2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38612L-Amino acid oxidases (LAAOs) are flavoenzymes catalyzing the oxidative deamination of an L-amino acid substrate to an α-keto acid along with the production of ammonia and hydrogen peroxide. Several other biological activities have also been reported, such as anti-bacterial, cytotoxic, immunomodulated, coagulative, anti-viral and microbial metabolism activity. And hence these enzymes have received attentions as potential diagnostic reagents and industrial biocatalysts. However, little is known about the heterologous expression, the catalytic mechanism and the protein structures derived from this group of genes. In this proposal, we would like to focus on L-lysine oxidase (LKO) from filamentous fungi Trichoderma pseudokoningii. LKO is a dimeric flavoprotein with a molecular mass of 110 kDa and has a highest substrate specific activity for L-lysine. This enzyme has been found with cytotoxicity on erythroleukemic cell line IW201 and IW32.4.1. We have cloned the LKO encoding gene, and its structural fold was predicted to be similar to C. rhodostoma LAAO (crLAAO) and human monoamine oxidase (huMAO). In spite of the apparent lack of sequence homology, LKO, LAAO, and MAO share a common structural topology. LKO can be classified as a member of the glutathione reductase (GR) subfamily. Here we will investigate the signal peptide sequence and regulation of optimal expression for LKO in bacteria and yeast. To ascertain the role of glycosylation and biologic function of LKO, the biochemical and cellular biological studies will be performed. Further analysis of the crystal structures of LKO would allow a better understanding of the structure-function relationship and allow as a basis for protein engineering. In this three-year project, we would like to study : The first year 1. To ascertain the signal peptide of LKO (in the first year) 2. Protein heterologous expression in E. coli and Pichia (in the first year) 3. Isolation of recombinant protein (in the first year) 4. Characterization of recombinant protein (in the first and second year) The second year 1. Investigate the role of glycosylation and biologic activity (in the second year) 2. Crystallization of the recombinant proteins (in the second year) 3. Protein structural determination (in the second and third year) The third year 1. Study of the structure-function relationship by protein engineering (in the third year) 2. Enzyme mechanism study (in the third year) Similar to the other LAAO enzymes purified from the venoms or other organisms, LKO is a cytotoxic protein with oxidase activity. It is secreted from fungi which only in Trichoderma species that with mycoparasitism. Little is known about the function of LAAOs from prokaryotes and lower eukaryotes and the biological activity suggested interesting. L-lysine is the only good substrate for LKO suggests that it may have great potential applicability. If we could express the enzyme on a large scale used to further analysis and using protein engineering techniques to improve enzyme function, the resultant knowledge should be applicable and useful in food processing industry.L-胺基酸氧化酶廣泛存在於自然界，這類酵素可與L-胺基酸反應，產生H2O2， 具有細胞毒殺、抗菌、免疫調節、凝血功能調節、抗病毒且與微生物的代謝相關，在 臨床檢驗與食品工業的應用值得期待，但是關於其基因表現、蛋白質立體結構、並 以蛋白質工程技術研究其酵素催化機制之解析研究卻不多見。 本計畫將研究有絲真菌 Trichoderma pseudokoningii 所分泌的L-離胺酸氧化 酶 (L-lysine oxidase, LKO)。該酵素是由二個單元體所構成之flavoprotein，分子量 為110 KDa，對L-lys 有最佳之反應性，可對紅血球癌細胞IW201 與IW32.4.1 產生細 胞毒殺作用。目前Trichoderma pseudokoningii 所分泌的之LKO 已完成基因選殖及 定序， 根據Phyre program 所作structural fold 的預測，LKO 與C. rhodostoma 所分 泌的蛇毒蛋白L-amino acid oxidase (crLAAO)以及人類之monoamine oxidase (MAO) 類似，儘管LKO、crLAAO 及MAO 彼此之間並沒有明顯的序列相似度(sequence homology)，但它們卻擁有非常類似的立體結構，包括FAD 結合區、基質結合區和 螺旋結構區。LKO 可被歸類於glutathione reductase (GR) family，且又細分屬於GR2 family，GR2 family 的成員中，序列相似性主要位於N 端區域，其乃構成Rossmann fold 而用於結合cofactor (FAD-binding domain)。 本計畫之目的為將已選殖出之LKO 基因，確定其訊息序列後，分別於原核 表現系統（大腸桿菌）與真核表現系統（酵母菌Pichia pastoris）中大量表現，建 立最佳醱酵條件、純化方法、分析基因重組生產之蛋白質特性，探討蛋白糖鏈及生 理活性功能，篩選蛋白質結晶條件，於最佳之養晶條件下生產蛋白質晶體，以便解 析蛋白質結構，並利用蛋白質工程技術改變功能性殘基或結構區域，建構各種突變 株，以分析蛋白結構與功能特性關係以及酵素催化機制，並測定其生理活性，作為 日後進一步改良酵素活性與增加酵素穩定性之基礎。 本計畫將執行三年，預定進行之研究項目如下： 第一年： 1. 決定LKO 之訊息序列。 2. 探討LKO 基因於大腸桿菌及Pichia 系統中之表現。 3. 酵素醱酵生產。 4. 重組酵素特性研究。 第二年： 1. 蛋白生理活性及糖鏈之生理功能特討。 2. 蛋白晶體培養及蛋白結晶條件最佳化 3. 蛋白立體結構解析(與陽明生科系廖淑惠教授合作)。 第三年： 1. 以突變技術探討蛋白結構與功能關係。 2. 酵素催化機制探討。 本計畫在學術上之價值在於本酵素（LKO）是真菌所分泌之毒殺蛋白，且具 有氧化酶活性的，僅於具有寄生黴菌特性(mycoparasitism)之Trichoderma sp.所發現， 與蛇毒及其他生物所分泌之抗菌毒殺蛋白類似，其生理意義極具意義，在產業應用 上本酵素之基質特異性非常特殊，在食品加工業與醫學檢驗上應有相當應用性，若 能大量表現，研究其酵素結構，並以蛋白質工程技術研究其酵素催化機制，必能有 重大成果呈現。L-離胺酸氧化?基因表現生理活性蛋白質糖鏈蛋白質結晶蛋白質結構解析蛋白質工程蛋白結構功能關係酵素催化機制L-lysine oxidase (LKO)gene expressionbiological activitiesglycosylationprotein crystalizationstructural determinationprotein engineeringstructure-function relationshipenzyme mechanismProject