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

Enzyme and Microbial Technology 32 (2003) 414–421

A putative proline iminopeptidase of Thermotoga maritima is a leucine aminopeptidese with lysine-p-nitroanilide hydrolyzing activity Sunil Ratnayake, Ponniah Selvarkumar, Kiyoshi Hayashi∗ Enzyme Laboratory, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan Received 15 July 2002; received in revised form 7 November 2002; accepted 11 November 2002

Abstract 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. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Leucine aminopeptidase; Thermotoga maritima; Cloning; Thermostability; Substrate specificity

1. Introduction Aminopeptidase plays critical role in the various kind of metabolic and industrial processes including the degradation of proteins and peptides in the cells, protein digestion in its terminal stage, regulation of hormone levels, selective or homeostatic protein turnover, plasmid stabilization and protein maturation [1]. The enzyme is widely distributed in prokaryotes and eukaryotes as either integral membrane or cystosolic proteins [2,3]. Aminopeptidase are generally classified in terms of their substrate specificities, i.e. preference for neutral, acidic, or basic amino acids at N-terminal position (P1). Aminopeptidase catalyzes the sequential removal of amino acids by hydrolyzing the peptide bonds from the unblocked N termini of peptidase and proteins. The assay of bacterial leucyl aminopeptidase (EC 3.4.11.10) involves the release of p-nitroaniline from the substrate of l-leucine-p-nitroanilide [4]. In general, eukaryotic leucyl aminopeptidase (EC 3.4.11.1) prefers l-leucyl ∗

Corresponding author. Tel.: +81-298-38-8071; fax: +81-298-38-7321. E-mail address: [email protected] (K. Hayashi).

residues at the N-terminal position (P1). However, peptides with leucine at P1 position and proline at the penultimate position (P1 ) are not hydrolyzed by eukaryotic leucyl aminopeptidase [5]. In contrast, the prokaryotic X-Pro aminopeptidase (EC 3.4.11.9) hydrolyzes the hydrogen bond at the N-terminal position (P1) amino acid residues from peptidase, which have a proline at the penultimate position (P1 ) [6]. Most aminopeptidases are metalloenzymes [3]. Metal ions of Mn2+ or Co2+ are required for the optimum activity of bacterial X-Pro aminopeptidase (EC 3.4.11.9) while EDTA was inhibitory to the enzyme activity [7]. Moreover, Maras et al. [8] reported that the aminopeptidase from Streptomyces griseus is a calcium-activated metalloenzyme that contains tightly bound zinc to the protein. Hyperthermophiles are a fascinating group of microorganism that has the remarkable property of growing at a temperature of 70 ◦ C or above. Because of the thermostability of the enzymes extracted from these organisms, there is a developing interest of utilizing thermoenzymes as biocatalysts for the industries [9–11]. Almost 20 different genera of hyperthermophiles are currently known [12]. The

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S. Ratnayake et al. / Enzyme and Microbial Technology 32 (2003) 414–421

