选择互补且各具优势的2株米曲霉A98和A33为出发菌株,研究了其原生质体制备和电融合条件.接种YPD液体培养基,30℃摇瓶培养11h获得菌丝.以0.6mol/L甘露醇为渗透压稳定剂,最佳破壁酶液为溶壁酶∶蜗牛酶∶纤维素酶=1∶1∶1,浓度为10mg/mL,28℃酶解2.5h时原生质体浓度达到108个/mL,再生率≥20％；最佳电融合条件为1MHz交流频率,交流场强300V/cm,直流场强6kV/cm,脉冲间隔60μs,脉冲个数3;在此条件下,电融合率达到10-5.对获得的融合子进行酱油发酵筛选,以原料收得率为关键考察指标,筛选获得了1株融合子F01-008,其原料收得率较亲本较高者提高了5.61％.%Using Aspergillus oryzae A98 and A33 as the original strains, the conditions for their protoplast preparation and electronic fusion were studied in this paper. The mycelium were cultured in YPD liquid medium under 30"C for 1 lh. Mannitol was used as the osmotic stabilizer with 0.6mol/L. The optimal enzyme for protoplast preparations were the mixture of lywallzyme, snailase and cellulase, which were mixed at 1:1:1. Their concentrations were lOmg/ml. The protoplast concentration reached 10* per milliliter when hydrolyzed for 2.5h under 28tl. The regeneration rates were more than 20%. The optimal parameters for the fusion were as followed: alternating current (AC) frequency 1MHz, AC voltage, 300V/cm, direct current (DC) 6kV/cm pulse interval 60|isec and 3 pulses. On these conditions, the fusion frequency reached 10"5 per protoplast pair. Fusants were screened by soy sauce fermentation using material utilization ratio as the key factor. One fusant Fm-008 was obtained, and its material utilization ratio was 5.61% higher than that of their parent.
展开▼
