Purification and Characterization of Bacterial Aryl Alcohol Oxidase from Sphingobacterium sp. ATM and Its Uses in Textile Dye Decolorization

Dhawal P. Tamboli,Amar A. Telke,Vishal V. Dawkar,Shekhar B. Jadhav,Sanjay P. Govindwar 한국생물공학회 2011 Biotechnology and Bioprocess Engineering Vol.16 No.4

Aryl alcohol oxidase (AAO) produced by dye decolorizing bacteria Sphingobacterium sp. ATM, was purified 22.63 fold to a specific activity of 21.75 μmol/min/mg protein using anion exchange and size exclusion chromatography. The molecular weight of the purified AAO was found to be 71 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),and confirmed by zymography of AAO using L-dopa. The enzyme showed substrate specificity towards veratryl alcohol, followed by n-propanol. The optimum pH and temperature of purified AAO were found to be 3.0 and 40°C, respectively. The K_m and V_(max) of AAO was 1.1615mM and 3.13 mM/min when veratryl alcohol was used as substrate. Sodium azide showed maximum inhibition while ethylenediamine tetra acetic acid (EDTA), L-cysteine and dithiothreitol showed slight inhibition. Metal ions also showed slight inhibition. HPLC analysis confirmed the degradation of Direct Red 5B. The metabolite obtained after decolorization of Direct Red 5B was characterized as 3 diazenyl 7 [-(phenyl carbonyl) amino] naphthalene-2-sulfonic acid using GC-MS analysis.

Peroxidase from Bacillus sp. VUS and Its Role in the Decolorization of Textile Dyes

Vishal V. Dawkar,Umesh U. Jadhav,Amar A. Telke,Sanjay P. Govindwar 한국생물공학회 2009 Biotechnology and Bioprocess Engineering Vol.14 No.3

Peroxidase was purified by an ion exchange chromatography followed by gel filtration chromatography from dye degrading Bacillus sp. strain VUS. The optimum pH and temperature of the enzyme activity was 3.0 and 65°C, respectively. This enzyme showed more activity with n-propanol than other substrates tested viz. xylidine, 3-(3,4-dihydroxy phenyl) Lalanine (L-DOPA), hydroxyquinone, ethanol, indole, and veratrole. Km value of the enzyme was 0.076 mM towards n-propanol under standard assay conditions. Peroxidase was more active in presence of the metal ions like Li²+ , Co²+ , K²+ , Zn²+, and Cu²+ where as it showed less activity in the presence of Ca²+ and Mn²+ . Inhibitors like ethylenediamine tetraacetic acid (EDTA), glutamine, and phenylalanine inhibited the enzyme partially, while sodium azide (NaN3) completely. The crude as well as the purified peroxidase was able to decolourize different industrial dyes. This enzyme decolourized various textile dyes and enhanced percent decolourization in the presence of redox mediators. Aniline was the most effective redox mediator than other mediators tested. Gas chromatography-Mass spectrometry (GC-MS) confirmed the formation of 7-Acetylamino-4-hydroxy-naphthalene-2-sulphonic acid as the final product of Reactive Orange 16 indicating asymmetric cleavage of the dye Peroxidase was purified by an ion exchange chromatography followed by gel filtration chromatography from dye degrading Bacillus sp. strain VUS. The optimum pH and temperature of the enzyme activity was 3.0 and 65°C, respectively. This enzyme showed more activity with n-propanol than other substrates tested viz. xylidine, 3-(3,4-dihydroxy phenyl) Lalanine (L-DOPA), hydroxyquinone, ethanol, indole, and veratrole. Km value of the enzyme was 0.076 mM towards n-propanol under standard assay conditions. Peroxidase was more active in presence of the metal ions like Li²+ , Co²+ , K²+ , Zn²+, and Cu²+ where as it showed less activity in the presence of Ca²+ and Mn²+ . Inhibitors like ethylenediamine tetraacetic acid (EDTA), glutamine, and phenylalanine inhibited the enzyme partially, while sodium azide (NaN3) completely. The crude as well as the purified peroxidase was able to decolourize different industrial dyes. This enzyme decolourized various textile dyes and enhanced percent decolourization in the presence of redox mediators. Aniline was the most effective redox mediator than other mediators tested. Gas chromatography-Mass spectrometry (GC-MS) confirmed the formation of 7-Acetylamino-4-hydroxy-naphthalene-2-sulphonic acid as the final product of Reactive Orange 16 indicating asymmetric cleavage of the dye

Purification and Characterization of Veratryl Alcohol Oxidase from Comamonas sp. UVS and Its Role in Decolorization of Textile Dyes

Umesh U. Jadhav,Vishal V. Dawkar,Dhawal P. Tamboli,Sanjay P. Govindwar 한국생물공학회 2009 Biotechnology and Bioprocess Engineering Vol.14 No.3

