Proteomic Analysis of Extracellular Proteins from Aspergillus oryzae Grown under Submerged and Solid-State Culture Conditions

Abstract

Filamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae because of its ability to produce heterologous proteins in solid-state culture. To provide an overview of protein secretion by A. oryzae in solid-state culture, we carried out a comparative proteome analysis of extracellular proteins in solid-state and submerged (liquid) cultures. Extracellular proteins prepared from both cultures sequentially from 0 to 40 h were subjected to twodimensional electrophoresis, and protein spots at 40 h were identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. We also attempted to identify cell wall-bound proteins of the submerged culture. We analyzed 85 spots from the solid-state culture and 110 spots from the submerged culture. We identified a total of 29 proteins, which were classified into 4 groups. Group 1 consisted of extracellular proteins specifically produced in the solid-state growth condition, such as glucoamylase B and alanyl dipeptidyl peptidase. Group 2 consisted of extracellular proteins specifically produced in the submerged condition, such as glucoamylase A (GlaA) and xylanase G2 (XynG2). Group 3 consisted of proteins produced in both conditions, such as xylanase G1. Group 4 consisted of proteins that were secreted to the medium in the solid-state growth condition but trapped in the cell wall in the submerged condition, such as ␣-amylase (TAA) and ␤-glucosidase (Bgl). A Northern analysis of seven genes from the four groups suggested that the secretion of TAA and Bgl was regulated by trapping these proteins in the cell wall in submerged culture and that secretion of GlaA and XynG2 was regulated at the posttranscriptional level in the solid-state culture.

Comparative proteomic analysis of an Aspergillus fumigatus mutant deficient in glucosidase I (AfCwh41)

“…Interestingly, some proteins showed the same pI value but different molecular mass, and the molecular mass of some proteins in the mutant was higher than that of their counterparts in the wild-type. Unfortunately, we identified only two of these proteins (spots 35 and 50, spots 70 and 22) by MS, which might be due to the technical difficulty of glycoprotein identification (Oda et al, 2006).…”

Proteomics Methodology Applied to the Analysis of Filamentous Fungi - New Trends for an Impressive Diverse Group of Organisms

“…This fungus is also an interesting host for enzyme production (homologous and heterologous) (Bouws et al, 2008). The extracellular proteome analysis of A. oryzae at different times in submerged and solid-state cultures containing wheat bran showed an impressive number of enzymes related to cell wall degradation (glucoamylase A, xylanase G2, -glucosidase A, cellulase B, -amylase, -glucosidase and xylanase F3) (Oda et al, 2006). Thus, A. oryzae is also used in modern biotechnology due to this array of secreted proteins (Machida et al, 2005).…”

Identification and characterization of filamentous fungi isolated from fermentation starters for Hong Qu glutinous rice wine brewing

“…Aspergillus, a dominant genus existing in Daqu, also occurred frequently in White Qu and Hong Qu in this study, probably due to the abundance of this genus in nature. The genus Aspergillus has the ability to secrete large amounts of a wide range of different enzymes into its environment (Oda et al, 2006), which may contribute to the saccharifi cation of the remaining starch. A. oryzae, included in the genus Aspergillus, has been used for more than a thousand years in the making of such traditional fermented food products such as miso (Japanese fermented soybean paste), soy sauce and sake (Japanese rice wine) (Machida et al, 2008;Wang et al, 2004).…”