l-Asparaginase from E. coli

l-Asparaginase [EC 3.5.1.1], antitumor enzyme, was purified to a crystalline form from the cell free extract of Escherichia coli A-l-3 KY3598, by ethanol fractionation and chromatographies on DEAE cellulose and CM Sephadex. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation: s_{20, w} was 7.87S.

The molecular weight was estimated to be 141,000 by the short column method. The pI of the enzyme protein was 4.75 according to isoelectric electrofocusing.

Amino acid analysis revealed the absence of cysteine or cystine residues in the molecule.

The enzyme exhibited optimal activity between pH 6 and 8. It was stable in the pH range 5.5 ~ 9.0.

The enzyme activity was cleared very slowly in the plasma of dog. Intravenous administration of the enzyme caused a complete regression of the Gardner lymphoma implanted in the C3H mice.     

Immobilization of tomato (Lycopersicon esculentum) pectinmethylesterase in calcium alginate beads and its application in fruit juice clarification

Clarity of fruit juices is desirable to maintain an aesthetically pleasing quality and international standards. The most commonly used enzymes in juice industries are pectinases. A partially-purified pectinmethylesterase from tomato was entrapped in calcium alginate beads and used for juice clarification. The activity yield was maximum at 1 % (w/v) CaCl₂ and 2.5 % (w/v) alginate. The immobilized enzyme retained ~55 % of its initial activity (5.7 × 10^?2 units) after more than ten successive batch reactions. The K_m, pH and temperature optima were increased after immobilization. The most effective clarification of fruit juice (%T₆₂₀ ~60 %) by the immobilized enzyme was at 4 °C with a holding time of 20 min. The viscosity dropped by 56 % and the filterability increased by 260 %. The juice remains clear after 2 months of storage at 4 °C.     

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⁺     

Studies on Tannin Acyl Hydrolase of Microorganisms

Tannin acyl hydrolase (Tannase) from Asp. oryzae No. 7 was purified. The purified enzyme was homogenous on column chromatography (DEAE-Sephadex A50, Sephadex G100), ultra centrifugation and electrophoresis.

The molecular weight of the enzyme estimated by gel filtration method was about 200,000.

The enzyme was stable in the range of pH 3 to 7.5 for 12 hr at 5°C, and for 25 hr at the same temperature in the range of pH 4.5 to 6. The optimum pH for the reaction was 5.5. It was stable under 30°C (over one day, in 0.05 M-citrate buffer of pH 5.5), and the optimum temperature was 30~40°C (reaction for 20min). The activity was lost completely at 55°C in 20 min at pH 5.5, or at 85°C in 10 min at the same pH.

Any metal salt tested did not activate the enzyme, Zink chloride and cupric chloride inhibited the activity or denatured the enzyme. The activity was lost completely by dialysis against EDTA-solution at pH 7.25, although it was not affected by dialysis against deionized water.     

Exopolygalacturonase in tomato fruit

A low level of polygalacturonase has been found in unripe tomato fruit. The enzyme was extracted with 0.5 M NaCl containing 0.05 M CaCl₂, concentrated by ultrafiltration and purified 150-fold by ion-exchange chromatography. The M, of the enzyme was 47 000. It was optimally active at pH 5 and required Ca²⁺ for activity, with an optimum concentration of 0.42 mM Ca₂₊. The enzyme has been characterized as an exopolygalacturonase that cleaves monomer units from the non-reducing ends of the substrate molecules. The optimum substrate size for the enzyme was that with a degree of polymerization of ca 13. The amount of exopolygalacturonase activity remained essentially constant during development and ripening of the fruit.     

Diurnal regulation of phosphoenolpyruvate carboxylase from crassula

Phosphoenolpyruvate carboxylase appears to be located in or associated with the chloroplasts of Crassula. As has been found with this enzyme in other CAM plants, a crude extract of leaves gathered during darkness and rapidly assayed for phosphoenolpyruvate carboxylase (PEPc) activity is relatively insensitive to inhibition by malate. After illumination begins, the PEPc activity becomes progressively more sensitive to malate. This enzyme also shows a diurnal change in activation by glucose-6-phosphate, with the enzyme from dark leaves more strongly activated than that from leaves in the light.

When the enzyme is partially purified in the presence of malate, the characteristic sensitivity of the day leaf enzyme is largely retained. Partial purification of the enzyme from dark leaves results in a small increase in sensitivity to malate inhibition.

