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  1. May 2019
    1. Preparation of electrocompetent cells (E. coli cells) A protocol was employed. The procedure was carried out in cold under sterile conditions as follows: •A single colony of E. coli DH10B/ DH5α/XL1blue was inoculated in 20 mL of LB medium and grown overnight at 30 °C. •500 mL LB medium was inoculated with 5mL of this overnight grown culture of the E. coli and incubated with vigorous shaking (250 rpm) at 30 °C until an A600of 0.5 - 0.8 was achieved. •The cells were chilled in ice for 10-15 min and transferred to prechilled Sorvall® centrifuge tubes and sedimented at 4,000 rpm for 20 min at 4 °C. •The supernatant was decanted and cells were resuspended in 500 mL of sterile ice-cold water, mixed well and centrifuged as described above. •The washing of the cells described above was repeated with 250 mL of sterile ice-cold water, following which cells were washed with 40 mL of ice-cold 10 % (v/v) glycerol and centrifuged at 4,000 rpm for 10 min. •The glycerol solution was decanted and the cell volume was recorded. The cells were resuspended in an equal volume of ice-cold 10 % glycerol. •Cells were then dispensed in 40 μL volumes and stored at -80 °C until required.
    2. The isolated DNA was diluted (1:100) with MQ. The concentration (mg mL-1) of the DNA [N] was determined spectrophotometrically by recording absorbance at 260 nm (A260) as: A260 = ε 260[N]where ε 260 is the extinction coefficient of DNA (50 for ds DNA) [N] = concentration (mg mL-1) of DNA The concentration of ds DNA [N] was calculated as [DNA] (mg mL-1) = A260/ε 260 [DNA] (μg mL-1) = A260 × 50 × dilution factor Purity of DNA was checked by measuring absorbance at 260 and 280 nm and calculating the A260/A280 ratio (Sambrook et al., 1989). A DNA sample was considered pure when A260/A280 ranged between 1.8-1.9. An A260/A280 < 1.7 indicated contamination of the DNA preparation with protein or aromatic substances such as phenol, while an A260/A230 < 2.0 indicated possible contamination of high molecular weight polyphenolic compounds like humic substances.
    3. Determination of DNA quantity and purity
    4. as well as commercial methods (MN kit, Germany; Mo-Bio kit, CA, USA; Zymo soil DNA kit, CA, USA) according to the manufacturer’s protocols and compared in terms of DNA yield and purity.
    5. The soil DNA from Pantnagar and Lonar soil samples were also extracted by various manual (Desai and Madamwar, 2007; Agarwal et al., 2001; Yamamoto et al., 1998
    6. Alternatively metagenomic DNA was extracted from the alkaline soil samples by using different commercial kits (UltraClean™, PowerSoil™ [Mo Bio Laboratories Inc., Carlsbad, CA, USA], Nucleospin kit [Macherey-Nagal, Germany] and Zymo soil DNA isolation kit [CA, USA]). The DNA was finally suspended in 100 μL of sterile Milli Q water for further analysis.
    7. Commercial kits
    8. Comparison of yield and purity of crude DNA
    9. Soil (1 gm) was suspended with 0.4 gm (w/w) polyactivated charcoal (Datta and Madamwar, 2006) and 20 μL proteinase K (10 mg mL-1) in 2 mL of modified extraction buffer [N,N,N,N cetyltrimethylammonium bromide (CTAB) 1% w/v, polyvinylpolypyrrolidone (PVPP) 2% w/v, 1.5 M NaCl, 100mM EDTA, 0.1 M TE buffer (pH 8.0), 0.1M sodium phosphate buffer (pH 8.0) and 100 μL RNaseA] [Zhou et al., 1996] in 20 mL centrifuge tubes to homogenize the sample and incubated at 37 °C for 15 min in an incubator shaker at 200 rpm. Subsequently, 200 μL of 10% SDS was added to the homogenate and kept at 60 °C for 2 h with intermittent shaking. DNA was precipitated by adding 0.5 V PEG 8000 (30 % in 1.6 M NaCl) and left at room temperature for an hour (Yeates et al., 1998). The precipitated DNA was collected by centrifugation at 8000 x g at 4 °C. The supernatant was discarded and pellet was dissolved in 1 mL of TE buffer (pH 8.0) and then100 μL of 5 M potassium acetate (pH 4.5) was added and incubated at 4 °C for 15 min. The supernatant was collected after centrifugation at 8000 x g and treated with equal volumes of phenol: chloroform (1:1) followed by chloroform: isoamylalcohol (24:1) at 8000 x g for 15 min
    11. Various strains of Escherchia coli (DH5α, XL1Blue, DH10B) were used as hosts for the propagation of recombinant vectors. In addition, Bacillus subtilis was used as a host for the expression of xylanase gene from the recombinant vector pWHMxyl. Different vectors used in this investigation are listed in
    13. Soil, sediment, effluent, and water samples have been collected from various hot and alkaline regions of India and Japan in sterile polyethylene bags/bottles. The samples were transported to the laboratory and preserved at 4 °C. Temperature and pH of the samples was recorded.
