Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.
Appl Microbiol Biotechnol. 2013 Feb;97(3):1113-20. doi: 10.1007/s00253-012-3986-3. Epub 2012 Mar 10.
A 1,965-bp fragment encoding a poly(vinyl alcohol) dehydrogenase (PVADH) from Sphingopyxis sp. 113P3 was synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris. The fragment was then amplified by polymerase chain reaction and inserted into the site between EcoRI and NotI sites in pPIC9K, which was under the control of the AOX1 promoter and α-mating factor signal sequence from Saccharomyces cerevisiae. The recombinant plasmid, designated as pPIC9K-PVADH, was linearized using SalI and transformed into P. pastoris GS115 by electroporation. The PVADH activity reached 55 U/mL in a shake flask and 902 U/mL in a 3-L bioreactor. Surprisingly, the sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and N-terminal sequencing indicated that the secreted PVADH was truncated, and it had only 548 amino acid residues (an 81-amino acid sequence from the secreted protein was cleaved). The optimum pH and temperature ranges for the truncated PVADH were 7.0-8.0 and 41-53 °C, respectively. The activation energy of the recombinant truncated PVADH was approximately 10.36 kcal/mol between 29 and 41 °C. Both Ca(2+) and Mg(2+) had stimulating effects on the activity of PVADH. With PVA1799 as the substrate, the truncated PVADH had a Michaelis constant (K (m)) of 1.89 mg/mL and a maximum reaction rate (V (max)) of 34.9 nmol/(min mg protein). To the best of our knowledge, this is the first report on the expression of PVADH in P. pastoris, and the achieved PVADH yield is the highest ever reported.
根据甲基营养酵母毕赤酵母的密码子偏爱性，合成了编码来自鞘氨醇单胞菌 113P3 的聚（乙烯醇）脱氢酶（PVADH）的 1965bp 片段。然后通过聚合酶链反应扩增该片段，并将其插入到 pPIC9K 中 EcoRI 和 NotI 位点之间，pPIC9K 受甲醇酵母的 AOX1 启动子和α-交配因子信号序列的控制。将该重组质粒命名为 pPIC9K-PVADH，用 SalI 线性化，然后通过电穿孔转化到毕赤酵母 GS115 中。在摇瓶中，PVADH 活性达到 55U/mL，在 3L 生物反应器中达到 902U/mL。令人惊讶的是，十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析和 N 端测序表明，分泌的 PVADH 被截断，只有 548 个氨基酸残基（从分泌蛋白中切割了 81 个氨基酸序列）。截断的 PVADH 的最适 pH 和温度范围分别为 7.0-8.0 和 41-53°C。重组截断的 PVADH 的活化能在 29-41°C 之间约为 10.36kcal/mol。Ca(2+)和 Mg(2+)对 PVADH 的活性均有刺激作用。以 PVA1799 为底物时，截断的 PVADH 的米氏常数（K(m)）为 1.89mg/mL，最大反应速率（V(max)）为 34.9nmol/(minmg 蛋白)。据我们所知，这是首次在毕赤酵母中表达 PVADH 的报道，并且达到的 PVADH 产量是迄今为止报道的最高产量。
