Purification and characterization of extracellular tannin acyl hydrolase from <Emphasis Type="Italic">Aspergillus heteromorphus</Emphasis> MTCC 8818

A tannase (E.C. 3.1.1.20) producing fungal strain was isolated from soil and identified as Aspergillus heteromorphus MTCC 8818. Maximum tannase production was achieved on Czapek Dox minimal medium containing 1% tannic acid at a pH of 4.5 and 30°C after 48 h incubation. The crude enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography. Diethylaminoethyl-cellulose column chromatography led to an overall purification of 39.74-fold with a yield of 19.29%. Optimum temperature and pH for tannase activity were 50°C and 5.5 respectively. Metal ions such as Ca²⁺, Fe²⁺, Cu¹⁺, and Cu²⁺ increased tannase activity, whereas Hg²⁺, Na¹⁺, K¹⁺, Zn²⁺, Ag¹⁺, Mg²⁺, and Cd²⁺ acted as enzyme inhibitors. Various organic solvents such as isopropanol, isoamyl alcohol, benzene, methanol, ethanol, toluene, and glycerol also inhibited enzyme activity. Among the surfactants and chelators studied, Tween 20, Tween 80, Triton X-100, EDTA, and 1, 10-o-phenanthrolein inhibited tannase activity, whereas sodium lauryl sulfate enhanced tannase activity at 1% (w/v).     

Identification of novel single nucleotide polymorphisms in the DGAT1 gene of buffaloes by PCR-SSCP

Diacylglycerol O-acyltransferase 1 (DGAT1) is a microsomal enzyme that catalyzes the final step of triglyceride synthesis. The DGAT1 gene is a strong functional candidate for determining milk fat content in cattle. In this work, we used PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) and DNA sequencing to examine polymorphism in the region spanning exon 7 to exon 9 of the DGAT1 gene in Murrah and Pandharpuri buffaloes. Three alleles (A, B and C) and four novel single-nucleotide polymorphisms were identified in the buffalo DGAT1 gene. The frequencies of the alleles differed between the two buffalo breeds, with allele C being present in Murrah but not in Pandharpuri buffalo. The allele variation detected in this work may influence DGAT1 expression and function. The results described here could be useful in examining the association between the DGAT1 gene and milk traits in buffalo.     

Production of tannase through solid state fermentation using Indian Rosewood (Dalbergia Sissoo)sawdust—a timber industry waste

The tannase producing strain Aspergillus heteromorphus MTCC 8818 was used in the present study for the production of tannase under solid state fermentation using Rosewood (Dalbergia sissoo) sawdust—a timber industry waste—as substrate. Various physico-chemical parameters were optimized for extracellular yield of tannase. Maximum tannase (1.84 U/g dry substrate) and gallic acid (5.4 mg/g ds) was observed at 30 °C after 96 h of incubation. Czapek dox medium was found to be the best moistening agent, with pH and relative humidity of 5.5 and 70 %, respectively. The constituents of Czapek dox medium were varied to enhance enzyme production. The optimum concentration of modified Czapek dox constituents contained 0.2 % NaNO₃, 0.05 % K₂HPO₄ and MgSO₄, 0.15 % KCl. Among the additional salts supplemented to Czapek dox medium, ZnSO₄ and CuSO₄ were found to have a stimulating effect, with a relative tannase activity of 116 and 111 %, respectively. Glucose as an external carbon source was found to be a repressor of enzyme production.     

Molecular differentiation of Peroxysome proliferator activated receptor coactivator-1 among different breeds of Bubalus bubalis

