Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40°C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu²⁺ and Cd²⁺. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or β-mercapto-ethanol, while Zn²⁺ or Co²⁺ restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (K_m, 0.55 mM) but that it can hydrolyze this substrate at a high rate (V_max, 30 μmol/min per mg of protein).     

Purification and characterization of a prolyl aminopeptidase from Debaryomyces hansenii

A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45 degrees C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg(2+), which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The K(m) for proline-7-amido-4-methylcoumarin was 40 micro M. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.     

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°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺) 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 μM, 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.     

Characterization of Native and Recombinant Forms of an Unusual Cobalt-Dependent Proline Dipeptidase (Prolidase) from the Hyperthermophilic Archaeon Pyrococcus furiosus

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co²⁺ ions (K_d, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co²⁺ could be replaced by Mn²⁺ (resulting in a 25% decrease in activity) but not by Mg²⁺, Ca²⁺, Fe²⁺, Zn²⁺, Cu²⁺, or Ni²⁺. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100°C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the K_m values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater V_max value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.     

Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N from Streptococcus thermophilus A

The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys–7-amino-4-methylcoumarin at pH 7 and 37°C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co²⁺, Zn²⁺, and Mn²⁺. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of the pepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.     

Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K_m s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.     

Purification and characterization of an aminopeptidase fromStreptococcus mitis ATCC 903

An aminopeptidase isolated from the cytoplasmic fraction of a cell extract ofStreptococcus mitis ATCC 903 was purified 330-fold by ion-exchange chromatography, gel filtration, and hydroxyapatite chromatography. The partially purified enzyme had a broad substrate specificity. Twelve aminoacyl-ß-naphthylamide substrates were hydrolyzed and also several di-, tri-, tetra-, and pentapeptides and bradykinin. The enzyme hydrolyzed arginine-ß-naphthylamide at the highest rate. Optimal conditions for activity were at pH 7.0–7.2 and at 37–40°C. The molecular weight of the enzyme was estimated to be 93,000. The enzyme was activated by Co²⁺ ions. Hg²⁺ inhibited the activity completely. SDS, EDTA, urea, and pCMB also inhibited activity. Inhibition by EDTA could be completely reversed by dialysis and addition of Co²⁺ ions. Reducing agents, sodium fluoride, and PMSF had no effect on the activity of the enzyme. The isoelectric point of the enzyme was at pH 4.3. High substrate concentrations inhibited activity. Substrate inhibition increased in the presence of high concentrations of Co²⁺ ions.     

A putative proline iminopeptidase of Thermotoga maritima is a leucine aminopeptidese with lysine-p-nitroanilide hydrolyzing activity

A putative aminopeptidase P gene (TM0042, Swissport Q9WXP9, GeneBank AAD35136) of Thermotoga maritima was cloned and expressed in Escherichia coli BL21 (RIL). The enzyme was purified by the combination of ion exchange chromatography; Q-Sepharose and Mono-Q column. The purified recombinant T. maritima aminopeptidase P enzyme, gave a homogenous protein band with an apparent molecular weight of 40 kDa in SDS-PAGE analysis. The enzyme was purified 23-fold with the specific activity of 16.5 unit/mg with the final recovery of 22%. The enzyme was thermostable up to 90 °C for 30 min. An optimal activity was observed at 90 °C at pH 7.5. The purified enzyme was stable between pH 6.5 and 8 at 80 °C with the optimum of pH 7.5. Based on the amino acid sequence, the enzyme belongs to M 24B family of metalloenzymes. None of the divalent cations enhance the activity of the enzyme while Pb²⁺, Cu²⁺, Co²⁺, Cd²⁺, and Zn²⁺ were inhibitory to the enzyme activity. Divalent cation of Mg²⁺ showed 100% enzyme activity, to a lesser extent, Ca²⁺ and Mn²⁺ whereas strong inhibition of enzyme activity was observed with Zn²⁺ and Cd²⁺. The enzyme designated as putative aminopeptidase P was very low activity in hydrolyzing proline-p-nitroanilide. Kinetic studies on the purified enzyme confirmed that the enzyme is a leucine aminopeptidase. Enzyme also hydrolyzes lysine-p-nitroanilide with efficiency comparable to that of leucine-p-nitroanilide. This is the first report of leucine aminopeptidase with lysine-p-nitroanilide hydrolyzing activity, which belongs to the M 24B family of metalloenzymes.     

