Purification and characterization of novel cutinase from nasturtium (Tropaeolum majus) pollen.

Cutinase from pollen grains of Tropaeolum majus was purified by Sephadex G-100 gel filtration, QAE-Sephadex chromatography, and isoelectric focusing. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be 40,000 by both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cutinase was found to be a glycoprotein containing about 7% carbohydrate and the isoelectric point of this enzyme was 5.45. It catalyzed hydrolysis of p-nitrophenyl esters of C₂ to C₁₈ fatty acids with similar K_m and V. The purified cutinase showed an optimum pH of 6.8 with cutin as the substrate, whereas with p-nitrophenyl esters of fatty acids the optimum pH was 8.0. This enzyme did not show any metal ion requirement. Unlike the previously studied fungal cutinases, the present pollen enzyme was strongly inhibited by thiol-directed reagents such as N-ethylmaleimide and p-hydroxymercuribenzoate whereas it was totally insensitive to the active serine-directed reagent, diisopropylfluorophosphate. The purified pollen cutinase showed preference for primary alcohol esters, but it did not catalyze hydrolysis of tripalmitoyl or trioleyl glycerol at significant rates. The properties of the pollen enzyme are, in general, in sharp contrast to those of the fungal cutinase, and the present results strongly suggest that the pollen enzyme belongs to a new class of cutinases. Another esterase which preferentially hydrolyzed p-nitrophenyl acetate was also found in the extracellular fluid. This enzyme, separated from cutinase, showed a pI of 5.6 and it was sensitive to diisopropylfluorophosphate, but not to SH-directed reagents.     

Discovery of a cutinase-producing Pseudomonas sp. cohabiting with an apparently nitrogen-fixing Corynebacterium sp. in the phyllosphere

A phyllospheric bacterial culture, previously reported to partially replace nitrogen fertilizer (B. R. Patti and A. K. Chandra, Plant Soil 61:419-427, 1981) was found to contain a fluorescent pseudomonas which was identified as Pseudomonas putida and a Corynebacterium sp. The P. putida isolate was found to produce an extracellular cutinase when grown in a medium containing cutin, the polyester structural component of plant cuticle. The Corynebacterium sp. grew on nitrogen-free medium but could not produce cutinase under any induction conditions tested, whereas P. putida could not grow on nitrogen-free medium. When cocultured with the nitrogen-fixing Corynebacterium sp., the P. putida isolate grew in a nitrogen-free medium, suggesting that the former provided fixed N2 for the latter. These results suggest that the two species coexist on the plant surface, with one providing carbon and the other providing reduced nitrogen for their growth. The presence of cutin in the medium induced cutinase production by P. putida. However, unlike the previously studied fungal systems, cutin hydrolysate did not induce cutinase. Thin-layer chromatographic analysis of the products released from labeled apple fruit cutin showed that the extracellular enzyme released all classes of cutin monomers. This enzyme also catalyzed hydrolysis of the model ester substrates, p-nitrophenyl esters of fatty acids, and optimal conditions were determined for a spectrophotometric assay with p-nitrophenyl butyrate as the substrate. It did not hydrolyze triacyl glycerols, indicating that the cutinase activity was not due to a nonspecific lipase. It showed a broad pH optimum between 8.0 and 10.5 with 3H-labeled apple cutin as the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a cutinase from Fusarium roseum culmorum and its immunological comparison with cutinases from F. solani pisi.

