amino acid. Dipeptidyl aminopeptidase IV was inactive if proline was also present in the third position from the N-terminus whereas aminopeptidase P was unable to remove the N-terminal glycyl, pyroglutamyl or prolyl residues even though proline was present in the second position. Soluble dipeptidyl aminopeptidase IV was differentiated from the previously reported particulate form by its sensitivity to p-chloromercuribenzoate, N-ethyl maleimide and puromycin. The metabolism of Leu-Pro-Pro-Ser by guinea-pig cytoplasm was investigated in the presence of inhibitors to evaluate the contribution by dipeptidyl aminopeptidase IV and aminopeptidase P to the hydrolysis of a peptide containing two consecutive proline residues. The results indicated that either dipeptidyl aminopeptidase IV or prolyl oligopeptidase were required along with aminopeptidase P and prolidase to achieve complete hydrolysis of this tetrapeptide.

Purification and characterization of a lysine-p-nitroanilide hydrolase, a broad specificity aminopeptidase, from the cytoplasm of Lactococcus lactis subsp. cremoris AM2.

A hydrolase activity that cleaves lysyl-p-nitroanilide (Lys-pNA) has been purified from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 by chromatography on DE52, DEAE Affi-Gel Blue Gel, Hydroxyapatite Bio-Gel HTP and Phenyl Sepharose. The purified aminopeptidase was found to have a native M(r) of 50,000-55,000 by gel filtration chromatography and by FPLC gel filtration on Superose 12 and to be composed of a single polypeptide chain following SDS-PAGE. Enzyme activity was almost completely inhibited by EDTA, amastatin, puromycin and bestatin, while the sulphydryl-reactive agents p-chloromercuribenzoate and iodoacetamide were inhibitory. The enzyme was found to be very unstable during the purification procedures at 4 degrees C and its stability was greatly improved when 10 ml glycerol/l and 2 mM-dithiothreitol were included in the purification buffers. The purified enzyme was found to hydrolyse a wide range of dipeptides, tripeptides and longer peptides provided that proline was not present in the penultimate position from the N-terminus or that a pyroglutamyl residue was not present at the N-terminus. While neither Asp-pNA nor Pro-pNA was hydrolysed by the purified enzyme, the release of N-terminal acidic residues from peptides was observed in addition to the release of N-terminal proline from Pro-Leu-Gly-NH2, Pro-Leu-Gly-Gly and Pro-His-Pro-Phe-His-Leu-Phe-Val-Tyr. This ability of Lys-pNA hydrolase to release N-terminal proline residues was employed in concert with a purified aminopeptidase P preparation to release alternate N-terminal amino acids from Tyr-Pro-Phe-Pro-Gly. The complementary action of these enzymes represents an alternative mechanism to that of post-proline dipeptidyl aminopeptidase for metabolism of proline-containing peptides.

Hydrolysis of alphas1- and beta-casein-derived peptides with a broad specificity aminopeptidase and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2.

Aminopeptidase hydrolysis of alpha(s)1 - and beta-casein-derived synthetic peptides containing non-consecutive and consecutive proline residues was characterised. Aminopeptidase P (Pep P) (EC 3.4.11.9) or post-proline dipeptidyl aminopeptidase (PPDA) (EC 3.4.14.5) along with lysine-paranitroanilide hydrolase (KpNA-H) (EC 3.4.11.1) activities are required in the degradation of peptides containing non-consecutive proline residues. However, both Pep P and PPDA along with KpNA-H are required for hydrolysis of peptides containing consecutive proline residues. The results demonstrate the mechanism by which combinations of purified general and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2 hydrolyse peptides containing proline residues.

Purification and characterization of aminopeptidase P from Lactococcus lactis subsp. cremoris.

Aminopeptidase P was purified 65.3-fold from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 with a 5.8% yield. The purified enzyme was found to consist of one polypeptide chain with a relative molecular mass of 41,600. Metal chelating agents were found to be inhibitory and Mn2+ and Co2+ stimulated activity 7-fold and 6-fold respectively. The purified enzyme removed the N-terminal amino acid from peptides only where proline (and in one case alanine) was present in the penultimate position. No hydrolysis was observed either with dipeptides even when proline was present in the C-terminal position or when either N-terminal proline or pyroglutamate was present preceding a proline residue in the penultimate position of longer peptides. On the basis of this substrate specificity either aminopeptidase P or post-proline dipeptidyl aminopeptidase are necessary along with a broad specificity aminopeptidase to effect complete hydrolysis of casein-derived peptides containing a single internally placed proline residue. However, both aminopeptidase P and post-proline dipeptidyl aminopeptidase would be required together with a broad specificity aminopeptidase in order to completely hydrolyse casein-derived peptides that contain two internally placed consecutive proline residues. As bitter casein-derived peptides are likely to contain either single prolines or pairs of prolines, aminopeptidase P appears to be an important enzyme for debittering.

