原生质体融合技术构建高效驱油细胞工程菌的研究

Construction of Cell Engineering Bacteria for MEOR through Protoplast Fusion

在采用原生质体融合技术构建MEOR用细胞工程菌的实验研究中 ,所用一种亲本菌为耐温度 72℃、耐 30 %NaCl、耐酸碱度 pH 5～ 9.4、可利用原油为碳源代谢生物表面活性剂的芽孢杆菌属菌Ⅰ ,另一种为可在 30℃利用糖蜜代谢水不溶性多糖聚合物的肠内杆菌属菌JD。以 1mg/mL溶菌酶处理处于对数生长后期的两株亲本 12 0min ,原生质体形成率 >95 %。融合率 >4 .5 % ;将得到的融合子进行许多次传代培养优选 ,最终获得 9株遗传性状稳定、代谢多糖性能优异的融合菌 ;其中有代表性的融合子FH9 17,代谢多糖的温度由 30℃提高到 4 5℃ ,盐度由3%NaCl提高到 10 %NaCl,pH范围由 6～ 9.5扩大到 6～ 10。表 3参 9。

In the purpose of constructing cell engineering bacteria for MEOR by the way of protoplast fusion, a thermophilic (72℃), salt tolerant (up to 30% NaCl), acid- and alkaline-resistant (pH range 3.5-9.4) bacterium Bacillus sp, notated as bacterium Ⅰ and producing biosurfactant on petroleum hydrocarbons and a bacterium Enterobacter sp, notated as bacterium JD and producing water insoluble microbial polysaccharide on molasses at 30℃ are taken as parent strains. The two parent bacteria at the later logarithmic phase of their growth are treated by 1 mg/mL lysozyme solution in 120 min and more than 95% bacterial cells are broken and more than 4.5% cells are fused; cultivation of these fusants generation after generation and strain screening result in obtaining 9 strains of bacteria-fusants with stabilized genetic characteristics and exellent polysaccharide producing capacity; a representative fusant, FH9-17, metabolizes polysaccharide at higher temperature of 45℃ instead of 30℃ in media of salinity of 10% NaCl instead of 3%NaCl and of wider pH range 6-10 instead of 6-9.5.