Partially purified enzyme is found by polyacrylamide gel electrophoresis analysis to have two bands of PEPc activity. In enzymes from dark leaves, the slower moving band predominates, but in the light, the faster moving band is preponderant. Both of these bands are shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be composed of the same subunit of 103,000 daltons.

The enzyme partially purified from night leaves has a pH optimum of 5.6, and is relatively insensitive to malate inhibition over the range from pH 4.5 to 8. The enzyme from day leaves has a pH optimum of 6.6 and is strongly inhibited by malate at pH values below 7, but becomes insensitive at higher pH values.

Gel filtration of partially purified PEPc showed two activity peaks, one corresponding approximately to a dimer of the single subunit, and the other twice as large. The larger protein was relatively insensitive to malate inhibition, the smaller was strongly inhibited by malate.

Kinetic studies showed that malate is a mixed type inhibitor of the sensitive, day, enzyme, increasing K_m for phosphoenolpyruvate and reducing V_max. With the insensitive, night, enzyme, malate is a K type inhibitor, reducing the K_m for phosphoenolpyruvate, but having little effect on V_max. The inhibition of the insensitive enzyme by malate appears to be hysteretic, taking several minutes to be expressed during assay, probably indicating a change in the conformation or aggregation state of the enzyme.

Activation by glucose-6-phosphate is of the mixed type for the day form of the enzyme, causing both a decreased K_m for phosphoenolpyruvate and an increased V_max, but the night, or insensitive, form shows only an increase in V_max in response to glucose-6-phosphate.

     

Studies on Biochemistry of the Thiobacilli

In the oxidation of thiosulfate at pH 4.5 tetrathionate was formed as an intermediate, and the thiosulfate-oxidizing enzyme was active in acidic pH range in contrast to the enzyme of T. thioparus and Thiobacillus X.

Phosphate did not seem to affect the oxidation of thiosulfate but rather affect the conversion of tetrathionate. In the absence of phosphate, tetrathionate, which was produced from thiosulfate oxidation, seemed to accumulate without undergoing further conversion.

Quantitative oxidation of tetrathionate to sulfate was achieved with freshly harvested cells of T. thiooxidans; pH optimum for the oxidation of tetrathionate by the washed cells was 2~3, and the activity fell markedly at pH above 3.5.

Tetrathionate might be enzymatically dismuted to pentathionate and trithionate under anaerobic conditions with crude extracts of T. thiooxidans; pH optimum for the reaction was about 2.7 and the activity fell strikingly at pH 4.7. The formed trithionate might be further hydrolyzed to thiosulfate and sulfate.     

Specificity of a Nuclease from Penicillium citrinum

The enzyme with high milk clotting activity produced by Irpex lacteus was partially purified by a CM-cellulose chromatography. Throughout the over-all process, the enzyme was purified approximately 9-fold from a crude powder with about 22.8% recovery of the original activity. The MCA/PU ratio of this fraction was 2.51 and the specific milk clotting activity was 188.7.

The purified enzyme is a sort of acid protease with optimum pH of 2.5 for casein digestion and 4.0 for hemoglobin digestion. The Lineweaver-Burk plot, when casein was used as a substrate, showed that the Km value of the enzyme was about 0.07% and the V_max value was 0.4. The molecular weight of the enzyme is about 34,000, the isoelectric point is pH 5.2 and a ultraviolet absorption maximum is at 277 mμ. The enzyme has not yet been crystalized but seems to be a sort of glycoprotein, because the Molish reaction was positive at the present purification stage.

Some enzymological properties of the enzyme was studied and compared with those of a calf rennet and Mucor rennet. In some respects such as pH optima, pH stability, thermostability and temperature optima, the enzyme is Mucor rennet alike. On the other hand, as to the increase in activity along with decrease in pH of milk and the increase in activity along with the addition of Ca ion, the enzyme is not very different from the calf rennet. However, proteolysis of milk casein by the enzyme was fairly higher than by the calf rennet.

As to the production of enzymes, I. lacteus can produce at least three types of proteases into liquid media. When, for example, R medium was used, only one type of protease, that is the fraction A, could mainly be produced and it was this enzyme that assumed to be a rennet like enzyme.     

Studies on Transglycosidation to Vitamin B6 by Microorganisms

Some properties of pyridoxine glucoside-synthesizing enzyme were studied using the partially and highly purified enzyme preparations from Micrococcus sp. No. 431.

