Characterization of an Aminopeptidase A from Tetragenococcus halophilus CY54 Isolated from Myeolchi-Jeotgal.

In this study, a pepA gene encoding glutamyl (aspartyl)-specific aminopeptidase (PepA; E.C. 3.4.11.7) was cloned from Tetragenococcus halophilus CY54. The translated PepA from T. halophilus CY54 showed very low similarities with PepAs from Lactobacillus and Lactococcus genera. The pepA from T. halophilus CY54 was overexpressed in E. coli BL21(DE3) using pET26b(+). The recombinant PepA was purified by using an Ni- NTA column. The size of the recombinant PepA was 39.13 kDa as determined by SDS-PAGE, while its optimum pH and temperature were pH 5.0 and 60°C, respectively. In addition, the PepA was completely inactivated by 1 mM EDTA, indicating its metallopeptidase nature. The Km and Vmax of the PepA were 0.98 ± 0.006 mM and 0.1 ± 0.002 mM/min, respectively, when Glu-pNA was used as the substrate. This is the first report on PepA from Tetragenococcus species.

Simple purification method for a recombinantly expressed native His-tag-free aminopeptidase A from Lactobacillus delbrueckii.

The aminopeptidase A (PepA; EC 3.4.11.7) is an intracellular exopeptidase present in lactic acid bacteria. The PepA cleaves glutamyl/aspartyl residues from the N-terminal end of peptides and can, therefore, be applied for the production of protein hydrolysates with an increased amount of these amino acids, which results in a savory taste (umami). The first PepA from a lactobacilli strain was recombinantly expressed in Escherichia coli in a recently published study and harbored a C-terminal His6-tag for easier purification. Due to the fact that a His-tag might influence the properties of an enzyme, a simple purification method for the non-His-tagged PepA was required. Surprisingly, the PepA precipitated at a very low ammonium sulfate concentration of 5%. Unusual for a precipitating step, the purity of PepA was over 95% and the obtained activity yield was 110%. The high purity allows biochemical characterization and kinetic investigation. As a result, the optimum pH (6.0-6.5) and temperature (60-65 °C) were comparable to the His6-tag harboring PepA; the KM value was at 0.79 mM slightly lower compared to 1.21 mM, respectively. Since PepA is a homo dodecamer, it has a high molecular mass of approximately 480 kDa. Therefore, a subsequent preparative size-exclusion chromatography (SEC) step seemed promising. The PepA after SEC was purified to homogeneity. In summary, the simple two-step purification method presented can be applied to purify high amounts of PepA that will allow the performance of experiments in the future to crystalize PepA for the first time.

Purification and functional characterisation of rhiminopeptidase A, a novel aminopeptidase from the venom of Bitis gabonica rhinoceros.

BACKGROUND: Snake bite is a major neglected public health issue within poor communities living in the rural areas of several countries throughout the world. An estimated 2.5 million people are bitten by snakes each year and the cost and lack of efficacy of current anti-venom therapy, together with the lack of detailed knowledge about toxic components of venom and their modes of action, and the unavailability of treatments in rural areas mean that annually there are around 125,000 deaths worldwide. In order to develop cheaper and more effective therapeutics, the toxic components of snake venom and their modes of action need to be clearly understood. One particularly poorly understood component of snake venom is aminopeptidases. These are exo-metalloproteases, which, in mammals, are involved in important physiological functions such as the maintenance of blood pressure and brain function. Although aminopeptidase activities have been reported in some snake venoms, no detailed analysis of any individual snake venom aminopeptidases has been performed so far. As is the case for mammals, snake venom aminopeptidases may also play important roles in altering the physiological functions of victims during envenomation. In order to further understand this important group of snake venom enzymes we have isolated, functionally characterised and analysed the sequence-structure relationships of an aminopeptidase from the venom of the large, highly venomous West African gaboon viper, Bitis gabonica rhinoceros. METHODOLOGY AND PRINCIPAL FINDINGS: The venom of B. g. rhinoceros was fractionated by size exclusion chromatography and fractions with aminopeptidase activities were isolated. Fractions with aminopeptidase activities showed a pure protein with a molecular weight of 150 kDa on SDS-PAGE. In the absence of calcium, this purified protein had broad aminopeptidase activities against acidic, basic and neutral amino acids but in the presence of calcium, it had only acidic aminopeptidase activity (APA). Together with the functional data, mass spectrometry analysis of the purified protein confirmed this as an aminopeptidase A and thus this has been named as rhiminopeptidase A. The complete gene sequence of rhiminopeptidase A was obtained by sequencing the PCR amplified aminopeptidase A gene from the venom gland cDNA of B. g. rhinoceros. The gene codes for a predicted protein of 955 amino acids (110 kDa), which contains the key amino acids necessary for functioning as an aminopeptidase A. A structural model of rhiminopeptidase A shows the structure to consist of 4 domains: an N-terminal saddle-shaped beta domain, a mixed alpha and beta catalytic domain, a beta-sandwich domain and a C-terminal alpha helical domain. CONCLUSIONS: This study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, it could have additional effects on the biological functions of other host proteins by cleaving their N-terminal amino acids. This study points towards the importance of complete analysis of individual components of snake venom in order to develop effective therapies for snake bites.