An activated by cobalt alkaline aminopeptidase from <Emphasis Type="Italic">Bacillus mycoides</Emphasis>

An intracellular arginine—specific aminopeptidase synthesized by Bacillus mycoides was purified and characterized. The purification procedure for studied aminopeptidase consisted of ammonium sulphate precipitation and three chromatographic steps: anion exchange chromatography and gel permeation chromatography. A molecular weight of ∼50 kDa was estimated for the aminopeptidase by gel permeation chromatography and SDS-PAGE. The optimal activity of the enzyme on arginyl-β-naphthylamide as a substrate was at 37°C and pH 9.0. The enzyme showed maximum specificity for basic amino acids: such as Arg and Lys but was also able to hydrolyze aromatic amino acids: Trp, Tyr, and Phe. Co²⁺ ions activated the enzyme, while Zn²⁺, Cu²⁺, Hg²⁺ and Mn²⁺ inhibited it. The enzyme is a metalloaminopeptidase whose activity is inhibited by typical metalloaminopeptidase inhibitors: EDTA and 1,10-phenanthroline. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to Amp S of Bacillus cereus and APII of B. thuringensis.     

Purification and characterization of human placental microsomal aminopeptidase: immunological difference between placental microsomal aminopeptidase and pregnancy serum cystyl-aminopeptidase

Human placental microsomal aminopeptidase (microsomal PAP) was purified 3,880-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100 and also trypsin digestion, and subjected to zinc sulfate fractionation, chromatographies with DE-52, hydroxylapatite, Sephacryl S-300 and lentil lectin-Sepharose 4B, and finally affinity chromatography with bestatin-Sepharose 4B. Microsomal PAP was separated from aminopeptidase A (AAP) by affinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 220,000 by high-performance liquid chromatography with an aqueous gel column. The purified enzyme gave almost a single band with a molecular mass of 140,000 by sodium dodecyl sulfate (SDS) gel electrophoresis. The isoelectric point of the enzyme was 5.2. The purified enzyme was most active at pH 8.0 with L-leucine-p-nitroanilide as substrate; the Km value for this substrate was 1.1 mmol/l. The microsomal PAP was immunologically different from the pregnancy serum cystyl aminopeptidase (serum PAP).     

Ambient pH Is a Major Determinant in the Expression of Cuticle-Degrading Enzymes and Hydrophobin by Metarhizium anisopliae

Secretion of proteolytic and chitinolytic enzymes is a hallmark of infection processes of Metarhizium anisopliae in response to host (insect) cuticular signals. The regulation of these enzymes (subtilisin-like proteases [Pr1a and Pr1b], trypsin-like proteases [Pr2], metalloproteases, aspartyl proteases, aminopeptidase, and chitinases) and a hydrophobin was investigated by Northern analysis and/or enzyme assay. The production of each enzyme showed a differential expression pattern in response to ambient pH; enzymes were synthesized only at pHs at which they function effectively, irrespective of whether the medium contained an inductive cuticle substrate. Three aspartyl proteases (pH optimum, 3), and chitinase (pH optimum, 5) showed maximal accumulation at acidic pHs. The highest level of aminopeptidase (pH optimum, 7) was detected at pH 7. The highest levels of five metalloproteases (pH optima, ca. 7) were detected over the pH range 6 to 8. Two trypsins and several subtilisin-like Pr1 isoforms with pH optima of ca. 8 were produced only under alkaline conditions. Northern analysis of RNA species corresponding to seven cDNA sequences encoding proteases and chitinase confirmed that the ambient pH played a major role in gene expression of secreted proteins. Hydrophobin was expressed almost equally at pHs 5 and 8 but was not expressed at pH 3. During fungal penetration, the pH of infected cuticle rises from about 6.3 to 7.7. Consistent with pH regulation of enzyme production, serine and metalloproteases were produced in situ during infection, but no production of aspartyl proteases was found. We propose that the alkalinity of infected cuticle represents a physiological signal that triggers the production of virulence factors.     

Isolation, Purification, and Some Properties of Penicillium chrysogenum Tannase

Tannase isolated from Penicillium chrysogenum was purified 24-fold with 18.5% recovery after ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. Optimum enzyme activity was recorded at pH 5.0 to 6.0 and at 30 to 40°C. The enzyme was stable up to 30°C and within the pH range of 4.0 to 6.5. The K_m value was found to be 0.48 × 10⁻⁴ M when tannic acid was used as the substrate. Metal salts at 20 mM inhibited the enzyme to different levels.     

