Development and properties of fructose 1,6-bisphosphatase in the endosperm of castor-bean seedlings.

1. The activity of fructose 1,6-bisphosphatase (EC 3.1.3.11) in the fatty endosperm of castor bean (Ricinus communis) increases 25-fold during germination and then declines. The developmental pattern follows that of catalase, a marker enzyme for gluconeogenesis in this tissue. 2. The enzyme at its peak of development was partially purified, and its properties were studied. It has an optimal activity at neutral pH (7.0-8.0). The apparent Km value for fructose 1,6-bisphosphate is 3.8 X 10(-5) M. The activity is inhibited by AMP allosterically with an apparent Ki value of 2.2 X 10(-4) M. The enzyme hydrolyses fructose 1,6-bisphosphate and not ribulose 1,5-bisphosphate or sedoehptulose 1,7-bisphosphate. 3. Treatment of the partially purified enzyme with acid leads to an 80% decrease in activity. The remaining activity is insensitive to AMP and has optimal activity at pH 6.7 and a high apparent Km value (2.5 X 10(-4) M) for fructose 1.6-bisphosphate. Enzyme extracted from the tissue with water instead of buffer has a similar modification. The effect of acid explains the discrepancies between this report and previous ones on the properties of the enzyme in this tissue. 4. The storage tissues of various fatty seedlings all contain a 'neutral' fructose 1,6-bisphosphatase. The activities of the enzyme from some of the tissues are inhibited by AMP. 5. The properties of the enzyme in fatty seedlings and in green leaves are discussed in comparison with that in animal tissues.     

Interaction of carboxypeptidase A with carbamate and carbonate esters

The carbamate ester N-(phenoxycarbonyl)-L-phenylalanine binds well to carboxypeptidase A in the manner of peptide substrates. The ester exhibits linear competitive inhibition toward carboxypeptidase A catalyzed hydrolysis of the amide hippuryl-L-phenylalanine (Ki = 1.0 X 10(-3) M at pH 7.5) and linear noncompetitive inhibition toward hydrolysis of the specific ester substrate O-hippuryl-L-beta-phenyllactate (Ki = 1.4 X 10(-3) M at pH 7.5). Linear inhibition shows that only one molecule of inhibitor is bound per active site at pH 7.5. The hydrolysis of the carbamate ester is not affected by the presence of 10(-8)-10(-9) M enzyme (the concentrations employed in inhibition experiments), but at an enzyme concentration of 3 X 10(-6) M catalysis can be detected. The value of kcat at 30 degrees C, mu = 0.5 M, and pH 7.45 is 0.25 s-1, and Km is 1.5 X 10(-3) M. The near identity of Km and Ki shows that Km is a dissociation constant. Substrate inhibition can be detected at pH less than 7 but not at pH values above 7, which suggests that a conformational change is occurring near that pH. The analogous carbonate ester O-(phenoxycarbonyl)-L-beta-phenyllactic acid is also a substrate for the enzyme. The Km is pH independent from pH 6.5 to 9 and has the value of 7.6 X 10(-5) M in that pH region. The rate constant kcat is pH independent from pH 8 to 10 at 30 degrees C (mu = 0.5 M) with a limiting value of 1.60 s-1. Modification of the carboxyl group of glutamic acid-270 to the methoxyamide strongly inhibits the hydrolysis of O-(phenoxycarbonyl)-L-beta-phenyllactic acid. Binding of beta-phenyllactate esters and phenylalanine amides must occur in different subsites, but the ratios of kcat and kcat/Km for the structural change from hippuryl to phenoxy in each series are closely similar, which suggests that the rate-determining steps are mechanistically similar.     

