1、酵母菌乙醇脫氫酶與乙醛脫氫酶之抽取及其在原核系統之表達Extraction of Alcohol Dehydrogenase and Aldehyde Dehydrogenase from Saccharomyces cerevisiae and their Expression by Prokaryotic System研究生：戴嘉慶 Jia-Chin Dai指導教授：林茂勇 Maw-Yeong Lin【摘要】Saccharomyces cerevisiae ( SC ) 麵包酵母菌体中含有許多具高經濟價值性且無毒性的酵素蛋白等物質，頗具經濟開發與利用效益。本試驗先以數種商業培養基培養市售
2、11 種麵包酵母菌，並測定其菌體之產量以為日後大量培養 SC 菌體之應用，結果篩選出 Angel 酵母菌 ( SC04 )。並以機械研磨充分打破 Angel 酵母菌體後，行蒸餾水抽取，再以丙酮及飽和硫酸銨次第純化乙醇脫氫酶 ( alcohol dehydrogenase；ADH )，另以檸檬酸及魚精蛋白次第純化乙醛脫氫酶 ( aldehyde dehydrogenase； ALDH )，利用 NAD+ 擷取酒精及乙醛中氫離子之特性在 340 nm 光譜可吸收NADH 之變化而測定 ADH 和 ALDH 之含量。另一方面，以聚合酶鏈連鎖反應 ( PCR ) 增幅出ADH ( 1062 bp )
3、及 ALDH ( 1536 bp ) 基因，經插入表現載體 ( pET32a ) 定序後，再轉殖到 BL21之大腸桿菌中進行表現各重組蛋白。結果以傳統方法抽取之每克酵母粉分別可獲取 33210 單位之ADH ( 47 kDa ) 及 97 單位之 ALDH ( 52 kDa )，另以原核系統所表達之 ADH ( 59 kDa ) 及 ALDH ( 76 kDa ) 雖可以西方轉漬法分別檢出其所表現的蛋白，但兩者均不具 ADH 及 ALDH 的活性。以本實驗進行之 E. coli 表現系統尚無法獲取傳統抽取方式所能具體獲得具酵素活性之 ADH 及ALDH。關鍵字：麵包酵母菌、乙醇脫氫酶、乙醛脫氫
4、酶【Abstract】Yeast cell containing a great deal of high cost nontoxic enzymes, proteins etc. provides a good source for further economic utilization. In this study, The Saccharomyces cerevisia ( SC ) of the Angel baking powder seed yeast ( SC04 ) gave the best growth in variant commercial and self-pre
5、pared medium for cultivating SC was chosed from 11 commercial baking powder seeds for further mass production. Futhermore, Alcohol dehydrogenase ( ADH ) were extracted from a complete mechanically breaking up SC cells with distilled water ( DW ), then purified with cold acetone and saturated ammoniu
6、m sulfate, and aldehyde dehydrogenase ( ALDH ) was then purified from the above DW extract, then further purified with pooled acid and protoamine solution. A 33210 units of ADH ( 47 kDa ) and 97 units of ALDH ( 52 kDa ) were harvested from the powder per gram as measuring by the spectrophotometer de
7、tecting the nicotinamide adenine dinucleotide ( NADH ) formation under 340 nm ultraviolet light. The whole length of ADH ( 1062 bp ) and ALDH ( 1536 bp ) gene were polymerase chain reaction ( PCR ) amplified and inserted in the pET32a vector individually. The recombinant plasmid was transformated in
8、to E. coli BL21 expression system. A recombinant ADH ( 59 kDa ) or ALDH ( 76 kDa ) was expressed from each system with Western blotting identified, but without providing any dehydrogenation activity to ethyl alcohol or acetoaldehyde. ADH and ALDH expressed by the E. coli expression system as done in this experiment is not able to produce the product with good enzymatic activity as the traditional extraction from the baking yeast does.Key words: Saccharomyces cerevisia, Alcohol dehydrogenase, Aldehyde dehydrogenase
