"神舟"4号空间飞行对搭载的转谷氨酰胺酶链霉菌选育的影响

Effect of "Shenzhou" 4 Spaceflight on Screening of Microbial Transglutaminase-producing Streptomyces Mutants

目的利用"神舟"4号无人飞船搭载生产微生物转谷氨酰胺酶(MTG)的链霉菌株,进行工业实用性微生物菌种的空间诱变育种研究,定向选育高产酶突变菌株。方法将产酶菌株链霉菌WZFF.L-M168(2.60 u/ml)以孢子和培养基中添加或不添加诱变剂亚硝基胍(NTG)的斜面培养形式搭载飞船后。采用蛋白质交联絮凝-沉淀实验方法初筛出正突变菌株,再采用摇瓶发酵产酶分析实验进行复筛驯化实验。结果实验首先发现在各种宇宙空间环境因素的综合作用下,搭载菌株的菌落形态发生明显变化,特定形态与产酶能力有一定正相关性。根据菌落形态特征从各搭载试管组中分离的733搭载突变株经系列选育驯化后,得到一系列MTG生产能力显著提高的优良搭载突变菌株。其中,最高产酶菌株在利用小型生化反应器进行发酵培养的产酶效果良好,酶活提高了40％以上,达到3.62 u/ml,超过现有国际最高水平。结论将产MTG菌种搭载"神舟"4号飞船,并进行严格的菌种驯化选育,获得了具优异性能的高产酶搭载突变株,表明空间诱变育种效果显著。

Objective To isolate the effective Streptomyces strains producing microbial transglutaminase (MTG) through space breeding techniquely loading microbial strains in 'Shenzhou' 4 spacecraft and screening productive mutants. Method Streptomyces sp. WZFF. L-M168 2.60 u/ml was selected by loading in the forms of spores and slant mycelia cultured with or without nitrosoguaidine as a mutagenic reagent. Result The systematic screening experimental results first showed that significant morphological changes of colonies caniul had occurred because of the complex effects of several space environmental factors, and that some of the typical changes had a direct relationship with the enzyme productivity. These properties were then used to isolate total 733 mutant strains, and they were screened for the purpose to increase the enzyme producing capacity by the method of directive breeding through the test of flocculation-precipitation of proteins by MTG cross-linking as the primary screening, and the successive examination test of MTG production through shaking culture as the secondary screening. Many potent mutant strains with high MTG activities were selected out. Among them, three special high efficient mutants also with strong stabilities of genetic characteristics could continuously give enzyme production of MTG higher than 3.5 u/ml, which showed a significant increase of 40 % higher than that of the original strain,and higher than the best up-to-date value reported. Conclusion These results demonstrate that the space breeding technique by loading in a spacecraft is effective.