Synthesis and degradation of phenylalanine ammonia-lyase of Rhodosporidium toruloides.

The regulation of the enzyme phenylalanine ammonia-lyase (PAL), which is of potential use in oral treatment of phenylketonuria, was investigated. Antiserum against PAL was prepared and was shown to be monospecific for the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme and two inactive mutant forms of the enzyme were purified to homogeneity by immunoaffinity chromatography, using anti-PAL immunoglobulin G-Sepharose 4B. Both mutant enzymes contained intact prosthetic groups. The formation of PAL catalytic activity after phenylalanine was added to yeast cultures was paralleled by the appearance of enzyme antigen. During induction, uptake of [3H]leucine into the enzyme was higher than uptake into total protein. Our results are consistent with de novo synthesis of an enzyme induced by phenylalanine, rather than activation of a proenzyme. The half-lives of PAL and total protein were similar in both exponential and stationary phase cultures. No metabolite tested affected the rate of enzyme degradation. Glucose repressed enzyme synthesis, whereas ammonia reduced phenylalanine uptake and pool size and so may repress enzyme synthesis through inducer exclusion. The synthesis of enzyme antigen by a mutant unable to metabolize phenylalanine indicated that this amino acid is the physiological inducer of the enzyme.     

Effects of phospholipids on L-lactate dehydrogenase from membranes of Escherichia coli. Activation and stabilization of the enzyme with phospholipids.

Membrane-bound L-lactate dehydrogenase was freed from the detergent used during purification. The detergent-free enzyme had about one-half the specific activity of the enzyme in 1.0% Tween 80, and was only partially sensitive to the specific antibody. This enzyme was activated about 3-fold with phosphatidylglycerol, cardiolipin, or a mixture of phospholipids. The phospholipid-activated enzyme had a similar Km value for L-lactate to that of the membrane enzyme and was completely inhibited by the specific antibody. On heat treatment, the phospholipid-activated enzyme was more stable than detergent-free enzyme and was as stable as membrane-bound enzyme. The alpha helical content of the enzyme increased 1.7-fold during preincubation with these lipids and the alpha helix became more stable during heat treatment than that of the detergent-free enzyme. These results suggest that the enzyme showed monomolecular dispersion in the lipid bilayer and that its conformation, including its active site and secondary structure, was different from that of the detergent-free enzyme. Phosphatidylethanolamine, dilauroyl lecithin and lecithin from egg yolk had none of the above effects on the activity or the secondary structure of the enzyme. On the other hand, mixtures of each of these lipids and cholate had essentially similar effects to phosphatidylglycerol.     

Enzymatic modification of vegetable protein: Immobilization of Penicillium duponti enzyme on reconstituted collagen and the use of the immobilized-enzyme complex for solubilizing vegetable protein in a recycle reactor

Penicillium duponti enzyme was immobilized on reconstituted collagen by macromolecular complication, impregnation, and covalent crosslinking techniques. The immobilization of the enzyme on collagen has a twofold purpose: (1) providing a protein microenvironment for the proteolytic enzyme; and (2) extending the useful life the enzyme once immobilized on the collagen matrix. Two types of collagen were used, one produced by the United States Department of Agriculture and the other produced by FMC. The USDA collagen contained unhydrolyzed telepeptide linkages and required pretreatment to reduce collagenaselike activity of the enzyme. Activity analysis of the immobilized enzyme complex showed that membranes with enzyme loading less than 10 mg enzyme/g of wet membrane in the reactor were dimensionally stable. The degree of crosslinking was an important parameter. Membranes with structural opening up to three times the initial dry thickness were found to be the maximum limit for controlled release of enzyme from the collagen membrane during enzymatic reaction. Higher activities and better stability of the enzyme in collagen membrane were found for covalent crosslinking of the enzyme to treated collagen films. The hydrolysis of soybean vegetable protein with the immobilized enzyme in a recycle reactor at enzyme loading of mg/g of wet membrane at 40°C, pH 3.4, produced 56.5% of soluble protein in 10h. The production is equivalent to 1.84 h total contact time between the substrate and the immobilized enzyme. The average productivity based on a stable enzyme activity and 20g of dry membrane was 329 mg of protein/g/mg of active enzyme immobilized. The productivity of the free enzyme in a batch reactor was 62.5 mg protein/h/mg enzyme.     

