Kinetic characterization of a novel acid ectophosphatase from Enterobacter asburiae.

Expression of acid ectophosphatase by Enterobacter asburiae, isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis, was strictly regulated by phosphorus ions, with its optimal activity being observed at an inorganic phosphate concentration of 7 mM. At the optimum pH 3.5, intact cells released p-nitrophenol at a rate of 350.76 ± 13.53 nmol of p-nitrophenolate (pNP)/min/10(8) cells. The membrane-bound enzyme was obtained by centrifugation at 100,000 × g for 1 h at 4 °C. p-Nitrophenylphosphate (pNPP) hydrolysis by the enzyme follows "Michaelis-Menten" kinetics with V = 61.2 U/mg and K0.5 = 60 µM, while ATP hydrolysis showed V = 19.7 U/mg, K0.5 = 110 µM, and nH = 1.6 and pyrophosphate hydrolysis showed V = 29.7 U/mg, K0.5 = 84 µM, and nH = 2.3. Arsenate and phosphate were competitive inhibitors with K i = 0.6 mM and K i = 1.8 mM, respectively. p-Nitrophenyl phosphatase (pNPPase) activity was inhibited by vanadate, while p-hydroxymercuribenzoate, EDTA, calcium, copper, and cobalt had no inhibitory effects. Magnesium ions were stimulatory (K0.5 = 2.2 mM and nH = 0.5). Production of an acid ectophosphatase can be a mechanism for the solubilization of mineral phosphates by microorganisms such as Enterobacter asburiae that are versatile in the solubilization of insoluble minerals, which, in turn, increases the availability of nutrients for plants, particularly in soils that are poor in phosphorus.

Characterization of a Thermotolerant Phytase Produced by Rhizopus microsporus var. microsporus Biofilm on an Inert Support Using Sugarcane Bagasse as Carbon Source.

The Rhizopus microsporus var. microsporus biofilm was able to produce increased levels of an extracellular thermotolerant phytase using polyethylene and viscose as an inert support in both modified NBRIP medium and modified Khanna medium containing sugarcane bagasse as the carbon source. The enzyme production was strictly regulated by the phosphorus content with optimal production at 0.5 mM of sodium phytate and KH2PO4. The extracellular phytase, RMPhy1, was purified 4.18-fold with 4.78 % recovery using DEAE-cellulose and CM-cellulose. A single protein band with a molecular mass of 35.4 kDa was obtained when the samples were subjected to 10 % SDS-PAGE. The optimum temperature for activity was 55 °C and the optimum pH was 4.5. R. microsporus var. microsporus phytase exhibited high stability at 30 and 40 °C with a half-life of 115 min at 60 °C. The enzyme activity increased in the presence of Ca (2+) and was inhibited by Zn(2+), arsenate, and sodium phosphate. Phytase demonstrated high substrate specificity for sodium phytate with K m = 0.72 mM and V max = 94.55 U/mg of protein and for p-NPP with K m = 0.04 mM and V max = 106.38 U/mg of protein. The enzyme also hydrolyzed ATP, AMPc, glucose 6-phosphate, glucose 1-phosphate, and UDPG. This is the first report on phytase characterization delivered with biofilm technology. The properties of the enzyme account for its high potential for use in biotechnology and the possibility of application in different industrial sectors as feed in the future.

Phytase production by Rhizopus microsporus var. microsporus biofilm: characterization of enzymatic activity after spray drying in presence of carbohydrates and nonconventional adjuvants.

Microbial phytases are enzymes with biotechnological interest for the feed industry. In this article, the effect of spray-drying conditions on the stability and activity of extracellular phytase produced by R. microsporus var. microsporus biofilm is described. The phytase was spray-dried in the presence of starch, corn meal (>150 µm), soy bean meal (SB), corn meal (<150 µm) (CM), and maltodextrin as drying adjuvants. The residual enzyme activity after drying ranged from 10.7% to 60.4%, with SB and CM standing out as stabilizing agents. Water concentration and residual enzyme activity were determined in obtained powders as a function of the drying condition. When exposed to different pH values, the SB and CM products were stable, with residual activity above 50% in the pH range from 4.5 to 8.5 for 60 min. The use of CM as drying adjuvant promoted the best retention of enzymatic activity compared with SB. Spray drying of the R. microsporus var. microsporus phytase using different drying adjuvants showed interesting results, being quite feasible with regards their biotechnological applications, especially for poultry diets.