Establishment of a High-Throughput Screening Method for Rhizomucor miehei Lipase Production by A. oryzae
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摘要: 为了快速、高效地筛选出高产米黑根毛霉脂肪酶（RML）的米曲霉菌株，利用常温常压等离子体诱变技术（ARTP）对米曲霉孢子进行诱变，优化了高通量检测RML酶活的双指示剂法，建立了24孔板米曲霉高通量培养体系。通过对诱变后的1200孔（36000~48000个孢子）进行高通量筛选，最终筛选出4株米曲霉高产突变株。经由摇瓶验证，突变株Ⅰ7-D6-7、Ⅱ4-4B-5、Ⅲ3-5C-1、Ⅴ7-6C-3酶活分别达到了265.98、240.90、253.52、293.50 U/mL，比出发菌株的酶活（176.52 U/mL）分别提高了50.68%、36.47%、43.61%、66.27%。Abstract: In order to quickly and efficiently screen out Aspergillus oryzae strains of high-yielding Rhizomucor miehei lipase (RML), the spores of Aspergillus oryzae (A. oryzae) were mutated by atmospheric room temperature plasma (ARTP) mutagenesis technology. The dual indicator method for high-throughput detection of RML enzyme activity was optimized, and a 24-well plate high-throughput culture system of A. oryzae was established.Through high-throughput screening of 1,200 wells (36,000-48,000 spores) after mutagenesis, 4 high-yielding mutants, which was designated Ⅰ 7-D6-7, Ⅱ 4-4B-5, Ⅲ 3-5C-1 and Ⅴ 7-6C-3, were finally obtained. In shake flask cultures, the enzyme activities of mutants reached 265.98, 240.90, 253.52 U/mL and 293.50 U/mL, which was increased by 50.68%, 36.47%, 43.61% and 66.27%, respectively, compared to the original strain (176.52 U/mL) .
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图 1 孔板中不同孔的吸光度随预热时间的变化
Figure 1. Variation of absorbance of different wells in the plate with preheating time
图 2 孔板中不同孔初始吸光度的差异
Figure 2. Difference of initial absorbance of different wells in the plate
图 3 不同计算方式下标准曲线的差异：（a）利用反应15 min后的吸光度绘制；（b）利用预热5 min后与反应15 min后的吸光度差值绘制
Figure 3. Difference of standard curve under different calculation methods: (a) The standard curve drawn by absorbance after reaction for 15 min; (b) The standard curve drawn by absorbance difference between preheating 5 min and reaction 15 min
图 4 不同培养条件下米曲霉24孔孔板生长情况对比
Figure 4. Comparison of the growth of 24-well plates of A. oryzae under different culture conditions
图 6 部分突变株初步筛选结果（红线为出发菌株酶活）
Figure 6. Preliminary screening results of some mutant strains（Red line is the lipase activity of the original strain）
表 1 双指示剂法与p-NPP法测量一致性的比较
Table 1. Comparison of double indicator method and p-NPP method on measuring consistency
Strain Enzyme activity/ (U·mL−1) p-NPP method Double indicator method Ⅲ3-5C-1 31.58±7.36 46.88±7.49 Ⅰ7-D6--7 29.36±0.99 42.08±10.89 Ⅱ4-4B-5 35.10±3.25 54.03±5.54 V7-6C-3 30.04±2.25 40.63±6.37 Original strain 23.81±1.78 37.47±5.02 
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