    1. lasmodium Jalciparum strain 3D7 (MR4, American Type Culture Collection) was i used for all the experiments except where gametocyte rich culture was required. I For generating gametocytes, 3D7 A a variant of 3D7 was used. The parasite was cultured as describerl below:
    2. lasmodiumfalciparum culture
    1. acrylamide, TEMED were obtained from Bio-Rad laboratories (California, USA). Coomassie Plus protein assay reagent was purchased from Pierce (111inois, USA). All other chemicals were at least of analytical grade and were from Qualigens . laboratories (Bombay, India). HSA was from alpha therapeutic corporation (California, USA). Bacto-tryptone, yeast extract, and bacto-agar were obtained from Difco laboratories (Detroit, USA).
    2. Agarose, ampici11in, ammonium acetate, ammonium persulfate, 1-acetyl 2-phenyl hydrazine, ~-mercaptuahanol, boric acid, calcium chloride, chloramphenicol, citric acid, coomassie blue 0250, creatine phosphate, creatine phosphokinase, DEPC, dialysis tubing, disodium hydrogen phosphate, dithioerythritol, dithiothreitol, DMPG, DOPG, DMPA, EDT A, ethidium bromide, glucose, glycerol, GSSG, guanidine hydrochloride, heparin, haemin., HEPES, IPTG, kanamycin, L-glycine, L-arginine, lithium chloride, magnesium acetate, magnesium sulfate, MES, PEG 8000, potassium acetate, potassium chloride, RNase free BSA, SDS, sucrose, sodium acetate, sodium dihydrogen phosphate, spermidine, sodium bicarbonate, sigmacote, Tris base, Triton X-100, urea and uridine were obtained from Sigma chemical Co. (St. Louis, USA). Trizol reagent, PCR buffer, magnesium chloride solution for PCR, RPMI-1640, leucine free RPMI, DMEM, trypsin, Fetal calf serum, antibiotic-antimycotic solution were purchased from Life Technologies (Maryland, USA). NTPs, dNTPs, cation exchange resins: S-sepharose and SP-sepharose were obtained from Pharmacia Biotech (Uppsala, Sweden). Bromophenol blue, xylene cyanol, acrylamide, bis
    3. Chemicals
    1. Radioisotopes: dCTP[a-32p] (3000 Ci/mM), dATP[a_35s] (1250 Ci/ mM) and 35s Met (I 000 Ci/ mM), were procured from NEN Life Sciences Products, Boston, MA, USA Others: Ni-nitrilo-tri-acetic acid (NTA) affinity resin from Qiagen; Membranes for Western blotting were obtained from BioRad; Hybond N and X-ray films were from Amersham; DNA and protein analysis softwares, PCgene from IntelliGenetics, Inc., Mountain View, CA, USA and Lasergene from DNASTAR Inc., Madison, Wisconsin, USA; Ultrafilteration assembly and YM5 membranes from Amicon Corp., Lexington, MA, USA.
