Studies on production and characterization of tannase (Tannin acyl hydrolase)
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Date
2015
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Publisher
YSPU
Abstract
Penicillium crustosum AN3 isolated from apple orchard soil is the first report from Penicillium genera
to produce maximum tannase which is an important industrial enzyme used in biodegradation and food industry. A total of eleven fungal isolates were obtained from apple orchard soil, pine forest soil and botanical garden soil, respectively. Out of these eleven isolates five isolates viz., three isolates from apple orchard soil, one isolate from botanical garden soil and one isolate from pine forest soil were showed zone of degradation on Czapdox minimal medium supplemented with 0.5% tannic acid confirming tannase activity. These isolates were rescreened qualitatively for maximum tannase production in submerged fermentation isolate A3 exhibited maximum tannase activity of 2.48Uml-1. Molecular characterization of these isolates was carried out using 18S rrna gene technology and in silico analysis of 18S rrna gene sequences lead to identification of these fungal isolates as Fusarium redolens AN1, Aspergillus fumigatus AN2, Penicillium crustosum AN3, P.restrictum AN4 and P. commune AN5. Tannase producing bacterial isolates were also obtained on nutrient agar (NA) medium supplemented with 0.5% tannic acid. A total of four bacterial isolates from tea garden soil, pine forest soil, ruminial fluid and sheep excreta were screened on the basis of their ability to utilize tannic acid and were characterized morphologically, biochemically and for tannase activity leads to identification as Klebsiella sp, Enterobacter sp, Staphylococcus sp and E.coli respectively. Molecular characterization two bacterial isolates was carried out using 16S rrna gene technology and in silico analysis of 16S rrna gene sequences lead to confirmation of these isolates as Klebsiella variicola AN1 and Enterobacter hormaechei AN2.
Penicillium crustosum AN3 produced maximum tannase (2.45Uml-1) in submerged fermentation was
selected for tannase production in SSF. The enzyme was partially purified from culture extract of enzyme.
Cultural conditions and process parameters i.e. type of substrate, incubation time, temperature, initial pH,
moisture level, substrate concentration, tannic acid concentration, carbon sources, peptone, yeast extract, urea, NH4NO3 were optimized using one factor at a time approach and activity obtained was 39.5Ug-1. Further optimization was carried out using central composite design following response surface methodology with three independent variables which resulted in increased in tannase production to 55.55 Ug-1. The enzyme was partially purified by acetone precipitation and gel filteration chromatography which showed 46.48 yield with 3.94 fold purification and had a molecular mass of 205kDa. Gallic acid the hydrolytic product in crude enzyme was detected by TLC, FTIR and HPLC using gallic acid as standard. Crude enzyme obtained was studied for its ability in pine needle degradation, tea colour decolourization, dye decolourization and removal of apple browning.
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Keywords
organic acids, productivity, fungi, acidity, enzymes, fermentation, poultry equipment, bacteria, genes, extraction