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Four mannanases (Mannanases I, II, III, and IV) were isolated from the culture filtrate of a Streptomyces sp. by ion exchange chromatography. Mannanase IV was the main component and accounted for 64.4% of the total activity of the four mannanases. Mannanase IV was further purified by gel filtration, and the purified Mannanase IV was homogeneous on disc-gel electrophoretic analysis.Optimum pH and temperature for the activity of Mannanase IV were 6.8 and 57°C, respectively. It was stable at temperatures up to 45°C when examined at pH 6.8 for 30min, and lost only 15% of its activity at 70°C for 30min at pH 6.8. The isoelectric point and molecular weight were pH 3.65 and 42,900, respectively. The enzyme was strongly inactivated by Al3+, Hg2+, Fe2+, Fe3+, Cd2+, Ag+, Sn2+, and Cu2+, and completely inhibited by iodoacetic acid and N-bromosuccinimide. The enzyme hydrolyzed mannotriose to mannose and mannobiose, but did not hydrolyze mannobiose.

A β-mannanase was purified from the culture filtrate of Penicillium purpurogenum No. 618 by 1st and 2nd DEAE-cellulose column chromatographies, and subsequent Ultro-gel chromatography. The final preparation thus obtained showed a single band on polyacrylamide disc-gel and SDS-polyacrylamide gel electrophoresis. The molecular weight and isoelectric point were determined to be 57,000 and pH 4.1 by SDS-polyacrylamide gel electrophoresis and isoelectric focusing, respectively. The purified mannanase contained the following amino acids: glycine > serine >glutamic acid > alanine > aspartic acid. The mannanase exhibited maximum activity at pH 5 and 70°C, and was stable in the pH range of 4.5 to 8 and at temperatures up to 65°C. The enzyme activity was not affected considerably by either metal compounds or ethyl- enediaminetetraacetic acid. Copra galactomannan (Gal: Man =1 :14) was finally hydrolyzed to galactose, mannose and β-1,4-mannobiose through the sequential actions of the purified mannanase and the α-gala...

Milk-clotting enzymes may be classified into two groups according to their degradation pattern of αs1-casein in solution at pH 6.0. On the one hand, calf chymosin and Mucor miehei enzyme produced only one degradation product corresponding to αs1-I under the conditions we employed. On the other hand, Irpex lacteus and Endothia parasitica enzymes produced several degradation products accompanied by a product corresponding to αs1-I. The Irpex milk-clotting enzyme hydrolyzed αs1-casein at the positions of His(8)-Gln(9), Phe(23)-Phe(24), Lys(103)-Tyr(104), and Phe(153)-Tyr(154). Irpex enzyme has only one common cleaving site with calf chymosin, that is, the Phe(23)-Phe(24) bond of αs1-casein.

To clarify the substrate specificity of the milk-clotting enzyme excreted by Irpex lacteus (IR), various peptide hormones were used as substrates to find the cleavage sites and to compare the specificity with those of other milk-clotting enzymes such as calf chymosin (CR), Mucor miehei enzyme (MR), and Endothia parasitica enzyme (ER).IR hydrolyzed substance P, physalaemin, β-neo-endorphin, neurotensin, angiotensin I, LHRH, and DSIP, but not bradykinin. Although IR generally require hydrophobic amino acids in the P1 and P1’ sites, it also hydrolyzed peptide bonds which have only one hydrophobic amino acid either in the P1 or P1’ site.CR did not hydrolyze either angiotensin I or DSIP, and MR did not hydrolyze DSIP, but both enzymes hydrolyzed the Tyr(5)–Glu(6) bond of LHRH slightly. On the other hand, ER hydrolyzed these substrates at several bonds. Besides the common cleavage sites with IR, ER hydrolyzed the His(6)–Pro(7) bond of angiotensin I, the Leu(7)–Arg(8) bond of LHRH, and the Ala(2)–Gly(3) and Gly(...

A coagulation test of soymilk-protein was carried out with various commercial proteinases originating from microorganisms, plants and animals. The soymilk-protein was coagulated by the following proteinases: Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus polymyxa, Streptomyces griseus, Streptomyces caespitosus, Aspergillus oryzae, Aspergillus sojae, Endothia parasitica, Rhizopus sp., Mucor miehei, bromelain, papain and trypsin. However, such proteinases as Aspergillus saitoi, rennin and pepsin did not coagulate the protein. The coagulability of enzymes decreased as the pH of the soymilk was increased from 5.9 to 6.7. The optimum temperature for soymilk-clotting activity varied widely with the enzyme origin; for example, 55°C in the case of proteinases of Bacillus polymyxa and Rhizopus sp., 65°C in that of Aspergillus oryzae, Aspergillus so jae and trypsin, 75°C in that of Bacillus amyloliquefaciens, Streptomyces griseus, Streptomyces caespitosus and Endothia paracitica, and 85°C in that of Bacill...

A rapid and large scale isolation of chymosin was achieved by an affinity column including pepstatin-aminohexylagarose with a column of DEAE-cellulose. The affinity column with 3 ml wet gel made it possible to isolate 90 mg pure chymosins (I, II and III) from 20 g crude rennet tablet. Isoelectric points of chymosin-I, -II and -III were 4.6, 4.6 and 4.5, respectively. Each of chymosins was homogeneous electrophoretically and chromatographically.

To find the primary sequence of Irpex lacteus aspartic proteinase (ILAP), a cDNA library of J. lacteus mRNA in pBR322 was constructed. A clone, which had an insert size of about 1.2 kilobase pairs, was found to contain the coding region of the mature enzyme. The deduced amino acid sequence showed that the enzyme consisted of 340 amino acid residues with a molecular weight of 35,000. Cysteine and methionine were not found in the enzyme and two putative /V-glycosylation sites were indicated. The lack of S-S bridges in the molecule is a striking feature of the enzyme. The alignment of the sequence of the enzyme against other aspartic proteinases revealed homology around the active site aspartic acid residues.

