The intracellular proteolytic system of Yarrowia lipolytica and characterization of an aminopeptidase

Intracellular proteases of Yarrowia lipolytica have been scarcely studied. These enzymes may play an important role in nitrogen metabolism, posttranslational processing, nutritional stress, dimorphism, etc.; biochemical and genetic control of these enzymes can help in obtaining high-level expression of recombinant proteins in heterologous systems. In this study, we report the presence of three proteases: aminopeptidase yylAPE, carboxypeptidase yylCP and dipeptidyl aminopeptidase yylDAP, measured under several nutritional conditions. Yarrowia lipolytica produced the highest level of intracellular proteolytic enzymes, i.e. yylAPE, yylCP and yylDAP, in media with peptone during stationary growth phase. When soluble extracts were subjected to PAGE, and the three activities were revealed in gels with the corresponding substrates, only one band of activity was detected for each one. The three enzymes were affected by serine protease inhibitors. Chelating agents affected mainly APE activity. The aminopeptidase was purified by selective fractionation with ammonium sulfate and three chromatographic steps (anion exchange, hydrophobic interaction and gel filtration chromatography). The enzyme had a molecular mass of 97 kDa; optimal pH and temperature were 7.0 and 37 degrees C, respectively. The aminopeptidase showed a preference for lysine in the N-position. The K(m) value was 0.86 microM and V(max) value was 990.8 micromoL min(-1) mg(-1) for Lys-pNA.     

Purification and characterization of a dipeptidase from Lactobacillus sake.

A dipeptidase was purified from cell extracts of Lactobacillus sake. This compound was a monomer having a molecular weight of 50,000 and a pI of 4.7 and exhibited broad specificity against all dipeptides except those with proline or glycine at the N terminus. The enzyme was inhibited by EDTA or 1,10-phenanthroline but could be reactivated with CoCl2 and MnCl2.     

Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1

Sourdough lactic acid bacteria, cultivated in wheat flour hydrolysate, produced antimould compounds. The antimould activity varied greatly among the strains and was mainly detected within obligately heterofermentative Lactobacillus spp. Among these, Lb. sanfrancisco CB1 had the largest spectrum. It inhibited moulds related to bread spoilage such as Fusarium, Penicillium, Aspergillus and Monilia. A mixture of acetic, caproic, formic, propionic, butyric and n-valeric acids, acting in a synergistic way, was responsible for the antimould activity. Caproic acid played a key role in inhibiting mould growth. Received: 20 January 1998 / Received revision: 17 April 1998 / Accepted: 27 April 1998     

Biochemical and molecular characterization of PepR, a dipeptidase, from Lactobacillus helveticus CNRZ32.

A dipeptidase with prolinase activity from Lactobacillus helveticus CNRZ32, which was designated PepR, was purified to gel electrophoretic homogeneity and characterized. The NH2-terminal amino acid sequence of the purified protein had 96% identity to the deduced NH2-terminal amino acid sequence of the pepR gene, which was previously designated pepPN, from L. helveticus CNRZ32. The purified enzyme hydrolyzed Pro-Met, Thr-Leu, and Ser-Phe as well as dipeptides containing neutral, nonpolar amino acid residues at the amino terminus. Purified PepR was determined to have a molecular mass of 125 kDa with subunits of 33 kDa. The isoelectric point of the enzyme was determined to be 4.5. The optimal reaction conditions, as determined with Pro-Leu as substrate, were pH 6.0 to 6.5 and 45 to 50 degrees C. The purified PepR had a Km of 4.9 to 5.2 mM and a Vmax of 260 to 270 mumol of protein per min/mg at pH 6.5 and 37 degrees C. The activity of purified PepR was inhibited by Zn2+ but not by other cations or cysteine, serine, aspartic, or metal-containing protease inhibitors or reducing agents. Results obtained by site-directed mutagenesis indicated that PepR is a serine-dependent protease. Gene replacement was employed to construct a PepR-deficient derivative of CNRZ32. This mutant did not differ from the wild-type strain in its ability to acidify milk. However, the PepR-deficient construct was determined to have reduced dipeptidase activity compared to the wild-type strain with all dipeptide substrates examined.     

