Crude Enzyme Solution Research Articles

DETERIORATION OF POLYURETHANE FILAMENTS WITH FUNGAL ENZYMES

Deterioration of polyurethane yarn was investigated with crude enzyme solution of fungi.Two types of polyurethane yarns including ether type only or ester type mainly in the part of soft structure were tested. Two fungi, Aspergillus niger FERM J-1 and Cladosporium cladosporioides FERM J-8 were employed. The test pieces of yarns were immersed in the enzyme solutions from the fungi at given pH (3.5, 4.0, or 6.5). The mixture was shaken at 30°C for two weeks. The incubated yarns were observed by scanning electron micrography and some physical properties of the yarns were examined. Furthermore, the chemical changes of the yarns and the liberations of carboxyl group and ammonia were measured.The tensile strength, viscoelasticity and extension behavior were remarkably changed. On the enzymatic digestion, various types of degradation were caused by the hydrolysis of several different type bonds in these polymers. Protease and esterase activities were in Asp. niger and Clad. cladosporioides. Urease was only detected in Asp. niger. These enzymes were inducible enzymes. Hydrolysis took place with enzymes of endo type.

A BETACYANINE DECOLORIZING ENZYME FOUND IN RED BEET TISSUE

ABSTRACTThe activity of a betacyanine decolorizing enzyme present in raw beet tissue has been confirmed. The enzyme is postulated to be bound to the inner membrane of the cell wall. Comparison between the rate of betacyanine degradation for solutions containing the crude enzyme extract and solutions of identical pH and temperature void of enzyme indicated optimum enzyme activity at pH 3.4 and 40°C. Sample evaluations were made from 22–55°C over a pH range 2.0–4.8. Minimal enzyme activity was noted at pH and temperature extremes. The activity of this enzyme must be considered as an important factor in the extraction of raw beets and during storage and food product utilization of beet juices in that residual enzyme activity may decolorize substantial amounts of the betacyanine pigments. The enzyme has been found present in diffusion‐type beet juice.

Purification of phospholipase C from Bacillus cereus by hydrophobic chromatography on palmitoyl cellulose.

Phospholipase C (phosphatidylcholine choline-phosphohydrolase, EC 3.1.4.E) from Bacillus cereus (IAM-1208) was adsorbed to palmitoyl cellulose from a crude enzyme solution at pH 5--9. The adsorption was not influenced by ionic strength up to 2 M NaCl. The adsorbed enzyme was eluted almost completely by washing the cellulose with a suitable detergent, such as Triton X-100, Adekatol SO-120, Cation DT-205, or sodium deoxycholate. The enzyme was then purified by column chromatography on a palmitoylated textile (palmitoylated gauze) with an overall recovery of 91% and a 467-fold increase in specific activity over that of enzyme in the crude culture supernatant. Subsequent fractionation with acetone and chromatography on a Sephadex G-75 column separated two nearly homogeneous phospholipase C's. The enzyme adsorbed on palmitoyl cellulose was active, although its activity was about one-fourth that of free phospholipase C. Therefore, the enzyme appeared to be adsorbed to the cellulose through a hydrophobic site that was distinct from the catalytic site on the enzyme molecule.

A debranching enzyme of rice seeds. I. A debranching enzyme of rice seeds at milky stage, its purification and substrate specificities.

A debranching enzyme was purified about 100-fold over the crude enzyme solution from non-glutinous rice seeds at the milky stage by using DEAE-, CM-cellulose, and then Sephadex G-100 column chromatography. This disc-electrophoretically homogeneous enzyme showed a specific activity of 16.5 pullulanase units/mg of protein (25°C) with its optimum pH at 5.6. The enzyme debranched the α-1, 6-linkages in pullulan or an α-amylolysis product from starch most favorably, several β-limit dextrins from starch or from amylopectin rapidly, and phytoglycogen β-limit dextrin and amylopectin moderately, while it was unable to debranch phytoglycogen. These substrate specificities were similar to those of the so-called “limit de0x-trinases” already reported with respect to several plant sources.

Purification of lipase from Chromobacterium viscosum by chromatography on palmitoyl cellulose.

Palmitoyl cellulose was used to adsorb the extracellular lipase [triacylglycerol acyl-hydrolase EC 3.1.1.3] of Chromobacterium viscosum from crude enzyme solution, and the adsorbed enzyme was eluted with a suitable detergent, such as Adekatol 45-S-8 or Triton X-100. The enzyme was then purified by chromatography on a palmitoylated gauze column with an overall recovery of 71% and an increase in the specific activity of 11-fold from the supernatant fluid of bacterial cultures. Further purification procedures included fractionation with acetone, and chromatography on Sephadex G-150 and G-75 columns. Two isoenzymes were obtained, each in a homogeneous state on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: one had a molecular weight of 120,000 and pI of 3.7 and the other a molecular weight of 30,000 with a pI of 7.3.