The enzyme was stable at pH 7.0 and between 0°C and 30°C. The maximal activity was obtained at pH 8.0 and 37°C. Besides sucrose, phenyl-α-d-glucoside and maltose served as glucosyl donor. Of vitamin B₆ compounds tested, only pyridoxine served as glucosyl acceptor. The enzyme activity was inhibited by PCMB and heavy metal ions, and the inhibition was prevented by 2-mercaptoethanol, indicating the enzyme would be a sulfhydryl enzyme. The activity was not affected by chelating agents and not activated by metal ions.     

Phosphodiesterase in Microsomes from Bovine Milk

Phosphodiesterase was solubilized from bovine milk microsomes and partially purified. The purified enzyme showed 20-fold specific activity compared with that of microsomes, and 1,500-fold with that of the original milk.

The properties of the enzyme were investigated by using NpT. The pH optimum was at 9.5. The enzyme was inhibited with EDT A and reactivated with the addition of magnesium or calcium ions. This enzyme was strongly inhibited with reducing reagents. K_m, value was 7.4 x 10^-4 M for NpT at pH 9.5.

RNA was hydrolyzed completely to 5′-mononucleotides, and this enzyme may be considered to show the exonucleolytic action for RNA.     

Purification and Some Properties of Melanoidin Decolorizing Enzymes,P-III and P-IV,from Mycelia of Coriolus versicolor Ps4a

Melanoidin decolorizing enzymes (MDE) were extracted from mycelia of Coriolus versicolor Ps4a and purified by DEAE-Sephadex, DEAE-Sephacel and Sephadex G-200 column chromatographies. MDE of this strain consisted of a main fraction, P-fraction, and a minor fraction, E-fraction, and the P-fraction was composed of at least five enzymes. P-III and P-IV in the P-fraction were picked as typical enzymes of this strain, and their enzymatic properties were investigated. P-III had a molecular weight of 48,400 ~ 50,000, an optimum pH of 5.5 and an optimum temperature of 30~35°C. P-III required glucose and 02 for the appearance of the activity, and was inhibited by p-CMB, N-BSI, Ag⁺ and o-phenanthroline.

On the other hand, P-IV had a molecular weight of 43,800 ~ 45,000, an optimum pH of 4.0~4.5 and an optimum temperature of 30~35°C. P-IV could decolorize melanoidin in the absence of glucose and O₂, and was inhibited weakly by Ag⁺, p-CMB and N-BSI. P-IV is the enzyme that attacks the melanoidin directly in comparison with P-III which attacks melanoidin indirectly as in the sub-reaction of sugar oxidase.

Incidentally, a multiplicative effect between P-III and P-IV for decolorization was observed.     

β-Glucosidase from Botrytis cinerea: Its Relation to the Pathogenicity of This Fungus

Cellulolytic enzyme activities in 11 strains of B. cinerea and their pathogenicity against apples, grapes, and lettuce were tested. A positive correlation was found between the β-glucosidase activity of B. cinerea cultured in PYA-medium and the pathogenicity, as expressed in the area of lesions. Further, this correlation was found to hold for the β-glucosidase activity of the apple fruit lesion caused by B. cinerea.

Since it was suggested that pathogenicity of B. cinerea depended on its β-glucosidase activity, the β-glucosidase was purified and characterized. The enzyme had its optimum pH at 3.0, and was very stable in a wide range of pH. These properties were extremely advantageous for B. cinerea to infect the apple fruit.     

Synthesis and Biological Activity of (±)2′,3′-Dihydroabscisic Acid

An enzyme which catalyzes the oxidation of poly(vinyl alcohol) (PVA) has been purified from a fraction adsorbed to DEAE-Sephadex at pH 7.0 from PVA-degrading enzyme activities produced by a bacterial symbiotic mixed culture in a culture broth when the culture was grown in a minimal medium where PVA served as a sole source of carbon and energy. The enzyme was separated from a coexisting oxidized PVA hydrolase by dye-ligand chromatography on Matrex Gel Blue A. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoreses in the absence and presence of SDS.

The enzyme is a single polypeptide with a molecular weight of about 40,000 and has an isoelectric point of 4.5. The amino acid composition of the enzyme has been determined and found to have no histidine. The N- and C-terminal amino acid residues are both alanine. The enzyme solution is pink and shows absorption maxima at 276, 364, and 469 nm. One atom of non-heme iron has been detected per molecule in the enzyme.