Fusarium roseum culmorum, grown on apple cutin as the sole source of carbon, was shown to produce a cutin depolymerizing enzyme. From the extracellular fluid of these F. roseum cultures, a cutinase and a nonspecific esterase were isolated utilizing Sephadex G-100, QAE-Sephadex, and SP-Sephadex chromatography. The homogeneity of the cutinase was verified by polyacrylamide disc gel electrophoresis. The molecular weight of the cutinase was estimated to be 24,300 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Electrophoretic mobility of this enzyme was between that of Cutinases I and II from Fusarium solani pisi. The F. roseum cutinase hydrolyzed p-nitrophenyl butyrate and cutin, but not p-nitrophenyl palmitate, while the nonspecific esterase hydrolyzed the long-chain esters. Amino acid composition of F. roseum cutinase was found to be similar to that of F. solani pisi Cutinase I except for differences in the number of serine, valine, and cysteine residues. The time-course, protein concentration dependence, substrate concentration dependence, and pH optimum (10.0 for cutin hydrolysis) of the F. roseum cutinase was similar to the cutinases from F. solani pisi. The F. roseum cutinase was inhibited by diisopropylfluorophosphate and paraoxon, and the [³H]diisopropylphosphate group was covalently attached to the enzyme upon treatment with tritiated diisopropylfluorophosphate. Therefore, it is concluded that catalysis by cutinase involves an “active serine.” Immunochemical studies with a rabbit antibody prepared against F. solani pisi Cutinase I demonstrated that Cutinase II from this organism was immunologically very similar to, but not identical to, Cutinase I. On the other hand, the cutinase from F. roseum was immunologically quite different from the cutinases isolated from F. solani pisi in that it did not cross-react with anticutinase I. However, all three cutinases were virtually identical in their sensitivity to inhibition by anticutinase I, and all three enzymes were virtually completely inhibited by the anticutinase I.     

Purification and characterization of 7 beta-hydroxysteroid dehydrogenase from Ruminococcus sp. of human intestine

7 beta-Hydroxysteroid dehydrogenase (7 beta-HSD) was produced by Ruminococcus sp. PO1-3 obtained from among human intestinal bacteria. The enzyme was purified from a crude extract by ammonium sulfate fractionation, and Butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A and Octyl-Sepharose chromatographies. The purified enzyme was obtained as a single band on polyacrylamide gel electrophoresis with enzyme activity staining and as one band corresponding to a molecular weight of 30,000 on SDS-polyacrylamide gel electrophoresis. On gel filtration, its apparent molecular weight was estimated to be 60,000. The enzyme had a sulfhydryl group(s) in its active site. Substrate specificity studies revealed that the enzyme showed absolute specificity for the beta-configuration of a hydroxyl group at the 7 position of bile acids, and required NADP+ and NADPH as cosubstrates. The Km values for ursodeoxycholic acid, 7-k etolithocholic acid, NADP+, and NADPH were 5.0, 8.5, 7.7, and 24 microM, respectively.     

Purification and properties of oxytocinase (cystine amino-peptidase) from monkey placenta.

Oxytocinase (cystyl-aminopeptidase) [EC 3.4.11.3] was isolated from monkey placenta in a purified form by a six-step prodedure comprising extraction from monkey placenta homogenate, ammonium sulfate fractionation, repeated chromatography on hydroxylapatite, chromatography on a column of DEAE-cellulose and gel filtration on a column of Sephadex G-200. The purified enzyme showed a single band on polyacrylamide disc electrophoresis. Oxytocin was inactivated by this enzyme preparation. The enzyme hydrolyzed several aminoacyl-beta-naphthylamides. A terminal amino group was required for enzyme activity. The molecular weight of the purified enzyme was estimated to be 87,000 by gel filtration and 83,000 by sodium dodecyl sulfate gel electrophoresis. Other properties of the enzyme, the effects of metal ions and various chemical reagents on the enzyme activity, the pH optimum, and Km values for a number of aminoacyl-beta-naphthylamides were also examined.     

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.     

Purification and properties of beta-lactamase from Proteus morganii.

The cephalosporin beta-lactamase was purified from a strain of Proteus morganii that showed resistance to beta-lactam antibiotics and produced the enzyme constitutively. The purified enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis and consisted of a single polypeptide of molecular weight 38,000 to 40,000 from gel filtration of Sephadex G-100 and sodium dodecyl sulfate-acrylamide gel electrophoresis, its isoelectric point being pH 7.2 No cysteine residue was found in its amino acid composition. The specific activity was 190 mumol/min per mg of the purified enzyme protein for the hydrolysis of cephaloridine, the optimal pH was about 8.5 and the optimal temperature was 50 degrees C. Antibodies against the purified beta-lactamase inhibited not only the enzyme activity of the purified preparation, but also the enzyme activity of all of the other strains of P. morganii so far tested, regardless of whether the modes of their production were inducible or constitutive. None of the beta-lactamases produced by beta-lactam antibiotic-resistant strains of other species of Proteus was affected at all by the antibodies, thus showing that the purified cephalosporin beta-lactamase was of the species-specific type. The enzymological properties of the preparation have been compared with those of beta-lactamases derived from other gram-negative enteric bacteria.     

