Analysis of the proteins secreted by Trichoderma harzianum P49P11 under carbon-limited conditions

Lucas Gelain,Martin Pabst,José Geraldo Da Cruz Pradella,Aline Carvalho Da Costa,Luuk Van Der Wielen,Walter M Van Gulik

doi:10.1016/j.jprot.2020.103922

Abstract

The wild type strain Trichoderma harzianum was able to synthesize enzymes that can catalyse the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (PNPGase) in glucose-limited chemostat cultures. Fructose/glucose and sucrose conditions provided low levels of PNPGase activity. To investigate whether under these conditions other enzymes were produced, a shotgun proteomics analysis of their supernatants was performed. The analysis has indicated that the different carbon sources used influenced the amounts of proteins secreted including 1,3-beta-glucanosyltransferase, alpha-1,2-mannosidase, alpha-galactosidase and glucan 1,3-beta-glucosidase. The analysis has also suggested the presence of beta-glucosidase, which could also be represented by PNPGase activity. Intracellular metabolites were quantified during PNPGase production for the condition using 20 g/L of glucose in the feed and differences were observed, indicating that intracellular glucose could be inhibiting PNPGase production. SignificanceThis work shows that sugars such as glucose, fructose/glucose and sucrose can be used as substrates for the continuous synthesis of different enzymes under carbon-limited conditions by Trichoderma harzianum. As far as we know, this is the first work about the continuous synthesis of enzymes under carbon-limited conditions suggesting that different easily assimilated carbon sources can be used to generate different enzymatic cocktails. Each enzyme or uncharacterized protein suggested by shotgun proteomics has the potential to become a promising product for biotechnological applications.

Highlights

Proteins are a diverse group of molecules containing different properties such as catalytic activity, molecular weight and solubility [1]
Shotgun proteomics and SDS-PAGE analysis were performed for the proteins present in the supernatant of carbon-limited chemostat cul tures using glucose, fructose/glucose and sucrose as carbon sources
The shotgun proteomics analysis has indicated that the different carbon sources used greatly influenced the amounts of secreted proteins, of which many of them are enzymes

Summary

Introduction

Proteins are a diverse group of molecules containing different properties such as catalytic activity, molecular weight and solubility [1]. A method called shotgun analysis can be used to identify proteins [2]. Proteolytic enzymes such as trypsin are used to digest the proteins, and due to the specificity of this enzyme, it generates peptides of a size that is more readily analysed in the mass spectrometer and facilitates the identification of the protein [1]. Liquid chromatography can be used to separate the peptides, and peptide sequencing can be performed by tandem mass spectro metry (MS/MS) [1] In this method, peptides are ionized and selected peptide ions are subjected to sequencing, which is determined by MS/

Methods

Results

Conclusion