納豆菌漆脢之異源表現

Heterologous expression of laccase gene from Bacillus subtilis natto

林軒立(Hsien-Li Lin)

指導教授 ： 李昆達

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00983

摘要

本研究使用選殖自Bacillus subtilis natto NTU18漆脢基因，分別轉殖到大腸桿菌的誘導型表現質體pQE-30 Xa和酵母菌Pichia pastoris X33的持續型表現質體pGAPZαA進行異源表現。在大腸桿菌表現系統方面，漆脢活性在Hinton氏搖瓶培養和7-L生物反應器批次培養中，分別達到0.084 U mL-1和0.87 U mL-1，菌體濃度分別為0.37 gDCW-1和4.40 gDCW L-1；但在7-L生物反應器中進行批次饋料培養，菌體濃度雖可達批次培養的13.3倍 (58.5 gDCW L-1)，但活性卻下降了30%，只達到0.61 U mL-1，推測應為大腸桿菌在快速且大量的增殖時，表現質體無法穩定存在於細胞內，造成活性無法如預期般的提升。而在P. pastoris X33表現系統方面，漆脢活性在Hinton氏搖瓶培養和7-L生物反應器饋料批次培養中，則分別可達到0.13 U mL-1和0.62 U mL-1，菌體濃度分別為55.7 gWCW L-1和392.2 gWCW L-1。而重組納豆菌漆脢的最適反應溫度為85℃，相對於雲芝漆脢之最適反應溫度為65℃，重組納豆菌漆脢的最適反應溫度較高；且重組納豆菌漆脢經過55℃處理24小時之後，活性沒有下降，顯示其具有良好的熱穩定性。酵素動力學分析結果顯示，兩酵素在對於ABTS的親和性與最大反應速率並沒有很大的差異。

關鍵字

納豆菌 ； 漆脢 ； 饋料批次培養

並列摘要

The laccase gene of Bacillus subtilis natto NTU18, which was isolated form commercial natto by previous graduated student, was cloned into pQE-30 Xa and pGAPZαA. In Escherichia coli expression system, the laccase activity reached 0.084 U mL-1 in Hinton’s flask, and 0.87 U mL-1 in 7-L bioreactor batch culture. And the biomass was reached to 0.37 gDCW L-1 and 4.40 gDCW L-1. However, in 7-L bioreactor fed-batch culture, the biomass was 13.3 times to which in batch culture, up to 58.5 gDCW L-1. But the laccase activity was reduced by 30%, only 0.61 U mL-1 was obtained. The low activity may due to the genetic instability of plasmid replication during E. coli reproduction. In Pichia pastoris expression system, the laccase activity reached 0.13 U mL-1 in Hinton’s flask, and 0.62 U mL-1 in 7-L bioreactor fed-batch culture. And the biomass was 55.7 gWCW L-1 and 392.2 gWCW L-1. The optimum reaction temperature of recombinant laccase was 85℃, higher than the optimum reaction temperature of T. versicolor laccase, which was 65℃. The recombinant laccase also showed high thermostability under the treatment of 55℃ for 24 h. And there is no significant difference of the enzyme kinetic constant Km and Vmax between recombinant and T. versicolor laccase.

並列關鍵字

Laccase ； Bacillus subtilis natto ； fed-batch culture

參考文獻

Alexandre, G., and Zhulin, I.B. (2000). Laccases are widespread in bacteria. Trends Biotechnol 18, 41-42.

Ash, C., Farrow, J.A.E., Wallbanks, S., and Collins, M.D. (1991). Phylogenetic Heterogeneity of the Genus Bacillus Revealed by Comparative-Analysis of Small-Subunit-Ribosomal Rna Sequences. Lett Appl Microbiol 13, 202-206.

Ashiuchi, M., Kamei, T., and Misono, H. (2003). Poly-gamma-glutamate synthetase of Bacillus subtilis. J Mol Catal B-Enzym 23, 101-106.

Baldrian, P. (2006). Fungal laccases - occurrence and properties. Fems Microbiol Rev 30, 215-242.

Barnun, N., Lev, A.T., Harel, E., and Mayer, A.M. (1988). Repression of Laccase Formation in Botrytis-Cinerea and Its Possible Relation to Phytopathogenicity. Phytochemistry 27, 2505-2509.