Purification and properties of polygalacturonase associated with the infection process of Colletotrichum truncatum CP2 in chilli

The plant cell wallis a major barrier to the establishment of fungal infection on a host. Most plant-pathogenic fungi produce a number of cell wall-degrading enzymes when grown in liquid culture containing pectin. One of these enzymes, PGhas been implicated routinely in facilitating the invasion and colonization of host tissue during pathogenesis of fungal pathogens (Choi et al., 2013). Highly purified PGfrom many fungal pathogens has been proved by several researchers to have the ability to cause cell maceration and kill tissues in a similar way to that seen in soft-rot diseases (Protsenko et al., 2010; Herbert et al., 2004; Oeser et al., 2002).

Many organisms produces polygalacturonases, for example, bacteria, parasites and yeast (Jurick et al., 2009; Latif & Sohail, 2012). Microbial PGfrom different microbial sources shows wide variety in their physicochemical and biological properties. Most of the PGwas found optimum at pH range of 3.5 to 5.5 and temperature between 30 to 50 °C. Molecular mass of the PGalso varies from 25 kDa to 85 kDa.

Microbial PGhas to be purified for the complete understanding of its properties. Numerous purification strategies have been reported for PGall with varying degree of success. The purification methods commonly employed include ammonium sulphate precipitation, ion-exchange chromatography, Sephadex G-25 gel filtration, ultrafiltration, gel permeation chromatography and ethanol precipitation (Deshmukh et al., 2012; Jurick et al., 2009; Thakur et al., 2010). Each purification method has its own drawback associated with low yield and purity, cost and the requirement for a skilled operator (Shaligram & Singhal, 2010).

The ATPS has been proposed as an ideal and versatile strategy for the extraction and purification of biomolecules because of its high productivity, environmental-friendly, simplicity, short processing time, cost effectiveness and ease of scaling-up (Naganagouda & Mulimani, 2008; Raja et al., 2012). ATPS which consist of PEG/ salt system has been generally employed for the bioseparation of proteins due to its availability at low cost and wide range of hydrophobic differences between the two phase systems which allow enhancement of the partition selectivity of the target protein (Mehrnoush et al., 2011). ATPS has been applied in the extraction and purification of various compounds such as enzymes, biopharmaceuticals adnatural products (Srinivas & Raghavarao, 2000).

Selection of ATPS as a purification method is usually dependent on the types of biomolecules and economic considerations. Since the polymer/polymer system is very costly, the aqueous two phase polymer/salt systems are often used compared to the polymer/polymer systems (Abbasiliasi et al., 2014). Moreover, polymer/salt systems have significant differences in density, greater selectivity, lower viscosity, lower cost and the larger relative size of the drops (Nadar et al., 2017). Phosphates and sulfates are commonly used salts in polymer/salt ATPS. But the use of these salts has contributes to environmental problems, especially high concentrations of phosphate and sulfates in the effluent streams. Currently, the use of citrate salts as one of the ATPS components with PEG is preferred since citrate salts are biodegradable and non-toxic (Glyk et al., 2015). In view of the fact that ATPS is an ideal purification technique for biomolecules such as enzymes, this study evaluated the partitioning efficiency of a PGproduced by C. truncatum CP2 using ATPS which comprised of PEG and sodium citrate. Since the partitioning mechanism in ATPS is still unknown, the effects of various parameters on the partitioning of the PG, such as the molecular weight, salt concentration, pH, crude load and addition of sodium chloride (NaCl) were investigated. The physico-chemical properties of partially purified PGwere also characterized.