The purification and characterization of a proline dipeptidase from guinea pig brain

A proline dipeptidase (EC 3.4.13.9) from guinea pig brain was purified to over 90% homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, calcium phosphate-cellulose chromatography, chromatofocusing, and gel filtration on Sephadex G-200. A purification factor of 2718-fold was obtained with a yield of 7%. The purified enzyme was found to have an apparent molecular weight of 132,000 and to consist of two dissimilar subunits of molecular weights 64,000 and 68,000. The substrate specificity of the enzyme is not that of a strict proline dipeptidase. Although it preferentially hydrolyzes proline dipeptides (Leu-Pro) it also hydrolyzes prolyl dipeptides (Pro-Leu) and dipeptides not containing proline (Leu-Leu). The purified enzyme preparation exhibited weak aminoacylproline aminopeptidase activity against Arg-Pro-Pro but it did not exhibit any post-proline dipeptidyl aminopeptidase, post-proline cleaving endopeptidase, proline iminopeptidase, prolyl carboxypeptidase or carboxypeptidase P activities when tested with a large variety of peptides and arylamides. With all of the proline and prolyl dipeptides examined the enzyme exhibited biphasic kinetics (two distinct slopes on Lineweaver-Burk plots). However, with Leu-Leu as substrate normal Michaelis-Menten kinetics were obeyed.     

Bacillus subtilis aminopeptidase: purification, characterization and some enzymatic properties.

The extracellular aminopeptidase from Bacillus subtilis was purified 300-fold by a simple procedure which gave a high recovery of enzyme. The native enzyme was shown to be a monomer of molecular weight 46,500 and to contain 1 g-atom of Zn²⁺ per mole of protein. Amino acid analyses demonstrated the protein to be rich in acidic residues and Lys, to possess about 3 residues of Met, and to be devoid of Cys. When activated with 5 mm Co(NO₃)₂ for 90 min the activity of the native enzyme was increased; the amount of activation depended on the identity of the substrate. Cobalt activation involved the reversible binding of 1 g-atom of Co²⁺ per mole of protein, without displacing the native Zn²⁺; K_Co was 1.25 mm. Zinc ions competed with Co²⁺ during activation, a process characterized by a _KZn of 28 μm. Ions other than Co²⁺ did not appreciably activate the enzyme.     

Purification and characterization of an arginine aminopeptidase from Lactobacillus sakei

An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37 degrees C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu(2+), Hg(2+), and Zn(2+)) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The K(m) values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 microM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.     

The proteolytic system of Penicillium roqueforti. V. -- Purification and properties of an alkaline aminopeptidase.

An alkaline aminopeptidase was isolated from the culture medium of Penicillium roqueforti. The enzyme was purified by ammonium sulfate precipitation, filtration on Bio-Gel P-100, chromatography on D.E.A.E.-cellulose and hydroxylapatite, filtration on Bio-Gel P-150 and electrofusing. The purified preparation was homogeneous on polyacrylamide gel electrophoresis at pH 8.5. The molecular weight of the enzyme was estimated to be about 35,000 daltons. The isoelectric point is 4.5. The optimum pH for L-leucine-p-nitroanilide hydrolysis is 8.0. At 35 degrees C the enzyme is stable between pH 6.0 and 7.0. Ethylenediamine tetraacetic acid and a sulfhydryl reagent (p-hydroxymercuribenzoate) inhibit the activity, but the enzyme is insensitive to diisopropylfluorophosphate. Hydrolysis of synthetic peptides shows that the enzyme releases apolar amino acids. Dipeptides are poorly hydrolyzed and Gly in penultimate or N-terminal position causes poor activity. The enzyme is able to cleave the N-terminal Arg-Pro bond of bradykinin.     

Isolation and characterization of an aminopeptidase from Lactobacillus acidophilus R-26

An intracellular aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1) isolated from cell extracts of Lactobacillus acidophilus R-26 was purified 634-fold to homogeneity. This enzyme, which was responsible for all of the N-terminal exopeptidase and amidase activities observed in crude extracts, had no detectable endopeptidase or esterase activity. Although a broad range of L-amino acid peptide, amide and p-nitroanilide derivatives possessing free alpha-amino termini are attacked, the enzyme favored substrates with hydrophobic N-terminal R groups. The native enzyme, which was found to be a tetramer of molecular weight 156000, contained 4 mol of tightly bound Zn2+. The catalytically inactive native zinc metalloenzyme was capable of being activated by either Zn2+, Co2+, Ni2+ or Mn2+. The shape of the log Vmax versus pH plot indicates that two active-center ionizable groups (pKES1 = 5.80; pKES2 = 8.00) may be involved in catalysis. Methylene-blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino-acid analysis indicated that this photooxidative loss of activity corresponds to the modification of one histidine residue per monomer of protein.     

