Pectinase enzyme-complex production by Aspergillus spp. in solid-state fermentation: A comparative study

Abstract

A comparative evaluation of three Aspergillus species according to their pectinase production in solid-state fermentation was performed. Solid-state fermentation offers several potential advantages for enzyme production by fungal strains. Utilization of agricultural by-products as low-cost substrates for microbial enzyme production resulted in an economical and promising process. The pectinolytic enzyme activities of two Aspergillus sojae strains were compared to a known producer, Aspergillus niger IMI 91881, and to A. sojae ATCC 20235, which was re-classified as Aspergillus oryzae. Evaluation of polymethylgalacturonase and polygalacturonase activity was performed as well as exo- vs. endo-enzyme activity in the crude pectinase enzyme-complex of the mentioned strains. Furthermore, a plate diffusion assay was applied to determine the presence and action of proteases in the crude extracts. A. sojae ATCC 20235 with highest polymethylgalacturonase activity and highest polygalacturonase activity both exo- and endo-enzyme activity, is a promising candidate for industrial pectinase production, a group of enzymes with high commercial value, in solid-state fermentation processes. Beside the enzymatic assays a protein profile of each strain is given by SDS-PAGE electrophoresis and in addition species-specific zymograms for pectinolytic enzymes were observed, revealing the differences in protein pattern of the A. sojae strains to the re-classified A. oryzae.

Introduction

The middle lamella and the primary cell wall of higher plants contain a complex heteropolysaccharide called pectin. These carbohydrate polymers support the cohesion of the other cell wall polysaccharides and proteins. Pectin is composed mainly of galacturonic acid residues (Willats et al., 2001). Pectinases include a number of related enzymes involved in the breaking down of pectic substances. Therefore, they can cause plant tissue maceration, cell lysis, and modification of cell wall structures, allowing other depolymerases to further degrade their product of decomposition (Collmer et al., 1988). Pectinolytic enzymes are extensively used in the food industry, e.g., as processing aids, the largest industrial application being in fruit juice extraction and clarification. Break down of pectin reduces the viscosity of pectin-rich crude juice and thus increases juice flow and reduces the press-time. Pectinases are also involved in clarification of wine, oil extraction, removal of citric fruit peels, and degumming fibres (Jayani et al., 2005, Mutlu et al., 1999, Silva et al., 2002). Depolymerizing enzymes like polygalacturonases are distinguished according to their substrate preference, whether they have preference for poly[α(1 → 4)-d-methylgalacturonic acid] (pectin-like substrates), which are termed as PMG in this study or poly[α(1 → 4)-d-galacturonic acid] (pectic acid-like substrates), which are termed as PG (Whitaker, 1984). Furthermore these enzymes are termed as exo- or endo-enzymes depending on the action pattern. Endo-PGs randomly attack the [1 → 4]α-glycosidic linkages of the polysaccharide chain producing a number of galacturonic acid oligomers, while exo-PGs specifically hydrolyses at the non-reducing end of polygalacturonic acid. Commercial pectic enzymes used in food industry normally contain a mixture of enzymes that split pectic compounds; traditionally mixtures consist of PG, PL (pectin lyase) and PME (pectin methylesterase), and are associated with cellulytic, proteolytic and other species of enzymes apart from the main pectinases (Del Cañizo et al., 1994). In some food processes, it is convenient to use only one type of pectinolytic enzymes, e.g., preparation of instant potato flakes and carrot juice for baby food requires the maceration, where vitamins, color and aroma have to be preserved and for these applications preparations that mainly contain PG activity are preferred (Lang and Dörnenburg, 2000).

Filamentous fungi especially Aspergillus niger (A. niger), are the major producers of acidic pectic enzymes used in fruit juice industries and wine production (Kashyap et al., 2001, Naidu and Panda, 1998). Products of A. niger as well as Aspergillus sojae and Aspergillus oryzae have obtained a GRAS (General Regarded As Safe) status, which has approved their use in the food industry. Usually pectolytic microorganisms produce a multiplicity of pectinolytic enzymes. The production of these enzymes is carried out in solid-state (SSF) and submerged fermentation (SmF).

The utilization of SSF processes is interesting for pectinase production by fungi, because of its capability to grow in low water activity (a_w), which is a dimensionless quantity used to estimate the amount of free water that is readily available for the microorganisms. This SSF process offers several potential advantages in comparison to SmF, e.g., higher product concentration, simpler fermentation technology, and reduced waste-water output (Pandey et al., 2000). SSF also holds a tremendous potential for enzyme production. In several comparative studies on fungal pectinases production in solid-state and submerged fermentation, SSF gave superior results compared to submerged conditions and the protease production was also extremely lower (Díaz-Godínez et al., 2001, Favela-Torres et al., 2006, Patil and Dayanand, 2006a). Moreover, simple and economic agricultural by-products like wheat bran and orange peel could be utilized so as to provide both nutritional and physical support during solid substrate cultivation. Wheat bran, which is composed predominantly of non-starch carbohydrates like arabinoxylans or cellulose, starch and crude proteins, has been a preferred substrate for the production of pectinolytic enzymes (Sun et al., 2008).

Previous attempts to produce the pectinolytic enzyme polygalacturonase (PG) by A. sojae ATCC 20235 have included submerged and surface cultivation (Tari et al., 2007, Ustok et al., 2007). These studies have demonstrated the presence of enzymes with exo-PG activity in the crude (or partially purified) fermentation broths. These studies have triggered an interest in understanding the potential of A. sojae for the production of PG, as well as an eagerness of knowledge on the characteristics and technical applications of pectinases. On the other hand, SSF has been successfully employed for the production of the pectinase-complex from other Aspergillus strains, notably A. niger (Favela-Torres et al., 2006, Pandey et al., 2000).

The aim of this study focused on the comparative evaluation of three Aspergillus species including A. niger IMI 91881, as a known producer, A. sojae ATCC 20235, which was reclassified as A. oryzae (Heerikhuisen et al., 2005), A. sojae CBS 100928, and A. sojae IMI 191303 for the production of pectinolytic enzymes. This general screening was performed in order to identify potential pectinase producers. The focus of this work was the evaluation of pectinase production of exo- vs. endo-PG activity, as well as exo- vs. endo-polymethylgalacturonase (PMG) activity in the crude extract obtained in SSF of the mentioned strains. To the best of our knowledge, there is no available information in the open literature related to the production of both pectinases by the two A. sojae strains employed in this study.