Mutagenesis for Improvement of Activity and Stability of Prolyl Aminopeptidase from Aspergillus oryzae

Liu, Dehua; Zhang, Dawei; Huang, Qinqin; Gu, Lili; Zhou, Nandi; Tian, Yaping

doi:10.1007/s12010-020-03277-y

Cited by 5 publications

(3 citation statements)

References 41 publications

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“…The introduction of mutants on these specific residues weakens the inter-domain interaction of the enzymes to reduce the restriction of bigger substrates to enter the active site [ 55 ]. The increased specificity of the enzyme activity was recorded when random and site-directed saturation mutagenesis was performed on a prolyl aminopeptidase derived from Aspergillus oryzae [ 106 ]. Mutagenesis at C185V allowed a higher resistance towards high temperature, broader acidity range, and longer half-life compared to the wild type, making it a good candidate for many industrial sectors [ 106 ].…”

Section: Protein Engineeringmentioning

confidence: 99%

Post-Proline Cleaving Enzymes (PPCEs): Classification, Structure, Molecular Properties, and Applications

Baharin

Ting

Goh

2022

Plants

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Proteases or peptidases are hydrolases that catalyze the breakdown of polypeptide chains into smaller peptide subunits. Proteases exist in all life forms, including archaea, bacteria, protozoa, insects, animals, and plants due to their vital functions in cellular processing and regulation. There are several classes of proteases in the MEROPS database based on their catalytic mechanisms. This review focuses on post-proline cleaving enzymes (PPCEs) from different peptidase families, as well as prolyl endoprotease/oligopeptidase (PEP/POP) from the serine peptidase family. To date, most PPCEs studied are of microbial and animal origins. Recently, there have been reports of plant PPCEs. The most common PEP/POP are members of the S9 family that comprise two conserved domains. The substrate-limiting β-propeller domain prevents unwanted digestion, while the α/β hydrolase catalyzes the reaction at the carboxyl-terminal of proline residues. PPCEs display preferences towards the Pro-X bonds for hydrolysis. This level of selectivity is substantial and has benefited the brewing industry, therapeutics for celiac disease by targeting proline-rich substrates, drug targets for human diseases, and proteomics analysis. Protein engineering via mutagenesis has been performed to improve heat resistance, pepsin-resistant capability, specificity, and protein turnover of PPCEs for pharmacological applications. This review aims to synthesize recent structure–function studies of PPCEs from different families of peptidases to provide insights into the molecular mechanism of prolyl cleaving activity. Despite the non-exhaustive list of PPCEs, this is the first comprehensive review to cover the biochemical properties, biological functions, and biotechnological applications of PPCEs from the diverse taxa.

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Section: Protein Engineeringmentioning

confidence: 99%

Post-Proline Cleaving Enzymes (PPCEs): Classification, Structure, Molecular Properties, and Applications

Baharin

Ting

Goh

2022

Plants

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“…Like gluten, collagen is rich in proline and 4-hydroxyproline. Thus, efficient hydrolysis of collagen by PIPs allows it to be used to treat wastewater, particularly in the meat industry, which produces a high amount of collagen [19]. The proteolysis of food proteins like soy protein, gluten, or casein often leads to the release of bitter peptides and negatively influences the products' taste.…”

Section: Introductionmentioning

confidence: 99%

“…In light of the proline iminopeptidase industrial benefits, a plethora of molecular characterisation studies have been geared towards the investigation of PIPs from bacteria and fungi, such as Xanthomonas campestri, Aspergillus oryzae, Aspergillus niger, and Phanerochaete chrysosporium [19][20][21][22]. Despite the presence of comparable PIP genes in the genomes of other green and blue moulds, the gene is absent from the genome of Saccharomyces cerevisiae, the most extensively used eukaryotic model organism.…”

Section: Introductionmentioning

confidence: 99%

Biochemical Characterisation and Structure Determination of a Novel Cold-Active Proline Iminopeptidase from the Psychrophilic Yeast, Glaciozyma antarctica PI12

et al. 2022

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Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively characterised in bacteria and filamentous fungi. However, no similar reports exist for yeasts. In this study, a protease gene from Glaciozyma antarctica designated as GaPIP was cloned and overexpressed in Escherichia coli. Sequence analyses of the gene revealed a 960 bp open reading frame encoding a 319 amino acid protein (35,406 Da). The purified recombinant GaPIP showed a specific activity of 3561 Umg−1 towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. GaPIP is a cold-active enzyme with an optimum activity of 30 °C at pH 7.0. The enzyme is stable between pH 7.0 and 8.0 and able to retain its activity at 10–30 °C. Although GaPIP is a serine protease, only 25% inhibition by the serine protease inhibitor, phenylmethanesulfonylfluoride (PMSF) was recorded. This enzyme is strongly inhibited by the presence of EDTA, suggesting that it is a metalloenzyme. The dimeric structure of GaPIP was determined at a resolution of 2.4 Å. To date, GaPIP is the first characterised PIP from yeasts and the structure of GaPIP is the first structure for PIP from eukaryotes.

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Alanine aminopeptidase from Bacillus licheniformis E7 expressed in Bacillus subtilis efficiently hydrolyzes soy protein to small peptides and free amino acids

Chen

Zhang

et al. 2022

LWT

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Mutagenesis for Improvement of Activity and Stability of Prolyl Aminopeptidase from Aspergillus oryzae

Cited by 5 publications

References 41 publications

Post-Proline Cleaving Enzymes (PPCEs): Classification, Structure, Molecular Properties, and Applications

Post-Proline Cleaving Enzymes (PPCEs): Classification, Structure, Molecular Properties, and Applications

Biochemical Characterisation and Structure Determination of a Novel Cold-Active Proline Iminopeptidase from the Psychrophilic Yeast, Glaciozyma antarctica PI12

Alanine aminopeptidase from Bacillus licheniformis E7 expressed in Bacillus subtilis efficiently hydrolyzes soy protein to small peptides and free amino acids

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