Subunit interaction: A molecular basis of heterosis

Acid phosphatase, a dimeric enzyme, in Drosophila malerkotliana was studied in isogenic flies to explore the molecular basis of heterosis. As the enzyme activity in heterozygotes is 34% more than that in the better parent (S/S), heterosis is indicated. Vmax, Km, and Ki values are 14.60, 3.6 X 10(-4) M, and 0.45 X 10(-4) M, respectively, for the enzyme from F/S flies and 11.80, 4.0 X 10(-4) M, and 0.37 X 10(-4) M, respectively, for the enzyme from S/S flies. Thus heterosis for enzyme activity results from a better enzyme in F/S flies. The higher efficiency and better quality of the enzyme in F/S flies were traced to the heterodimeric allozyme, present only in heterozygotes. Enzyme activity, Vmax, Km, and Ki values are 0.739, 42.1; 3.6 X 10(-4) M, and 0.50 X 10(-4) M, respectively, for the heterodimeric and 0.513, 36.8; 4.1 X 10(-4) M, and 0.37 X 10(-4) M, respectively, for the better parental homodimeric allozyme. On an equimolar basis the enzyme activity of the heterodimer is 44% higher than that of the better homodimer. The better performance of the heterodimer is probably a reflection of superior conformation resulting from interaction between component subunits (F and S polypeptides).     

Purification and properties of inosine monophosphate oxidoreductase from nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp)

Using ammonium sulfate precipitation, gel filtration, and affinity chromatography, inosine monophosphate (IMP) oxidoreductase (EC 1.2.1.14) was isolated from the soluble proteins of the plant cell fraction of nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp). The enzyme, purified more than 140-fold with a yield of 11%, was stabilized with glycerol and required a sulfydryl-reducing agent for maximum activity. Gel filtration indicated a molecular weight of 200,000, and sodium dodecyl sulfate-gel electrophoresis a single subunit of 50,000 Da. The final specific activity ranged from 1.1 to 1.5 mumol min-1 mg protein-1. The enzyme had an alkaline pH optimum and showed a high affinity for IMP (Km = 9.1 X 10(-6) M at pH 8.8 and NAD levels above 0.25 mM) and NAD (Km = 18-35 X 10(-6) M at pH 8.8). NAD was the preferred coenzyme, with NADP reduction less than 10% of that with NAD, while molecular oxygen did not serve as an electron acceptor. Intermediates of ureide metabolism (allantoin, allantoic acid, uric acid, inosine, xanthosine, and XMP) did not affect the enzyme, while AMP, GMP, and NADH were inhibitors. GMP inhibition was competitive with a Ki = 60 X 10(-6) M. The purified enzyme was activated by K+ (Km = 1.6 X 10(-3) M) but not by NH+4. The K+ activation was competitively inhibited by Mg2+. The significance of the properties of IMP oxidoreductase for regulation of ureide biosynthesis in legume root nodules is discussed.     

Further Characterization of a Myelin-Associated Neuraminidase: Properties and Substrate Specificity

A neuraminidase activity in myelin isolated from adult rat brains was examined. The enzyme activity in myelin was first compared with that in microsomes using N-acetylneuramin(alpha 2----3)lactitol (NL) as a substrate. In contrast to the microsomal neuraminidase which exhibited a sharp pH dependency for its activity, the myelin enzyme gave a very shallow pH activity curve over a range between 3.6 and 5.9. The myelin enzyme was more stable to heat denaturation (65 degrees C) than the microsomal enzyme. Inhibition studies with a competitive inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, showed the Ki value for the myelin neuraminidase to be about one-fifth of that for the microsomal enzyme (1.3 X 10(-6) M versus 6.3 X 10(-6) M). The apparent Km values for the myelin and the microsomal enzyme were 1.3 X 10(-4) M and 4.3 X 10(-4) M, respectively. An enzyme preparation that was practically devoid of myelin lipids was then prepared and its substrate specificity examined. The "delipidated enzyme" could hydrolyze fetuin, NL, and ganglioside substrates, including GM1 and GM2. When the delipidated enzyme was exposed to high temperature (55 degrees C) or low pH (pH 2.54), the neuraminidase activities toward NL and GM3 decreased at nearly the same rate. Both fetuin and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid inhibited NL and GM3 hydrolysis. With 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, inhibition of NL was greater than that of GM3; however, the Ki values for each substrate were almost identical. GM3 and GM1 also competitively inhibited the hydrolysis of NL and NL similarly inhibited GM3 hydrolysis by the enzyme. These results indicate that rat brain myelin has intrinsic neuraminidase activities toward nonganglioside as well as ganglioside substrates, and that these two enzyme activities are likely catalyzed by a single enzyme entity.     

