Memories of late professor Hideaki Yamada, a giant in enzyme engineering, and successive activities stemmed from his philosophy

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Basics and applications of gut bacterial lipid-metabolizing enzymes- A tribute to the late Professor Hideaki Yamada

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Arachidonic acid production by the oleaginous fungus Mortierella alpina 1S-4 : A review

The filamentous fungus Mortierella alpina 1S-4 is capable of accumulating a large amount of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs). Indeed, triacylglycerol production by M. alpina 1S-4 can reach 20 g/L of culture broth, and the critical cellular signaling and structural PUFA arachidonic acid (ARA) comprises 30%–70% of the total fatty acid. The demonstrated health benefits of functional PUFAs have in turn encouraged the search for rich sources of these compounds, including fungal strains showing enhanced production of specific PUFAs. Screening for mutants and targeted gene manipulation of M. alpina 1S-4 have elucidated the functions of various enzymes involved in PUFA biosynthesis and established lines with improved PUFA productivity. In some cases, these strains have been used for indistrial-scale production of PUFAs, including ARA. In this review, we described practical ARA production through mutant breeding, functional analyses of genes encoding enzymes involved in PUFA biosynthesis, and recent advances in the production of specific PUFAs through molecular breeding of M. alpina 1S-4

多価不飽和脂肪酸を２位に結合するホスファチジルコリンの炭素アナローグの合成

Carbon analogues of Phosphatidylcholines having linoleic or arachidonic acid at the 2-position were synthesized. The synthetic route involves conversion of the polyunsaturated fatty acid iodination. The derivatives were converted to diols by LiAIH4 reduction and submitted to lipase-catalyzed monostearoylation in isopropylether. The mono-ester was converted to phoshatidylcholines by the usual phosphodiester synthesis.自然界に広く存在するホスフォリパーゼA2はグリセロリン脂質の2位のエステル結合を選択的に切断する酵素であり,消化,アラキドン酸カスケードの起動,リン脂質過酸化物の代謝等,生理作用に広く関わっている。本研究ではホスフォリパーゼA2の基質ミメテイックとしてホスファチジルコリンの2位エステル結合が炭素-炭素結合に置き換わった化合物をアラキドン酸とステアリン酸を出発原料としてリパーゼ触媒によるアシル化反応及び有機化学反応によって合成した

Residue size at position 87 of cytochrome P450 BM-3 determines its stereoselectivity in propylbenzene and 3-chlorostyrene oxidation

AbstractWe report here oxidation of propylbenzene and 3-chlorostyrene by wild-type cytochrome P450 BM-3 with high turnover (479 nmol 1-phenyl-1-propanol/min/nmol P450 and 300 nmol 3-chlorostyrene oxide/min/nmol P450). Furthermore, the residue size at position 87 of P450 BM-3 was found to play critical roles in determining stereoselectivity in oxidation of propylbenzene and 3-chlorostyrene. Replacement of Phe87 with Val, Ala and Gly resulted in decreases in optical purity of produced (R)-(+)-1-phenyl-1-propanol from 90.0 to 37.4, 26.0 and −15.6% e.e., respectively, and in increases in those of produced (R)-(+)-3-chlorostyrene oxide from −61.0 to −38.0, 67.0 and 94.6% e.e., respectively

Sn-1位に多価不飽和脂肪酸を結合する2-0-methoxyethoxymethylglycerolの立体配置決定

A correlation of optical rotation, optical purity and configuration of the asymmetric center was done for 2-0-methoxyethoxymethylglycerol bearing polyunsaturated fatty acyl group at sn-1 position. This compound was enzymatically prepared and is an important starting material for the syntheses of optical active glycerophospholipids naving polyunwaturated fatty acyl groups.自然界に広く存在し，重要な生理機能を担っている多価不飽和脂肪酸結合リン脂質の化学的合成に必要な出発原料としての2-0-methoxyethoxymethylglycerolの立体配置と光学純度を，立体配置・光学純度既知の物質に化学的に誘導し，それらの比旋光度をお互いに比較する事により決定した

Construction of microbial platform for an energy-requiring bioprocess: practical 2′-deoxyribonucleoside production involving a C−C coupling reaction with high energy substrates

