Sujit Sadashiv Jagtap

Decolorization of adsorbed textile dyes by developed consortium of Pseudomonas sp. SUK1 and Aspergillus ochraceus NCIM-1146 under solid state fermentation.

The objective of this study was to develop consortium using Pseudomonas sp. SUK1 and Aspergillus ochraceus NCIM-1146 to decolorize adsorbed dyes from textile effluent wastewater under solid state fermentation. Among various agricultural wastes rice bran showed dye adsorption up to 90, 62 and 80% from textile dye reactive navy blue HE2R (RNB HE2R) solution, mixture of textile dyes and textile industry wastewater, respectively. Pseudomonas sp. SUK1 and A. ochraceus NCIM-1146 showed 62 and 38% decolorization of RNB HE2R adsorbed on rice bran in 24h under solid state fermentation. However, the consortium of Pseudomonas sp. SUK1 and A. ochraceus NCIM-1146 (consortium-PA) showed 80% decolorization in 24h. The consortium-PA showed effective ADMI removal ratio of adsorbed dyes from textile industry wastewater (77%), mixture of textile dyes (82%) and chemical precipitate of textile dye effluent (CPTDE) (86%). Secretion of extracellular enzymes such as laccase, azoreductase, tyrosinase and NADH-DCIP reductase and their significant induction in the presence of adsorbed dye suggests their role in the decolorization of RNB HE2R. GCMS and HPLC analysis of product suggests the different fates of biodegradation of RNB HE2R when used Pseudomonas sp. SUK1, A. ochraceus NCIM-1146 and consortium PA.

Saccharification of poplar biomass by using lignocellulases from Pholiota adiposa

A basidiomycetous fungus, identified as Pholiota adiposa SKU0714 on the basis of morphological and phylogenetic analyses, was found to secrete efficient lignocellulose degrading enzymes. The strain showed maximum endoglucanase, cellobiohydrolase and β-glucosidase activities of 26, 32 and 39 U/mL, respectively and also secreted xylanase, laccase, mannanase, and lignin peroxidase with activities of 1680, 0.12, 65 and 0.41 U/mL, respectively when grown with rice straw as a carbon source. Among the various plant biomasses tested for saccharification, poplar biomass produced the maximum amount of reducing sugar. Response surface methodology was used to optimize hydrolysis parameters. A maximum saccharification yield of 83.4% (667 mg/g-substrate), the highest yield from any plant biomass, was obtained with Populus biomass after 24h of hydrolysis. P. adiposa was proven to be a good choice for the production of reducing sugars from cellulosic biomass.

Enzymatic hydrolysis of aspen biomass into fermentable sugars by using lignocellulases from Armillaria gemina

A white rot fungus, identified as Armillaria gemina SKU2114 on the basis of morphological and phylogenetic analyses, was found to secrete efficient lignocellulose-degrading enzymes. The strain showed maximum endoglucanase, cellobiohydrolase, and β-glucosidase activities of 146, 34, and 15 U/mL, respectively, and also secreted xylanase, laccase, mannanase, and lignin peroxidase with activities of 1270, 0.16, 57, and 0.31 U/mL, respectively, when grown with rice straw as a carbon source. Among various plant biomasses tested for saccharification, aspen biomass produced the maximum amount of reducing sugar. Response surface methodology was used to optimize the hydrolysis of aspen biomass to achieve the highest level of sugar production. A maximum saccharification yield of 62% (429 mg/g-substrate) was obtained using Populus tomentiglandulosa biomass after 48 h of hydrolysis. A. gemina was shown to be a good option for use in the production of reducing sugars from lignocellulosic biomass.

Cloning and characterization of a galactitol 2-dehydrogenase from Rhizobium legumenosarum and its application in D-tagatose production.

Galactitol 2-dehydrogenase (GDH) belongs to the protein subfamily of short-chain dehydrogenases/reductases and can be used to produce optically pure building blocks and for the bioconversion of bioactive compounds. An NAD(+)-dependent GDH from Rhizobium leguminosarum bv. viciae 3841 (RlGDH) was cloned and overexpressed in Escherichia coli. The RlGDH protein was purified as an active soluble form using His-tag affinity chromatography. The molecular mass of the purified enzyme was estimated to be 28kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 114kDa by gel filtration chromatography, suggesting that the enzyme is a homotetramer. The enzyme has an optimal pH and temperature of 9.5 and 35°C, respectively. The purified recombinant RlGDH catalyzed the oxidation of a wide range of substrates, including polyvalent aliphatic alcohols and polyols, to the corresponding ketones and ketoses. Among various polyols, galactitol was the preferred substrate of RlGDH with a Km of 8.8mM, kcat of 835min(-1) and a kcat/Km of 94.9min(-1)mM(-1). Although GDHs have been characterized from a few other sources, RlGDH is distinguished from other GDHs by its higher specific activity for galactitol and broad substrate spectrum, making RlGDH a good choice for practical applications.

Role of the non-conserved amino acid asparagine 285 in the glycone-binding pocket of Neosartorya fischeri β-glucosidase

Neosartorya fischeri β-glucosidase (NfBGL595) is distinguished from other BGLs by its high turnover for p-nitrophenyl β-D-glucopyranoside (pNPG) and flavones. The role of non-conserved amino acids in the glycone-binding pocket of NfBGL595 was studied using sequence alignment and homology modeling, followed by site-directed mutagenesis. Nine amino acids (Y223, I224, I225, V283, L284, N285, M359, H361, and V428) were identified as variable residues around the active site residues, E221 and E430, and selected for mutagenesis. Mutation of the residues to Ala resulted in a drastic alteration in the kcat and Km values when compared to the wild type NfBGL595. Among these nine residues, mutation of N285 to Ala resulted in a complete loss of activity toward pNPG and flavonoid glucosides. Further mutation, structural, and docking analyses revealed that residue N285 is crucial in maintaining the pKa and polarity around E221, which is surrounded by non-polar residues. This study suggests the importance of the pKa and microenvironment around the active site pocket for BGL catalysis.

Microbial conversion of xylose into useful bioproducts

Microorganisms can produce a number of different bioproducts from the sugars in plant biomass. One challenge is devising processes that utilize all of the sugars in lignocellulosic hydrolysates. D-xylose is the second most abundant sugar in these hydrolysates. The microbial conversion of D-xylose to ethanol has been studied extensively; only recently, however, has conversion to bioproducts other than ethanol been explored. Moreover, in the case of yeast, D-xylose may provide a better feedstock for the production of bioproducts other than ethanol, because the relevant pathways are not subject to glucose-dependent repression. In this review, we discuss how different microorganisms are being used to produce novel bioproducts from D-xylose. We also discuss how D-xylose could be potentially used instead of glucose for the production of value-added bioproducts.