    2. from Gibco BRL; ampicillin, kanamycin and neutral red were from Sigma; FCS, was obtained from Biological Industries, Hibbutz Beit, Haemek, Israel. Bacterial Strains and Plasmids: M15[pREP4] and SG13009[pREP4] from Qiagen GmbH, Hilden, Germany, DH5a, BL21 (DE3) and BL21(pLysS) from Stratagene, La Jolla, USA, DF5 cells were kindly provided by Prof. K. Dharmalingam, Madurai Kamraj University, Madurai, Tamil Nadu, India. pBluescriptll SK( +) vector from Stratagene, pQE30 from Qiagen, pBacPAK8 vector from Clonetech Laboratories Inc., Palo Alto, CA, USA and pAcSecG2T vector, from Pharmingen, San Diego, CA, USA were obtained. Kits: Poly AT® tract mRNA isolation system and Riboclone® eDNA synthesis system from Promega Inc.; Geneclean® II kit from Bio 101 Inc., La Jolla, USA;Plasmid Midi kit and QIAexpress™ from Qiagen GmbH, Hilden, Germany; Sequenase version 2.0 DNA sequencing kit and Multiprime DNA labeling system from Amersham, Little Chalfont, Buckinghamshire, UK; BacPAK™ baculovirus expression system from Clonetech and Baculogold™ transfection kit from Pharmingen. Primers: Various oligonucleotide primers used were custom made by Rama Biotechnologies India Pvt. Ltd., Secunderab_ad, AP, India. Enzymes: Various restriction enzymes used and Vent DNA polymerase were procured from New England Biolabs, Beverly, MA, USA. Taq DNA polymerase was obtained from Stratagene. Antibodies and Conjugates: Goat anti-GST Ab was obtained from Pharmacia Biotech, Uppsala, Sweden. The following secondary revealing Abs were used: i) goat anti-mouse immunoglobulin G (lgG)-horse radish peroxidase (HRPO) from BioRad; ii) goat anti-rabbit IgG-HRPO (Pierce Chemical Co.); iii) goat anti-monkey IgG-HRPO (Sigma); iv) anti-rabbit-FITC and v) anti-goat-HRPO (Reagent Bank, Nil, New Delhi) vi) anti-mouse FITC (Dakopatts a/s, Glostrup, Denmark).
    3. Chemicals: Tris, glycine, acrylamide, N', N'-methylene bisacrylamide, sodium dodecyl sulfate (SDS), N', N', N', N'-tetramethylethylene diamine (TEMED), ammonium persulfate (APS), ~-mercaptoethanol (BME), 4-chloronaphthol, urea, guanidine HCl, guanidine isothiocyanate (GITC), sarcosyl, sodium citrate, phenol, ficoll, polyvinylpyrrolidone (PVP), agarose, bromophenol blue, Coomassie brilliant blue, ethidium bromide, calcium chloride and bicinchoninic acid (BCA) were obtained from Sigma Chemical Co., St. Louis, MO, USA. LMP agarose and isopropyl ~-D thiogalactopyranoside (IPTG) were from Amresco, Solon, USA. Molecular weight standards were obtained from Gibco-BRL, Grand Island, NY, USA, or Bio-Rad Laboratories, Hercules, CA, USA. Reagents for enzyme immunoassays viz., bovine serum albumin (BSA), orthophenylene diamine (OPD) were procured from Sigma, while Tween-20 was obtained from Amresco. Reagents for conjugation and immunization, viz. diphtheria toxoid (DT) and tetanus toxoid (TT) were from Serum Institute, Pune, India while glutaraldehyde, L-lysine, 2, 6, 10, 15, 19, 23-hexamethyl-2, 6, 10, 14, 18, 22-tetracosa-hexane (Squalene) and mannide monooleate (Arlacel A) were procured from Sigma; Pergonal® was obtained from Laboratoires Serono S.A., Aubonne, Switzerland; Sodium phthalyl derivative of lipopolysaccharide (SPLPS) was kindly provided by the lmmunoendocrinology Laboratory, National Institute of Immunology (Nil), New Delhi. Reagents used in the estimation of progesterone such as gelatin, charcoal, dextran, 3H-progesterone and anti-progesterone Ab were provided by the WHO Matched Reagent Assay Programme while diphenoxazole (PPO), 1-4 bis (5-phenyl-2-oxazolyl) benzene (POPOP), and mercury-[(o-carboxyphenyl)thio]ethyl sodium salt (Thimerosal) were obtained from Sigma. Media and Antibiotics: Bacto-tryptone, bacto yeast extract and bacto agar were from Difco Laboratories, Detroit, USA; Grace's insect cell medium and antibiotic-antimycotic
    1. The bacterial strains used in this study were ~.coli Kl2 strains, HBlOl ( F-, hsd S20 ( rB-, mB-) , supE44, ara14, ~-, galK2, lacYl, proA2, rpsL20, xyl-5, mtl-1, recA13 ] (Boyer et al., 1969 ), and JM105 ( thi, rpsL, endA, sbcB15, hsdR4, ( lac-proAB ), {F1, traD36, proAB, laciqZ M15 } ]. The mammalian cell lines used are listed in Table 1. Rat-2 is an established rat fibroblast cell line. FWIL (Larrick et al., unpublished) is a human myeloma cell line derived from the fusion of U266 IgE myeloma cells with WIL-2 lymphoblastoid cells. Rat - 2 and FWIL cell lines were kindly provided by Dr. J.W. Larrick, Cetus Corporation, USA. The other four cell lines were obtained from American Type Culture Collection ( ATCC ). The plasmids used in this study are described in Table 2.
