Purification and biochemical characterization of phytase from Bacillus cereus isolated from gastrointestinal tract of African giant snail (Achatina fulica) / Purificación y caracterización bioquímica de fitasa de Bacillus cereus aislada del tracto gastrointestinal de caracol gigante africano (Achatina fulica)

Phytases are specialized enzymes meant for phytic acid degradation. They possess ability to prevent phytic acid indigestion, including its attendant environmental pollution. This study was aimed at investigating biochemical properties of purified phytase of B. cereus isolated from Achatina fulica. Phytase produced from Bacillus cereus that exhibited optimal phytate degrading-ability of all the bacteria isolated was purified in a three-step purification. The biochemical properties of the purified enzyme were also determined. The phytase homogeny of approximately 45 kDa exhibited 12.8-purification fold and 1.6% yield with optima phytate degrading efficiency and maximum stability at pH 7 and 50 °C. Remaining activity of 52 and 47% obtained between 60 and 70 °C after 2 h further established thermostability of the purified phytase. Mg2+ and Zn2+ enhanced phytate hydrolysis by the enzyme, while Na+ showed mild inhibition but Hg2+ severely inhibited the enzymatic activity. Km and Vmax were estimated to be 0.11 mM and 55.6 μmol/min/mL, displaying enzyme-high substrate affinity and catalytic efficiency, respectively. Phytase purified from Bacillus cereus, isolated from African giant snails, has shown excellent characteristics suitable for phytic acid hydrolysis and could be employed in industrial and biotechnological applications.(AU)

Purification and biochemical characterization of phytase from Bacillus cereus isolated from gastrointestinal tract of African giant snail (Achatina fulica).

Phytases are specialized enzymes meant for phytic acid degradation. They possess ability to prevent phytic acid indigestion, including its attendant environmental pollution. This study was aimed at investigating biochemical properties of purified phytase of B. cereus isolated from Achatina fulica. Phytase produced from Bacillus cereus that exhibited optimal phytate degrading-ability of all the bacteria isolated was purified in a three-step purification. The biochemical properties of the purified enzyme were also determined. The phytase homogeny of approximately 45 kDa exhibited 12.8-purification fold and 1.6% yield with optima phytate degrading efficiency and maximum stability at pH 7 and 50 °C. Remaining activity of 52 and 47% obtained between 60 and 70 °C after 2 h further established thermostability of the purified phytase. Mg2+ and Zn2+ enhanced phytate hydrolysis by the enzyme, while Na+ showed mild inhibition but Hg2+ severely inhibited the enzymatic activity. Km and Vmax were estimated to be 0.11 mM and 55.6 µmol/min/mL, displaying enzyme-high substrate affinity and catalytic efficiency, respectively. Phytase purified from Bacillus cereus, isolated from African giant snails, has shown excellent characteristics suitable for phytic acid hydrolysis and could be employed in industrial and biotechnological applications.

Engineered Alcaligenes sp. by chemical mutagen produces thermostable and acido-alkalophilic endo-1,4-ß-mannanases for improved industrial biocatalyst.

This study reported physicochemical properties of purified endo-1,4-ß-mannanase from the wild type, Alcaligenes sp. and its most promising chemical mutant. The crude enzymes from fermentation of wild and mutant bacteria were purified by ammonium sulfate precipitation, ion exchange and gel-filtration chromatography followed by an investigation of the physicochemical properties of purified wild and mutant enzymes. ß-mannanase from wild and mutant Alcaligenes sp. exhibited 1.75 and 1.6 purification-folds with percentage recoveries of 2.6 and 2.5% and molecular weights of 61.6 and 80 kDa respectively. The wild and mutant ß-mannanase were most active at 40 and 50 °C with optimum pH 6.0 for both and were thermostable with very high percentage activity but the wild-type ß-mannanase showed better stability over a broad pH activity. The ß-mannanase activity from the parent strain was stimulated in the presence of Mn2+, Co2+, Zn2+, Mg2+ and Na+. Vmax and Km for the wild type and its mutant were found to be 0.747 U//mL/min and 5.2 × 10-4 mg/mL, and 0.247 U/mL/min and 2.47 × 10-4 mg/mL, respectively. Changes that occurred in the nucleotide sequences of the most improved mutant may be attributed to its thermo-stability, thermo-tolerant and high substrate affinity- desired properties for improved bioprocesses.

Properties of a neutral, thermally stable and surfactant-tolerant pullulanase from worker termite gut-dwelling Bacillus safensis as potential for industrial applications.

The gut of termite has been observed to host communities of bacteria which exhibited pullulan-degrading ability. Bacillus safensis displayed maximum pullulanase (a debranching enzyme) activity and it was therefore selected for production, purification and characterization of pullulanase which was the aim of the study. The crude enzyme obtained from the pullulanase production medium was subjected to ammonium sulphate precipitation, ion exchange and gel-filtration chromatography and the physicochemical properties of the purified was thereafter characterized. A purified pullulanase with the yield of 13% and 24-fold purification was obtained and its homogeneity was established by molecular weight of 42 kDa. The optimum pH 7 and 60 °C were obtained while the enzyme was stable between 40-60 °C and pH 4-5 and 7-8 respectively with significant amount of residual activities recorded. The purified pullulanase was stimulated in the presence of Ca2+, urea and SDS while Al3+, Fe2+, Co2+, Cu2+, Mg2+ and chelating agent, EDTA mildly inhibited the activity of the enzyme in a concentration-dependent manner. The K m and V max were found to be 0.324 µmol/ml/min and 6.85 mg/ml respectively. The exceptional physicochemical properties of B. safensis pullulanase could find application in several industrial processes.

Biochemical studies of enzyme-induced browning of African bush mango (Irvingia gabonensis) fruit pulp.

The purpose of this study was to examine the biochemical properties of African bush mango (Irvingia gabonensis) pulp PPO. PPO was purified from I. gabonensis fruit pulp in three steps and characterized. A purification fold of 343 with specific activity of 216 U/mg and 13% recovery were obtained as well as molecular weight of 32.67 kDa was observed. The optimum pH and temperature were found to be pH 7.0 and 50 °C respectively while the enzyme showed instability at low pH 2-4 with total inactivation at pH 2 but maximal at pH 5-9 with remaining residual activity of 60-90%, whereas, total enzyme activity inactivation was observed at 90 °C. However, Cu2+, Fe2+ and Mg2+ enhanced the PPO activity but inhibited by Ca2+, Ba2+, K+ and Na+. Notably, purified PPO was inactivated completely by urea at concentration above 10 mM while Km and Vmax values were estimated to be 7.34 mM and 0.36 U/min for catechol, 10.76 mM and 0.30 U/min for L-DOPA, and 14.90 mM and 0.26 U/min for tyrosine, respectively. The activity of PPO in I. gabonensis fruit and its juicy product could be controlled at high temperature in acidified medium.