本土多黏類芽孢桿菌Bs83β-葡萄糖??之分離純化與特性分析

Purification and characterization of β-glucosidase from Paenibacillus polymyxa Bs83

袁秀榕(Hsiu-Jung Yuan)

指導教授 ： 高穗生

朝陽科技大學/理工學院/應用化學系生化科技碩博士班/碩士(2010年)

摘要

本研究利用Mandel-Reese平板培養基自144株本土芽孢桿菌屬篩選具纖維分解酵素活性之菌株，挑選出9株具較佳纖維素水解能力之菌株。之後挑選具最高纖維分解酵素活性之菌株，利用16S rDNA及Biolog方法辨別其種名，鑑定結果為多黏類芽孢桿菌(Paenibacillus polymyxa)。利用硫酸銨沉澱方法和離子交換層析純化出β-葡萄糖??，其純化倍數為108.71，回收率為5.08 %。以SDS-PAGE電泳分析純化之β-葡萄糖??分子量約為52 kDa。純化之β-葡萄糖??能水解纖維雙醣(cellobiose)、纖維三醣(cellotriose)和纖維五醣(cellopentaose)，但對結晶纖維素(Avicel)、木聚糖(xylan)、氫氧基乙基纖維素(hydroxyethyl-cellulose)、甲基纖維素(methyl-cellulose)、羧甲基纖維素鈉(carboxymethyl-cellulose sodium salt, CMC)、昆布多糖(laminarin)、地衣澱粉(lichenan)及葡聚糖(curdlan)皆無水解活性。純化後酵素在溫度35oC及pH 6.5有最佳之酵素活性；而在20oC~35oC時溫度穩定性最佳及在pH 3-pH 10時pH穩定性最佳。利用薄層層析(TLC)分析純化酵素與纖維寡醣作用後之生成物，依據其結果推論從多黏類芽孢桿菌(P. polymyxa)純化出的酵素應為β-葡萄糖??。

關鍵字

β-葡萄糖?? ； 纖維分解酵素 ； 多黏類芽孢桿菌

並列摘要

One hundred and fourty four local Bacillus spp. isolates were screened for their cellulase activity by Mandel-Reese agar plate. Nine isolates with higher cellulolytic activity were selected for further testing. Local isolate Bs83 with the highest cellulase activity was selected and identified as Paenibacillus polymyxa by analysis of 16S rDNA and Biolog. The β-glucosidase was purified 108.71-fold by ammonium sulphate precipitation and ion exchange chromatography, with an overall recovery of 5.08 %. With the analysis of SDS–PAGE, the molecular weight of the purified β-glucosidase was estimated to be 52 kDa. The purified β-glucosidase can hydrolyze cellobiose, cellotriose and cellopentaose, but not Avicel, xylan, hydroxyethyl-cellulose, methyl-cellulose, carboxymethyl-cellulose sodium salt (CMC), laminarin, lichenan and curdlan. Optimal temperature and pH for the purified enzyme activity were determined to be 35oC and 6.5, respectively. The thermal and pH stability of purified enzyme were 20oC -35oC and 3.0-10.0, respectively. The action patterns of purified enzyme on cellooligosaccharides were examined by thin-layer chromatography (TLC). Results suggested that the enzyme produced by P. polymyxa was β-glucosidase.

並列關鍵字

β-glucosidase ； cellulase ； Paenibacillus polymyxa

參考文獻

陳威廷，「纖維素水解菌之培養策略與纖維素水解酵素之鑑定」，碩士論文，國立成功大學化學工程學系，台南 (2004)。

Ash, C., Farrow, J. A. E., Wallbanks, S., and Collins. M. D.,“Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences,” Lett. Appl. Microbiol., Vol. 13, pp.202-206 (1991).

Ash, C., Priest, F. G., and Collins, M. D.,“Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks, and Collins) using a PCR probe test,” Antonie van Leeuwenhoek, Vol. 64, pp.253-260 (1993).

Beguin, P., and Aubert, J. P.,“The biological degradation of cellulose,”FEMS Microbiol. Rev., Vol. 13, pp.25-58 (1994).

Bellamy, W. D.,“Single cell proteins from cellulosic wastes,”Biotechnol. Bioeng.,Vol. 16, pp.869-880 (1974).