Purification of membrane-bound galactosyltransferase from rat liver microsomal fractions

1. Rat liver microsomal preparations incubated in 1% Triton X-100 at 37°C for 1h released about 60% of the membrane-bound UDP-galactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. The supernatant galactosyltransferase which was solubilized but not purified by this treatment had a higher molecular weight than the serum enzyme as shown by Sephadex G-100 column chromatography. 2. The galactosyltransferase present in the high-speed supernatant was purified 680-fold by an affinity-column-chromatographic technique by using a column of activated Sepharose 4B coupled with α-lactalbumin. The galactosyltransferase ran as a single band on polyacrylamide gels and contained no sialyltransferase, N-acetylglucosaminyltransferase or UDP-galactose pyrophosphatase activities. 3. The purified membrane enzyme had properties similar to serum galactosyltransferase. It had an absolute requirement for Mn²⁺ that could not be replaced by Ca²⁺, Mg²⁺, Zn²⁺ or Co²⁺, and was active over a wide pH range (6–8) with a pH optimum of 6.5. The apparent K_m for UDP-galactose was 10.8μm. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose. 4. The molecular weight of the membrane enzyme was 65000–70000, similar to that of the purified serum enzyme. Amino acid analyses of the two proteins were similar but not identical. 5. Sephadex G-100 column chromatography of the purified membrane enzyme showed a small peak (2–5%) of higher molecular weight than the purified serum enzyme. Inclusion of 1mm-ε-aminohexanoic acid in the isolation procedures increased this peak to as much as 30% of the total enzyme recovered. Increasing the ε-aminohexanoic acid concentration to 100mm resulted in no further increase in this high-molecular-weight fraction.     

Purification and Partial Characterization of a Prolyl-Dipeptidyl Aminopeptidase from Lactobacillus helveticus CNRZ 32

X-prolyl-dipeptidyl aminopeptidase, which hydrolyzed Gly-Pro-p-nitroanilide (relative activity [RA] = 100%) and Arg-Pro-p-nitroanilide (RA, 130%), was purified to homogeneity from the cell extract of Lactobacillus helveticus CNRZ 32. The enzyme also hydrolyzed Ala-Pro-Gly (RA, 11%) and Ala-Ala-p-nitroanilide (RA, 2%) but was not active on Ala-Leu-Ala, dipeptides, and endopeptidase and carboxypeptidase substrates. The enzyme was purified 145-fold by streptomycin sulfate precipitation, ammonium sulfate fractionation, and a series of column chromatographies on DEAE-cellulose, arginine-Sepharose 4B, and glycyl-prolyl-AH-Sepharose 4B. The purified enzyme appeared as a single band on native polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses and had a molecular weight of 72,000. Optima for activity by the purified enzyme were pH 7.0 and 40°C. The enzyme was incubated at 40°C for 15 min with various metal ions. It was activated by Mg²⁺ (2.5 mM), Ca²⁺ (0.1 to 2.5 mM), Na⁺ (10 to 50 mM), and K⁺ (10 to 50 mM) and was inhibited by Hg²⁺ (0.1 to 2.5 mM), Cu²⁺ (0.1 to 2.5 mM), and Zn²⁺ (0.1 to 2.5 mM). Enzyme activity was partially inhibited by EDTA (1.0 mM, 20 h at 40°C), 1,10-phenanthroline (1.0 mM, 15 min at 40°C), phenylmethylsulfonyl fluoride (1.0 mM), N-ethylmaleimide (1.0 mM), and iodoacetate (1.0 mM). It was completely inhibited by diisopropyl fluorophosphate (1.0 mM, 2 h at 40°C) and p-chloromercuribenzoate (1.0 mM, 15 min at 40°C). The enzyme was not affected by dithioerythritol (1.0 to 10 mM).     

Partial Purification and Characterization of a 3'- Phosphoadenosine 5' -Phosphosulfate: Desulfoglucosinolate Sulfotransferase from Cress (Lepidium sativum)

