Juichiro Fukumoto

Studies on Mold Protease:Part I. Purification, Crystallization and Some Enzymatic Properties of Acid Protease of Rhizopus chinensis

An acid protease of Rhizopus chinensis was purified by sequential chromatographies on columns of Duolite A-2, Sephadex G-100 and CM-cellulose, and crystallized from aqueous acetone solution. The preparation was shown to be monodisperse on column chromatography of ion-exchange sephadex and on ultracentrifugal analysis. The enzyme was most active at pH values between 2.9 and 3.3 and was stable over the range of pH 2.8 to 6.5. The protease was markedly inactivated by ferric ions and sodium lauryl sulfate, whereas it was affected by neither sulfhydryl reagents nor metal-chelating agents. In milk-clotting activity, the acid protease was shown to be one of the most potent enzymes among those of fungal origin. Substrate specificity experiments on several synthetic peptides indicated that the peptide bonds susceptible to the action of the enzyme were mainly those involving amino groups of aromatic amino acids.

Studies of Hemicellulolytic Enzymes of Bacillus subtilis :Part I. Purification, Crystallization and Some Properties of Arabinogalactanase

Many strains of Bacillus subtilis were found to secrete several hemicellulolytic enzymes such as arabinoxylanase, galactomannanase, arabinogalactanase, etc. Chemical and enzymatic properties of certain strains of the bacterium were comparatively investigated. An arabinogalactanase was purified and obtained in a crystalline state. Its molecular weight and isoelectric point were estimated to be 3.7×104 and 8.39, respectively. The enzyme showed an optimal pH for reactions at 6.0, and was stable in a pH range of 5.0 to 9.5 at 30°C. It required no metallic ions for its activity and hydrolyzed soybean arabinogalactan, forming galactobiose as the main product. No liberation of arabinose was observed in the hydrolysate. Also, the enzyme did not attack coffee bean arabinogalactan. Some implications of the experimental results are discussed.

Studies on Mold Proteases:Part II. Substrate Specificity of Acid Protease of Rhizopus chinensis

The substrate specificity of the crystalline acid protease obtained from Rhizopus chinensis was determined using B-chain of oxidized beef insulin and numerous synthetic peptides, comparing with tha...

Bitter Peptides in Cow Milk Casein Digests with Bacterial Proteinase: Part I. Isolation and Determination of Amino Acid Sequence of a Bitter Peptide

Isolation and determination of amino acid sequence of the bitter peptides formed in the digestion of cow milk casein with alkaline proteinase of Bacillus subtilis were investigated. The casein digest with the enzyme was extracted with butanol and the extracted bitter peptides were fractionally purified by treating with several other organic solvents followed by subjecting to chromatography and gel-filtration. The amino acid sequence of one of the bitter peptides was determined as follows: Arg-Gly-Pro-Pro-Phe-Ileu-Val. Liberation of N-terminal Arg with trypsin or bacterial aminopeptidase did not affect the bitterness. Also, splitting off of Val and Ileu or Ileu-Val at the C-terminus by carboxypeptidase, or a bacterial neutral proteinase gave no influence on the bitterness. However, liberation of Arg and Gly from the peptide with bacterial aminopeptidase gave rise to a non bitter peptide.

Studies on the Formation of Uricase by Streptomyces :Part II. The Induced Formation of Uricase by the Resting Cells

A strain of Streptomyces sp. produced little of uricase in the cells when they were grown in a medium consisted of peptone, glucose and inorganic salts, even in the presence of urate. The cells, however, formed a large amount of the enzyme, when they were incubated with urate in K-phosphate buffer. The amount of uricase thus formed was maximum by the cells which were harvested at the middle logarithmic phase of the preliminary growth. The induced formation of uricase required K ions in addition to Mg ions and was accelerated by glucose and some other carbon sources. The enzyme formation was inhibited completely by chloramphenicol at a low concentration. An equimolar allantoin to urate decomposed by the cells was accumulated in the incubation mixture. More than 3.0 units of uricase per g of wet cells were produced under the best conditions known from the present experiments. The derepression of uricase formation in the resting cells incubated in the phosphate buffer was discussed.

