Makoto Kaneda

Cucumisin-like protease from the latex of Euphorbia supina

A protease has been purified from the latex of Euphorbia supina Rafin by two steps of chromatography. The Mr was estimated by SDS-PAGE to be 80 kDa. Its activity was inhibited strongly by diisopropyl fluorophosphate, but not by EDTA, pepstatin, or cysteine protease inhibitors, indicating that the enzyme is a serine protease. The specificity of the protease is broad, but the preferential cleavage sites were C-terminal sites of hydrophobic amino acid residues. The N-terminal sequence of the first fifteen residues was determined and six of the residues match those in cucumisin [EC 3.4.21.25], a protease from the sarcocarp of melon fruit (Cucumis melo L. var. Prince). The results indicate that the E. supina protease is a cucumisin-like serine protease.

Cucumisin like protease from the sarcocarp of Benincasa hispida var. Ryukyu

A protease has been purified from the sarcocarp of Benincasa hispida (Thunb.) Cogn. var. Ryukyu by two steps of chromatography. Its M(r) was estimated by SDS-PAGE to be about 67,000. The enzyme was strongly inhibited by diisopropyl fluorophosphate, but not by EDTA and cysteine protease inhibitors. The substrate having alanine at the position of P1 was the best among the Ala-Ala-Pro-X-pNAs (X = Ala, Lys, Phe, Glu, and diaminopropionic acid (Dap)). The N-terminal sequence of the first 33 residues was determined and 25 of the residues agreed with that of cucumisin [EC 3.4.21.25], a protease from the sarcocarp of melon fruit (Cucumis melo L. var. Prince). The results indicated that the B. hispida protease is a cucumisin like serine protease.

Isolation and characterization of a serine protease from the sprouts of Pleioblastus hindsii Nakai

An endopeptidase has been purified from sprouts of bamboo (Pleioblastus hindsii Nakai) to electrophoretic homogeneity by four purification steps. Its Mr was estimated to be 82 kDa by SDS-PAGE. Enzyme activity was inhibited strongly by diisopropyl fluorophosphate, and weakly by p-chloromercuriphenylsulfonic acid, but not at all by EDTA or pepstatin, indicating that it was a serine protease. The preferential cleavage sites for this protease were found to be large hydrophobic and amide residues at the P1 position. The specificity of the bamboo serine protease differed from that of cucumisin [EC 3.4.21.25], which cleaved the charged amino acid residues at the P1 position.

Milk-clotting activity of cucumisin, a plant serine protease from melon fruit

Cucumisin (EC 3.4.21.25) isolated from prince melon fruit is a plant serine protease. Its milk-clotting activity was compared with plant cysteine proteases such as papain (EC 3.4.22.2) and ficain (EC 3.4.22.3). Cucumisin was more stable than papain under the condition of pH 7.1, 37‡C for 24 h. The milk-clotting activity of cucumisin was the same to that of papain and was half value of that of ficain.

Proteases from the sarcocarp of yellow snake-gourd

Abstract Two serine proteases, A and B, have been purified from the sarcocarp of Trichosanthes kirilowii . Each protease has a M r , of ca 50 000 (gel filtration) and an optimum pH in the range of 10–11 at 35° using casein as substrate. The optimum temperature (at pH 7.2) for A was 60° and for B 70°. The enzymes are inhibited by diisopropyl fluorophosphate but not by metal-chelating reagents and cysteine proteinase inhibitors. The results indicated that the yellow snake gourd proteases are similar to cucumisin from the sarcocarp of melon fruit ( Cucumis melo L. var. Prince).

Isolation and characterization of a proteinase from white gourd

Abstract A proteinase from the sarcocarp of Benincasa cerifera was purified. ItsMW was estimated by two different methods to be about 50000. The maximum activity was found in the alkaline pH region against casein as a substrate. The enzyme was strongly inhibited by di-isopropyl fluorophosphate and not inhibited by EDTA and p -chloromercuribenzoic acid.

Purification and Some Properties of a Protease from the Sarcocarp of Musk Melon Fruit

A protease has been purified from sarcocarp of musk melon, Cucumis melo ssp. melo var. reticulatus Naud. Earls Favourite. The protease was mostly present in the placenta part of the fruit and next in the inside mesocarp. The molecular mass of the enzyme was estimated to be about 62kDa on SDS-PAGE. The enzyme had a carbohydrate moiety. The optimum pH of the enzyme was 11 at 35°C using casein as a substrate. The enzyme was stable between pH 6 and 11. The enzyme was strongly inhibited by diisopropyl fluorophosphate, but was not inhibited by EDTA or cysteine protease inhibitors. From the digestion of Ala-Ala-Pro-X-pNA (X = Phe, Leu, Val, Ala, Gly, Lys, Glu, Pro, and diaminopropionic acid (Dap) substrates the specificity of the protease was found to be approximately broad, but the preferential cleavage sites were C-terminal sites of hydrophobic or acidic amino acid residues at P, position. It was proved that the enzymatic properties of musk melon protease are similar to those of cucumisin [EC 3.4.21.25]. The enzyme was not inhibited by typical proteinous inhibitors such as STI or ovomucoid. Therefore, this enzyme seems to be a useful protease for the food industries.

Protease D from the sarcocarp of honeydew melon fruit

A serine protease was purified from commercially available honeydew melon fruit (Cucumis melo L. var. inodorus Naud) by five steps including CM-Sepharose CL-6B column chromatography. At the first CM-Sepharose CL-6B chromatography four active fractions appeared. No difference in enzymatic properties and M(r) in each fraction was found. Finally, honeydew melon protease D was isolated from the major active fraction D. A M(r) of 50,000 was determined for protease D on SDS-PAGE and gel filtration. Protease D was a glycoprotein. The protease exhibited a remarkable stability, even in 8 M urea or 2 M guanidine hydrochloride for 24 hr. The optimum pH of the enzyme was 11 at 35 degrees using casein as substrate. The enzyme was strongly inhibited by diisopropyl fluorophosphate and was not inhibited by metal-chelating reagents or cysteine protease inhibitors. The enzymatic properties of honeydew melon protease D were similar to those of cucumisin [EC 3.4.21.25], except for its stability at high temperature, wide pH range and against detergents.

Protease from the sarcocarp of Trichosanthes bracteata.

A protease has been purified from sarcocarp of Trichosanthes bracteata (Lam.) Voigt by four steps of chromatography. Its M(r) was estimated by SDS-PAGE to be ca 67,000. The optimum pH of the enzyme was 11 at 35 degrees using casein substrate. The enzyme was strongly inhibited by di-isopropyl fluorophosphate, but not by EDTA and cysteine protease inhibitors. The oxidized insulin B-chain was cleaved at the peptide bonds of Cys7 (SO3H)-Gly8, Glu13-Ala14, Try16-Leu17 by the enzyme for 1 min. The results indicated that the T. bracteata protease is serine protease, similar to cucumisin from the sarcocarp of melon fruit (Cucumis melo L. var. Prince).

Isolation and characterization of a protease from Phytolacca americana

Abstract A cysteine protease of M r 26 000 has been isolated in homogeneous form from the fruit juice of Phytolacca americana . The enzyme, named phytolacin, has a maximum activity in the pH range 7.5–9.0 and contains carbohydrate moiety. The substrate specificity of phytolacin differs from that of papain.