Bacillus subtilis DB428,DB430 醱酵系統之研究

Abstract

枯草桿菌 (Bacillus subtilis) 為格蘭氏陽性菌，由於可以產生大量胞 外蛋白而易於進行純化，並且具有容易保存、培養及不具致病性等特性， 因此可利用在生產基因重組蛋白質的醱酵工業上。由於培養基條件會直接 影響到菌體的生長情況，因此本論文研究了不同培養基組成、培養條件與 培養方式對於枯草桿菌生長的影響。此外也探討不同氮源、碳源對於質體 穩定性及產孢情形的影響，並且計畫將菌株進行突變以獲得更適合之菌種 ，因為此研究主要目的在找到最適培養基與最適培養條件，以得到最高的 菌體濃度，並將此系統應用在大量表現外來蛋白質，所以本實驗首先以搖 莫養枯草桿菌，得知其最適生長溫度為 37℃、 pH 值為 7。實驗結果也 發現培養基的溶氧量會影響菌體生長，較高的溶氧量可幫助菌體生長。此 外，培養基的最適氮源為 soytone 與 yeast extract，最適碳源則為葡 萄糖，以此二者組合可得較高的菌體生長濃度。在醱酵槽中以上述條件大 量培養枯草桿菌時，發現饋料式培養可得到最高的菌體濃度 (OD600) 與 菌體乾重 (biomass)，其中最適氮、碳源組合為 yeast extract 加上葡 萄糖，所得到的菌液吸光值可高達 60，乾重可達 15 g/l。但是不同氮、 碳源組合及培養方式對於 VP3 蛋白質的表現量則沒有明顯影響。在載體 的選擇，不同的質體在不同的培養基成分中所測得的穩定性有所差異，其 中以質體 pUB110 的穩定性最高， pE194 最低，以 tryptone 作為氮源 時則會降低質體之穩定性。此外，在不同氮、碳源及培養方式培養下，枯 草桿菌DB428 及 DB430產孢情形皆不明顯，顯示培養基中的養分能繼續提 供菌體生長。而構築枯草桿菌 recE 突變株亦可幫助大量表現外來蛋白質 時質與量的穩定。 Abstract Bacillus subtilis, one of the gram-positive bacteria, is a potential microorganism to express recombinant proteins. The advantage of B. subtilis includes the characteristics of nonpathogenic, easy to cultivate, store and secrete proteins. To develop a process for recombination protein production, the effect of medium component on cell growth, plasmid stability and sporulation were studied in flask and fermentor. In addition, in order to increase expression of stable heterologous gene products in B. subtilis, the recE mutants were also constructed. The experimental results showed that 37℃, pH 7.0 and high agitation were the optimal conditions to cultivate B. subtilis DB428 and DB430. When B. subtilis DB428 grew in the medium containing soytone, yeast extract and glucose, the cell density was higher than they were grown in the medium containing other carbon and nitrogen sources. This result suggested that soytone and yeast extract were better nitrogen sources, and glucose was a better carbon source. The plasmid stability studies demonstrated that pUB110 was the most stable plasmid whereas pE194 was the lowest stable plasmid. However, medium with different nitrogen and carbon sources did not affect sporulation of B. subtilis DB428 and DB430. When B. subtilis DB428 grew in 2.5 L fermentor containing yeast extract and glucose as medium, the cell density was as high as 60 (OD600), and the cell dry weight achieved to 15 g/l. Expression of recombinant VP3 protein did not vary at different combination of nitrogen and carbon sources. The highest expression of VP3 was 760 mg/l in fed-batch culture.