Biochemical Characterization and Potential for Textile Dye Degradation of Blue Laccase from Aspergillus ochraceus NCIM-1146

Amar A. Telke,Avinash A. Kadam,Sujit S. Jagtap,Jyoti P. Jadhav,Sanjay P. Govindwar 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.4

In our study, we produced intracellular blue laccase by growing the filamentous fungus Aspergillus ochraceus NCIM-1146 in potato dextrose broth. The enzyme was then purified 22-fold to a specific activity of 4.81 U/mg using anion-exchange and size exclusion chromatography. The molecular weight of purified laccase was estimated as 68 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme showed maximum substrate specificity toward 2,2'-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid than any other substrate. The optimum pH and temperature for laccase activity were 4.0 and 60ºC, respectively. The purified enzyme was stable up to 50ºC, and high laccase activity was maintained at pH 5.0 ~ 7.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol, and L-cysteine. Purified laccase decolorized various textile dyes within 4 h in the absence of redox mediators. HPLC and FTIR analysis confirmed degradation of methyl orange. The metabolite formed after decolorization of methyl orange was characterized as p-N,N'-dimethylamine phenyldiazine using GCMS.

Enzymatic analysis, structural study and molecular docking of laccase and catalase from <i>B. subtilis</i> SK1 after textile dye exposure

Kadam, Suhas K.,Tamboli, Asif S.,Sambhare, Susmit B.,Jeon, Byong-Hun,Govindwar, Sanjay P. Elsevier 2018 Ecological Informatics Vol.48 No.-

<P><B>Abstract</B></P> <P>The textile dye decolorizing efficiency of <I>Bacillus subtilis</I> SK1 against 70 mg/L each of Malachite Green, Methyl Orange, Rubine GFL and Red HE3B was observed as 71.7, 73.6, 74.4 and 82.6%, respectively within 3 h. UV–Vis spectroscopy, GC–MS and HPTLC analysis confirmed mineralization of model dyes into its metabolites. Physico-chemical characterization confirmed acidic and hydrophilic nature of both laccase and catalase enzymes. Both enzymes contain dominant random coiled secondary structure (SOPMA tool) and intracellular location (CELLO_v.2.5), however, laccase alone contains two disulfide bridges (CYS_REC tool). The validation of constructed 3D structure (Modeller 9.19) of laccase and catalase enzymes revealed, RAMPAGE- 96.3 and 95.8% residues in favoured region, ProSA- Z score −8.2 and −9.6, respectively and ERRAT-Overall quality factor > 68. Potential energies −1.328 × 10<SUP>6</SUP> kJ/mol and −2.685 × 10<SUP>6</SUP> kJ/mol remained constant after 1395 and 1545 steps for laccase and catalase, respectively in energy minimization. Molecular docking results showed interaction of Methyl Orange with laccase (Thr260) and catalase (Lys 48), Rubine GFL with laccase (Thr 262) and catalase (His 176) and Red HE3B with laccase (Asn 264, Thr 418, Gly 321, Thr 262 and Gly 378) and catalase (Gln 258). This study provides dye degrading potential of <I>Bacillus subtilis</I> strain SK1 with structurally different textile dyes and vital role of the polar amino acids of laccase and catalase in these interactions.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Bacillus subtilis</I> SK1 showed efficient remediation of different textile dyes. </LI> <LI> Laccase and catalase are accounted for degradation of structurally different dyes. </LI> <LI> Enzymes model fall in favourable region and has good quality of structure. </LI> <LI> Polar amino acids play a vital role in biotransformation of dyes. </LI> </UL> </P>

Pretreatment of microalgal biomass for enhanced recovery/extraction of reducing sugars and proteins

Eldalatony, M. M.,Kabra, A. N.,Hwang, J. H.,Govindwar, S. P.,Kim, K. H.,Kim, H.,Jeon, B. H. Springer Science + Business Media 2016 BIOPROCESS AND BIOSYSTEMS ENGINEERING Vol.39 No.1

