College of Life and Physical Science, Sichuan Agricultural University, Ya'an 625014, China.
Appl Biochem Biotechnol. 2012 Feb;166(3):549-62. doi: 10.1007/s12010-011-9447-0. Epub 2011 Nov 19.
The mutant acid phytase (phyA ( m )) gene was modified by random mutagenesis to improve enzymatic activity by using an error-prone PCR (ep-PCR) strategy. The mutated gene was linearized and inserted into plasmid vector pPIC9K and transformed by electroporation into Pichia pastoris GS115. A single transformant, PP-NP(ep)-6A, showing the strongest phytase activity from among the 5,500 transformants, was selected for detailed analyses. Southern blot analysis of the mutant yeast transformant showed that phyA ( ep ) gene was integrated into the chromosome genome through single crossover with one copy of phyA. The kinetic parameters indicated that the mutant one showed 61% higher specific activity and 53% lower k (m) value than that of PP-NP(m)-8 (P < 0.05). In addition, the overall catalytic efficiency (k (cat)/k (m)) of the mutant one was 84% higher (P < 0.05) than that of PP-NP(m)-8. Nine bases were altered in the mutant sequences, which resulted in three amino acid changes, namely, Glu156Gly, Thr236Ala, and Gln396Arg. The structural predictions indicated that the mutations generated by ep-PCR somehow reorganized or remodeled the active site, which could lead to increasing catalytic efficiency.
通过易错 PCR（ep-PCR）策略，对突变酸性植酸酶（phyA（m））基因进行修饰，以提高酶活性。将突变基因线性化并插入质粒载体 pPIC9K 中，并通过电穿孔转化为毕赤酵母 GS115。在 5500 个转化子中，选择具有最强植酸酶活性的单个转化子 PP-NP（ep）-6A 进行详细分析。突变酵母转化子的 Southern blot 分析表明，phyA（ep）基因通过单交换与一个 phyA 拷贝整合到染色体基因组中。动力学参数表明，突变体的比活度比 PP-NP（m）-8 高 61%，而 k（m）值低 53%（P<0.05）。此外，突变体的总催化效率（k（cat）/k（m））比 PP-NP（m）-8 高 84%（P<0.05）。突变序列中有 9 个碱基发生改变，导致 3 个氨基酸变化，即 Glu156Gly、Thr236Ala 和 Gln396Arg。结构预测表明，ep-PCR 产生的突变以某种方式重新组织或重塑了活性位点，从而提高了催化效率。
