Biochemical Characterization, Thermal Stability, and Partial Sequence of a Novel Exo-Polygalacturonase from the Thermophilic Fungus Rhizomucor pusillus A13.36 Obtained by Submerged Cultivation

Abstract

This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45°C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5–47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61°C. It presents thermal stability between 30 and 60°C, has 70% activation in the presence of Ca2+, and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%. E a(d) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and ΔG (d) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus. Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot.

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

“…In comparison to Penicillium notatum, both enzymes were purified three times, but the PG of P. notatum presented a specific activity of 27Á79 U mg À1 (Amin et al 2017). 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).…”

“…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 pH stability was evaluated by incubating the purified PG in the absence of the substrate, in McIlvaine buffer, adjusted to different pHs (3Á0-8Á0), at 4°C for 24 h, and after the pH was adjusted to 5 the residual activity was determined. (Bennamoun 2016) and an exo-PG from Rhizomucor pusillus A13Á36 (Trindade et al 2016).…”

“…Pectinases have several applications in industry, especially in the extraction and clarification of wine and fruit juice, vegetable fibre degumming, vegetable oil extraction, coffee fermentation, degumming and waste water treatment and in the animal feed industry (Quiroga et al ; Dey and Banerjee ; Garg et al ; Trindade et al ). Of commercial pectinases, most enzymes are produced from fungal sources such as Aspergillus sp.…”

Biochemical characterization of an isolated 50 kDa beta-glucosidase from the thermophilic fungus Myceliophthora thermophila M.7.7

“…The thermodynamic analysis of BGL50 thermal denaturation was done using pNPG. The calculation of the activation energy E a , the temperature coefficient Q 10 , half-life T 1/2 parameters of the enzyme, as well as those related to the thermal denaturation (the activation energy E a(D) , ΔH (D) , ΔG (D) and ΔS (D) ) followed the method proposed in the literature (Saqib et al, 2012(Saqib et al, , 2010 and done in a previous study (Trindade et al, 2016). It is assumed that the irreversible denatured "I" state is evaluated using N ↔ D → I, where "N" represents the native conformation and "D" the reversible denatured conformation.…”