1. The role of polymeric chains as a protective environment for improving the stability and efficiency of fluorogenic peptide substrates
Ana Arnaiz, Marta Guembe-García, Estefanía Delgado-Pinar, Artur J M Valente, Saturnino Ibeas, José M García, Saúl Vallejos Sci Rep. 2022 May 25;12(1):8818. doi: 10.1038/s41598-022-12848-4.
We have faced the preparation of fully water-soluble fluorescent peptide substrate with long-term environmental stability (in solution more than 35 weeks) and, accordingly, with stable results in the use of this probe in determining the activity of enzymes. We have achieved this goal by preparing a co-polymer of the commercial N-vinyl-2-pyrrolidone (99.5% mol) and a fluorescent substrate for trypsin activity determination having a vinylic group (0.5%). The activity of trypsin has been measured in water solutions of this polymer over time, contrasted against the activity of both the commercial substrate Z-L-Arg-7-amido-4-methylcoumarin hydrochloride and its monomeric derivative, prepared ad-hoc. Initially, the activity of the sensory polymer was 74.53 ± 1.72 nmol/min/mg of enzyme, while that of the commercial substrate was 20.44 ± 0.65 nmol/min/mg of enzyme, the former maintained stable along weeks and the latter with a deep decay to zero in three weeks. The 'protection' effect exerted by the polymer chain has been studied by solvation studies by UV-Vis spectroscopy, steady-state & time resolved fluorescence, thermogravimetry and isothermal titration calorimetry.
2. Comparison of protease and aminopeptidase activities in meconium: A pilot study
Ewa Skarżyńska, Paulina Wilczyńska, Bartosz Kiersztyn, Joanna Żytyńska-Daniluk, Artur Jakimiuk, Barbara Lisowska-Myjak Biomed Rep. 2020 Aug;13(2):7. doi: 10.3892/br.2020.1314. Epub 2020 Jun 9.
The successive accumulation of proteases and aminopeptidases in meconium are important physiological components of the intrauterine environment in which a fetus develops. The aim of the present study was to assess the changes in the activities of these enzymes in meconium of healthy infants, and to investigate whether there were any statistically significant associations between activity of the enzymes of interest and the mode of delivery. The activities of proteases and aminopeptidases were determined in meconium portions (n=110) using the substrates BODIPY FL casein and L-leucine-7-amido-4-methylcoumarin hydrochloride, respectively. Serial meconium samples (2-5 per neonate) were collected from healthy infants born vaginally (n=14), and by a cesarean section (n=16). Protease activity (104 RFU/h) was lower in the first meconium sample compared with the final sample from the same infant (3.99±2.03 vs. 5.76±2.24, respectively, mean ± standard deviation; P=0.004). Conversely, there was no significant difference in aminopeptidase activity (103 nM/l/h) between consecutive meconium samples (P=0.702). The ratios of the first-meconium sample enzyme activity to the last-meconium sample enzyme activity were lower for proteases compared with aminopeptidases (0.76±0.48 vs. 1.35±1.04, respectively mean ± standard deviation; P=0.014), and sustained in the infants born by a cesarean section (P=0.008). Spearman's correlation coefficient analysis between the first and last meconium samples showed the correlation increased in the infants born vaginally compared with the rest of the infants (proteases, R=0.618 vs. R=0.314; aminopeptidases, R=0.688 vs. R=0.566). Aminopeptidase activity did not exhibit any notable dynamic changes during meconium accumulation in the fetal intestine. In infants born vaginally compared with those born by a cesarean section, the activity of both proteases and aminopeptidases in the first meconium sample showed an improved correlation with the activity of the final meconium sample. This may suggest that in the intrauterine environment, during accumulation of meconium in the digestive tract of the fetus, the activity and/or levels of these enzymes and the substrates they catalyze were more stable in newborns born vaginally compared with infants born by caesarean section.
3. Purification, molecular cloning, and biochemical characterization of subtilisin JB1 from a newly isolated Bacillus subtilis JB1
Ji Hea Sung, Sang Jung Ahn, Na Young Kim, Soo-Kyoung Jeong, Joong Kyun Kim, Joon Ki Chung, Hyung Ho Lee Appl Biochem Biotechnol. 2010 Oct;162(3):900-11. doi: 10.1007/s12010-009-8830-6. Epub 2009 Nov 10.
An extracellular gelatinolytic enzyme obtained from the newly isolated Bacillus subtilis JB1, a thermophilic microorganism relevant to the aerobic biodegradation process of fish-meal production, was purified via ammonium sulfate precipitation, Sephadex G-200 Gel filtration chromatography, and one-dimensional gel electrophoresis separation and subsequently identified via peptide mass fingerprinting and chemically assisted fragmentation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The subtilisin JB1 gene was sequenced and its recombinant protein prosubtilisin JB1 was expressed in Escherichia coli, and the purified prosubtilisin JB1 (62 kDa) protein was digested with gelatin, bovine serum albumin, azocasein, fibrinogen, and the fluorogenic peptide substrate Ala-Ala-Phe-7-amido-4-methylcoumarin hydrochloride, whereas the serine protease inhibitors phenylmethylsulfonyl fluoride and chymostatin completely inhibited its enzyme activity at an optimal pH of 7.5. Thus, our results show that subtilisin JB1 may serve as a potential source material for use in industrial applications of proteolytic enzymes and microorganisms for fishery waste degradation and fish by-product processing.