In the present work, we have purified veratryl alcohol oxidase (VAO) enzyme from Comamonas sp. UVS to evaluate its potential to decolorize textile dyes. VAO was purified (13.9 fold) by an ion exchange followed by the size exclusion chromatography. Molecular weight of the VAO was estimated to be about 66 kDa by SDS-PAGE. The optimum pH and temperature of oxidase were 30°C and 65°C, respectively. VAO showed maximum activity with n-propanol among the various substrates (n-propanol, veratryl alcohol, L-dopa, tryptophan, etc.). Under standard assay conditions, Km value of the enzyme was 2.5 mM towards veratrole. The enzyme activity was completely inhibited by 0.5 mM sodium azide. L-cysteine, dithiothreitol, and the metal chelator, EDTA had a slight inhibitory effect. The purified enzyme was able to decolorize textile dyes, Red HE7B (57.5%) and Direct Blue GLL (51.09%) within 15 h at 40 μg/mL concentration. GC-MS analysis of the metabolites suggested oxidative cleavage and desulphonation of these dyes In the present work, we have purified veratryl alcohol oxidase (VAO) enzyme from Comamonas sp. UVS to evaluate its potential to decolorize textile dyes. VAO was purified (13.9 fold) by an ion exchange followed by the size exclusion chromatography. Molecular weight of the VAO was estimated to be about 66 kDa by SDS-PAGE. The optimum pH and temperature of oxidase were 30°C and 65°C, respectively. VAO showed maximum activity with n-propanol among the various substrates (n-propanol, veratryl alcohol, L-dopa, tryptophan, etc.). Under standard assay conditions, Km value of the enzyme was 2.5 mM towards veratrole. The enzyme activity was completely inhibited by 0.5 mM sodium azide. L-cysteine, dithiothreitol, and the metal chelator, EDTA had a slight inhibitory effect. The purified enzyme was able to decolorize textile dyes, Red HE7B (57.5%) and Direct Blue GLL (51.09%) within 15 h at 40 μg/mL concentration. GC-MS analysis of the metabolites suggested oxidative cleavage and desulphonation of these dyes

Parasitism by Chelonus blackburni (Hymenoptera) affects food consumption and development of Helicoverpa armigera (Lepidoptera) and cellular architecture of the midgut

Yogita Sanap,Vishal V. Dawkar,Ashok P. Giri,Avalokiteswar Sen,Radhakrishna S. Pandit 한국응용곤충학회 2016 Journal of Asia-Pacific Entomology Vol.19 No.1

Biological control agents are vital components of an integrated pest management strategy, and this is frequently referred to as natural control. Natural enemies of insect pests include predators, parasitoids, and pathogens. Among them, a parasitoid, Chelonus blackburni (Cameron), was found to be the best biological control agent for the polyphagous pest, Helicoverpa armigera (Hübner). C. blackburni alters the feeding performance of H. armigera larvae upon parasitism and as a result severely affects growth and development. Moreover, it shortens the feeding period of H. armigera and increases mortality. Furthermore, total hemocyte count (THC) was significantly decreased in parasitized larvae than control. Parasitized H. armigera had 26% less number of blood cells compared to healthy larvae. Histological studies showed that the structure of midgut of H. armigera is drastically affected by C. blackburni leading to reduced food consumption, which ultimately led to larval death. The present study provides an insight to changes involved in H. armigera due to parasitism by C. blackburni, a parasite that could be used as an effective biocontrol agent to manage H. armigera.

Biodegradation of Disperse Dye Brown 3REL by Microbial Consortium of Galactomyces geotrichum MTCC 1360 and Bacillus sp. VUS

S. U. Jadhav,U. U. Jadhav,V. V. Dawkar,S. P. Govindwar 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.2

The consortium-GB (Galactomyces geotrichum MTCC 1360 and Bacillus sp. VUS) exhibited 100% decolorization ability with the dye Brown 3REL within 2 h at shaking condition with optima of pH 7 and at 50℃. However, G. geotrichum MTCC 1360 showed 39% decolorization within 24 h and Bacillus sp. VUS took 5 h for 100% decolorization, when incubated individually. Additional carbon and nitrogen sources like, starch, peptone, and urea were found to enhance decolorization. Induction in lignin peroxidase, tyrosinase, and riboflavin reductase was observed in consortium as that of individual organisms. GCMS identification showed different metabolites formed using consortium (2-(6,8-dichloro-quinazolin-4yloxy)-acetyl-urea and 2-(6,8-dichloro-quinazolin-4yloxy)-acetyl-formamide) and Bacillus sp. VUS (6,8-dichloro-4 methoxy-quinazoline) after 2 h of incubation with Brown 3REL. G. geotrichum MTCC 1360 showed minor modifications in structure of Brown 3REL. Phytotoxicity revealed non toxic nature of metabolites. This consortium-GB was also able to decolorize various industrial dyes.