BACKGROUND: Reproduction and sustainability are important for future society, and bioprocesses are one technology that can be used to realize these concepts. However, there is still limited variation in bioprocesses and there are several challenges, especially in the operation of energy-requiring bioprocesses. As an example of a microbial platform for an energy-requiring bioprocess, we established a process that efficiently and enzymatically synthesizes 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase. This method consists of the coupling reactions of the reversible nucleoside degradation pathway and energy generation through the yeast glycolytic pathway. RESULTS: Using E. coli that co-express deoxyriboaldolase and phosphopentomutase, a high amount of 2′-deoxyribonucleoside was produced with efficient energy transfer under phosphate-limiting reaction conditions. Keeping the nucleobase concentration low and the mixture at a low reaction temperature increased the yield of 2′-deoxyribonucleoside relative to the amount of added nucleobase, indicating that energy was efficiently generated from glucose via the yeast glycolytic pathway under these reaction conditions. Using a one-pot reaction in which small amounts of adenine, adenosine, and acetone-dried yeast were fed into the reaction, 75 mM of 2′-deoxyinosine, the deaminated product of 2′-deoxyadenosine, was produced from glucose (600 mM), acetaldehyde (250 mM), adenine (70 mM), and adenosine (20 mM) with a high yield relative to the total base moiety input (83%). Moreover, a variety of natural dNSs were further synthesized by introducing a base-exchange reaction into the process. CONCLUSION: A critical common issue in energy-requiring bioprocess is fine control of phosphate concentration. We tried to resolve this problem, and provide the convenient recipe for establishment of energy-requiring bioprocesses. It is anticipated that the commercial demand for dNSs, which are primary metabolites that accumulate at very low levels in the metabolic pool, will grow. The development of an efficient production method for these compounds will have a great impact in both fields of applied microbiology and industry and will also serve as a good example of a microbial platform for energy-requiring bioprocesses

リン脂質過酸化物の生物有機化学的合成

Chemoenzymatic synthesis of 1-stearoyl-2-hydropeoxyacyl-sn-glycerophospholipids including phosphatidylcholine (PC-OOH), phosphatidic acid (PA-OOH), phosphatidylethanolamine (PE-OOH), phosphatidylglycerol (PG-OOH) and phosphatidylserine (PS-OOH). The hydroperoxy acyl moieties were prepared via hydroperoxidation of linoleic, dihomo-γ-linolenic and arachidonic acids by soybean, potate lipoxygenase or autoxidation. Their hydroperoxy group was protected as a dimethylperacetal before condensation with lysophosphatidylcholine. Optically active lysophosphatidylcholine was prepared via short pathway involving lipase-catalyzed direct enantioselective stearoylation of 2-O-benzylglycerol and choline phosphate synthesis. Peroxy fatty acids and lysophosphatidylcholine thus obtained were condensed using dicyclohexylcarbodiimide in chloroform. Removing the peracetal group in the product and purification by reverse-phase chromatography afforded the desired PC-OOH’s. PA-OOH, PG-OOH, PE-OOH and PS-OOH were obtained by phospholipase-D catalyzed transphosphatidylation from PC-OOH. As a reference compound for biological studies of hydroperoxy phopholipid, PC-OH's were also prepared in which hydroxy unsaturated fatty acyl group was linked to the sn-2 position of the glycerophospholipids.健康正常人の血液などの組織中にはリン脂質過酸化物が極微量で存在し，疾病や老化によってその濃度が顕著に上昇する事が知られている．その事が明らかにされた当初は，極めて複雑な混合物をなす生体脂質中に含まれる不安定な極微量の過酸化脂質を単離・構造決定する事は殆ど不可能と考えられていた．現在もなお，そのような脂質過酸化物を生体組織から純粋に取り出し，構造決定したという報告は無い．従ってそのような分子種の化学的・生理学的性質は不明であったが，脂肪酸過酸化物が毒性を示す事から，リン脂質過酸化物もおそらく毒性を示すだろうと考えられてきた．このような漠然とした推定を科学的に明らかにするためには，化学合成によらざるを得ない．我々はこの未知の合成に取りかかった．しかし，従来の化学的手法のみでは不可能である事も明らかであった．その中で予想された困難の一つは極めて不安定なヒドロペルオキシ基を不飽和脂肪酸のある特定の位置にどのように導入するかという問題と，ヒドロペルオキシ基に影響を与える事なく合成中間体をどのように化学変換するかであった．第一の問題に対する解決策として，不飽和脂肪酸に大豆リポキシゲナーゼを作用させる事で解決する事ができた．植物に広く分布する酵素であるリポキシゲナーゼは植物中でリノール酸に作用して過酸化し，その生成物にヒドロペルオキシドリアーゼという酵素が作用して種々のアルデヒドが精製し，これは植物の青臭みを与える．第二の問題に対しては，リノール酸に導入された不安定なヒドロペルオキシ基をパーアセタールとしての保護する事により解決した．この保護基は，中間体から最終生成物に至るまでの反応条件，例えばDCCによるアシル化反応に対して安定である事が明らかとなった．この二つの問題を解決する事によって，リン脂質過酸化物の一つであるホスファチジルコリン過酸化物を世界に先駆けて成功した．さらにこのホスファチジルコリン過酸化物に微生物由来のホスフォリパーゼDを作用させる事によってホスファチジルエタノールアミン過酸化物，ホスファチジルセリン過酸化物やホスファチジルグリセロール過酸化物の合成にも成功した．また，トリグリセリド過酸化物の合成も可能になった．これらの脂質過酸化物が化学的に実態のあるものとして認識されてから，その生理作用に関する研究が広範に行われている．しかし，生体組織に存在するリン脂質過酸化物の生理学的役割は依然として明らかになっていない．ある種のリン脂質過酸化物が動物の免疫系を活性化するという報告もあり，必ずしも生体に対して悪い作用をするばかりではないようである