    2. Bacterial strains, cell lines and plasrnids.
    1. Franklin Lakes, NJ, USA. Glass micro-slides and coverslips were purchased from Blue Star, Polar Industries and Co., India.
    2. immunoglobulins-FITC conjugates were obtained from Pierce, Rockford, IL, USA. Anti-. rabies monoclonal antibody conjugated to FITC was purchased from Centocor Inc, Malvern, P A, USA. Others: Sodium chloride was purchased from S. D. fine-chem. Ltd., Mumbai, India, Lipofectamine and Trizol were obtained from Gibco-BRL, Rockville, MD, USA. UM-449 cell line was a kind gift by Prof. Nirbhay Kumar (TheW. Harry Feinstone Department of Molecular Microbiology and Immunology, John Hopkins University-School of Public Health, N. Wolfe Street, Baltimore, MD 21205, USA), COS-7 cell line was kindly provided by Dr. Chetan Chitnis (Malaria Research Group, International Center for Genetic Engineering and Biotechnology, New Delhi, India), MNA cell line, CVS-11 rabies virus and Standard Rabies Immune Globulin were kindly gifted by Dr. Charles E. Rupprecht (Rabies section, Division of Viral and Rickettsial Diseases, Centres for Disease Control and Prevention, Atlanta, Georgia 30333, USA), Nickel-nitrilotriacetic acid (Ni-NTA) was procured from QIAGEN, ultrafiltration assembly and YM30 membrane from Amicon Corp., Lexington, MA, USA. Bicinchoninic acid (BCA) was purchased from Pierce. Complete and incomplete Freund's adjuvants were procured from Difco laboratories. Nitrocellulose membrane, Tefzel tubing, gold microcarriers and Helios gene gun assembly were obtained from Bio-Rad. T -25, T -75 tissue culture flasks, 6-well tissue culture plates, 8-well tissue culture chamber slide and 96-well microtitration plates were procured from Nunc a/s, Rosakilde, Denmark. The 24-well tissue culture plates were procured from Corning glass works, Corning, NY, USA. The 96-well tissue culture plates were purchased from Becton Dickinson and Co.,
    3. Bacterial strains and plasmids: DH5a and BL2l(DE3)pLysS strains of E. coli were purchased from Stratagene, La Jolla, CA, USA. SG13009[pREP4] E. coli strain was obtained from QIAGEN GmbH, Hilden, Germany. Expression vector, VRI 020 was a kind gift from VICAL Incorp., San Diego, CA, USA, pKB3-JE-13 clone was procured from ATCC, Rockville, MD, USA, pQE30 vector was procured from QIAGEN and pRSET-A vector was acquired from Invitrogen Corp., Carlsbad, California, USA. Kits: PCR-Script™ Amp cloning kit was obtained from Stratagene. Plasmid DNA purification mega kit was purchased from QIAGEN. The pGEM-T Easy cloning kit was purchased from Promega, Madison, WI, USA. Primers and Enzymes: Various oligonucleotide primers were custom made by Rama Biotechnologies, India Pvt. Ltd., Secundrabad, AP, India, Sigma-Genosys Ltd, New Delhi, India and Microsynth GmbH, Hilden, Germany. Restriction enzymes were obtained from New England BioLabs (NEB), Beverly, MA, USA and Promega, Taq DNA polymerase and T4 DNA ligase were bought from Promega. Shrimp Alkaline Phosphatase was bought from Amersham. Molecular weight markers: pGEM DNA markers were procured from Promega, /..DNA-Hind III digest and 1 kb DNA ladder were purchased from NEB. Prestained SDS-PAGE standards were obtained from Bio-Rad, Hercules, CA, USA. Antibodies and Conjugates: Rabbit anti-mouse IgG (whole molecule) conjugated to horseradish peroxidase (HRPO) was procured from Dako A/S, Glostrup, Denmark. Goat anti-mouse IgG-FITC conjugate was procured from Sigma. Mouse monoclonal antibody isotyping kit was also purchased from Sigma. Goat anti-mouse immunoglobulins-HRPO, goat anti-rabbit immunoglobulins-HRPO, rabbit anti-goat IgG-HRPO and goat anti-rabbit
