Characterization Of Aspartic Protease Research Articles

Purification and characterization of aspartic protease from Aspergillus niger and its efficient hydrolysis applications in soy protein degradation

BackgroundAdding acid protease to feed can enhance protein digestibility, boost feed utilization, and stimulate the growth of animals in breading industry. In order to obtain an acid protease with high hydrolysis efficiency to plant protein, in this study, an aspartic protease from Aspergillus niger was heterologous expressed in Pichia pastoris (P. pastoris). The enzymatic properties and application in soybean protein degradation were also studied.ResultsIn our investigation, the high aspartic protease (Apa1) activity level of 1500 U/mL was achieved in 3 L bioreactor. After dialysis and anion exchange chromatography, the total enzyme activity and specific enzyme activity were 9412 U and 4852 U/mg, respectively. The molecular weight of the purified protease was 50 kDa, while the optimal pH and temperature were 3.0 and 50 °C, respectively. It was stable at pH 2.0–5.0 and 30–60 °C. Apa1 was used to hydrolyze soybean isolate protein (SPI) at 40 °C and pH 3.0, and a high hydrolysis degree (DH) of 61.65% was achieved. In addition, the molecular weight distribution of SPI hydrolysis products was studied, the result showed that the hydrolysis products were primarily oligopeptides with molecular weights of 189 Da or below.ConclusionsIn this study, Apa1 was successfully expressed in P. pastoris and high expression level was obtained. In addition, the highest protein hydrolysis rate to SPI degradation so far was achieved. The acid protease in this study provides a new protease that is suitable for the feed industry, which will be very helpful to improve the feed utilization and promote the development of the breeding industry.

Purification and characterization of a milk-clotting aspartic protease from Withania coagulans fruit

Withania coagulans fruit has traditionally been used as milk coagulant. The present study reports the purification and characterization of an aspartic protease from W. coagulans fruit. The enzyme was purified via fractional ammonium sulfate precipitation and cation exchange chromatography. SDS-PAGE analysis revealed the presence of a monomeric protein with molecular weight of 31kDa. Proteolytic activity (PA) of the protease was evaluated using casein, and the milk-clotting activity (MCA) was analyzed by skim milk. The Km and Vmax values of the enzyme for casein were obtained to be 1.29mg/ml and 0.035μmol Tyr/min, respectively. Optimal temperature and pH were 65°C and 5.5, respectively. After incubation of enzyme at 65°C for 1h, 73% of PA was remained which demonstrated high thermal stability of the enzyme. Mass spectrometry analysis of the purified protease and enzyme assays in the presence of protease inhibitors indicated that aspartic protease was the only responsible enzyme in milk coagulation. Furthermore, by investigating the effect of salts on enzyme activity, it was observed that both NaCl and CaCl2 reduced enzyme activity. These characteristics of the protease suggest that the enzyme may be suitable for producing low salt content cheeses.

Bioinformatic characterization of aspartic protease (AP) enzyme in seed plants

Aspartic proteases (APs; EC: 3.4.23) are a catalytic type of protease enzymes that use an activated water molecule bound to one or more aspartate residues for catalysis of their peptide substrates. They have been extensively studied and are widely distributed among vertebrates, plants, yeast, nematodes, parasites, fungi, and viruses. In this study, bioinformatic analyses of AP enzyme were performed in seed plants protein sequences, including 33 species of 14 different families. According to the conserved motifs obtained by MEME and MAST tools, two motifs were common in all seed plants. The structural and functional analyses of six selected monocots and dicots were investigated by ProtParam, SOPMA, SignalP 4.1, TMHMM 2.0 and ProDom tools in ExPASy database. Tertiary structure of Arabidopsis thaliana as a sample of seed plants, was predicted by Phyre2 server using “d1oewa” model (PDB accession code: 1oew) and its quality was verified by PROCHECK and ProSA-web servers. The protein sequences were aligned with ClustalW algorithm by MEGA 6.06 software and phylogenetic tree was constructed using the Neighbor-joining (NJ) method. In protein–protein interactions analysis by STRING 9.1 tool, 12 and 135 significant protein interaction groups were identified in Arabidopsis thaliana and other species, respectively. According to the results, there is a high identity among APs of different species in seed plants, so that they should be derived from a common ancestor. The approaches and results reported here would be useful to prioritize candidate genes for further functional proteomic studies of this enzyme in seed plants.