利用Escherichia coli 和 Bacillus subtilis作為宿主來表現Bacillus sp. NCHU-5的幾丁聚醣酵素基因以及幾丁聚醣酵素在Rhizopus oryzae轉型上的應用

Abstract

Chitosan oligosaccharides of hexamer to decamer in chain length perform more potent antimicrobial, antitumor, and immunopotentiating activities, than shorter oligosaccharides. Chitosan digested with an endo-type chitosanase have the opportunity to obtain hexamer to decamer oligosaccharides. In this study chitosanase gene from Bacillus sp. NCHU-05 was cloned to expression vectors, and expressed in Escherichia coli NovaBlue or Bacllus subtilis WB800. Expressed recombinant chitosanases were purified by Ni-column chromatography. The molecular mass of the chitosanase was estimated to be 32 kDa by means of sodium dodecyl sulfate -polyacrylamide gel electrophoresis. Purified chitosanase established the optima temperature and pH value were with 40 ℃ and pH 5.6, respectively. The specific activities of recombinant chitosanases were 5231 U/mg from E. coli NovaBlue and 5823 U/mg from B. subtilis WB800. Production of chitosanases from E. coli NovaBlue were 150 mg/l, and from B. subtilis WB800 wre 100 mg/l. Purified chitosanase could be used in the preparation of R. oyzae protoplasts for gene transformation with transformation efficiency of 0.45 transformant/μg DNA.

Chitosan的水解產物往往具有生物活性。一般而言，分子量為hexamer ~ decamer的chitosan oligosaccharide，其抗菌、抗癌及調節免疫機能的能力高於分子量較小的oligochitosan。利用endo形式的 chitosanase進行水解，有機會獲得hexamer到decamer的oligosaccharide。本研究將Bacillus sp.NCHU-5的chitosanase基因分別選殖入表現載體並以Escherichia coli NovaBlue和Bacllus subtilis WB800為宿主進行chitosanase基因之表現。表現生產的重組的chitosanase，可利用 Ni-column純化。純化的chitosanase經sodium dodecyl sulfate -polyacrylamide gel electrophoresis電泳圖分析為32kDa。純化的chitosanase其最適反應溫度為40 ℃與最適反應pH為5.6。由重組E. coli NovaBlue純化的chitosanase比活性為5231 U/mg，而重組Bacllus subtilis WB800 純化的chitosanse活性為5823 U/mg。E. coli NovaBlue生產的chitosanase，每升產量為150 mg。B. subtilis WB800所生產的chitosnase，每升產量為100 mg 。純化的chitosanase也成功地利用在Rhizopus oryzae原生質體的製備與轉型，其轉型效率為0.45 transformant/μg DNA。