Thermostable ficin from jelly fig (Ficus pumila var. awkeotsang) latex: purification, identification and characterization.

BACKGROUND: The achenes/seeds of endemic jelly fig (Ficus pumila var. awkeotsang) fruit have been applied to prepare a traditional beverage in Taiwan. Upon fruit harvest, jelly fig latex exuded from stalks was discarded. Protease activity was monitored in its latex. Proteases capable of hydrolyzing proteins have many application aspects based on diverse characteristics. Commercial plant proteases are frequently from latex. RESULTS: The latex protease of jelly fig, termed FaFicin, was purified to homogeneity with a molecular mass of ~32 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. According to liquid chromatographic-tandem mass spectrometric analyses, the expected protein band of protease was matched to ficin A, ficin B or chymopapain from common fig or papaya. Iodoacetamide, an inhibitor of cysteine protease, inhibited its protease activity completely. Hence FaFicin was identified as a papain-like cysteine protease (PLCP), exhibiting more than 80% and 70% activity as assayed at pH 5-8 and 40-70 °C, respectively. It maintained ~89% of initial activity after 120 min at 55 °C and pH 7. Moreover, FaFicin could degrade the myosin and actin of meat, and clot milk. CONCLUSION: The ficin FaFicin was obtained, purified and identified as a PLCP member from agricultural waste: jelly fig latex. It possessed activity under a wide range of pH values and temperature, and exhibited excellent thermostability. Based on its initial evaluation as a meat tenderizer and milk clotting reagent, the application of FaFicin was possible, which may extend utilization of jelly fig. © 2022 Society of Chemical Industry.

Purification, identification and characterization of Nag2 N-acetylglucosaminidase from Trichoderma virens strain mango.

BACKGROUND: N-acetylglucosaminidase (NAGase) could liberate N-acetylglucosamine (GlcNAc) from GlcNAc-containing oligosaccharides. Trichoderma spp. is an important source of chitinase, particularly NAGase for industrial use. nag1 and nag2 genes encoding NAGase, are found in the genome in Trichoderma spp. The deduced Nag1 and Nag2 shares ~ 55% homology in Trichoderma virens. Most studies were focus on Nag1 and nag1 previously. RESULTS: The native NAGase (TvmNAG2) was purified to homogeneity with molecular mass of ~ 68 kDa on SDS-PAGE analysis, and identified as Nag2 by MALDI/MS analysis from an isolate T. virens strain mango. RT-PCR analyses revealed that only nag2 gene was expressed in liquid culture of T. virens, while both of nag1 and nag2 were expressed in T. virens cultured on the plates. TvmNAG2 was thermally stable up to 60 °C for 2 h, and the optimal pH and temperature were 5.0 and 60-65 °C, respectively, using p-nitrophenyl-N-acetyl-ß-D-glucosaminide (pNP-NAG) as substrate. The hydrolytic product of colloidal chitin by TvmNAG2 was suggested to be GlcNAc based on TLC analyses. Moreover, TvmNAG2 possesses antifungal activity, inhibiting the mycelium growth of Sclerotium rolfsii. And it was resistant to the proteolysis by papain and trypsin. CONCLUSIONS: The native Nag2, TvmNAG2 was purified and identified from T. virens strain mango, as well as enzymatic properties. To our knowledge, it is the first report with the properties of native Trichoderma Nag2.