Characteristics of an Acidic Phytase from Aspergillus aculeatus APF1 for Dephytinization of Biofortified Wheat Genotypes.

Phytases are the special class of enzymes which have excellent application potential for enhancing the quality of food by decreasing its inherent anti-nutrient components. In current study, a protease-resistant, acidic phytase from Aspergillus aculeatus APF1 was partially purified by ammonium sulfate fractionation followed by chromatography techniques. The molecular weight of partially purified phytase was in range of 25-35 kDa. The purified APF1 phytase was biochemically characterized and found catalytically active at pH 3.0 and 50 °C. The Km and Vmax values of APF1 phytase for calcium phytate were 3.21 mM and 3.78 U/mg protein, respectively. Variable activity was observed with metal ions and among inhibitors, chaotropic agents and organic solvents; phenyl glyoxal, potassium iodide, and butanol inhibited enzyme activity, respectively, while the enzyme activity was not majorly influenced by EDTA, urea, ethanol, and hexane. APF1 phytase treatment was found effective in dephytinization of flour biofortified wheat genotypes. Maximum decrease in phytic acid content was noticed in genotype MB-16-1-4 (89.98%) followed by PRH3-30-3 (82.32%) and PRH3-43-1 (81.47%). Overall, the study revealed that phytase from Aspergillus aculeatus APF1 could be effectively used in food and feed processing industry for enhancing nutritional value of food.

ß-Propeller phytases: Diversity, catalytic attributes, current developments and potential biotechnological applications.

Phytases are phosphatases which stepwise remove phosphates from phytic acid or its salts. ß-Propeller phytase (BPPhy) belongs to a special class of microbial phytases that is regarded as most diverse, isolated and characterized from different microbes, mainly from Bacillus spp. BPPhy class is unique for its Ca2+-dependent catalytic activity, strict substrate specificity, active at neutral to alkaline pH and high thermostability. Numerous sequence and structure based studies have revealed unique attributes and catalytic properties of this class, as compared to other classes of phytases. Recent studies including cloning and expression and genetic engineering approaches have led to improvements in BPPhy which provide an opportunity for extended utilization of this class of phytases in improving animal nutrition, human health, plant growth promotion, and environmental protection, etc. This review describes the sources and diversity of BPPhy genes, biochemical properties, Ca2+ dependence, current developments in structural elucidation, heterogeneous expression and catalytic improvements, and multifarious applications of BPPhy.

Identification of pregnancy-associated glycoproteins by peptide mass fingerprinting in water buffalo (Bubalus bubalis).

Ruminant placentas synthesize pregnancy-associated glycoproteins (PAGs) during pregnancy, which serve as biomarkers of pregnancy. The present study was conducted to verify, whether PAGs are expressed in buffalo placenta by using lectin-based affinity chromatography and peptide mass finger printing (PMF). Fetal cotyledonary tissues were collected from gravid uteri procured from slaughtered house. Proteins were extracted and subjected to wheat germ agglutinin (WGA) lectin affinity chromatography to isolate the PAGs. The isolated glycoproteins were separated by one-dimensional SDS-PAGE. PMF results of the 75 kDa protein revealed presence of two PAGs (PAG-7 and -11). The PAG-7 consisted of about 170 mass signals, of which 16 were assigned to corresponding/translated cDNA sequences of buffalo PAG-7, leading to sequence coverage of 40%. PMF result of PAG-11 showed 170 mass signals, of which 15 were assigned to buffalo PAG-11, leading to sequence coverage of 34%. In conclusion, the glycoprotein isolated from placental extract corresponding to 75 kDa band on SDS PAGE gel was a mixture of PAG-7 and -11, which may help in development of suitable diagnostics for pregnancy in buffalo.

Insulin stimulates progesterone secretion to a greater extent than LH in early pregnant buffalo luteal cells cultured in vitro.