Purification and properties of beta-D-glucosidase (linamarase) from the butter bean, Phaseolus lunatus

A beta-D-glucosidase (linamarase) was purified 11,700-fold from the butter bean, Phaseolus lunatus L., by means of successive procedures including extraction, ammonium sulfate fractionation, acetone treatment, and chromatographies on CM-Sephadex, DEAE-Sephadex, and Sephadex G-200. The final preparation gave a single protein band on both disc polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. In spite of its electrophoretic purity, the final enzyme preparation showed four glycosidase activities; beta-D-glucosidase, beta-D-galactosidase, beta-D-fucosidase, and beta-D-xylosidase. The molecular weight of the enzyme was determined to be 124,000 +/- 9,000 by Sephadex G-200 gel filtration, and 59,000 +/- 2,400 by SDS-disc gel electrophoresis. The enzyme showed a pH optimum in the range of 5.1 to 6.0 with p-nitrophenyl beta-D-glucoside, 4-methylumbelliferyl beta-D-glucoside, and linamarin. Among natural substrates containing a beta-glucosyl terminal, linamarin, prunasin, and salicin were hydrolyzed by the enzyme from butter beans, but amygdalin, cellobiose, gentiobiose, and laminarin were hardly hydrolyzed.     

Purification and properties of 3 alpha-hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum from human intestine

3 alpha-Hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum, isolated from human intestinal bacteria, was capable of converting 3-ketoglycyrrhetic acid to 3 alpha-hydroxyglycyrrhetic acid. The enzyme was purified to homogeneity by means of butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A, Toyopearl HW-55S, and isoelectric focusing column chromatographies. The purified enzyme showed a specific activity of 156 mumol/min.mg toward 3 alpha-hydroxyglycyrrhetic acid, and showed a single band on SDS-polyacrylamide gel electrophoresis. The apparent molecular weight was 53,000, as estimated by gel filtration, and 30,000, as judged by SDS-polyacrylamide gel electrophoresis. Its isoelectric point was 5.2. The enzyme showed absolute specificity for the 3 alpha-hydroxyl and 3-ketonic groups of 18 alpha- or 18 beta-glycyrrhetic acid and required NADP+ and NADPH as cosubstrates. The enzyme did not act on any 3 alpha-hydroxyl or 3-ketonic group of steroids or bile acids. The enzyme is a novel type of enzyme, defined as 3 alpha-hydroxy-glycyrrhetinate dehydrogenase, being quite different from 3 alpha-hydroxysteroid dehydrogenase [EC 1.1.1.50].     

Synthesis and characterization of a new cutinase substrate, 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate

Phytopathogenic fungi penetrate plants by breaking down the cuticular barrier with cutinase. Cutinases are extracellular hydrolytic enzymes that degrade cutin, a polyester composed of hydroxy and epoxy fatty acids. Until now, cutinase has been recognized by its ability to release labeled cutin monomers or by a non-specific esterase assay based on the hydrolysis of p-nitrophenyl esters of short fatty acids. In this work, an insoluble p-nitrophenyl derivative was synthesized and purified, and its structure was determined to be 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate (pNMSEH) by nuclear magnetic resonance (H+ NMR) analysis. pNMSEH was tested as a new cutinase substrate with Pseudomonas mandocino cutinase and porcine liver esterase. While a linear release over time of p-nitrophenol (pNP) was recorded in the presence of cutinase, no response was obtained with the esterase. The calculated kinetic parameters of pNMSEH hydrolysis by cutinase revealed a high specificity (Km=1.8mM), albeit a low catalytic rate (Vmax=10.5 micromol min(-l)l(-1)). This new synthetic substrate may be helpful for detecting and assaying cutinase activity in mixed solutions, such as crude fungal extracellular extracts.     