<i>Streptococcus mutans</i> が産生する不溶性グルカンを分解する酵素について

An attempt was made to search for the enzymes that degrade insoluble glucans (IG) produced by Streptococcus mutans strain JC2. By screening many soil microorganisms and a veriety of fungi strains, it was found that Streptomyces nigrifaciens ISP 5071 produces these enzymes.Culture conditions for the enzyme production were tested with the Streptomyces. The enzyme was produced only in the culture containing IG as an inducer. A maximum production of the enzymes was obtained in the culture supernatant when the spore was inoculated in the basic medium containing 0.3% IG, 0.4% glycerol and cultured at 27°C for 4 days under rotary shake of 160 per min.The crude enzyme solution was prepared from the culture supernatant by precipitation on 30-50% saturation of ammonium sulfate. The degradation activity of the crude enzyme solution toward some glucans was studied.The results were as follows.1) The crude enzyme solution readily degraded IG and a modified-IG which consisted only of α-(1→3) glucosidic bond.2) The crude enzyme solution split IG synergistically in combination with dextranase.3) Paper chromatographic analysis of the enzymatic digests from IG and modified-IG showed that nigerose was the main reaction product, and that practically no isomaltose and its oligosaccharides were present.4) The activity of dextranase was not detected in the crude enzyme solution. These results strongly suggested that the crude enzyme solution contains a certain α-(1→3) glucanohy drolase and not α(1→6) glucanohydrolase.

Alkaline protease in the larvae of the army worm, Spodoptera litura

The alkaline protease activity in the gut of Spodoptera litura was found to increase with the development of larvae and decreased with the onset of pupation. Fasting of the 5th instar larvae caused a slight increase in protease activity at 4 hr, which declined consistently on further starvation. The optimum pH for the gut protease was 11.0, with a shoulder between pH 8.0 and 9.0. The protease was inactivated upon dialysis of the crude enzyme solution at room temperature but not at 4°C. Incubation of the crude enzyme solution at pH 11.0 and 37°C for 22 hr resulted in a three-fold rise in specific activity of the alkaline protease which declined on further incubation. The three-fold purified preparation, obtained by incubation of the crude enzyme solution, was passed through Sephadex G-75 to give a seven-fold purification and 70% yield. The preparation was not completely homogeneous and showed three clearly separable protein bands by polyacrylamide gel electrophoresis. The partially purified protease exhibited no shoulder between pH 8 to 9, like the crude preparation.

Variation in the virulence of Xanthomonas oryzae. II. Difference in protease activity of X. oryzae isolates.

Protease activity of various X. oryzae isolates including N7301 mutant clones and several wild types was investigated in relation with their virulence. Activity was assayed by halo-colony (H-C) ratio method on casein media, and also by ordinary Casein-Lowry method on crude enzyme solution obtained from culture filtrate.It was found that X. oryzae showed marked protease activity. The protease activity in crude enzyme solution was enhanced along with bacterial multiplication, and decreased after stationary stage of bacterial growth. The activity was stable at pH ranging from 6 to 9, optimum at pH 9.3, and seemed an alkaline protease. Several other properties of the enzyme were also investigated.Among wild-type isolates of X. oryzae, H-C ratio was considerably correlated with the virulence. Virulent isolates showed higher H-C ratio than less virulent ones, and also halos of virulent isolates were much clearer. Among N7301 mutant clones, however, the correlationship was not distinct. According to Casein-Lowry method, no close relationship was found between protease activity and bacterial virulence.

Subunit heterogeneity of acetylcholinesterase

Several different preparations of purified 11 S acetylcholinesterase have been examined for structural heterogeneity. While no contaminant protein was observed in any of the preparations, minor isozymic forms with catalytic activity were observed in addition to the major component both in polyacrylamide gel electrophoresis and in isoelectric focusing. Major differences in the relative composition of the disulfide-reduced polypeptides among the preparations were found by gel electrophoresis in sodium dodecyl sulfate. Several characteristics of these differences strongly suggest that they derive from a proteolytic fragmentation of a single subunit species. In particular, the apparent fragmentation in the crude enzyme solution is inhibited by benzethonium chloride, an inhibitor of proteolysis which also prevents the conversion of 18, 14, and 8 S acetylcholinesterase species to the 11 S form in fresh electric tissue extracts. No significant differences in the enzyme specific activity are observed among the preparations, an observation which indicates that fully active native enzyme molecules are composed of subunits which are heterogeneous with respect to discrete points of polypeptide cleavage.

