Cloning and Expression of Pectobacterium carotovorum Endo-polygalacturonase Gene in Pichia pastoris for Production of Oligogalacturonates

Abstract

A bacterial endo-polygalacturonase (endo-PGase) gene from the plant pathogen Pectobacterium carotovorum was cloned into pGAPZαA vector and constitutively expressed in Pichia pastoris. The recombinant endoPGase secreted by the Pichia clone showed a 1.7 fold increase when the culture medium included glycerol in replacement of glucose as the carbon source. The enzyme had optimum activity at pH 5.5 and 40 °C with stability between pH 5.0 and 8.0 and at temperatures up to 50 °C. The enzyme activity was enhanced by 41% with the addition of 1 mM Co ++ , and inhibited by Fe ++ with a 63% reduction. The mode of the enzyme action showed internal cleavage of α-1,4 glycoside bonds of polygalacturonic acid and citrus peel pectin. Trigalacturonate and hexagalacturonate were the main hydrolysis products, with a yield of 0.44±0.01 and 0.21±0.01 mg released per mg polygalacturonic acid substrate, respectively. This represents the first report of a microbial endo-PGase that produced trimer and hexamer uniquely as the end products of hydrolysis, in contrast to mixtures of mono-, di-, and trigalacturonates commonly observed for the action of fungal enzymes. Pectic oligosaccharides generated from native carbohydrate polymers offer the potential application as building blocks for value-added products.

Metabolic engineering ofBacillus subtiliswith an Endopolygalacturonase gene Isolated fromPectobacterium. carotovorum; a Plant Pathogenic Bacterial Strain

“…While the complex/higher oligosaccharides form a smear on TLC plate and were not further degraded into smaller oligosaccharides. These results confirmed our previously reported analysis of recombinant enzyme from Pichia pastoris [26]. HPLC chromatograms (Fig 13(b).…”

“…At the same time one control sample without having any metal cations was also included. The enzyme activity was measured by DNS method as previously described [26,28].…”

“…Previously we cloned and expressed endo-PGase gene from P. carotovorum into Pichia pastoris [26]. with encouraging results.…”

Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain

“…At the same time, one control sample without having any metal cations was also included as blank. The enzyme activity was again measured by DNS method as previously described [ 26 , 29 ].…”

“…Previously, we cloned and expressed endo-PGase gene from P . carotovorum into Pichia pastoris [ 26 ] with encouraging results. Here, for the first time, we report the expression of an endo-PGase gene from Pectobacterium carotovorum into Bacillus subtilis using pHT43 vectorH.…”