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.     

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.     

Continuous-flow enrichment of a strain ofErwinia carotovora having specificity for highly methylated pectin

A pectinolytic bacterium was isolated from a mixed microbial population by means of a chemostat enrichment procedure. The bacterium, which was identified asErwinia carotovora, grew only on highly methylated pectin and produced a pectin lysase which released unsaturated monomer and dimer from 71% esterified citrus pectin. The pectin lyase was inducible only by pectins having a high methyl content and in pectin-limited chemostats its synthesis passed through a maximum at a dilution rate close to 0.04h^-1.     

Pectinolytic activity of bacteria isolated from soil and two fungal strains during submerged fermentation

Seven different strains were selected for their ability to degrade citrus pectin. Alkaline pectinases were produced by five bacterial soil isolates, whereas two fungal strains produced pectinase in an acidic environment. The bacteria were isolated from soil of a plum orchard in Northern Ireland. These isolates produced significant amounts of pectin lyase (PL) and polygalacturonase (PG) with maximum activities of 30.1 and 29.1 U/ml respectively. Fungal strains Aspergillus sp. and PN-1 produced four different pectinolytic activities; endo-PG, exo-PG, pectin esterase (PE) and PL. The Aspergillus sp. produced higher amounts of pectinase than PN-1. The Aspergillus sp. excreted highly stable pectinases, which may be of importance for industrial applications.     

Production,purification and characterization of pectinase from a Bacillus sp. DT7

A soil isolate, Bacillus sp. DT7 has been found to produce significant amounts of an extracellular pectinase subsequently characterized as pectin lyase (EC 4.2.2.10). By optimizing growth conditions, Bacillus sp. DT7 produced higher amount of pectin lyase (53 units/ml) than that has been reported in the literature. Using gel filtration and ion exchange chromatography, this enzyme was purified and found to have a molecular mass of 106 kDa. The purified enzyme exhibited maximal activity at a temperature of 60 C and pH 8.0. The presence of 100 mM concentrations of CaCl₂ and mercaptoethanol significantly enhanced pectinase activity of the purified enzyme. This pectinase has tremendous applications in textile industry, plant tissue maceration and fruit juice wastewater treatments.     

Heterologous expression of a Penicillium purpurogenum pectin lyase in Pichia pastoris and its characterization

Lignocellulose is the major component of plant cell walls and it represents a great source of renewable organic matter. One of lignocellulose constituents is pectin. Pectin is composed of two basic structures: a ‘smooth’ region and a ‘hairy’ region. The ‘smooth’ region (homogalacturonan) is a linear polymer of galacturonic acid residues with α-(1→4) linkages, substituted by methyl and acetyl residues. The ‘hairy’ region is more complex, containing xylogalacturonan and rhamnogalacturonans I and II. Among the enzymes which degrade pectin (pectinases) is pectin lyase (E.C. 4.2.2.10). This enzyme acts on highly esterified homogalacturonan, catalysing the cleavage of α-(1→4) glycosidic bonds between methoxylated residues of galacturonic acid by means of β-elimination, with the formation of 4,5-unsaturated products. In this work, the gene and cDNA of a pectin lyase from Penicillium purpurogenum have been sequenced, and the cDNA has been expressed in Pichia pastoris. The gene is 1334 pb long, has three introns and codes for a protein of 376 amino acid residues. The recombinant enzyme was purified to homogeneity and characterized. Pectin lyase has a molecular mass of 45 kDa as determined by SDS-PAGE. It is active on highly esterified pectin, and decreases 40 % the viscosity of pectin with a degree of esterification ≥85 %. The enzyme showed no activity on polygalacturonic acid and pectin from citrus fruit 8 % esterified. The optimum pH and temperature for the recombinant enzyme are 6.0 and 50 °C, respectively, and it is stable up to 50 °C when exposed for 3 h. A purified pectin lyase may be useful in biotechnological applications such as the food industry where the liberation of toxic methanol in pectin degradation should be avoided.     

