摘要
本文将赖氨酸产生菌——北京棒杆菌1134株进行标记,同时将枯草芽孢杆菌BR151菌株进一步标记后,进行科间原生质体融合的探讨,揭示了科间原生质体融合的可能性。经适宜的条件培养和预处理,再在适宜的条件下,酶解制备亲本株的原生质体,用PEG(MW6000)为聚合剂,经低温短时间处理,将1134衍生株与151衍生株的原生质体进行融合;并在改良的DM_3平皿上加入适量的细胞壁碎片(作引物)与适量的人血清白蛋白制剂,使其再生,结果棒杆菌(在酶解后2.5h.)与枯草芽孢杆菌(酶解后20min)的制备率均已达到99.98％以上,再生率分别达21.3％与90.8％以上。经适温培养48h后,从融合平皿上随机挑出若干个菌落。点种于CM母平皿上,再分别影印于10套选择培养基平皿上,进行鉴定,其融合率达到了3.9×10^(-4)。从菌落形态和菌体形态上可大体分为近1134类型,近151类型和中间类型三种。经连续传代五代后,从融合子中筛选山了几株在摇床上以甜菜糖蜜为原料。产酸水平捉高近一倍的稳定高产融合子。其中Q4413株的产酸率平均达6.56％,10立升小罐发酵产酸率6.52％,糖酸转化率为40.25％,经进一步验证,该菌株的产酸能力和遗传性均是稳定的,为远缘工业微生物育种开辟了新的可行途径。
ln this paper, the possibility of protoplast fusion of interfamilial bacteria was studied through the protoplast fusion between Corynebacterium pekinense 1134 producing lysine and Bacillus subtilis BR151 after marking respectively. After the proper culture and pretreatment to the cells, the two parental protoplasts were prepared in the improved HM solution under suitabie conditions, and fused by the treatment with PEG (MW 6000) under low temperature, and then regenerated on solid medium DM_3 plus appropriate human serum albumin and regeneration primers of cell wall. The results obtained showed that the protoplast preparation rates of C. pekinense 1134 (exposed to lysozyme for2. 5h.) and B. subtilis BR 151 (for 20min.) were both above 99.98%, and the regeneration rates about 21.3 and 90.8% respectively. A certain number of colonies picked randomly frcm the fusion plates after the culture under suitable temperature for 48h. were dibbled on the GM to prepare the master plates, and following incubation 10 sets of selection plates were replicated with the master plates separately for the identification. According to the statistics and the calculation, the fusion rate was 3.9×10^(-4). A few stable high—yield fusants which used sugar beet molasses with lysine output being twice as much as of parent 1134 were selected from more than one thousand fusants having grown in succession for 5 generations. Among them, the producing lysine rate of the fusion strain 04413 avcraged 6.56 and 6.52% in shake flask and small fermenter (10L) respectively, and the transformation rate from sugar to lysine was 40.25%.
出处
《微生物学杂志》
CAS
CSCD
1991年第2期29-34,共6页
Journal of Microbiology
关键词
科间
原生质体融合
细菌
Bacillus subtilis
Corynebacterium pekinense
Interfamilial protoplast fusion