metabolic enzymes from this species investigated so far show a very high intrinsic thermostability [13–16]. Thermotoga maritima is a hyperthermophilic eubacterium, which has the ability to grow in very high temperatures of up to 90 ◦ C with the optimum of 80 ◦ C which was originally isolated from a geo thermally heated marine sediments at volcano in Italy [17]. The complete genome sequence of T. maritima has been determined [18]. The extremely thermophilic bacterium T. maritima was shown to produce an aminopeptidase P but its potential has not been investigated yet. This enzyme is classified as a putative aminopeptidase P, proline iminopeptidase, under family M 24B of metalloenzymes. The enzyme consists of 359 amino acids with a molecular weight of 39,939 Da. Most of the enzymes isolated from T. maritima spp. showed that high thermostability which possibly be advantageous for biotechnological applications. Accordingly, in the current study the putative aminopeptidase P gene (TM0042, Swissport Q9WXP9, GeneBank AAD35136) from T. maritima was cloned and expressed in Escherichia coli BL21 (RIL) and the expressed protein was purified and characterized. 2. Materials and methods 2.1. Bacterial strains, cloning vectors, and plasmids Prof. Karl O. Stetter and Prof. Robert Huber, in Universitaet Regensburg, Germany, kindly provided the genomic DNA of T. maritima. The E. coli BL21 (RIL) strain (hsd sgal (cIts 857 ind 1 sam 7 nin5lac UV5-T7 gene 1)) was used as host for the recombinant plasmid. The plasmid pET28a(+) (Novagen, Madison, WI) used for subcloning, DNA sequencing, and expression. Recombinent DNA techniques and agarose gel electrophoresis were performed as described by Sambrook et al. [19]. Digestion by restriction enzymes was carried out in the appropriate buffer concentrations of 1–10 unit/␮g of DNA for 4–16 h at appropriate temperatures. Completion of the reaction was confirmed by agarose gel electrophoresis. QIAEX Agarose Gel Extraction Kit (Qiagen, Hilden, Germany) was used for the extraction and purification of DNA from agrose gels. All reagents used in this study were of analytical grade. 2.2. Cloning Aminopeptidase P gene from T. maritima was amplified by employing Polymarase Chain Reaction (PCR) using GeneAmp PCR System 9600 (Perkin-Elmer, Norwalk, CT) using two primers with restriction sites (underlined) of NcoI and EcoRI having the nucleotide sequences of CCA TGG ACA GAT CAG AAA GAT TG and AGA TCT ATC TTC GTC GTC TGA GAA TTC, respectively. Two primers were designed based on the nucleotide sequence of the aminopeptidase P gene. Amplification was performed using following conditions: 98 ◦ C for 5 min, 98 ◦ C for 30 s, 55 ◦ C for 30 s,

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and 68 ◦ C for 1.3 min for 25 cycles. DNA polymerase from Pyrococcus kodakaraensin KODdash (TOYOBO, Osaka, Japan) was used at 1 unit/50 ␮l of reaction mixture. The amplified PCR products were treated with Tag DNA Polymerase (5 unit/50 ␮l reaction) at 72 ◦ C for 10 min. The amplified PCR product was purified from agarose gels using a QIAquick gel extraction kit (QIAGEN, Germany). Resulted DNA fragments were cloned into pCR-TOPO-XL vector using pCR-TOPO cloning kit (Invitrogen, Carlsbad, CA). The resulting recombinent plasmid (TM-APP-TOPO-XL) was subjected to DNA sequencing to check the nucleotide sequences to ensure that no mutations were present in the amplified gene. DNA encoding aminopeptidase P gene was sequenced by the dideoxy chain-termination procedure [20] using the Bigdye Terminator cycle sequencing kit (Perkin-Elmer Applied Biosystems, Foster city, CA,) on a 310 Genetic Analyser (Perkin-Elmer Applied Biosystems). The sequence data were analyzed with the GENETIX program (Software Development Co., Tokyo, Japan). The aminopeptidase P gene, absence of any aminoacid sequence mutation was digested by restriction enzymes NcoI and EcoRI and ligated with the expression vector pET28a(+) (previously digeted with same restriction enzymes), resulting in plasmid TM-APP-pET28. During subcloning, ligation-High T4 DNA ligase (TOYOBO, Osaka, Japan) was used for gene-vector ligation. 2.3. Expression and purification For the expression of recombinent plasmid, TM-APPpET28 was transformed in to E. coli BL21 (RIL) competent cells which has isopropyl-␤-d-thiogalactopyranoside (IPTG) inducible expression of T7 DNA Polymerase and grown in LB medium (1 l containing 50 ␮l/ml kanamycin) at 25 ◦ C using rotary shaker. After reaching an optical density of 0.4–6 at 600 nm, the target protein production was induced by the addition of IPTG (0.4 mM). Incubation was then continued for 16 h and then E. coli cells were harvested by centrifugation (8000 × g, 20 min at 4 ◦ C) and the residue was suspended in 50 mM Tris–HCl buffer (pH 8.0) and sonicated (Branson Sonifier Model 250D, Japan). The cell lysate was then centrifuged (8000 × g, 10 min at 4 ◦ C) and the supernatant collected. The supernatent was applied to ion exchange chromatography on a Q-Sepharose Fast Performance column (Amersam Phamacia Biotech, Uppsala, Sweden), which was previously equilibrated with 20 mM Tris–HCl buffer (pH 8.0). The column was eluted with a linear gradient of NaCl (0–0.5 M) in the same buffer at a flow rate of 3 ml/min. Fractions were collected and assayed for aminopeptidase activity. Active fractions were pooled and the combined fractions were applied again to Q-Sepharose Fast Performance column. The column was eluted with same gradient of NaCl (0–0.5 M). Active fractions were dialyzed (2–3 h at 4 ◦ C) separately using membrane tubes (8000 Da molecular weight cut off) then diluted two-fold with the 20 mM Tris–HCl buffer (pH 8.0)

