Purification and Properties of Polygalacturonase Produced by Thermophilic Fungus Thermoascus aurantiacus CBMAI-756 on Solid-State Fermentation

Eduardo Da Silva Martins,Roberto Da Silva,Rodrigo Simões Ribeiro Leite,Eleni Gomes

doi:10.1155/2013/438645

Abstract

Polygalacturonases are enzymes involved in the degradation of pectic substances, being extensively used in food industries, textile processing, degumming of plant rough fibres, and treatment of pectic wastewaters. Polygalacturonase (PG) production by thermophilic fungus Thermoascus aurantiacus on solid-state fermentation was carried out in culture media containing sugar cane bagasse and orange bagasse in proportions of 30% and 70% (w/w) at 45°C for 4 days. PG obtained was purified by gel filtration and ion-exchange chromatography. The highest activity was found between pH 4.5 and 5.5, and the enzyme preserved more than 80% of its activity at pH values between 5.0 and 6.5. At pH values between 3.0 and 4.5, PG retained about 73% of the original activity, whereas at pH 10.0 it remained around 44%. The optimum temperature was 60–65°C. The enzyme was completely stable when incubated for 1 hour at 50°C. At 55°C and 60°C, the activity decreased 55% and 90%, respectively. The apparent molecular weight was 29.3 kDa, K m of 1.58 mg/mL and V max of 1553.1 μmol/min/mg. The presence of Zn+2, Mn+2, and Hg+2 inhibited 59%, 77%, and 100% of enzyme activity, respectively. The hydrolysis product suggests that polygalacturonase was shown to be an endo/exoenzyme.

Highlights

Pectinases are a heterogeneous group of enzymes that hydrolyze the pectic substances present in plant material
The polygalacturonases catalyze the hydrolysis of glycosidic α-1-4 linkages in pectic acid and are of two types: endo-polygalacturonases, which act by hydrolysis of internal glycosidic bonds α-1-4 of polygalacturonic acid at random form, resulting in molecule depolymerization with release of oligogalacturonic acids, and exopolygalacturonases which hydrolyse alternate α-1-4 glycosidic linkages of polygalacturonic acid from the nonreducing end, releasing unsaturated mono- or digalacturonic acids [2, 3]
The present study aimed to purify polygalacturonase produced by thermophilic fungus Thermoascus aurantiacus in solid-state fermentation and compare its properties with those of the purified polygalacturonase produced by the same fungus in submerged fermentation (SmF), in a work reported by Martins et al [10]

Summary

Introduction

Pectinases are a heterogeneous group of enzymes that hydrolyze the pectic substances present in plant material. The polygalacturonases catalyze the hydrolysis of glycosidic α-1-4 linkages in pectic acid and are of two types: endo-polygalacturonases (endo PG, EC 3.2.1.15), which act by hydrolysis of internal glycosidic bonds α-1-4 of polygalacturonic acid at random form, resulting in molecule depolymerization with release of oligogalacturonic acids, and exopolygalacturonases (exo PG, EC 3.2.1.67) which hydrolyse alternate α-1-4 glycosidic linkages of polygalacturonic acid from the nonreducing end, releasing unsaturated mono- or digalacturonic acids [2, 3] This group of enzymes has been widely used in the food industry process such as clarification and viscosity reduction of fruit juices, preliminary treatment of grape juice for wine industries, tomato pulp extraction, oil extraction, and tea fermentation and in the textile industry in fibers degumming [4, 5]. The present study aimed to purify polygalacturonase produced by thermophilic fungus Thermoascus aurantiacus in solid-state fermentation and compare its properties with those of the purified polygalacturonase produced by the same fungus in SmF, in a work reported by Martins et al [10]

Materials and Methods

Results and Discussion

Conclusions