Proteases are important for decomposition of proteins to generate peptides or amino acids and have a broad range of applications in different industries. Herein, a gene encoding an alkaline protease (AprBcp) from Bacillus circulans R1 was cloned and bioinformatics analyzed. In addition, a series of strategies were applied to achieve high-level expression of AprBcp in Bacillus subtilis. The maximum activity of AprBcp reached 165,870 U/ml after 60 h fed-batch cultivation in 50 l bioreactor. The purified recombinant AprBcp exhibited maximum activity at 60°C and pH 10.0, and remained stable in the range from pH 8.0 to 11.0 and 30 to 45°C. Metal ions Ca2+, Mn2+, and Mg2+ could improve the stability of AprBcp. Furthermore, the recombinant AprBcp displayed great potential application on the recovery of protein from soybean dregs. The results of this study will provide an effective method to prepare AprBcp in B. subtilis and its potential application on utilization of soybean dregs.
Pigment proteins play a vital role in the red colour change of the red swamp crayfish (Procambarus clarkii) shell after cooking. In this study, two red-change-related pigment proteins with molecular weights of approximately 170 and 43 kDa—denoted as F1 and F2, respectively—were purified by ammonium sulphate salting-out and size exclusion chromatography. F1 and F2 entirely comprised homomultimeric protein complexes composed of 21 kDa subunits. LC-MS/MS analysis showed that the 21 kDa protein subunit belonged to the crustacyanin family, named P. clarkii crustacyanin A2 (PcCRA2). The full-length cDNA of PcCRA2 was cloned, which encoded 190 amino acid residues and was highly homologous (91.58%) with Cherax quadricarinatus crustacyanin A. The predicted 3D structure showed that PcCRA2 had a β-barrel structure for pigment encapsulation. The colour change of F1 was first detected at 40 °C, and the red change occurred upon heating above 60 °C. Additionally, with increasing temperature, its β-sheet content increased, and its α-helix content reduced. Correlation analysis showed that the redness value of F1 was significantly related to the heating temperature and the β-sheet content.
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