| Summary: | 博士 === 國立臺灣海洋大學 === 生物科技研究所 === 94 === Abstract
Candida rugosa lipase (CRL), an important industrial biocatalyst, has been widely employed to catalyze various chemical reactions such as nonspecific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications. Several isozymes encoded by the lip gene family, namely lip1 to lip7, possess distinct thermal stability and substrate specificity. In this study, we utilize PCR to remove an unnecessary linker of pGAPZ�哸 vector and use overlap extension PCR-based multiple site-directed mutagenesis to convert several nonuniversal CTG-serine codons into universal TCT-serine codons and successfully express a highly active recombinant C. rugosa LIP1 and LIP3 in the Pichia pastoris expression system.
To improve the expression efficiency of recombinant LIP1 and LIP3 in P. pastoris, we adopted (I) the Response surface methodology (RSM) and 4-factor-5-level central composite rotatable design (CCRD) to evaluate the effects of growth parameters, such as temperature (21.6 to 38.4 �aC), glucose concentration (0.3 to 3.7%), yeast extract (0.16 to 1.84%), and pH (5.3 to 8.7) on the lipolytic activity of LIP1 and biomass of P. pastoris and to obtain the optimum production condition. Based on ridge max analysis, the optimum LIP1 production conditions were: temperature 24.1 �aC, glucose concentration 2.6%, yeast extract 1.4%, and pH 7.6. The predicted value of lipolytic activity was 246.9�b39.7 U/mL and the actual value was 253.3�b18.8 U/mL. The lipolytic activity of the recombinant LIP1 resulting from the present work is twofold higher than that achieved by a methanol induction system; (II) developed a predictive model for Pichia pastoris expression of highly active recombinant Candida rugosa LIP1 by combining the Gompertz function and response surface methodology (RSM) to evaluate the effect of yeast extract concentration (0.16 to 1.84%), glucose concentration (0.3 to 3.7%), temperature (21.6 to 38.4 �aC), and pH (5.3 to 8.7) on specific responses of P. pastoris growth kinetics. Based on ridge max analysis, the optimum population density conditions were: temperature 24.4 �aC, glucose concentration 2.0%, yeast extract 1.5%, and pH 7.6. The optimum specific growth rate conditions were: temperature 28.9 �aC, glucose concentration 2.0%, yeast extract 1.1%, and pH 6.9. The optimum protein concentration conditions were: temperature 24.2 �aC, glucose concentration 1.9%, yeast extract 1.5%, and pH 7.6. Based on ridge minimum analysis, the minimal lag phase conditions were: temperature 32.3 �aC, glucose concentration 2.1%, yeast extract 1.1%, and pH 5.4. For the predicted value, the maximum population density, specific growth rate, protein concentration, and minimum lag phase duration were 15.7 mg/mL, 3.4 h-1, 0.78 mg/mL, and 4.2 h, and the actual values were 14.3 �b 3.5 mg/mL, 3.6 �b 0.6 h-1, 0.72 �b 0.2 mg/mL, and 4.4 �b 1.6 h, respectively; (III) we also reconstruct a regional synthetic gene fragment of lip1 or lip3 near the 5’ end of a transcript to match P. pastoris-preferred codon usage for simple scale-up fermentation. More detail biochemical properties of the purified recombinant LIP1and LIP3 for further industrial applications are also determined and discussed in detail in this study. For LIP1 case, our present results show that the production level (152 mg/ l) of codon-optimized lip1 (colip1) has an overall improvement of 4.6 fold relative to that (33 mg/L) of noncodon optimized lip1 with only one-half the cultivation time of P. pastoris. This finding demonstrates that the regional codon optimization lip1 gene fragment at the 5’ end can greatly increase the expression level of recombinant LIP1 in the P. pastoris system. The optimal pH and temperature for purified recombinant LIP1 are pH 7.0 and 40 °C, respectively. After incubation at various pH and temperatures, the enzyme was stable at pH 4.0–6.0 and temperature 30–40 °C. Among numerous substrate tests, the p-nitrophenyl (p-NP) caprylate (C8), trilaurin (C12) and cholesteryl oleate (C18:1) were the favorite substrates for recombinant LIP1. The enzyme was greatly inhibited by SDS but little affected by other detergents tested. It retained 95% of its residual activity after incubation in 30% acetone for 16 h. The enzyme activity increased 33%, 35%, and 2.5-fold in the presence of 10 mM CaCl2, MnCl2 and CuSO4, respectively. These distinct biochemical properties suggest it is very useful for further industrial applications.
For LIP3 case, our results show that the production yield (0.687 U/mL) of N-fused lip3 (nflip3) has an overall improvement of 69-fold relative to that (0.01 U/mL) of lip3, and of 52-fold (0.47 U/mL) of codon-optimized lip3 (colip3) relative to that (0.01 U/mL) of noncodon-optimized lip3, with the cultivation time set at five days. This finding demonstrates that the N-terminus peptide and the regional codon optimization of the lip3 gene fragment at the 5’ end can greatly increase the expression level of recombinant LIP3 in the P. pastoris system. The optimal pH and temperature ranges for purified recombinant LIP3 are pH 4.0–6.0 and 20–50 °C, respectively. After incubation at various pH and temperatures, the enzyme was stable at pH 5.0–8.0 and temperature 37–60 °C. Among numerous substrate tests, the p-nitrophenyl (p-NP) caprate (C10), tricaprylin (C8) and cholesteryl oleate (C18:1) were the favorite substrates for recombinant LIP3. The enzyme was greatly inhibited by SDS and 1% CHAPS but little affected by other detergents tested. It retained 99% of its residual activity after incubation in 30% DMSO for 16 h. The enzyme activity increased 2.3-fold in the presence of 10 mM MnCl2. These distinct biochemical properties suggest it might be useful for other industrial applications.
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