<P>Microalgae species including Chlamydomonas mexicana, Micractinium reisseri, Scenedesmus obliquus and Tribonema aequale were cultivated in batch cultures, and their biochemical composition was determined. C. mexicana showed the highest carbohydrate content of 52.6 % and was selected for further study. Sonication pretreatment under optimum conditions (at 40 kHz, 2.2 Kw, 50 A degrees C for 15 min) released 74 +/- A 2.7 mg g(-1) of total reducing sugars (TRS) of dry cell weight, while the combined sonication and enzymatic hydrolysis treatment enhanced the TRS yield by fourfold (280.5 +/- A 4.9 mg g(-1)). The optimal ratio of enzyme [E]:substrate [S] for maximum TRS yield was [1]:[5] at 50 A degrees C and pH 5. Combined sonication and hydrolysis treatment released 7.3 % (27.1 +/- A 0.9 mg g(-1)) soluble protein of dry cell weight, and further fermentation of the dissolved carbohydrate fraction enhanced the soluble protein content up to 56 % (228.4 mg g(-1)) of total protein content. Scanning and transmission electron microscopic analyses indicated that microalgae cells were significantly disrupted by the combined sonication and enzyme hydrolysis treatment. This study indicates that pretreatment and subsequent fermentation of the microalgal biomass enhance the recovery of carbohydrates and proteins which can be used as feedstocks for generation of biofuels.</P>

Degradation and Detoxification of Disperse Dye Scarlet RR by Galactomyces geotrichum MTCC 1360

S. U. Jadhav,G. S. Ghodake,A. A. Telke,D. P. Tamboli,S. P. Govindwar 한국미생물 · 생명공학회 2009 Journal of microbiology and biotechnology Vol.19 No.4

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.

Insights into microalgae mediated biodegradation of diazinon by Chlorella vulgaris: Microalgal tolerance to xenobiotic pollutants and metabolism

Kurade, M.B.,Kim, J.R.,Govindwar, S.P.,Jeon, B.H. Elsevier B.V 2016 Algal research Vol.20 No.-

<P>Diazinon is one of the most widely used organophosphorus insecticides for agricultural activities, and it is highly toxic to mammals and other non-target organisms. The present study demonstrated the effective removal of diazinon from the aqueous phase by a freshwater, green microalga, Chlorella vulgaris. Among the four screened species (Scenedesmus obliquus, Chlamydomonas mexicana, Chlorella vulgaris and Chlamydomonas pitschmannii), C. vulgaris showed the highest removal capacity (94%) of diazinon at 20 mg L-1. The growth of C. vulgaris was significantly affected above 40 mg L-1 of diazinon, showing >30% growth inhibition after 12 days of cultivation. Significant enhancement of the microalgal growth in the exponential growth phase suggested a less/non-toxic nature of the diazinon by-products. Biochemical properties, including carotenoid, chlorophyll and antioxidant enzymes of C. vulgaris were influenced by diazinon at relatively high concentrations. The degradation rate constant (k) and the half-life (T1/2) of diazinon (0.5-100 mg L-1) ranged between 0.2304-0.049 d(-1) and 3.01-14.06 d, respectively. Gas chromatography mass spectroscopic (GC-MS) study suggested the formation of a less toxic by-product, 2-isopropyl-6-methyl-4-pyrimidinol (IMP) as a result of microalgal metabolism of diazinon. This study demonstrated that C. vulgaris is highly tolerant of diazinon, which could be voluntarily involved in the removal of traces of diazinon from contaminated wastewater and has potential application in the removal of such artificial toxins using algae. (C) 2016 Elsevier B.V. All rights reserved.</P>

Degradation of Sulfonated Azo Dyes by the Purified Lignin Peroxidase from Brevibacillus laterosporus MTCC 2298

Sushama S. Gomare,Jyoti P. Jadhav,Sanjay P. Govindwar 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.2