Purification of inactivated photoresponsive nitrile hydratase

Photoresponsive nitrile hydratase from Rhodococcus sp. N-771 was purified in its inactivated form. The enzyme had a molecular weight of approximately 60 kDa and consisted of 2 subunits each having molecular weight of 27.5 and 28 kDa. The enzyme also contained 2 iron atoms/enzyme as a cofactor. The enzyme was more stable in its inactivated form, rather than the activated during storage in the dark. The enzyme was most stable in the temperature region of 0-35 degrees C, and lost its activity above 40 degrees C. The enzyme was most stable in the pH region of 6-8. The optimum temperature and pH for the enzyme activity was 30 degrees C and 7.8, respectively. The enzyme showed wide substrate specificity, and most of the metal ions did not affect enzyme activity significantly. The absorption spectrum revealed the presence of some cofactor which changed form after photoirradiation.     

Immobilization of Aspergillus oryzae tannase and properties of the immobilized enzyme

Tannase enzyme from Aspergillus oryzae was immobilized on various carriers by different methods. The immobilized enzyme on chitosan with a bifunctional agent (glutaraldehyde) had the highest activity. The catalytic properties and stability of the immobilized tannase were compared with the corresponding free enzyme. The bound enzyme retained 20·3% of the original specific activity exhibited by the free enzyme. The optimum pH of the immobilized enzyme was shifted to a more acidic range compared with the free enzyme. The optimum temperature of the reaction was determined to be 40 °C for the free enzyme and 55 °C for the immobilized form. The stability at low pH, as well as thermal stability, were significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilized enzyme retained about 85% of the initial catalytic activity, even after being used 17 times.     

Purification and characterization of precarthamin decarboxylase from the yellow petals of Carthamus tinctorius L

Carthamin, a red quinochalcone pigment in safflower (Carthamus tinctorius L.), is enzymatically converted from a yellow precursor, precarthamin. The enzyme, which catalyzes the oxidative decarboxylation of precarthamin to carthamin, was purified to apparent homogeneity from yellow petals of safflower and named precarthamin decarboxylase. The molecular mass of the denatured enzyme was estimated as 33 kDa by SDS-PAGE. The molecular mass of the native enzyme was determined by gel filtration chromatography to be 24 kDa; thus, the native enzyme is a monomer. The optimum pH of the enzyme was 5.0. The enzyme activity was inhibited by Mn2+, Fe2+, and Cu2+ and sharply decreased at temperatures higher than 50 degrees C for 10 min. The activation energy and the Arrhenius frequency factor of the enzyme reaction were 19.7 kcal mol(-1) and 9.94 x 10(11) s(-1), respectively. The saturation curve of precarthamin showed that the enzyme follows Michaelis-Menten kinetics. The Km and Vmax of the enzyme were calculated as 164 microM and 29.2 nmol/ min, respectively. The turnover number (kcat) of the enzyme was calculated as 1.42 x 10(2) s(-1). The enzyme activity was severely inhibited by reducing agents such as glutathione and DTT at pH 5.0, suggesting that a disulfide bond may play an important role in enzyme function.     

Characterization of rat muscle fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphatase has been purified from rat muscle. Although the specific activity of the enzyme in the crude extract of rat muscle was extremely low, purification by the present procedure is highly reproducible. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The subunit molecular weight of the muscle enzyme was 37,500 in contrast to 43,000 in the case of the liver enzyme. Immunoreactivity of the muscle enzyme to anti-muscle and anti-liver fructose 1,6-bisphosphatase sera was clearly distinct from that of the liver enzyme. All one-dimensional peptide mappings of the muscle enzyme with staphylococcal V8 protease, chymotrypsin, and papain showed different patterns from those of the liver enzyme. When incubated with subtilisin, the extent of activation of muscle fructose 1,6-bisphosphatase at pH 9.1 was smaller than that of the liver enzyme. The subtilisin digestion pattern of the muscle enzyme on SDS-polyacrylamide gel electrophoresis was distinct from that of the liver enzyme. The AMP-concentration giving 50% inhibition of the muscle enzyme was 0.54 microM, whereas that of the liver enzyme was 85 microM. The concentrations of fructose 2,6-bisphosphate that gave 50% inhibition of rat muscle and liver enzymes were 6.3 and 1.5 microM, respectively. Fructose 1,6-bisphosphatase protein was not detected in soleus muscle by immunoelectroblotting with anti-muscle fructose 1,6-bisphosphatase serum.     