カツオ筋肉の5'-ヌクレオチダーゼに関する研究-I

The crude enzyme solution was obtained by homogenizing bonito muscle in water, dialysing with water and centrifuging. The optimum pH of the crude enzyme solution was 5.0 and 9-10, and the activity at pH 6.0 was remarkably increased by the addition of 0.01M Mg2+. 5'-Nucleotidase was separated and purified from bonito muscle by extraction with water, ammonium sulfate fractionation, DEAE-cellulose column chromatography and Sephadex G-200 gel filtration. By ammonium sulfate fractionation (30-70% saturation), 54% of the total activities of 5'-nucleotidase were recovered. On purification by DEAE-cellulose chromatography, high specific activity found in the fraction of 0.005M Tris-acetate buffer containing 0.2MNaCI. Combined fractions were concentrated with 0.7 saturated (NH4)2SO4, purified by re-chromatography on DEAE-cellulose and gel filtration (Sephadex G-200). It was found that the purified 5'-nucleotidase of bonito muscle had increased about 215 fold in specific activity, and was recognized to be homogeneous on disc electrophoretic analysis.

Azo dye binding proteins as interfering factors in enzyme assays based on azo coupling

The azo coupling method used in the estimation of aminopeptidase and esterase activity is criticized. When N- l-aminoacyl-2-naphthylamines are used as substrates, certain proteins present in crude enzyme solutions as well as the amino acids liberated during the enzymic reaction severely interfere with the activity assay. Conditions are studied in which the azo dye binding caused by hemoglobin and albumin present in enzyme preparations produce the greatest interference. The effects described can lead to false fractionation patterns in column chromatography of amino-peptidases and esterases. The errors can be avoided when the azo dye is used in excessive amounts and if only small quantities of the proteins are present.

Studies on selective herbicide, CMMP (solan)

These studies were designed to obtain some informations on the mode of action of a selective herbicide, CMMP (solan) for tomato, carrot and mitsuba (Cryptotaenia japonica HASSK.).1. As CMMP-chloroplast complex was formed readily, CMMP was made to react with chlorophyll, one of the chloroplast components. The reaction of CMMP with chlorophyll took place apparently.2. CMMP may be able to react with the porphyrin ring of chlorophyll, because the reaction of CMMP with chlorophyllin was recognized.3. Reactions of CMMP with extracts from several resistant and susceptible etiolated plants were investigated. It was therefore assumed that CMMP reacted with protein or protochlorophyll in plants. Besides, CMMP apparently reacted with bovine alubumin from blood.4. The selective action of CMMP for some plants was examined by detecting the difference of combining ability of crude enzyme solution with CMMP or CMA (3-chloro-4-methylaniline). It was reaffirmed that the detoxic hydrolysis of CMMP in the enzyme solution did not take place at all even in resistant plants.5. The recovery of injury by application of chlorophyllin after spraying with CMMP was much more remarkable for resistant plant (tomato) than for susceptible plant (egg plant).6. The application of chlorophyllin after spraying with CMMP was more effective than the application of glucose, sucrose or glucuron for recovery of injury by CMMP.

Studies on cutin-esterase II. Characteristics of cutin-esterase from Botrytis cinerea and its activity on tomato-cutin

The activity of cutin-esterase, cutinase, was detected in the mycelial homogenate of Botrytis cinerea cultured in a peptone-sucrose medium at 25°C for 7 days. The crude enzyme solution was prepared from the homogenate by centrifugation at 106,600×g, treatment with (NH4)2SO4 at 70% saturation, and dialysis against 0.01 M phosphate buffer. The optimum pH, temperature and assay duration for enzyme activity were 5.0, 25°C and 18 hr, respectively. Specific activity was 255 mµmoles/mg protein/18 hr as palmitate under optimum conditions. 83% of the activity was lost by heating the enzyme solution (pH 7.6) for 4 min at 95°C. Palmitic, stearic, oleic, 9, 10-dihydroxystearic or linoleic, dihydroxyeicosanoic and octadecanedioic acids were recognized in the enzymic hydrolysate of tomato-cutin using gas-liquid chromatography. Among these fatty acids, palmitic, oleic and octadecanedioic acids were readily liberated by the enzyme, but dihydroxyeicosanoic acid, the major component of tomato-cutin, was isolated only in small amounts. The enzyme is, therefore, an exo-type cutinase which hydrolyses minor side chains of fatty acids bound to the major structure of cutin. Cutinesterase may facilitate cuticular invasion by fungi as a result of reduction in mechanical strength of the cuticle by the enzyme

