Institute for Molecular Biology and Genetics, Center for Fungal Pathogenesis, Chonbuk National University, Jeonju, Chonbuk 561-756, Korea.
BMC Biotechnol. 2010 Feb 24;10:18. doi: 10.1186/1472-6750-10-18.
A tannic acid-inducible and mycoviral-regulated laccase3 (lac3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae.
Laccase activity in the culture broth of transformants measured using a laccase-specific substrate suggested that the lac3 gene was successfully expressed and the corresponding protein product secreted into the culture media. In addition, activity staining and Western blot analysis of a native gel revealed that the enzyme activity co-existed with the protein product specific to anti-laccase3 antibody, confirming that the cloned lac3 gene is responsible for the laccase activity. When transformants were grown on plates containing tannic acid-supplemented media, brown coloration was observed around transformed cells, indicating the oxidation of tannic acid. However, the enzymatic activity was measurable only in the selective ura- media and was negligible in nonselective nutrient-rich culture conditions. This was in part because of the increased plasmid instability in the nonselective media. Moreover, the protein product of lac3 appears to be sensitive to the cultured nonselective nutrient-rich broth, because a rapid decline in enzymatic activity was observed when the cultured broth of ura- media was mixed with that of nonselective nutrient-rich broth. In addition, constitutive expression of the lac3 gene resulted in a reduced cell number of the lac3 transformants compared to that of vector-only transformed control. However, the presence of recombinant vector without lac3 induction did not affect the growth of transformants.
The results suggest that expression of the lac3 gene has an inhibitory effect on the growth of transformed S. cerevisiae and that the controlled expression of lac3 is appropriate for the possible application of recombinant yeast to the treatment of phenolic compounds.
栗疫病菌中的单宁酸诱导和真菌病毒调节漆酶 3（lac3）最近被鉴定出来，但由于单宁酸补充剂会导致蛋白质产物沉淀，进一步的表征受到了阻碍。本研究利用酵母酿酒酵母对功能性 lac3 进行了异源表达。
用漆酶特异性底物测量转化体培养物中的漆酶活性表明，lac3 基因已成功表达，相应的蛋白质产物分泌到培养基中。此外，天然凝胶的活性染色和 Western blot 分析显示，酶活性与抗 lac3 抗体的特异性蛋白产物共存，证实克隆的 lac3 基因负责漆酶活性。当转化体在含有单宁酸补充培养基的平板上生长时，观察到转化细胞周围出现棕色着色，表明单宁酸被氧化。然而，只有在选择性 ura-培养基中才能测量到酶活性，在非选择性营养丰富的培养条件下则微不足道。这部分是由于非选择性培养基中质粒的不稳定性增加所致。此外，lac3 的蛋白质产物似乎对培养的非选择性营养丰富的肉汤敏感，因为当将 ura-培养基的培养肉汤与非选择性营养丰富的肉汤混合时，观察到酶活性迅速下降。此外，lac3 基因的组成型表达导致 lac3 转化体的细胞数量比仅转化载体的对照减少。然而，没有 lac3 诱导的重组载体的存在并不影响转化体的生长。
结果表明，lac3 基因的表达对转化酿酒酵母的生长有抑制作用，并且 lac3 的受控表达适合将重组酵母应用于酚类化合物的处理。