The role of an essential histidine residue of yeast alcohol dehydrogenase.

1. Inactivation of yeast alcohol dehydrogenase for diethyl pyrocarbonate indicates that one histidine residue per enzyme subunit is necessary for enzymic activity. The inactivated enzyme regains its activity over a period of days. 2. Enzyme modified by diethyl pyrocarbonate can form the binary enzyme - NADH complex with the same maximum NADH-binding capacity as that of native enzyme. Modified enzyme cannot form normal ternary complexes of the type enzyme - NADH - acetamide and enzyme - NAD+ - pyrazole, which are characteristic of native enzyme. 3. The rate constant for the reaction of enzyme with diethyl pyrocarbonate has been determined over the pH range 5.5--9. The histidine residue involved has approximately the same pKa as free histidine, but is 10-fold more reactive than free histidine.     

Immunological characterization of maize starch branching enzymes

Highly purified fractions of three starch branching enzymes from developing maize (Zea mays L.) endosperm were used to prepare antisera in rabbits. In double diffusion experiments, no immunoprecipitate was observed when branching enzyme IIa or IIb was tested against branching enzyme I antiserum. No immunoprecipitate was formed when branching enzyme I was tested against branching enzyme IIa or IIb antiserum. Increasing amounts of antisera in the above combinations also failed to inhibit enzyme activity. Branching enzyme IIa antiserum cross-reacted and formed spurs with branching enzyme IIb when compared with branching enzyme IIa antigen. Comparison of branching enzyme IIb antiserum with branching enzyme IIa also resulted in an immunoprecipitate. Increasing levels of branching enzyme IIa antiserum inhibited branching enzyme IIb as did the reciprocal combination. The data indicated that branching enzymes IIa and IIb are immunologically similar while branching enzyme I is distinct. The data supports the classification of starch branching enzymes based on genetic, kinetic, and chromatographic properties.     

Substrate-dependent dissociation of malate thiokinase.

Malate thiokinase has been purified to apparent homogeneity by employing conventional purification techniques along with affinity chromatography. The enzyme is composed of two nonidentical subunits (alpha subunit Mr=34,000, beta subunit Mr=42,500) to yield an alpha 4 beta 4 structure for the native enzyme. Phosphorylation of the enzyme by ATP occurs exclusively on the alpha subunit. The phosphorylated enzyme is acid labile and base stable consistent with phosphorylation of a histidine residue. Dephosphorylation of the enzyme is promoted by ADP, succinate, malate, and coenzyme A plus inorganic phosphate. Phosphorylation of the enzyme leads to a reversible change in the sedimentation properties of the enzyme; the native enzyme exhibits an S20,w of approximately 10, whereas the phosphoenzyme exhibits an S20,w of approximately 7. Formation of the 7 S form of the enzyme is also observed when coenzyme A and succinyl-CoA interact with the enzyme. The ratio of alpha to beta subunits in both the 10 S and 7 S forms of the enzyme is approximately 1.0, suggesting that the 7 S form of the enzyme has an alpha 2 beta 2 structure.     

