Laboratoire Of Genie Enzymatique Et Of Microbiologie

Sfax, Tunisia

Laboratoire Of Genie Enzymatique Et Of Microbiologie

Sfax, Tunisia
SEARCH FILTERS
Time filter
Source Type

Bougatef A.,Laboratoire Of Genie Enzymatique Et Of Microbiologie | Balti R.,Laboratoire Of Genie Enzymatique Et Of Microbiologie | Nasri R.,Laboratoire Of Genie Enzymatique Et Of Microbiologie | Jellouli K.,Laboratoire Of Genie Enzymatique Et Of Microbiologie | And 2 more authors.
Journal of Agricultural and Food Chemistry | Year: 2010

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 Nα-p-tosyl-l-arginine methyl ester hydrochloride (TAME) that was four times greater than its amidase-specific activity on Nα-benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the trypsin activity were pH 8.5 and 50 °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 °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 (Km) and catalytic constant (Kcat) 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, Km and Kcat were 0.156 ± 0.01 mM and 59.15 ± 2.2 s-1, respectively. © 2010 American Chemical Society.


PubMed | Laboratoire Of Genie Enzymatique Et Of Microbiologie
Type: Journal Article | Journal: Journal of agricultural and food chemistry | Year: 2010

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.


PubMed | Laboratoire Of Genie Enzymatique Et Of Microbiologie
Type: Journal Article | Journal: Applied biochemistry and biotechnology | Year: 2011

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 12,000U/mL were obtained with 80g/L SWP and 135g/L FSW, respectively. The optimum temperature and pH for protease activity were 60C 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 3h hydrolysis at 40C with an E/S ratio of 0.5 and 5U/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 -chitin. These results suggest that enzymatic deproteinization of shrimp waste by A2 protease could be applicable to the chitin production process.

Loading Laboratoire Of Genie Enzymatique Et Of Microbiologie collaborators
Loading Laboratoire Of Genie Enzymatique Et Of Microbiologie collaborators