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.     

Another Pectate Lyase Produced by Streptomyces nitrosporeus

Another pectate lyase was purified to a nearly homogeneous state from the culture filtrate of Streptomyces nitrosporeus. The molecular weight was estimated to be about 41,000. Iso-electric point was pH 4.6. The enzyme was most active at pH 10.0 and 50°C, and was relatively stable at a pH range of 4–11 (at 2°C for 48 hr) and below 40°C (at pH 7.0 for 10 min). Ca²⁺ was required for maximum activity. The enzyme was an endo-pectate lyase which was more active on low methoxyl pectin than on polygalacturonic acid and had macerating activity on potato tissue and Ganpi bark.     

Production, Purification of Exo-Polygalacturonase from Soil Isolate Paecilomyces variotii NFCCI 1769 and Its Application

The aim of the present study was to produce exo-polygalacturonase from potent soil isolate by submerged fermentation and its application for fruit juice treatment. Pectinase producing strains were selectively isolated from pectin industry waste. A selected isolate C2 was found to produce significant amount of exo-polygalacturonase. The isolate was identified as Paecilomyces variotii on the basis of morphological characteristics and 18S rRNA gene sequence analysis. The exo-polygalacturonase produced by the isolate was purified by ammonium sulphate precipitation, size exclusion chromatography and ion exchange chromatography. The purified enzyme had MW of 39.4 kD based on SDS PAGE. Under partially optimized conditions, purified exo-polygalacturonase showed specific activity of 98.49 U/mg protein at pH 6.0 and 30°C. The enzyme was comparatively stable from 10 to 30°C and the activity decreased with increasing temperature. Purified enzyme brought about considerable reduction in viscosity of fruit juice samples.     

Purification and characterization of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes sp. nov.

Summary Membrane-bound alcohol dehydrogenase (ADH) was purified from the membrane fraction of an industrial-vinegar-producing strain, A. polyoxogenes sp. nov. NBI1028 by solubilization using Triton X-100 and subsequent column chromatography on DEAE-Sepharose CL-6B and hydroxyapatite. The purified enzyme was homogeneous on polyacrylamide disc gel electrophoresis (PAGE). Upon sodium dodecyl sulphate-PAGE, the enzyme showed the presence of two subunits with a molecular mass of 72 000 daltons and 44 000 daltons, respectively. The small subunit was identified as cytochrome c. In addition, absorption and fluorescence spectra showed the the presence of pyrroloquinoline quinone in the purified ADH. The ADH preferentially oxidized aliphatic alcohols with a straight carbon chain except for methanol. Formaldehyde and acetaldehyde were also oxidizable substrates. The apparent K _m for ethanol was 1.2 mM. The optimum pH and temperature were 5.0–6.0 and 40°C, respectively. p-Chloromercuribenzoic acid and heavy metals such as CuSO₄ were inhibitory to the enzyme activity. Ferricyanide was effective as an electron acceptor.Offprint requests to: M. Fukaya     

Purification and characterization of membrane-bound aldehyde dehydrogenase from Acetobacter polyoxogenes sp. nov.

Summary Membrane-bound aldehyde dehydrogenase (ALDH) was purified from the membrane fraction of an industrial-vinegar-producing strain, Acetobacter polyoxogenes sp. nov. NBI1028 by solubilization with Triton X-100 and sodium N-lauroyl sarcosinate and subsequent column chromatography on DEAE-Sepharose CL-6B and hydroxyapatite. The purified enzyme was homogeneos on polyacrylamide disc gel electrophoresis. Upon sodium dodecyl sulphate-polyacrylamide gelelectrophoresis, the enzyme showed the presence of two subunits with a molecular mass of 75 000 daltons and 19 000 daltons, respectively. From the absorption and fluorescence spectra, the absence of cytochrome c and the presence of pyrroloquinoline quinone in the purified enzyme were demonstrated. The ALDH preferentially oxidized aliphatic aldehyde with a straight carbon chain except for formaldehyde. The apparent K _m for acetaldehyde was 12 mM. The optimum pH and temperature were 7.0 and 50°–60°C, respectively. The enzyme remained active after storage at 4°C for 20 days. p-Chloromercuribenzoic acid and heavy metal salts such as CuSO₄ were inhibitory to the enzyme. Ferricyanide was effective as an electron acceptor.Offprint requests to: M. Fukaya     

Endo-polygalacturonate lyase of Cytophaga johnsonii

Summary An extracellular endopolygalacturonate lyase of Cytophaga johnsonii was purified from the culture filtrate. It appeared to be homogeneous as judged by polyacrylamide gel electrophoresis at pH 8.6 as well as pH 4.3. The purified enzyme had a pH optimum around 9.0 and required Ca⁺⁺ ions for its maximum activity. The apparent K _mfor polygalacturonic acid was found to be 0.22%. Both paper and column chromatography indicated formation and accumulation of an unsaturated monomer along with unsaturated di-, tri-, tetra- and pentamers from polygalacturonic acid by the enzyme action, indicating that the enzyme cleaved the substrate randomly in a non-hydrolytic manner. The glycosidic linkage next to the non-reducing end of polygalacturonic acid was not resistant to attack by this enzyme unlike in other known polygalacturonate lyases.Abbreviations PG lyase Polygalacturonate lyase - Tris Tris (hydroxymethyl) aminomethane     

Cloning and expression in Escherichia coli of pectinase genes of Erwinia carotovora subsp. carotovora.