A comparative investigation was carried out on functional properties of three kinds of soymilk curds, which were prepared with an enzyme, calcium chloride and an acid. The enzyme-curd showed high emulsion stability over wide ranges of pH and temperature, whereas the Ca- and acidcurds produced lower values at pH 4-6 and temperatures higher than 40°C. The foam stability of each soymilk curd was low, but the enzyme-curds prepared from defatted soymilk had higher foam stability than the others over wide ranges of pH and temperature. The lyophilized enzyme soymilk curd solubility in water was higher than the others, although its water-holding capacity was lower than the others. None of the soymilk curds showed gelation. The experimental results suggest that enzyme-curd could be widely applicable to various food items with its properties of a remarkably high emulsion stability and a very smooth texture.

Enzymes I and II, which have a high soymilk-clotting activity, produced from K-295G-7 were purified by chromatographies on Sephadex G-100, CM-cellulose, hydroxylapatite, and 2nd Sephadex G-100. The two purified enzymes were found to be homogeneous by polyacrylamide gel electrophoresis (PAGE) at pH 4.3. The molecular weights of enzymes I and II were 28, 000 and 29, 500 by SDS-PAGE, and their isoelectric points were 9.22 and 9.45, respectively. Enzymes I and II coagulated soymilk optimally at 65°C and were stable up to 45°C. Both enzymes were most active at pH 5.8, for soymilk coagulation between pH 5.8 to 6.7, and were stable with about 50-100% of the original activity from pH 5 to 10. Each of the purified enzymes was a serine protease with an optimum pH of 9.0 for soy protein isolate (SPI) and casein digestions, because these enzymes were inhibited completely by diisopropylfluoro-phosphate (DFP). The soymilk-clotting activity to proteolytic activity ratio of the enzyme II was 3 times higher than that of enzyme I. Enzymes I and II were more sensitive to the calcium ion concentration in soymilk than bromelain is.

A soymilk-clotting efficiency test was carried out with various commercial proteinases. The efficiency was relatively high in the cases of proteinases originating from Bacillus amyloliquefaciens, Bacillus subtilis, Aspergillus oryzae, Aspergillus sojae, Bacillus thermoproteolyticus and bromelain, while it was relatively low in those of Endothia parasitica, Rhizopus sp., Mucor miehei, papain and trypsin. Of the enzymes investigated, proteinases showing high proteolytic activity as to soymilk-protein also exhibited high soymilk-clotting efficiency. Such coagulation of soymilk-protein increased with the addition of CaCl2 or MgCl2 to the soymilk. The coagula, formed with enzymes showing high soymilk-clotting efficiency, could easily be separated into soymilk-curd and whey by centrifugation at 1600 × g for 2min. The volume ratio, after centrifugation of coagula formed at 5 min incubation with the enzymes, of soymilk-curd to whey was about 1:1, and the volume of the curd had a tendency to increase with increasing incubation time. The experimental results suggest that alkaline and neutral proteinases originating from microorganisms are suitable for making soymilk-curd on an industrial scale.

The milk-clotting enzyme fraction from Irpex lacteus (IR) was obtained by affinity chromatography. To evaluate IR as a calf rennet substitute, Cheddar cheese-making trials were done. There was no difference in cheese yield, protein recovery, or fat recovery between cheese made with calf rennet (CR) and that made with IR. Although IR cheese showed a slightly higher extent of proteolysis in comparison to the control during ripening, IR cheese did not develop a bitter taste even after 6 months of ripening. These facts indicate that IR is a promising rennet substitute for cheese-making.

Some microorganisms, including some bacteria isolated from soil, were found to secrete an extracellular soymilk-clotting enzyme. Among them, strain No. K-295G-7 showed the highest soymilk-clotting activity and stability of the production of the soymilk-clotting enzyme. The enzyme system (culture filtrate) coagulated protein in soymilk, a curd being formed at pH 5.8~6.7 and at 55~75°C. The optimum temperature for the soymilk-clotting activity was 75°C and the enzyme system was stable at temperatures below 50°C down to 35°C. About 80~100% of the original activity remained after 1 hr at pH 5~7 and 35°C.

A pepstatin-insensitive carboxyl proteinase of Polyporus tulipiferae (formerly Irpex lacteus) was purified by methods including affinity chromatography with chymostatin as the ligand.Although the enzyme’s maximum proteolytic activity on hemoglobin was at pH 2.8, it was not affected by carboxyl proteinase inhibitors such as DAN, EPNP, and pepstatin. On the other hand, the enzyme was inhibited by chymostatin competitively and its Ki value was estimated to be 1.6×10−5 m. The enzyme was very heat labile and was inactivated completely at pH 4.6 by heating at 45°C for 15min. Polyporus enzyme and Scytalidium lignicolum enzyme A1 hydrolyzed the same peptide bond of Z-tetrapeptides, but their primary specificities were slightly different. Polyporus enzyme as well as Ganoderma lucidum enzyme contains histidine, and the amino acid compositions of Polyporus enzyme and other pepstatin-insensitive carboxyl proteinases resembled each other.

Etude de l'activite de 2 enzymes de Bacillus sur la sous-unite acide isolee de farine de soja delipidee pour expliquer leur mode d'action dans la coagitation. Mesure de l'evolution de la durabilite et de la degradation a la proteine par electrophorese sur gel, en presence de SDS

(1988). Coagulation of Proteins in Leguminous Milk by Commercial Proteinases. Agricultural and Biological Chemistry: Vol. 52, No. 5, pp. 1317-1318.