Studies on the mechanism of action of ACTH on triglyceride synthesis in fat cells.

It was found that ACTH greatly reduced lipogenesis in fat cells in the presence of calcium ion, but not in the absence of calcium ion. Of the enzymes involved in triglyceride synthesis from fatty acid in lipid micelle membranes, only acyl-CoA synthetase was inhibited by calcium ion, the apparent Ki value of calcium ion being 4.2 X 10(-4) M. The Km values of the enzyme for palmitate and ATP were 2.0 X 10(-4) M and 2.5 X 10(-4) M, respectively and calcium ion caused non-competitive inhibition with both palmitate and ATP. The acyl-CoA synthetase activity of lipid micelle membranes was inhibited by treatment with phospholipase A or C, but not by treatment with phospholipase D. The mechanism of inhibition of triglyceride synthesis by ACTH is discussed on the basis of these results.     

Differences in catalytic properties between native isoenzymes of xyloglucan endotransglycosylase (XET)

Four isoenzymes of xyloglucan endotransglycosylase (XET; EC 2.4.1.207) were isolated from sprouting mung bean seedlings (M35, M45, M55a, M55b) and two from cauliflower florets (C30, C45). Purification in each case was by ammonium sulphate precipitation, reversible formation of a covalent xyloglucan-enzyme complex, and cation-exchange chromatography. The isoenzymes differed in pH optimum (range 5.0-6.5), Km for the nonasaccharide XLLGol (Gal2.Xyl3.Glc3.glucitol) as acceptor substrate, ability to utilise diverse oligosaccharides as acceptor substrate, and ability to bind to carboxymethyl-cellulose (and thus possibly to other polyanions such as pectin in the cell wall). None of the isoenzymes was particularly cold-tolerant, unlike one XET (TCH4) of Arabidopsis. The two cauliflower isoenzymes had higher Km values for XLLGol (70-130 microM) than the four mung bean isoenzymes (16-35 microM). We suggest that this difference is related to the major roles of the XETs in these two tissues: integration of new xyloglucan into the walls of the densely cytoplasmic cauliflower florets, and re-structuring of existing wall material in the rapidly vacuolating bean shoots.     

Interaction between 1,4-thiazine derivatives and D-amino-acid oxidase

Aminoethylcysteine-ketimine (2H-1,4-thiazine-5,6-dihydro-3-carboxylic acid) strongly inhibits D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3). The inhibition is purely competitive (Ki = 3.3 X 10(-7) M). Aminoethylcysteine-ketimine modifies the visible spectrum of the enzyme: the absorption maxima of bound FAD shift from 375-455 nm to 385-445 nm with a definite shoulder at 465 nm; the appearance of a large absorption band centered at 750 nm may be due to a charge-transfer complex formation. The dissociation constant for the aminoethylcysteine-ketimine-enzyme complex, calculated by a photometric procedure (4 X 10(-7) M), is in good agreement with kinetic data. The dicarboxylic analogue of this inhibitor (lanthionine-ketimine) is ineffective in D-amino-acid oxidase inhibition and does not produce any spectral modification of the enzyme. These results confirm structural requirements for D-amino-acid oxidase inhibitor reported by other researchers. Ketimine reduced forms (thiomorpholine-2-carboxylic acid and thiomorpholine-2,6-dicarboxylic acid) are chemically synthesized and checked as D-amino-acid oxidase substrates: only thiomorpholine-2-carboxylic acid is oxidized to aminoethylcysteine-ketimine (Km = 2 X 10(-4) M).     