    4. Chemicals: Tris, glycine, acrylamide, N, N'-Methylene-bisacrylamide, sodium dodecyl sulfate (SDS), P-mercaptoethanol, N, N, N', N'-Tetramethylethylenediamine (TEMED), phenol, ethidium bromide, ethylenediaminetetraacetic acid (EDTA), agarose, Bromophenol blue, Coomassie brilliant blue-R250, calcium chloride, sodium acetate, glucose, lysozyme, glycerol, chloroform, lithium chloride, phenylmethyl sulphonyl fluoride (PMSF), spermidine, saponin, paraformaldehyde were procured from Sigma Chemical Co., St. Louis, MO, USA. Low melting point (LMP) agarose, isoamyl alcohol, sodium deoxycholate, oxidized glutathione, reduced glutathione, dimethyl sulfoxide (DMSO), 4-chloro-1-naphthol, isopropyl-P-D-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside (X-gal), were purchased from Amresco, Solon, Ohio, USA. Ammonium persulfate and guanidine hydrochloride were purchased from Amersham, Cleveland, Ohio, USA. Alum was procured from Superfos Biosector, Elsenbakken, Frederikssund, Denmark. Reagents for Enzyme Immunoassay: Bovine serum albumin (BSA) and orthophenylene diamine (OPD) were purchased from Sigma. Tween-20 (polyoxyethylene-20-sorbitan monolaurate) was obtained from Amresco. Media and Antibiotics: Bacto tryptone, bacto yeast extract and bacto agar were obtained from Difco laboratories, Detroit, USA, Dulbecco's Modified Eagle's Medium (DMEM) and RPMI-1640 media were purchased from Sigma. Antibiotics such as gentamycin sulfate, ampicillin (sodium salt) and kanamycin were also obtained from Sigma. Fetal calf serum (FCS) was procured from Biological Industries, Hibbutz Beit, Haemek, Israel.
    1. Agarose, ampicillin, ammonium acetate, Tris Base, EDTA, SDS, sodium-acetate, potassium-acetate, boric acid, disodium-hydrogen-phosphate, sodium-dihydrogen-phosphate, sodium chloride ethidium bromide, urea, ammonium persulphate, MOPS, glycerol, sodium bicarbonate, Triton X-100, dithiothreitol, magnesium chloride, BSA, IPTG, Orange G, DEPC, Tween-20, acrylamide, calcium chloride, trypsin, EDTA, sodium citrate, bromophenol blue, xylene cyanol FF, were obtained from Sigma-Aldrich Co. (Missouri, U.S.A.). X-gal, NTP and dNTP, sodium chloride, bis-acrylamide, TEMED, PCR buffer and Magnesium chloride for PCR, DNA markers, were from Promega Biotech Co. (Madison, U.S.A.). All other chemicals were at least of analytical grade and were from Qualigens laboratories (Bombay, India) or Merck (New Jersey, U.S.A.) Trizol reagents, DMEM, lipofectin, lipofectamine 2000, antimycotic-antibiotic, gentamicin, RNase-DNase free water were obtained from Invitrogen-GffiCO/BRL (Maryland, U.S.A.). Fetal bovine serum was obtained from Biological Industries (Beit Haemek, Israel). Luria Bertini medium and Luria Miller agar for bacterial culture were obtained from Difco Laboratories (Detroit, U.S.A.). Pre-stained rainbow protein markers, nylon and nitro-cellulose membranes, ECL reagent, all were obtained from Amersham Biosciences (Buckinghamshire, U.K.).
