Purification and Properties of API–2b→API–2c Converting Protease from Streptomyces griseoincarnatus Strain No. KTo–250, an API–2 Producer

Among three alkaline protease inhibitors (API-2a, -2b, -2c) produced by Streptomyces griseoincarnatus strain No. KTo-250, API-2b was converted to API-2c in the growing system.

The cultural conditions were examined exclusively for the production of API-2b→ API-2c converting protease in the culture filtrate. The protease was purified about 1080-folds by salting-out with ammonium sulfate, column chromatography on DEAE-cellulose and gel filtration on Sephadex G–100.

The optimal and maximal caseinolytic activities of the protease were around pH 9.0 and at 28°C, respectively. The protease activity was inhibited by EDTA and DFP, but not by PCMB, o-phenanthroline, TPCK, TLCK, AP-I and S-SI. The protease was a DFP and EDTA-sensitive alkaline protease, and it required Ca²⁺ ion for its activity and stability.     

Partial Purification of Intra- and Extra-cellular Cellulases from Pyricularia oryzae: with Reference to Optimum pH at Alkaline Side

In order to elucidate the relation between the difference in cellulase activity among various strains of P. oryzae and the optimum pH at alkaline side, and also to know the relation between the intra- and extra-cellulases, the elution patterns of the enzymes from the Sephadex G–100 column were compared and the occurrence of the enzyme fractions showing the optimum pH at alkaline side was investigated.

The elution patterns of the intracellular cellulases were shown to be relatively constant, but those of the extracellular enzymes did not. The peak e appeared comparatively constant, but the peak c was considered to undergo some change during the excretion into the medium.

The optimum pH at alkaline side was shown to occur in the peak e among five peaks on Sephadex G–100 of the partially purified intra- and extra-cellular cellulases. The peak seems to be significant for P. oryzae.     

Purification and Some Properties of ATP: Nucleotide Pyrophospho-transferase of Streptomyces adephospholyticus

ATP: nucleotide pyrophosphotransferase was purified from culture filtrate of Streptomyces adephospholyticus A–4668 about 13,000 fold by the method including ammonium sulfate fractionation, Amberlite IRC–50 treatment and column chromatography with DEAE-cellulose, DEAE-Sephadex A–25, SP-Sephadex C–25 and Sephadex G–75. The purified enzyme was homogenous on disk gel electrophoresis and ultracentrifugation and the specific activity was 915 units per mg protein, The molecular weight was determined as 28,000 by gel filtration on Sephadex G–75. The enzyme was found to be stable in the pH range of 5.5 to 10.5. More than 80% of the activity was remained after heating at 60°C for 30 min. The enzyme exhibited maximum activity at 50°C.     

Purification and Some Properties of Trametes sanguinea Rhodanese

A fungal rhodanese from the spray-dried powder of a culture filtrate of Trametes sanguinea was purified to 142-fold by ammonium sulfate precipitation and DEAE-cellulose and Sephadex G–100 column chromatography. The purified rhodanese (pI 5.10) showed a single band on disc electrophoresis, and its molecular weight was estimated to be 51,700 by gel filtration technique. The enzyme had a broad pH optimum between 7.5 and 8.5 and was stable at pH values from 4 to 8 at 30°C for 44 hr. Its activity was inhibited by p-chloromercuribenzoate at pH 9.5, but not at pH 8.0, and was inhibited by cysteine, β-mercaptoethanol and sodium borohydride at pH 8.0. Both thiosulfate and cyanide showed substrate inhibition at high concentrations. Dihydrolipoate and benzenethiosulfonate were also good substrates.     

Complex Formation of Microbial Alkaline Protease Inhibitor (S-SI) with Subtilisin BPN′ and Its Properties

Microbial alkaline protease inhibitor, S–SI, was investigated on the interaction with subtilisin BPN′ Inhibitory equivalent of S–SI to subtilisin BPN′ was determined that one molecule of S–SI (MW = 23,000) inhibited two molecules of subtilisin BPN′ (MW = 27,700). The S–SI-subtilisin BPN′ complex was isolated by gel filtration on Sephadex G–100 and rhombic crystals were obtained. DIP- and ZAGPCK-subtilisin BPN′ did not form such complex with S–SI. Homogeneity of the complex was determined by disc electrophoresis. The isoelectric point of the complex was pH 5.5. Assay of S–SI dissociated and amino acid analysis of the complex indicated that one subunit (a half molecule) of S–SI was combined with one molecule of subtilisin BPN′ From molecular weight determination, it was clarified that the complex was composed of one molecule (consist of 2 subunits) of S–SI and two molecules of subtilisin BPN′.     