Limited proteolysis of maize NADP-malic enzyme

The incubation of maize malic enzyme at 37 degrees C with trypsin at a ratio of 150:1 of malic enzyme to trypsin caused rapid and complete inactivation of enzyme activity. The inactivation was caused by fairly specific cleavage of the enzyme monomer (62 kDa) into 40 kDa and 20 kDa fragments. The intensity of 40 kDa band increased with the time of treatment of enzyme with trypsin from 2 to 30 min. Substrates, especially NADP (25 microM) provided almost total protection against trypsin inactivation of the enzyme activity. The studies carried out with various other endoproteases indicated that endoprotease Lys-C was most effective in inactivating malic enzyme activity. The kinetic properties of the truncated enzyme have been studied. The Km value for malate in case of native and modified enzyme was found to be identical. Km NADP for the modified enzyme was slightly higher indicating that after proteolysis the enzyme affinity for NADP had decreased. Limited proteolysis with trypsin did not show any appreciable change in fluorescence properties of the modified enzyme. Binding of NADPH to the enzyme was not affected after modification.     

Presence of angiotensin-converting enzyme in follicular fluids of porcine ovaries and its possible involvement in the intrafollicular breakdown of bradykinin

The follicular fluid of porcine ovaries contains a metalloenzyme capable of hydrolyzing the synthetic substrate, benzyloxycarbonyl-Val-Lys-Met-MCA. This enzyme was purified by ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose, CM-cellulose, Zn(2+)-chelating Cellulofine, and Diol-300 gel-filtration columns. The molecular weight of the purified enzyme was estimated to be 170,000 by SDS-PAGE and 400,000 by gel-filtration analysis, suggesting that the native enzyme is a dimer of the 170-kDa subunit polypeptide. The enzyme activity was drastically enhanced by the presence of chloride ion, and strongly inhibited by captopril and bradykinin potentiator B. A 9-residue peptide containing a processing site of human amyloid precursor protein was degraded by its dipeptidyl carboxypeptidase activity. Furthermore, the purified protein was recognized by specific antibody raised against human angiotensin-converting enzyme. The enzyme rapidly degraded bradykinin in vitro. These results indicate that benzyloxycarbonyl-Val-Lys-Met-MCA-hydrolyzing enzyme is a porcine angiotensin-converting enzyme, and that the enzyme may play a role in bradykinin turnover within the follicles of porcine ovaries.     

Substitution of glutamine for lysine at the pyridoxal phosphate binding site of bacterial D-amino acid transaminase. Effects of exogenous amines on the slow formation of intermediates

In bacterial D-amino acid transaminase, Lys-145, which binds the coenzyme pyridoxal 5'-phosphate in Schiff base linkage, was changed to Gln-145 by site-directed mutagenesis (K145Q). The mutant enzyme had 0.015% the activity of the wild-type enzyme and was capable of forming a Schiff base with D-alanine; this external aldimine was formed over a period of minutes depending upon the D-alanine concentration. The transformation of the pyridoxal-5'-phosphate form of the enzyme to the pyridoxamine-5'-phosphate form (i.e. the half-reaction of transamination) occurred over a period of hours with this mutant enzyme. Thus, information on these two steps in the reaction and on the factors that influence them can readily be obtained with this mutant enzyme. In contrast, these reactions with the wild-type enzyme occur at much faster rates and are not easily studied separately. The mutant enzyme shows distinct preference for D- over L-alanine as substrates but it does so about 50-fold less effectively than the wild-type enzyme. Thus, Lys-145 probably acts in concert with the coenzyme and other functional side chain(s) to lead to efficient and stereochemically precise transamination in the wild-type enzyme. The addition of exogenous amines, ethanolamine or methyl amine, increased the rate of external aldimine formation with D-alanine and the mutant enzyme but the subsequent transformation to the pyridoxamine-5'-phosphate form of the enzyme was unaffected by exogenous amines. The wild-type enzyme displayed a large negative trough in the circular dichroic spectrum at 420 nm, which was practically absent in the mutant enzyme. However, addition of D-alanine to the mutant enzyme generated this negative Cotton effect (due to formation of the external aldimine with D-alanine). This circular dichroism band gradually collapsed in parallel with the transformation to the pyridoxamine-5'-phosphate enzyme. Further studies on this mutant enzyme, which displays the characteristics of the wild-type enzyme but at attenuated rates, may yield information on the factors controlling the stereochemistry of the reaction as well as on the catalytic steps of the transaminase pathway.     