Genes coding for an endo-pectate lyase, an exo-pectate lyase, and an endopolygalacturonase of Erwinia carotovora subsp. carotovora Ecc71 were cloned in Escherichia coli HB101, using the cosmid pHC79. The products of the cloned pectinase genes paralleled their counterparts in strain Ecc71 in isoelectric mobility, mode of substrate degradation, and ability to macerate potato tuber tissue.     

Cloning and expression in Escherichia coli of pectinase genes of Erwinia carotovora subsp. carotovora

Genes coding for an endo-pectate lyase, an exo-pectate lyase, and an endopolygalacturonase of Erwinia carotovora subsp. carotovora Ecc71 were cloned in Escherichia coli HB101, using the cosmid pHC79. The products of the cloned pectinase genes paralleled their counterparts in strain Ecc71 in isoelectric mobility, mode of substrate degradation, and ability to macerate potato tuber tissue.     

Hydrolysis of orange peel by a pectin lyase-overproducing hybrid obtained by protoplast fusion between mutant pectinolytic Aspergillus flavipes and Aspergillus niveus CH-Y-1043

Pectin lyases cleave the internal glycosidic bonds of pectin by β-elimination, producing non-saturated galacturonic oligomers. Genetic improvement of pectin lyase-overproducing strains is still necessary to improve industrial processes based on this enzyme. In the present study hybrids were obtained by protoplast fusion between mutant pectinolytic Aspergillus flavipes and Aspergillus niveus CH-Y-1043 strains. Prototrophic segregants showed different isoenzymatic profiles and produced increased levels of pectin lyase in cultures containing lemon peel as a sole carbon source. Hybrid HZ showed an increase of 450% and 1300% in pectin lyase production compared with that of A. niveus CH-Y-1043 and A. flavipes, respectively. Pectin lyase produced by the hybrid HZ was partially purified and used for the hydrolysis of orange peel. Pectin lyase was able to hydrolyze 56% of orange peel biomass. However, addition of 2 RFU and 20 U of endo- and exo-polygalacturonase, respectively, induced the hydrolysis of 92% of orange peel solids. In conclusion HZ is a pectin lyase-overproducing hybrid with potential applications in the pectin industry.     

Requirement for two or more Erwinia carotovora subsp. carotovora pectolytic gene products for maceration of potato tuber tissue by Escherichia coli.

Several genes encoding enzymes capable of degrading plant cell wall components have been cloned from Erwinia carotovora subsp. carotovora EC14. Plasmids containing cloned EC14 DNA mediate the production of endo-pectate lyases, exo-pectate lyase, endo-polygalacturonase, and cellulase(s). Escherichia coli strains containing one of these plasmids or combinations of two plasmids were tested for their ability to macerate potato tuber slices. Only one E. coli strain, containing two plasmids that encode endo-pectate lyases, exo-pectate lyase, and endo-polygalacturonase, caused limited maceration. The pectolytic proteins associated with one of these plasmids, pDR1, have been described previously (D. P. Roberts, P. M. Berman, C. Allen, V. K. Stromberg, G. H. Lacy, and M. S. Mount, Can. J. Plant Pathol. 8:17-27, 1986) and include two secreted endo-pectate lyases. The second plasmid, pDR30, contains a 2.1-kilobase EC14 DNA insert that mediates the production of an exo-pectate lyase and an endo-polygalacturonase. These enzymes are similar in physicochemical properties to those produced by EC14. Our results suggest that the concerted activities of endo-pectate lyases with endo-polygalacturonase or exo-pectate lyase or both cause maceration.     

