Structural analysis, and antioxidant and antibacterial properties of chitosan-poly (vinyl alcohol) biodegradable films.

The development and characterization of biodegradable blend films based on chitosan and poly (vinyl alcohol) for possible use in a variety of biological activities are reported. Fourier transform infrared spectroscopy (FTIR) spectra of chitosan-poly (vinyl alcohol) (Ch/PVA) films showed characteristics peaks shifting to a lower frequency range due to hydrogen bonding between -OH of PVA and -NH2 of chitosan. The chitosan and PVA polymers presented good compatibility. The morphology study of chitosan and composite films showed a compact and homogenous structure. The tensile strength and elongation at break increased with PVA content. In fact, the highest tensile strength and elongation at break (53.58 MPa and 454 %) occurs with pure PVA film. The results showed that PVA incorporation in the blends contributes to increase the intermolecular interactions, thus improving the mechanical properties. In addition, the prepared films demonstrated high antioxidant activities monitored by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging, reducing power, and ß-carotene bleaching activity. Nevertheless, PVA addition reduced antioxidant and antibacterial activities against Gram-positive and Gram-negative bacteria tested.

Cytotoxicity of chitosans with different acetylation degrees and molecular weights on bladder carcinoma cells.

The purpose of this research was to evaluate the cytotoxicity of chitosans with different degrees of acetylation (DA) and molecular weights (MW), as well as the effect of their positive ionic charges controlled by pH on bladder carcinoma cells (RT112 and RT112cp) using the tetrazolium salt colorimetric (MTT) assay. Our data showed that all chitosan samples were cytotoxic on RT112 and RT112cp cells with a higher cytotoxicity obtained at lower pH. Further, it was found that the toxicity increased with increasing DA. However, no significant difference in cytotoxicity between chitosans with different molecular weights was observed. Annexin V-FITC staining test was then used to study and quantify the induction of apoptosis. Data shows that chitosans induce apoptosis of RT112 and RT112cp cells with the same dependence with DA.

Optimization of proteins and minerals removal from shrimp shells to produce highly acetylated chitin.

Chitin and derivatives used for biomedical or pharmaceutical applications require a high level of purity and quality that are difficult to achieve. In this study, we propose to optimize the extraction of chitin in order to obtain pure product keeping a structure as close as possible to the native form. Thus, demineralization step was firstly optimized using response surface methodology. In the optimized conditions predicted by the model, the obtained chitin has an acetylation degree (DA) and a demineralization degree (DM) equal to 99% and 100%, respectively. Then, different microbial and fish crude alkaline proteases were tested for their efficiency in deproteinization. Crude alkaline proteases giving the highest deproteinization degrees (DP), Bacillus mojavensis A21 and Scorpaena scrofa, were selected for chitin extraction. The obtained DP was 88±2% and 83±1%, respectively. At the end, effect of the use of mixed enzymatic treatment with the two selected crude enzymes and the order of demineralization/deproteinization steps were tested. The results demonstrated that two separated steps in enzymatic treatments realized on demineralized sample give the best DP (96%) preserving the DA (99%).

Characterization and In Vitro Evaluation of Cytotoxicity, Antimicrobial and Antioxidant Activities of Chitosans Extracted from Three Different Marine Sources.

Chitins in the α and ß isomorphs were extracted from three Tunisian marine sources shrimp (Penaeus kerathurus) waste, crab (Carcinus mediterraneus) shells and cuttlefish (Sepia officinalis) bones. The obtained chitins were transformed into chitosans, the acid-soluble form of chitin. Chitosans were characterized and their biological activities were compared. Chitosan samples were then characterized by Fourier transform infrared spectroscopy (FTIR). The results showed that all chitosans presented identical spectra. Antimicrobial, antioxidant, and antitumor activities of the extracted chitosans were investigated. In fact, cuttlefish chitosan showed the highest DPPH radical-scavenging activity (83 %, 5 mg/ml), whereas it was 79 % and 76 % for shrimp and crab chitosans, respectively. However, in linoleate-ß-carotene system, cuttlefish and crab chitosans exerted higher antioxidant activity (82 % and 70 %, respectively), than shrimp chitosan (49 %). Chitosans were tested for their antimicrobial activities against three Gram-negative and four Gram-positive bacteria and five fungi. Chitosans markedly inhibited growth of most bacteria and fungi tested, although the antimicrobial activity depends on the type of microorganism and on the source of chitin. In addition, chitosans showed high antitumor activity which seemed to be dependent on the chitosan characteristics such as acetylation degree and especially the molecular weight.