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and then applied to an anion-exchange chromatography on a Mono-Q column (HR 5/5, Phramcia LKB) which was previously equilibrated with 20 mM Tris–HCl buffer (pH 8.0). The absorbed proteins were eluted with linear gradient of NaCl (0–0.5 M) in the same buffer at a flow rate of 0.5 ml/min. The active fractions were used as a purified aminopeptidase, eluted as a single protein peak. The purity of the protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). 2.4. SDS-PAGE SDS-PAGE was conducted on 1 mm thick 10% slab of acrylamide gel. The samples were incubated in boiling water for 3 min with the Tris–HCl loading buffer (1% SDS, w/v; 20% glycerol, v/v; and 2% 2-mercaptoethnol, v/v). Proteins were stained with Coomassie Brilliant blue R 250 (1%, w/v) in a solution mixture of methanol:acetic acid:water (50:10:40, v/v/v). Then the destaining was performed using same solution mixture of methanol:acetic acid:water (50:10:40, v/v/v). The protein ladder of 10 kDa (Life Technologies, Gibco BRL, Rockville) was used as molecular weight marker. 2.5. Assay of aminopeptidase The enzyme activity of aminopeptidase was determined by calculated the amount of p-nitroaniline release from the l-leucine-p-nitroanilide at 80 ◦ C. The assay mixture of 0.5 ml consisting of 2 mM l-leucine-p-nitroanilide and 0.1 mM MgCl2 in 50 mM N-[2-hydroxyethyl]piperazine-N [2-ethanesulfonic acid] (HEPES) buffer (pH 7.5) was incubated with the enzyme for 20 min at 80 ◦ C. The reaction was stopped by the addition of 0.5 ml of acetic acid (1N). The amount of p-nitroaniline released was determined by measuring the absorbance at 405 nm. One unit of the aminopeptidase activity was defined as the amount of enzyme required to release 1 ␮mol p-nitroaniline per minute under defined assay condition. 2.6. Effect of temperature and pH The optimum temperature of the enzyme was determined using the standard assay condition over the temperatures ranging 30–100 ◦ C. The thermal stability of the enzyme was determined by incubating the enzyme for 30 min at pH 7.5 over the temperatures ranging 30–100 ◦ C. After cooling the reaction mixture on ice for 5 min, the remaining activity was determined by using standard assay procedure. The effect of pH on the enzyme activity was measured at 80 ◦ C in 50 mM concentration of various buffers including sodium citrate (pH 2.18–4.21), sodium acetate (pH 3.76–5.73), 2-[N-morpholino]ethanesulfonic acid (MES) (pH 5.15–7.13), 3-[N-morpholino]propanesulfonic acid (MOPS) (pH 6.22–8.20), HEPES (pH 6.54–8.54), Tris [hydroxymethyl]aminomethane (Tris–HCl) (pH 7.20–9.00)