Isolation and characterization of human liver guanine deaminase

Guanine deaminase (EC 3.5.4.3, guanine aminohydrolase [GAH]) was purified 3248-fold from human liver to homogeneity with a specific activity of 21.5. A combination of ammonium sulfate fractionation, and DEAE-cellulose, hydroxylapatite, and affinity chromatography with guanine triphosphate ligand were used to purify the enzyme. The enzyme was a dimer protein of a molecular weight of 120,000 with each subunit of 59,000 as determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing gave a pI of 4.76. It was found to be an acidic protein, as evidenced by the amino acid analysis, enriched with glutamate, aspartate, alanine and glycine. It showed a sharp pH optimum of 8.0. The apparent Km for guanine was determined to be 1.53 X 10(-5) M at pH 6.0 and 2 X 10(-4) M for 8-azaguanine as a substrate at pH 6.0. The enzyme was found to be sensitive to p-hydroxymercuribenzoate inhibition with a Ki of 1.53 X 10(-5) M and a Ki of 5 X 10(-5) M with 5-aminoimidazole-4-carboxamide as an inhibitor. The inhibition with iodoacetic acid showed only a 7% loss in the activity at 1 X 10(-4) M and a 24% loss at 1 X 10(-3) M after 30 min of incubation, whereas p-hydroxymercuribenzoate incubation for 30 min resulted in a 91% loss of activity at a concentration of 1 X 10(-4) M. Guanine was the substrate for all of the inhibition studies. The enzyme was observed to be stable up to 40 degrees C, with a loss of almost all activity at 65 degrees C with 30 min incubation. Two pKa values were obtained at 5.85 and 8.0. Analysis of the N-terminal amino acid proved to be valine while the C-terminal residue was identified as alanine.     

Kinetics of membrane-bound aldehyde dehydrogenase from Acinetobacter calcoaceticus

In recent investigations we were able to demonstrate that the NADP-dependent aldehyde dehydrogenase of Acinetobacter calcoaceticus is an inducible enzyme localized in intracytoplasmic membranes limiting alkane inclusions. Long-chain aliphatic hydrocarbons and alkanols are inducers of the enzyme. It was purified by us and now kinetically characterized using the enzyme-micelle form, which contains bacterial phospholipids and a detergent (sodium cholate), too. The pH optimum of aldehyde dehydrogenase was determined to be at pH 10. The enzyme showed substrate inhibition (by aldehyde excess). The Ks and Km values of the leading substrate NADP+ were found to be 8.6 X 10(-5) and 10.3 X 10(-5)M independent of the chain-length of the aldehydes. The Km values of the aldehydes decreased depending on increasing chain-length (butanal: 1.6 X 10(-3), decanal: 1.5 X 10(-6)M). The Ki values (for inhibition by aldehyde excess) showed a similar behaviour (butanal: 7.5 X 10(-3), decanal: 3.5 X 10(-5)M) as well as the optimal aldehyde concentrations inducing the "maximal" reaction velocity (butanal: 5mM, decanal: 6 microM). The number of inhibiting aldehyde molecules per enzyme-substrate complex was determined to be n = 1. NADPH showed product inhibition kinetics (Ki(NADPH) = 2.2 X 10(-4)M), fatty acids did not. We were unable to measure a reverse reaction. The following ions and organic compounds were non-competitive inhibitors of the enzyme: Sn2+, Fe2+, Cu2+, BO3(3-), CN-, EDTA, o-phenanthroline, p-chloromercuri-benzoate, mercaptoethanol, phenylmethylsulfonyl fluoride, and diisopropylfluorophosphate; iodoacetate did not influence enzyme activity. Chloral hydrate was a competitive inhibitor of the aldehydes. Ethyl butyrate activates the enzyme, dependent on the chain-length of the aldehyde substrates.     