Purification and properties of GM1 ganglioside beta-galactosidases from bovine brain

Two GM1-beta-galactosidases, beta-galactosidases I, and II, have been highly purified from bovine brain by procedures including acetone and butanol treatments, and chromatographies on Con A-Sepharose, PATG-Sepharose, and Sephadex G-200. beta-Galactosidase I was purified 30,000-fold and beta-galactosidase II 19,000-fold. Both enzymes appeared to be homogeneous, as judged from the results of polyacrylamide disc gel electrophoresis. Enzyme I had a molecular weight of 600,000-700,000 and enzyme II one of 68,000, as determined on gel filtration. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under denaturing conditions, enzyme II gave a single band with a molecular weight of 62,000, while enzyme I gave two minor bands with molecular weights of 32,000 and 20,000 in addition to the major band at 62,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide but not from galactosylceramide. Enzyme I showed a pH optimum of 4.0 and was heat stable, while enzyme II showed a pH optimum of 5.0 and lost 50% of its activity in 15 min at 45 degrees C. Enzyme I showed a pI of 4.2 and enzyme II one of 5.9.     

Purification and properties of acid phosphatase from cultured tobacco cells

One component of acid phosphatase was purified from cultured tobacco cells. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The enzyme possesses high activity toward nucleoside di- and triphosphate, much less activity toward nucleoside monophosphates and sugar esters. The MWs of the phosphatase determined by Sephadex G-100 gel filtration and dodecyl sulfate gel electrophoresis were 74000 and 76000, respectively. The phosphatase showed high affinity for concanavalin A-Sepharose and single superimposed bands of protein and carbohydrate on gel electrophoresis, suggesting that it is a glycoprotein.     

Production of a Novel Extracellular Cutinase by the Pollen and the Chemical Composition and Ultrastructure of the Stigma Cuticle of Nasturtium (Tropaeolum majus)

Germinating nasturtium pollen (Tropaeolum majus) is shown to excrete an enzyme(s) which hydrolyzes all types of monomers from biosynthetically labeled cutin and p-nitrophenyl esters, which are model substrates for fungal cutinases. The pollen cutinase showed an optimum pH near 6.5 and was inhibited by thiol-directed reagents such as p-hydroxymercuribenzoate and N-ethyl maleimide but not by diisopropyl-fluorophosphate, an “active serine”-directed reagent indicating that the pollen enzyme is an “-SH cutinase” unlike the fungal enzyme which is a serine cutinase. Excretion of the pollen cutinase into the extracellular fluid was complete within 4 to 6 hours at 30 C. Since actinomycin D and cycloheximide showed little effect on the level of cutinase excreted, it appears that cutinase is an enzyme synthesized prior to germination. Release of cutinase into the medium did not require germination. Electron microscopy revealed the presence of a continuous cutin layer on mature stigma with extensive folds, which are proposed to play a role similar to that played by the cellular papillae found in the stigma of other plants. Chemical analysis of stigma cutin by depolymerization and combined gas-liquid chromatography and mass spectrometry showed that this cutin consists of mainly the C₁₆ family of acids. The major (70%) components were dihydroxy C₁₆ acids which consisted of 10,16- (64%), 9,16- (16%), 8,16- (12%), and 7,16- (8%) dihydroxy plamitic acid. Deuterium-labeling studies showed the presence of 16-oxo-9-hydroxy C₁₆ acid and 16-oxo-10-hydroxy C₁₆ acid in this cutin. The biochemical and ultrastructural studies indicate that the pollen tube may gain entry into stigma using cutinase excreted by the pollen.     

A peptidase activity exhibited by human serum pseudocholinesterase

The identity of a peptidase activity with human serum pseudocholinesterase (PsChE) purified to apparent homogeneity was demonstrated by co-elution of both peptidase and PsChE activities from procainamide-Sepharose and concanavalin-A--Sepharose affinity chromatographic columns; comigration on polyacrylamide gel electrophoresis; co-elution on Sephadex G-200 gel filtration and coprecipitation at different dilutions of an antibody raised against purified PsChE. The purified enzyme showed a single protein band on gel electrophoresis under non-denaturing conditions. SDS gel electrophoresis under reducing conditions, followed by silver staining, also gave a single protein band (Mr approximately equal to 90,000). Peptidase activity using different peptides showed the release of C-terminal amino acids. Blocking the carboxy terminal by an amide or ester group did not prevent the hydrolysis of peptides. There was no evidence for release of N-terminal amino acids. Potent anionic or esterase site inhibitors of PsChE, such as eserine sulphate, neostigmine, procainamide, ethopropazine, imipramine, diisopropylfluorophosphate, tetra-isopropylpyrophosphoramide and phenyl boronic acid, did not inhibit the peptidase activity. An anionic site inhibitor (neostigmine or eserine) in combination with an esterase site inhibitor (diisopropylfluorophosphate) also did not inhibit the peptidase. However, the choline esters (acetylcholine, butyrylcholine, propionylcholine, benzoylcholine and succinylcholine) markedly inhibited the peptidase activity in parallel to PsChE. Choline alone or in combination with acetate, butyrate, propionate, benzoate or succinate did not significantly inhibit the peptidase activity. It appeared that inhibitor compounds which bind to both the anionic and esteratic sites simultaneously (like the substrate analogues choline esters) could inhibit the peptidase activity possibly through conformational changes affecting a peptidase domain.     

