In a culture medium, the Rhizopus oryzae strain produces only one form of lipase, ROL32. When the concentrated culture medium was stored at 0°C during several months or kept at 6°C during a few days, we noticed the appearance of a second shorter form of ROL32 lacking its N-terminal 28 amino acid (ROL29). ROL29 was purified to homogeneity and its 21 N-terminal amino acid residues were found to be identical to the 29-49 sequence of ROL32. The cleavage of the N-terminal peptide reduced the specific activity of ROL29 by 50% using either triolein or tributyrin as substrates. In order to explain this decrease of the specific activity of ROL29, we measured its critical surface pressure of penetration into phosphatidyl choline from egg yolk films which was found to be 10 mN/m, in contrast to a value of 23 mN/m found in ROL32. A kinetic study on the surface pressure dependency, stereoselectivity and regioselectivity of ROL29 was performed using the three dicaprin isomers spread as monomolecular films at the air-water interface. Our results showed that in contrast to ROL32, ROL29 presented a preference for the distal ester groups of one diglyceride isomer (1,3-sn-dicaprin). Furthermore, ROL32 was markedly more stereoselective than ROL29 for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. A structural explanation of the enhanced penetration capacity as well as the catalytic activity of ROL32 was proposed by molecular modeling. We concluded that the N-terminal peptide of ROL32 can play an important role in the specific activity, the regioselectivity, the stereoselectivity and the binding of the enzyme to its substrate.

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A fibrinolytic enzyme producing bacterium was isolated and identified as Bacillus subtilis A26 on the basis of the 16S rRNA gene sequence. The fibrin zymography analysis reveals the presence of at least three fibrinolytic enzymes. The crude enzyme exhibited maximal activity at 60 degrees C and pH 8.0. Medium composition and culture conditions for the enzyme production by B. subtilis A26 were optimized using two statistical methods. The Plackett-Burman statistical design was applied to find the key ingredients and conditions for the best yield of enzyme production. Five significant variables (hulled grain of wheat, casein peptone, NaCl, CaCl2, and initial pH) were selected for the optimization studies. The response surface methodological approach was used to determine the optimal concentrations and conditions. The optimized medium contained 40.0 g.L-1 hulled grain of wheat, 3.53 g.L-1 casein peptone, 4.0 g.L-1 CaCl2, 3.99 g.L-1 NaCl, 0.01 g.L-1 MgSO4, and 0.01 g.L-1 KH2PO4, pH 7.78. The medium optimization resulted in a 4.2-fold increased level of fibrinolytic production (269.36 U.mL-1) compared with that obtained with the initial medium (63.45 U.mL-1). A successful and significant improvement in the production of protease by the A26 strain was accomplished using inexpensive carbon substrate (hulled grain of wheat), allowing a significant reduction in the cost of medium constituents.

Medium composition and culture conditions for the acid protease production byAspergillus nigerI1 were optimized by response surface methodology (RSM). A significant influence of temperature, KH2PO4, 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) withR2=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; KH2PO41.0; NaCl 0.3; MgSO4(7H2O) 0.5; CaCl2(7H2O) 0.4; ZnSO40.1; Na2HPO41.6; shrimp peptone (SP) 1.0. The pH was adjusted at 5 and the temperature at30°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.

To explore proteolytic activity of endophytic fungi inhabiting date palm roots, a Penicillium bilaiae isolate, displaying the highest level of protease production, has been recovered. Response surface methodology (RSM) was applied to optimize culture conditions for protease production by the fungus. Plackett-Burman design allowed for screening of variables effective in protease production. Results indicated that temperature, initial pH and glucose concentration dramatically affect protease yield. These factors were further optimized using a Box-Behnken design and RSM. A combination of initial pH (6.26), temperature (24.5 °C), glucose (13.75 g/L), NaNO3 (1.5 g/L), MgSO4 (0.2 g/L), KH2PO4 (0.5 g/L) and KCl (0.5 g/L) were optimum for maximum production of protease. A 1086-fold enhancement of protease production was gained after optimization. Biochemical properties of fungal protease including the effect of pH and temperature on the activity and the stability of proteolytic enzyme were determined. Moreover, the influence of carbon and nitrogen sources, metal ions, detergents as well as enzyme inhibitors was investigated. Our results highlighted that protease of Penicillium bilaiae isolate TDPEF30 could be considered as a promising candidate for industrial applications.