Chitin extraction from crab shells by Bacillus bacteria. Biological activities of fermented crab supernatants.

Crab shells waste were fermented using six protease-producing Bacillus species (Bacillus subtilis A26, Bacillus mojavensis A21, Bacillus pumilus A1, Bacillus amyloliquefaciens An6, Bacillus licheniformis NH1 and Bacillus cereus BG1) for the production of chitin and fermented-crab supernatants (FCSs). In medium containing only crab shells, the highest demineralization DM was obtained with B. licheniformis NH1 (83±0.5%) and B. pumilus A1 (80±0.6%), while the highest deproteinization (DP) was achieved with A1 (94±1%) followed by NH1 (90±1.5%) strains. Cultures conducted in medium containing crab shells waste supplemented with 5% (w/v) glucose, were found to remarkably promote demineralization efficiency, and enhance slightly deproteinization rates. FTIR spectra of chitins showed the characteristics bands of α-chitin. FCSs showed varying degrees of antioxidant activities which were in a dose-dependent manner (p<0.01). In fact, FCS produced by B. amyloliquefaciens An6 exhibited the highest DPPH free radical-scavenging activity (92% at 4 mg/ml), while the lowest hydroxyl radical-scavenging activity (60% at 4 mg/ml) was obtained with B. subtilis A26 hydrolysates. However, the highest reducing power (OD700nm=2 at 0.5 mg/ml) was obtained by B.amyloliquefaciens An6 hydrolysates. These results suggest that crab hydrolysates are good sources of natural antioxidants. Further, FCSs were found to exhibit antibacterial activity against Gram-positive and Gram-negative bacteria.

Use of a fractional factorial design to study the effects of experimental factors on the chitin deacetylation.

Chitosan is obtained by deacetylation of chitin. Chitosan versatility is directly related to the polymer's characteristics depending on the deacetylation process. The aim of this research was to study the parameters influencing deacetylation and to elucidate their effect on acetylation degree (DA) and molecular weight (MW). The effect on chitosan DA was investigated using a fractional factorial design 2(7-3) with seven factors and two variation levels. The tested factors were: X1=number of successive baths, X2=reaction time, X3=temperature, X4=alkali reagent, X5=sodium borohydride, X6=the atmospheric conditions and X7=alkali concentration. A mathematical model was investigated corresponding to the following relation y=7.469-1.344X1-1.094X2-3.094X3+1.906X4+0.656X5+0.906X6-1.031X7+0.469X1X2-0.781X3X4+0.906X1X3X4 with R2=0.99. This model allows fixing experimental conditions for each desired DA. To study the effect on chitosan MW, only atmospheric conditions and use of sodium borohydride as an oxygen scavenger were investigated. The use of sodium borohydride and nitrogen atmosphere was found to have a protective effect against chitosan degradation during deacetylation.

Chitin extraction from shrimp shell using enzymatic treatment. Antitumor, antioxidant and antimicrobial activities of chitosan.

Chitin was recovered through enzymatic deproteinization of the shrimp processing by-products. Different microbial and fish viscera proteases were tested for their deproteinization efficiency. High levels of protein removal of about 77±3% and 78±2% were recorded using Bacillus mojavensis A21 and Balistes capriscus proteases, respectively, after 3h of hydrolysis at 45°C using an enzyme/substrate ratio of 20U/mg. Therefore, these two crude proteases were used separately for chitin extraction and then chitosan preparation by N-deacetylation. Chitin and chitosan samples were then characterized by 13 Cross polarization magic angle spinning nuclear magnetic resonance (CP/MAS)-NMR spectroscopy and compared to samples prepared through chemical deproteinization. All chitins and chitosans showed identical spectra. Chitosans prepared through enzymatic deproteinization have practically the same acetylation degree but higher molecular weights compared to that obtained through chemical process. Antimicobial, antioxidant and antitumoral activitities of chitosan-M obtained by treatment with A21 proteases and chitosan-C obtained by alkaline treatment were investigated. Results showed that both chitosans inhibited the growth of most Gram-negative, Gram-positive bacteria and fungi tested. Furthermore, both chitosans exhibited antioxidant and antitumor activities which was dependent on the molecular weight.

Influence of acetylation degree and molecular weight of homogeneous chitosans on antibacterial and antifungal activities.