and 2-[N-cyclohexylamino]ethanesulfonic acid (CHES) (pH 8.17–10.14). The pH stability of the enzyme was determined by preincubating the enzyme in the same buffers for 30 min at 80 ◦ C and the remaining activity was determined by the standard assay procedure. 2.7. Effect of metal ions on the enzyme The effect of metal ions on the activity of aminopeptidase was measured at 80 ◦ C in 50 mM HEPES buffer (pH 7.5) containing various divalent cations including Cd2+ , Co2+ , Ca2+ , Cu2+ , Pb2+ , Mg2+ , Mn2+ , and Zn2+ by the standard assay procedure. 2.8. Kinetic parameters The kinetic parameters of the enzyme were determined at 80 ◦ C in 50 mM HEPES buffer (pH 7.5) using various substrates such as l-leucine-p-nitroanilide, l-alanin-p-nitroanilide, l-proline-p-nitroanilide, and l-lysine-p-nitroanilide. The data was obtained by measuring the initial rate of hydrolysis by incubating the enzyme with appropriate concentrations of the substrate. An initial rate of hydrolysis was determined at six different substrate concentrations ranging from approximately 0.5–2.0 times of the Km value. Km and kcat and their standard errors were calculated by using the nonlinear regression analysis program Grafit (Grafit Version 3.09b, Erithacus software, UK, 1996). 3. Results and discussion 3.1. Cloning of putative aminopeptidase P and sequence similarity with other aminopeptidases Putative aminopeptidase P gene (TM0042, Swissport Q9WXP9, GeneBank AAD35136) was amplified from T. maritima genome and cloned by using specific forward (NcoI) and reverse (EcoRI) primers in to pET28a(+) expression vector. The whole nucleotide sequence of the inserted fragment of plasmid pET28a(+) was confirmed by the dideoxy chain-termination procedure. The putative aminopeptidase P from T. maritima displays significant pair wise aminoacid homology with Xaa-Pro aminopeptidase from Thermoanaerobacter tengcongensis (49%) [21], metallopeptidase from Bacillus anthracis (44%) [22], Xaa-Pro dipeptidase from Pyrococcus furiosus (42%) [23], Bacillus halodurans (41%) [24], Staphylococcus aureus (41%) [25], and aminopeptidase P from Listeria monocytogenes (41%) [26]. According to the evolutionary tree for M 24B family of metalloenzymes, the putative aminopeptidase P from T. maritima belongs to the group of aminopeptidase that consisting of X-pro dipeptidase (archaea) from Pyrococcus abyssi (Swissport: E75088), Pyrococcus horikoshii (Swissport: O58885) and P. furiosus (Swissport: P81535) (www.merops.co.uk).

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Table 1 Summary of the purification of aminopeptidase from Thermotoga maritima Purification steps

Total protein (mg)

Total activity (unit)

Specific activity (unit/mg)

Fold of purification

Recovery (%)

Crude extract First Q-sepharose Second Q-sepharose Mono-Q

736 76.8 42.5 6.9

524 232 157 114

0.71 3.02 3.69 16.5

1.00 4.25 5.20 23.2

100 44.3 30.0 21.8

3.2. Purification of the enzyme The production of aminopeptidase protein in E. coli (RIL) was induced successfully using IPTG and the maximum induction was obtained in the culture incubated for 16 h at 25 ◦ C. The activity of T. maritima aminopeptidase was recognized in the crude extract of E. coli by enzyme assay at 80 ◦ C, which eliminated the activity of aminopeptidase from host cell. The appearance of large protein band, assessed by SDS-PAGE, corresponding to the size of T. maritima aminopeptidase (39.9 kDa) confirmed the induction of target protein. The T. maritima aminopeptidase was purified from the crude cell lysate using ion exchange column chromatography on a Q-Sepharose and Mono-Q. The primary purification step (Q-Sepharose column) removed most of the proteins including host aminopeptidase. Subsequent purification steps resulted 23-fold pure protein (Table 1). The specific activity of purified enzyme was 16.5 U/mg while the total recovery was 21.8%. About 6.9 mg of pure aminopeptidase enzyme was isolated from 2 l cultivation. The purity and the molecular mass of T. maritima aminopeptidase were assessed by SDS-PAGE as shown in Fig. 1.

