利用基因重組大腸桿菌以大量表現來自嗜鹼性Bacillus halodurans的內切型木聚醣酶

Over-Expression of Endoxylanase from Alkaliphilic Bacillus halodurans in Recombinant Escherichia coli

薛琮翰(Tsung-Han Hsueh)

指導教授 ： 李文乾

國立中正大學/工學院/化學工程學系/碩士(2014年)

摘要

本研究利用含有質體pET29a之重組大腸桿菌E.coli BL21 以不同誘導條件過量表現xyn45基因以分泌內切型木聚糖水解酶 (endo-1,4-β -xylanase; Xyn45) 到胞外。實驗上利用不同培養基 (LB、PM medium)、誘導劑種類 (IPTG、Lactose)和濃度高低，在搖瓶中比較Xyn45在胞內及胞外的蛋白質量及活性，並以饋料高密度發酵進行大量表達。在培養基的比較上， Xyn45在PM medium分泌至胞外的活性比起LB高出1.31倍。在誘導劑的比較上，低濃度IPTG誘導產生的Xyn45活性為Lactose的5.69倍，在誘導蛋白活性上IPTG皆優於Lactose。在誘導濃度的比較上，胞外Xyn45於PM以低濃度0.05 mM IPTG誘導比起高濃度1 mM，活性提升了5.96倍。在胞內及胞外的Xyn45活性比較上，以高濃度IPTG誘導時，胞外的Xyn45活性與胞內活性相近；而以低濃度IPTG誘導時，胞外的Xyn45活性比胞內活性高出4.4倍。推測以低濃度緩和的方式誘導，可提升Xyn45摺疊及生成融合蛋白的正確性，進而提升活性。在搖瓶小量培養時，最佳誘導條件為：PM中0.05 mM IPTG誘導，可得胞外最佳活性8.2 ± 2.9 U/ml及81.7 ± 14.9 U/mg，而胞外Xyn45占總分泌蛋白質之20.8%。大量培養則進行兩組不同誘導濃度之高密度發酵：第一組在PM中以IPTG 0.05 mM/OD600 (final conc. = 1.77 mM) 誘導，可得胞外Xyn45活性為11.7 U/ml (胞外與胞內活性相近)，OD600測得值為47.8，胞外Xyn45占總分泌蛋白質之37.4 %；第二組在PM中以 IPTG 0.05 mM (final conc. ) 誘導，可得胞外Xyn45活性為18.4 U/ml (胞外比胞內活性高出4.38倍)OD600測得值為62.67，而胞外Xyn45占總分泌蛋白質之37 %。以鹼液萃取麥麩所得之半纖維素，其中arabinose與xylose的比值為0.52，所含的木醣及阿拉伯醣總合約為16~17%，由麥麩原料所能萃取出的xylan比率為90.2%。

關鍵字

高密度發酵 ； 大腸桿菌 ； 酵素

並列摘要

Extracellular endo-1,4-β-xylanase encoded by a gene xyn45 from Bacillus halodurans was over-expressed in recombinant Escherichia coli BL21 (DE3) using plasmid pET29a as the vector. The influence of culture medium (LB or PM medium) and concentration of inducer (IPTG or lactose in different concentration levels) on the production of this extracellular endoxylanase was studied. Using low concentration of IPTG as the inducer, the total activity of endoxylanase was higher in PM medium than that in LB medium. As the cultivation was in PM medium, the use of IPTG led to 5.7 times of enzymatic activity compared with the use of lactose as the inducer. Also, the use of low concentration (0.05 mM) of IPTG resulted in much higher endoxylanase activity in extracellular medium than the use of high concentration (1 mM) of IPTG. In PM medium containing 0.05 mM IPTG for induction, an activity of 8.2 ± 2.9 U/ml in the medium with a specific activity of 81.7 ± 14.9 U/mg was obtained. Furthermore, about 20.8% of over-expressed endoxylanase was found to be extracellular protein. The low concentration of inducer also resulted in a higher ratio of extracellular to intracellular activities. The recombinant E. coli was also cultivated to high-cell-density in fed-batch fermentation in PM medium and IPTG was used to induce the synthesis of extracellular endoxylanase. For large scale fermentation, PM medium containing IPTG with 0.05 mM/OD600 (final concentration = 1.77 mM) was used. As results, activity of extracellular Xyn45 was 11.7 U/ml (similar to activity of intracellular), OD600 = 47.8, and the yield of Xyn45 in total extracellular proteins is 37.4%. In the second group, PM medium containing IPTG 0.05 mM (final concentration) was used, the activity of extracellular Xyn45 was 18.4 U/ml (4.38 times higher than intracellular), OD600 = 62.67, and the yield of Xyn45 in total extracellular proteins is 37%.

並列關鍵字

enzyme ； HCDC ； E.coli

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