Identification of amino acid residues critical for. Jun 05, 2008 read expression of pectate lyase a from aspergillus nidulans in bacillus subtilis, world journal of microbiology and biotechnology on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. The polypeptide backbone folds into a large righthanded cylinder, termed a parallel b helix. Among the known pectinases, lyases cleave a1,4 bonds between galacturonic acid residues by transelimination to produce 4,5unsaturated galacturonide. The threedimensional structure of a complex between the pectate lyase c pelc r218k mutant and a plant cell wall fragment has been determined by xray diffraction techniques to a resolution of 2. Aspergillus niger is also cultured for the extraction of the enzyme, glucose oxidase, used in the design of glucose biosensors, due to its high affinity for.
In this study, a novel pectate lyase appel1 was identified and characterized from aspergillus parasiticus. Identification of fungi of the genus aspergillus hydrolytic enzymes like lipases and amylases 1, 26. This message will disappear when all data is loaded. The appel1 hydrolysed oligogalacturonides ogs effectively and produced 4,5unsaturated ogs from lowmethoxyl lm pectin, with dp 2 to dp 5 as the major products. A pectin lyase, named pliii, was purified to homogeneity from the culture filtrate of aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. The current study was conducted for the production and characterization of pectin lyase from aspergillus niger using citrus fruits peel as a substrate. Kester, d jaap visser, d and frances jurnak a,3 a department of physiology and biophysics, 346d med sci i, university of california, irvine.
The aspergillus niger plya gene encoding pectate lyase a ec 4. The variability in the number of pectate lyase genes among different species of aspergillus with a maximum of eight pl genes in a. Aspergillus flavus nrrl 3357, aspergillus niger, aspergillus nidulans, aspergillus fumigatus af293, aspergillus oryzae, aspergillus. Pectin and pectinases the open biotechnology journal. Crystallization and preliminary xray analysis of pectin. In addition, the acidic pectin lyase pnlzj5a can effectively decreased the viscosity. Pectin lyase acts on highly methylated and nonesteri. Identification and characterization of a thermostable. The j b c 1999 by the american society for biochemistry.
Pectate lyase antisera were produced and used to identify pectate lyase clones in a cdna expression library. Aspergillus nidulans pectate lyase was purified from culture filtrates. An adi not specified was established at the 35th jecfa 1989. The refinement alternated between manual building and rebuilding of the. The j b c 2002 by the american society for biochemistry. The j b c 2002 by the american society for biochemistry and. So far pectin lyases have been isolated and identified mostly in fungi, like neurospora, botrytis, penicillia, and aspergilli. It shows approximately 20% identity with the bacterial pectate lyases and 4560% identity with the other isozymes found in aspergillus. Pectate lyase pl was produced by the filamentous fungus penicillium viridicatum rfc3 in solidstate cultures of a mixture of orange bagasse and wheat bran 1. Among pectinolytic enzymes, pectin lyase is the most important in depolymerization of pectin, since it cleaves internal glycosidic bonds of highly methylated pectins.
The pectin lyase gene pnlzj5a from aspergillus niger zj5 was identified and expressed in pichia. Protein engineering of lyases specific for particular patterns of methylation, will yield modified pectins of high value to the food and pharmaceutical industries. Pectate lyase aspergillus sp ammonium sulphate enzyme megazyme to be able to use megazyme in full range, we recommend activating javascript in your browser. Commercial preparations are mixtures of polygalacturonases, pectate lyases and pectin esterases. Production of pectate lyase by penicillium viridicatum. An acidic pectin lyase from aspergillus niger society for. Pectate lyases, cell wall degradation and fruit softening. Biotechnological valorization of pectinolytics and. In enzymology, a pectin lyase also known as pectolyase is a naturally occurring pectinase a type of enzyme that degrades pectin. Plb shares 46 to 65% amino acid sequence identity with other a. Three bacterial pectate lyases, a pectin lyase from aspergillus niger, the structures of rhamnogalacturonase a from aspergillus aculeatus, rgase a, and the p22phage tailspike protein, tsp, display the righthanded parallel bhelix architecture.
Pectate lyase aspergillus sp ammonium sulphate enzyme. Review article enzymatic extraction and clarification of. Evolution and functional characterization of pectate lyase. Identification of amino acid residues critical for catalysis and stability in aspergillus niger family 1 pectin lyase a. Production and partial purification of pectin lyase by aspergillus niger grown on. Pdf characterization of aspergillus niger pectate lyase a. The threedimensional structure of aspergillus niger pectin lyase b at 1. The gene encoding aspergillus nidulans pectate lyase a pela without signal peptide was fused with a sixhis tag at its cterminus in the expression vector vbszqx containing sac b signal peptide. The aspergillus niger strain is genetically modified to contain several copies of the wild type aspergillus nigerphytase gene and is referred to as aself cloned microorganism. Pliii was able to digest apple pectin and citrus pectins with di erent degrees of methyl esteri cation.
