大量生產木聚醣水解酶重組蛋白以將廢棄農作物 轉化為木寡醣

Overproduction Of Recombinant Xylanases For Generating Oligoxylose From Agro-wastes

李育錚(Lee, Yu Jheng)

指導教授 ： 曾銘仁

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2012年)

摘要

嗜鹼性 Bacillus halodurans 可分泌出木聚醣水解酶，選殖出兩個全長木聚醣水解酶的DNA片段並定序序列無誤，從ORF開始分子量依序分別為45和23 kDa。而另一株Bacillus pumilus NCCU也可分泌出木聚醣水解酶， 並選殖出DNA片段且定序序列無誤，從ORF開始分子量為26 kDa。因此將上述所選殖出的木聚醣水解酶依分子量分別命名為xyn45、xyn23和BPxynA
，將其DNA片段次選殖於pET29a表現載體中，於C段接至His6-tag，轉染於E. coli BL21 (DE3) 做大量蛋白表現。
分析Xyn45-His6其結果得知，最佳誘導條件為25 oC下最終濃度0.05 mM IPTG和誘導時間為18小時，即可觀察出超過一半的酵素會分泌到間質體和培養液中，降低Xyn45-His6蛋白堆積於菌體中形成包涵體。利用NTA親合管柱結合Xyn45-His6並純化此蛋白。分析Xyn45-His6物化特性，酵素具有寬廣的pH值活性，於50℃下最適pH值為9.0。Xyn45-His6酵素屬熱穩定蛋白，將此蛋白於50℃下預培養24小時候，仍可偵測到與初始酵素相同的活性能力，且於72℃下可偵測到最高活性溫度。Xyn45-His6酵素對樺木木聚醣為基質的酵素動力分析，於 50℃與pH 9的條件下，Km為 0.52 ± 0.06 mg ，而 Vmax 為 0.32 ± 0.01 μmole min-1 μg-1。利用HPLC (HTX87H) 分析Xyn45-His6酵素 (0.225 U) 與不同的受質 (樺木木聚醣、NaOH前處理的稻稈和NaOH前處理的玉米芯) 作用24小時後，分析其水解產物木寡糖之產量
。結果顯示主要產物為木二糖、木三糖、長鏈的木寡糖和少量的木一糖。其酵素將樺木木聚醣、NaOH前處理的稻稈和NaOH前處理的玉米芯的轉換成木寡糖能力高達75%。
將xyn23和 BPxynA也利用於大腸桿菌系統大量表現蛋白，但結果顯示兩個蛋白皆表現於包涵體。更換為pET29a為表現載體，並將xyn45 的 signal peptide置換Xyn23和BPxynA中的signal peptide，再利用大腸桿菌系
統大量生產蛋白。但結果顯示Xyn23-His6仍然在包涵體，且誘導BPxynA-His6 蛋白時，菌落數急速降低且酵素活性卻隨時間增加，因此推測BPxynA- His6蛋白或酵素活性對E. coli BL21 (DE3) 有嚴重的毒害效果。

關鍵字

木聚醣水解酶

並列摘要

An alkalophilic Bacillus halodurans and Bacillus pumilus NCCU produces two thermostable xylanases and one secretion xylanase, respectively. The full-length DNA fragments of three xylanases have been cloned, sequenced and the molecular weights of deduced open reading frames (ORF) were 45, 23 and 26 kDa, respectively. The ORF of 45, 23 and 26 kDa xylanases, order of designated xyn45, xyn23 and BPxynA was subcloned into pET29a expression vector to overproduce xyn45, xyn23 and BPxynA with C-terminal His6-tag in E. coli BL21 (DE3).
For Xyn45-His6, the best induction condition for protein overproduction was using 0.05 mM IPTG for 18 hr at 25℃. Over half of the Xyn45-His6 proteins were secreted into both periplasma and media which lower the concentration of Xyn45-His6 proteins and avoid the formation of inclusion bodies in cytosol. A Ni-affinity chromatography was used to purify Xyn45-His6 proteins. The enzyme was relatively stable over a broad pH range and the optimal pH was 9.0 at 50℃. The Xyn45-His6 protein was thermostable and retained the same enzymatic activity after preincubation at 50℃ for 24 hr and showed the best enzymatic activity at 72℃. The Km of Xyn45-His6 for birchwood xylan was 0.52 ± 0.06 mg and the Vmax was 0.32 ± 0.01 mmole min-1 μg-1 at pH 9 and 50℃. The production of xylo-oligosaccharides (XOS) was analyzed by HPLC (HTX87H), various substrates (2%), birchwood xylan, NaOH-pretreated rice straw or corn cobs were incubated with Xyn45-His6 (0.225U) at pH 9.0 and 50℃ for 24 hr. The main XOS produced by Xyn45-His6 on these substrates were xylobiose, xylotetriose, xylotetraose and larger XOS and a small amount of xylose. The enzymatic conversions of birchwood xylan, NaOH-pretreated rice straw or corn cobs to XOS were over 75%.
We also tried to use pET29a expression vector to overexpress Xyn23-His6 and BPxynA-His6 in E. coli, but the result showed that proteins were in inclusion
bodies. We substituted Xyn45 signal peptide to xyn23 and BPxynA signal peptides for protein overexpression, respectively. Unfortunately, Xyn23-His6 still formed in inclusion bodies. On the other hand, BPxynA-His6 could not be detected in coomassie blue stain. We discovered colonies numbers of E. coli overproduces BPxynA-His6 would decrease and these decreasing might be due to xylanase activity while we detected increase of xylanases activity progressively.

並列關鍵字

xylanase

參考文獻

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