Extraction and Characterization of Chitin,Chitosan, and Protein Hydrolysates Prepared from Shrimp Waste by Treatment with Crude Protease from <Emphasis Type="Italic">Bacillus cereus</Emphasis> SV1

Chitin is a polysaccharide found in abundance in the shell of crustaceans. In this study, the protease from Bacillus cereus SV1 was applied for chitin extraction from shrimp waste material of Metapenaeus monoceros. A high level of deproteinization 88.8% ± 0.4 was recorded with an E/S ratio of 20. The demineralization was completely achieved within 6 h at room temperature in HCl 1.25 M, and the residual content of calcium in chitin was below 0.01%. ¹³C CP/MAS-NMR spectral analysis of chitin prepared by the enzymatic deproteinization of shrimp wastes was found to be similar to that obtained by alkaline treatment and to the commercial α-chitin. The degree of N-acetylation, calculated from the spectrum, was 89.5%. Chitin obtained by treatment with crude protease from B. cereus was converted to chitosan by N-deacetylation, and the antibacterial activity of chitosan solution against different bacteria was investigated. Results showed that chitosan solution at 50 mg/mL markedly inhibited the growth of most Gram-negative and Gram-positive bacteria tested. Furthermore, the antioxidant potential of the protein hydrolysates obtained during enzymatic isolation of chitin was evaluated using various in vitro assays. All the samples exerted remarkable antioxidant activities. These results suggest that enzymatic deproteinization of the shrimp shell wastes, using B. cereus SV1 protease, could be applicable to the chitin production process.     

Quantum Mechanics/Molecular Mechanics Studies of the Mechanism of Cysteine Proteases Inhibition by Dipeptidyl Nitroalkenes

In this work a computational study of the mechanism of inhibition of cruzain, rhodesain, and cathepsin L cysteine proteases by the dipeptidyl nitroalkene Cbz-Phe-Ala-CH=CH-NO₂ has been carried out by means of molecular dynamics simulations with hybrid QM/MM potentials. The free-energy surfaces confirmed that the inhibition takes place by the formation of a covalent bond between the protein and the β-carbon atom of the inhibitor. According to the results, the tested inhibitor should be a much more efficient inhibitor of cruzain than of rhodesain, and little activity would be expected against cathepsin L, in total correspondence with the available experimental data. The origin of these differences may lie in the different stabilizing electrostatic interactions established between the inhibitor and the residues of the active site and S2 pocket of these enzymes. These results may be useful for the rational design of new dipeptidyl nitroalkenes with higher and more selective inhibitory activity against cysteine proteases.     

A Solvent-Stable Metalloprotease Produced by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> A2 Grown on Shrimp Shell Waste and Its Application in Chitin Extraction

A solvent-stable protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa A2. The strain was found to produce high level of protease activity when grown in media containing only fresh shrimp waste (FSW) or shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. Maximum protease activities 17,000 and 12,000 U/mL were obtained with 80 g/L SWP and 135 g/L FSW, respectively. The optimum temperature and pH for protease activity were 60 °C and 8.0, respectively. The crude protease, at different enzyme/substrate (E/S) ratio, was tested for the deproteinization of shrimp waste to produce chitin. The crude enzyme of P. aeruginosa A2 was found to be effective in the deproteinization of shrimp waste. The protein removals after 3 h hydrolysis at 40 °C with an E/S ratio of 0.5 and 5 U/mg protein were about 56% and 85%, respectively. ¹³C CP/MAS-NMR spectral analysis of the chitin prepared by treatment with the crude protease was carried out and was found to be similar to that of the commercial α-chitin. These results suggest that enzymatic deproteinization of shrimp waste by A2 protease could be applicable to the chitin production process.     

Quality of Speech Reproduction Using Stochastic Digital Systems of Information Transfer with its Statistical Compaction

Russian Physics Journal - Quality of oral speech reproduction using stochastic digital systems of information transfer at its statistical compaction is analyzed (on the example of Russian and...     

Elucidating the Dual Mode of Action of Dipeptidyl Enoates in the Inhibition of Rhodesain Cysteine Proteases

A computational study of the two possible inhibition mechanisms of rhodesain cysteine protease by the dipeptidyl enoate Cbz-Phe-Leu-CH=CH−CO₂C₂H₅ has been carried out by means of molecular dynamics simulations with hybrid QM/MM potentials. The low free energy barriers confirm that the Cys25 residue can attack both C_β and C1 atoms of the inhibitor, confirming a dual mode of action in the inhibition of the rhodesain by enoates. According to the results, the inhibition process through the Cys25 attack on the C_β atom of the inhibitor is an exergonic and irreversible process, while the inhibition process when Cys25 attacks on the C1 atom of the inhibitor is and exergonic but reversible process. The interactions between the inhibitor and rhodesain suggest that P2 is the most important fragment to consider in the design of new efficient inhibitors of rhodesain. These results may be useful for the design of new inhibitors of rhodesain and other related cysteine proteases based on dipeptidyl enoates scaffolds.     

