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

1.植物醛肟-腈途径的筛选及其应用：(1)优化了植物醛肟-腈途径的提取方法，使其扩展到植物醛肟-腈途径的筛选成为可能。建立了高效液相色谱法测定HNL活性的新方法。(2)与富山植物园（Botanic gardens of Toyama）合作，从大约200种植物中筛选了HNL。(S)-HNL分别在巴利ospermum monum中发现，(R)-HNL分别在西葫芦、枇杷、木瓜、山梨、李子和桃李中发现。(3)合成了巴西橡胶树（Hevea brasilliensis）和木香树（Manihot esculanta）的(S)-HNL基因。(4)从蒙古巴利精子中合成cDNA，并根据纯化的蒙古巴利精子HNL的n端氨基酸序列信息合成DNA低聚物。通过杂交从cDNA文库中克隆出HNL基因。研究了该基因在大肠杆菌中的表达。(5)研究了枇杷（Eriobotrya japonica）、山梨（balaliospermum monmonum）、李子（Prunus mume）中HNL的底物特异性。微生物醛肟-丁腈途径的筛选及其利用：(1)明确了微生物醛肟脱水酶基因与腈化酶、腈水合酶、酰胺酶等丁腈代谢酶基因的连锁，证明了其在微生物中的普遍分布。我们还发现醛肟脱水酶可能是证明醛肟- nitirle通路发生的关键酶。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.