Pectin lyase from Aspergillus sp. CH-Y-1043

Aspergillus sp. CH-Y-1043 synthesizes pectin lyase when grown on citrus pectin at 37° C. Production is favoured by increased esterification degree of the pectin used as carbon source. This enzyme displays higher activity at pH values of 8.5–8.8 and temperatures of 40–45° C. The optimal substrate for the enzyme was highly esterified pectin and no enzymatic activity was registered on polygalacturonic acid. The activity is stimulated by, though not dependent on, divalent cations (Ca²⁺, Mg²⁺, Mn²⁺, Ba²⁺ and Co²⁺) and inhibited by Zn²⁺, and it is not sensitive to the addition of EDTA. The enzyme is very stable when exposed to pH variations: at 4° C it preserves more than 95% of its activity at pHs ranging from 2.0 to 10.0, and at 30° C stability is preserved at pHs ranging from 4.0 to 8.0. At a constant pH of 5.0, the enzyme conserves its stability at temperatures ranging from 4 to 50° C and at pH 8.0 sensitivity to temperature increased. The results on the endo-exo nature of the enzyme suggest that this is an exo-pectin lyase. Correspondence to: G. Aguilar     

Overexpression of the <Emphasis Type="Italic">plg</Emphasis>1 gene encoding pectin lyase in <Emphasis Type="Italic">Penicillium griseoroseum</Emphasis>

The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar cane juice showed PL activities of 4,804 or 5,202 U ml⁻¹ respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately 40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to express pectin lyase at levels comparable to, or exceeding, previously reported data.     

Viscometric method for assaying of total endodepolymerase activity of pectinases

An improved method for assaying of the total endodepolymerase activity of pectinases has been developed. The method is based on the determination of the viscosity of a citrus pectin solution in the presence of the enzyme using an Ostwald viscometer. The depolymerizing activity of different pectinases can be detected including polygalacturonase, polymethylgalacturonase, pectin lyase, and pectate lyase. One unit of the endodepolymerase activity corresponds to the activity resulting in 50% decrease in the relative viscosity of 0.5% citrus pectin solution for 5 min at 40°C and the appropriate pH. Depending on the pH-optima of the enzymes, two modifications of the method are described: 1) for acid pectinases at pH 5.0, and 2) for neutral (mildly alkaline) pectinases at pH 8.0. The modifications differed in the control and in the calculation of the activity. Six enzyme preparations were used to demonstrate the applicability of the method. The parameter used for the calculation of the enzymatic activity was directly proportional to the enzyme concentration (the dependence was linear in the range of at least 10-fold change in the enzyme concentration). The relative error of the method did not exceed 10%.     

Purification and characterization of polygalacturonase produced by thermophilic Thermoascus aurantiacus CBMAI-756 in submerged fermentation

An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C. The apparent K _m with citrus pectin was 1.46 mg/ml and the V _max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity at pH 9.0. Metal ions, such as Zn⁺², Mn⁺², and Hg⁺², inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing mono, di and tri-galacturonic acids within 10 min of hydrolysis.     

Isolation and properties of pectinases from the fungus Aspergillus japonicus

Using anion-exchange chromatography on different carriers and phenyl-Sepharose hydrophobic chromatography, five pectolytic enzymes were isolated from the culture liquid of a mutant strain of Aspergillus japonicus: two endo-polygalacturonases (I and II, 38 and 65 kD, pI5.6 and 3.3), pectin lyase (50 kD, pI3.8), and two pectinesterases (I and II) with similar molecular weights (46 and 47 kD) and the same pI(3.8). The pectinesterases apparently represent two isoforms of the same enzyme. All purified enzymes were homogenous according to SDS-PAGE and polyacrylamide gel-IEF, except for endo-polygalacturonase II that gave two bands on isoelectric focusing, but one band on electrophoresis. All enzymes had maximal activity in an acid medium (at pH 4.0-5.5). The pectin lyase and pectinesterase were stable at 40-50°C. The thermal stability of both endo-polygalacturonases was much lower (after 3 h of incubation at 30°C, endo-polygalacturonases I and II lost 40 and 10% of the activity, respectively). The activity of endo-polygalacturonases I and II towards polygalacturonic acid strongly depended on NaCl concentration (optimal concentration of the salt was 0.1-0.2 M); the enzymes were also capable of reducing the viscosity of pectin solution, but rather slowly. The pectin lyase had no activity towards polygalacturonic acid. The activity of the pectin lyase increased with increasing degree of methylation of pectins. Both endo-polygalacturonases demonstrated synergism with the pectinesterase during the hydrolysis of highly methylated pectin. On the contrary, in the mixture of pectin lyase and pectinesterase an antagonism between the two enzymes was observed.     

Expression,purification and characterization of pectate lyase A from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pectate lyase A (PelA) of Aspergillus nidulans was successfully expressed in Escherichia coli and effectively purified using a Ni²⁺-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 U ml⁻¹ medium. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5–10 at 50°C. Mn²⁺, Ca²⁺, Fe²⁺, Mg²⁺ and Fe³⁺ ions stimulated the pectate lyase activity, but Cu²⁺ and Zn²⁺ inhibited it. The recombinant PelA had a V _max of 77 μmol min⁻¹ mg⁻¹ and an apparent K _m of 0.50 mg ml⁻¹ for polygalacturonic acid. Low-esterified pectin was the optimum substrate for the PelA, whereas higher-esterified pectin was hardly cleaved by it. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.     

Cloning of the<Emphasis Type="Italic"> pelA</Emphasis> gene from<Emphasis Type="Italic"> Bacillus licheniformis</Emphasis> 14A and biochemical characterization of recombinant,thermostable, high-alkaline pectate lyase

The pectate lyase gene pelA from alkaliphilic Bacillus licheniformis strain 14A was cloned and sequenced. The nucleotide sequence corresponded to an open reading frame of 1,026 bp that codes for a 39 amino acid signal peptide and a mature protein with a molecular mass of 33,451 Da. The mature PelA showed significant homology to other pectate lyases belonging to polysaccharide lyase family 1, such as enzymes from different Bacillus spp. and Erwinia chrysanthemi. The pelA gene was expressed in Escherichia coli as a recombinant fusion protein containing a C-terminal His-tag, allowing purification to near homogeneity in a one-step procedure. The values for the kinetic parameters K _m and V _max of the fusion protein were 0.56 g/l and 51 µmol/min, respectively. The activity of purified PelA^His was inhibited in the presence of excess substrate. Characterization of product formation revealed unsaturated trigalacturonate as the main product. The yields of unsaturated trigalacturonic acids were further examined for the substrates polygalacturonic acid, citrus pectin and sugar-beet pectin.     

Pectinase production by Penicillium frequentans

High activities of extracellular pectinase with viscosity-diminishing and reducing groups-releasing activities were produced by Penicillium frequentans after 48 h at 35°C, in agitated cultures supplemented with 0.5% citrus pectin and initial pH of 2.5. Under these conditions the fungus also produced high activity of pectinesterase. At an initial pH of 7.0 or 8.0, pectin lyase activity was also detected. Enzyme activity releasing reducing sugars was more stable at 50°C than viscosity-diminishing activity. Both activities were maximal at pH 2.5 to 5.2 and at 55°C.The authors are from the Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Avenida do Café, s/n^o, Bairro Monte Alegre, 14.049 Ribeirão Preto, S.P., Brazil.     

Purification and characterization of an exo-polygalacturonase from Pycnoporus sanguineus

The present work describes the purification and characterization of a novel extracellular polygalacturonase, PGase I, produced by Pycnoporus sanguineus when grown on citrus fruit pectin. This substrate gave enhanced enzyme production as compared to sucrose and lactose. PGase I is an exocellular enzyme releasing galacturonic acid as its principal hydrolysis product as determined by TLC and orcinol-sulphuric acid staining. Its capacity to hydrolyze digalacturonate identified PGase I as an exo-polygalacturonase. SDS-PAGE showed that PGase I is an N-glycosidated monomer. The enzyme has a molecular mass of 42 kDa, optimum pH 4.8 and stability between pH 3.8 and 8.0. A temperature optimum was observed at 50–60 °C, with some enzyme activity retained up to 80 °C. Its activation energy was 5.352 cal mol⁻¹. PGase I showed a higher affinity towards PGA than citric pectin (Km = 0.55 ± 0.02 and 0.72 ± 0.02 mg ml⁻¹, respectively). Consequently, PGase I is an exo-PGase, EC 3.2.1.82.     

Growth and protopectinase production of Geotrichum klebahnii in batch and continuous cultures with synthetic media

Geotrichum klebahnii ATCC 42397 produces a protopectinase (PPase-SE) with polygalacturonase (PGase) activity. The microorganism was aerobically cultivated in synthetic media. Glucose, fructose and xylose yielded the highest enzyme levels (10–11 PGase units ml⁻¹). Galacturonic acid repressed enzyme production and no growth was obtained with disaccharides and pectin. Specific enzyme activity obtained in an O₂-limited culture was similar to that found in nonlimited ones. A growth yield (Y _x/s) of 0.49 g of cell dry weight per gram of glucose consumed was obtained in a typical batch bioreactor culture. Enzyme production was growth associated, and no major products other than biomass and CO₂ were detected. The volumetric enzyme activity reached a maximum around D=0.3–0.4 h⁻¹ in glucose-limited continuous cultures. However, it varied strongly (together with microorganism morphology) even after retention times ≥8 at any D tested (0.035–0.44 h⁻¹) though the rest of the culture variables remained fairly constant. No correlation between morphology and enzyme activity could be obtained. Enzyme production was poor in urea- and vitamin-limited continuous cultures. In all cases, biomass and CO₂ accounted for ≅100% of carbon recovery though Y _x/s values were different. Journal of Industrial Microbiology & Biotechnology (2000) 25, 260–265. Received 20 April 2000/ Accepted in revised form 15 September 2000     

Characterization of a pectinolytic activity inAzospirillum irakense

A fifth and newAzospirillum species,A. irakense, a nitrogen fixing and pectinolytic bacterium was found associated with roots and rhizosphere of rice in the region of Diwaniya (Qadisya), Iraq. This species produces pectate lyase and pectin methylesterase activities and can fix nitrogen when pectin is the sole carbon source. The four other species ofAzospirillum fail to show a pectinolytic activity.     

Purification and characterization of thermostable pectate-lyases from a newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov.

A novel thermophilic spore-forming anaerobic microorganism (strain Ab9) able to grow on citrus pectin and polygalacturonic acid (pectate) was isolated from a thermal spa in Italy. The newly isolated strain grows optimally at 70°C with a growth rate of 0.23 h⁻¹ with pectin and 0.12 h⁻¹ with pectate as substrates. Xylan, starch, and glycogen are also utilized as carbon sources and thermoactive xylanolytic (highest activity at 70°–75°C), amylolytic as well as pullulolytic enzymes (highest activity at 80°–85°C) are formed. Two thermoactive pectate lyases were isolated from the supernatant of a 300-l culture of isolate Ab9 after growth on citrus pectin. The two enzymes (lyases a and b) were purified to homogeneity by ammonium sulfate treatment, anion exchange chromatography, hydrophobic chromatography and finally by preparative gel electrophoresis. After sodium dodecylsulfate (SDS) gel electrophoresis, lyase a appeared as a single polypeptide with a molecular mass of 135 000 Da whereas lyase b consisted of two subunits with molecular masses of 93 000 Da and 158 000 Da. Both enzymes displayed similar catalytic properties with optimal activity at pH 9.0 and 80°C. The enzymes were very stable at 70°C and at 80°C with a half-life of more than 60 min. The maximal activity of the purified lyases was observed with orange pectate (100%) and pectate-sodium salt (90%), whereas pectin was attacked to a much lesser extent (50%). The K _m values of both lyases for pectate and citrus pectin were 0.5 g·l⁻¹ and 5.0 g·l⁻¹, respectively. After incubation with polygalacturonic acid, mono-, di-, and tri-galacturonate were detected as final products. A 2.5-fold increase of activity was obtained when pectate lyases were incubated in the presence of 1 mM Ca²⁺. The addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These heat-stable enzymes represent the first pectate-lyases isolated and characterized from a thermophilic anaerobic bacterium. On the basis of the results of the 16S rRNA sequence comparisons and the observed phenotypic differences, we propose strain Ab9 as a new species of Thermoanaerobacter, namely Thermoanaerobacter italicus sp. nov. Received: May 25, 1997 / Accepted: June 5, 1997     

Polygalacturonate lyase production byThermomonospora curvata on protein-extracted lucerne fiber

Summary Protein-extracted lucerne fiber was used as carbon and energy source for production of extracellular polygalacturonate lyase byThermomonospora curvata. The optimal fiber concentration was 1.5% (w/v); peal lyase activity in culture fluid occurred after 3 days growth at 53°C. During that time, lyase biosynthesis was controlled through induction; production was accelerated by adding small amounts of pectin or by grinding the fiber to 40-mesh particle size to release more inducer. After 3 days growth, lyase activity decreased; inactivation of the enzyme was delayed by the presence of 1 mM Ca or by inhibition of serine proteases with 0.05 mM phenylmethylsulfonyl fluoride. The molecular weight of the lyase produced during growth on the fiber was 35 kDa compared to 56 kDa for the enzyme produced on pure pectin. TheK _m of the 35-kDa form was 0.54% pectin compared to 0.06% for the 56-kDa form. The smaller form was rapidly inactivated at 60°C, the optimal temperature for activity of the larger form.