Kazuyuki Morihara

Comparative study of various serine alkaline proteinases from microorganisms: Esterase activity against N-acylated peptide ester substrates

Abstract Hydrolyses of N -acylated peptide ester substrates by various serine alkaline proteinases from bacterial and mold origin were compared using Ac- or Z-(Ala) m -X-OMe (m = 0-2 or 0-3; X = phenylalanine, alanine, and lysine) as esterase substrates. The results indicated that the esterase activities of these enzymes were markedly promoted by elongating the peptide chain from P 1 to P 2 or P 3 with alanine, irrespective of the kind of the amino acid residue at the P 1 -position (amino acid residues in peptide substrates are numbered according to the system of Schechter and Berger (1)). The effect of the kind of amino acid residue at the P 2 -position was further determined using Z-X-Lys-OMe (X = glycine, alanine, leucine, or phenylalanine) as esterase substrates. Alanine was the most efficient residue as X with subtilisins and Streptomyces fradiae Ib enzyme, while leucine or phenylalanine were most efficient with the enzymes from Streptomyces fradiae II, Aspergillus sojae , and Aspergillus melleus. All the serine alkaline proteinases tested in this study were sensitive to Z-Ala-Gly-PheCH 2 Cl, the dependence of inhibition on the inhibitor concentration differed among the enzymes.

Comparative specificity of microbial acid proteinases for synthetic peptides: Primary specificity with Z-tetrapeptides

Abstract The substrates Z-X Leu-(Ala) 2 and Z-Phe X-(Ala) 2 (Z = benzyloxycarbonyl, X = various amino acid residues) were synthesized in order to investigate the primary specificity of acid proteinases from molds and yeasts. Since these peptides are mainly susceptible to cleavage by the enzymes at the peptide bonds shown by the arrows, it was possible to determine the specificity with respect to the amino acid residues on both sides of the splitting point. Pepsin was used for comparison. The results indicated that the microbial acid proteinases exhibit specificity for aromatic or hydrophobic amino acid residues on both sides of splitting point in peptide substrates, as does pepsin. However, the microbial enzymes showed somewhat broader specificity than pepsin. The former enzymes, which possess trypsinogen-activating ability, show specificity for a lysine residue, while pepsin or Mucor rennin-like enzyme does not. Although pepsin is very specific for a tyrosine residue on the imino side of the splitting point, the microbial enzymes do not show such stringency.

Studies on the Protease of Pseudomonas: Part IV. Factors Affecting the Enzyme Production, Especially on the Effect of CalciumPart V. On the Role of Calcium in the Enzyme Production

The extracellular proteinase of Ps. myxogenes sp. was found to be produced by cell suspension in a phosphate buffer of pH. 7.0 containing a carbon source and calcium ion under aerobic conditions.Various factors having affect on the proteinase production were studied, and calcium was found to be essential in its production. However, in cells prepared from a synthetic medium without calcium, the enzyme producing ability was possessed similarly as in those from the medium containing calcium, and the production was not altered by amino acid analogues. Thus, it might be assumed that calcium is necessary in the later stages, from the more complex precursor state than the amino acid-stage, in the biosynthetic reaction of proteinase. In the presence of available calcium, the balance between the concentrations of the carbon source and cells was important in production, and by maintaining the balance of both factors the enzymatic activity produced was almost parallel to the cdl concentrations. Furthermore, the addi...

Milk-clotting or Blood-coagulating Activities of Some Proteinases and their Substrate Specificities

インシュリンB鎖に対する基質特異性の判明している数種の蛋白分解酵素について,凝乳および凝血作用を測定し,それらの活性と基質特異性との間に関連性があるかどうかを検討した.凝乳作用はインシュリンB鎖のフェニルアラニンをC-端とするペプチド結合の開裂能力を欠く酵素にはその活性を検出しえなかったが,フェニルアラニン・ペプチド開裂能力と凝乳作用とは必ずしも一致しなかった.一方,凝血作用はインシュリンB鎖に対してトロンビンと同じ特異性を示す酵素のうちトリプシンにわずかの活性を認めたが,他は全く活性を示さなかった.以上より,諸種蛋白分解酵素の凝乳,凝血作用とインシュリンB鎖に対する基質特異性とは必ずしも関連しないと結論した.

Studies on the Protease of Pseudomonas :Part III, Specific Action of the Protease

The specific action of the crystalline protease obtained from Ps. myxogenes sp., was examined from its action on various proteins, determination of the N-terminal residues of the enzymatically degraded gelatin — which was most readily digested by the enzyme — and comparison with other proteinases, such as trypsin, papain and pepsin. It was shown that the enzyme might be classified as a collagenase and the mode of digestion of gelatin had a more close resemblance to papain than trypsin or pepsin.

Studies on the Protease of Pseudomonas :Part I. The Production of the Enzyme under Various Cultural Conditions

The protease production of Ps. myxogenes sp. was found to be profoundly affected by some components of the cultural medium: Ca was indispensable for the protease production and a high concentration of glucose in the shaking culture was found to promote production remarkably, although they were not effective for growth of the organism, while Mg or organic nitrogen was effective for growth but suppressive for production of the enzyme.Therefore, to obtain a high yield of protease, each of these influential components should be adjusted to their optimal concentration in the medium, so that the organism will be able to consume glucose in an adequately high concentration, in the presence of Ca.