Therapeutic and biotechnological applications of substrate specific microbial aminopeptidases.

Aminopeptidases (EC 3.4.11.) belongs to exoprotease family, which can catalyze the cleavage of peptide bond which connects the N-terminal amino acid to the penultimate residue in a protein. Aminopeptidases catalyze the process of removal of the N-terminal amino acids of target substrates by sequential cleavage of one amino acid residue at a time. Microbial aminopeptidase are of great acceptance as industrial enzymes with varying applications in food and pharma industry since these enzymes possess unique characteristics than aminopeptidases from other sources. This review describes the various applications of microbial aminopeptidases in different industrial sectors. These enzymes are widely used in food industry as a debittering agent as well as in the preparation of protein hydrolysates. In baking, brewing, and cheese making aminopeptidases are extensively used for removing the bitterness of peptides. The inhibitors of these enzymes are found great clinical applications against various diseases such as cancer, diabetes, and viral infections. Aminopeptidases are widely used for the synthesis of biopeptides and amino acids, and found to be efficient than chemical synthesis. These enzymes are capable of hydrolyzing organophosphate compounds, thus having biological as well as environmental significance.Key Points• Cleaves the amino-terminal amino acid residues from proteins and peptides.• Microbial aminopeptidase are of great acceptance as both therapeutic and industrial enzyme.• Review describes the potential applications of microbial aminopeptidases.

Molecular advances in microbial aminopeptidases.

Aminopeptidases are exopeptidases that catalyze the hydrolysis of amino acid residues from the N terminus of peptides and proteins. They are widely and diversely used for protein hydrolysis in industrial and research applications. They form a large enzyme family in microorganisms and most of the sequenced microbial genomes contain several aminopeptidase coding genes. Various approaches are being used to enhance the yield and desired properties of these enzymes to make it more suited for industrial applications. Novel aminopeptidases are being developed by site directed mutagenesis and recombinant DNA technology with improved substrate specificity and stability. This review focuses on its classification and recent advancements in the molecular studies pertaining to this enzyme.

Unveiling aminopeptidase P from Streptomyces lavendulae: molecular cloning, expression and biochemical characterization.

Presence of proline residues in the second position of the N-terminus in peptides restricts the usage of many aminopeptidases; however, aminopeptidase P (APP) is capable of removing this blockage. Based on the N-terminal amino acid sequences of APP from Streptomyces lavendulae, app gene was cloned in pET28a(+) and over expressed as a His-tagged protein with a molecular weight of ≈60 kDa in Escherichia coli BL21 (DE3). Nucleotide sequencing revealed a 1467 bp open reading frame encoding 488 amino acids (NCBI Accession No: GenBank: KC292272.1). The substrate specificity of the recombinant APP was analyzed by the hydrolysis of the Xaa-Pro bond in Gly-Pro dipeptide and bradykinin. K(m) and V(max) of the enzyme were found to be 0.4697 mmol l⁻¹ and 0.6396 µmol min⁻¹, respectively. APP activity was enhanced in the presence of metal ions such as Co²âº, Mn²âº, Mg²âº and Cu²âº ions and was inhibited by 1,10-phenanthroline, EDTA, PMSF and DTT. The atomic absorption studies revealed the presence of Mn²âº in the protein as a co-factor. This substrate specific metalloenzyme was found to be a tetramer and optimally active at pH 8 and 37 °C.

Arginine specific aminopeptidase from Lactobacillus brevis

The proteolytic system of lactic acid bacteria contribute to the development of flavor during the ripening of cheese through the generation of short peptides and free amino acids, which directly or indirectly act as flavor precursors. Newly isolated lactic acid bacteria (LAB) as well as those procured from culture collection centers were screened for the production of various substrate specific aminopeptidases. Among all the strains screened, L. brevis (NRRL B-1836) was found to produce quantifiable amount of intracellular arginine specific aminopeptidase (EC 3.4.11.6). The productivity of arginine aminopeptidase in 5 L fermentor was 36 IU/L/h. The Luedeking and Piret model was tested for intracellular production of aminopeptidase and the data seemed to fit well, as the correlation coefficient was 0.9964 for MRS. The αAP and βAP was 0.4865 and 0.0046, respectively in MRS medium indicating that the yield was predominantly depended on growth. The culture produced lactic acid and also tolerated pH 2.0-3.0 and 0.3-0.5 percent bile salts, the most important probiotic features.

Proline-specific extracellular aminopeptidase purified from Streptomyces lavendulae.

Aminopeptidases catalyze the cleavage of specific amino acids from the amino terminus of protein or peptide substrates. A proline-specific aminopeptidase was purified to homogeneity from the culture-free extract of Streptomyces lavendulae ATCC 14162 in sequential steps comprising ammonium sulfate precipitation, ultra-filtration, and column chromatography on Q-sepharose and Sephadex G-100. The purified protein showed approximately 60 kDa in SDS-PAGE and was optimally active at pH 6.5 and 40 °C. Kinetic studies showed a K(m) and V(max) of 0.23 mM and 0.087 µmol/min, respectively, using Pro-p-NA, the substrate with maximum specificity. Enzyme activity was inhibited by PMSF and ions like Zn²+, Co²+, and Ni²+. However, unlike other aminopeptidases, the activity was enhanced in the presence of DTT, 1,10-phenanthroline, EDTA, amastatin, and bestatin. Ions like Ca²+, Mg²+, and Mn²+ also enhanced the activity.

Arginine specific aminopeptidase from Lactobacillus brevis

The proteolytic system of lactic acid bacteria contribute to the development of flavor during the ripening of cheese through the generation of short peptides and free amino acids, which directly or indirectly act as flavor precursors. Newly isolated lactic acid bacteria (LAB) as well as those procured from culture collection centers were screened for the production of various substrate specific aminopeptidases. Among all the strains screened, L. brevis (NRRL B-1836) was found to produce quantifiable amount of intracellular arginine specific aminopeptidase (EC 3.4.11.6). The productivity of arginine aminopeptidase in 5 L fermentor was 36 IU/L/h. The Luedeking and Piret model was tested for intracellular production of aminopeptidase and the data seemed to fit well, as the correlation coefficient was 0.9964 for MRS. The αAP and βAP was 0.4865 and 0.0046, respectively in MRS medium indicating that the yield was predominantly depended on growth. The culture produced lactic acid and also tolerated pH 2.0-3.0 and 0.3-0.5 percent bile salts, the most important probiotic features.