Laccase Production Under Optimized Parameters by Aspergillus oryzae, an Endophytic Fungus and their Application to Waste Water Treatment
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Objective: To isolate an endophytic fungal laccase producer from the Ziziphus mauritiana plant leaves in the surroundings of paper mill effluents and to study its role in the decolorization of synthetic dyes, removal of COD and phenol from industrial effluents. Methods: Laccase-producing endophytic fungi were isolated and screened by using an agar plate method. The positive isolates were identified, and laccase activity was determined by using spectrophotometric methods to monitor the oxidation of guaiacol and optimizing various parameters affecting laccase production. The molecular mass of the purified laccase enzyme was determined by using 12% SDSgel electrophoresis. Industrial effluents were treated with laccase to remove phenol, decolorize dyes, and reduce chemical oxygen demand. For the analysis, a spectrophotometric method was employed. Findings: One of the most effective endophytic fungal isolates, Aspergillus oryzae, was screened as a maximum laccase producer. The optimal pH of 6, temperature of 35 oC, inoculation period of 8 days, and the inoculum number of 3 discs/100 ml of Czapek Dox Broth in submerged culture were determined for the maximum laccase production. Sucrose and sodium nitrate, as carbon and nitrogen sources, considerably assisted laccase production. The molecular weight of the isolated laccase from A. oryzae was 66 kDa. The greatest activity was determined to be 64.2 U/mL, which is two times more than under unoptimized conditions. After the fifth day of exposure, the A. oryzae laccase decolorizes the synthetic dyes Bromophenol blue, Congo red, Methyl orange, and Phenol red.Chemical oxygen demand and phenolic pollutants’ clearance rates were 38–43% and 60% from coal and textile effluents during their exposure times, respectively. Novelty: A. oryzae was discovered to be a potent natural laccase producer endophytic fungus from paper mill effluents, which may be used for decolorizing non-textile dyes, treatment of various industrial effluents, and other industrial purposes. Keywords: Endophytic Fungi; Aspergillus oryzae; Laccase; Chemical oxygen demand; Synthetic dyesKeywords:
Guaiacol
Aspergillus oryzae
Bromophenol blue
Guaiacol
Sawdust
ABTS
Biotransformation
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Laccase is an important enzyme in terms of its versatile applicability, but its commercial use is limited by factors such as high production cost, low activity and/or stability under given conditions. The objective of this study was to screen xylophagic bacteria isolated from termites for the production of extracellular and intracellular laccases. Six laccase-positive strains were isolated, namely CA, A3, A5, A6, A7 and A8. They were molecularly identified by sequence analysis of 16S rRNA and classified under the genera Bacillus (A7, A8, CA) and Pseudomonas (A3, A5, A6). Laccase was produced by these bacterial isolates by submerged fermentation and was optimized at 37°C, pH 5.5, 6.2 and 7.0, with agitation and 0.5 mM guaiacol (as carbon source). Laccase activity was determined by measuring the oxidation of guaiacol and ABTS (2,21-azino bis[3-ethylbenzthiazoline-6-sulfonate]). Strain A5 produced extracellular laccase titers ranging from 123 to 168 U ml-1. Guaiacol was identified as a better substrate for the quantification of laccase. In conclusion, bacteria harboring the gut of termites can produce extracellular laccase with activity at medium to moderate acidity.
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Laccases are versatile biocatalysts for the bioremediation of various xenobiotics, including dyes and polyaromatic hydrocarbons. However, current sources of new enzymes, simple heterologous expression hosts and enzymatic information (such as the appropriateness of common screening substrates on laccase engineering) remain scarce to support efficient engineering of laccase for better "green" applications. To address the issue, this study began with cloning the laccase family of Lentinula edodes. Three laccases perfectio sensu stricto (Lcc4A, Lcc5, and Lcc7) were then expressed from Pichia pastoris, characterized and compared with the previously reported Lcc1A and Lcc1B in terms of kinetics, stability, and degradation of dyes and polyaromatic hydrocarbons. Lcc7 represented a novel laccase, and it exhibited both the highest catalytic efficiency (assayed with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS]) and thermostability. However, its performance on "green" applications surprisingly did not match the activity on the common screening substrates, namely, ABTS and 2,6-dimethoxyphenol. On the other hand, correlation analyses revealed that guaiacol is much better associated with the decolorization of multiple structurally different dyes than are the two common screening substrates. Comparison of the oxidation chemistry of guaiacol and phenolic dyes, such as azo dyes, further showed that they both involve generation of phenoxyl radicals in laccase-catalyzed oxidation. In summary, this study concluded a robust expression platform of L. edodes laccases, novel laccases, and an indicative screening substrate, guaiacol, which are all essential fundamentals for appropriately driving the engineering of laccases towards more efficient "green" applications.