Alanine aminopeptidase of guinea-pig brain: a broad specificity cytoplasmic enzyme capable of hydrolysing short and intermediate length peptides.

Alanine aminopeptidase is reported to be a broad specificity aminopeptidase acting on peptides of different lengths. In this study we wish to define the properties of the activity from guinea-pig brain and compare these properties with previous findings. Alanine amino-peptidase was purified from cytoplasm of guinea-pig brain by a four-step procedure involving chromatography on DE-52, hydroxylapatite, Sephacryl S-200 and DEAE-Sephacryl. Relative molecular mass was determined by chromatography on Sephacryl S-200 column and subunit size determined by SDS-PAGE under denaturing conditions. Cations which reactivate the enzyme were determined with EDTA treated enzyme. Substrate specificity was determined by TLC and kinetic parameters were derived from Lineweaver-Burk plots. A 216-fold purification was achieved by the above procedures. The purified enzyme was found to consist of one polypeptide chain with a relative molecular mass of 104,000. Its activity was inhibited by chelating agents, sulphydryl reactive agents, puromycin, bestatin and amastatin but stimulated over 6-fold by dithiothreitol. Some dipeptides and all tripeptides and longer peptides containing up to 16 amino acids tested were hydrolysed provided neither Glp or Pro occurred at the N-terminus or that Pro did not occur in the penultimate position from the N-terminus. The enzyme preferred bulky non-polar residues at the N-terminal and penultimate positions and was found to hydrolyse three dipeptidyl methyl coumarin amides used in detecting dipeptidyl aminopeptidases. Alanine aminopeptidase is thus a broad specificity amino-peptidase acting on short and intermediate length peptides whose affinity for substrates increases with increasing peptide length. Its properties are well suited to a role in peptide turnover in brain cytoplasm.

Dipeptidyl aminopeptidase III of guinea-pig brain: specificity for short oligopeptide sequences.

A dipeptidyl aminopeptidase III-type activity has been purified from the cytoplasm of guinea-pig brain using arginyl-arginyl-7-amido-4 methylcoumarin as substrate. The enzyme was purified 754-fold relative to the crude homogenate and with a 12.7% recovery. The purified enzyme was found to have a relative molecular weight of 85,000 and consists of one polypeptide chain of relative molecular weight 80,000, on the basis of its migration on calibrated sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel. It is highly sensitive to the presence of chelating agents, sulphydryl reactive agents, and the dipeptide Tyr-Tyr. Dithiothreitol (1 mM) reduced activity by 28%, and 36 and 65% inhibition was noted with phenylmethylsulphonyl fluoride and puromycin (both at 1 mM), respectively. Little or no inhibition was observed with bestatin, bacitracin, captopril, amastatin, and arphamenine B. The purified enzyme released dipeptide moieties from a wide range of peptides including enkephalin sequences and also angiotensin sequences up to the octapeptide angiotensin II. These sequences inhibited the hydrolysis of arginyl-arginyl-7-amido-4-methylcoumarin by dipeptidyl aminopeptidase III with Ki values in the micromolar range. No hydrolysis was observed with angiotensin I or with peptide sequences containing more than 10 amino acids. No hydrolysis was observed also with peptide sequences containing a Pro residue on either side of the sissile bond. Peptides containing less than four amino acids were not hydrolysed.

Dipeptidyl aminopeptidase activities of guinea-pig brain.

1. The subcellular distribution of dipeptidyl aminopeptidase activities in guinea-pig brain was investigated. Our studies show that DAP I (Gly-Arg-NH-Mec hydrolase) type activity was found to have an acidic optimum and was associated with the nuclear pellet. 2. No DAP II (Lys-Ala-NH-Mec hydrolase) type activity could be detected. Apparent hydrolysis was mainly due to aminopeptidase activity. 3. DAP III (Arg-Arg-NH-Mec hydrolase) type activity is largely cytoplasmic, but there was evidence of a membrane form associated with the synaptosomes. 4. DAP IV (Gly-Pro-NH-Mec hydrolase) type activity is present on the synaptosomal membrane, and also enriched in the microsomes. A soluble form of Gly-Pro-NH-Mec hydrolase activity is also present in the cytoplasm. Whether this activity is a DAP II or IV type activity is still yet to be determined.

Degradation of thyrotropin-releasing hormone and luteinising hormone-releasing hormone by enzymes of brain tissue.

In this article, the enzymes of brain and associated tissues that can degrade thyrotropin-releasing hormone (TRH) and luteinising hormone-releasing hormone (LH-RH) are reviewed. As both TRH and LH-RH are considered to act as neurotransmitters or neuromodulators in the CNS, attention is paid to the subcellular location of the enzymes described and how their topographies and substrate specificities fit them to playing roles as inactivating agents for TRH and LH-RH or as regulators of intracellular concentrations of TRH and LH-RH. Consideration is also given to enzymes involved in biotransformation of TRH to secondary metabolites that exhibit biological activity and to enzymes involved in the metabolism of secondary metabolites.