米曲霉3.951菌株与RIB40菌株高产原生质体的制备及再生条件的研究

Mass Preparation and Regeneration Conditions of Protoplast from Aspergillus oryzae AS 3.951 and RIB40

针对2株米曲霉进行原生质体制备条件的研究,比较了制备材料、菌龄、渗透压稳定剂、复合酶配比、酶解时间以及酶解温度对米曲霉原生质体形成和再生的影响。结果表明,2株米曲霉原生质体制备的最适宜条件稍有差异,其中米曲霉3.951菌株原生质体制备和再生的适宜条件是:以菌丝为制备材料,菌龄为14 h,渗透压稳定剂为0.8 mol/L NaCl,复合酶配比为1.0%纤维素酶、1.0%裂解酶、0.l%蜗牛酶,酶解时间为3 h,酶解温度为35℃;米曲霉RIB40的适宜条件是:以菌丝制备材料,菌龄为12 h,渗透要稳定剂为0.8 mol/L NaCl,复合酶配比是为1.0%纤维素酶、1.0%裂解酶、0.l%蜗牛酶,酶解时间为3 h,酶解温度是35℃。在优化条件下米曲霉3.951菌株释放的原生质体达6.43×107个/g,再生率可达23.5%,米曲霉RIB40释放的原生质体可达4.24×107个/g,再生率达22.41%。

Formation and regeneration conditions of protoplasts for two Aspergillus strains were optimized, the effect of material, cell age, osmotic stabilizer, enzyme proportion, enzyme acting time and acting temperature on the formation and regeneration of protoplasts from Aspergillus oryzae 3.951 and Asperigullus oryzae RIB40 were studied. The optimized conditions of A. oryzae 3. 951 were： mycelial as the preparation material and its age was 14 h, 0.8 mol/L NaCI as the osmotic stabilizer, enzyme solution has 1.0 % （w/v） cellulase, 1.0 % （w/v） lysing enzyme and 0.1% （w/v） snailase, enzyme acting time 3 h and enzyme acting temperature 35 ~C. The optimized conditions of A. oryzae RIB40 were the same as A. oryzae 3. 951 but a better age of its mycelial was 12 h. Under optimized conditions, A. oryzae 3. 951 released 6.43 x 107 protoplast/g with the regeneration rate of 23.5 % , and A. oryzae RIB40 released 4.24 x 107 protoplast/g with a regeneration rate of 22.41%.