Comparative Biochemistry of the Aldoxime-Nitrile Pathway of Plant and Microbial Origins

1.植物醛-腈途径的筛选及其利用：(1)优化了植物HNLS的提取方法，使其有可能推广到植物HNLS的筛选。(2)与富山植物园合作，从大约200种植物中筛选出HNL。(3)从巴西橡胶树和木瓜中合成了(S)-hnL基因。(4)从巴厘树中合成了hnl基因，并根据纯化的氨基酸序列合成了DNA寡聚体。通过杂交的方法，从文库中克隆了HNL基因。(5)研究了河蟹、文昌、梅花HNL的底物特异性。2.微生物醛-腈途径的筛选及其利用：(1)明确了微生物醛-腈脱水酶基因与硝酸酶、丁腈水合酶、酰胺酶等腈代谢酶基因连锁，并证明了其在微生物中的普遍分布。我们还表明，醛肟脱水酶可能是证明醛肟-NItirle途径中发生的关键酶。从微生物中克隆了多种醛肟脱水酶基因。

1.Screening for plant aldoxime-Nitrile pathway and its utilization :(1)We have optimized the method of extraction of plant HNLs and made it possible to extend to plant screening for HNLs. A new method of HNL activity measurement by the use of HPLC was established.(2)In collaboration with the Botanic gardens of Toyama, HNL was screened from around 200 species of plants. (S)-HNL was discovered in Baliospermum montanum, (R)-HNL was found in P assiflora edulis, Eriobotrya japonica, Chaenomles sinensis, Sorbus aucuparia, Prunus mume, and Prunus persica.(3)(S)-HNL genes from Hevea brasilliensis and Manihot esculanta were synthesized.(4)cDNA was synthesized from Baliospermum montanum and DNA oligomers were synthesized according to the information from N-terminus amino acid sequence of the purified HNL from Baliospermum montanum. By hybridization, the HNL gene was cloned from the cDNA library. Expression the gene in E.coil was studied.(5)Substrate specificities of HNL from Eriobotrya japonica, Baliospermum montanum, Prunus mume were studied.2.Screening for microbial aldoxime-Nitrile pathway and its utilization :(1)We clarified that microbial aldoxime dehydratase gene is linked with the gene for nitrile metabolizing enzymes such as nitirlase, nitirile hydratase, and amidase, and proved its general distribution among microorganisms. We also shown that aldoxime dehydratase can be the key enzyme to prove the occurrence of in aldoxim-NItirle pathway. Various aldoxime dehydratase genes were cloned from microorganism.