Microorganisms

Microorganisms are referred as predominant source of commercial enzyme (Wiwapat et al., 2002, Kvesitadze, Kvesitadze 1990, Kutateladze et al., 2009). Due to diverse spectrum of applications enzyme production now became a multi-billion dollar business (Bhat, 2000). The market of the technical enzymes showed great diversity both in terms of the applications as well as the consumption. It has been evaluated that global sale of enzymes was $1.7–2 billion in 2005 which is expected to grow in the forthcoming years exponentially with the increase of industrialization. Typically, pectinases shares a huge market value up to $75 million (Godfrey and West, 1996).

Pectinases is one of the hydrolytic enzymes that have diverse degree of applications in biotechnological industry. Pectinase is one of the 20 enzymes that have been globally marketed. Pectinase catalyzing the degradation of pectin containing compounds are of huge industrial significance (Spanga et al., 1995). Pectinases are now considered to be central part of complex processing of juice in food industry and removal of sizing agents in textile industries (Kashyap et al., 2001). They cause a drastic increase in filtration efficacy of juices and increase yield by aiding in clarification. (Joslyn et al., 1952) (Brawman 1981). They are also used as wood preservative (Fogarty 1973) and in liquefaction, maceration and extraction of vegetable tissues (Charley 1969 and Bohdziewiez and Bodzek 1994). Different extraction procedures like extraction of oils, pigments and flavors also employ the help of the pectinases (Castilho et al., 1999). They also got their role in tea and coffee fermentations (Taragano et al., 1997) and novel applications in the production of oligogalacturonides as useful food components (Hang and Dornenburg 2000).  

Pectinases used synthetic and natural pectic substances as substrates. Pectic substances are complex high molecular mass glycosidic macromolecules found in middle lamella of higher plants (Whitaker, 1984). D-galacturonic acid units in the pectic substances are joined by α-1, 4 linkages and can be classified as protopectin, pectin and pectic acid (Kertesz, 1951). These compounds are characterized by their capacity to form gels and their presence contributes in providing structure and firmness to plants (Sathyanarayana and Panda, 2003). Many microorganisms like bacteria, yeast, and fungi produce variety of pectinases (Rombouts et al., 1988). However, searching for effective producers and establishing their optimum cultivation conditions are effective tools to enhance the level of product significantly. A range of organisms including; plants, filamentous fungi, yeast and bacteria have been exploited for the production of pectinolytic enzymes, however, microbial sources are considered economically more feasible (Silva 2005; Prade 1999; Behere 1993).

From analytical point of view, the enzyme must be purified to examine its chemical and physical nature and working efficacy. Pectinases of diversified microbial sources have been purified using different chromatographic procedures, however, these techniques have different ration of productivity (Gummadi and Panda, 2003).

MATERIALS AND METHODS

Chemicals

Pectin (Sigma-Aldrich chemicals), DNS (Sigma-Aldrich chemicals), Sephadex G-75 (Sigma-Aldrich chemicals).

Microorganism

An already screened pectinolytic strain from Microbiology Research Lab was selected for the production of enzyme. The culture was stored and maintained on potato dextrose agar slants at 4 °C. Identification of the strain was done on the basis of microscopy. The strain was already optimized for production of enzyme under submerged fermentation conditions. Crude enzyme produced under optimized conditions was subjected to different purification techniques.

Purification of Pectinase

Fungal samples were collected after 4 days by filtering through a filter paper (Whattman No 4) aseptically and cells were harvested by centrifugation at 10,000 rpm for 10 minutes at 4oC. Following steps were carried out for purification of pectinase from cell free supernatant.

Precipitation

Gel permeation chromatography

Lyophilization    

Precipitation

Precipitation of pectinase was accomplished by applying various concentrations of ammonium sulfate. To flocculate protein, cell free supernatant was subjected to rising concentration of ammonium sulfate (40-80%). Finely grinded ammonium sulfate was added with constant stirring. When final fraction of salt was added then stirring was ceased and the mixture was allowed to stand for overnight to allow stabilization. The protein was centrifuged at 10,000 rpm for 0°C, for 15 minutes. Precipitates were centrifuged at 10000 rpm at 4°C. The pellet obtained after centrifugation was dissolved in 0.05M citrate buffer, at pH 4.8. Precipitated proteins were stored at 4°C and used for further analysis.