A 3′ -phosphoadenosine 5′ -phosphosulfate (PAPS):desulfoglucosinolate sulfotransferase (EC 2.8.2-) was extensively purified from light-grown cress (Lepidium sativum L.) seedlings by gel filtration and concanavalin A-Sepharose 4B, Matrex Gel Green A, and Mono Q fast protein liquid chromatography. The purified enzyme, which required bovine serum albumin for stabilization, had a native molecular weight of 31,000 ± 5,000 and an apparent isoelectric point of 5.2. Using PAPS (K_m 60 micromolar) as sulfur donor, it catalyzed the sulfation of desulfobenzylglucosinolate (K_m 82 micromolar), desulfo-p-hydroxybenzylglucosinolate (K_m 670 micromolar), and desulfoallylglucosinolate (K_m 6.5 millimolar) at an optimal pH of 9.0. All other potential substrates tested, including flavonoids, flavonoid glycosides, cinnamic acids, and phenylacetaldoxime, were not sulfated. Sulfotransferase activity was stimulated by MgCl₂, MnCl₂ and reducing agents and inhibited by ZnCl₂, PbNO₃ NiCl₂ and the reaction product PAP. The thiol reagents N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, and 5,5′ -dithio-bis-(2-nitrobenzoic acid) were also potent inhibitors, but the enzyme was protected from covalent modification by β-mercaptoethanol. The kinetics of desulfobenzylglucosinolate sulfation were consistent with a rapid equilibrium ordered mechanism with desulfobenzylglucosinolate binding first and PAPS second.     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.     

Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp.

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and β-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg²⁺, Co²⁺, and Mn²⁺ were required for maximum activity of the enzyme. The K_m values for D-xylose and D-glucose at 80°C and pH 7.5 were 6.66 and 142 mM, respectively, while K_cat values were 2.3 × 10² s^-1 and 0.5 × 10² s^-1, respectively.     

Optimized expression of prolyl aminopeptidase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its characteristics after glycosylation

Prolyl aminopeptidases are specific exopeptidases that catalyze the hydrolysis of the N-terminus proline residue of peptides and proteins. In the present study, the prolyl aminopeptidase gene (pap) from Aspergillus oryzae JN-412 was optimized through the codon usage of Pichia pastoris. Both the native and optimized pap genes were inserted into the expression vector pPIC9 K and were successfully expressed in P. pastoris. Additionally, the activity of the intracellular enzyme expressed by the recombinant optimized pap gene reached 61.26 U mL^?1, an activity that is 2.1-fold higher than that of the native gene. The recombinant enzyme was purified by one-step elution through Ni-affinity chromatography. The optimal temperature and pH of the purified PAP were 60 °C and 7.5, respectively. Additionally, the recombinant PAP was recovered at a yield greater than 65 % at an extremely broad range of pH values from 6 to 10 after treatment at 50 °C for 6 h. The molecular weight of the recombinant PAP decreased from 50 kDa to 48 kDa after treatment with a deglycosylation enzyme, indicating that the recombinant PAP was completely glycosylated. The glycosylated PAP exhibited high thermo-stability. Half of the activity remained after incubation at 50 °C for 50 h, whereas the remaining activity of PAP expressed in E. coli was only 10 % after incubation at 50 °C for 1 h. PAP could be activated by the appropriate salt concentration and exhibited salt tolerance against NaCl at a concentration up to 5 mol L^?1.     

Purification and Characterization of a Tripeptidase from Lactococcus lactis subsp. cremoris Wg2

A tripeptidase from a cell extract of Lactococcus lactis subsp. cremoris Wg2 has been purified to homogeneity by DEAE-Sephacel and phenyl-Sepharose chromatography followed by gel filtration over a Sephadex G-100 SF column and a high-performance liquid chromatography TSK G3000 SW column. The enzyme appears to be a dimer with a molecular weight of between 103,000 and 105,000 and is composed of two identical subunits each with a molecular weight of about 52,000. The tripeptidase is capable of hydrolyzing only tripeptides. The enzyme activity is optimal at pH 7.5 and at 55°C. EDTA inhibits the activity, and this can be reactivated with Zn²⁺, Mn²⁺, and partially with Co²⁺. The reducing agents dithiothreitol and β-mercaptoethanol and the divalent cation Cu²⁺ inhibit tripeptidase activity. Kinetic studies indicate that the peptidase hydrolyzes leucyl-leucyl-leucine with a K_m of 0.15 mM and a V_max of 151 μmol/min per mg of protein.     

Partial Purification and Characterization of a Thermostable Actinomycete β-Amylase

A thermostable amylase, possibly a β-amylase from Thermoactinomyces sp. no. 2 isolated from soil, is reported. The enzyme was purified 36-fold by acetone precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration, and the molecular weight was estimated at 31,600. The enzyme was characterized by demonstration of optimum activity at 60°C and pH 7 and by retention of 70% activity at 70°C (30 min). It was stimulated by Mn²⁺ and Fe²⁺ but strongly inhibited by Hg²⁺. Maltose was the only detectable product of hydrolysis of starches and was quantitatively highest in plantain starch hydrolysate.