嗜熱菌 Geobacillus thermocatenulatus NTU 03 高溫下逆境反應及其熱穩定脂肪酶性質研究與應用

Abstract

嗜熱菌由於其生存於高溫中，故成為熱穩定性酵素很好的來源，除了工業上的應用外，這些熱穩定性蛋白質對於蛋白質結構的研究亦有很大的幫助。此外，由物理層面來看，高溫與常溫下各種因子的性質，如黏稠度，溶解度等必有所不同。此可推論生存於高溫環境下的嗜熱菌，為應變高溫環境溫度改變而產生的反應，應與嗜中溫菌有所不同。 在本研究中，以脂解酵素活性為菌種分離策略，自不同環境中，分離具有脂解酵素活性的嗜熱菌。以生化特性與基因鑑定法，對表現最高脂解酵素活性菌株進行鑑定，將此分離株命名為 Geobacillus thermocatenulatus NTU 03。以此菌株為目標，以蛋白質體學為主要方法，研究其在高溫下的相關逆境後發現，除了為因應高溫所引發的蛋白質變性、DNA 構形改變、細胞膜流動性及半透性改變外，高溫對此菌株也引發其氧化還原平衡被干擾以及輕微的氧缺乏反應。高溫、氧化壓力與氧缺乏，對此菌株主要的影響在於呼吸作用、電子傳遞鏈等能量的代謝程序，也因此提升細胞內活性氧的產生，但未進一步造成嚴重的氧化逆境。此外，硝酸呼吸作用也因為氧缺乏與 NADH 的累積而被啟動，雖然平衡了胞內的電子平衡，也因此改變胞內氨的相關代謝過程。因此，嗜熱菌對於高溫下的逆境反應，必須也要考慮由熱引發的種種物化因子改變。 除了逆境反應外，G. thermocatenulatus NTU 03 所表現的脂解酵素活性是另一個研究的方向。重組G. thermocatenulatus NTU 03 脂肪酶 (r03Lip) 為鹼性熱穩定脂肪酶，其最適反應溫度為 55°C。55°C 之最適反應 pH 值為9。Ca2+ 與 Na+ 對 r03Lip 結構有穩定作用可因此提高其反應活性。非極性的有機溶劑對r03Lip 活性有增益效果。以易錯傾向聚合酶鏈反應對 r03Lip 進行突變，篩選得到在 lid domain 有一個胺基酸取代 (E189I) 的突變脂肪酶，雖然熱穩定性下降，但活性上升 79 倍，主要的影響因素為 E189 會與 K185形成鹽橋，且此鹽橋作用力可與 lid domain C 端的 AXXXA motif 形成穩定結構。另外，E189I 提升了 lid domain的親脂性，而增加了與脂肪酸基質的親和力，是影響活性提升的重要因子。應用突變脂肪酶製成大腸桿菌全菌體生物催化劑 (whole-cell biocatalyst)，並應用於降低脂肪酸異丙酯生質柴油製程成本的研究。結果顯示，全菌體生物催化劑的確可以降低製程成本，但酵素再利用率與反應效率的提升是未來應用必要的研究方向。

Thermophiles have been reported to be a good thermostable enzyme source due to their living environment. Thermostable enzymes not only exhibit potential for industrial applications but are also used as protein structure models in studies on protein thermostability. In addition, because of the high temperature of these environments, many of their physical and chemical properties differ from those of lower-temperature environments. Hence, to deal with elevated temperature-induced changes in high temperature environmental conditions, the stress responses of thermophiles should differ from mesophiles. In this study, a thermophile (isolate NTU 03) was isolated in Taiwan on the basis of its lipolytic activity. This isolate was classified as Geobacillus thermocatenulatus NTU 03 on the basis of analysis pertaining to biochemical characteristics and 16S rRNA, recA, and rpoB sequence homology. The stress response of G. thermocatenulatus NTU 03 at elevated temperatures was investigated by proteomic analysis. Two-dimensional (2-D) gel analysis showed that heat stress induced modulation of protein expression, imbalance in the redox state, and a sudden decrease in the oxygen supply. The elevated temperatures adversely affected cellular energy metabolism, including respiration and electron transport, and subsequently elevated the reactive oxygen species level. However, the induced reactive oxygen species level was not high enough to cause oxidative stress. Transient activation of nitrate respiration due to heat-induced insufficiency in oxygen supply modulated ammonium metabolism. Our results support the view that both heat stress and heat-induced stress should be considered together when investigating the stress responses of thermophiles. A thermoalkalophilic lipase was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant NTU 03 lipase (r03Lip) showed optimal reaction activity at 55°C and pH 9. r03Lip exhibited increased activity and stability in the presence of sodium and calcium ions; this effect was caused by the structure stabilization effect of these ions. r03Lip exhibited good tolerance to various organic solvents under experimental conditions, and the non-polar organic solvent used enhanced r03Lip activity. Error-prone polymerase chain reaction was used to create more thermoactive and/or thermostable variants of thermoalkalophilic lipases. A variant of the α6 helix (lid domain) with a Glu to Ile substitution at residue 189 (E189I) lost its thermostability but exhibited 79-fold higher activity than its wild-type predecessor (r03Lip) did. Glu189 could form a salt bridge interaction with K185, and the salt bridge interaction could interact with the AXXXA motif at the C-terminal of the α6 helix to stabilize the helical structure. Furthermore, E189I increased the local hydrophobicity of the α6 helix and then increased its activity by improving its substrate affinity with fatty acids. To develop a cost-efficient process for production biodiesel, we developed a whole-cell biocatalyst harboring mutated lipase E189I. The process developed could reduce the cost of producing fatty acid isopropyl ester; however, the recycling and reaction rates of the biocatalyst need to be improved for industrial application of this kind of biocatalyst.