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate

Maleate cis-trans isomerase in Alcaligenes faecalis IB–14 was induced by malonate and purified about 100-fold over the crude cell-free extract by treatments of ammonium sulfate fractionation, Sephadex G–100 gel filtration, DEAE-cellulose and DEAE-Sephadex A–50 column chromatography. The preparation was shown to be monodisperse on ultracentrifugal analysis and Svedberg value was found to be 3.84 S.

The enzyme was most active at pH value around 8.3 and was stable over the range of pH 5.0 to 7.0 in the presence of dithiothreitol (DTT) for a few weeks, but in the absence of it, the enzyme activity was markedly decreased, especially in the alkaline region. The enzyme activity was inhibited by various sulfhydryl reagents and oxidizing agents, whereas it was not affected by metal chelating agents. The inhibition by Hg²⁺ and PCMB was overcome by the addition of sulfhydryl compounds such as DTT, 2-mercaptoethanol, l-cysteine and glutathione. It was observed that the enzyme did not require co-factor for its function.

Kinetic studies showed that Michaelis constant for maleate was 2.8×10^?3 m and the enzyme did not catalyze the reverse reaction.     

Isolation and Identification of Spawning Inhibitors from Ovaries of the Starfish,Asterias amurensis

An extracellular alkaline proteinase produced by Candida lipolytica was purified through iso-propanol and ammonium sulfate precipitation, decolorization with DEAE-cellulose, gel filtration with Sephadex G–100 and ion-exchange chromatography on DEAE-Sephadex A–50. The optimum pH of its caseinolytic activity was 9.0, and this activity was completely inactivated with DFP but not with chelating reagents, PCMB, STI, TLCK, TPCK, or SSI. This enzyme also hydrolyzed salmin and synthetic esters, such as Bz. Arg. OEt, Bz. His. OMe, Tos. Lys. OMe or Ac. Tyr. OEt, and the optimum pH of its esterase activity was 8.0. The molecular weight of the enzyme was estimated to be about 30,000 by the gel filtration method. These facts indicated that this enzyme was distinguishable from other microbial alkaline proteinases so far studied.     

Purification and Properties of Lipase Activator Produced by Streptomyces sp. Strain No. BR-1381

To obtain actinomycetes capable of producing new enzyme affectors such as enzyme inhibitors or activators, a screening test was carried out. Streptomyces sp. strain No. BR-1381 isolated in our laboratory produced a proteinous lipase activator abbreviated as LAV. LAV was purified from the culture filtrate by salting-out with ammonium sulfate, DEAE-cellulose column chromatography and gel filtration on Sephadex G-100. LAV was stable in the pH range from 3 to 7 at 37°c for 20 hr and in a wider range of pH at 4°C for 5 days. LAV itself was very stable against heat treatment, but LAV did not have any effect on the thermal stability of Phycomyces nitens lipase. LAV activated several microbial lipases, but did not activate pancreatic or rice bran lipases. LAV particularly showed strong activation for Phycomyces nitens lipase.     

Purification and Some Properties of β-Xylosidase from Malbranchea pulchella var. sulfurea No. 48

A β-xylosidase of a thermophilic fungus, Malbranchea pulchella var. sulfurea No. 48, was purified 99-fold from the culture filtrate after ammonium sulfate fractionation, DEAE-cellulose column chromatography, column electrophoresis and gel filtration on Sephadex G–200. The purified enzyme was found to be homogeneous upon ultracentrifugal analysis, disc electrophoresis and gel filtration. The molecular weight of the enzyme was estimated to be 26,000 by gel filtration, and the sedimentation coefficient was calculated to be 2.78S. at 280 nm in phosphate buffer (pH 6.7) was 13.2. The optimum pH was found to be in the range of 6.2~6.8, and the optimum temperature was 50°C.     

A thermophilic extracellular -amylase from Bacillus licheniformis

A strain of Bacillus licheniformis isolated from soil produced an extracellular α-amylase(s) with unusual characteristics. The enzyme was purified 126-fold by starch adsorption, DEAE-cellulose treatment, and CM-cellulose column chromatography. Four active protein bands were detected by disc electrophoresis in poly-acrylamide gel although the enzyme behaved as a single peak during both ultracen-trifugation and chromatography using CM-cellulose and Sephadex G-100. The enzyme showed a very broad pH-activity curve and had substantial activity in the alkaline range. The optimal temperature was 76 °C at pH 9.O. The enzyme was stable between pH 6 and 11 at 25 °C, and below 60 °C at pH 8.0. Using Sephadex G-100 gel filtration, a molecular weight of 22,500 was estimated for the enzyme. The action pattern on amylose and amylopectin is unique in that the predominant product during all stages of hydrolysis is maltopentaose.     

