1993 Fiscal Year Annual Research Report

新規微生物酵素フェニルアラニン脱水素酵素の構造、機能および応用に関する研究

Research Project

Project/Area Number	05044138
Research Institution	Toyama Prefectural University
Principal Investigator	浅野泰久富山県立大学, 工学部, 助教授 (00222589)
Co-Investigator(Kenkyū-buntansha)	加藤康夫富山県立大学, 工学部, 助手 (20254237) クーパーアーサー.J. 米国コーネル大学, 医学部・生化学科, 教授
Keywords	Amino Acid / Immunotitration / Amino Acid Dehydrogenase / Primary Structure / Enzyme Purification / Phenylalanine Dehydrogenase / Screening / Sequence Homology
Research Abstract	フェニルアラニン脱水素酵素産生菌として、土壌中よりスクリーニングされたBacillus spaericus及びSporosarcina ureae由来のフェニルアラニン脱水素酵素を、本酵素遺伝子を有する組み換え大腸菌より数十mg単位で高度に精製した。また、Sporosarcina ureae野性株より本酵素を高度に精製した。フェニルアラニン脱水素酵素産生菌として、保存菌株中よりスクリーニングされたBacillus badiusの無細胞抽出液よりフェニルアラニン脱水素酵素を結晶状に単離した。酵素標品をリジルエンドペプチダーゼで分解後、得られた部分的ペプチド断片をHPLCで分取し、プロテインシーケンサーを用いて酵素内部のアミノ酸配列を決定した。すでに全一次構造の明らかになっているBacillus sphaericus由来のフェニルアラニン脱水素酵素の配列と比較検討を行ったところ、両酵素間で優位なホモロジーが確認された。Bacillus sphaericusの本酵素遺伝子配列を参考にして、Bacillus badiusの塩基配列決定用のDNAプローブを合成した。結晶状に精製したBacillus spaericus由来のフェニルアラニン脱水素酵素を用いてポリクロナール抗体を作成した。本抗体を用いて、他の菌株由来のフェニルアラニン脱水素酵素との免疫交差反応を行ったところ、Sporosarcina ureae由来の酵素のみならず、Bacillus spaericusと同属であるBacillus badius由来の酵素とも免疫反応は陰性であった。

Research Products
(15 results)

All Other

All Publications (15 results)

[Publications] M.Ueda: "Purification and Chracterization of Maleate Hydratase from Arthrobacter sp.strain MCI2612" Bioscience Biotechnology Biochemistry. 57. 1545-1548 (1993)
[Publications] Y.Asano: "Occurrence of 3-Methylaspartate ammonia-lyase in Facultative Anaerobes and Their Application to Synthesis of 3-Substituted(S)-Aspartic Acids." Bioscience Biotechnology Biochemistry. 58. 223-224 (1994)
[Publications] M.Ueda: "Production of S-(+)-Citramalate from Itaconateby Resting Cells of Alcaligenes denitrificans strain MCI2775" Applied Microbiology and Biotechnology. 40. 466-469 (1993)
[Publications] Y.Asano: "Microbial Production of Theobromine from Caffeine" Bioscience Biotechnology Biochemistry. 57. 1286-1289 (1993)
[Publications] Y.Asano: "Microbial Production of D-Malate from Maleate" Applied and Environmental Microbiology. 59. 1110-1113 (1993)
[Publications] Y.Kato: "Enzymatic Synthesis of L-β-chloroalanine Using Amino Acid Dehydrogenase" Applied Microbiology and Biotechnology. 39. 301-304 (1993)
[Publications] A.J.L.Cooper: "Enzymology of cysteine S-conjugate beta-lyases" Advances in Pharmacology. 27. 73-115 (1994)
[Publications] A.J.L.Cooper: "High activities of glutamine transaminase K(dichloro vinylcysteine beta-lyase)and omega-amidase in the choroid plexus of rat brain" Journal of Neurochemistry. 61. 1731-1751 (1993)
[Publications] A.J.L.Cooper: "Ammonia metabolism in mammals:Interorgan relationships." Adv.Exp.Biol.Med.341. 21-37 (1993)
[Publications] T.K.Makar: "Vitamin E,glutathione,glutathione disulfide and enzymes of glutathione metabolism in cultures of chick astrocytes and neurons:Evidence that astrocytes play an important role in antioxidative processes in the brain." Journal of Neurochemistry. 62. 45-53 (1994)
[Publications] T.K.Makar: "Glutamine transaminase K and omega-amidase in primary cultures of astrocytes and neurons and in embryonic chick forebrain.Marked induction of brain glutamine transaminase K at time of hatching." Journal of Neurochemistry. 62. 1983-1988 (1994)
[Publications] Y.Asano: "A New Enzyme D-Aminopeptidase:Structure,Function and Application to Organic Synthesis in Frontiers and New Horizons in Amino Acid Research" Elsevier, (1992)
[Publications] A.J.L.Cooper: "Glutathione in the brain;disorders of glutathione metabolism.In:The Molecular and Genetic Basis of Neurological Disease" Butterworth-Heinemman,Boston,Massachusetts, (1993)
[Publications] Q.R.Smith: "Amino acid transport in brain.In:Mammalian Amino Acid Transport:Mechanism and Control" Plenum Press,New York, (1993)
[Publications] W.A.Pulsinelli: "Metabolic encephalopathies and coma.,In:Basic Neurochemistry:Molecular,Cellular and Medical Aspects,5th Edition" Raven Press,New York, (1994)

