Purification and Properties of a Pectate Lyase in Erwinia aroideae

A strain of Erwinia aroideae produced an extracellular pectolytic enzyme under growth conditions with pectin or pectic acid as the inducer. This strain also produced a pectin lyase when nalidixic acid is added to a culture medium. The pectolytic enzyme produced under the growth conditions was purified approximately 40-fold from the culture fluid by carboxy- methyl cellulose and Sephadex G-75 gel column chromatographies. The purified enzyme was almost homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis, having a molecular weight of about 36,000 to 38,000. This enzyme, with optimal activity at pH 9.0 to 9.2, produced reaction products which had a strong absorption at 230 nm indicating a lyase type of the reaction. The enzyme activity was markedly stimulated by calcium ion and completely inhibited by cobalt and mercuric ions and by ethylenediaminetetraacetate. Pectic acid or pectin with lower methoxyl content was a good substrate for this enzyme, while no significant activity was observed when pectin with higher methoxyl content was used as a substrate. It was concluded that the enzyme produced under the normal growth conditions is an endo-pectate lyase and differs from the pectin lyase induced by nalidixic acid.     

Distribution of Tyrosine Phenol Lyase in Microorganisms

The distribution of tyrosine phenol lyase activity in microorganisms was studied with intact cells in a synthetic reaction mixture containing l-serine and phenol or pyrocatechol. This activity was found in various bacteria, most of which belonged to the Enterobacteriaceae; especially to the genera Escherichia, Proteus and Erwinia. Cells of Erwinia herbicola ATCC 21434 were selected as a promising source of enzyme.

Intact cells of Erwinia herbicola ATCC 21434 prepared from a broth cultured for 24 hr contained markedly high enzymic activity and catalyzed the synthetic reaction of l-tyrosine or 3,4-dihydroxyphenyl-l-alanine (l-dopa) from l-serine and phenol or pyrocatechol in significantly high yields.

Results of the isolation and identification of the products showed that the amino acid synthesized by this enzymatic method was identical with l-tyrosine or l-dopa.     

酪氨酸酚裂解酶的固定化与转化条件的优化

以欧文氏菌(Erwinia herbicola)来源的酪氨酸酚裂解酶的重组大肠埃希菌Escherichia coli BL21为研究对象,研究固定化大肠埃希菌生产L-酪氨酸的条件。以海藻酸钠为载体,采用单因素实验分别考察了载体材料、明胶浓度、反应时间、苯酚浓度和辅助剂(二氧化硅、硅藻土和碳酸钙)等因素对L-酪氨酸生产的影响,发现明胶浓度、反应时间、苯酚和碳酸钙等因素的影响较为显著,进而通过正交实验探索最优条件。结果表明,生产L-酪氨酸的最优条件:载体为4%海藻酸钠与6%明胶的混合载体,苯酚浓度0.08 mol/L,反应时间8 h,于载体中添加0.6%碳酸钙。此条件下,连续反应9次后L-酪氨酸的产量达到64.5 g/L,比优化前提高了451.3%。     

Elimination,Replacement and Isomerization Reactions by Intact Cells Containing Tyrosine Phenol Lyase

Tyrosine phenol lyase catalyzes a series of α,β-elimination, β-replacement and racemization reactions. These reactions were studied with intact cells of Erwinia herbicola ATCC 21434 containing tyrosine phenol lyase.

Various aromatic amino acids were synthesized from l-serine and phenol, pyrocatechol, resorcinol or pyrogallol by the replacement reaction using the intact cells. l(d)-Tyrosine, 3,4-dihydroxyphenyl-l(d)-alanine (l(d)-dopa), l(d)-serine, l-cysteine, l-cystine and S-methyl-l-cysteine were degraded to pyruvate and ammonia by the elimination reaction. These amino acids could be used as substrate, together with phenol or pyrocatechol, to synthesize l-tyrosine or l-dopa via the replacement reaction by intact cells. l-Serine and d-serine were the best amino acid substrates for the synthesis of l-tyrosine or l-dopa. l-Tyrosine and l-dopa synthesized from d-serine and phenol or pyrocatechol were confirmed to be entirely l-form after isolation and identification of these products. The isomerization of d-tyrosine to l-tyrosine was also catalyzed by intact cells.

Thus, l-tyrosine or l-dopa could be synthesized from dl-serine and phenol or pyrocatechol by intact cells of Erwinia herbicola containing tyrosine phenol lyase.     