Production and Purification of a New Chillproofing Enzyme and Its Properties

Chillproofing enzyme was obtained from broth cultures of Serratia marcescens B–103. This extracellular enzyme, tentatively, named the S-enzyme was highly purified from the culture supernatant by ammonium sulfate precipitation, ethanol fractionation, gel filtration on Sephadex G–200 and column chromatography on DEAE-Sephadex A–50.

The purified preparation appeared homogeneous on a ultracentrifugation with a sedimentation coefficient of 3.14 S and a molecular weight of 38,000~45,000 determined by the method of Whitaker.

The S-enzyme hydrolyzed various proteins at pH 4~6 and at low temperature hydrolyzed nitrogenous substances which may cause chill haze in beer. So the chillproofing activity of the S-enzyme may be due to its proteolytic activity.

The S-enzyme was stable at 4°C at pH 5~10.5. It was completely inactivated by heating at 60°C for 10 min, and was inactivated by Hg²⁺ and Pb²⁺ and activated by Mn²⁺, Ca²⁺. Mg²⁺ and Zn²⁺     

Purification of an β-D-(1→3)-Glucanase from Rhizopus niveus

An β-D-(l→3)-glucanase has been purified from the culture medium of Rhizopus niveus. The purification involves calcium acetate treatment, polyethylene glycol 6000 fractionation, CM-cellulose batch treatment, DEAE-cellulose column chromatography and gel filtration on Sephadex G–150.

The final preparation is homogenous on the basis of discelectrophoresis on acryl amide gel, sedimentation in the ultracentrifuge.

Some properties of the purified enzyme have been also tested.     

Purification and Properties of Acid Carboxypeptidase IV from Aspergillus oryzae

Acid carboxypeptidase IV from Aspergillus oryzae was purified from the rivanol precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A–50, hydroxylapatite and P-cellulose and gel filtration through Sephadex G–100. The optimum pH is at pH 3.0 for carbobenzoxy-l-glutamyl-l-tyrosine. The enzyme activity was inhibited by sulfhydryl reagents and diisopropylphosphorofluoridate, but was not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 43,000 by gel filtration method.     

Purification,Crystallization and Some Properties of Extracellular Pullulanase from Aerobacter aerogenes

Extracellular pullulanase was purified and crystallized from the culture fluid of Aerobacter aerogenes. Pullulanase was purified by means of ammonium sulfate fraction, DEAE-cellulose column chromatography and Sephadex column chromatography. Crystalline pullulanase was formed when saturated ammonium sulfate solution was added to the purified enzyme solution. The crystalline enzyme appeared as colorless fine rods. On ultracentrifugation analysis, the enzyme showed a single sharp and symmetrical Schlieren peak. The sedimentation coefficient, s_20,w was 4.39S. Polyacrylamide gel electrophoresis at pH 8.4 gave a main band with two sub-bands and the molecular weight of the main enzyme was estimated to be 66,000 from Polyacrylamide gel electrophoresis and to be 58,000 from sedimentation equilibrium. The optimum pH and temperature for the enzyme action were pH 6.5 and 50°C, respectively.     

Serratia Protease

A strain of Serratia, isolated from an intestinal canal of a silkworm, produced a large quantity of protease. The enzyme was extracellular and was named Serratiopeptidase, tentatively. Protease production of this strain was over 3 times as much as that of Serratia marcescens which was known as a protease-producing organism. The highly purified enzyme was prepared from the culture supernatant through ammonium sulfate precipitation, acetone fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-75.

The purified enzyme moved homogeneously with a sedimentation constant, s_20,w of 3.8 S in ultracentrifugation and the molecular weight was determined to be 6.0 × 10₄ by the Archibald method. Determination of the ultraviolet absorption spectrum indicated the E^1%_{280 mμ,1 cm} was 13.0. Neither carbohydrate nor sulfur-containing amino acid was detected in the purified enzyme preparation. The enzyme showed maximal activity at pH 9.0 and at 40°C, and was stable under lower temperatures over the pH range from 5 to 10, whereas it was unstable at 37°C in alkaline conditions. The enzyme was completely inactivated by heating at 55°C for 15 min.     

The Isolation of Collagenase and Its Enzymological and Physico-chemical Properties

A collagenolytic enzyme specific for native collagen and gelatin was isolated from Pseudomonas marinoglutinosa by DEAE-cellulose column chromatography, Sephadex G–150 gel filtration and by disc electrophoresis on polyacrylamide gel.