    2. Chemicals
    1. 3.0–5.0, phosphate buffer for pH 6.0–8.0 and Tris-HCl buffer for pH 9.0) were used. •pH stability: The pH stability of the selected tannases was examined in the range of 3.0–9.0 by incubating the enzyme samples for 6 h in different buffers. Tannase activity was estimated under standard assay conditions. •Temperature tolerance: Temperature tolerance of the tannases was examined by assaying their activity at different temperatures in the range of 20 to 80ºC. •Temperature stability: Temperature stability of the tannases was determined by incubating them in the temperature range of 20 to 70 ºC for 6 h. After the incubation tannase activity (%) was determined under standard assay conditions. •Organic solvent stability: In order to determine the suitability of the selected tannases for organic synthesis, their stability was determined in different organic solvents. Experimentally, 10 mg of each of the crude lyophilized tannase from the selected cultures were mixed with 1.0 ml of the following organic solvent: a) Hexane b) Methanol c) Propanol d) Isoamyl alcohol e) Petroleum ether f ) Chloroform The mixture was incubated for 6 h at optimal temperature and the organic solvents were then decanted and the residues were dried in a vacuum desiccator. These dried samples were dissolved in 1.0 ml of citrate phosphate buffer (50 mM, pH 5.0) and the tannase activity was determined under standard assay conditions. The tannase activity thus obtained from each culture were compared with initial tannase activity. Finally, on the basis of tannase titres produced per ml and desirable biochemical properties, the best tannase producer was selected for further investigations
    2. The procedure of Sharma et al. (2000) was used to estimate the gallic acid in the culture filtrate. Reagents: Methanolic rhodanine solution (0.667% w/v): Prepared by dissolving 0.667 g of rhodanine in 100 ml of methanol.Potassium hydroxide (0.5 N): 2.8 gpotassium hydroxide dissolved in100 ml of distilled water.
    3. The procedure of Hagerman and Butler (1978) was used to estimate the tannin content in different tannin sources. Reagents: Bovine serum albumin (BSA) 1.0 mg/ml: 10.0 mg of bovine serum albumin was dissolved in 10.0 ml of 0.2 M acetate buffer, pH 5.0, containing 0.17 M sodium chloride. Sodium dodecyl sulfate (SDS)-triethanolamine solution: The solution contained 1.0% SDS and 5.0% (v/v) triethanolamine in distilled water. Ferric chloride reagent (0.01 M): 1.62 g of ferric chloride was dissolved in 1.0 L of 0.01 N hydrochloric acid.