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ABTS
Lentinula
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To discover novel laccases potential for industrial applications.Fungi were cultivated on solid media containing indicator compounds that enabled the detection of laccases as specific colour reactions. The indicators used were Remazol Brilliant Blue R (RBBR), Poly R-478, guaiacol and tannic acid. The screening work resulted in isolation of 26 positive fungal strains. Liquid cultivations of positive strains confirmed that four efficient laccase producers were found in the screening. Biochemical characteristics of the four novel laccases were typical for fungal laccases in terms of molecular weight, pH optima and pI. The laccases showed good thermal stability at 60 degrees C.Plate-test screening based on polymeric dye compounds, guaiacol and tannic acid is an efficient way to discover novel laccase producers. The results indicated that screening for laccase activity can be performed with guaiacol and RBBR or Poly R-478.Laccases have many potential industrial applications including textile dye decolourization, delignification of pulp and effluent detoxification. It is essential to find novel, efficient enzymes to further develop these applications. This study showed that relatively simple plate test screening method can be used for discovery of novel laccases.
Guaiacol
Tannic acid
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Abstract: Laccase (E.C. 1.10.3.2) are enzyme belonging to the group of blue multi-copper oxidases. Laccases catalyze the oxidation of variety of phenolic compounds, diamines and aromatic amines. Fungal isolate producing laccase was screened on Potato Dextrose Agar medium enriched with 0.02% guaiacol from soil as well as decaying wood samples collected from farm/forest site. Confirmation of laccase production was done by Bavendam test which is a plate assay technique using 3mM guaiacol. The isolate was identified and found to belong genera Aspergillus and was tested for producing laccase enzyme. An attempt was made to isolate, screening and production of laccase enzyme produced from Aspergillus niger. Isolate giving best result was processed further for production of laccase using Submerged fermentation. Guaiacol and sodium citrate buffer were used to assay laccase production. Characterization of fungal isolate was done by morphological analysis. This study showed that enzyme production can be increased by media manipulation in the fungal cultures. Optimization of the media condition can maximize the desired enzyme production. Application or Dye decolorization assay was been performed using 0.1ml crystal violet solution, where dye decolorization efficiency showed the ability of laccase to decolorize dyes. It is present in Ascomycetes, Deuteromycetes and Basidiomycetes and abundant in lignin degrading white / brown rot fungi. In the recent years, these enzymes have gained application in the field of textile, pulp and paper and food industries. Recently, it is also used in the design of biosensors, biofuel cells as a medical diagnostics tool and bioremediation agent to clean up herbicides, pesticides and certain explosive in soil. Laccases have received attention of researchers in the last few decades due to their ability to oxidize both phenolic and non phenolic lignin related compound as well as highly recalcitrant environmental pollutants.
Guaiacol
Potato dextrose agar
Yeast extract
Lignin peroxidase
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The sixty-five fungi strains that were isolated from the alpine grassland soil in the eastern Qilian Mountains were researched as the object in this paper.The fungi initially selected through culturing on the medium of guaiacol-PDA andα-naphthol-PDA medium.The fungi with the function to produce laccases was screened(identified as No.310b)based on the growth and colony sizes on the culture mediums of guaiacol, caechol,and o-benzyl aniline,the activities of redox reaction catalyzed by the laccases in these fungi grown,as well as the activity of laccases in these fungi cultured on the stems of oil rape.By rDNA-ITS sequence analysis, the fungus was identified as Marasmius tricolor.Then elementary study on the conditions under which laccases were produced by the fungus(No.310b)was carried out.These results showed that the activity of laccases in the fungus reached the peak when it was cultured under the condition:25℃,initial pH 4.0,sucrose and peptone that were used as carbon and nitrogen resources,respectively.Few non-trophic organic chemicals showed different effects on laccase activity in the fungus.α-naphthol and Tween-80did not have significant effects on laccase activity in the fungus,but guaiacol,tannic acid,and indole acetic acid inhibited the activity of laccases, especiall indole acetic acid,which significantly inhibited laccase activity(P0.01).When Cu2+concentrations ranged from 0.001-0.025g/L,laccase activity increased as the concentrations of Cu2+increased,reaching the peak at 0.025g/L.With the increase of inoculum sizes,laccase activity increased.Ranging from 60to 180 r/min,laccase activity increased.When the amount of the powder of oil rape stems ranged from 0to 1g,laccase activity increased,but when the amount was higher than 1,the activity decreased.
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