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The endo-galacturonase A gene (pgaA) was cloned using the cDNAs synthesized from total RNA of Aspergillus niger JL-15 by reverse transcription as template. The open reading frame (ORF) of pgaA was 1113bp, encoding a peptide of 370 amino acids with the predicted molecular mass of 38.8kDa. The pgaA was successfully expressed in Pichia pastoris GS115 under the control of AOX1 promoter. After induction by methanol for 96h, the activity of the recombinant endo-galacturonase A (rePgaA) in culture supernatant was 2091.0U/mg. SDS-PAGE analysis showed that the molecular mass of rePgaA was about 40.0kDa. Enzymatic properties assays showed that the optimum temperature and pH for rePgaA were 50°C and pH 5.0, respectively. The Michaelis-Menten constant (Km) and maximal velocity (Vmax) of rePgaA for citrus pectin were 3.20mgml(-1) and 40.97μmolmin(-1)ml(-1), respectively. The rePgaA mediated a rapid decrease in viscosity of pectin solution with release of small amount of reducing sugar. High performance liquid chromatography (HPLC) analysis revealed that digalacturonate (G2) and trigalalcturonate (G3) were the main hydrolysis products released from pectin by rePgaA. The rePgaA showed very low activity on G2 and G3, which suggested it was a typical endo-acting enzyme., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was purified. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme was purified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS-PAGE analysis, enzyme was found to be a heterodimer of 34 and 69 kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106 kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45 °C, pH of 4.8, reaction time of 15 min. The enzyme was stable within the pH range of 4.0-5.5 for 1 h. At 4 °C it retained 50% activity after 108 h but at room temperature it lost its 50% activity after 3h. The addition of Mn(2+), K(+), Zn(2+), Ca(2+) and Al(3+) inhibited the enzyme activity; it increased in the presence of Mg(2+) and Cu(2+) ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The K(m) and V(max) values of the enzyme were found to be 0.083 mg/ml and 18.21 μmol/ml/min. The enzyme was used for guava juice extraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%. Addition of purified enzyme increased the %T(650) from 2.5 to 20.4 and °Brix from 1.9 to 4.8. The pH of the enzyme treated juice decreased from 4.5 to 3.02., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We have succeeded in the expression of Stereum purpureum endopolygalacturonase I (EndoPG I) using the Pichia expression system and in purification of the three kinds of recombinant EndoPG I, which have one to three sugar chains by using CM52 column chromatography. The sugar chains which were added to EndoPG I were the M8, M9, and/or M10 high-mannose type. The results of LC-MS analysis showed that recombinant EndoPG Is were randomly glycosylated at four N-glycosylation sites. From the thermal denaturation curves of the recombinant enzymes, it was suggested that EndoPG I differing in thermal stability was included in the sample after purification. Therefore, we investigated the disulfide bonds of recombinant EndoPG I by LC-MS analysis. As a result, peptides without a second or third disulfide bond were detected. This result is the first indicating that there are incomplete enzymes in terms of disulfide bonds in the Pichia expression system.

Endo-polygalacturonase (PG) may be a critical virulence factor secreted by several fungi upon plant invasion. The single-copy gene encoding PG in Fusarium verticillioides and in eight other species of the Gibberella fujikuroi complex (F. sacchari, F. fujikuroi, F. proliferatum, F. subglutinans, F. thapsinum, F. nygamai, F. circinatum, and F. anthophilum) was functionally analyzed in this paper. Both the nucleotide and amino acid sequences were highly similar among the 12 strains of F. verticillioides analyzed, as well as among those from the G. fujikuroi complex. The PGs were not inhibited by the polygalacturonase-inhibiting proteins (PGIPs) from the monocot asparagus and leek plants, but were inhibited to variable extents by bean PGIP. PGs from F. verticillioides, F. nygamai and one strain of F. proliferatum were barely inhibited. Residue 97 within PG was demonstrated to contribute to the different levels of inhibition. Together these findings provide new insights into the structural and functional relationships between the PG from the species of the G. fujikuroi complex and the plant PGIP.

Endo-polygalacturonases (endoPGs) belong to the glycoside hydrolase family 28 and hydrolyze the alpha-1,4 glycosidic bond present in the smooth regions of pectins. Pectic substances are among the principal macromolecular components of the primary plant cell walls and are subjected to enzymatic degradation not only in the course of important physiological processes such as plant senescence and ripening, but also during infection events by plant pathogens. Here we report, for the first time, the isolation and the purification of an endoPG (PehA) from the supernatant of the plant pathogen Burkholderia cepacia strain ATCC 25416. In order to obtain adequate amounts of protein required for structural and functional studies, the gene coding for pehA was PCR-amplified and cloned in Escherichia coli cells. The recombinant protein was purified to homogeneity and characterized. PehA exhibited a pI value of 8.0 and an optimal activity at pH 3.5. Far-UV circular dichroism (CD) measurements show that PehA assumes a beta-helix fold super-secondary structural motif.

Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 A resolution with synchrotron radiation.

Affinity precipitation is a simple, single plate separation process in which the complex of a smart macroaffinity ligand with the target protein (from a crude broth) can be selectively precipitated by application of a suitable stimulus. Alginate is a copolymer of guluronic acid and mannuronic acid residues and precipitates with Ca(2+) ions. It was found to bind to pectinase present in a commercial preparation of Aspergillus niger, Pectinex Ultra-SPL. Microwave pretreatment of alginate at 75 degrees C was found to enhance the selectivity of the affinity precipitation. Using microwave-treated alginate, 83% of the enzyme activity with 20-fold purification could be recovered. SDS-PAGE upon silver staining confirmed the enhanced selectivity of affinity precipitation when microwave-treated alginate was used.