Expression of an erwinia pectate lyase in three species of. Plb consists of 359 amino acids with a molecular weight of 37,820 calculated from the amino acid sequence. An acidic pectin lyase from aspergillus niger with favourable ef. Regulation of the aspergillus nidulans pectate lyase gene pela. It is produced commercially for the food industry from fungi and used to destroy residual fruit starch, known as pectin, in wine and cider. Purification and characterization of a unique pectin lyase. Mode of action of pectin lyase a of aspergillus niger on differently. A gene fusion between the saccharomyces cerevisiae actin gene promoter and the cdna of the fusarium solani f. It shows approximately 20% identity with the bacterial pectate lyases and 4560% identity with the other isozymes found in aspergillus spp.
Read onlinepdf 82 kb go to volume 39, issue 50 biochemistry get e alerts. Pectate lyases catalyze the eliminative cleavage of deesterified pectin, which is a major component of the primary cell walls of many higher plants. To gain an understanding of the carbohydrate recognition strategy and catalysis by belimination in pectinolytic enzymes, we have determined the xray crystal structures of pectin lyase a from two strains of aspergillus niger n400 and 4m147 to a res. The isolation of the species of the nigri section on creatine sucrose agar crea enabled to distinguish the aspergillus sp species, which was characterized by the lack of sporulation and by the. Structure of a plant cell wall fragment complexed to pectate. In silico analysis of pectin lyase and pectinase sequences. Pectate lyase aspergillus sp aqueous glycerol enzyme megazyme to be able to use megazyme in full range, we recommend activating javascript in your browser. Pectate lyases cleave glycosidic linkages preferentially in polygalacturonic acid, forming an unsaturated product. The alkaline pectate lyase according to claim 9, which has the following enzymatic properties.
Exo pectate lyases are members of family pl2 and pl9. Microbial pectin and pectate lyases are virulence factors that degrade the pectic. The structure of pectin lyase a, described here, has established the structural basis for the substrate specificity of these two lyases and provides an understanding of the ph and. The threedimensional structure of aspergillus nigerpectin. The identified enzyme, named aspela, is orthologous to pela from a. Replacing a suite of commercial pectinases with a single. Purification and characterization of an alkaline pectin lyase from. Aspergillus giganteus produces a pectinolytic complex which includes at least one polygalacturonase and. Zj5a can effectively decreased the viscosity and improved the yield of apple juice and light transmittance. Furthermore, the multiple sequence alignments, structure model and phylogenetic analyses of the appel1 indicated that its. Three threedimensional structures have been solved, two structures of pectin lyase a pla and one from pectin lyase b plb, all from aspergillus niger. Construction of a recombinant wine yeast strain expressing.
Transgenic filamentous fungi of the species aspergillus niger, a. Two crystal structures of pectin lyase a from aspergillus. Pectinases play important roles in widespread industrial applications. Aspergillus niger pectate lyase pectin lyase polygalacturonic acid pectin methylesterase these keywords were added by machine and not by the authors. Cloning of a novel constitutively expressed pectate lyase. In silicoanalysis of pectin lyase and pectinase sequences 1053 biochemistry moscow vol. The crystal structures of pectin lyase a from two strains of aspergillus niger, n400 and 4m147, have been determined at ph6. Pmc free article gonzalezcandelas l, kolattukudy pe. Enzyme activity of the recombinant pela could reach 360 u ml. The threedimensional structure of aspergillus niger pectin. Production of pectate lyase by penicillium viridicatum rfc3. Structure of a plant cell wall fragment complexed to pectate lyase c robert d. Pectinolytic enzymes are used in diverse industrial applications.
Isolation and analysis of a novel inducible pectate lyase gene from the phytopathogenic fungus fusarium solani f. Recently, comparative studies revealed significant gene copy number expansion of the polysaccharide lyase 1 pl1 pectin pectate lyase gene family in the clonostachys rosea genome, while only low numbers were found in trichoderma species. Pliii was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Aspergillus niger growing from goldmining solution contained cyanometal complexes, such as gold, silver, copper, iron, and zinc. The threedimensional structure of aspergillus niger pectin lyase b plb has been determined by crystallographic techniques at a resolution of 1. The threedimensional structure of aspergillus niger pectin lyase. Aspergillus niger, n400 and 4m147, have been determined at ph 6. Three threedimensional structures have been solved, two structures of pectin lyase a pla 7 and one from pectin lyase b plb 8, all from aspergillus niger. Zj5a was purified by ultrafiltration, anion exchange and gel chromatography. A pectin lyase, named pliii, was puri ed to homogeneity from the culture ltrate of aspergillus giganteus growninsubmerged culture containing orange peel waste as carbon source.