The p-nitrophenyl phosphatase activity of ubiquitin from bovine erythrocytes

Ubiquitin, a unique protein with esterase and carbonic anhydrase activity, has been found to have also a p-nitrophenyl phosphatase activity. This phosphomonoesterase activity of ubiquitin has an acidic pH optimum; its true substrate appears to be the phosphomonoanion, with a Km of 1.8 X 10(-3) M. It is competitively inhibited by the typical acid phosphatase inhibitors, arsenate (Ki = 1.3 X 10(-3) M), molybdate (Ki = 1.2 X 10(-6) M), and phosphate (Ki = 1.4 X 10(-3) M). These inhibitors have no effect on the CO2 hydration and p-nitrophenyl acetate esterase activities of the ubiquitin. Acetazolamide slightly inhibited the p-nitrophenyl phosphatase activity.     

Effect of LAB 173 711, an ABA analogue,on low-temperature resistance of mung bean seedlings

The effect of LAB 173 711, a synthetic analogue of abscisic acid, has been evaluated on chilling-sensitive mung bean (Vigna radiata L. cv. Local V.) seedlings. Electrical conductivity was used for assessing the degree of chilling injury. Exposure of 8-day-old mung bean seedlings to 4°C for 35 h resulted in a 50% electrolyte leakage and induced irreversible chilling injury. The seedlings gained the best protection against chilling injury by pretreatment with LAB 173 711 (5 × 10^–4 M) for 3 days. The protection effect could be sustained for 4 days. The LAB 173 711 pretreatment at 28°C did not cause a significant difference in the electrolyte leakage over the ambient temperature (28°C) control. Application of LAB 173 711 at 28°C reduced visible injury and the treated seedlings had higher ethylene production and respiration rate over the untreated control. LAB 173 711 helped maintain the integrity of the cell membrane and thus reduced the leakage of soluble sugar and amino acids. These combined effects led to a higher chilling tolerance in the mung bean seedlings.     

Purification and characterization of phosphodiesterase from the venom of Trimeresurus mucrosquamatus

Phosphodiesterase was isolated from the venom of Trimeresurus mucrosquamatus from Taiwan using gel filtration on a Sephadex G-100 column, followed by anion or cation exchange chromatography. Phosphodiesterase was homogeneous as established by a single band on acrylamide gel electrophoresis and immunodiffusion. Phosphodiesterase activity was inhibited by ethylenediamine tetraacetic acid (EDTA), o-phenanthroline, thioglycolic acid or p-chloromercuribenzoate (PCMB) but not by soybean trypsin inhibitor (SBTI) or benzamidine. The molecular weight of this enzyme was determined to be approximately 140,000 and the isoelectric point was found to be pH 7.4 by isoelectric focusing with carrier ampholyte. The Michaelis constant (Km) of this enzyme for p-nitrophenyl thymidine-5'-phosphate and inhibition constant (Ki) for PCMB were found to be 5.6 X 10(-3) and 7.6 X 10(-4) M, respectively.     

Carbamate kinase of Lactobacillus buchneri NCDO110. I. Purification and properties

Carbamate kinase has been prepared from Lactobacillus buchneri NCDO110. An approximately 91-fold increase in the specific activity of the enzyme was achieved. The purified extract exhibited a single band following polyacrylamide gel electrophoresis. The apparent molecular weight as determined by gel electrophoresis was about 97,000. The enzyme is stable for 2 weeks at -20 degrees C. Maximum enzymatic activity was observed at 30 degrees C and pH 5.4 in 0.1 M acetate buffer. L. buchneri carbamate kinase requires Mg2+ or Mn2+; its activity is higher with Mn2+. The activation energy of the reaction was 4078 cal mol-1 for the reaction with Mn2+ and 3059 cal mol-1 for the reaction with Mg2+. From a Dixon plot a pK value of 4.8 was calculated. The apparent Km values for ADP with Mg2+ or Mn2+ were 0.71 X 10(-3) and 1.17 X 10(-3) M, respectively, and the apparent Km values for carbamyl phosphate with Mg2+ or Mn2+ were 1.63 X 10(-3) and 1.53 X 10(-3) M, respectively. ATP and CTP acted as inhibitors of this reaction and the following values were obtained: Ki (ATP)Mg2+ = 9.4 mM, Ki (ATP)Mn2+ = 6.2 mM, and Ki (CTP)Mg2+ = 4.4 mM.     

