共表达透明颤菌血红蛋白提高脂肪酶在毕赤酵母中的表达.pdfVIP

现代食品科技 Modern Food Science and Technology 2014, Vol.30, No.6 共表达透明颤菌血红蛋白提高脂肪酶在 毕赤酵母中的表达 卢俊裕，林影，梁书利，叶燕锐 （华南理工大学生物科学与工程学院，广东广州 510006） 摘要：为了克服巴斯德毕赤酵母高密度发酵过程中的溶氧限制，提高外源蛋白的表达量，本文以启动子YPT 介导透明颤菌血红 蛋白（VHb）在培养过程中组成型表达。同时以启动子AOX1 介导的南极假丝酵母脂肪酶B （CALB）基因作为报告基因，使CALB 在甲醇诱导阶段表达。另外，还将绿色荧光蛋白在过氧化物表面表达，将红色荧光蛋白在过氧化物基质中表达以检测信号肽能否定位 在相应的位置。最后，将VHb 分别在过氧化物酶体表面和基质两个位置表达，以观察表达位置的不同所产生的影响。通过SDS 和Western Blot 分析表明，CALB 和VHb 在重组菌中成功实现了共表达。在限氧条件下，重组菌株GS115/ExVHb 、GS115/VHbSKL 的外源蛋白CALB 的表达量比对照菌株GS115/CALB 分别提高了27.57%和20.52%。这说明VHb 与外源基因共表达可以改善酵母在 发酵过程中的摄氧情况，提高外源蛋白的表达量。 关键词：透明颤菌血红蛋白；脂肪酶；氧限制；毕赤酵母；分泌表达 文章篇号：1673-9078(2014)6-110-115 Expression of Vitreoscilla Hemoglobin Improves CALB Production in Pichiapastoris LU Jun-yu, LIN Ying, LIANG Shu-li, YE Yan-rui (College of Biological Science and Engineering, South China Universityof Technology, Guangzhou 510006, China) Abstract: To alleviate the dissolved oxygen (DO) limitation and improve Candida antarctica lipase B (CALB) production during high-cell density fermentation, the CALB gene and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichiapastoris under the AOX1 and YPT promoter control, respectively. In addition, to detect the effect of signal peptide, GFP was expressed on the surface of peroxide, while RFP was expressed in the peroxide matrix. Finally, GFP or RFP was replaced by VHb protein to f ind out the impaction of the different positions. SDSand Western Blot analysis results indicated that VHb and CALB were successfully co-expressed in recombinant strains. Compared with the control strain, expression of CALB in GS115/ExVHb and GS115/VHbSKL under oxygen limitation was improved by 27.57% and 20.52%, respec