Evaluation of the biotechnological potential of a novel purified protease BS1 from Bacillus safensis S406 on the chitin extraction and detergent formulation.

An extracellular alkaline stable protease BS1 from a new bacteria strain, Bacillus safensis S406, isolated from the Sfax solar saltern, was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-75 gel filtration, Mono-Q anion-exchange chromatography and ultrafiltration, with a 12.70-fold increase in specific activity and 20.29% recovery. The enzyme has a molecular weight of 29kDa and appeared as a single band on native-PAGE. The optimum pH and temperature values of its proteolytic activity were pH 11.0 and 60°C, respectively. BS1 was tested for the deproteinization of shrimp wastes to extract chitin. An enzyme-protein ratio of 10U/mg of proteins allows to eliminate 93% of protein linked to the chitin after 3h hydrolysis at 45°C. Being very active in alkaline conditions, the potential application of BS1 in laundry formulation was investigated. The enzyme showed high stability in the presence of non-ionic surfactants and some commercial liquid and solid detergents, suggesting its eventual use in detergent formulations.

Alkaline proteases from a newly isolated Micromonospora chaiyaphumensis S103: Characterization and application as a detergent additive and for chitin extraction from shrimp shell waste.

The present study was undertaken to characterize the extracellular thermostable serine alkaline proteases from newly actinomycete strain Micromonospora chaiyaphumensis S103 and to describe their evaluation in commercial detergents and shrimp waste deproteinization. This proteolytic crude extract was active and stable in alkaline solution. It was extremely stable in the pH range of 5.0-12.0. The optimum pH and temperature were 8.0 and 70°C, respectively, using casein as a substrate. The thermoactivity and thermostability of proteases were enhanced by the addition of 5mM Ca2+. Proteases from S103 were also used for shrimp wastes deproteinization in the process of chitin preparation. The percent of protein removal after 3h hydrolysis at 45°C with an enzyme/substrate ratio of 20U/mg had reached 93%. Furthermore, S103 crude enzyme was stable towards several organic solvents and retained 100% of its original activity after 90days of incubation in the presence of methanol, hexane, acetone, and DMSO. These properties make S103 proteases an ideal choice for application in detergent formulations, chitin production, and enzymatic peptide synthesis.

New acidic proteases from Liza aurata viscera: Characterization and application in gelatin production.

The present study reports the extraction and characterisation of acidic protease from the viscera of golden grey mullet (LACAP), and its use in gelatin preparation. The optimum pH for the crude extract activity was 3.0, with high stability over a pH range from 3.0 to 7.0. The enzymatic extract lost about 79% of its activity by pepstatin A. Due to its high activity under acidic conditions, gelatin was extracted from the skin of L. aurata using different levels of LACAP (0, 5 and 10U/g of skin, named GGSG0, GGSG5 and GGSG10). The extraction yield of GGSG0 was only 3.3% and the addition of acidic proteases increased the yields, which reached 5% and 8.2% at 5 and 10U/g of skin, respectively. In addition, Fourier transform infrared (FT-IR) and SDS-page profiles of gelatins indicated that the structure was affected by enzymatic pre-treatment. The results of functional properties showed that the emulsion stability and activity indexes (ESI and EAI) of GGSG0 were higher than those of GGSG5 and GGSG10. Furthermore, foam expansion (FE) and foam stability (FS) increased as the concentration of gelatin increased. The results showed that L. aurata by-products can be a potential source of gelatin and protease.

Digestive alkaline proteases from thornback ray (Raja clavata): Characteristics and applications.