N-hydroxy-2-acetylaminofluorene as effective inhibitor of aldehyde oxidase

N-Hydroxy-2-acetylaminofluorene has been found to be an effective inhibitor of aldehyde oxidase. At concentrations of 1 X 10(-6) M and 1 X 10(-5) M, 38% and 88% inhibition was observed on the oxidase activity towards N1-methylnicotinamide. The inhibition was of noncompetitive type and had a Ki value of 4.4 X 10(-6) M. In contrast, little inhibition of the enzyme was observed with 2-aminofluorene, 2-acetylaminofluorene and acetohydroxamic acid even at a concentration of 1 X 10(-4) M.     

Thymidylate synthetase from Diplococcus pneumoniae, properties and inhibition by folate analogs.

Thymidilate synthetase (methylenetetrahydrofolate:dUMP C-methyltransferase) in crude extract from Diplococcus pneumoniae exhibits a partial but variable requirement for Mg-2+ depending upon the buffer. Optimum Mg-2+ concentration is between 0.014 and 0.02 M. The optimum pH for activity in a variety of buffers occurred as a broad peak between 7.0 and 7.7. In Tris/acetate buffer, but not in potassium phosphate buffer, the pH optimum was different in the presence and absence of Mg-2+. Methylation of uridylate, cytidylate and deoxycytidylate could not be demonstrated over a pH range of 5.0-8.0. The enzyme exhibited an apparent Km for deoxyuridylate of 3.08 - 10-5 M and an apparent Km for L-(+)(minus)-5,10-methylene tetrahydrofolate of 2.66 - 10-4 M. During molecular-sieve chromatography and sucrose density-gradient centrifugation, the enzyme was detectable only as a single catalytically active form of Mr 34 000-38 000. 2,4-Diamino quinazoline antifolates were better competitive inhibitors (Ki = 3-8 -10-6 M) of thymidylate synthetase than 2,4-diamino pteridines (Ki = 3- 10-5 M). 2-Amino-4-hydroxy-quinazolines were the best inhibitors (Ki = 1.3-2.9 - 10-6 M). All of the 2,4-diamino quinazolines and pteridines inhibited dihydrofolate reductase from D. pneumoniae in a nearly stoichiometric fashion (Ki = less than 10-10 M). The 2-amino-4-hydroxy-quinazolines were poor inhibitors of this enzyme (Ki = 10=5 M).     

Phosphoprotein phosphatase activity of human prostate acid phosphatase

Human prostate acid phosphatase (EC 3.1.3.2) has been shown to dephosphorylate different phosphoproteins with the maximum rate at pH 4.0-4.5. The activity with phosvitin is distinctly higher than with beta-casein, casein and most of all than with riboflavin-binding protein. The native phosvitin is homogeneous on isoelectric focusing with pI value of 2.1, whereas phosvitin partially dephosphorylated (in about 15%) by the prostate acid phosphatase shows multiple bands with pI values of 3.5 - 6.8 or higher. The phosphate groups bound to serine residues are removed enzymatically twice as fast as phosphothreonine residues. The apparent Km value for phosvitin was 2.4 X 10(-7) M, and is by three orders of magnitude lower than Km of p-nitrophenyl phosphate (2.9 X 10(-4) M). The competitive inhibitors of prostate acid phosphatase, fluoride and L(+)-tartrate, show the same Ki values for phosvitin and p-nitrophenyl phosphate.     