The results given in the literature are conflicting when considering the relationship between antimicrobial activity and chitosan characteristics. To be able to clarify, we prepared fifteen homogeneous chitosans with different acetylation degrees (DA) and molecular weights (MW) by reacetylation of a fully deacetylated chitin under homogeneous conditions. They were tested at different pH values for their antimicrobial activities against four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi), four Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis and Micrococcus luteus) and three fungi (Aspergillus niger, Fusarium oxysporum and Alternaria solani). Chitosans markedly inhibited growth of most bacteria and fungi tested, although the inhibitory effect depends on the type of microorganism and on the chitosan characteristics (DA and MW) with minimum inhibitory concentrations in the range of 0.001 to 0.1 w%. Considering chitosan efficiency on bacteria, our series of data clearly show that the lower DA and the lower pH give the larger efficiency. Antibacterial activity was further enhanced for Gram-negative bacteria with decreasing MW, whereas, opposite effect was observed with the Gram-positive. Concerning the antifungal activity, the influence of chitosan characteristics was dependent on the particular type of fungus. Fungal growth decreased with increasing MW for F. oxysporum and decreasing DA for A. solani, but no MW or DA dependences were observed with A. niger.

Structural differences between chitin and chitosan extracted from three different marine sources.

Three marine sources of chitin from Tunisia were investigated. Structural differences between α-chitin from shrimp (Penaeus kerathurus) waste, crab (Carcinus mediterraneus) shells, and ß-chitin from cuttlefish (Sepia officinalis) bones were studied by the (13)C NMR, FTIR, and XRD diffractograms. The (13)C NMR analysis showed a splitting of the C3 and C5 carbon signals for α-chitin, while that of ß-chitin was merged into a single resonance. The bands contour of deconvoluted and curve-fit FTIR spectra showed a more detailed structure of α-chitin in the region of O-H, N-H and CO stretching regions. IR and (13)C NMR were used to determine the chitin degree of acetylation (DA). XRD analysis indicated that α-chitins were more crystalline polymorph than ß-chitin. Shrimp chitin was obtained with a good yield (20% on raw material dry weight) and no residual protein and salts. Chitosans, with a DA lower than 20% and relatively low molecular masses were prepared from the wet chitins in the same experimental conditions. They were perfectly soluble in acidic medium. Nevertheless, chitin and chitosan characteristics were depending upon the chitin source.

Optimization of chitin extraction from shrimp waste with Bacillus pumilus A1 using response surface methodology.

Chitin extraction from shrimp shells by biological treatment, using the Bacilli Bacillus pumilus A1, is a non-polluting method and offers the opportunity to preserve the exceptional qualities of chitin and its derivatives. However, the major disadvantage of the fermentative way is the low efficiency of demineralization and deproteinization. The aim of this study is to improve the yield of extraction which depends on many factors, such as the medium composition and the physical parameters. In order to look for the optimal conditions, a Plackett and Burman design was carried out to screen eight factors influencing the deproteinization and demineralization efficiencies. The four most influencing variables were then examined to achieve the optimization using a central composite design. The results obtained showed that the optimal conditions were: shrimp shell concentration of 70 g/l, glucose concentration of 50 g/l, pH of 5.0 incubated with 0.225 OD of B. pumilus A1 inoculum, at 35 °C and 150 rpm for 6 days in 500 ml flask containing 100 ml of working volume. These conditions led to 88% of demineralization and 94% of deproteinization. (13)C CP/MAS NMR spectral analysis of the chitin prepared was carried out and was found to be similar to that of the commercial α-chitin.

Pseudomonas aeruginosa A2 elastase: purification, characterization and biotechnological applications.

An extracellular protease from Pseudomonas aeruginosa A2 grown in media containing shrimp shell powder as a unique source of nutriments was purified and characterized. The enzyme was purified to homogeneity from culture supernatant by ultrafiltration, Sephadex G-100 gel filtration and Sepharose Mono Q anion exchange chromatography, with a 2.23-fold increase in specific activity and 64.3% recovery. The molecular mass of the enzyme was estimated to be 34 kDa. Temperature and pH with highest activity were 60 °C and 8.0, respectively. The protease activity was inhibited by EDTA suggesting that the purified enzyme is a metalloprotease. The enzyme is stable in the presence of organic solvents mainly diethyl ether and DMSO. The lasB gene, encoding the A2 elastase, was isolated and its DNA sequence was determined. The A2 protease was tested for shrimp waste deproteinization in the process of chitin preparation. The percent of protein removal after 3 h hydrolysis at 40 °C with an enzyme/substrate (E/S) ratio of 5 U/mg protein was about 75%. Additionally, A2 proteolytic preparation demonstrated powerful depilating capabilities of hair removal from bovine skin. Considering its promising properties, P. aeruginosa A2 protease may be considered a potential candidate for future use in several biotechnological processes.

