Although laccase has been recognized as a wonder molecule and green enzyme, the use of low yielding fungal strains, poor production, purification, and low enzyme kinetics have hampered its large-scale application. Thus,this study aims to select high yielding fungal strains and optimize the production, purification, and kinetics of laccase of Aspergillus sp. HB_RZ4. The results obtained indicated that Aspergillus sp. HB_RZ4 produced a significantly large amount of laccase under meso-acidophilic shaking conditions in a medium containing glucose and yeast extract. A 25 μM CuSO 4 was observed to enhance the enzyme yield. The enzyme was best purified on a Sephadex G-100 column. The purified enzyme resembled laccase of A. flavus. The kinetics of the purified enzyme revealed high substrate specificity and good velocity of reaction,using ABTS as a substrate. The enzyme was observed to be stable over various pH values and temperatures. The peptide structure of the purified enzyme was found to resemble laccase of A. kawachii IFO 4308. The fungus was observed to decolorize various dyes independent of the requirement of a laccase mediator system. Aspergillus sp. HB_RZ4 was observed to be a potent natural producer of laccase, and it decolorized the dyes even in the absence of a laccase mediator system. Thus, it can be used for bioremediation of effluent that contains non-textile dyes.

Laccase enzyme has gained tremendous demand in various industries, waste water treatment, bioremediation and nanobiotechnology. This compels the availability of enzyme in greater yields that can be achieved by employing potential laccase producing cultures and statistical optimization. Use of Plackett-Burman design (PBD) that evaluates various medium components and having two level factorial design help to determine the factor and its level to increase the yield of product. Present paper reports the screening of laccase producting fungus identified as Aspergillus sp. by 18S rDNA of internal transcribed spacer regions and the large subunit. Aspergillus sp. HB_RZ4 isolated from tree bark scrapping showed enhanced production of laccase in minimal medium by applying two-stage statistical approach, as PBD and Response Surface Methodology using central composite design (CCD). In the first stage of optimization, out of 8 variables of minimal medium, glucose, yeast extract, CuSO 4 and temperature were found as significant components that influenced laccase production. Aspergillus sp. HB_RZ4 produced highest amount (7.27 Uml −1 ) of extracellular laccase. Further optimization using CCD revealed that 34 °C temperature, 2.1 gL −1 of glucose (carbon source), 2.5 gL −1 of yeast extract (nitrogen source) and 0.0025 gLCuSO 4 (inducer) were the optimum values that yielded maximum laccase. The optimization by PBD statistical design studies revealed optimum laccase yield with conditions being; pH, 6.0; temperature, 34 °C; incubation, 9 days; glucose, 2.1 gL −1 ; yeast extract, 2.5 gL −1 and CuSO 4 ., 0.0025 gL −1 .

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Although laccase has been recognized as a wonder molecule, and green enzyme, the use of low yielding fungal strains, poor production, purification, and low enzyme kinetics have hampered its larger-scale applications. Hence the present research was aimed to select high yielding fungal strains and to optimize the production, purification, and kinetics of laccase of Aspergillus sp. HB_RZ4. Aspergillus sp. HB_RZ4 produced a copious amount of laccase on under meso-acidophillic shaking conditions in a medium containing glucose and yeast extract. A 25 µM of CuSO 4 enhanced the enzyme yield. The enzyme was best purified on Sephadex G-100 column. Purified enzyme resembled with the laccase of A. flavus. Kinetics of purified enzyme revealed the high substrate specificity and good velocity of reaction with ABTS as substrate. The enzyme was stable over a wide range of pH and temperature. The peptide structure of the purified enzyme resembled with the laccase of A. kawachii IFO 4308. The fungus decolorized various dyes independent of the requirement of a laccase mediator system (LMS). Aspergillus sp. HB_RZ4 came out as a potent natural producer of laccase, it decolorized the dyes even in absence of LMS and thus can be used for bioremediation.