Inorganic Pyrophosphase Activities of Serum and Human Tissue Extracts

The inorganic Pyrophosphatase activities of sera from human and rat were assayed as substrate 3.0mM sodium pyrophosphatase in 50mM Tris-HCl-1mM MgCl2, pH 8.5.The result of this work was reported in the 42nd Congress of the Japanese Orthopedic Association in April, 1969.In the present paper, the inhibitions of pyrophosphatase activity by heat, urea and EDTA-2Na were examined with reference to alkalne phosphatase activity, employing the homogenates of human tissue extracts as the crude enzyme solution. Heat inactivation: the enzyme solution with MgCl2 and H2O was incubated for 5, 10, 15min. at 56°C. After 3min. cooling in ice bath, the remaining activity was assayed. Effects of urea and EDTA: the enzyme solution was preincubated with urea or EDTA-2Na of various concentration for 15min. at 37°C, and then activity was assayed. The results;The pyrophosphatase activities in both patient serum and tissue extracts were parallel with those of alkaline phosphatase.The alkaline phosphatase/pyrophosphatase ratios of itssue extracts were 7 to 9 except for intestine extract. In heat inactivation and effect of urea, pyrophosphatase activities of placental extract and serum of pregnant woman were most stable, whereas those of bone extract and serum of skeletal disease were extremely inactivated.As compared with alkaline phosphatase, pyrophosphatase activity was more stable by heating and more inhibited by EDTA treatment. The result shows that the increased activity of inorganic pyrophosphatase in serum of bone diseases acts as a marker of bone metabolism.

ペクチン分解酵素に関する研究(第22報)

Botrytis cinerea was cultivated in the media containing pectin, peptone, NaNO3, KH2PO4, MgSO4•7H2O and trace of FeCl3. Crude enzyme solution was prepared by salting out and dialysis, and the influence of pH value on the enzymatic action was shown. The optimum pH of endo-PG was found to be 5.4 and 3.6, and peaks of macerating action were found at pH 5.0 and 3.0 for potato disks, at pH 4.5 and 1.5 for barks of Ganpi. The macerating action at pH 4.5_??_5.0 seems to be resulted from the joint action of endo-PG and pectinesterase.

魚類筋肉の解糖作用に関する酵素学的研究-V

Activity of muscle phosphorylase of seabass was determined on the crude extracts which were prepared from the muscles at different parts of body and from the muscles of different freshness. The highest enzyme activity was observed in the extract from caudal muscle, and the lowest in the extract from ventral muscle. Change of the enzyme activity due to different freshness of muscle was very slow, but cold storage did not inhibit completely the loss of the enzyme activity. The enzyme reaction was accelerated by the addition of urethane and sodium chloride of low concentration, and inhibited by nitrofurazone, chlortetracyclin and sodium chloride of high concentration. The crude enzyme solution was found to be comparatively resistant to proteolytic activity of pancreatin.

Application of Chromatography. XXXIX. Biosynthesis of Riboflavin. (3).

4-Ribitylamino-5-aminouracil (II), which is regarded as an intermediate in the biosynthesis of riboflavin, was synthesized and the formation of 6, 7-dimethylribolumazine (III) and riboflavin (IV) was examined in three cases, in which a mixture of (II) and a crude enzyme solution prepared from Er. ashbyii, a mixture of (II) and acetoin, and a mixture of (II), acetoin, and a crude enzyme solution were each incubated at 37°. In the first two cases, the formation of (III) and (IV) was not observed, but in the lase case the formation of a green fluorescent substance and increase of (IV) were recognized. The green fluorescent substance was confirmed to be (III) from its Rf values and ultraviolet spectrum. From these results, it seems reasonable to think that riboflavin which is an isoalloxazine derivative is biosynthesized in the mycelium of Er. ashbyii from a pyrimidine compound (II) through a pteridine compound (III).

Application of Chromatography. XXXVI. Biosynthesis of Riboflavin. (2). Riboflavin-synthesizing Enzyme extracted from Eremothecium ashbyii.

The crude enzyme solution, prepared by extracting the ground mycelium of Er. ashbyii with a phosphate buffer and dialyzing the extract against the same buffer solution, was incubated with 6, 7-dimethylribolumazine at 37°. Paper partition chromatography of the reaction mixture gave a yellow and a purple fluorescent spot. The former spot (Rf 0.3 ; solvent-system : EtOH·BuOH·H2O) was cut out and extracted with hot water, and the ultraviolet spectrum of the extract was measured, showing that it is identical with that of riboflavin. The riboflavin was determined by bioassay with L. casei and by various chemical methods such as the lumiflavin-fluorescence method and others. Next, investigation was made on relationship between quantity of the resulting riboflavin and reaction time, quantity of the crude enzyme solution added, and concentration of the substrate, and further on the optimal pH and temperature for the action of the enzyme solution. Lastly, the above purple-fluorescent spot (Rf 0.23 ; solvent-system : EtOH·BuOH·H2O) was proved to be that of 6-methyl-7-hydroxyribolumazine, and a new route was proposed for the formation of this compound. Further, the fact that the lumazine derivative was not affected by the crude enzyme solution made more certain the authors'previous assumption that the compound is a final product in the metabolism by Er. ashbyii.