Ali Fazil Yenidunya

Production and immobilization of a novel thermoalkalophilic extracellular amylase from bacilli isolate

A Thermoalkalophilic amylase was produced from an environmental bacterial isolate. The enzyme was then immobilized through its amino groups onto the epoxy rings of magnetic poly glycidyl methacrylate [m-poly (GMA)] beads. The free enzyme was active within a large pH range, between 7 and 12 and displayed the optimum activity at 95°C and pH 10. The immobilization appeared to increase the stability of the enzyme as its bound form showed optimum activity at 105°C and pH 11.0. Kinetic studies demonstrated that immobilized enzyme had higher K(m) and lower V(max) values. The activity of the free and bound enzyme was determined, at 37°C and pH 10.0 and pH 11.0, respectively, in the presence of various organic solvents and detergents (5%, v/v). Results obtained indicated that detergents, sodium dodecyl sulfate (SDS) and TritonX-100, caused six fold increase and that various organic solvents also increased the activity of the amylase.

Synthesis, characterization, and assessment of cytotoxic, antiproliferative, and antiangiogenic effects of a novel procainamide hydrochloride-poly(maleic anhydride-co-styrene) conjugate

Poly(maleic anhydride-co-styrene) (MAST) was synthesized by a free-radical polymerization reaction. A bioactive molecule, procainamide hydrochloride (PH), was then conjugated to MAST. The conjugation product was named as MAST/PH. Structural characterization of MAST and MAST/PH was carried out by Fourier Transform Infrared and Nuclear Magnetic Resonance spectroscopy. Their molecular weights were determined by size-exclusion chromatography. A mechanism was then suggested for the conjugation reaction. The results of the cytotoxicity assay, employing a mouse fibroblast cell line (L929), indicated that MAST/PH had no cytotoxicity at concentrations 62 μg mL−1 (p > 0.05). Antiproliferative activities of MAST/PH and PH were determined by the BrdU cell proliferation ELISA assay, using C6 and HeLa cell lines. In the experiment, two anticancer chemotherapy drugs, cisplatin and 5-fluorouracil, were included as positive control. Antiproliferative activity results demonstrated that MAST/PH yielded the highest suppression profile (approximately 42%) at 20 μg/ml, while free PH exerted the same activity at 100 μg/ml. Interestingly, both MAST/PH and PH suppressed the proliferation of only one of the cell lines, C6 cells. Both cisplatin and 5-fluorouracil yielded approximately 60% antiproliferative activity on C6 cells at 20 and 100 μg/ml concentrations. Antiangiogenic capacity of both MAST and MAST/PH was also investigated by using the chicken chorioallantoic membrane assay. Results obtained indicated that while MAST/PH could be included into the category of good antiangiogenic substances, the activity score of MAST was within the weak category.

Production, purification, and characterization of metalloprotease from Candida kefyr 41 PSB

A thermostable metalloprotease, produced from an environmental strain of Candida kefyr 41 PSB, was purified 16 fold with a 60% yield by cold ethanol precipitation and affinity chromatography (bentonite-acrylamide-cysteine microcomposite). The purified enzyme appeared as a single protein band at 43kDa. Its optimum pH and temperature points were found to be 7.0 and 105°C, respectively. Km and Vmax values of the enzyme were determined to be 3.5mg/mL and 4.4μmolmL-1min-1, 1.65mg/mL and 6.1μmolmL-1min-1, using casein and gelatine as the substrates, respectively. The activity was inhibited by using ethylenediamine tetraacetic acid (EDTA), indicating that the enzyme was a metalloprotease. Stability of the enzyme was investigated by using thermodynamic and kinetic parameters. The thermal inactivation profile of the enzyme conformed to the first order kinetics. The half life of the enzyme at 95, 105, 115, 125 and 135°C was 1310, 610, 220, 150, and 86min, respectively.

Amoebicidal Effect of Poly (Maleic Anhydride-Co-Vinyl Acetate) Copolymer on Entamoeba Histolytica Trophozoites

Poly (maleic anhydride-co-vinyl acetate) (MAVA) copolymer was synthesized by free-radical copolymerization in methyl ethyl ketone (MEK) at 80oC, using benzoyl peroxide (BPO) as the initiator. The radical chain copolymerization was confirmed by Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (1H-NMR) spectra. Copolymer surface morphology was visualized by scanning electron microscopy (SEM). After these characterization studies, the amoebicidal effect of MAVA was investigated on E.histolytica trophozoites using differing copolymer concentrations and periods of time. Results indicated that MAVA killed all the trophozoites at 32 mg/mL concentration in 3 h.

Characterization of thermostable β-amylase isozymes from Lactobacillus fermentum

A strain of Lactobacillus fermentum producing two isozymes of a 20kDa β-amylase was isolated from the faecal sample of a newborn. The starin was identified by sequencing its 16S rRNA gene. The two β-amylase isozymes were resolved and visualized by two dimensional protein gel electrophoresis (2-D gel electrophoresis). Some of the physical and biochemical properties of the enzymes were characterized. The β-amylase displayed two optimum pH s, 5.0 and 10.0 and two optimum temperatures, 45°C and 37°C, respectively. The isozymes hydrolyzed different substrates: glycogen at pH 5.0, and corn starch at pH 10.0. The activity did not require Ca2+, though the activity at pH 10.0 was enhanced in the presence of 5.0mM and 10.0mM CaCl2, 110% and 130%, respectively.