Isolation, Purification, and Crystallization of Aspartate Aminotransferase from Wheat Grain

A procedure for isolation and purification of aspartate aminotransferase from wheat grain includes chromatography on DEAE cellulose, acidification-alkalization, precipitation with protamine sulfate, fractionation with ammonium sulfate, and chromatography on hydroxyapatite. The yield of protein was 27% with 95% purity. Crystals of the enzyme (0.05 x 0.025 x 0.015 mm3) were obtained from ammonium sulfate solution.     

Purification,Crystallization and Properties of Tryptophanase from Proteus rettgeri

An inducible tryptophanase was crystallized from the cell extract of Proteus rettgeri grown in a medium containing l-tryptophan. The purification procedure included ammonium sulfate fractionation, heat treatment, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystals were obtained from solutions of the purified enzyme by the addition of ammonium sulfate.

The crystalline enzyme preparation was homogeneous by the criteria of ultracentrifugation and zone electrophoresis. The molecular weight was determined to be approximately 210,000.

The crystalline enzyme catalyzed the degradation of l-tryptophan into indole, pyruvate and ammonia in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from 5-hydroxy-l-tryptophan, 5-methyl-l-tryptophan, S-methyl-l-cysteine and l- cysteine. l-, d-Alanine, l-phenylalanine and indole inhibited pyruvate formation from these substrates.     

Proferrioxamine profiles of Erwinia herbicola and related bacteria

Two strains of Erwinia herbicola effective in the biocontrol of E. amylovora, the etiological agent of fire blight, were screened for proferrioxamine siderophores by on-line liquid chromatography-electrospray mass spectrometry (LC-MS). Type strains of E. herbicola and Pantoea species were included in this study for taxonomic comparisons. Proferrioxamine profiles similar to that previously described for E. amylovora, including tri- and tetrameric hydroxamates and diaminopropane-containing proferrioxamines, were observed for P. agglomerans, but not for other E. herbicola-like species. Biocontrol activity was not correlated with proferrioxamine synthesis. The results of this study are consistent with the notion that some, but not all, biocontrol strains may inhibit E. amylovora via competition for iron. Further studies into the link between biocontrol of fire blight and siderophores are thus warranted. This study also revealed limitations of standard nutrient utilization and fatty acid profile analyses for the differentiation of P. agglomerans, P. dispersa and other E. herbicola-like species from each other. Given these limitations, LC-MS may become a much needed additional diagnostic tool for the identification of E. herbicola-like strains at the species level.     

Purification,Crystallization and Some Properties of Lipase from Geotrichum candidum Link

Lipase (EC 3.1.1.3) of Geotrichum candidum Link was purified by means of ammonium sulfate fractionation, DEAE-Sephadex column chromatography, gel-filtration on Sephadex G–100 and Sephadex G–200, and was finally crystallized in concentrated aqueous solution. It was confirmed that the crystallized preparation was homogeneous electrophoretically and ultracentrifugally.

It was estimated with the crystalline enzyme that the sedimentation constant (s₂₀, w) was 4.0, the isoelectric point was pH 4.33, and the molecular weight was 53,000~55,000. From the result of amino acid analysis, none of sulfur containing amino acid was detected in the enzyme. It was also recognized that the crystalline preparation contained about 7% of the carbohydrate and very small amount of lipid. It was characterized that the lipase was the most active at pH 5.6~7.0 on olive oil, at 40°C and was stable in the range of pH 4.2 to 9.8 at 30°C for 24 hr, and was stable below 55°C for 15 min.     

Purification,Crystallization and Some Properties of Intracellular Pullulanase from Aerobacter aerogenes

Intracellular pullulanase was entirely extracted with sodium dodecylsulfate from the cells and was purified by means of ammonium sulfate fractionation and DEAE-cellulose and Sephadex chromatography. Crystalline pullulanase was precipitated with saturated ammonium sulfate solution. Intracellular pullulanase was purified over 150 fold in 17% yield to a final specific activity of 7000 per mg protein from the enzyme solution obtained by SDS-extraction. On ultracentrifugation analysis, the enzyme showed a symmetrical peak. The sedimentation coefficient, s_{20, w} was 6.29 S. Polyacrylamide disc electrophoresis gave a main band and a sub-band, and both showed activity. Molecular weight of intracellular pullulanase was estimated to be (8±1) × 10,000 from gel filtration with Sephadex G-200 and to be (9±1) × 10,000 from sedimentation equilibrium. These values were higher than that (6~7 × 10,000) of extracellular pullulanase. Both enzymes differed slightly in thermal- and pH-stabilities.     

