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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Bioproducts Research » Research » Publications at this Location » Publication #329233

Research Project: Technologies for Improving Industrial Biorefineries that Produce Marketable Biobased Products

Location: Bioproducts Research

Title: Cloning and expression of Pectobacterium carotovorum endo-polygalacturonase gene in Pichia pastoris for production of oligogalacturonates

item Wong, Dominic
item RAFIQUE, NAGINA - University Of Azad Jammu & Kashmir
item TABASSUM, ROMANA - National Institute Of Biotechnology And Genetic Engineering (NIBGE)
item AWAN, SIDDIQUE - University Of Poonch Rawalakot
item Orts, William

Submitted to: BioResources
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/10/2016
Publication Date: 4/26/2016
Citation: Wong, D., Rafique, N., Tabassum, R., Awan, S., Orts, W.J. 2016. Cloning and expression of Pectobacterium carotovorum endo-polygalacturonase gene in Pichia pastoris for production of oligogalacturonates. BioResources. 11(2):5204-5214.

Interpretive Summary: Plant cell walls are rich in pectic substances which are heterogeneous mixtures of branched and highly hydrated polysaccharides. Microbial enzymes are found to degrade pectins, resulting in softening of plant cell tissues during maturation and storage. These enzymes have been used for commercial applications in fruit and vegetable juice processing and in the paper and pulp industry. Applications are also found in engineering texture and rheological properties of biomass fibers. Most pectinolytic enzymes are derived from fungal origin. Our laboratory extends the classic industrial repertoire of pectinolytic enzymes to include the study of bacterial pectinases. The endo-polygalacturonase peh gene of Pectobacterium carotovorum was heterologously cloned and expressed in Pichia pastoris. The properties of the recombinant enzyme were analyzed, and the oligogalacuronate reaction products were determined and quantified. This report presents a bacterial recombinant enzyme for the production of the types of pectic oligosaccharides with functional characteristics that are not commonly found in pectic hydrolysis products using fungal endo-polygalacturonases or commercial enzyme preparations.

Technical Abstract: A bacterial endo-polygalacturonase (endo-PGase) gene from the plant pathogen Pectobacterium carotovorum was cloned into pGAPZaA 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 40oC with stability between pH 5.0 - 8.0 and at temperatures up to 50oC. 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 a-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.