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

Serratiopeptidase, a bacterial metalloprotease known for its pain-relieving and anti-inflammatory properties, can be produced through fermentation with S. marcescens. This study aimed to identify key factors related to nutrient composition and physicochemical conditions for production in Erlenmeyer flasks and to scale up the mixture to a bioreactor to obtain the maximum proteolytic activity. A Plackett-Burman design was used to determine whether the presence of silkworm pupae (at 1.5%) was a significant parameter for serratiopeptidase production. Along with the variables pH, temperature, and time, they were optimized using a Taguchi experimental design, resulting in values of 7, 25 °C, and 36 h, respectively. Scaling up with a kLa of 25.45 ± 3.12 h-1 showed the highest serratiopeptidase production at 24 h. A factorial design was used for ultrafiltration, resulting in an LMH (liters per square meter per hour) of 960 L/m2h, a TMP (transmembrane pressure) of 15 psi, and a concentration factor of five, with a specific activity of 24,325.81 ± 1515.69 U/mg. Afterward, the retentate was purified using strong anion exchange chromatography and ultrafiltration, yielding a 19.94 ± 3.07% recovery and a purification factor of 1.59 ± 0.31. In conclusion, waste from the sericulture industry can be used for serratiopeptidase production.

Performance of Colombian Silk Fibroin Hydrogels for Hyaline Cartilage Tissue Engineering.

The development and evaluation of scaffolds play a crucial role in the engineering of hyaline cartilage tissue. This work aims to evaluate the performance of silk fibroin hydrogels fabricated from the cocoons of the Colombian hybrid in the in vitro regeneration of hyaline cartilage. The scaffolds were physicochemically characterized, and their performance was evaluated in a cellular model. The results showed that the scaffolds were rich in random coils and ß-sheets in their structure and susceptible to various serine proteases with different degradation profiles. Furthermore, they showed a significant increase in ACAN, COL10A1, and COL2A1 expression compared to pellet culture alone and allowed GAG deposition. The soluble portion of the scaffold did not affect chondrogenesis. Furthermore, they promoted the increase in COL1A2, showing a slight tendency to differentiate towards fibrous cartilage. The results also showed that Colombian silk could be used as a source of biomedical devices, paving the way for sericulture to become a more diverse economic activity in emerging countries.

Purification and characterization of a metalloprotease produced by the C8 isolate of Serratia marcescens using silkworm pupae or casein as a protein source.

Serratiopeptidase, a metalloprotease produced by Serratia marcescens, is produced through a fermentation process using carbohydrates and proteins as carbon and nitrogen sources. However, some byproducts of the silk industry could be an alternative source for serratiopeptidase production. Therefore, the present work is focused on the purification and characterization of a serratiopeptidase produced from the C8 isolate of Serratia marcescens and obtained from a Colombian silkworm hybrid using casein or silkworm pupae. The protease was purified using ultrafiltration, anion-exchange, and size-exclusion chromatography. The purified enzyme showed a molecular weight of ~50 kDa with a purity above 96%, an isoelectric point of ~4.6, optimum pH and temperature of 6 and 50 °C, and stability at 4 °C for one month. The kinetic constants using azocasein as substrate were 0.63 mM (Km), 2,016 µM/min (Vmax), 41.41 s-1 (Kcat), and 6.56 × 107 M-1 s-1 (Kcat/Km). Inhibition by 5 mM EDTA or 1,10-phenanthroline was recovered by adding Zn2+ at the same concentration. Mass spectrometry analysis indicated 94% homology with the sequence of serratiopeptidase produced by the E-15 strain. We purified and characterized a serratiopeptidase produced by the C8 isolate of S. marcescens in a culture medium based on a renewable source from the silk industry.