Journal of Scientific & Industrial Research Vol. 77, December 2018, pp. 713-722 Application of Experimental Designs for Optimization the Production of Alcaligenes Faecalis Nyso Laccase Soad A Abdelgalil 1 , Ahmed M Attia 2 , Reyed M Reyed 1 , Nadia A Soliman 1 * and Hesham A El Enshasy 1,3 1 Bioprocess Development Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technological Applications, Alexandria, Egypt 2 Institute of Graduate Studies and Research, Alexandria University, Environmental Studies Department 3 Institute of Bioproducts Development (IBD), Universiti Teknologi Malaysia (UTM), Skudai, Johor, Malaysia Received 14 December 2017; revised 10 July 2018; accepted 30 October 2018 A sequential optimization strategy based on statistical experimental designs was implemented in order to enhance laccase production by a local isolate Alcaligenes faecalis NYSO in a submerged culture. To screen the parameters significantly influencing the laccase productivity, a 2-level Plackett-Burman design was applied. Among the studied variables, the pH, yeast extract, (NH 4 ) 2 SO 4 , glucose, and CuSO 4 .5H 2 O were selected based on their high positive significant effect on laccase productivity. In order to find out the combination among the most significant variables that brings maximum yield, Response Surface Methodology was applied, where a 3-level Box-Behnken design was utilized to create a polynomial quadratic model correlating the relationship between the five variables and the laccase productivity. The optimal combination of the major medium constituents for laccase production was evaluated using the JMP program, was as follows: yeast extract, 0.896%; (NH 4 ) 2 SO 4 , 0.035%; CuSO 4 .5H 2 O, 0.0075%; FeSO 4 .7H 2 O, 0.000133%; glucose, 0.0943%, pH 10.6 and 30 o C for 24 hrs. The predicted optimum laccase activity was 791U ml -1 min -1 , which was 700 times the activity with basal medium. In addition, the further optimization for both pH, CuSO 4 .5H 2 O concentration lead the yield to be 2435 U ml - 1 min at pH 11.0, 200 mg CuSO 4 .5H 2 O which achieved after 18 hrs incubation time. Keywords: Laccase, Fractional Factorial Design, Response Surface Methodology, Alcaligenes faecalis NYSO Introduction The need to the maintenance of ecosystem and environment is essential concern nowadays and problems associated with environmental pollution demonstrate the necessity to improve remediation processes for detoxification of a wide variety of xenobiotic compounds which continuously discharged into the environment. Green chemistry technology is an environmental friendly novel trend; it was evaluated to overcome the obstacles associated with the traditional methods and improve the products quality 1 . Laccases (p-benzenediol: oxygen oxidoreductase, 1.10.3.2) are blue multicupper oxidases had a unique structure, it is a dimeric or tetrameric glycoprotein, which usually contains four cupper atoms per monomer distributed in three redox sites. The exploitation of laccase in biotechnological processes requires the production of high amounts of the enzyme at low cost and hence the current research focused and oriented towards the identification and optimization of such an efficient production system 2 . An isolation and identification of environmental friendly bacteria for lignin bioremediation become an essential because all the previous researchers concentrated on using fungal treatments. Bacterial laccases have great potential as biocatalysts due to their intrinsic properties of high thermal and alkaline pH stability than fungi 2 . The major obstacle to bacterial laccase commercial application is the lack of sufficient enzyme stocks. In view of the importance of laccases, the main objective of this work directed to design a new strategy for production of bacterial laccase. Material and Methods Sample collection and isolate sources Sludge, slime, black liquor and wastewater samples used in this study were collected aseptically from different discharged sites of paper and pulp industries and tanning & leather factories, Alexandria, Egypt. Stone and soil samples were collected from Muzdalifah and Mina, Saudi Arabia, the temperature of samples was 48 °C at the time of collection. The ——————— *Author for Correspondence E-mail: nadiastuttgart@yahoo.com