Immunoenzymic studies on testicular hyaluronidase from rhesus monkeys (Macaca mulatta)

Hyaluronidase from rhesus monkey testes was purified by detergent extraction, ammonium sulphate fractionation, Sephadex G-200 column chromatography and concanavalin A-Sepharose affinity chromatography. The purified hyaluronidase showed one protein band on acrylamide gel electrophoresis. Antibodies to the purified hyaluronidase were raised in rabbits and showed a single precipitin line by Ouchterlony gel diffusion. The enzyme had a molecular weight of 62,000. The Km was 0.5 mg/ml for hydrolysis of hyaluronic acid at 37 degrees C. The optimum pH for the enzyme was 5.0 but activity was present over a broad pH range. The hyaluronidase was inhibited by HgCl2, CuSO4, FeSO4 and p-chloromercuribenzoate all at a concentration of 2 x 10(-4) M. Cysteine protected the enzyme against HgCl2 inhibition.     

Purification and properties of a second enzyme catalyzing the splitting of carbon-mercury linkages from mercury-resistant Pseudomonas K-62.

An enzyme (splitting enzyme 2) which catalyzes the splitting of carbon-mercury linkage of arylmercury compounds was found in extracts of mercury-resistant Pseudomonas K-62. This enzyme was purified about 725-fold by treatment with streptomycin, precipitation with ammonium sulfate, and successive chromatography on Sephadex G-75 and diethylaminoethyl-cellulose. A purified preparation of the enzyme showed a single band in electrophoresis either on polyacrylamide or sodium dodecyl sulfate-containing polyacrylamide gels. The molecular weight of the enzyme was estimated to be 20,000 (determined by Sephadex G-75 gel filtration) 17,000 (determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis). The enzyme showed a Km of 180 micron and a Vmax of 3.1 mumol/min per mg for p-chloromercuribenzoic acid and a Km of 250 micron and a Vmax of 20 mumol/min per mg for phenylmercuric acetate. The optimum temperature and pH for the reaction were 40 degrees C and 5.0, respectively.     

Isolation and characterization of an acyl-CoA thioesterase from dark-grown Euglena gracilis

An acyl-CoA hydrolase from dark-grown Euglena gracilis Z was purified 700-fold by subjecting the 105,000g supernatant of the cell-free extract to (NH4)2SO4 precipitation, acid precipitation, calcium phosphate gel treatment, gel filtration on Sephadex G-100, and chromatography on QAE-Sephadex, hydroxylapatite, and CM-Sephadex. Polyacrylamide disc gel electrophoresis of the purified enzyme showed a major protein band (greater than 80%) which contained thioesterase activity and a minor protein band with no thioesterase activity. Molecular weight estimated by gel filtration was 37,000 and sodium dodecyl sulfate-electrophoresis showed one major band (greater than 80%) corresponding to a molecular weight of 37,000 and a minor band of molecular weight 32,000, suggesting that the enzyme was monomeric. The pH optimum of the purified enzyme progressively increased with the chain length of the substrate, with hexanoyl-CoA showing a pH optimum at 4.5 and stearoyl-CoA at 7.0. The rate of hydrolysis of acyl-CoA showed a nonlinear dependence on protein concentration, and bovine serum albumin overcame this effect as well as stimulated the rate. The extent of stimulation by albumin increased with chain length of the substrate up to lauroyl-CoA and then decreased as chain length increased; albumin inhibited the hydrolysis of stearoyl-CoA. This enzyme hydrolyzed CoA esters of C6 to C18 fatty acids with a maximal rate of 17 mumol min-1 mg protein-1 for C14. Typical substrate saturation patterns were obtained with all substrates except that high concentrations were inhibitory. Studies on the effect of pH on the apparent Km and Vmax values for octanoyl-CoA, lauroyl-CoA, and palmitoyl-CoA showed that in all cases Vmax was greatest and Km was lowest at the respective pH optima. Active-serine-directed reagents severely inhibited the thioesterase activity, suggesting the participation of an active serine residue in catalysis; thiol-directed reagents were not effective inhibitors. Diethylpyrocarbonate also inhibited the enzyme and hydroxylamine reversed this inhibition, suggesting the involvement of a histidine residue in catalysis as expected for enzymes containing active serine. This thioesterase did not affect the chain length distribution of the products generated by the Euglena fatty acid synthase I.     