The enzyme catalyzes the oxidation of PVA and also of various low molecular weight secondary alcohols to the corresponding ketones with the production of H₂0₂ and the consumption of 0₂. The molar ratio of these ketones, H₂0₂ and 0₂ is 1:1:1. The most effective electron acceptor is 0₂, while 2,6-dichlorophenolindophenol and nitro blue tetrazolium also serve as the acceptor with efficiencies to 0₂ of about 31 and 16%, respectively. The enzyme is, therefore, considered to be a secondary alcohol oxidase.

The enzyme is most active at pH 7.0 and at 45°C and is stable between pH 5.0 and 9.0 and at temperatures below 45°C. The activity is inhibited by Hg²⁺ and is restored by the addition of reduced glutathione, although p-chloromercuribenzoate has no effect.

The enzyme shows a common antigenicity in immunodiffusion and neutralization reactions with antisera to a secondary alcohol oxidase previously isolated from another fraction adsorbed on SP-Sephadex at pH 7.0 of the PVA-degrading enzyme activities [Agric. Biol. Chem., 43, 1225 (1979)]. The relations between these two secondary alcohol oxidases are discussed.     

Vitamin B12-dependent Methionine Synthetase in Photosynthetic Bacteria Partial Purification and Properties

Vitamin B₁₂-dependent methionine synthetase (N⁵-methyItetrahydrofolate-homocysteine Bi2-methyltransferase; EC 2.1.1.13) was partially purified from two different types of photo-synthetic bacteria, Chromatium D and Rhodospirillum rubrum.

Chromatium D, which does not produce vitamin B₁₂, possessed apomethionine synthetase when grown in the absence of the vitamin. Partially purified apoenzyme was converted to holoenzyme efficiently with CH₃B₁₂ or OHB₁₂. Holo-methionine synthetase was purified 244 fold with 56.4 % recovery from Chromatium D cells grown with vitamin B₁₂ added. The partially purified enzyme required reductants but was only partially dependent on S-adenosylmethionine.

On the other hand, Rsp. rubrum methionine synthetase which was always present as holoenzyme, in contrast with that of Chromatium D, was purified 40 fold with 2.8% recovery. The obtained preparation required S-adenosylmethionine and reductants for the enzyme activity. The optimal pH of Chromatium D enzyme and of Rsp. rubrum enzyme was in the range of 7.5~7.8 and 6.5~6.75, respectively.     

Sucrose Synthase in Wild Tomato, Lycopersicon chmielewskii, and Tomato Fruit Sink Strength

Here it is reported that sucrose synthase can be readily measured in growing wild tomato fruits (Lycopersicon chmielewskii) when suitable methods are adopted during fruit extraction. The enzyme also was present in fruit pericarp tissues, in seeds, and in flowers. To check for novel characteristics, the wild tomato fruit sucrose synthase was purified, by (NH₄)₂SO₄ fraction and chromatography with DE-32, Sephadex G-200, and PBA-60, to one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The following characteristics were obtained: native protein relative molecular weight 380,000; subunit relative molecular weight 89,000; K_m values with: sucrose 53 millimolar, UDP 18.9 micromolar, UDP-glucose 88 micromolar, fructose 8.4 millimolar; pH optima between 6.2 to 7.3 for sucrose breakdown and 7 to 9 for synthesis; and temperature optima near 50°C. The enzyme exhibited a high affinity and a preference for uridylates. The enzyme showed more sensitivity to divalent cations in the synthesis of sucrose than in its breakdown. Sink strength in tomato fruits also was investigated in regard to sucrose breakdown enzyme activities versus fruit weight gain. Sucrose synthase activity was consistently related to increases in fruit weight (sink strength) in both wild and commercial tomatoes. Acid and neutral invertases were not, because the published invertase activity values were too variable for quantitative analyses regarding the roles of invertases in tomato fruit development. In rapidly growing fruits of both wild and commercially developed tomato plants, the activity of sucrose synthase per growing fruit, i.e. sucrose synthase peak activity X fruit size, was linearly related to final fruit size; and the activity exceeded fruit growth and carbon import rates by at least 10-fold. In mature, nongrowing fruits, sucrose synthase activities approached nil values. Therefore, sucrose synthase can serve as an indicator of sink strength in growing tomato fruits.     

Purification and Characterization of Soymilk-clotting Enzymes from Bacillus sp. K-295G-7

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 elec-trophoresis (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.     