In the present study, effect of insulin alone or in combination with LH on modulation of progesterone production by early pregnant buffalo luteal cells was reported. Luteal cells were isolated using collagenase and subsequently cultured in Ham'F-12 at 37 °C in an atmosphere of 5% CO2 and 95% humidified air. Small luteal cells (SLC, 12-23 µ) appeared as spindle shaped with eccentrically placed irregular nucleus, however, large luteal cells (LLC, 25-55 µ) were polyhedral or spherical in shape with centrally placed large round nucleus having one or two nucleoli. There was an abundance of cytoplasmic lipid droplets and a greater cytoplasmic to nuclear ratio as compared to SLC. Both small and large luteal cells were positive to 3 ß-HSD, a marker for steroidogenic capacity. Luteal cells attached to surface within 24h of culture and appeared typical of epithelial cells with numerous cytoplasmic lipid droplets within the cytoplasm. These cells maintained the morphological characteristics throughout the culture period. Luteal cells were treated with insulin (0.05 IU/ml) and LH (10 ng/ml) alone or in combination for 7 days to study the effect on progesterone production. Morphology of luteal cells did not change with the addition of LH and insulin. Addition of insulin enhanced (P<0.01) basal as well as LH stimulated progesterone production and also minimized loss of cell number by maintaining greater cell populations throughout the culture period as compared to control and LH treatment. In the absence of tropic stimulation, progesterone secretion decreased rapidly in the control group while addition of insulin greatly decreased the rate of decline. The findings of the present study reveal insulin enhances progesterone secretion by the luteal cells indicating its possible role to modulate corpus luteum function in buffalo.

Cellular and functional characterization of buffalo (Bubalus bubalis) corpus luteum during the estrous cycle and pregnancy.

In the present paper, cellular composition of buffalo corpus luteum (CL) with its functional characterization based on 3ß-HSD and progesterone secretory ability at different stages of estrous cycle and pregnancy was studied. Buffalo uteri along with ovaries bearing CL were collected from the local slaughter house. These were classified into different stages of estrous cycle (Stage I, II, III and IV) and pregnancy (Stage I, II and III) based on morphological appearance of CL, surface follicles on the ovary and crown rump length of conceptus. Luteal cell population, progesterone content and steroidogenic properties were studied by dispersion of luteal cells using collagenase type I enzyme, RIA and 3ß-HSD activity, respectively. Large luteal cells (LLC) appeared as polyhedral or spherical in shape with a centrally placed large round nucleus and an abundance of cytoplasmic lipid droplets. However, small luteal cells (SLC) appeared to be spindle shaped with an eccentrically placed irregular nucleus and there was paucity of cytoplasmic lipid droplets. The size of SLC (range 12-23µm) and LLC (range 25-55µm) increased (P<0.01) with the advancement of stage of estrous cycle and pregnancy. The mean progesterone concentration per gram and per CL increased (P<0.01) from Stage I to III of estrous cycle with maximum concentration at Stage III of estrous cycle and pregnancy. The progesterone concentration decreased at Stage IV (day 17-20) of estrous cycle coinciding with CL regression. Total luteal cell number (LLC and SLC) also increased (P<0.01) from Stage I to III of estrous cycle and decreased (P<0.05), thereafter, at Stage IV indicating degeneration of luteal cells and regression of the CL. Total luteal cell population during pregnancy also increased (P<0.01) from Stage I to II and thereafter decreased (P>0.05) indicating cessation of mitosis. Increased (P<0.05) large luteal cell numbers from Stage I to III of estrous cycle and pregnancy coincided with the increased progesterone secretion and 3ß-HSD activity of CL. Thus, proportionate increases of large compared with small luteal cells were primarily responsible for increased progesterone secretion during the advanced stages of the estrous cycle and pregnancy. Total luteal cells and progesterone content per CL during the mid-luteal stage in buffalo as observed in the present study seem to be less than with cattle suggesting inherent luteal deficiency.