Shrimp waste fermentation with Pseudomonas aeruginosa A2: optimization of chitin extraction conditions through Plackett-Burman and response surface methodology approaches.

A Box-Bhenken design with four variables (shrimp shell concentration (SSC), glucose concentration, incubation time and inoculum size) and three levels was used for the determination of the deproteinization and demineralization efficiencies in fermented shrimp shells by Pseudomonas aeruginosa A2. The fermentation variables were selected in accordance with Plackett-Burman design. Maximum demineralization of 96%, with about 89% of protein removal occurs under the following conditions: SSC 50 g/l, glucose 50 g/l, 5 days and inoculum of 0.05 OD. This environment friendly method (biological treatment) can be considered as an effective pretreatment to produce a high-quality chitin.

A solvent-stable metalloprotease produced by Pseudomonas aeruginosa 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. (13)C 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.

Purification and characterization of three trypsin isoforms from viscera of sardinelle (Sardinella aurita).

Three trypsin isoforms A, B and C were purified to homogeneity from the viscera of sardinelle (Sardinella aurita). Purification was achieved by ammonium sulfate precipitation (20-70% (w/v)), Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography. The molecular weights of these purified enzymes were estimated to be 28.8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the native PAGE and casein-zymography, each purified trypsin appeared as a single band. Trypsins A and C exhibited the maximal activity at 55°C, while trypsin B at 50°C. All isoforms showed the same optimal pH (pH 9.0) using Nα-benzoyl-DL: -arginine-p-nitroanilide (BAPNA) as a substrate. The three trypsins were stable at temperatures below 40°C and over a broad pH range (7.0-11.0). The activities of the three isoforms were strongly inhibited by soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, a serine protease inhibitor, and partially inhibited by ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. Kinetic constants of trypsins A, B and C for BAPNA were evaluated at 25°C and pH 9.0. The values of K (m) and k (cat) were 0.125, 0.083 and 0.10 mM, and 2.24, 1.21 and 5.76 s(-1), respectively. The N-terminal sequences of the first 10 amino acids were "I V G G Y E C Q K Y" for trypsin A and "I V G G Y E A Q S Y" for trypsins B and C. These sequences showed highly homology to other fish trypsins.

Comparative Study on Biochemical Properties and Antioxidative Activity of Cuttlefish (Sepia officinalis) Protein Hydrolysates Produced by Alcalase and Bacillus licheniformis NH1 Proteases.

Antioxidative activities and biochemical properties of protein hydrolysates prepared from cuttlefish (Sepia officinalis) using Alcalase 2.4 L and Bacillus licheniformis NH1 proteases with different degrees of hydrolysis (DH) were determined. For the biochemical properties, hydrolysis by both enzymes increased protein solubility to above 75% over a wide pH range. The antioxidant activities of cuttlefish protein hydrolysates (CPHs) increase with increasing DH. In addition, all CPHs exhibited antioxidative activity in a concentration-dependent manner. NH1-CPHs generally showed greater antioxidative activity than Alcalase protein hydrolysates (P < 0.05) as indicated by the higher 1,1-diphenyl-1-picryhydrazyl (DPPH) radical scavenging activity and ferrous chelating activity. Both Alcalase and NH1 protein hydrolysates were able to retard lipid peroxidation and ß-carotene-linoleic acid oxidation. Alcalase-CPH (DH = 12.5%) and NH1-CPH (DH = 15%) contained 75.36% and 80.11% protein, respectively, with histidine and arginine as the major amino acids, followed by glutamic acid/glutamine, serine, lysine, and leucine. In addition, CPHs have a high percentage of essential amino acids made up 48.85% and 50.04%. Cuttlefish muscle protein hydrolysates had a high nutritional value and could be used as supplement to poorly balanced dietary proteins.

Cathepsin D from the hepatopancreas of the cuttlefish (Sepia officinalis): purification and characterization.

