Aspergillus oryzae与Aspergillus niger原生质体的制备、再生与非对称灭活工艺研究

为提高菌丝原生质体的释放量和活力，分别考查了培养基碳源、菌龄、酶浓度、酶解时间及再生培养基种类对Aspergillus oryzae和Aspergillus niger 原生质体释放和再生的影响，并研究了两亲本原生质体的非对称灭活参数。结果表明:（1）以MPY液体培养基为菌丝培养基，按105个孢子/ml培养基的接种量接入新鲜孢子悬液，30 ℃/100 rpm分别振荡培养18和21 h，总浓度15 mg/ml的复合酶液（溶壁酶：蜗牛酶：纤维素酶＝1:1:1）按1 g湿菌丝球/10 mL酶液的比例，于30 ℃/70 rpm条件下酶解2.5 h，然后以高渗豆汁固体培养基为再生培养基，在上述制备工艺参数下两亲本原生质体的释放量和再生率均可达到107个/ml和30％以上；（2）通过灭活曲线确定A. oryzae HN3042原生质体于15 W紫光灯垂直距离13 cm处照射15 min，A. niger CICC2377原生质体于65 ℃水浴10 min，此灭活剂量可使99％以上双亲原生质体非对称失活。

Release, regeneration and asymmetric inactivation of protoplasts from Aspergillus oryzae and Aspergillus niger

To enhance the quantity and viability of protoplasts released from mycelia of Aspergillus oryzae and Aspergillus niger, the effects of carbon resource, mycelia age, enzyme concentration, incubation time and regeneration medium on release and regeneration of protoplasts were investigated and the parameters for asymmetric inactivation were also examined. The results showed that: (1) the optimal conditions for protoplast preparation were as follows: cultivation in a submerged medium consisting of 1.0 % maltose, 1.0 % polytephone and 0.5 % yeast extract for 18 and 21 h at 30 ℃ and 100 rpm, and then incubating the mycelia with an enzyme cocktail, total concentration of 15 mg/ml, consisting of lywallzyme, cellulose R-10 and snailase with the ratio of 1:1:1, for 2.5 h at 30 ℃ and 70 rpm, and then regeneration on the high osmotic soybean extract solid medium. Under these conditions for protoplast preparation, the release and regeneration efficiency of protoplasts from A.oryzae HN3042 and A.niger CICC2377 reached 107 /mL and above 30%, respectively; (2) according to the time curse for asymmetric inactivation, the dose of UV irradiation treatment with a 15 W ultraviolet lamp to A. oryzae protoplasts was 15 min under the light with distance of 13 cm and for the heat treatment towards A. niger the parameter was for 10 min at 65 ℃ water incubation.