The aspergillus nigerplya gene encoding pectate lyase a ec. Several cycles of manual rebuilding in o 25 and positional. Aspergillus sp ufla dca 01, despite not having been totally effective in elucidating species related to a. The gene encoding aspergillus nidulans pectate lyase a pela without signal peptide was fused with a sixhis tag at its cterminus in the expression vector vbszqx containing sac b signal peptide and p43 promotor. Pectinolytic enzyme consist of four classes of enzymes. Request pdf purification and characterization of an alkaline pectin lyase. The plya gene was overexpressed using a promoter fusion with the a. Only for pectate lyase has a threedimensional structure in complex with a galpa. Prepared at the 55th jecfa 2000 and published in fnp 52 add 8 2000, superseding tentative specifications prepared at the 31st jecfa 1987 and published in fnp 38 1988 and in fnp 52 1992. Biochemical characterization and evolutionary analysis of. Mode of action of pectin lyase a of aspergillus nigeron. The structures and active sites of pectinases springerlink.
Correct processing of the preenzyme was achieved using the a. The pectate lyase is an endo enzyme, requiring calcium ions for its activity. They are all endoacting enzymes and have been classified together with the majority of pectate lyases within family 1 of the polysaccharide lyases. Embl4 library using the aspergillus nidulans pectate lyase encoding gene dean, r. Pectate lyase is responsible for the eliminative cleavage of pectate, yielding oligosaccharides with 4deoxy. Expression, purification and characterization of pectate. The polypeptide backbone folds into a large righthanded. Pdf production and partial purification of pectin lyase by. Aspergillus nigeratcc16404 strain by deliberately adding increasing concentration of pectin in their growth media and studied its effect on their enzyme production capabilities. Characterization of aspergillus niger pectate lyase a. Pectate lyase aspergillus sp aqueous glycerol enzyme.
Aspergillus niger when fermented with distillers yeast 19. Characterization of aspergillus niger pectate lyase a, biochemistry. The activity of pectin lyase a is ph dependent with an optimum activity at ph 5. Effect of ph on commercially applicable pectinase enzyme. An acidic pectin lyase from aspergillus niger with. The protein is maximally expressed late in pollen development. Rna extraction from powdered mycelia according to the manual. Kinetic characterization of aspergillus niger n400. Computational assessment of predicted three dimensional. To date, no threedimensional structures in complex with substrate are available. It has been suggested that the pollen expression of pectate lyase. Favors pectate, the anion, over pectin, the methyl ester by similarity. An acidic pectin lyase from aspergillus niger with favourable. Two crystal structures of pectin lyase a from aspergillus reveal a ph.
The km and vmax values determined using citrus pectin were 066 mg ml. The model, with all 359 amino acids and 339 water molecules, refines to a final crystallographic r factor of 16. Regulation of the aspergillus nidulans pectate lyase gene. Aspergillus niger atcc16404 strains were grown in to 5 sets of pectin enriched pda media with pectin concentration as 0.
The aspergillus nigerplya gene encoding pectate lyase a ec 4. This process is experimental and the keywords may be updated as the learning algorithm improves. Isolation and analysis of a novel inducible pectate lyase. Recently, the crystal structure of endopolygalacturonase from the bacterium erwinia carotovora was solved 10. Pectate lyases can act on the esterified pectin while the polygalacturonases act on the desesterified pectin. In plant cell culture, it is used in combination with the enzyme cellulase to generate protoplasts by degrading the plant cell walls. Characterization of pectate lyase a from aspergillus niger. Aspergillus niger pectin lyase a and bacillus subtilis pectate lyase show 17%. Interestingly, the pliii activity was stimulated in the presence of some divalent cations including pb 2.
Chy1043 article pdf available in applied microbiology and biotechnology 394. The expressed pela exhibited its optimum level of activity over the range of ph 7. We sought to isolate a pectate lyase from aspergillus luchuensis var. Pectate lyases are produced by many bacteria, where they are the largest group of pectinolytic enzymes and are directly involved in plant. Favors pectate, the anion, over pectin, the methyl ester. The threedimensional structure of aspergillus nigerpectin lyase b plb has been determined by crystallographic techniques at a resolution of 1. The polypeptide backbone folds into a large righthanded cylinder, termed a parallel. Thirteen of 14 clones identified immunologically crosshybridized. Plb belongs to a multigene family of pectin lyases produced by aspergillus niger kustervan someren et al. Structure and evolution of parallel bhelix proteins.
The k m and v max values determined using citrus pectin were 066 mg ml. Both of these fungal genera are widely known for their ability to parasitize and kill other fungi. Aspergillus niger pectin lyase a and bacillus subtilis pectate lyase show 17% sequence identity after structural alignment, and represent two distinct specificities. The lyases have 7 complete coils while rgaseaand tsp have 11 and 12. The threedimensional structure of aspergillus niger. Research article purification and characterization of a. Pdf characterization of aspergillus niger pectate lyase. Biochemical characterization and evolutionary analysis of a. The enzyme catalyzed a random eliminative cleavage reaction, had an apparent molecular weight of 40,000, and a pl of 4.
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