Furthermore, the pH optima and stability of membrane bound aminopeptidase P from bovine lung demonstrates the pH optimum at 6.5–7.0 and most stable in the basic pH range [28]. 3.4. Effect of metal ions on enzyme activity The activity of aminopeptidase belongs to the M 24B family of metalloenzymes was measured under standard assay conditions in the presence of various divalent cations and the results were summarized in Table 2. None of the experimented 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+ . Likewise, Rusu and Yaron [29] reported that aminopeptidase P from human leucocytes was inhibited markedly by Ni2+ , Cd2+ , and Zn2+ while Mn2+ caused maximum activation. Nakanishi et al. [30] reported that the Zn2+ and Cu2+ demonstrate the strong inhibition of the activity of leucine aminopeptidase from placental tissues. Furthermore, the activity of aminopep-

3.3. Effect of temperature and pH on enzyme activity The distinct properties of this aminopeptidase were its high optimum temperature and its thermostability, which are similar to those from the most of the other enzymes isolated from T. maritima [13–16]. The enzyme activity at various temperatures (30–100 ◦ C) illustrated that the optimum activity at 90 ◦ C at pH 7.5 and 80 and 42% of the original activity was retained while it was incubated at 95 and 100 ◦ C for 20 min, respectively (Fig. 2). The curve of relative enzyme activity against temperature illustrates that the enzyme is stable at the temperatures up to 90 ◦ C at pH 7.5 (Fig. 2). The enzyme activity was determined at series of various pH buffers at 80 ◦ C. The maximum pH activity was observed at pH 7.5 as shown in Fig. 3. The stability of the aminopeptidase at different levels of pH indicated that it was stable in the pH range of 6.5–8 at 80 ◦ C for 30-min incubation and only 30% of the original activity was remained at pH 10 (Fig. 4). In contrast, Yoshimoto et al. [27] reported that aminopeptidase P from E. coli HB101 showed the pH optimum at 8.5 and the stability at pH 8.0–9.0 at 50 ◦ C.

Fig. 1. SDS-PAGE of the purified aminopeptidase. Molecular weight marker (lane 1) and purified aminopeptidase P (lane 2).

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Fig. 2. Effect of temperature and thermal stability on aminopeptidase activity. The effect of temperature (䊊) was determined at various temperatures using standard assay. For the thermal stability (䊉), the remaining activity was measured after preincubation of the enzyme for 30 min at different temperatures by using the standard assay.

tidase P from E. coli HB101 was inhibited by EDTA and activated five-fold with Mn2+ [31]. In the present study, the EDTA inactivation of enzyme with different incubation temperatures for various time showed that the enzyme was completely lost its activity by incubating with 0.1 mM EDTA at 30 ◦ C for 10 min. EDTA-treated enzyme was effectively reactivated by the addition of 1 mM Mg2+ , Ca2+ , and Mn2+ (after removal of EDTA) with relative activities of 80, 70, and 65%, respectively. These results collectively suggested that T. maritima aminopeptidase is a metallopeptidase, preferably magnesium containing aminopeptidase. Similarly, the reported human leucine aminopeptidase (EC 3.4.11.1) [32] and animal erepsin leucine aminopeptidase [33] are activated by Mg2+ and Mn2+ .

Table 2 Effect of metal ions on aminopeptidase activitya Metal ions (0.1 mM)

Relative activity (%)

Control MgCl2 CaCl2 MnCl2 PbCl2 CuCl2 CoCl2 CdCl2 ZnCl2 EDTA

100 99.1 55.1 29.3 15.5 14.1 13.7 7.2 6.9 0.0

a The activity was measured using leucine-p-nitroanilide at 80 ◦ C, pH 7.5 for 20 min.

Fig. 3. Effect of pH on aminopeptidase activity. The consequence of pH on the enzyme activity was measured at 80 ◦ C in 50 mM concentration of various buffers including sodium citrate (䊊), sodium acetate (䉱), MES (), MOPS (䊉), HEPES (䊐), Tris–HCl (×), and CHES (䊏).