Overexpression of alginate lyase of <Emphasis Type="Italic">Pseudoalteromonas elyakovii</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>, purification,and characterization of the recombinant alginate lyase

The alyPEEC gene encoding alginate lyase from marine bacterium Pseudoalteromonas elyakovii IAM 14594 was subcloned into pBAD24 with arabinose promoter and sequenced, and overexpressed in TOP10 strain of E. coli after arabinose induction. Expression levels of alyPEEC gene in E. coli cells were over 39.6-fold higher than those in P. elyakovii IAM 14594 cells. The molecular mass of purified alginate lyase from the engineered E. coli cells was estimated to be 32.0 kDa. Optimum pH and temperature of the alginate lyase activity were 7.0 and 30 °C, respectively. The enzyme was unstable on heating and in acidic and alkaline solution. The enzyme activity was stimulated by the MgCl₂, NaCl, KCl, CaCl₂, BaCl₂ and MnCl₂, but was inhibited by the addition of 1.0 mM of EGTA, EDTA, SDS, ZnSO₄, AgNO₃, and CoCl₂. All the alginate, polyM and polyG could be converted into oligosaccharides with more than tetrasaccharides by the purified recombinant alginate lyase, suggesting that the recombinant alginate lyase produced by the engineered E. coli has highly potential application in seaweed genetics, food and pharmaceutical industries.     

Sugar Synthesis in Leptospira

The presence and some properties of the key enzymes of the glyoxylate cycle, isocitrate lyase (threo-D_s-isocitrate glyoxylate-lyase, EC 4.1.3.1) and malate synthase (L-malate glyoxylate-lyase (CoA-acetylating) EC 4.1.3.2), were investigated in Leptospira biflexa. Isocitrate lyase activity was found for the first time in the organism. The enzyme was induced by ethanol but not by acetate. The optimum pH was 6.8. The activity was inhibited by phosphoenolpyruvate, a specific inhibitor of isocitrate lyase. The optimum pH of malate synthase of L. biflexa was about 8.5. The K_m value for glyoxylate was 3.0 × 10^?3 M and the activity was inhibited by glycolate, the inhibitor. The results strongly suggested the presence of a glyoxylate cycle in Leptospira. The possibility that the glyoxylate cycle plays an essential role in the synthesis of sugars, amino acids and other cellular components as an anaplerotic pathway of the tricarboxylic acid cycle in Leptospira was discussed.     

Enzymatic production of microthecin by aldos-2-ulose dehydratase from 1,5-anhydro-D-fructose and stability studies of microthecin

Microthecin (2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one), which has anti-microbial activity, is one of the end products of an alternative glycogen and starch degrading pathway, the anhydrofructose pathway. It is formed from 1,5-anhydro-d-fructose (AF), a product of α-1,4-glucan lyase (EC 4.2.2.13) from glycogen and starch by aldos-2-ulose dehydratase (AUDH; EC 4. 2.1.110). In the current study, the yield and purity of microthecin was examined with respect to pH and buffers using AUDH purified from the fungus Phanerochaete chrysosporium. It was found that AUDH had a K_m of 5.4 and 4.9 mM towards AF and its natural substrate glucosone, respectively, while its V_max with AF was 5.5 times higher than that with glucosone. Higher molar conversion of 90% was obtained in a reactor with pH controlled around 5.0 and 24°C with de-ionized water as the solvent. Microthecin was found to be most stable in de-ionized water at a pH around 7.0 and stable in freeze-dried form. Under acidic conditions microthecin was degraded to 2′-furyl-2-hydroxymethylketone (FHMK). A ¹³C-NMR method was established to simultaneously monitor the reaction components including AF, microthecin and its intermediates. A mechanism of microthecin formation from AF via the intermediate ascopyrone M (APM) and the degradation of microthecin to FHMK are proposed based on the NMR data obtained.     

Effect of pectin on pectinases in autolysis of Botrytis cinerea

Pectic activity in autolyzed cultures of Botrytis cinerea in a medium with and without pectin was similar, but in the medium with pectin maximal activities occurred in younger cultures. The pectic activities found were polygalacturonase, polymethylgalacturonase, endo activity (pectin as substrate) and pectin lyase. The molecular weights of polygalacturonase, polymethylgalacturonase and endo activity (pectin as substrate) were 36000, 33000 and 30200 daltons respectively, and the molecular weight of pectin lyase was 18200 daltons. By gel electrophoresis four different pectic activities were detected, three in the top of the gel and one in the bottom. Two enzymes were characterized, the polygalacturonase activity (first band in the top) inhibited by Ca⁺⁺ and the pectin lyase activity (in the bottom) which was not inhibited by Ca⁺⁺. These enzymes are not induced by the presence of pectin in the medium during degradation of Botrytis cinerea.     

