Optimization of Fermentation Conditions for Laccase Production by Recombinant Pichia pastoris GS115-LCCA Using Response Surface Methodology and Its Application to Dye Decolorization

Qi Li, Jingcong Xie, Linguo Zhao, Qiuxia Xue, Jianjun Pei


In this work, recombinant Pichia pastoris GS115-lccA was cultured and expressed with the highest laccase activity of 2357 U/L with ABTS (2, 2'-nazinobis-(3-ethylbenzthiazoline-6-sulphonate)) as reacting substrate. To achieve the higher laccase activity, multiple key factors were screened (using a Plackett-Burman design of experiments methodology) related to the fermentation conditions for producing GS115-lccA. Subsequently, a Box-Behnken response surface methodology was employed for further optimization. The optimum fermentation conditions of the fermentation were obtained as follows: 0.603% methanol added into the culture every 24 h, medium optimal initial pH 7.1, and liquid medium volume of 20.4% provided the highest enzyme activity of 5235 U/L. The decolorization experiments of dyes (Reactive Blue KN-R and Acid Red 35) were carried with the laccase cultured by recombinant P. pastoris GS115-lccA. This purified enzyme showed excellent decolorization capacity. After 24 h, the decolorization of Reactive Blue KN-R with 100 mg/L at 50 °C, pH 4.5 using 20 mM acetate buffer with 2 U/mL purified enzyme was 91.33%, and for Acid Red 35, the decolorization was 78.96%. All results suggested that this laccase may be suitable for the wastewater treatment of similar azo and anthraquinone dyes from the deinking and dyeing industries.


Laccase; Response surface methodology; Dye decolorization; Reactive Blue KN-R; Acid Red 35

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