1993 Fiscal Year Annual Research Report

新規微生物酵素フェニルアラニン脱水素酵素の構造、機能および応用に関する研究

Principal Investigator

浅野 泰久 富山県立大学, 工学部, 助教授 (00222589)

Research Products

[Publications] M.Ueda: "Purification and Chracterization of Maleate Hydratase from Arthrobacter sp.strain MCI2612" Bioscience Biotechnology Biochemistry. 57. 1545-1548 (1993)

[Publications] Y.Asano: "Occurrence of 3-Methylaspartate ammonia-lyase in Facultative Anaerobes and Their Application to Synthesis of 3-Substituted(S)-Aspartic Acids." Bioscience Biotechnology Biochemistry. 58. 223-224 (1994)

[Publications] M.Ueda: "Production of S-(+)-Citramalate from Itaconateby Resting Cells of Alcaligenes denitrificans strain MCI2775" Applied Microbiology and Biotechnology. 40. 466-469 (1993)

[Publications] Y.Asano: "Microbial Production of Theobromine from Caffeine" Bioscience Biotechnology Biochemistry. 57. 1286-1289 (1993)

[Publications] Y.Asano: "Microbial Production of D-Malate from Maleate" Applied and Environmental Microbiology. 59. 1110-1113 (1993)

[Publications] Y.Kato: "Enzymatic Synthesis of L-β-chloroalanine Using Amino Acid Dehydrogenase" Applied Microbiology and Biotechnology. 39. 301-304 (1993)

[Publications] A.J.L.Cooper: "Enzymology of cysteine S-conjugate beta-lyases" Advances in Pharmacology. 27. 73-115 (1994)

[Publications] A.J.L.Cooper: "High activities of glutamine transaminase K(dichloro vinylcysteine beta-lyase)and omega-amidase in the choroid plexus of rat brain" Journal of Neurochemistry. 61. 1731-1751 (1993)

[Publications] A.J.L.Cooper: "Ammonia metabolism in mammals:Interorgan relationships." Adv.Exp.Biol.Med.341. 21-37 (1993)

[Publications] T.K.Makar: "Vitamin E,glutathione,glutathione disulfide and enzymes of glutathione metabolism in cultures of chick astrocytes and neurons:Evidence that astrocytes play an important role in antioxidative processes in the brain." Journal of Neurochemistry. 62. 45-53 (1994)

[Publications] T.K.Makar: "Glutamine transaminase K and omega-amidase in primary cultures of astrocytes and neurons and in embryonic chick forebrain.Marked induction of brain glutamine transaminase K at time of hatching." Journal of Neurochemistry. 62. 1983-1988 (1994)

[Publications] Y.Asano: "A New Enzyme D-Aminopeptidase:Structure,Function and Application to Organic Synthesis in Frontiers and New Horizons in Amino Acid Research" Elsevier, (1992)

[Publications] A.J.L.Cooper: "Glutathione in the brain;disorders of glutathione metabolism.In:The Molecular and Genetic Basis of Neurological Disease" Butterworth-Heinemman,Boston,Massachusetts, (1993)

[Publications] Q.R.Smith: "Amino acid transport in brain.In:Mammalian Amino Acid Transport:Mechanism and Control" Plenum Press,New York, (1993)

[Publications] W.A.Pulsinelli: "Metabolic encephalopathies and coma.,In:Basic Neurochemistry:Molecular,Cellular and Medical Aspects,5th Edition" Raven Press,New York, (1994)

浅野泰久富山県立大学, 工学部, 助教授 (00222589)