Studies on the Formation of Uricase by Streptomyces :Part IV The Uptake of Urate by the Cells

Urate uptake by a strain of Streptomyces sp. was studied using the cells previously induced uricase with urate. Optimum pH’s for the urate uptake were 6.5 to 8.5 and optimum temperatures 30 to 40°C. Urate was not accumulated in the cells but released into the medium as allantoin and the urate uptake seemed to be correlated with uricase activity of the cells. Urate uptake activity of the cells, however, differed from uricase activity extracted from the cells in the following points: 1) the urate uptake showed a relatively broad pH-activity curve, 2) it was not inhibited by the substrate in higher concentrations and followed a saturation kinetics, 3) it was not affected by borate ion, and 4) it was inhibited by propionate, iodoacetate, uranyl acetate and toluene which did not inhibit the enzyme activity. It was also inhibited by KCN and competitively by xanthine. The uptake activity was not affected by energy sources and by inhibitors of energy metabolism. It was suggested that the urate uptake might be med...

Intracellular Peptidase of Bacillus subtilis :Part III. Effects of Metal Ions on Activity and Specificity of Aminopeptidase of Bacillus subtilis

An electrophoretically pure preparation of aminopeptidase was isolated from the cells of a strain of Bacillus subtilis which secreted saccharifying α-amylase. The purified peptidase was active only in the presence of manganese and cobaltous ions. Both the metallic ions were effective to a similar degree for the enzyme on most of the peptides used as the substrate. However, for hydrolysis of leucine amide by the enzyme was effective only cobaltous ion and, in this case, manganese ion showed to act as a competitive inhibitor. On the other hand, for hydrolysis of certain other peptides such as glycyl proline, quite the opposite relationship was observed between those metallic ions. In the present paper is also described a new micro-assay method of amidase activity shown by the peptidase.

The Actions of the Two Types of Bacterial α-Amylase on Maltodextrin

Sacillus subtilis属細菌群の分泌する細菌液化型および細菌糖化型アミラーゼ(BLAおよびBSA)を還元性末端に放射性を有するオリゴ糖に作用せしめ,初期分解物を分析すると,それぞれ特定の結合位置が分解を受けることが知られている. さらに高分子の糖への反応について検討したところ, (1)重合度23付近の直鎖デキストリンにBSAを作用した場合,ほぼオリゴ糖の場合と同様に非還元性来端より3番目以上内側の結合が最も作用を受けやすい傾向を示した. (2) BSAは反応中間物として生ずるオリゴ糖のうちG4, G5に対して高い作用性を示し,速やかにG1～G3を生ずることが明らかとなった. (3) Starch, β-limit dextrinのBSA分解物はmaltodextrinの場合とほぼ同様で,これら多糖体分解の際もオリゴ糖に見出されたような規則性がある程度成立するものと推察された.

Studies on Uricase Part II

Streptomycesに属する1菌株を塚酸を含む培地で培養し,菌体内のウリカーゼを超音波処理によって抽出した.この抽出液よりウリカーゼを,硫安分画,アセトン分画,DEAE-セファデツクスカラムクロマトグラフィー,セファデツクG-200ゲル濾過等の方法により,約600倍精製した. 精製酵素の反応最適PHは,硼酸緩衝液中ではpH 8.0,燐酸およびトリス緩衝液中ではpH9.0であり,その活性は,pH 8.0の硼酸緩衝液中で最高であった.反応の最適温度は,30～50°Cであった.精製酵素は,pH 6.0以上,また50°C以下では極めて安定であった.硼酸塩を含まない反応液中では,290mμの吸光度で測定した尿酸の減少に遅れが認められたが,これは紫外部に吸収のある反応中間産物の影響によるものと考えられた.放線菌のウリカーゼは,哺乳動物の酵素と同様に,高濃度の基質によって阻害され,またキサンチンにより拮抗的に阻害された.精製酵素の尿酸に対するKmは2.50×10-5M,キサンチンに対するKi1.33×10-4Mであった.

Studies on Bacterial Protease: Part XVIII. Proteolytic Specificity of Neutral Protease of Bacillus subtilis var. amylosacchariticus

Substrate specificity of the crystalline neutral protease of B. amylosacchariticus was investigated using the B-chain of oxidized beef insulin as the substrate, and the results were compared with those of proteases obtained from other strains of Bacillus subtilis. The neutral protease split the B-chain at eleven sites of the peptide linkages, indicating the narrow specificity as compared with subtilopeptidase A, The results also indicated that the peptide bonds susceptible to the action of the neutral protease were mainly those involving amino group of hydrophobic amino acids and tyrosine, with a few exception. The enzyme showed potent activities in casein digestion at near neutrality and in milk clotting at pH 5.6, whereas it was completely inert on esters and keratin, and only slightly active toward elastin.