The molecular weight of the enzyme was approximately 74,000 and its isoelectric point was found to be around 4.5. The optimum pH and temperature for Z–GPLGP hydrolysis were around 7.6 and 38°C, respectively. The enzyme was rather stable up to 50°C and in the range between pH 5.0 and 10.0, and was stabilized by Ca²⁺ to some extent. Some chelating agents and metal ions such as Hg²⁺, Pb²⁺, Zn²⁺, Ni²⁺ and Fe²⁺ inactivated the enzyme, but diisopropyl phosphofluoridate, sulfhydryl agents and some trypsin inhibitors did not affect the activity.

The EDTA-inactivated enzyme was restored its activity by added Ca-salt to almost completely and very slightly by Co-, Mn- and Sr-salt.

Metal analysis showed the enzyme contained 1 g atom of zinc and 4 g atoms of calcium per mole.     

Purification and some properties of trehalase from Chaetomium aureum MS-27

The trehalase of Chaetomium aureum was purified about 196-fold with a yield of 51% from the culture filtrate by ammonium sulfate fractionation, DEAE-cellulose column chromatography, acetone fractionation, and Sephadex G-100 gel filtration. The enzyme preparation was homogeneous on disc electrophoresis. The enzyme was most active at pH 4.0 and 50°C. The enzyme was stable from pH 4.0 to 9.0 on 12 h incubation at 37°C. The molecular weight of the enzyme was estimated to be 450,000 by gel filtration on a column of Sepharose 6B, and 115,000 by SDS polyacrylamide gel electrophoresis. This indicated that the enzyme might consist of 4 subunits. The isoelectric point of the enzyme was pH 4.0. The enzyme was active specifically on trehalose and not active on the other disaccharides tested.     

Purification,Crystallization and Some Properties of Lipase from Geotrichum candidum Link

Lipase (EC 3.1.1.3) of Geotrichum candidum Link was purified by means of ammonium sulfate fractionation, DEAE-Sephadex column chromatography, gel-filtration on Sephadex G–100 and Sephadex G–200, and was finally crystallized in concentrated aqueous solution. It was confirmed that the crystallized preparation was homogeneous electrophoretically and ultracentrifugally.

It was estimated with the crystalline enzyme that the sedimentation constant (s₂₀, w) was 4.0, the isoelectric point was pH 4.33, and the molecular weight was 53,000~55,000. From the result of amino acid analysis, none of sulfur containing amino acid was detected in the enzyme. It was also recognized that the crystalline preparation contained about 7% of the carbohydrate and very small amount of lipid. It was characterized that the lipase was the most active at pH 5.6~7.0 on olive oil, at 40°C and was stable in the range of pH 4.2 to 9.8 at 30°C for 24 hr, and was stable below 55°C for 15 min.     

Studies on α-Glucosidase in Rice

Two kinds of αglucosidase which were homogeneous in disc electrophoretic and ultra-centrifugal analysis were isolated from rice seeds by means of ammonium sulfate fractionation and CM-cellulose, Sephadex G–100 and DEAE-cellulose column chromatography and designated as α-glucosidase I and α-glucosidase II.

Both α-glucosidases hydrolyzed maltose and soluble starch to glucose and showed same optimal pH (4.0) on the both substrates. In addition, both enzymes acted on various α-linked gluco-oligosaccharides and soluble starch but little or not on α-linked hetero-glucosides and α-l,6-glucan (dextran).

Activity of the enzymes on maltose and soluble starch was inhibited by Tris and erythritol. α-Glucosidase II was more sensitive to the inhibitors than α-glucosidase I.

Km value for maltose was 1.1 mM for α-glucosidase I and 2.0 mM for α-glucosidase II.     

Isolation and Crystallization of Extracellular 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum nov. sp.

Among about 500 strains tested, a newly isolated soil bacterium, Brevibacterium sterolicum nov. sp. KY 3463 (ATCC 21387) showed the highest potency in production of 3β-hydroxysteroid oxidase in the culture fluid.

The 3β-hydroxysteroid oxidase was purified from the culture filtrate by a procedure involving ammonium sulfate fractionation, DEAE-cellulose and hydroxyapatite column chromatographies and Sephadex G–75 gel filtration. Crystals of the enzyme were obtained from solutions of the purified preparation by the addition of ammonium sulfate. The crystals appeared as fine rods, with a bright yellow color.

The enzyme is homogeneous by disc gel electrophoresis and ultracentrifugation. Sedimentation velocity yields a value of . It exhibits a typical flavoprotein spectrum of absorption maxima at 280, 390, and 470 mμ.