This study describes the characterization of a crude protease extract from thornback ray (Raja clavata) and its evaluation in liquid detergent and in deproteinizattion of shrimp waste. At least five clear caseinolytic proteases bands were observed in a zymogram. The crude protease showed optimum activity at pH 8.0 and 50 °C, and it was highly stable over pH range from 8.0 to 11.0. Proteolytic enzymes were very stable in non-ionic surfactants and in the presence of oxidizing agents, maintaining 70% of their activity after incubation for 1 h at 30 °C in the presence of 1% sodium perborate. In addition, they showed high stability and compatibility with various liquid laundry-detergents available in the Tunisian market. The crude extract retained 100% of its activity after preincubation for 60 min at 30 °C in the presence of Nadhif Perfect, Textil and Carrefour laundry detergents. Further, proteases from R. clavata viscera were used for shrimp waste deproteinization in the process of chitin preparation. The percent of protein removal after 3 h hydrolysis at 45 °C with an enzyme/substrate ratio of 30 U/mg of proteins was 74%. These results suggest that enzymatic deproteinization of shrimp wastes by fish endogenous alkaline proteases could be applicable to the chitin production process.

A cysteine protease isolated from the latex of Ficus microcarpa: purification and biochemical characterization.

A plant protease named microcarpain was purified from the latex of Ficus microcarpa by acetonic (20-40 % saturation) precipitation, Sephadex G-75 filtration, and Mono Q-Sefinose FF chromatography. The protease was purified with a yield of 9.25 % and a purification factor of 8. The molecular weight of the microcarpain was estimated to be 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme showed maximum activity at pH 8.0 and at a temperature of 70 °C. Proteolytic activity was strongly inhibited by dithio-bis-nitrobenzoic acid (DTNB), Hg(2+), and Cu(2+). The N-terminal amino acid sequence of the purified microcarpain "VPETVDWRSKGAV" showed high homology with a protease from Arabidopsis thaliana. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine peptidases family.

Zebra blenny (Salaria basilisca) viscera as a source of solvent-stable proteases: characteristics, potential application in the deproteinization of shrimp wastes and evaluation in liquid laundry commercial detergents.

The present study describes the characterization of crude protease extract from zebra blenny (Salaria basilisca) and its evaluation in liquid detergent and shrimp waste deproteinization. At least five caseinolytic proteases clear bands were observed in zymogram. The crude alkaline protease showed optimum activity at pH 8.0 and 60 °C, and it was highly stable over a wide range of pH from 6.0 to 11.0. Proteolytic enzymes showed extreme stability towards non-ionic surfactants (5 % Tween 80 and 5 % Triton X-100) and oxidizing agents (1 % sodium perborate), and relative stability towards anionic surfactant (1 % Sodium dodecyl sulfate (SDS)). They also showed high stability and compatibility with various laundry liquid detergents from Tunisian market. Furthermore, the crude enzyme was stable towards several organic solvents and retained more than 50 % of its original activity after 30 days of incubation at 30 °C in the presence of 50 % (v/v) dimethylsulfoxide (DMSO). Further, proteases from zebra blenny viscera were found to be effective in the deproteinization of shrimp wastes. The protein removal after 3 h at 40 °C with an enzyme/substrate ratio (E/S) of 5 U/mg protein was about 77 %.

Optimization of acid protease production by Aspergillus niger I1 on shrimp peptone using statistical experimental design.

Medium composition and culture conditions for the acid protease production by Aspergillus niger I1 were optimized by response surface methodology (RSM). A significant influence of temperature, KH(2)PO(4), and initial pH on the protease production was evaluated by Plackett-Burman design (PBD). These factors were further optimized using Box-Behnken design and RSM. Under the proposed optimized conditions, the experimental protease production (183.13 U mL(-1)) closely matched the yield predicted by the statistical model (172.57 U mL(-1)) with R(2) = 0.914. Compared with the initial M1 medium on which protease production was 43.13 U mL(-1), a successful and significant improvement by 4.25 folds was achieved in the optimized medium containing (g/L): hulled grain of wheat (HGW) 5.0; KH(2)PO(4) 1.0; NaCl 0.3; MgSO(4)(7H(2)O) 0.5; CaCl(2) (7H(2)O) 0.4; ZnSO(4) 0.1; Na(2)HPO(4) 1.6; shrimp peptone (SP) 1.0. The pH was adjusted at 5 and the temperature at 30°C. More interestingly, the optimization was accomplished using two cheap and local fermentation substrates, HGW and SP, which may result in a significant reduction in the cost of medium constituents.