Production and properties of three pectinolytic activities produced byAspergillus niger in submerged and solid-state fermentation

Three extracellular pectinases were produced byAspergillus niger CH4 by submerged and solid-state fermentation, and their physicochemical and kinetic properties were studied. The highest productivities of endo- and exo-pectinase and pectin lyase were obtained with solid-state fermentation. The kinetic and physicochemical properties of these enzymes were influenced by the type of culture method used. All activities were very different in terms of pH and temperature optima, stability at different pH and temperature values and affinity for the substrate (K _m values). In solid-state fermentation, all pectinase activities were more stable at extreme pH and temperature values but theK _m values of endo-pectinase and pectin lyase were higher with respect to those activities obtained by the submerged-culture technique. The pectin lyase activity obtained by the submerged-culture technique showed substrate inhibition but the enzyme obtained by solid-state fermentation did not. Electrophoresis, using sodium dodecyl sulphate/polyacrylamide gel with enzymatic extracts obtained for both culture methods, showed the same number on protein bands but some differences were found in their electrophoretic position. The results obtained in this work suggest that the culture method (submerged or solid-state) may be responsible for inducing changes in some of the pectinolytic enzymes produced byA. niger.     

Purification and properties of alcohol oxidase from Tanacetum vulgare

Alcohol oxidase (alcohol: O₂ oxidoreductase) from leaves of Tanacetum vulgare has been purified 5150-fold to homogeneity on disc electrophoresis and gel electrofocussing. The enzyme which is probably flavoprotein, has molecular weight 180 000 daltons and is comprised of two sub-units of 94 000 and 75 000 daltons. It is active over a broad range (pH 5–9) and best accepts primary aliphatic alcohols with 6 to 10 carbons, especially those with a 2-ene group. K_m values for hex-trans-2-ene-1-ol, geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol) and n-octanol were 0.19, 1.56 and 0.49 mM respectively. The significance of the enzyme in the formation of leaf aldehyde (hex-trans-2-ene-1-al) and in terpene metabolism is discussed.     

Purification and characterization of pectin lyase secreted by <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The molecular mass determined by SDS-PAGE was 31 kDa. The K _m and k _cat values were found to be 1 mg/ml and 76 sec⁻¹, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 985–992.     

Selenocysteine lyase activity in a cysteine-requiring mutant ofEscherichia coli K-12

Selenocysteine lyase activity was detected in crude extracts from a cysteine-requiring mutant ofEscherichia coli K-12. The level of activity was the same whether cells had been grown aerobically or anaerobically, with or without selenocysteine. Selenocysteine lyase catalyzes the conversion of selenocysteine to alanine and elemental Se, a reaction that is followed by a nonenzymatic reduction of the Se to hydrogen selenide. Both of these end products were identified in this study. With cysteine as the substrate, alanine and H₂S were formed, but only at levels 50% less than the products formed from selenocysteine. Selenocysteine lyase has been identified in a number of mammals and bacteria; its presence in a cysK mutant ofE. coli K-12 suggests a common route whereby hydrogen selenide, derived from selenocysteine, can then be assimilated into selenoproteins.     

Physical properties of pectic polysaccharidases ofPseudomonas solanacearum from Nigeria

Pseudomonas solanacearum (obtained from Nigeria) produced certain pectic polysaccharidases when grown in aerobic batch cultures containing pectic substances. The pH optima of the enzymes were different. The optimum for polygalacturonase EC 3.2.2.15 was 5.5, and for pectate lyase EC 4.2.99.3 it was 8.5. The -1,4-glycosidic bonds between galacturonide units were cleaved at random, indicating the endo character of the enzymes. The pectic polysaccharidases were purified by (NH₄)₂SO₄ fractionation and by electrofocusing. Highest polygalacturonase activity and pectate lyase activity were obtained in the fractions at 41%–60% and 61%–80% (NH₄)₂SO₄ saturation, respectively. Polygalacturonase was resolved into two components with isoelectric points of 5.0 and 7.5; the isoelectric point of pectate lyase was 8.1.     

Purification of Sulfated Fucoglucuronomannan Lyase from Bacterial Strain of <Emphasis Type="Italic">Fucobacter marina</Emphasis> and Study of Appropriate Conditions for Its Enzyme Digestion

A marine bacterial strain, Fucobacter marina, produced extracellular sulfated fucoglucuronomannan (SFGM) lyase when cultivated in the presence of crude SFGM obtained from fucoidan of Kjellmaniella crassifolia (brown algae) by cetyl pyridinium chloride fractionation. For the SFGM lyase assay, SFGM fraction separated from K. crassifolia fucoidan by anion exchange column chromatography was used as the substrate. The extracellular SFGM lyase was purified to homogeneity on an electrophoresis gel with 4240-fold purity at 13.8% yield. The enzyme proved to be a monomer, since gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis gave the same relative molecular mass of 67,000. The enzyme specifically digested SFGM but did not digest any other uronic-acid-containing polysaccharides tested. The optimum conditions for the enzyme reaction were around pH 7.5, 43°C, and 0.4 M NaCl concentration. The enzyme was strongly inhibited by CuCl₂ and ZnCl₂, and also by some sulfhydryl reagents.