1. Encapsulation and immobilization of ficin extract in electrospun polymeric nanofibers
Andrea S Rojas-Mercado, Iván E Moreno-Cortez, Raúl Lucio-Porto, Luis López Pavón Int J Biol Macromol. 2018 Oct 15;118(Pt B):2287-2295. doi: 10.1016/j.ijbiomac.2018.07.113. Epub 2018 Jul 18.
In this work, ficin extract (3.4.22.3, >1.0 u/mg) was immobilized for the first time in poly (vinyl alcohol) (PVA) electrospun nanofibers in order to preserve its tridimensional molecular structure. The analysis of the morphology of the electrospun nanofibers was carried out using scanning electron microscopy (SEM) showing a diameter in the range of 124-194 nm. The interaction of the ficin extract with the nanofibers structure was analyzed by infrared spectroscopy (FTIR) analysis. The immobilization step was achieved through crosslinking involving the exposure to glutaraldehyde (GA) vapor. The enzyme catalytic behavior was followed by ultraviolet spectroscopy (UV) using the Earlanger method using Nα-benzoyl-l-arginine-4-nitroanilide hydrochloride (BAPA) as substrate. The maximum catalytic activity was reached with 1 h of crosslinking, 20% of enzyme loading and pH 8. The immobilized ficin extract showed 92% of the enzyme activity of the crude ficin extract. The enzymatic activity of the immobilized ficin extract was conserved after a total of nine reuse cycles and maintained after being stored for 25 days. Finally, both the glass transition (Tg) and heat of fusion (Hf) were affected by number of enzyme molecules inside the polymeric nanofibers matrix according to the study of the thermal properties by differential scanning calorimetry (DSC).
2. Comparative analyses of proteolytic activities in seven species of synanthropic acaridid mites
Tomas Erban, Jan Hubert Arch Insect Biochem Physiol. 2010 Nov;75(3):187-206. doi: 10.1002/arch.20388.
Microplate assays with 96 wells were optimized to screen proteolytic activities in mite homogenates. Whole-mite extracts of Acarus siro, Aleuroglyphus ovatus, Tyrophagus putrescentiae, Tyroborus lini, Carpoglyphus lactis, Lepidoglyphus destructor, and Dermatophagoides farinae exhibited non-specific proteolytic activity in buffers from pH 2 to 12, and three peaks of highest activity at pH 3, 5-6, and 10 were distinguished. The reducing agent Tris(2-carboxyethyl)phosphine hydrochloride decreased general proteolytic activity on azocasein at pH 5 and 6. The results obtained on two non-specific substrates, azocasein and azoalbumin, showed highly different ranks of the species at pH 5 and 6. Proteolytic activities toward N(α)-Benzoyl-D,L-arginine 4-nitroanilide hydrochloride, N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-nitroanilide, N-Succinyl-L-alanyl-L-alanyl-L-alanine 4-nitroanilide, Benzyloxycarbonyl-L-arginine-L-arginyl 4-nitroanilide, and N-Methoxysuccinyl-L-alanyl-L-alanyl-L-prolyl-L-methionine 4-nitroanilide (MAAPMpNA) were highest at alkaline pH, but the activity toward MAAPMpNA was also high at pH 5 and 6. In contrast, N-Succinyl-L-alanyl-L-alanyl-L-phenylalanine 4-nitroanilide (AAPpNA) and L-arginyl 4-nitroanilide (ArgpNA) had the highest activity recorded at pH 6. The high activities observed on AAPpNA, ArgpNA, and MAAPMpNA at digestive pH suggest that enzymes present in these extracts could have the majority of proteolysis in the mite gut. Evidence of the presence of proteolytic activities on all tested substrates and in all the tested mite homogenates suggests that the proteolytic activities may contribute to allergenicity. Poor or undetected hydrolytic activities of mite extracts toward substrates for keratin and collagen at digestive pH underline the importance of ecological interactions between mites and microorganisms in the utilization of such substrates.
3. An anomalous behavior of trypsin immobilized in alginate network
Chrystelle Ganachaud, Diana Bernin, Dan Isaksson, Krister Holmberg Appl Microbiol Biotechnol. 2013 May;97(10):4403-14. doi: 10.1007/s00253-012-4333-4. Epub 2012 Aug 12.
Alginate is a biopolymer used in drug formulations and for surgical purposes. In the presence of divalent cations, it forms solid gels, and such gels are of interest for immobilization of cells and enzymes. In this work, we entrapped trypsin in an alginate gel together with a known substrate, N α-benzoyl-L-arginine-4-nitroanilide hydrochloride (L-BAPNA), and in the presence or absence of D-BAPNA, which is known to be a competitive inhibitor. Interactions between alginate and the substrate as well as the enzyme were characterized with transmission electron microscopy, rheology, and nuclear magnetic resonance spectroscopy. The biocatalysis was monitored by spectrophotometry at temperatures ranging from 10 to 42 °C. It was found that at 37 and 42 °C a strong acceleration of the reaction was obtained, whereas at 10 °C and at room temperature, the presence of D-BAPNA leads to a retardation of the reaction rate. The same effect was found when the reaction was performed in a non-cross-linked alginate solution. In alginate-free buffer solution, as well as in a solution of carboxymethylcellulose, a biopolymer that resembles alginate, the normal behavior was obtained; however, with D-BAPNA acting as an inhibitor at all temperatures. A more detailed investigation of the reaction kinetics showed that at higher temperature and in the presence of alginate, the curve of initial reaction rate versus L-BAPNA concentration had a sigmoidal shape, indicating an allosteric behavior. We believe that the anomalous behavior of trypsin in the presence of alginate is due to conformational changes caused by interactions between the positively charged trypsin and the strongly negatively charged alginate.
