A new carboxypeptidase from Aspergillus niger with good thermostability, pH stability and broad substrate specificity

Abstract

A new serine carboxypeptidase gene, capA, was identified in Aspergillus niger CBS 513.88 by reading genomic information and performing sequence alignment, and the gene was cloned and expressed in Pichia pastoris GS115. In a shake flask, the enzyme activity of the recombinant strain GS115 (pPIC9K-capA) reached 209.3 U mg−1. The optimal temperature and pH for enzyme activity were determined to be 45 °C and 6.0, respectively. After incubation at 40–50 °C or at pH 4.0–8.0 for 1 h, the enzyme retained more than 80% or 60% of its initial activity. The presence of 1–10 mmol L−1 Mg2+ enhanced the activity of CapA, whereas 1–10 mmol L−1 Cu2+, Fe2+, or Co2+, 10 mmol L−1 Mn2+, or 1–10 mmol L−1 phenylmethylsulfonyl fluoride (PMSF) significantly inhibited its activity. CapA had a broad substrate specificity and preferred the hydrophobic amino acids Leu and Lys at the C terminus of proteins, and N-benzyloxycarbonyl-l-phenylalanyl-l-leucine (Cbz-Phe-Leu) was the optimal substrate, for which CapA exhibited Km 0.063 mmol L−1 and kcat/Km 186.35 mmol L−1 s−1. The good thermostability, pH stability and hydrolysis characteristics of CapA provide a solid foundation for application in the food and biotechnology fields.

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

“…So the data outlined above are evidence of the successful use of K. phaffii for the heterologous production of almost all the major enzymes important for increasing the nutritional value of feed, including the most popular ones: thermostable phytases [ 198 ], glucoamylases [ 229 , 230 , 231 , 232 , 233 , 234 ], and α-amylases [ 223 , 224 , 225 ], including the co-expression of glucoamylase and α-amylase genes [ 230 ] as well as for the heterologous synthesis of xylanase [ 246 , 247 , 253 ], mannanase [ 276 , 277 , 279 , 281 , 282 , 283 , 285 , 332 ], laccase [ 314 ], cellulases [ 316 ], and proteases [ 326 , 327 , 328 , 329 , 330 , 331 ].…”

“…It was stated above that thermostable and acidophilic enzymes are desirable as feed additives. The source of acid proteases is mainly various species of filamentous fungi Aspergillus, and some of these enzymes are not only resistant to high temperatures [ 325 , 326 , 327 ] but also have exceptionally high specific activity [ 328 ].…”

“…Regarding enzymes that hydrolyze plant proteins, K. phaffii has also been successfully utilized to create strains that synthesize thermostable proteases that will be helpful in animal husbandry for feed manufacture and the improvement of feed quality. An example is the K. phaffii strains secreting recombinant proteases from A. niger [ 326 , 327 , 328 ], Bispora sp. MEY-1 [ 329 ], Aspergillus pseudotamarii [ 330 ], or Tritirachium album [ 331 ].…”

In Silico Structural Analysis of Serine Carboxypeptidase Nf314, a Potential Drug Target in Naegleria fowleri Infections

“…Furthermore, this biochemical reaction is a post-translational modification that plays a critical role in the degradation, processing, and modulation of intracellular proteins. Based on the catalytic mechanism, a typical classification sorts them into serine, metal, and cysteine carboxypeptidases [ 1 , 2 , 3 , 4 ].…”

“…Most of those studied are involved in the intracellular turnover of polypeptide substrates, but some also release amino acids from extracellular proteins or peptides [ 1 , 12 , 13 , 14 , 15 ]. Interestingly, those isolated from fungi are single polypeptides, whereas those from animal cells comprise two polypeptides linked by disulfide bonds [ 1 , 3 , 7 , 9 , 16 , 17 , 18 ].…”