    4. The total protein content in the culture filtrate was estimated by Lowry’s method as described below: Reagents: Solution A: 2.0% Na2CO3 in 0.1 N NaOHSolution B: 0.5 % CuSO4 in 1.0 % Sodium potassium tartarate Solution C: 50.0 ml of solution A was mixed with 1.0 ml of solution B Folin Ciocalteau’s reagent
    5. In this method, tannase activity was estimated through spectrophotometric method by determining the concentration of the end product i.e., gallic acid, by estimating the absorbance at 260 nm. Reagents: •Tannic acid (1.0%): The solution was prepared by dissolving 1.0 g of tannic acid in 100 ml of citrate-phosphate buffer of the desired pH.•Bovine serum albumin (BSA): BSA (2.0%) was prepared in citrate phosphate buffer (pH 5.0)
    6. For bacterial isolates, a single colony from a nutrient agar slant was inoculated into 50 ml of nutrient broth in a 250 ml Erlenmeyer flask. These flasks were incubated at 37±1°C in a incubator shaker till an optical density of 0.6 at 660nm. Now these cultures were used to inoculate 50 ml of the tannase production medium in 250 ml Erlenmeyer flasks using 2% v/v inoculum. These flasks were incubated at 37±1°C in an incubator shaker (Multitron AG-27; Switzerland) at 200 rpm for 72h. The experiments were carried out in triplicates. Samples (2.0 ml for bacteria and same for fungi) were withdrawn at regular intervals of 12h upto 72 h. The samples thus obtained were centrifuged at 10,000 rpm in a refrigerated centrifuge (SIGMA 4K15 Germany) for 10 min at 4°C. The supernatant/s were analyzed for tannase activity
    7. For fungal cultures, spores were harvested from 72 hour old cultures grown on PDA/Tannic acid agar slants by adding 10 ml of sterilized normal saline and a few drops of sterilized Tween-80 followed by vortexing. The spore suspension was filtered through sterile cotton filter to ensure that mycelial filaments are removed. The spores were counted using a haemocytometer (Neubaeur). Approximately, 5X106 spores were inoculated in 50 ml of tannase production medium in 250 ml Erlenmeyer flasks. These flasks were then incubated at 30±1 and 37±1°C in an incubator shaker (model G25KC, New Brunswick Scientific, NJ, USA) at 200 rpm
    8. Pharmacia (Uppsala, Sweden).Column chromatography matrices (Sephadex G series), DEAE-Cellulose E-Merck Germany Silica Thin Layer Chromatography Plates 60 F254, All solvents used in the present investigation were purchased from E-Merck Sisco Research Laboratories (SRL), SD fine chemicals Ltd, Qualigens, Central Drug House (CDH), Thomas BakerChemicals/components used in the preparation of various media were obtained from these companies Local commercial sourcewheat bran, rice bran, wheat straw, corn cob, acacia arabicajambula leaves, aamla, Indian plum, jowari, black tea, kangra orthodox black tea Double distilled water (DDW) was used for preparation of reagents, stock solutions, buffers and different media. All glass and plastic wares used were from Borosil, Schott Duran and Qualigens.
    9. Tannic acid, DEAE-cellulose, phenyl sepharose, acrylamide, bisacrylamide, TEMED, ampholine PAG plates, bromophenol blue, chitosan, chitin, silica, celite, DEAE-sephadex, amberlite XAD-7, glutaraldehyde, cholic acid, saponin, sodium taurocholeate, SDS, tauro cholic acid, sodium choleate, triton X-100, Tween-80, EDTA, phenyl methyl sulfonyl fluoride, p-Chloromercuric benzoic acid, N bromosuccinimide, Phenyl boronic acid, O-phenanthrolin, sodium deoxycholate, phenanthrolin, N-ethylmaleimide, dithiothreitol, β- Mercaptoethanol, bromoacetic acid. gallic acid and its esters (methyl, propyl, ethyl, butyl gallate), epigallocatechin gallate , epigallocatechin, caffeine, epicatechin, epicatechin gallate, 2,2-diphenyl-1-picrylhydrazyl (DPPH) (E)-2-hexenal (Z-3-hexenol), 1-penten-3-ol, 3,7-dimethyl-1,5,7-octatrien-3-ol, linalool, linalool oxides (furanoid), geraniol, methylsalicylate, epoxylinalol, α-irone (2,5-dimethyl-α-ionone),2,7-epoxymegastigma-4,8-diene and 1,3-dioxolane
    10. Chemicals and Reagents
    1. a-Genotype designations are as in (Berlyn, 1998).b-Strains DH5α, MC4100andMG1655 were from the laboratory stock collection.FRT-indicates FLP recognition target, a sequence that is recognized by FLP recombinase and is left as a scar at the indicated sites after excision of an antibiotic marker that is flanked by the FRT sites
    2. The E. coli strains used in this study with their genotypes are shown in the table below. Bacterial strains were routinely stored on solid agar plates at 4ºC and also as thick suspensions in 20% glycerolat –80ºC. Plasmid harbouring strains were freshly prepared by transformation of the required plasmid. The bacteriophage P1kc was used for routine transduction to move a locus from one strain to another and is referred to as P1 throughout this thesis.Bacterial Strains Table 2.1: E. coli strains used and constructed in this study: