Pectin Lyase Production Research Articles

Production of pectin lyase by Penicillium griseoroseum in bioreactors in the absence of inducer

Penicillium griseoroseum was grown in bioreactors on mineral medium supplemented with yeast extract and sucrose. The influence of inoculum and carbon source concentrations, aeration and pH on pectin lyase (PL) production, as well as the capacity of P. griseoroseum to produce PL when grown on sugar cane syrup as carbon source were evaluated. Inoculum concentration did not influence PL production. Production was higher in non-aerated than in aerated medium. The best results were obtained using 60 mM sucrose at pH 6.3-7.2. Production using cane syrup 25% (v/v), without yeast extract supplement, was equal to that obtained under the conditions cited above.

Multiresponse analysis of microbiological parameters affecting the production of pectolytic enzymes by Aspergillus niger: a statistical view

The effect of microbiological parameters, viz., slant age, inoculum age and amount of inoculum were studied using response surface methodology for the production of pectolytic enzymes, polymethylgalacturonase, polygalacturonase and pectinlyase. The central composite design was used to determine the individual optimum values. Individual optimum values of slant age, inoculum age and amount of inoculum were 49.5 h, 52.3 h and 11.2%, respectively, for polymethylgalacturonase; 62.4 h, 60.5 h and 14.4%, respectively, for polygalacturonase, and 78.3 h, 52.5 h and 13.4%, respectively, for pectinlyase. In order to develop a two-stage inoculum possessing the best conditions for the maximum production of all three enzymes, a multiresponse analysis was carried out using a generalized distance approach. The microbiological parameters were optimized simultaneously for maximum production of enzymes. The simultaneous optimal values of slant age, inoculum age and amount of inoculum were 81.4 h, 66.7 h and 15.8%, respectively, for all the three enzymes. After optimization, the fermentation time was reduced from 144 to 120 h.

Application of Doehlert designs for water activity, pH, and fermentation time optimization for Aspergillus niger pectinolytic activities production in solid-state and submerged fermentation

The Doehlert design was applied to optimize water activity, pH, and fermentation time conditions for Aspergillus niger 148 pectinolytic activities production in solid-state (SSF) and submerged (SmF) fermentation. The fermentation technique had a great influence on the composition of pectinases produced by the fungus. Production of polygalacturonase was 5-fold higher in SmF than in SSF. However, pectin lyase production was 3 times higher in SSF than in SmF. Pectinesterase was only a 30% higher in SSF than in SmF. Optimization of the process was based on minimum pectinesterase production and maximal pectin lyase production. The optimal conditions to obtain the aforecited composition by a SSF process were initial pH 6.5–7, initial a w 0.93–0.94, and 2 days of fermentation. For the SmF process they were initial pH 5.5–6.2, initial a w above 0.99, and 3 days of fermentation. The results suggest that A. niger 148 overproduces pectin lyase under the optimized SSF culture conditions, as the concentration in the fermentation extract was around 0.25 g/l, representing ≈65% of total extracellular proteins produced by the fungus.

Cyclic AMP and low molecular weight effector(s) present in yeast extract are involved in pectin lyase production by Penicillium griseoroseum cultured on sucrose.

Pectin lyase (PL) induction by organic and inorganic components of yeast extract (YE) was evaluated in Penicillium griseoroseum, cultured in a mineral medium containing sucrose, by determining PL activity (A235) and mycelial growth (mycelial dry weight). The lowest YE concentration that promoted significant PL induction without acting as a carbon source for the fungus corresponded to 0.0075%. Neither calcined YE nor a nutrient solution containing micronutrients induced PL production, indicating that the inducer was an organic compound. Vitamins, phospholipid components, amino acids, and nitrogenous bases were tested in place of YE and promoted no significant PL induction. A PL inducer compound was found to be soluble in the nucleotide fraction obtained during extraction of YE. The inducer was shown to be a thermostable polar substance dialyzable at 2000 Daltons, hydrolyzable by HCl, and activated by boiling for up to 60 min. Cyclic AMP (cAMP) exogenously added to the culture medium at 5 and 10 mM was capable of inducing PL in P. griseoroseum grown on sucrose, suggesting that at least one compound may be present in YE acting in a cooperative fashion for the maintenance of high levels of cAMP into the cell. PL activity and the level of cAMP inside the fungal cells increased after the addition of YE to the culture medium, suggesting the participation of this messenger in this enzyme's synthesis.

Sugar-cane juice induces pectin lyase and polygalacturonase in Penicillium griseoroseum

The use of other inducers as substitutes for pectin was studied aiming to reduce the production costs of pectic enzymes. The effects of sugar-cane juice on the production of pectin lyase (PL) and polygalacturonase (PG) by Penicillium griseoroseum were investigated. The fungus was cultured in a mineral medium (pH 6.3) in a rotary shaker (150 rpm) for 48 h at 25oC. Culture media were supplemented with yeast extract and sucrose or sugar-cane juice. Sugar-cane juice added singly to the medium promoted higher PL activity and mycelial dry weight when compared to pectin and the use of sugar-cane juice and yeast extract yielded levels of PG activity that were similar to those obtained with sucrose-yeast extract or pectin. The results indicated that, even at low concentrations, sugar-cane juice was capable of inducing pectin lyase and polygalacturonase with no cellulase activity in P. griseoroseum.

Activation of the Erwinia carotovora subsp. carotovora pectin lyase structural gene pnlA: a role for RdgB.

The activation of pectin lyase (Pnl) production in Erwinia carotovora subsp. carotovora strain 71 occurs upon DNA damage via a unique regulatory circuit involving recA, rdgA and rdgB. In a similar Pnl-inducible system reconstituted in Escherichia coli, the rdgB product was found to activate the expression of pnlA, the structural gene for pectin lyase. The kinetic data presented here also show that transcription of pnlA followed that of rdgB in Er. carotovora subsp. carotovora, indicating a temporal order of gene expression. By deletion analysis we have localized the promoter/regulatory region within a 66 bp DNA segment upstream of the pnlA transcriptional start site. This region contains the -10 consensus sequence but not the sequences corresponding to the E. coli -35 region. For DNA-binding studies, rdgB was overexpressed in E. coli and a 14 kDa polypeptide was identified as the gene product. RdgB from crude extracts or a purified preparation caused an identical gel mobility shift of a 164 bp DNA segment containing the pnlA promoter/regulatory region. Utilizing DNase I protection assay the RdgB-binding site was localized between nucleotides -29 and -56, i.e. overlapping the position of the putative -35 box. The findings reported here, taken along with our previous observation that the rdgE product is required for pnlA expression, establishes that rdgB encodes a transcriptional factor which specifically interacts with the pnlA promoter/regulatory region.

Improvement of pectinase production by interspecific hybrids of Aspergillus strains

Protoplast fusion induced by polyethylene glycol and Ca 2+ , was performed between auxotrophic mutants of pectinolytic fungi Aspergillus sp. CH-Y-1043 (A13) ade- and Aspergillus flavipes ATCC-16795 (F7) lys-. Prototrophic colonies were developed on minimal medium with a fusion frequency of 1.0 x 10 -2 . The reversion frequency of the mutation in spores and protoplasts was low and ranged from 20 to 40 x 10 -7 . Four prototrophic hybrids (HH, HE, HF and HJ) exhibited enhanced production of endo-pectinase and pectin-lyase. The highest production was observed in HJ; maximum activities were 150 and 160% respectively, whereas the exo-pectinase production was similar to the wild-type strain Aspergillus sp. CH-Y-1043. Hybrid HJ showed the greatest growth; nevertheless, specific endo-pectinase and pectin-lyase activities were higher in all hybrids than those produced by the wild-type strains.

RecA relieves negative autoregulation of rdgA, which specifies a component of the RecA–Rdg regulatory circuit controlling pectin lyase production in Erwiniacarotovora ssp. carotovora

The production of pectin lyase (Pnl) in Erwinia carotovora ssp. carotovora strain 71 is induced by DNA-damaging agents such as mitomycin C (MC). This induction requires functions of recA, rdgA and rdgB genes. Based upon sequence homology, rdgA was predicted to encode a repressor and rdgB was presumed to specify a transcriptional activator. To elucidate the function of rdgA, the gene has been over-expressed in Escherichia coli, and the 30 kDa product purified by ammonium-sulphate precipitation, heparin-agarose chromatography and gel filtration. The results of gel mobility-shift and DNase I protection assays revealed that purified RdgA specifically binds the rdgA operator sequence located between the -10 and -35 boxes. The expression of a rdgA-lacZ gene fusion in E. coli MC4100 is suppressed upon overproduction of RdgA from a Ptac-rdgA construct induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). However, the suppression of rdgA-tacZ expression is relieved by MC in the RecA+ E. coli strain MC4100, but not in its RecA- derivative, MC4160. An immunoblot analysis revealed RecA-dependent in vivo cleavage of the 30 kDa RdgA protein upon MC treatment. These results demonstrate that the transcription of rdgA is autoregulated, and strongly support the idea that proteolytic activity of RecA* is responsible for the derepression of rdgA expression.

Pectin lyase production by Penicillium griseoroseum: Effect of tea extract, caffeine, yeast extract, and pectin

Mycelial growth and production of extracellular pectin lyase by Penicillium griseoroseum at different concentrations of inducers were investigated. The fungus was cultured in mineral medium using sucrose as a carbon source and caffeine, yeast extract, tea extract or pectin as inducers. Caffeine, yeast extract and tea extract in the presence of sucrose, and tea extract alone were capable of inducing pectin lyase in P. griseoroseum, even at low concentrations.

Protoplasts from pectinolytic fungi: isolation, regeneration and pectinolytic enzyme production

S. Solis, M.E. FLORES AND C. HUITRON. 1996. Protoplast release in pectinolytic strain mutants of Aspergillus sp. CH-Y-1043 (A13) and Aspergillus flavipes ATCC-16795 (F7) is described. Optimum yield of protoplasts A13 was obtained in a lapse of 1 h when commercially lytic enzymes of Trichoderma harzanium (2 mg ml−1) were added in 0.05 mol 1−1 citrate-phosphate buffer pH 5.0 containing 0.7 mol 1−1 KCl and 10 mg ml−1 BSA. Best results in F7 were obtained when the protoplasting system of A13 was supplemented with 10 mg ml−1Aureobasidium sp. lytic enzymes. Isolated protoplasts in A13 and F7 were capable of a high regeneration frequency of 87% and 53% when 0.7 mol 1−1 KCl and sorbitol were used as osmotic stabilizers. Endo-P, Exo-P and pectin lyase production were not modified during the process of regeneration.

Nucleotide sequence, organization and expression of rdgA and rdgB genes that regulate pectin lyase production in the plant pathogenic bacterium Erwinia carotovora subsp. carotovora in response to DNA-damaging agents.

In most soft-rotting Erwinia spp., including E. carotovora subsp. carotovora strain 71 (Ecc71), production of the plant cell wall degrading enzyme pectin lyase (Pnl) is activated by DNA-damaging agents such as mitomycin C (MC). Induction of Pnl production in Ecc71 requires a functional recA gene and the rdg locus. DNA sequencing and RNA analyses revealed that the rdg locus contains two regulatory genes, rdgA and rdgB, in separate transcriptional units. There is high homology between RdgA and repressors of lambdoid phages, specially phi 80. RdgB, however, has significant homology with transcriptional activators of Mu phage. Both RdgA and RdgB are also predicted to possess helix-turn-helix motifs. By replacing the rdgB promoter with the IPTG-inducible tac promoter, we have determined that rdgB by itself can activate Pnl production in Escherichia coli. However, deletion analysis of rdg+ DNA indicated that, when driven by their native promoters, functions of both rdgA and rdgB are required for the induction of pnlA expression by MC treatment. While rdgB transcription occurs only after MC treatment, a substantial level of rdgA mRNA is detected in the absence of MC treatment. Moreover, upon induction with MC, a new rdgA mRNA species, initiated from a different start site, is produced at a high level. Thus, the two closely linked rdgA and rdgB genes, required for the regulation of Pnl production, are expressed differently in Ecc71.

Production of pectin lyase by Penicillium griseoroseum cultured on sucrose and yeast extract for degumming of natural fibres

Sucrose, a non-pectic carbon source, with yeast extract (YE) added was able to support the production of pectin lyase (PL) by Penicillium griseoroseum Dierckx. However, in the absence of YE, the fungus did not produce PL but grew and caused a marked reduction in culture medium pH. Furthermore, in the absence of YE, only a culture medium with a high buffering capacity permitted the production of PL in the presence of pectin. On the other hand, in the presence of 0.06% YE and of 0.1% pectin, the fungus produced maximum growth and specific PL activity during a 48-h period of culture, with a small variation in medium pH. In the absence of sucrose, YE concentrations from 0 to 0.6% did not support enzyme production, indicating synergism between sucrose and YE for production of the enzyme.

Production of pectin lyase by Penicillium griseoroseum as a function of the inoculum and culture conditions

Optimum activity of an extracellular pectin lyase produced by Penicillium griseoroseum in submerged culture was after 120 h using 0.1% (w/v) citrus pectin as substrate. Sucrose at 0.1% (w/v) stimulated enzyme production and citrus pectin gave the highest activity of enzyme per unit growth.

Kinetic of the production of polygalacturonase and pectin lyase by two closely related Formae speciales of Fusarium oxysporum

The kinetic of the in vitro production of polygalacturonase and pectin lyase of two closely related fungi, Fusarium oxysporum f.sp. lycopersici and F. oxysporum f.sp. radicis-lycopersici, was examined under various culture conditions such as the source of carbon, the pH, and the age of cultures. Over a 5-day period, the production of these enzymes by various isolates of the same forma specialis (f. sp.) of F. oxysporum was not significantly different ( P ≥ 0.05). However, the amount of the enzymes produced differed markedly between both f. sp. The different carbon sources added to the culture media, such as citrus pectin, apple pectin, tomato cell wall fragments, and d-galacturonic acid, proved to be higher pectinase inducible substrates than sucrose and glucose. For both fungi, polygalacturonase and pectin lyase activities were optimal at pH 5.0 and 8.0, respectively. Furthermore, pectin lyase production had a partial Ca 2+ requirement in contrast to polygalacturonase production which was limited by Ca 2+. In most experiments performed, the production of polygalacturonase appeared superior with F. oxysporum f.sp. radicislycopersici than with F. oxysporum f.sp. lycopersici. On the other hand, pectin lyase production of F. oxysporum f.sp. lycopersici was approximately 10-fold greater than that by F. oxysporum f.sp. radicis-lycopersici in media supplemented with d-galacturonic acid.

Changed pectinase synthesis by aggregated mycelium of some Aspergillus niger mutants

By multistep mutagenesis (UV, MNNG, 80°C) of Aspergillus niger K 69/26 and a halo-screening procedure, some mutants growing in an aggregated mycelium structure were isolated that show a changed pectinase spectrum. Mutants that are also usually reduced in growth as Se 1 were obtained with approximately 100% increase in specific exo- and endopolygalacturonase activity in comparison to the parent strain. Furthermore, several mutants with different levels of exo- and endopolygalacturonase, pectinesterase, and pectinlyase were identified. By temperature treatment of a mutant reduced in polygalacturonase and pectinlyase production and negative in pectinesterase synthesis, some revertants were obtained, but the yield of the different pectinases was below that of the control strain. The mutants can be used to produce enzyme preparations that are specifically suited for different application purposes.

Production of extracellular pectinase enzymes by a mutant (Pol6) of Penicillium occitanis

Penicillium occitanis strain Pol6, a mutant developed for hyperproduction of cellulase and pectinase enzymes was used for the study of extracellular pectinase production when pectins from different sources (apple and citrus) and with varying degree of esterification (DE) were used as inducers. Highly esterified citrus pectins were found to be suitable substrates for polygalacturonase, pectinase and pectin methyl esterase production, while low esterified citrus pectin favoured pectin lyase (PL) production. Apple pectins induced other hydrolytic enzymes (e.g., β-1,3-glucanase, β-glucosidase, β-galactosidase), in addition to pectolytic enzymes. Moreover, the combination of high and low esterified citrus pectins induced the production of a complete pectinase complex. The extent of degradation of the substrate and the affinity for PL decreased with decreasing DE irrespective of the source. There was no evidence of PL activity in this strain. No significant effect of cations (Ca++, Mn++, Na+) on PL activity was observed. However, EDTA (100 mm) inhibited 50% of the activity, when tested on highly esterified (rapid set citrus) pectin.

Molecular cloning and characterization of an Erwinia carotovora subsp. carotovora pectin lyase gene that responds to DNA-damaging agents

recA-mediated production of pectin lyase (PNL) and the bacteriocin carotovoricin occurs in Erwinia carotovora subsp. carotovora 71 when this organism is subjected to agents that damage or inhibit the synthesis of DNA. The structural gene pnlA was isolated from a strain 71 cosmid gene library following mobilization of the cosmids into a moderate PNL producer, strain 193. The cosmid complemented pnl::Tn5 but not ctv::Tn5 mutations. A constitutive level of PNL activity was detected in RecA+ and RecA- Escherichia coli strains carrying the pnlA+ gene on the high-copy-number plasmid pBluescript SK-. Mappings of Mu dI1734 (Km lac'ZYA) insertions in pnlA and unidirectional deletion analyses allowed localization of the gene to approximately 1.4 kilobases of DNA. A typical pnlA-lacZ transcriptional fusion was inducible in a RecA+ but not a RecA- derivative of strain 71. In contrast, the pnlA-lacZ fusion was not inducible in a RecA+ E. coli strain. DNA sequences homologous to pnlA were detected in E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica strains and in one of four Erwinia rhapontici strains but not in Erwinia chrysanthemi.

Pectin Lyase Production by a Penicillium italicum Strain

Growth and concomitant production of an extracellular pectin lyase (PL) [poly(methoxylgalactosiduronate) endolyase; EC 4.2.2.10] were investigated in a group of 16 fungi grown in liquid medium containing pectin as a supplementary carbon source. Culture filtrates of both Penicillium italicum (CECT 2294) and P. expansum (CECT 2275) showed the highest PL activity and contained polygalacturonase but not pectinesterase activity. The effect of the inoculum size, the carbon source (sucrose and glucose syrup), and the presence of pectin on the production of PL by P. italicum was studied. The presence of 2.6 mM glycerophosphate in the culture medium enhanced the appearance of PL but was not inhibitory for the in vitro activity. However, glycerol inhibited the enzyme nearly 50% at such a concentration.

Selection and characterization of pectinase-deficient mutants of the vascular wilt pathogen Verticillium albo-atrum

Three putative pectinase-deficient mutants of the vascular parasite Verticillium albo-atrum which produced, 3, 9 and 43% of wild type pectin lyase were isolated on solid selection media specific for polygalacturonase and pectin lyase detection. The mutants (which were identical to the wild type in growth, morphology and sporulation, apart from 34i which grew poorly on pectic polymers) were grown on pectin, host cell walls, galacturonic acid and glucose to determine changes in production and regulation of pectin lyase, polygalacturonase, cellulase (C x ), β- d -galactosidase, β- d -glucosidase and l -leucine arylamidase. Analysis of culture fluids revealed isolate C23 to be defective in secretion of all enzymes and pectin lyase accumulated intracellularly; isolate 34i was unable to utilize galacturonides and was therefore jointly deficient in production of inducible polygalacturonase and pectin lyase. Analysis of isozyme patterns revealed that mutant 24d produced reduced levels of five of the six pectin lyase isozymes.

Production and properties of pectin lyase in Pseudomonas marginalis induced by mitomycin C.

Production and properties of pectin lyase in Pseudomonas marginalis induced by mitomycin C.