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Present study demonstrated the isolation of most promising β-galactosidase producing bacterial strain SB from soil. Morphological, biochemical, and 16s rRNA sequence analysis identified the bacterial strain as Arthrobacter oxydans. Several chemicals, including SDS, Triton X-100, Tween 20, isoamyl alcohol, and toluene-acetone mixture, were applied for extraction of intracellular β-galactosidase from the bacterial strain Arthrobacter oxydans. Among these, Tween 20 was recorded to be most effective. Role of pH, temperature, and shaker speed on production of β-galactosidase was evaluated using Box-Behnken design of response surface methodology. According to Box-Behnken analysis, optimum production of β-galactosidase (21.38 U (mg–1 protein)) is predicted at pH 6.76, temperature 36.1 °C, and shaker speed 121.37 r.p.m. The parameters are validated with the nearest value.

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