【作者】 熊欢；

【导师】 陈守文；

【作者基本信息】 华中农业大学 ， 发酵工程， 2008， 硕士

【摘要】 聚γ-谷氨酸（polyγ-glutamic acid;γ-PGA）是一种应用前景良好的生物高分子材料。地衣芽胞杆菌（Bacillus licheniformis）WX-02是γ-PGA的产生菌,其发酵液由于较粘稠而导致溶氧很差,溶氧成为γ-PGA产量提高的限制性因素之一。透明颤菌血红蛋白（VHb）是一种来源于原核生物的氧结合蛋白。本研究旨在B.licheniformisWX-02中表达透明颤菌血红蛋白基因（vgb）来改善其发酵过程中氧的利用效率,进而改善菌体生长,提高产量。本研究构建了能整合于芽胞杆菌染色体上的表达载体pDG1730-vgb,其含有芽胞杆菌α-淀粉酶基因的整合位点,通过Spizizen感受态转化导入B.licheniformisWX-02中,PCR验证表明vgb成功整合到其染色体上;经CO差光光谱分析,重组菌株B.licheniformis M2有VHb表达活性;摇瓶发酵实验结果显示,摇床转速为180r/min,发酵36 h的条件下,250 mL三角瓶中发酵液装液量为50 mL时,M2的生物量和γ-PGA产量分别为4.5 g/L和27.71 g/L,高于出发菌株WX-02 6.43%和6.74%;装液量为100 mL时,M2发酵的生物量和γ-PGA产量分别为4.17 g/L和26.59g╱L,高于出发菌株WX-02 15.02%和21.7%。vgb的表达促进了菌体和γ-PGA产量的增长,产物比生物量的增长明显,促进效果随着供氧条件的降低而增加。3 L发酵罐分批发酵结果显示,搅拌转速为600 r/min,通气量为2 L/min时,M2的生物量(OD₆₀₀)达到7.39,比出发菌株WX-02提高了25.5%,γ-PGA产量达到33.5g╱L,比WX-02提高了20%,进一步证实VHb蛋白的表达对γ-PGA产量和菌体生长的促进作用。整合载体pDG1730-vgb在高产γ-PGA的枯草芽胞杆菌W003中的整合表达已有初步结果,本研究还构建了另一个整合载体psacB-vgb,含有枯草芽胞杆菌果聚糖酶基因（sacB）的整合位点,其在高产γ-PGA的枯草芽胞杆菌W003中的整合表达有待进一步研究。更多还原

【Abstract】 Polyγ-glutamic acid(γ-PGA) is a biopolymer material that has potential applications in a wide range of industry and agriculture.Bacillus licheniformis WX-02 is a kind of areobacterium that can produceγ-PGA,the accumulation ofγ-PGA in the culture medium causes a significant viscosity increase that reduces dissolved oxygen(DO) in the fermentation broth.As a result,DO becomes one of the restricted factors that affects the increase ofγ-PGA yield and biomass.Vitreoscilla hemoglobin(VHb) is an oxygen-binding proteins of prokaryotic origin.This study was carried out to improve the transmission of oxygen by expression of Vitreoscilla hemoglobin gene(vgb) in Bacillus licheniformis WX-02 in order to promote synthesis ofγ-PGA and cell growth.An homologous recombination expression vector pDG1730-vgb which contains theα-amylase gene(amyE) for chromosomal intergration,was introduced into Bacillus licheniformis WX-02 by Spizizen natural genetic transformention,PCR analysis indicated that vgb has been successfully intergrated into the chromosome;CO binding difference spectrum analysis revealed the recombinate strain Bacillus licheniformis M2 has expressed Vitreoscilla hemoglobin with bio-activation;Shask flask fermentation experiments preliminarily showed that:the strains was cultivated at 37℃,180 r/min for 36 h,the biomass andγ-PGA yield of M2 were 4.5 g/L and 27.71 g/L,which were 6.43%and 6.74%higher than those of WX-02 when 50mL culture broth was in the 250 mL flask, comparatively the biomass andγ-PGA yield of M2 were 4.17 g/L and 26.59 g/L,then 15.02%and 21.7%higher than WX-02 with 100mL culture broth in the 250 mL flask, these results demonstrate the biomass andγ-PGA yield was improved by the vgb expression,and the increase on the latter were more obvious.The improve effects were enhanced following the oxygen supply decreased.It was shown by the results of batch cultures in a 3 L bioreactor that biomass andγ-PGA obtained in the recombinant M2 were 25.5%and 20%higher than those of the control respectively when agitation speed was 600 r/min and ventilation volume was 2 L/min,which further comferms the positive promotion effects of VHb on cell growth andγ-PGA producing.Another integrative expression vector psacB-vgb which contains the levansucrase gene(sacB) for chromosomal intergration was constructed,it will be integrated into the chromosome of Bacillus subtilis 003 in the next experimentation.更多还原