Helianti Is, Nurhayati Niknik, Ulfah Maria, Wahyuntari Budiasih, Setyahadi Siswa
Center for Bioindustrial Technology, Agency for Assessment and Application of Technology, Jl MH Thamrin no. 8, Jakarta, Indonesia.
J Biomed Biotechnol. 2010;2010. doi: 10.1155/2010/980567. Epub 2010 Sep 21.
A xylanolytic bacterium was isolated from the sediment of an aquarium. Based on the 16S rDNA sequence as well as morphological and biochemical properties the isolate was identified and denoted as Bacillus subtilis (B. subtilis) AQ1 strain. An endoxylanase-encoding gene along with its indigenous promoter was PCR amplified and after cloning expressed in E. coli. In E. coli the recombinant enzyme was found in the extracellular, in the cytoplasmic, and in the periplasmic fraction. The specific activity of the extracellular AQ1 recombinant endoxylanase after 24-hour fermentation was very high, namely, 2173.6 ± 51.4 and 2745.3 ± 11 U/mg in LB and LB-xylan medium, respectively. This activity was clearly exceeding that of the native B. subtilis AQ1 endoxylanase and that of 95% homologous recombinant one from B. subtilis DB104. The result shows that the original AQ1 endoxylanase promoter and the signal peptide gave a very high constitutive extracellular expression in E. coli and hence made the production in E. coli feasible.
从水族箱沉积物中分离出一株木聚糖分解细菌。基于16S rDNA序列以及形态学和生化特性，该分离株被鉴定并命名为枯草芽孢杆菌（B. subtilis）AQ1菌株。通过PCR扩增出一个编码内切木聚糖酶的基因及其天然启动子，并在克隆后在大肠杆菌中表达。在大肠杆菌中，重组酶存在于细胞外、细胞质和周质部分。24小时发酵后，细胞外AQ1重组内切木聚糖酶的比活性非常高，在LB培养基和LB-木聚糖培养基中分别为2173.6±51.4和2745.3±11 U/mg。该活性明显超过天然枯草芽孢杆菌AQ1内切木聚糖酶以及来自枯草芽孢杆菌DB104的95%同源重组酶的活性。结果表明，原始的AQ1内切木聚糖酶启动子和信号肽在大肠杆菌中实现了非常高的组成型细胞外表达，因此使得在大肠杆菌中的生产成为可能。
