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

Nagina Rafique, Romana Tabassum, Muhammad Siddique Awan, William Orts, Dominic W. S. Wong

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 endo-PGase 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.

Keywords


Endo-polygalacturonase; Oligogalacturonate; Pectobacterium carotovorum; Pichia pastoris; Gene cloning

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