枯草桿菌J1 α-L-阿拉伯糖苷酶的特性

Characterization of α-L-arabinofuranosidase (abfA) Gene from Bacillus subtilis J1

艾思亞(Nani Aisyah Putri)

指導教授 ： 陳又嘉博士

國立屏東科技大學/農學院/生物科技系/碩士(2020年)

https://doi.org/10.6346/NPUST202000374

摘要

植物細胞壁的降解需要幾種酶活性的作用。 α-L-阿拉伯呋喃糖苷酶（AbfA）作為脫支酶具有降解農業廢棄物中半纖維素的能力。該酶與木聚醣酶和木糖苷酶協同作用，以切割木聚醣上阿拉伯呋喃糖苷的側鏈。枯草芽孢桿菌是一種能夠產生AbfA的細菌，但是這種酶的活性仍然難以捉摸，需要深入研究。利用大腸桿菌生產重組酶是生產和鑑定枯草芽孢桿菌J1 AbfA的有前途的方法。從枯草芽孢桿菌J1基因組克隆了abfA，並將其連接到pET21a（+）質粒中。大腸桿菌BL21（DE3）用於產生和表徵添加礦物質以增加其溶解度的異丙基β-D-1-硫代吡喃半乳糖苷（IPTG）的最佳條件。該酶的最佳條件是在35℃的檸檬酸緩衝液中的pH 6.5，在pH 5-8範圍內穩定。該酶的分子量約為59.7 kDa。該酶對p-硝基苯基-α-L-阿拉伯呋喃糖苷（pNPA），阿拉伯木聚醣和阿拉伯聚醣底物具有活性，並且AbfA與Xyn的混合酶增加了阿拉伯木聚醣和木聚醣底物中的還原糖

關鍵字

α-L-阿拉伯呋喃糖苷酶 ； abfA ； 枯草芽孢桿菌 ； 基因克隆

並列摘要

The degradation of the plant cell wall requires the action of several enzyme activities. α-L-arabinofuranosidase (AbfA) displays ability as a debranching enzyme to degrade hemicellulose from agricultural waste. This enzyme works synergistically with xylanase and xylosidase to cut the side chain of arabinofuranose on xylan. Bacillus subtilis is one bacterium that is capable to produce AbfA, but the activity of this enzyme remains elusive and need to be studied profoundly. The production of recombinant enzymes by utilizing E. coli is a promising way to produce and characterize B. subtilis J1 AbfA. The abfA was cloned from B. subtilis J1 genome and ligated in pET21a(+) plasmid. E. coli BL21 (DE3) was used to produce and characterize the optimum condition of isopropyl β-D-1-thiogalactopyranoside (IPTG) induction with mineral addition to increase its solubility. This enzyme optimum condition was in citric acid buffer pH 6.5 at 35 ºC. It was stable in a broad pH ranges 5–8 and inactive in temperature above 55 °C. The enzyme had a molecular weight of approximately 59.7 kDa. This enzyme was active against para-nitrophenyl-α-L-arabinofuranoside (pNPA), arabinoxylan and arabinan substrate, and the mixture enzyme of AbfA with Xylanase has increased the reducing sugar in arabinoxylan and xylan substrate

並列關鍵字

α-L-arabinofuranosidase ； abfA ； Bacillus subtilis ； gene cloning

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