Purification,Crystallization and Some Properties of Extracellular Pullulanase from Aerobacter aerogenes

Extracellular pullulanase was purified and crystallized from the culture fluid of Aerobacter aerogenes. Pullulanase was purified by means of ammonium sulfate fraction, DEAE-cellulose column chromatography and Sephadex column chromatography. Crystalline pullulanase was formed when saturated ammonium sulfate solution was added to the purified enzyme solution. The crystalline enzyme appeared as colorless fine rods. On ultracentrifugation analysis, the enzyme showed a single sharp and symmetrical Schlieren peak. The sedimentation coefficient, s_20,w was 4.39S. Polyacrylamide gel electrophoresis at pH 8.4 gave a main band with two sub-bands and the molecular weight of the main enzyme was estimated to be 66,000 from Polyacrylamide gel electrophoresis and to be 58,000 from sedimentation equilibrium. The optimum pH and temperature for the enzyme action were pH 6.5 and 50°C, respectively.     

Purification and Properties of Fatty Acid Hydroperoxide Lyase from Green Bell Pepper Fruits

Fatty acid hydroperoxide lyase (HPO lyase) was purified to apparentlyhomogeneity state from immature fruits of green bell pepper(Capsicum annuum L.) by differential centrifugation, ion-exchangechromatography, hydroxylapatite chromatography and gel filtration.The enzymatic activity was separated into two fractions (HPOlyases I and II) during the chromatography on hydroxylapatite.Both the isoforms were deduced to be trimers of 55-kDa subunitsand have similar enzymatic properties. Peptide maps revealedonly slight differences between them. Furthermore, immunoblotanalysis showed that an antibody raised against HPO lyase Ireacted with HPO lyase II as strongly as with the original antigen.These results indicate that there is only limited heterogeneityin terms of amino acid sequence and/or post-translational modification.The activities of both HPO lyases were considerably inhibitedby lipophilic antioxidants, such as nordihydroguaiaretic acidand     

Immunological characterization of ice nucleation proteins from Pseudomonas syringae, Pseudomonas fluorescens, and Erwinia herbicola.

Antibodies were raised against the InaW protein, the product of the ice nucleation gene of Pseudomonas fluorescens MS1650, after protein isolation from an Escherichia coli clone. On Western blots (immunoblots), these antibodies recognized InaW protein and InaZ protein (the ice nucleation gene product of Pseudomonas syringae S203), produced by both E. coli clones and the source organisms. The InaZ protein appeared in P. syringae S203 during stationary phase; its appearance was correlated with the appearance of the ice nucleation-active phenotype. In contrast, the InaW protein occurred at relatively constant levels throughout the growth phases of P. fluorescens MS1650; the ice nucleation activity was also constant. Western analyses of membrane preparations of P. syringae PS31 and Erwinia herbicola MS3000 with this antibody revealed proteins which were synthesized with development of the nucleating phenotype. In these species the presence or absence of the nucleating phenotype was controlled by manipulation of culture conditions. In all nucleation-positive cultures examined, cross-reacting low-molecular-weight bands were observed; these bands appeared to be products of proteolytic degradation of ice nucleation proteins. The proteolysis pattern of InaZ protein seen on Western blots showed a periodic pattern of fragment sizes, suggesting a highly repetitive site for protease action. A periodic primary structure is predicted by the DNA sequence of the inaZ gene.     

Partial purification and properties of a beta-glucosidase from Erwinia herbicola Y46.

A constitutive beta-glucosidase of Erwinia herbicola Y46 was studied as a prerequisite to an assessment of its significance in the release of bacteriotoxic aglycones from plant beta-glucosides, and the possible effects of the aglycones on the course of such plant diseases as "fire-blight". The enzyme was purified 86.5-fold from crude extracts of cells grown on yeast beef broth. Ammonium sulfate precipitation, DEAE-cellulose fractionation, and gel filtration through Sephadex G-100 resulted in a preparation having one peak of activity on isoelectrofocussing, on gel filtration through Sephadex G-200, and on polyacrylamide gel electrophoresis. The latter techniques demonstrated, in addition to the major protein band associated with activity, a single minor impurity. The enzyme was active against p-nitrophenyl-beta-glucoside (p-NPG) and phloridzin, but showed only very slight activity against salicin and arbutin, and no detectable activity against beta-methyl-D-glucoside, cellobiose, lactose, and esculin. The production of beta-glucosidase was maximum at the late log phase of growth on yeast beef broth medium and declined somewhat thereafter. The incorporation of inducers (carbohydrates) in defined basal medium resulted in only small variations in specific activity in the resulting cells; The activity (p-NPG substrate) was not inhibited by D-glucose, phloretin, esculin, salicin, arbutin, lactose, or cellobiose, but was slightly inhibited by 1.0 mM phloridzin. Slight inhibition was observed in the presence of sulfhydryl reagents (iodoacetamide, p-chloromercuribenzoate), but sodium azide, ethylene-diaminetetraacetic acid, Cu2+, and Zn2+ ions produced no effect. The activity was stable, in both crude and purified preparations, over the pH ranges 6.0-7.5 (100% activity) and 4.5-greater than 8.5 (50% activity). The enzyme retained 80% activity after 30 min at 50 degrees C, but only 25% after 30 min at 60 degrees C. The enzyme had a mean K-m value (phloridzin) of 1.35 times 10-4 M, an isoelectric point of 4.75, a molecular weight, determined by Sephadex G-200 gel filtration, of about 122 000, and an optimum pH for activity of 6.5-7.0.     

