Enhanced Production Process of Recombinant Mature Serratiopeptidase in Escherichia coli Using Fed-Batch Culture by Self-Proteolytic Activity of Fusion Protein

Abstract

Microbial enzymes are increasingly finding applications as therapeutics due to their targeted activity and minimal side effects. Serratiopeptidase, also known as a miracle enzyme, has already proved its potential as an anti-inflammatory, mucolytic, fibrinolytic, analgesic in many studies. A cost effective, bioreactor level production process has been described here comprising of the fed-batch fermentation to produce recombinant serratiopeptidase protein expressed as a fusion construct. High yield of cell mass as well as protein was obtained by the optimization of bioreactor parameters. The downstream solubilization and purification processes were also optimized to achieve maximum yield of pure, active serratiopeptidase protein. A final yield of 2.5 ± 0.764 g L−1 of protein was obtained, having 8382 ± 291 U mg−1 of specific caseinolytic activity. Additionally, a novel, unexpected self-proteolytic activity of the enzyme that cleaves the N-terminal 6× His-SUMO fusion tag along with the enzyme propeptide, thus yielding a mature serratiopeptidase, was also found.

Scale-Up of the Fermentation Process for the Production and Purification of Serratiopeptidase Using Silkworm Pupae as a Substrate

“…The combined application of these analytical techniques not only confirmed the association between increased proteolytic activity and serratiopeptidase production but also provided valuable insights into the structural integrity and purity of the enzymes, which are essential aspects for further downstream applications and industrial-scale processes. Notably, these results are consistent with reported findings on various metalloproteases isolated from distinct strains of S. marcescens, which have molecular weights within the range of 43 to 60 kDa [26,[54][55][56][57][58][59].…”

Research Progress of Fermented Functional Foods and Protein Factory-Microbial Fermentation Technology

“…Fermentation has been used for ages as a safe technique for food preservation, and it uses minimal resources [2]. Some essential commercial enzymes used in fermented foods/beverages are: Protease, Catalase and Lactase for dairy; Amylase, Protease, Glucose oxidase, Pentosanase, Phytase, Pullulanase, Xylanase, Lipases, B-glucanase, Amyloglucosidase, A-acetolactate-decarboxylase, Cellulase and Pectinase for cereals; Glucose oxidase and Tannase for beverages; and Papain and Protease for meat [78][79][80][81]. Microorganisms involved in the fermentation of dairy products are Lactobacillus bulgaricus, Lactococcus lactis, L. acidophilus, L. cremoris, L. thermophilus, L. casei, L. paracasei, L. kefiri, L. caucasicus, Penicillium camembreti, Acetobacter lovaniensis, P. roqueforti, Kluyveromyces lactis and Saccharomyces cerevisiae [80].…”