Production, purification and biochemical characterization of an exo-polygalacturonase from Aspergillus niger MTCC 478 suitable for clarification of orange juice

Abstract

Polygalacturonases (PG) represent an important member of pectinases group of enzymes with immense industrial applications. A fungal strain Aspergillus niger MTCC478 was used for the production of polygalacturonase both under submerged and solid-state fermentation condition. Further its production was optimized under solid-state fermentation condition with media comprising of wheat bran and tea extract. Purification of an exo-PG was achieved by acetone precipitation (60-90%) and CMcellulose column chromatography revealing 15.28-fold purification with a specific activity of 33.47 U/mg protein and 1.2% yield. A relative molecular mass of purified PG was approximately 124.0 kDa. The pH and temperature optimum was found to be 4 and 50°C, respectively. The k cat and K m value for degradation of PGA by the purified enzyme was found to be 194 s -1 and 2.3 mg/mL, respectively. Cu 2? was found to enhance the PG activity while Ag ? completely inhibited the enzyme activity. The application of the purified PG in orange juice clarification was elucidated.

Biotechnological potential of an exo‐polygalacturonase of the new strain Penicillium janthinellum VI2R3M: biochemical characterization and clarification of fruit juices

“…The determination of optimum temperature of PG from P. janthinellum VI2R3M reveal similar results reported for the PG of P. notatum , Aspergillus niger , Aspergillus awamori and Fusarium graminearum (Dey and Banerjee ; Ortega et al ; Amin et al ; Anand et al ). However, the enzyme shows an optimum temperature less than that of the exo‐PG form Bacillus licheniformis (Evangelista et al ), a PG from Aureobasidium pullulans (Bennamoun ) and an exo‐PG from Rhizomucor pusillus A 13·36 (Trindade et al ).…”

“…The determination of optimum temperature of PG from P. janthinellum VI2R3M reveal similar results reported for the PG of P. notatum, Aspergillus niger, Aspergillus awamori and Fusarium graminearum (Dey and Banerjee 2014;Ortega et al 2014;Amin et al 2017;Anand et al 2017). However, the enzyme shows an Figure 3 Effect of temperature and pH on PG activity and stability.…”

“…The value of K m shown by polygalacturonase from P. janthinellum VI2R3M polygalacturonase was greater than of an exo‐PG of P. janthinellum (1·74 mg ml −1 ) described by Ma et al () and an exo‐PG of Penicillium frequentans (0·059 mg ml −1 ) (Anand et al ). However, it was a very close to K m reported for an exo‐PG of A. niger (2·3 mg ml −1 ).…”

“…The relative molecular mass of our enzyme (102Á0 kDa) was higher than the PG of P. janthinellum sw09 of 66Á2 kDa (Ma et al 2016) and exo-PG of Rhizomucor pusillus A13Á36 (Trindade et al 2016) described in the literature. The PGs of different micro-organisms have been reported with molecular weights ranging from 24 to 124 kDa (Gomes et al 2011;Anand et al 2017).…”

“…Degradation of pectin is facilitated mainly by the group of enzymes called pectinases. These include pectin methyl esterase (EC 3.1.1.11), pectin lyase (EC 4.2.2.10), endo‐polygalacturonase (EC 3.2.1.15) and exo‐polygalacturonase (EC 3.2.1.67) (Yuan et al ; Anand et al ). When demethylated, pectin turns to polygalacturonic acid (PGA) also known as pectic acid, which is the preferred substrate for most polygalacturonases (PGs) (Sassi et al ).…”