Cathepsin D from the hepatopancreas of cuttlefish ( Sepia officinalis ) was purified to homogeneity by precipitation with ammonium sulfate (30-60%, w/v), Sephadex G-100 gel filtration, Mono-S cation-exchange chromatography, Sephadex G-75 gel filtration, and Mono-S FPLC with a 54-fold increase in specific activity and 17% recovery. The molecular weight of the purified cathepsin D was estimated to be 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of the native-PAGE and hemoglobin zymography, the purified protease appeared as a single band. The optimum pH and temperature for the cathepsin D activity were pH 3.0 and 50 °C, respectively, using hemoglobin as a substrate. The purified enzyme was completely inhibited by pepstatin A; however, no inhibition was observed with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Moreover, the activity was strongly inhibited by SDS and molybdate and enhanced by ATP. The purified cathepsin D was activated by Mg(2+), Ni(2+), Zn(2+), Cu(2+), Cd(2+), Sr(2+), and Co(2+) ions, whereas it was not affected by Na(+), K(+), and Ca(2+) ions. The N-terminal amino acid sequence of the first 13 amino acids of the purified cathepsin D was APTPEPLSNYMDA. S. officinalis cathepsin D, which showed high homology with cathepsin D from marine vertebrates and invertebrates, had a Pro residue at position 6 and a Ser residue at position 8, where Thr and Lys are common in all marine vertebrates cathepsins D. S. officinalis cathepsin D showed high efficiency for the hydrolysis of myofibrillar proteins extracted from cuttlefish muscle.

A highly thermostable antimicrobial peptide from Aspergillus clavatus ES1: biochemical and molecular characterization.

Antimicrobial peptides (AMPs) are extremely attractive candidates as therapeutic agents due to their wide spectrum of antimicrobial activity and mechanism of action, which differs from that of small-molecule antibiotics. In this study, a 6.0-kDa antimicrobial peptide from Aspergillus clavatus ES1, designated as AcAMP, was isolated by a one-step heat treatment. AcAMP was sensitive to proteolytic enzymes, stable between pH 5.0 and 10.0, and heat resistant (15 min at 100 degrees C). The acamp gene encoding AcAMP peptide was isolated by reverse-transcriptase polymerase chain reaction (RT-PCR) and cloned in pCRII-TOPO vector. Sequence analysis of the complementary DNA (cDNA) acamp gene revealed an open reading frame of 282 bp encoding a peptide of 94 amino acid residues consisting of a 21-aa signal peptide, a 22-aa pro-peptide, and a 51-aa mature peptide. The deduced amino acid sequence showed high identity with other ascomycete antifungal peptides. AcAMP belongs to the group of small, cysteine-rich, basic proteins with antimicrobial activity. In addition to its antifungal activity, AcAMP is the first fungal peptide exhibiting antibacterial activity against several Gram-positive and Gram-negative bacteria. Based on all these features, AcAMP can be considered as a promising new member of the restraint family of ascomycete antimicrobial peptides that might be used in biological control of plant diseases and also for potential applications in food preservation.

Biochemical properties of anionic trypsin acting at high concentration of NaCl purified from the intestine of a carnivorous fish: smooth hound (Mustelus mustelus).

Trypsin from the intestine of smooth hound (Mustelus mustelus) was purified by fractionation with ammonium sulfate, Sephadex G-75 gel filtration, and DEAE-cellulose ion exchange chromatography, with a 65-fold increase in specific activity and 15% recovery. The molecular weight of the purified trypsin was estimated to be 24 kDa using size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme showed esterase-specific activity on N(alpha)-p-tosyl-L-arginine methyl ester hydrochloride (TAME) that was four times greater than its amidase-specific activity on Nalpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the trypsin activity were pH 8.5 and 50 degrees C, respectively, using TAME as a substrate. The enzyme was extremely stable in the pH range of 7.0-9.0 and highly stable up to 40 degrees C after 1 h of incubation. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and N-p-tosyl-1-lysine chloromethyl ketone (TLCK), specific inhibitors for trypsin. In addition, smooth hound trypsin showed higher proteolytic activity at high NaCl concentration, demonstrating its potential for protein hydrolysis at high salt content. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECKPHSQ. This sequence showed high homology with trypsins from marine vertebrates and invertebrates. Purified trypsin had a Michaelis-Menten constant (K(m)) and catalytic constant (K(cat)) of 0.387 +/- 0.02 mM and 2.62 +/- 0.11 s(-1), respectively, when BAPNA was used as a substrate. For the hydrolysis of TAME, K(m) and K(cat) were 0.156 +/- 0.01 mM and 59.15 +/- 2.2 s(-1), respectively.

Extraction and characterization of chitin, chitosan, and protein hydrolysates prepared from shrimp waste by treatment with crude protease from Bacillus cereus 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%. (13)C 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 alpha-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.

An oxidant- and solvent-stable protease produced by Bacillus cereus 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 degrees C, pH 6-9.5, and <55 degrees 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 degrees 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.