Purification of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli strain 080

Purification studies of 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) (EC 1.1.1.159) from Escherichia coli 080 showed that 1.59-fold purification could be achieved by heating (60 degrees C for 10 min) the ultracentrifuged enzyme preparation, and 6.46-fold purification was achieved by subsequent precipitation with ammonium sulfate. Further purification on Sephadex G-100 gel gave 10.1-fold purification. After pooling and concentrating the active fractions obtained from the Sephadex G-100 filtration, an 11.1-fold purification was achieved using DEAE-cellulose chromatography. The purified enzyme produced a single band on polyacrylamide gel electrophoresis and its molecular weight was determined to be 54,000. The enzyme was immunogenic and showed immunoprecipitation with homologus antisera.     

Isolation of a Fusarium solani mutant reduced in cutinase activity and virulence.

Fusarium solani isolate T-8 produces an extracellular enzyme, cutinase, which catalyzes the degradation of cutin in the plant cuticle. Cutinase activity can be measured by the hydrolysis of either the artifical substrate, p-nitrophenylbutyrate (PNB), or radioactive cutin containing [14C]palmitic acid. In the present study, the culture filtrate contained basal levels of cutinase when T-8 was grown on acetate as a sole source of carbon. After mutagenesis, a cutinase-defective mutant (PNB-1) was identified by screening acetate-grown colonies for a loss of PNBase activity. The mutant possessed an 80 to 90% reduction in cutinase activity when grown for 3 to 5 days on acetate- or cutin-containing medium. Induction of cutinase by cutin or hydrolyzed cutin after growth on glucose medium was similarly reduced. Kinetic analysis indicated that cutinase from the mutant possessed a near normal Km for PNB and a 92% reduction in Vmax. Fluorography and Western blotting of 15% sodium dodecyl sulfate-polyacrylamide gels of separated 35S-labeled proteins from cutin induction medium revealed that in the mutant the 22,000-molecular-weight band corresponding to cutinase was reduced approximately 85%. The virulence of the mutant in a pea stem bioassay was decreased by 55% and was restored to nearly the parental level by the addition of purified cutinase. The data suggest that the mutant synthesizes reduced quantities of a functional and immunoreactive cutinase enzyme and that cutinase plays a critical role in infection. The PNB1 mutation may be within a regulatory gene or a promoter for cutinase.     

Purification and Properties of Pyranose Oxidase from Coriolus versicolor

Coriolus versicolor KY2912 grown on a medium containing glucose, sucrose or glycerol produced pyranose oxidase. Pyranose oxidase (glucose-2-oxidase) was purified by HPA-75 chromatography, Sepharose 4B and Sephadex G-100 gel filtration, and hydroxyapatite chromatography. The purified enzyme preparation showed a single protein band on acrylamide gel electrophoresis. The highest activity was obtained when D-glucose was employed as substrate and molecular oxygen as electron acceptor. The enzyme was most active at pH 6.2 and 50°C, stable in the pH region between 5.0 and 7.4, and the activity was completely lost above 70°C. The activity was inhibited by Ag⁺ , Cu²⁺ and PCMB. The enzyme contained FAD covalently bound to the polypeptide chain. The enzyme consisted of identical subunits with a molecular weight of 68,000, and showed a total molecular weight of 220,000.