利用重組 Pichia pastoris 生產 Candida rugosa 脂肪酶 3 之生產菌株再改良與培養條件最適化

Abstract

Pichia pastoris GS115/CRL3, constructed by Prof. Shaw’s lab, Institute of Plant and Microbial Biology, Academia Sinica, was used to produce extracellular Candida rugosa lipase 3 (CRL3). Production strain was further improved by screening and selection on a series of agar plates in stepwise increase of Zeocin concentrations. The highest-yield strain was finally obtained and named as Pichia pastoris GS115/CRL3/Z1500. Using 500-mL Hinton`s flasks, response surface methodology (RSM) based on a three-factor three-level Box-Behnken design of experiment was used to optimize the culture conditions for CRL3 production by P. pastoris GS115/CRL3/Z1500. The optimal culture conditions were found as follows: C/N ratio in FM22, 12; inoculums size, 8.8%; temperature, 15℃. Under the optimal conditions, CRL3 activity of 8.47 U/mL was achieved after 120 h cultivation. Then, batch cultures were carried out in a 5-L bench-top fermentor under the optimal conditions. It was found that pH controlled at 5 was better than pH uncontrolled during cultivation. And a CRL3 activity of 26.77 U/mL was obtained after 96 h cultivation. Based on the result obtained, fed-batch cultures were further performed in order to attain the high cell density culture. While constant feeding rate at 11.25 mL/h was operated, OD600 of 627.6 and CRL3 activity of 451.7 U/mL were obtained, respectively after 216 h cultivation. However, a large amount of glycerol was accumulated in culture broth. While pseudo-exponential feeding mode was used instead, glycerol accumulation was minimized and CRL3 activity of 423.2 U/mL was achieved after 168 h cultivation. Productivity of 2.52 U/mL/h was obtained which was 20% higher than that of constant feeding mode.