Abstract

Plackett-Burman design and response surface methodology were employed to optimize the medium components for endochitinase production by Pichia pastoris. A Plackett- Burman design of seven factors with 12 runs was applied to evaluate the effects of different medium components. Yeast extract, oleic acid and Tween-80 were found to have significant influence on endochitinase production. The optimal concentrations of three factors were investigated by the response surface methodology using Box- Behnken design. The optimal medium components obtained for achieving the maximum activity of the endochitinase were as follows: Yeast extract 24.36 g/l, tryptone 20 g/l, YNB 5.0 g/l, potassium phosphate 100 mM, methanol 5 ml/l, oleic acid 1.758 ml/l, Tween-80 6.2 ml/l, Pichia trace metals (PTM₁) 4.0 ml/ l and biotin 4.00 × 10^-4 g/l. Under these conditions, endochitinase activity was up to 88.26 µ/ml, which was about 1.14-fold higher than using the original medium (77.62 µ/ml). This work will be very helpful for large-scale production of endochitinase for future industrial application.

Key words: Recombinant endochitinase, Plackett-Burman design, response surface methodology, fermentation.

Abbreviation

Abbreviations: RSM, Response surface methodology; 4-MU, 4-methylumbe–lliferyl; 4-MUChT, 4-methylumbelliferyl β-D-N, N’, N’’-triacetyl-chitotrioside; PTM₁, pichia trace metals; YNB, yeast nitrogen base.