Replacement of Trypsin by Proteases for Medical Applications

Cell culture has a crucial role in many applications in biotechnology. The production of vaccines, recombinant proteins, tissue engineering, and stem cell therapy all need cell culture. Most of these activities needed adherent cells to move, which should be trypsinized several times until received on a large scale. Although trypsin is manufactured from the bovine or porcine pancreas, the problem of contamination by unwanted animal proteins, unwanted immune reactions, or contamination to pathogen reagents is the main problem.

This study investigated microbial proteases as a safe alternative for trypsin replacement in cell culture experiments for the detachment of adherent cells.

The bacteria were isolated from the leather industry effluent based on their protease enzymes. After sequencing their 16S ribosomal deoxyribonucleic acid, their protease enzymes were purified, and their enzyme activities were assayed. The alteration of enzymatic activities using different substrates and the effect of substrate concentrations on enzyme activities were determined. The purified proteases were evaluated for cell detachment in the L929 fibroblast cells compared to trypsin. The separated cells were cultured again, and cell proliferation was determined by the MTT assay.

The results showed that the isolated bacteria were Bacillus pumilus, Stenotrophomonas sp., Klebsiella aerogenes, Stenotrophomonasmaltophilia, and Bacillus licheniformis. Among the isolated bacteria, the highest and the lowest protease activity belonged to Stenotrophomonas sp. and Klebsiella aerogenes, with 60.34 and 11.09 U/mL protease activity, respectively. All the isolated microbial proteases successfully affected L929 fibroblast cells’ surface proteins and detached the cells. A significant induction in cell proliferation was observed in the cells treated with Klebsiella aerogenes protease and Bacillus pumilus protease, respectively (P < 0.05).

The obtained results suggested that microbial proteases can be used as safe and efficient alternatives to trypsin in cell culture in biopharmaceutical applications.