Partial purification and characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) active aminopeptidase secreted in midgut.

We report the partial purification to apparent homogeneity of a soluble aminopeptidase (EC 3.4.11.1) from midgut of Helicoverpa armigera larvae, which preferentially degraded Leucine p-nitroanilide (LpNA). After midgut isolation, extraction and precipitation of soluble proteins with acetone, proteins were purified in two consecutive steps including gel filtration and ion-exchange chromatographies. Aminopeptidase activity was increased 8.95 fold after gel filtration chromatography. The purified enzyme appeared as single band with a molecular mass of approximately 112 kDa in SDS-PAGE, with a pH optimum of 7.0. Zymogram analysis revealed two enzymatically active proteinases using LpNA as substrate. The optimal temperature of aminopeptidase activity was 50-60 degrees C. The enzyme was characterized as metalloprotease as it was strongly inhibited by 1,10 phenanthroline. Strong inhibition was also being observed using the specific aminopeptidase inhibitor bestatin. Heavy metal ions, EDTA and cysteine strongly inhibited the enzyme, while Ca(+2), Mn(+2) and Mg(+2) somewhat stimulated aminopeptidase activity. Besides LpNA, the purified aminopeptidase also cleaved with decreasing activity ApNA, VpNA and BApNA. Study could be helpful to understand the mechanism of action of N-terminal degrading enzymes and also important is to further study the differential interaction of Bacillus thuringiensis cry insecticidal toxin with midgut receptor of insects.

Efficient production and partial characterization of aspartyl aminopeptidase from Aspergillus oryzae.

AIMS: Aspartyl aminopeptidase (DAP) has a high degree of substrate specificity, degrading only amino-terminal acidic amino acids from peptides. Therefore, attention is focused here on the efficient production of this enzyme by a recombinant Aspergillus oryzae and characterization of its biochemical properties. METHODS AND RESULTS: The gene encoding DAP was overexpressed under a taka-amylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a Co(2+)-containing medium. The enzyme was extracted from the mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kDa. This enzyme displayed high reactivity towards peptide substrate rather than synthetic substrates. CONCLUSIONS: Recombinant A. oryzae DAP was purified to homogeneity with an increased specific activity, when cultivated in a Co(2+)-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help in increasing the specific activity of other metalloproteases and their functional analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Recombinant DAP produced using a cobalt ion in culture media of A. oryzae and purification process allow high yield of the enzyme activity.

Human brain aminopeptidase A: biochemical properties and distribution in brain nuclei.

Aminopeptidase A (APA) generated brain angiotensin III, one of the main effector peptides of the brain renin angiotensin system, exerting a tonic stimulatory effect on the control of blood pressure in hypertensive rats. The distribution of APA in human brain has not been yet studied. We first biochemically characterized human brain APA (apparent molecular mass of 165 and 130 kDa) and we showed that the human enzyme exhibited similar enzymatic characteristics to recombinant mouse APA. Both enzymes had similar sensitivity to Ca(2+). Kinetic studies showed that the K(m) (190 mumol/L) of the human enzyme for the synthetic substrate-l-glutamyl-beta-naphthylamide was close from that of the mouse enzyme (256 mumol/L). Moreover, various classes of inhibitors including the specific and selective APA inhibitor, (S)-3-amino-4-mercapto-butyl sulfonic acid, had similar inhibitory potencies toward both enzymes. Using (S)-3-amino-4-mercapto-butyl sulfonic acid, we then specifically measured the activity of APA in 40 microdissected areas of the adult human brain. Significant heterogeneity was found in the activity of APA in the various analyzed regions. The highest activity was measured in the choroids plexus and the pineal gland. High activity was also detected in the dorsomedial medulla oblongata, in the septum, the prefrontal cortex, the olfactory bulb, the nucleus accumbens, and the hypothalamus, especially in the paraventricular and supraoptic nuclei. Immunostaining of human brain sections at the level of the medulla oblongata strengthened these data, showing for the first time a high density of immunoreactive neuronal cell bodies and fibers in the motor hypoglossal nucleus, the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the Roller nucleus, the ambiguus nucleus, the inferior olivary complex, and in the external cuneate nucleus. APA immunoreactivity was also visualized in vessels and capillaries in the dorsal motor nucleus of the vagus and the inferior olivary complex. The presence of APA in several human brain nuclei sensitive to angiotensins and involved in blood pressure regulation suggests that APA in humans is an integral component of the brain renin angiotensin system and strengthens the idea that APA inhibitors could be clinically tested as an additional therapy for the treatment of certain forms of hypertension.

Characterization of Aspergillus oryzae aspartyl aminopeptidase expressed in Escherichia coli.

To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.