电融合法选育高温高产二羧酸酵母菌的研究

STUDIES ON SCREENING THERMOTOLERANT HIGH-YIELDING DIOIC ACIDS YEAST BY ELECTROFUSION

研究了热带假丝酵母1045的两个突变株HYU_m和SD_1的原生质体电融合及其在高温下高产二羧酸菌株的选育。利用灭活的高温产酸HYU_m原生质体和28℃产酸、高温丧失产酸功能的SD_1原生质体,在1.2MHz、800V/cm高频交变电场下,施加脉冲宽度为25μs,强度6～12kV/cm,脉冲个数3、5或10,间隔0.5s的电脉冲,获得48株融合子,表观融合率为(0.95～5.05)×10^(-4)。对融合子进行菌落形态、拮抗、酯酶同工酶、DNA含量实验。筛选出高温产DC_(12)为亲株HYU_m的1.43倍、SD_1的3.4倍的融合菌株2株。还提出了用超声处理测定细胞DNA含量的新方法。

The protoplast electrofusion of mutant HYU_m and SD_1 of candida tropicalis 1045 and screening thermotolerant high-producing dioic acids fusants were studied. Inactivated thermotolerant HYU_m protoplasts were fused with protoplasts of unthermotolerant SD_1; 48 fusants were obtained under 39±0.5℃. Protoplasts were aligned in pearl chains in a A. C. fieldwith a frequency of 1.2 MHz and a field strength of 800 V/cm. Fusion was irriated by application of 3, 5 or 10 pulses, duration 25μs with intervals of 0.5s, and a field strength of 6～12kV/cm, having apparant fusion frequency of 0.95×10^(-4)～5.05×10^(-4). Colonies morpha, antagnistic action, esterase izozyme and DNA contents were determined. Two of the fusants producing DC_(12) more 0.43 times than HYU_m and 2.4 times than SD_1 were screened out by fermentation. A new simple and convenient DNA contents testing method, ultrosonic treatment method, was applied in this paper.