酵母菌表達小枯草桿菌衍生木聚醣酶

Expression system of a paenibacillus- derived Xylanase in pichia pastoris.

呂彥廷(Ian-Ting Lu)

指導教授 ： 柯淳涵

國立臺灣大學/生物資源暨農學院/森林環境暨資源學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10298

摘要

木聚醣為木材及植物生質中的重要主成分，而木聚醣水解酶可水解木聚醣，降低其分子量，並廣泛應用於製漿、畜牧、食品產業中。本研究將之胺基酸T44A取代之變異Paenibacillus木聚醣水解酶基因xylX-H1，轉殖於酵母菌Pichia pastoris上。以Pichia pastoris做為蛋白質表現系統，具有將蛋白質正確摺疊、轉錄後修飾並分泌等特性，以利於商業上大量生產。本研究利用Pichia pastoris表現系統表達基因，在原本的DNA上接上引子，引入外來基因的表現載體pPIC9K，再轉殖入Pichia pastoris SMD1168中。本研究以小量搖瓶培養探討重組Pichia pastoris生產木聚醣水解酶(XylX-H1)，並透過活性分析並找出最佳培養條件。結果顯示加入 1% (w/v)甲醇誘導96 hr後，可產生最佳的酵素活性。比較兩株不同載體的轉形株，以pPIC9K為載體的轉形株活性為9171 U/mg，較pPICZaA為載體的轉型株4461 U/mg高。搖瓶培養分析結果以溶氧量20 ppm、起始誘導菌量數OD600 30為最佳培養條件。以相同條件生產五公升發酵槽生產的木聚糖酶活性高達7693 U/mg。而轉殖後的XylX-H1水解樺木木聚醣產物，以木二醣為最多占水解產物42%，木三醣次之占39%。此外，分泌出來的酵素在置於酸性條件pH3.5下1hr後，保有84%的酵素活性，置於於pH2.5下1 hr，仍然可以保持62%的酵素活性，顯示此酵素具有耐酸的特性。未來可望應用於食品以達到腸胃保健功效，或應用於飼料上提升養分消化率。

關鍵字

木聚醣酶 ； 重組蛋白 ； 發酵

並列摘要

Xylan is one of the major components of wood and plant biomass. Xylanases hydrolyze xylanase and are widely applied in pulp & paper, animal feeding and food industries. xylX-H1, a T44A amino acid substitution mutant derived from Paenibacillus xylX, has been cloned in Pichia pastoris. Protein expression systems using Pichia pastoris can correctly fold, conduct post-translation modification and secretion. The above systems could be easily expended to a commercial scale. This study constructed a Pichia pastoris expression system, ligate a primer on the original DNA, clone the ligated DNA into the vector pPIC9K and then transform into Pichia pastoris SMD1168. This study investigated production of recombinant xylanase (XylX-H1) by shake flask fermentations, and optimal production conditions were analyzed. Results showed that the optimal enzyme activities were induced by 1% methanol induction after 96 hours. Transformed Pichia strains cloned with pPIC9K and pPICZaA produced 9171 and 4461 and U xylanase/mg protein. Other optimal parameters found were starting dissolved oxygen at 20 ppm and starting OD600 at 30 during shake flask fermentation runs. 5 L fermentation runs produced expressed XylX-H1 at 7693 U/mg. Hydrolyzing birchwood xylan by expressed XylX-H1 for 24 hours produced 42 % xylobiose and 39 % xylotriose. Expressed XylX-H1 retained 84 and 62 % activities after being subjected to pH 2.5 and 3.5 over 1 hour. Hence, it showed XylX-H1 is acid-tolerant and could be gastronomically applied in human and animals for probiotic and enhancing meat yields purposes.

並列關鍵字

Paenibacillus ； xylanase ； Pichia ； fermentation ； recombinant protein

參考文獻

Beg, Q. K., Kapoor, M., Mahajan L., Hoondal, G. S. (2001). Microbial xylanases and their industrial applications: a review. Applied Microbiology and Biotechnology 56(3 - 4):326-338.

Biely, P. (1985) Microbial xylanolytic systems. Trends in Biotechnology 3(11): 286 - 90.

Cereghino, J., Cregg, JM. (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 24, 45-66.

Cregg, JM., Madden, KR., Barringer, KJ., Thill, GP., Stillman, CA. (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol. Cell. Biol. 9: 1316-1323.

Collins, T., C. Gerday and G. Feller (2005). Xylanases, xylanase families and extremophilic xylanases.Fems Microbiology Reviews 29(1): 3 - 23.