Properties and quaternary structure of ribulosediphosphate carboxylase from the leaves of Elymus (Psathyrosachys) junceus

The enzyme ribulosdiphosphate carboxylase was isilated from the leaves of Elymus (Psathyrosachys) junceus. The enzyme was found homogenous during disc-electrophoresis in polyacrylamide gel and analytical ultracentrifugation. The sedimentation coefficient for the enzyme is 17,4S. The enzyme molecular weight as determined by the sedimentation equilibrium technique is equal to 540000. The enzyme molecule consists of 2 types of subunits, i.e. the larger subunit has m.w. of 55000, the smaller one--12900. The number of large subunits is 8, that of small ones--8. The specific activity of the homogenous enzyme makes up to 2,45 mkmoles of CO2 per min per mg of protin (pH 8,0, 30 degrees). The purified enzyme was stable in Mg2+- and dithiothreitol-containing buffers for 3--4 weeks at 4 degrees and for 5--6 months at --20 degrees. The amino acid composition of the enzyme molecule is similar to that of the enzyme from spinach leaves.     

Purification and characterization of beta-glucosidase of Alcaligenes faecalis

A cellobiose-utilizing bacterium isolated from sugar cane bagasse and identified as a strain of Alcaligenes faecalis (ATCC 21400) produced an inducible beta-glucoside-splitting enzyme. The enzyme was purified by a series of streptomycin and ammonium sulfate fractionations and by Sephadex and diethylaminoethyl column chromatography. The final preparation was purified 130-fold, with a recovery of about 10% of the initial enzyme activity. The enzyme had a wide pH range, with optimal activity at pH 6.0 to 7.0. The enzyme was stable in solution at pH 6.5 to 7.8 when kept at 30 C for 2 hr, but it was destroyed by temperatures above 55 C. At 58 and 60 C, the time required to inactivate 90% of the initial activity was 16 and 6.5 min, respectively. An activation energy of 9,500 cal/mole and a K(m) of 1.25 x 10(-4)m were obtained by using p-nitrophenyl beta-glucoside as a substrate. The K(i) value and hydrolysis of cellobiose by the enzyme indicated a high affinity of the enzyme for the cellobiose. The enzyme had its specificity on beta-glucosidic linkage and the rate of hydrolisis of glucosides depended upon the nature of the aglycon moiety. The inactivation studies showed the presence of sulfhydryl groups in the enzyme. The activity of the enzyme was easily destroyed by the Cu(++) and Hg(++) ions. The Michaelis-Menton relationship and the rate of heat inactivation indicated the presence of one type of noninteracting active site in the bacterial beta-glucosidase. Molecular weight of the enzyme was estimated by gel filtration (Sephadex G-200) and sucrose density gradient, and a value of 120,000 to 160,000 was obtained.     

Acetyl-coenzyme-A carboxylase of Candida Lipolytica. 1. Purification and properties of the enzyme.

Acetyl-coenzyme-A carboxylase has been isolated in homogeneous form from Candida lipolytica. The homogeneity of the enzyme preparation is evidenced by analytical ultracentrifugation, dodecyl-sulfate-polyacrylamide gel electrophoresis and Ouchterlony double-diffusion analysis. The purified enzyme exhibits a specific activity of 8.0 U/mg protein at 25 degrees C and contains 1 mol biotin/263000 g protein. The sedimentation coefficient (S20,W) of the enzyme is 18 S. It has been shown by dodecyl-sulfate-polyacrylamide gel electrophoresis that the enzyme possesses only one kind of subunit with a molecular weight of 230000. This finding, together with the biotin content, indicates that the C. lipolytica enzyme has a highly integrated subunit structure. The C. lipolytica enzyme is very labile, but is stabilized by glycerol. The enzyme is markedly activated by poly(ethyleneglycol), the activation being due principally to a decrease in the Km values for substrates. Even in the presence of this activator, the Km value for acetyl-CoA of the C. lipolytica enzyme is much higher than that of the enzyme from Saccharomyces cerevisiae and animal tissues. The C. lipolytica enzyme, unlike the enzyme from animal tissues, is not activated by citrate.     