Peroxysome proliferator activated receptor coactivator-1 gene (PPARGC1A) is a positional and functional candidate gene for milk fat yield. It has key role in energy, fat and glucose metabolism. Single nucleotide polymorphisms (SNPs) in Exon-8 of PPARGC1A are reported to be associated with milk fat yield in dairy cattle. In the present investigation PPARGC1A was partially amplified (around 767bp) by designing gene specific primer and confirm by sequencing the amplicon and its comparison with the PPARGC1A gene of bovine. Comparative study of PPARGC1A among different breeds of buffaloes reveals different level of mutations with respect to its gene sequence 0.013-1.69% and protein sequence 0.42% to 2.99%, Similarly the protein structures modeled from their sequences were compared by structural superposition that shows variations (RMSD) from 0.736 to 1.507. Furthermore, the sequences were used to generate a dendrogram. It reveals that Murrah and reference are very close to each other, similarly Toda, Bhadawari and Surti are closely related, whereas Pandharpuri is separated from both the cluster. Especially the variations are more at the binding site of this protein that may be the cause that different breeds have different percentage of milk fat. Further study is underway to detect polymorphism and associate them with milk fat related traits in buffalo.     

Characterization of genetic diversity and population structure in wheat using array based SNP markers

Genetic diversity is crucial for successful adaptation and sustained improvement in crops. India is bestowed with diverse agro-climatic conditions which makes it rich in wheat germplasm adapted to various niches. Germplasm repository consists of local landraces, trait specific genetic stocks including introgressions from wild relatives, exotic collections, released varieties, and improved germplasm. Characterization of genetic diversity is done using morpho-physiological characters as well as by analyzing variations at DNA level. However, there are not many reports on array based high throughput SNP markers having characteristics of genome wide coverage employed in Indian spring wheat germplasm. Amongst wheat SNP arrays, 35K Axiom Wheat Breeder’s Array has the highest SNP polymorphism efficiency suitable for genetic mapping and genetic diversity characterization. Therefore, genotyping was done using 35K in 483 wheat genotypes resulting in 14,650 quality filtered SNPs, that were distributed across the B (~?50%), A (~?39%), and D (~?10%) genomes. The total genetic distance coverage was 4477.85 cM with 3.27 SNP/cM and 0.49 cM/SNP as average marker density and average inter-marker distance, respectively. The PIC ranged from 0.09 to 0.38 with an average of 0.29 across genomes. Population structure and Principal Coordinate Analysis resulted in two subpopulations (SP1 and SP2). The analysis of molecular variance revealed the genetic variation of 2% among and 98% within subpopulations indicating high gene flow between SP1 and SP2. The subpopulation SP2 showed high level of genetic diversity based on genetic diversity indices viz. Shannon’s information index (I)?=?0.648, expected heterozygosity (He)?=?0.456 and unbiased expected heterozygosity (uHe)?=?0.456. To the best of our knowledge, this study is the first to include the largest set of Indian wheat genotypes studied exclusively for genetic diversity. These findings may serve as a potential source for the identification of uncharacterized QTL/gene using genome wide association studies and marker assisted selection in wheat breeding programs.

     

Loss of bone minerals and strength in rats with aldosteronism

Congestive heart failure (CHF) is a clinical syndrome with origins rooted in a salt-avid state largely mediated by effector hormones of the circulating renin-angiotensin-aldosterone system. Other participating neurohormones include catecholamines, endothelin-1, and arginine vasopressin. CHF is accompanied by a systemic illness of uncertain causality. Features include the appearance of oxidative/nitrosative stress and a wasting of tissues including bone. Herein we hypothesized that inappropriate (relative to dietary Na+) elevations in plasma aldosterone (Aldo) contribute to an altered redox state, augmented excretion of divalent cations, and in turn, a loss of bone minerals and strength. In uninephrectomized rats that received chronic Aldo and 1% NaCl treatment for 4-6 wk, we monitored plasma alpha1-antiproteinase activity, which is an inverse correlate of oxidative/nitrosative stress; plasma concentrations of ionized Mg2+ and Ca2+; urinary Mg2+ and Ca2+ excretion; and bone mineral composition and strength to flexure stress. Compared with controls, we found reductions in plasma alpha1-antiproteinase activity and ionized Mg2+ and Ca2+ together with persistently elevated urinary Mg2+ and Ca2+ excretion, a progressive loss of bone mineral density and content with reduced Mg2+ and Ca2+ concentrations, and a reduction in cortical bone strength. Thus the hypermagnesuria and hypercalciuria that accompany chronic Aldo-1% NaCl treatment contribute to the systemic appearance of oxidative/nitrosative stress and a wasting of bone minerals and strength.