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Fig. 4. Effect of pH on aminopeptidase stability. The pH stability of the enzyme was determined by preincubating the enzyme in the various buffers such as sodium citrate (䊊), sodium acetate (䉱), MES (), MOPS (䊉), HEPES (䊐), Tris–HCl (×), and CHES (䊏) for 30 min at 80 ◦ C and the remaining activity was determined by the standard assay procedure.

3.5. Substrate specificity The substrate specificity of the putative aminopeptidase P from T. maritima was measured with a various peptidyl-p-nitroanilide derivatives at 80 ◦ C under standard conditions (Table 3). The enzyme designated, as putative aminopeptidase P was very low activity in hydrolyzing proline-p-nitroanilide as reflected in a lower kcat value for this substrate than other substrates tested. In contrast to the present study, the most of the reports designated that the prolyl-p-nitroanilide is the substrate for most eukaryotic and prokaryotic aminopeptidase P [27–29,34–36]. It is interesting to note that the putative aminopeptidase P from T. maritima is not efficiently hydrolyze proline-p-nitroanilide even though it showed the high homology with the sequence of other X-pro dipeptidase, proline peptidase, or aminopeptidase P [21–26] which all belongs to the M 24B family of metalloenzymes. Higher kcat value of the enzyme for leucine-p-nitroanilide (0.285 s−1 ) than other substrates tested, suggests that the enzyme has higher efficiency in hydrolyzing leucine-p-nitroanilide. Relatively low kcat value of the present study is comparable to that of with the reported

aminopeptidases isolated from P. horikoshii (0.116 s−1 ) [37] and Aspergillus niger (0.49 ␮kat/mg) [38]. Moreover, the remarkably low kcat value (0.0018 s−1 ) was reported for the ␣-glucuronidase [39] isolated from T. maritima which is the same organism used to isolate the aminopeptidase in the current study. Interestingly, the enzyme was also seemed to hydrolyze lysine-p-nitroanilide with comparable efficiency to that of leucine-p-nitroanilide. In view of this observation, further kinetic study was carried out with leucine- and lysine-p-nitroanilide as a substrate at 35 ◦ C. The enzyme showed higher hydrolyzing efficiency for leucine-p-nitroanilide than lysine-p-nitroanilide at 35 ◦ C, as before that of showed at higher temperature (80 ◦ C) (Table 3). These results collectively suggest that the enzyme, aminopeptidase from T. maritima is a leucine aminopeptidase. In general lysine aminopeptidase known to have leucine-p-nitroanilide hydrolyzing activity [38,40–42], but only one leucine amino peptidase studied so far reported to have lysine-p-nitroanilide hydrolyzing activity [43] which supported the present study. The family M 24B comprises of Xaa-proaminopeptidase, X-pro dipeptidase and aminopeptidase P. To date, no leucine

Table 3 Kinetic parameters of aminopeptidase from Thermotoga maritima Substrate

Km ± S.E (mM)

80 ◦ C Leucine pNA Lysine pNA Alanine pNA Proline pNA

12.7 8.9 24.7 13.1

35 ◦ C Leucine pNA Lysine pNA

± ± ± ±

0.1 0.3 0.9 0.6

6.3 ± 0.1 5.0 ± 0.2

kcat ± S.E (s−1 ) 28.5 19.4 7.8 2.1

× × × ×

10−2 10−2 10−2 10−2

± ± ± ±

kcat /Km (mM−1 s−1 ) 0.1 0.5 0.2 0.5

× × × ×

10−2 10−2 10−2 10−2

1.57 × 10−2 ± 0.04 × 10−2 1.04 × 10−2 ± 0.03 × 10−2

2.24 2.18 0.32 0.16

× × × ×

10−2 10−2 10−2 10−2

0.25 × 10−2 0.21 × 10−2

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aminopeptidases are reported that belong to the family M 24B as they often classified in family M17. Accordingly, this appears to be the first report of leucine aminopeptidase with lysine-p-nitroanilide hydrolyzing activity, which belongs to the M 24B family of metalloenzymes [8,30,44–46].

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