拮抗性臘狀芽孢桿菌菌株28-9幾丁質水解酵素系統研究

Abstract

利用幾丁質培養基自百合根圈土壤分離出一幾丁質分解性臘狀芽胞桿菌菌株28-9，經對峙培養、切離葉等防治試驗得知此菌株具有防治百合灰黴病的潛力。為了解幾丁質水解能力與拮抗百合灰黴病菌的關係，分別從蛋白質與基因組方向進行，得到兩個幾丁質水解酵素基因，命名為chiCH及chiCW。其演譯之蛋白質除訊息序列外，ChiCH具有一催化功能區，ChiCW則具一催化功能區，一fibronectin type III相似功能區及一幾丁質結合功能區。分別自大腸桿菌表現及純化出ChiCH和ChiCW兩蛋白質，經抑菌試驗分析得知ChiCH僅輕微地抑制百合灰黴病菌分生孢子萌芽，而ChiCW可有效地抑制百合灰黴病菌分生孢子萌芽，推測ChiCW是菌株28-9拮抗百合灰黴病菌之重要因子。此外，本研究亦發展出利用融合glutathione-S-transferase的重組幾丁質水解酵素可在大腸桿菌中大量表現幾丁質水解酵素，經動力學、水解產物及基質結合力分析可知ChiCW為內切型而ChiCH為外切型幾丁質水解酵素。最後，本研究對ChiCW的C端區域在酵素功能上的影響進行分析，相對於ChiCW，在高溫及高酸鹼值的條件下， ChiCWΔFC對醣酐幾丁質有較高之活性，動力學分析結果則顯示ChiCW較ChiCWΔFC容易水解寡分子及聚分子基質，推測菌株28-9將ChiCW修飾成ChiCWΔFC可以提升自身在不同環境下分解幾丁質的效率，效地將大分子基質水解成更小的寡分子基質。

Bacillus cereus 28-9 is a chitinolytic bacterium isolated from rhizosphere of lilies. This bacterium showed antagonistic effect against Botrytis elliptica, the pathogen of lily leaf blight, by detached leaf assays. To investigate the involvement of the chitinolytic activity in antagonism, two chitinase-encoding genes, chiCH and chiCW, have been cloned. ChiCH consists of a signal peptide followed by a catalytic domain. ChiCW consists of a signal peptide, a catalytic domain, a fibronectin type III-like domain, and a chitin-binding domain. ChiCH and ChiCW are slightly and effectively inhibitory to conidial germination of B. elliptica, respectively, indicating that ChiCW may contribute to the antagonistic activity of B. cereus 28-9 against B. elliptica. In addition, an ideal method for high-level expression of chitinases in E. coli as glutathione-S-transferase fusion proteins was established in this study. According to the results of kinetics, hydrolysis products, and binding activities, ChiCW is an endo-chitinase and ChiCH is an exo-chitinase. Finally, the functions of the C-terminal region on ChiCW activity were investigated. Compared with ChiCW, ChiCWΔFC exhibited higher activity at high temperature and pH although both enzymes had the same optimal temperature and pH for enzyme activities. The kinetic properties of ChiCW and ChiCWΔFC indicate that ChiCW hydrolyzes oligomeric and polymeric substrates more efficiently than ChiCWΔFC. Based on the results, we suggest that B. cereus 28-9 proteolytically modifying ChiCW to ChiCWΔFC could improve efficiency of chitin degradation in different environments.