本土多黏類芽孢桿菌PaenibacilluspolymyxaBs83 β-澱粉?之分離純化與特性分析

Purification and characterization of β-amylase from Paenibacillus polymyxa Bs83

吳源崧(Yuan-Sung Wu)

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

本研究自144株本土芽孢桿菌屬菌株 (Bacillus spp.)中，利用Yeast extract-soluble starch agar平板挑選出10株具有較佳澱粉?活性之菌株。接著挑選具有最高活性之菌株編號Bs83利用16S rDNA 以及BIOLOG 進行菌種鑑定後發現此菌株為多黏類芽孢桿菌(Paenibacillus polymyxa)，之後以硫酸銨沉澱、 DEAE sepharose FF與Superdex TM 200 10/300 GL層析管柱進行純化工作，進而得到一純化之β-澱粉?。經由SDS-PAGE電泳分析蛋白質，證實此澱粉?已被純化，其純化倍率為34倍，比活性為28.64 Unit/mg，回收率為0.15%，分子量約為66 kDa。接著利用膠體酵素活性電泳分析(zymogram)分析此澱粉?，其蛋白質活性位置大約在132 kDa，因此推測為同源二聚體。純化過後β-澱粉?水解可溶性澱粉後的反應生成物為麥芽糖(maltose)，可推測此酵素為外切形式的澱粉?。當溫度於50oC、pH值為7.0時發現此酵素具有最高的活性，於20-50oC時有較佳的熱穩定性，而在pH 4-8有較好的pH值安定性。

關鍵字：多黏類芽孢桿菌、β-澱粉?、外切型澱粉?

關鍵字

外切型澱粉? ； 多黏類芽孢桿菌 ； β-澱粉?

並列摘要

Among 144 local Bacillus spp. isolates screened, 10 candidates with higher amylase activity assayed with yeast-soluble starch agar plate methods were selected for further testing. Local isolate with highest activity was selected out and identifical by 16S rDNA and BIOLOG as Paenibacillus polymyxa. An extracellular β-amylase from P. polymyxa was purified by ammonium sulfate precipitation, DEAE sepharose FF anion exchange and SuperdexTM 200 10/300 GL gel filtration chromatography with a 34-fold of purification and 28.64 U/mg in specific activity and 0.15% recovery. The molecular weight of the purified β-amylase was estimated to be 66 kDa and 132 kDa by sodium dodecyl sulphate-polyacrylamide gel electorphoresis (SDS-PAGE) and zymogram, respectively. The action patterns of purified β-amylase on soluble starch were examined by TLC. Results suggested that the enzyme produced by P. polymyxa was exo-amylase. The optimal pH and temperature of the purified enzyme were 7 and 50oC, respectively. The purified enzyme thermal and pH stability were 20-50 oC and 4.0-8.0, respectively.
Keywords: Paenibacillus polymyxa、β-amylase、exo-amylase

並列關鍵字

β-amylase ； exo-amylase ； Paenibacillus polymyxa

參考文獻

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