The consensus sequence of ice nucleation proteins from Erwinia herbicola, Pseudomonas fluorescens and Pseudomonas syringae

The consensus sequence of three bacterial ice nucleation proteins was determined by extrapolation from the nucleotide (nt) sequences of three ice nucleation-encoding genes, iceE (presented here), inaW and inaZ. The three proteins possess considerable similarity, so that a preferred amino acid is shown in most positions of the consensus. The corresponding genes show considerable divergence in the third nt positions of synonymous codons, suggesting that the proteins' conserved features have been maintained by selection. Therefore, the consensus sequence is likely to represent the components of primary structure most important to the ice nucleation function.     

Purification and Properties of Isocitrate Lyase from Pupas of the Butterfly Papilio machaon L.

Key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were identified in pupas of the butterfly Papilio machaon L. The activities of these enzymes in pupas were 0.056 and 0.108 unit per mg protein, respectively. Isocitrate lyase was purified by a combination of various chromatographic steps including ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl, and gel filtration. The specific activity of the purified enzyme was 5.5 units per mg protein, which corresponded to 98-fold purification and 6% yield. The enzyme followed Michaelis-Menten kinetics (Km for isocitrate, 1.4 mM) and was competitively inhibited by succinate (Ki = 1.8 mM) and malate (Ki = 1 mM). The study of physicochemical properties of the enzyme showed that it is a homodimer with a subunit molecular weight of 68 +/- 2 kD and a pH optimum of 7.5 (in Tris-HCl buffer).     

Production of L-DOPA from pyrocatechol and DL-serine by bioconversion using immobilized Erwinia herbicola cells

Summary Immobilized cells of Erwinia herbicola were used for L-DOPA production from pyrocatechol and DL-serine. Optimal conditions have been defined and utilized in batch and continuous reactors. A maximal volumetric productivity of 0.46 g/l.h in L-DOPA was obtained with a conversion yield of 18% (L-DOPA concentration 2.3 g/l).     

Partial Purification of Isocitrate Lyase from Candida tropicalis and Some Kinetic Properties of the Enzyme

Isocitrate lyase was purified partially from n-alkane-grown cells and glucose-grown cells of Candida tropicalis by means of ammonium sulfate fractionation and DEAE-cellulose column chromatography. The preparation from alkane-grown cells showed one peak of the enzyme activity, while that from glucose-grown cells showed two distinct peaks of the activity, on DEAE-cellulose column chromatography. These enzymes, having the similar pH optima (around 7.0) and Km values with dl-isocitrate (1.2 ~ 1.7 mm), were inhibited by various metabolic intermediates, such as 6-phosphogluconate and phosphoenolpyruvate.

Time-course changes in the activities of isocitrate lyase and isocitrate dehydrogenases of C. tropicalis during the growth indicated that the lyase would participate preferentially in alkane assimilation and NAD-linked isocitrate dehydrogenase in glucose utilization of the yeast.

Regulation of isocitrate metabolism in C. tropicalis through glyoxylate cycle and tricarboxylic acid cycle is discussed based on the kinetic properties, cellular localization and time- course changes in the levels of isocitrate lyase and NAD-linked and NADP-linked isocitrate dehydrogenases.     

Partial Purification and Some Properties of Tyrosine Phenol-Lyase from Aeromonas phenologenes ATCC 29063

Tyrosine phenol-lyase was purified 32-fold from Aeromonas phenologenes ATCC 29063, the organism that produces phenol in refrigerated haddock. The purification procedure included ammonium sulfate fractionation, protamine sulfate treatment, and column chromatography with Sephadex G-200, diethyl-aminoethyl-cellulose, and hydroxyapatite. The enzyme was found to be thermally inactivated at temperatures above 40 degrees C. The optimum pH of the enzyme was found to be pH 8.5. The Michaelis constants for l-tyrosine and pyridoxal phosphate were 2.3 x 10 M and 3.2 x 10 M, respectively. The molecular weight of tyrosine phenol-lyase was found by gel filtration and electrophoresis to be approximately 380,000.