Trichoderma pseudokoningii L-離胺酸氧化酶之選殖與表現分析

本實驗所探討之L-離胺酸氧化酶(tp-KOD) 為自土壤分離菌Trichoderma pseudokoningii之麩皮培養基中分離純化所得，為分泌型的糖蛋白質，在原菌株中表現量偏低，且生產tp-KOD需進行麩皮固體醱酵，母株菌體才能分泌此酵素，對自動化生產極為不利。因此將酵素的基因選殖於不同的宿主中，希望建立一套能夠大量生產，且易於純化的酵素生產系統。在結構序列比對中，tp-KOD較其他LAAO在C 端缺失了一段保留性高的序列，於是利用Site-Finding PCR與選殖cDNA進一步確認序列的完整性，而C端的確缺失一段70 a.a.長度的序列。在此本實驗室利用結構比對分析N端序列長度，並將N端不同起始序列之tp-KOD基因，分別選殖於真核與原核系統，探討重組蛋白的表現與活性。共構築preKO、KO8、KO39、KO50、KO82與KO87於pET-24d、pPICZαA以及pPICZB載體。結果發現C端序列缺失的24d-KO8可在E. coli大量表現，而完整序列的24d-preKO、24d-KO8與24d-KO87也可表現但表現量較低；真核系統的αA-preKO、αA-KO50與αA-KO82卻未偵測到蛋白表現與酵素活性，而ZB-preKO可表現於菌體內，推測tp-KOD的訊息胜肽會干擾P. pastoris的蛋白分泌路徑，或P. pastoris的蛋白分泌路徑系統對KOD蛋白無法做有效的蛋白摺疊。

L-Amino acid oxidases (LAAOs) have received attentions as potential diagnostic reagents and industrial biocatalysts. In this study, the L-lysine oxidase (tp-KOD) was a glycoprotein purified from filamentous fungi-Trichoderma pseudokoningii cultured in wheat bran-containing solid medium.The KOD expression from Trichoderma was low and need solid culture. Therefore, we cloned the tp-KOD gene to prokaryotic and yeast expression systems, expect to establish a great quantity expression and easy purification procedure. In this study, we confirmed the gene sequence integrity and analyzed the N-terminal sequence based on structural comparison. The different starting sequences of tp-KOD genes were cloned in eukaryotic and prokaryotic systems and further investigate the protein expression and enzyme activity. We cloned the presequence-containing gene ,G39-, G50- , L82- and V87-starting sequence to pET-24d, pPICZαA and pPICZB vectors. We observed the C-terminal truncated 24d-KO8 could be over expressed by prokaryotic system, the integral 24-preKO, 24d-KO8 and 24d-KO87 were also expressed without enzyme activity. The secreted and intracellular expression of αA-preKO、αA-KO50 and αA-KO82 recombinants both were unsuccessfully. However, the expression and intracellular location of ZB-preKO was observed. It is suggested that the signal peptide of tp-KOD may interfere the protein trafficking in Pichia pastoris, or the folding system in secretory pathway is not suitable for tp-KOD.