Lignin peroxidase (EC 1.11.1.14) was purified from the Brevibacillus laterosporus MTCC 2298 by ion exchange chromatography. The Km value of the purified lignin peroxidase (using n-propanol as substrate) was 1.6 mM. The MW of purified enzyme determined with the help of MW-standard markers was approximately 205 kDa. Purity of the enzyme was confirmed by native polyacrylamide gel electrophoresis (PAGE) and the activity staining using a substrate L-DOPA. Sulfonated azo dyes such as Methyl orange and Blue-2B were degraded by the purified lignin peroxidase. Degradation of the dyes was confirmed by HPLC, GC-MS, and FTIR spectroscopy. The mainly elected products of Methyl orange were 4-substituted hexanoic acid (m/z = 207), 4-cyclohexenone lactone cation (m/z = 191), and 4-isopropanal-2, 5-cyclohexa-dienone (m/z = 149) and for Blue-2B were 4-(2-hexenoic acid)-2, 5-cyclohexa-diene-one (m/z = 207; M - 1 = 206) and dehydro-acetic acid derivative (m/z = 223).

Monitoring the gradual biodegradation of dyes in a simulated textile effluent and development of a novel triple layered fixed bed reactor using a bacterium-yeast consortium

Kurade, M.B.,Waghmode, T.R.,Patil, S.M.,Jeon, B.H.,Govindwar, S.P. Elsevier 2017 Chemical engineering journal Vol.307 No.-

Textile industry effluents contain a variety of dyes, which are normally resistant to biodegradation. A bacterial-yeast consortium (Brevibacillus laterosporus and Galactomyces geotrichum) was used for decolorization of two real textile effluents (RTE) and a simulated synthetic effluent (SSE). It showed enhanced decolorization compared to that of individual microorganisms with decolorization efficiency of 89, 60 and 69% for RTE-1, RTE-2 and SSE respectively, within 48h. The cumulative action of oxido-reductive enzyme in the consortium was responsible for improved decolorization. Spectroscopic analysis suggested effective biodegradation of dyes present in the SSE by the consortium contrarily to the individual strains. The gradual biodegradation of each dye present in the SSE was monitored using high performance thin layer chromatography (HPTLC). The consortium biodegraded all of the dyes within 1has compared to that of partial biodegradation by the individual microorganisms. A novel, triple layered fixed bed reactor was designed for continuous decolorization of effluent. It showed >80% decolorization (at 100mLh<SUP>-1</SUP>flow-rate), for a period of 7days, along with ~78% reduction in COD. The reproducibility of the bioreactor could be maintained for three consecutive cycles (7dayseach).

Decolorization of Dyehouse Effluent and Biodegradation of Congo Red by Bacillus thuringiensis RUN1

( Olukanni,O. D ),( A A Osuntoki ),( A O Awotula ),( D C Kalyani ),( G O Gbenle ),( S P Govindwar ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.6

A dye-decolorizing bacterium was isolated from a soil sample and identified as Bacillus thuringiensis using 16S rRNA sequencing. The bacterium was able to decolorize three different textile dyes, namely, Reactive blue 13, Reactive red 58, and Reactive yellow 42, and a real dyehouse effluent up to 80-95% within 6 h. Some non-textile industrially important dyes were also decolorized to different extents. Fourier transform infrared spectroscopy and gas chromatography-mass spectrometer analysis of the ethyl acetate extract of Congo red dye and its metabolites showed that the bacterium could degrade it by the asymmetric cleavage of the azo bonds to yield sodium (4- amino-3-diazenylnaphthalene-1-sulfonate) and phenylbenzene. Sodium (4-amino-3-diazenylnaphthalene-1-sulfonate) was further oxidized by the ortho-cleavage pathway to yield 2- (1-amino-2-diazenyl-2-formylvinyl) benzoic acid. There was induction of the activities of laccase and azoreductase during the decolorization of Congo red, which suggests their probable role in the biodegradation. B. thuringiensis was found to be versatile and could be used for industrial effluent biodegradation.