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Nattokinase (NK, also known as subtilisin NAT) (EC 3.4.21.62) is one of the most considerable extracellular enzymes produced by Bacillus subtilis natto. The main interest about this enzyme is due to its direct fibrinolytic activity. Being stable enough in the gastrointestinal tract makes this enzyme a useful agent for the oral thrombolytic therapy. Thus, NK is regarded as a valuable dietary supplement or nutraceutical. Proven safety and ease of mass production are other advantages of this enzyme. In addition to these valuable advantages, there are other applications attributed to NK including treatment of hypertension, Alzheimer's disease, and vitreoretinal disorders. This review tends to bring a brief description about this valuable enzyme and summarizes the various biotechnological approaches used in its production, recovery, and purification. Some of the most important applications of NK, as well as its future prospects, are also discussed.

No abstract

Various advantages of protein secretion have prompted scientists to search for secretory production of heterologous proteins. Signal peptides are one of the most important factors for prosperous secretion of the recombinant proteins. The aim of this study was to evaluate 23 different signal peptides and theoretically determine suitable ones for secretory production of the human growth hormone (hGH) in the E. coli host. The signal peptide sequences and the precise location of their cleavage sites were predicted using SignalP 4.0 server. Accordingly, six of the signal peptides, including hGH, major outer membrane lipoprotein, protease VII, protein TolB, periplasmic protein TorT and beta-lactamase TEM were excluded from the further study, due to their inappropriate cleavage scores. Different physico-chemical properties, which are essential for selecting a proper signal peptide, were evaluated using ProtParam and Solpro as the most accurate and reliable servers. Computational analysis of the abovementioned factors, indicates that outer membrane protein C, fimbrial chaperone SfmC, outer membrane protein F and disulfide interchange protein DsbA can theoretically be suitable signal peptides for hGH secretion.

Several investigations are being pursued to enhance the efficacy and specificity of fibrinolytic therapy. In this regard, microbial fibrinolytic enzymes attracted much more medical interests during these decades. Subtilisin, a member of subtilases (the superfamily of subtilisin-like serine proteases) and also a fibrinolytic enzyme is quite common in Gram-positive bacteria, and Bacillus species stand out in particular, as many extracellular and even intracellular variants have been identified. In the present work, the subtilisin gene from Bacillus subtilis PTCC 1023 was cloned into the vector pET-15b and expressed in Escherichia coli strain BL21 (DE3). Total genomic DNA were isolated and used for PCR amplification of the subtilisin gene by means of the specific primers. SDS-PAGE and enzyme assay were done for characterizing the expressed protein. A ~1,100 bp of the structural subtilisin gene was amplified. The DNA and amino acid sequence alignments resulting from the BLAST search of subtilisin showed high sequence identity with the other strains of B. subtilis, whereas significantly lower identity was observed with other bacterial subtilisins. The recombinant enzyme had the same molecular weight as other reported subtilisins and the E. coli transformants showed high subtilisin activity. This study provides evidence that subtilisin can be actively expressed in E. coli. The commercial availability of subtilisin is of great importance for industrial applications and also pharmaceutical purposes as thrombolytic agent. Thus, the characterization of new recombinant subtilisin and the development of rapid, simple, and effective production methods are not only of academic interest, but also of practical importance.