Comparative Biochemical Studies of α-Glucosidases

The properties of brewer’s yeast α-glucosidase have been investigated. The enzyme was capable of hydrolyzing various α-glucosides and was active especially on aryl-α-glucosides in comparison with other α-glucosides and sugars. The rate of hydrolysis decreased in following order: phenyl-α-glucosides, sucrose, matlose and isomaltose.

The range of opt. temp, was 40~45°C and opt. pH, 6.5~7.0.

Cu⁺⁺ and Hg⁺⁺ inhibited strongly the enzyme activity and Zn⁺⁺, moderately. The enzyme was suggested to be a sulfhydryl enzyme from the inhibition experiments by SH-reagents and the effects of glutathione on the activity.

The enzyme synthesized some oligosaccharides from maltose. As the transglucosidation products, nigerose, isomaltose, kojibiose and maltotriose were detected by paperchromatography.

Pure nigerose was separated by splitting maltose with amyloglucosidase from the mixture of maltose and nigerose and by use of successive carbon column chromatography.     

Studies on the Decomposition of Raffinose by α-Galactosidase of Actinomycetes

α-Galactosidase was isolated from the culture broth of Streptomyces olivaceus and was partially purified by chromatography on a DEAE-sephadex column. The optimum pH of the preparation was found to be 5.2 for raffinose and the preparation was inactivated completely by maintaining it at 60°C for 15 minutes. p-Chloromercuribenzoate, HgCl₂ and AgNO₃ caused complete inhibition of the enzyme activity at 2 × 10^?5 M concentration. The preparation showed transglycosylase activity. A sugar spot, chromatographically identical with that of stachyose, appeared in the digest of raffinose. However, the preparation hydrolyzed raffinose completely into galactose and sucrose after a prolonged incubation.

A simple raffinose estimation method was developed using the enzyme preparation, and it was found that the method allowed to estimate 125~500 μg of raffinose with an accuracy of ±5%. The method was applied to the estimation of raffinose in beet molasses.     

Substrate Specificity and Some Properties of Crystalline Mold Maltase

The substrate specificity of crystalline mold maltase was investigated.

The enzyme acts upon various α-heteroglucosides or saccharides. Aryl-α-glucosides were hydrolyzed much faster than alkyl-α-glucosides. The enzyme acts on the maltose derivatives whose reducing groups have been masked. But among glucosylfructoses turanose, maltulose and isomaltulose were attacked with a slow rate while the enzyme was quite inert to sucrose. Malto- and isomalto-oligosaccharides were also hydrolyzed and the enzyme ceased its action at seven to eight units of hexose in both series of oligosaccharides.

The opt. pH range of Takamaltase was 4.2~4.6 and opt. temp., 50~55°C. Cu⁺⁺ and Hg⁺⁺ strongly inhibited the enzyme activity but other metal ions tested had no effects. It is suggested that the enzyme is not a sulfhydryl enzyme because of the lack of effects of SH-reagents on the activity.     

Preparation of Zeaxanthin from Berries of Boxthorn,Lycium chinensis

Purification and properties of a new alkaline protease of rat skeletal muscle have been reported. The purification procedure of the enzyme is as follows: skeletal muscle tissue was extracted successively with Hasselbach-Schneider solution, 5 m urea solution and 2% sodium deoxycholate solution. After then, the enzyme was extracted from the residue with 1.1 m potassium iodide solution. This enzyme solution was treated with n-butanol, and dialyzed against water. The enzyme precipitated during dialysis was collected and dissolved in 1.1 m potassium iodide solution. The enzyme solution was fractionated with acetone, and chromatographed on Sephadex G-200. The final preparation showed over 20,000 times of purity.

The optimum pH range of the enzyme activity is 9.5~10.5, and the maximum reaction rate occurs at 47~57°C. The enzyme is stable below 47°C at pH 7.3. At 37°C, the enzyme is stable during 30 min at least, in the pH range of 5.5~10.0. Below pH 5.0, it is relatively labile. Hg²⁺, Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, and Zn²⁺ scarcely affect the enzyme activity at the concentration of 1 mm. Ethylenediaminetetraacetate shows little effect on the activity at the concentration of 10 mm, and iodoacetamide, 2,4-dinitrophenol, p-chloromercuribenzoate show the similar effect at the concentration of 1 mm. Diisopropyl-flurophosphate inhibits the enzyme activity. From the results obtained, this enzyme is presumed to be responsible for the activity of autolytic breakdown of rat skeletal muscle proteins in the alkaline pH range.