藉由error-prone PCR改良endo-1, 4-β-xylanase之抗熱性

Improvement of thermostability of endo-1, 4-β-xylanase by error-prone PCR

陳德祥(Te-Hsiang Chen)

指導教授 ： 黎耀基

國立清華大學/生命科學院/生物科技研究所/碩士(2007年)

https://doi.org/10.6843/NTHU.2007.00572

摘要

木聚醣水解酵素目前被廣泛地應用在領域中，包括了食品工業、畜牧業、造紙以及紙漿工業等等，並且仍被認為尚有許多應用的潛力。而在其眾多不同的類型中，尤其以endo-1, 4-β-xylanase最為被廣泛應用也最被看好其潛力。於之前的研究中我們從製漿漂前黑液(58℃，pH 9.83)中分離出耐高溫高鹼的Paenibacillus sp. (Isolate BL11)。該菌株擁有分泌endo-1, 4-β-xylanase的能力，此酵素被命名為xylanase X。在最佳反應條件下(60℃、pH 7)xylanase X具有2392 IU/mg的酵素活性。本實驗的目的為利用error-prone PCR建立xylanase X的library，並且篩選出擁有更強抗溫能力的突變株，以符合工業應用的條件。最終我們分別得到了木聚醣分解能力上升的胺基酸取代株pXcm-H1(T44A)及後段缺失株pXcm-L1(胺基酸序列270-290因base deletion而由TSTHSFSLRGASNNANMARVD轉變為 LALIVSHCGGRRTMPTWPGWT)。pXcm-H1在67.5℃至70℃擁有原本xylanase的兩倍活性；而pXcm-L1在67.5℃至70℃擁有原本xylanase的三倍以上活性。且還篩選到了和pXcm-H1突變在同一胺基酸卻導致抗溫能力下降的pXcm-M2(T44M)。由此我們推論：第44號胺基酸Thr在xylanase X中可能是影響其抗高溫能力的關鍵胺基酸。

關鍵字

木聚醣水解脢 ； 抗熱性

並列摘要

Xylolytic hydrolases are generally applied in many kinds of fields, including food industry, animal husbandry, paper and pulp industry and are considered still very potential for application. In industry endo-1, 4-β-xylanase is mostly applied enzyme among the xylolytic hydrolases, and its potential is desirously expected. In our last research, we have isolated a thermo-alkaline Paenibacillus sp. (Isolate BL11) from black liquor (58℃, pH 9.83). The bacterium can secret an endo-1, 4-β-xylanase, named xylanase X. At optimal reaction condition (60℃ pH 7), xylanase X demonstrates an enzyme activity of 2392 IU/mg. The research goal of this experiment is to create a mutagenesis library of xyl X using error-prone PCR and isolate mutants of improved thermostability by screening the library for industry application. At the end of this experiment we have got two activity-improved mutants, an amino acid substitution mutatant pXcm-H1(T44A) and a downstream deletion mutant pXcm-L1(amino acid sequence 270-290 from TSTHSFSLRGASNNANMARVD changed to LALIVSHCGGRRTMPTWPGWT by a nucleotide deletion). Between 67.5℃ and 70℃ pXcm-H1 showed twice activity then xylanase X whereas pXcm-L1 had triple activity. Besides, we have got a thermostability-decreased mutatant pXcm-M2(T44M). The mutant carries a different amino acid substitution at the site of pXcm-H1, but its thermostability decreased distinctly. According to this result, we suggest that the Thr-44 is a critical amino acid affecting the thermostability of xylanase X.

並列關鍵字

endo-1, 4-β-xylanase ； thermostability

參考文獻

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