Purification and characterization of a protease from Aspergillus sydowii URM5774: Coffee ground residue for protease production by solid state fermentation.

Solid state fermentation is a promising technology largely used in biotechnology process and is a suitable strategy for producing low-cost enzymatic products. At the present study, a novel enzyme obtained through solid state fermentation using Aspergillus sydowii was herein purified and characterized. The fermentations used coffee ground residue as substrate and the crude enzyme was submitted through further purification steps of: acetonic precipitation, DEAE-Sephadex and Superdex G-75 column. Both crude and purified enzymes were submitted to biochemical characterization of their thermostability, optimal temperature and pH, effects of inhibitors and metal ions. A purified protease was obtained with yield of 5.9-fold and 53% recovery, with maximal proteolytic activity of 352.0 U/mL. SDS-PAGE revealed a band of protein at 47.0 kDa. The enzyme activity was abolished in the presence of phenyl-methyl sulfonyl fluoride and partially inhibited against Triton X-100 (78.0%). The optimal activity was found in pH 8.0 at 45°C of temperature. Besides, the enzyme showed stability between 35°C and 50°C. It was possible to determine appropriate conditions to the obtainment of thermostable proteases with biotechnological interest associated with a method that concomitantly shows excellent production levels and recovery waste raw material in a very profitable process.

Ultrasound-Assisted Enzyme-Catalyzed Hydrolysis of Collagen to Produce Peptides With Biomedical Potential: Collagenase From Aspergillus terreus UCP1276.

Ultrasound has been applied for varied purposes as it provides additional mechanical energy to a system, and is still profitable and straightforward, which are advantages for industrial applications. In this work, ultrasonic treatments were applied to purified collagenase fractions from a fermented extract by Aspergillus terreus UCP 1276 aiming to evaluate the potential effect on collagen hydrolysis. The physical agent was evaluated as an inductor of collagen degradation and consequently as a producer of peptides with anticoagulant activity. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses were also carried out to compare the hydrolysis techniques. The ultrasound (40 kHz, 47.4 W/L) processing was conducted under the same conditions of pH and temperature at different times. The ultrasound-assisted reaction was accelerated in relation to conventional processing. Collagenolytic activity was enhanced and tested in the presence of phenylmethanesulfonyl fluoride inhibitor. Underexposure, the activity was enhanced, reaching more than 72.0% of improvement in relation to the non-exposed enzyme. A period of 30 min of incubation under ultrasound exposure was enough to efficiently produce peptides with biological activity, including anticoagulation and effect on prothrombin time at about 60%. The results indicate that low-frequency ultrasound is an enzymatic inducer with likely commercial applicability accelerating the enzymatic reaction. Bioelectromagnetics. 2020;41:113-120. © 2019 Bioelectromagnetics Society.

CgTI, a novel thermostable Kunitz trypsin-inhibitor purified from Cassia grandis seeds: Purification, characterization and termiticidal activity.

Cassia grandis trypsin inhibitor (CgTI) is a novel plant serine proteinase inhibitor. This study sets out to purify a thermostable inhibitor from the seeds of Cassia grandis and to provide biochemical information about a novel peptide belonging to the Kunitz family. Moreover, toxicity assays against Artemia, Aedes aegypti larvae-L4 and Nasutitermes corniger are evaluated. The purification process was performed using acetone precipitation, Trypsin-Sepharose-CL4B and Superdex-G75. The inhibitor showed an apparent molecular mass of around 19.8 kDa on Superdex-G75 gel filtration, and a mass of around 19.0 kDa visualized by SDS-PAGE under reducing conditions, and it also showed the protein consists of two polypeptide chains. N-terminal sequencing by Edman's degradation of 16 residues revealed a sequence of amino acids SVVLDTSGEPIRNGGG. 2D-electrophoresis identified a pI value of 6.3 and a 1:1 stoichiometric ratio was noted during CgTI-trypsin complex formation. The inhibitor retained the inhibitory activity over a broad range of pH (5-10) and showed thermostable activity at temperatures 30-80 °C. Furthermore, in vivo assays showed no lethality effect against Artemia and Aedes aegypti larvae, but mortality against Nasutitermes corniger with termiticidal activity LC50 of 0.685 mg/mL on workers and 0.765 mg/mL on soldiers. Preliminary investigations of CgTI revealed it to be a promising biotechnological and biomedical candidate.