Schistosoma mansoni: purification and characterization of a membrane-associated leucine aminopeptidase

Membrane-associated leucine aminopeptidase (EC 3.4.11.1, LAP) has been purified to homogeneity from Schistosoma mansoni egg homogenates by a combination of ultracentrifugation, chromatofocusing, and molecular sieve chromatography. A 260-fold increase in specific activity was observed after purification. This is a metalloenzyme, containing carbohydrate moieties. Optimal enzyme activity was found at neutral pH. Enzyme activity was measured using L-leucine-7-amino-4-trifluoromethylcoumarin (L-Leu-AFC); in addition, schistosome egg LAP hydrolyzed a variety of other aminopeptidase substrates. Hydrolysis of L-Leu-AFC was inhibited by a number of aminopeptidase inhibitors, including 1,10-phenanthroline, bestatin, and amastatin.     

Thermostable dipeptidase from Bacillus stearothermophilus: its purification, characterization, and comparison with aminoacylase

Dipeptidase (dipeptide hydrolase [EC 3.4.13.11]) has been purified to homogeneity and crystallized from the cell extract of Bacillus stearothermophilus IFO 12983. The enzyme has a molecular weight of about 86,000, and is composed of two subunits identical in molecular weight (43,000). The enzyme contains 2 g atoms of zinc per mol of protein. A variety of dipeptides consisting of glycine or only L-amino acids serve as substrates of the enzyme; Km and Vmax values for L-valyl-L-alanine are 0.5 mM and 68.0 units/mg protein, respectively. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturants such as urea and guanidine hydrochloride. The enzyme also catalyzes hydrolysis of several N-acylamino acids with Vmax values 3-30% of those for the hydrolysis of dipeptides. The thermostable dipeptidase shares various properties with bacterial aminoacylase [EC 3.5.1.14]: their subunit molecular weight, metal content and requirement, amino acid composition, and amino acid sequence in the N-terminal region are very similar.     

The purification and characterization of a glomerular-basement-membrane-degrading neutral proteinase from rat mesangial cells.

A neutral proteinase, capable of degrading gelatin, has been found in both an active and a latent form in the medium from the culture of rat mesangial cells. The latent form had an Mr of 80,000-100,000 and could be activated with either 4-aminophenylmercuric acetate or prolonged incubation at neutral pH. The active form of the enzyme was extensively purified. The estimated Mr of the purified enzyme on gel filtration was approximately 200,000, indicating that the active enzyme formed aggregates. However, analysis by SDS/polyacrylamide-gel electrophoresis under reducing conditions showed two protein bands, with Mr 68,000 and 66,000. Both proteins were found to contain proteolytic activity when run on SDS/substrate gels. The enzyme was inhibited by EDTA and 1,10-phenanthroline, but not by inhibitors for cysteine, serine or aspartic proteinases. The enzyme did not digest fibronectin, bovine serum albumin, proteoglycan or interstitial collagen. The enzyme degraded pepsin-solubilized placental type V collagen at 31 degrees C, whereas similarly solubilized type IV collagen was only degraded at higher temperatures. In addition, the neutral proteinase degraded native soluble type IV collagen. It also had activity on insoluble type IV collagen of glomerular basement membrane. The above properties suggest that the mesangial neutral proteinase belongs to the gelatinase group of metalloproteinases and that it may play a role in the normal turnover of extracellular glomerular matrix.     

Mechanism of maltose uptake and glucose excretion in Lactobacillus sanfrancisco.

Lactobacillus sanfrancisco LTH 2581 can use only glucose and maltose as sources of metabolic energy. In maltose-metabolizing cells of L. sanfrancisco, approximately half of the internally generated glucose appears in the medium. The mechanisms of maltose (and glucose) uptake and glucose excretion have been investigated in cells and in membrane vesicles of L. sanfrancisco in which beef heart cytochrome c oxidase had been incorporated as a proton-motive-force-generating system. In the presence of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), and cytochrome c, the hybrid membranes facilitated maltose uptake against a concentration gradient, but accumulation of glucose could not be detected. Similarly, in intact cells of L. sanfrancisco, the nonmetabolizable glucose analog alpha-methylglucoside was taken up only to the equilibration level. Selective dissipation of the components of the proton and sodium motive force in the hybrid membranes indicated that maltose is transported by a proton symport mechanism. Internal [14C]maltose could be chased with external unlabeled maltose (homologous exchange), but heterologous maltose/glucose exchange could not be detected. Membrane vesicles of L. sanfrancisco also catalyzed glucose efflux and homologous glucose exchange. These activities could not be detected in membrane vesicles of glucose-grown cells. The results indicate that maltose-grown cells of L. sanfrancisco express a maltose-H+ symport and glucose uniport system. When maltose is the substrate, the formation of intracellular glucose can be more rapid than the subsequent metabolism, which leads to excretion of glucose via the uniport system.