A probe of the active site acidity of carboxypeptidase A

The substrate analogue 2-(1-carboxy-2-phenylethyl)-4-phenylazophenol is a potent competitive inhibitor of carboxypeptidase A. Upon ligation to the active site, the azophenol moiety undergoes a shift of pKa from a value of 8.76 to a value of 4.9; this provides an index of the Lewis acidity of the active site zinc ion. Examination of the pH dependence of Ki for the inhibitor shows maximum effectiveness in neutral solution (limiting Ki = 7.6 X 10(-7) M), with an increase in Ki in acid (pK1 = 6.16) and in alkaline solution (pK2 = 9.71, pK3 = 8.76). It is concluded that a proton-accepting enzymic functional group with the lower pKa (6.2) controls inhibitor binding, that ionization of this group is also manifested in the hydrolysis of peptide substrates (kcat/Km), and that the identity of this group is the water molecule that binds to the active site metal ion in the uncomplexed enzyme (H2OZn2+L3). Reverse protonation state inhibition is demonstrated, and conventional concepts regarding the mechanism of peptide hydrolysis by the enzyme are brought into question.     

Purification and characterization of two components of acid alpha-glucosidase from pig liver

Acid alpha-glucosidase [EC 3.2.1.3] was purified from pig liver by a procedure including Sephadex G-100 affinity chromatography. Electrophoresis on SDS-polyacrylamide gel of the purified enzyme indicated the presence of two components with molecular weights of 73K and 64K. The two components of the enzyme were completely separated, in reasonable yield, by chromatography on a DEAE-5PW column. Both components catalyzed the hydrolysis of the alpha-1,4 and alpha-1,6 linkages of glycogen, maltose, isomaltose, dextrin, and a synthetic glucoside at acid pH. The pH optima of both components were 4.3 for maltase and glucoamylase, and 4.8 for isomaltase and dextrinase. But as to the activity on 4MU-alpha-Glc, the pH optimum of the larger component was 4.8 and that of the smaller component 5.3. The Km values of both components for 4MU-alpha-Glc, maltose, glycogen, isomaltose, and dextrin were 1.0 X 10(-4) M, 9.1 X 10(-3) M, 16.7 mg/ml, 6.7 X 10(-2) M, and 12.5 mg/ml, respectively. Erythritol, Tris, and turanose inhibited the two components competitively. The Ki values of the larger component were 5.0 X 10(-2) M, 13.3 X 10(-3) M, and 3.2 X 10(-3) M, and those of the smaller component were 2.5 X 10(-2) M, 6.1 X 10(-3) M, and 4.7 X 10(-3) M, for erythritol, Tris, and turanose, respectively.     

Production of pinoresinol diglucoside,pinoresinol monoglucoside,and pinoresinol by <Emphasis Type="Italic">Phomopsis</Emphasis> sp. XP-8 using mung bean and its major components

Phomopsis sp. XP-8 is an endophytic fungus that has the ability to produce pinoresinol diglucoside (PDG) in vitro and thus has potential application for the biosynthesis of PDG independent of plants. When cultivated in mung bean medium, PDG production was significantly improved and pinoresinol monoglucoside (PMG) and pinoresinol (Pin) were also found in the culture medium. In this experiment, starch, protein, and polysaccharides were isolated from mung beans and separately used as the sole substrate in order to explore the mechanism of fermentation and identify the major substrates that attributed to the biotransformation of PDG, PMG, and Pin. The production of PDG, PMG, and Pin was monitored using high-performance liquid chromatography (HPLC) and confirmed using HPLC-MS. Activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) were analyzed and tracked during the cultivation. The reaction system contained the compounds isolated from mung bean in the designed amount. Accumulation of phenylalanine, cinnamic acid, p-coumaric acid, PDG, PMG, and Pin and the activities of PAL, C4H, and 4CL were measured during the bioconversion. PMG was found only when mung bean polysaccharide was analyzed, while production of PDG and Pin were found when both polysaccharide and starch were analyzed. After examining the monosaccharide composition of the mung bean polysaccharide and the effect of the different monosaccharides had on the production of PMG, PDG, and Pin, galactose in mung bean polysaccharide proved to be the major factor that stimulates the production of PMG.