Reaction Mechanisms and Application of New Bacterial Enzymes

(1)苯丙氨酸脱氢酶（Phenylalanine dehydrogenase, PheDH）：从大肠杆菌转化体中大规模纯化球形芽孢杆菌（Bacillus sphaericus）、巴氏芽孢杆菌（b.b hadius）、脲孢杆菌（Sporosarcina ureae）中的PheDH，并送到engel教授（爱尔兰都柏林大学学院）和rice教授（英国谢菲尔德大学）进行x射线结晶学分析。根据谷氨酸脱氢酶的x射线晶体学信息，构建了改变底物特异性的PheDH嵌合酶。(2)甲基天冬氨酸解氨酶（MAL）：对Enterobacter、Citrobacter、Proteus等肠杆菌的MAL进行了纯化，并对其酶学性质进行了详细分析。用内切肽酶酶切amalonaticcitrobacter YG-1002菌株MAL，测定n端氨基酸序列。基于这些信息，合成了DNA探针。利用PCR和Southern杂交技术，从amalonaticus菌株YG-1002的基因组文库中克隆出该酶的基因。将该基因序列与严格厌氧菌破伤风梭菌的序列进行了比较。MAL结晶后送到rice教授那里进行x射线晶体学的研究。(3) Opine dehydrogenase (ODH)：从Arthrobacter sp.IC中克隆出ODH基因，并在大肠杆菌中表达。这种酶被大规模提纯后送到赖斯教授那里，并开始对其进行x射线研究。来自Arthrobacter sp.菌株1C的（D，L）超家族成员N-(1-D-Carboxylethyl)-L-norvaline dehydrogenase的第一个结构已被解析到1.8*分辨率，并确定了结合辅酶的位置。(4)成果在一些国际学术会议上发表。

(1) Phenylalanine dehydrogenase (PheDH) : PheDHs from Bacillus sphaericus, B.badius, Sporosarcina ureae were purified from Escherichia coli transformants in large scales and sent to Prof.Engel (University College Dublin, Ireland) and Prof.Rice (University of Sheffield, U.K.) for X-ray crystallography. Based on informations of X-ray crystallography of glutamate dehydrogenase, chimeric enzymes of PheDH with altered substrate specificities were constructed.(2) Methylaspartate ammonia-lyase (MAL) : MALs from Enterobacteria such as Enterobacter, Citrobacter, Proteus were purified to homogeneities and their enxymological properties were analyzed in detail. MAL from Citrobacter amalonaticus strain YG-1002 was digested with endo peptidases and N-terminal amino acid sequences were determined. Based on the information, DNA probes were synthesized. The gene for the enzyme was cloned form the genomic library of c.amalonaticus strain YG-1002 by PCR and Southern hybridization. The sequence of the gene was compared with that of a strict anaerobe Clostridium tetanomorphum. MAL was crystallized and sent to Prof.Rice for studies of X-ray crystallography.(3) Opine dehydrogenase (ODH) : The gene for ODH from Arthrobacter sp.IC was cloned and expressed in E.coli. The enzyme was purified in a large scale and sent to Prof.Rice, and on its X-ray studies were started. The first structure of a (D,L) superfamily member, N-(1-D-Carboxylethyl)-L-norvaline dehydrogenase from Arthrobacter sp.strain 1C,has been solved to 1.8* resolution and the location of the bound coenzyme determined.(4) Results were presented in some international academic meetings.