Institute of Pharmaceutical Biotechnology, Fuzhou University, Fuzhou, Fujian 350108, China.
J Biosci Bioeng. 2011 Oct;112(4):321-5. doi: 10.1016/j.jbiosc.2011.06.015.
Hydroxynitrile lyase gene (hnl) from Eriobotrya japonica was successfully amplified using the method of SEFA PCR (Self-Formed Adaptor PCR). The complete sequence was 5.5 kbp in length, including 3100 bp of the upstream promoter region, 1659 bp of the coding sequence, three introns and 315 bp of the downstream transcription terminator. The phylogenetic analysis illustrated that the obtained hnl exhibited 66-70% identity to the reported isozymes from almond, black cherry and Japanese apricot. The EjHNL had 552 amino acids including a 25 amino acid-long signal peptide. The conserved characteristic structures of HNLs, such as FAD-binding motif, N-glycosylation sites and active sites were observed. The coding sequence of the hnl was inserted into pPIC9K vector for heterologous expression in Pichia pastoris. The HNL activity of the culture supernatant reached 15 U/ml after 96 h of induction by methanol. The specific activity of the recombinant HNL was about 197 U/mg. The enantiomeric excess value of the product R-mandelonitrile attained 98.6% and the value of K(m) of the recombinant HNL was determined to be 0.47 mM based on the kinetic data. The optimum temperature and pH of the recombinant HNL were 40°C and 6.0 respectively. The experimental data indicated that the obtained recombinant HNL showed similar catalytic characteristics with the natural EjHNL. The expression of the recombinant HNL in P. pastoris could present another available biocatalyst for the synthesis of R-selective cyanohydrins.
采用 SEFA PCR（自形成接头 PCR）方法成功扩增了来自枇杷的羟腈裂解酶基因（hnl）。完整序列长 5.5 kbp，包括 3100 bp 的上游启动子区、1659 bp 的编码序列、三个内含子和 315 bp 的下游转录终止子。系统发育分析表明，获得的 hnl 与杏仁、黑樱桃和日本甜樱中报道的同工酶具有 66-70%的同源性。EjHNL 含有 552 个氨基酸，包括一个 25 个氨基酸长的信号肽。观察到 HNL 的保守特征结构，如 FAD 结合基序、N-糖基化位点和活性位点。hnl 的编码序列被插入 pPIC9K 载体中，用于在毕赤酵母中异源表达。经甲醇诱导 96 h 后，培养上清液中的 HNL 活性达到 15 U/ml。重组 HNL 的比活约为 197 U/mg。产物 R-扁桃腈的对映体过量值达到 98.6%，根据动力学数据确定重组 HNL 的 K(m)值为 0.47 mM。重组 HNL 的最适温度和 pH 分别为 40°C 和 6.0。实验数据表明，获得的重组 HNL 表现出与天然 EjHNL 相似的催化特性。在毕赤酵母中表达重组 HNL 可为合成 R-选择性氰醇提供另一种可用的生物催化剂。