An Oxidant- and Solvent-Stable Protease Produced by <Emphasis Type="Italic">Bacillus cereus</Emphasis> SV1: Application in the Deproteinization of Shrimp Wastes and as a Laundry Detergent Additive

The current increase in amount of shrimp wastes produced by the shrimp industry has led to the need in finding new methods for shrimp wastes disposal. In this study, an extracellular organic solvent- and oxidant-stable metalloprotease was produced by Bacillus cereus SV1. Maximum protease activity (5,900 U/mL) was obtained when the strain was grown in medium containing 40 g/L shrimp wastes powder as a sole carbon source. The optimum pH, optimum temperature, pH stability, and thermal stability of the crude enzyme preparation were pH 8.0, 60 °C, pH 6–9.5, and <55 °C, respectively. The crude protease was extremely stable toward several organic solvents. No loss of activity was observed even after 60 days of incubation at 30 °C in the presence of 50% (v/v) dimethyl sulfoxide and ethyl ether; the enzyme retained more than 70% of its original activity in the presence of ethanol and N,N-dimethylformamide. The protease showed high stability toward anionic (SDS) and non-ionic (Tween 80, Tween 20, and Triton X-100) surfactants. Interestingly, the activity of the enzyme was significantly enhanced by oxidizing agents. In addition, the enzyme showed excellent compatibility with some commercial liquid detergents. The protease of B. cereus SV1, produced under the optimal culture conditions, was tested for shrimp waste deproteinization in the preparation of chitin. The protein removal with a ratio E/S of 20 was about 88%. The novelties of the SV1 protease include its high stability to organic solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis. In addition, the enzyme may find potential applications in the deproteinization of shrimp wastes to produce chitin.     

Aerosol Synthesis of Highly Dispersed Y3Al5O12:Ce3+ Phosphor with Intense Photoluminescence

Physics of the Solid State - A simple method of pyrolysis of an aerosol solution of yttrium, aluminum, and cerium nitrates with the addition of urea or citric acid, followed by brief annealing, is...     

Estimating the Analytical Performance of Raman Spectroscopy for Quantification of Active Ingredients in Human Stratum Corneum

Confocal Raman microscopy (CRM) has become a versatile technique that can be applied routinely to monitor skin penetration of active molecules. In the present study, CRM coupled to multivariate analysis (namely PLSR—partial least squares regression) is used for the quantitative measurement of an active ingredient (AI) applied to isolated (ex vivo) human stratum corneum (SC), using systematically varied doses of resorcinol, as model compound, and the performance is quantified according to key figures of merit defined by regulatory bodies (ICH, FDA, and EMA). A methodology is thus demonstrated to establish the limit of detection (LOD), precision, accuracy, sensitivity (SEN), and selectivity (SEL) of the technique, and the performance according to these key figures of merit is compared to that of similar established methodologies, based on studies available in literature. First, principal components analysis (PCA) was used to examine the variability within the spectral data set collected. Second, ratios calculated from the area under the curve (AUC) of characteristic resorcinol and proteins/lipids bands (1400–1500 cm⁻¹) were used to perform linear regression analysis of the Raman spectra. Third, cross-validated PLSR analysis was applied to perform quantitative analysis in the fingerprint region. The AUC results show clearly that the intensities of Raman features in the spectra collected are linearly correlated to resorcinol concentrations in the SC (R² = 0.999) despite a heterogeneity in the distribution of the active molecule in the samples. The Root Mean Square Error of Cross-Validation (RMSECV) (0.017 mg resorcinol/mg SC), The Root Mean Square of Prediction (RMSEP) (0.015 mg resorcinol/mg SC), and R² (0.971) demonstrate the reliability of the linear regression constructed, enabling accurate quantification of resorcinol. Furthermore, the results have enabled the determination, for the first time, of numerical criteria to estimate analytical performances of CRM, including LOD, precision using bias corrected mean square error prediction (BCMSEP), sensitivity, and selectivity, for quantification of the performance of the analytical technique. This is one step further towards demonstrating that Raman spectroscopy complies with international guidelines and to establishing the technique as a reference and approved tool for permeation studies.     

Extinction Coefficient of Optical Fibers Irradiated by Thermal Neutrons and Compressed

Russian Physics Journal - Results of experimental studies of the extinction coefficient of optical fibers preliminary irradiated by thermal neutrons and compressed are presented. In this case,...