Immobilization of the exo-maltohexaohydrolase by the irradiation method

Exomaltohexaohydrolase (E.C.3.2.1.98) was immobilized by radiocopolymerization of some synthetic monomers which were mixed in various combinations. Irradiation was carried out while the mixture of monomers and enzymes was frozen in petroleum ether-dry-ice bath. Recovery of the immobilized enzyme was 44-75%.The optimum pH of the enzyme slightly shifted to the acidic side. The pH stability was improved remarkably by immobilization. The enzyme was stable retaining more than 90% of its original activity in the range pH 4-11. The optimum reaction temperature of the enzyme increased about 2 degrees C. Heat stability was also improved by immobilization, and that the enzyme retained about 40% of its original activity after treatment at 75 degrees C for 15 min. The immobilized enzyme was stable to the repeated use of 20 cycles. The K(m) value of the enzyme for short-chain amylose was almost the same as that of native enzyme. When soluble starch was used as the substrate, the K(m), value of the enzyme was three times as large as that of native enzyme. Effects of various metal ions and inhibitors on the immobilized enzyme were also studied compared to the native enzyme.     

宫川蜜柑根际土壤酶活性与土壤养分含量相关性的研究

研究了不同肥力水平的宫川蜜柑根际土壤酶的活性及其与土壤农化特性的关系。结果表明 :高产园的土壤酶活性显著高于低产园的土壤酶活性。经统计分析 ,土壤酶活性与养分含量均呈极显著相关。而且酶的活性在土壤中的分布有一定的规律性。其水平分布是在树冠内半径的 4 /5处至树冠滴水线范围内 ,酶的活性最高 ,由此处向内向外酶的活性逐渐降低 ;其垂直分布是 0～ 2 0 cm土层酶的活性最高 ,随土层的加深而逐渐降低     

Immobilization of Bacillus licheniformis 5A1 milk-clotting enzyme and characterization of its enzyme properties

Milk-clotting enzyme from Bacillus licheniformis 5A1 was immobilized on Amberlite IR-120 by ionic binding. Almost all the enzyme activity was retained on the support. The immobilized milk-clotting enzyme was repeatedly used to produce cheese in a batch reactor. The production of cheese was repeated 5 times with no loss of activity. The specific activity calculated on a bound-protein basis was slightly higher than that of free enzyme. The free and immobilized enzyme were highly tolerant to repeated freezing and thawing. The optimum temperature for milk-clotting activity was 70 °C with the free enzyme whereas, it was ranged from 70 to 80 °C with the immobilized milk-clotting enzyme. The activation energy (E _A) of the immobilized milk-clotting enzyme was lower than the free enzyme (E _A = 1.59 and 1.99 Kcal mol⁻¹ respectively). The immobilized milk-clotting enzyme exhibited great thermal stability. The milk-clotting optimum pH was 7.0 for both free and immobilized enzyme. The Michaelis constant K _m of the immobilized milk-clotting enzyme was slightly lower than the free enzyme.     

Isolation and properties of rat plasma lecithin-cholesterol acyltransferase

Lecithin-cholesterol acyltransferase was purified from rat plasma and the properties of this enzyme during the purification procedures and those of the purified enzyme were investigated in comparison with the human enzyme. The rat enzyme was not adsorbed on hydroxyapatite, which was employed for the purification of the human enzyme. When purified human enzyme was incubated at 37 degrees C in 0.1 mM phosphate buffer (pH 7.4; ionic strength, 0.00025), no alteration of enzyme activity was observed for up to 6 h. In the case of the rat enzyme, however, approximately 40% of the enzyme activity was lost under the same conditions. The human enzyme and rat enzyme were both retained on a Sepharose 4B column to which HDL3 was covalently linked, in 39 mM phosphate buffer, pH 7.4. Although the human enzyme was eluted from the column in 1 mM phosphate buffer, the rat enzyme was dissociated from the column at a lower buffer concentration (0.1 mM phosphate buffer). These findings indicate that the rat enzyme effectively associated with HDL3 in 39 mM phosphate buffer, pH 7.4, but the association was more sensitive to increase of ionic strength compared with that of the human enzyme.