Identification of a trypsin-like serine protease from Trichoderma reesei QM9414

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Abstract

In the present work the genetically modified Trichoderma reesei strain QM9414 was used to produce full-length Cel7B (endoglucanase I, EGI) under the control of the constitutive Aspergillus nidulans gpdA promoter in the presence of glucose. However, the full-length Cel7B enzyme was found to be truncated to lower molecular weight components in the culture broth. Truncation of recombinant proteins produced in fungi may be due to protease activity. In order to identify major sectreted proteases, protease activity was assessed in culture filtrate of the T. reesei QM9414 recombinant. Zymogram analysis revealed the presence of proteolytic activity corresponding to one protein, which was subsequently purified by a combination of ion exchange and size exclusion chromatography. The protein has a molecular mass of 25 kDa, and an isoelectric point of 7.3. By matching tryptic peptide fragments analyzed by tandem mass spectrometry to fungal proteins available in databases as well as to expressed sequence tag (EST) sequences, and comparing the coded amino acids to full-length amino acid sequences, the purified protein was found to be homologous to several trypsin-like fungal serine proteases, with the highest homology to the protease P27 from Trichoderma harzianum. The purified protein was further characterized using benzoyl-arginyl-p-nitroanilide (BApNA) as substrate. It was found to have maximum activity at pH 8 and 50 °C, with a km-value of 0.3 mM.

Introduction

The soft-rot fungus Trichoderma reesei (also known as Hypocrea jecorina) is one of the most studied cellulolytic organisms because of its ability to produce a complete set of efficiently secreted cellulolytic enzymes. T. reesei is known to produce two exoglucanases (cellobiohydrolases, CBHs, EC 3.2.1.91) Cel6A (CBHII) and Cel7A (CBHI), five endoglucanases (EGs, EC 3.2.1.4) Cel5A (EGII), Cel7B (EGI), Cel12A (EGIII), Cel45A (EGV) and Cel61A (EGIV), as well as two β-glucosidases (BGLs, EC 3.2.1.21) Cel1A (BGLII) and Cel3A (BGLI) for cellulose degradation [1]. Many of these cellulases, e.g. Cel7B, has a modular structure with a catalytic module and a cellulose binding module (CBM) separated from each other by a linker region. The expression of the genes encoding cellulases have been shown to be glucose repressed [1], [2], [3]. Thus, a specific cellulase enzyme can be expressed under a constitutive promoter and cultivated at high glucose concentration, thereby minimizing the need for removal of other undesired cellulase components in the broth [4]. In the present work, this strategy was used for production of the full-length Cel7B.

An important consideration in production of any protein is the potential problem of proteolytic degradation [5], [6], [7]. Undesired degradation of cellulases has been observed in culture filtrates and commercial enzyme preparations from Trichoderma [8], [9], [10], [11], [12]. Although filamentous fungi have attracted increasing attention as host organisms for protein production, extracellular proteolytic degradation of homologous and heterologous proteins is still a major issue [7]. Since heterologous proteins often tend to be degraded more than homologous proteins, protease-deficient host strains have been isolated and developed for improved protein yield [13], [14].

Given the tentative huge importance of T. reesei as a host organism for large-scale production of cellulases used in bioethanol production, surprisingly few studies have been published concerning proteases secreted by T. reesei. In previous studies some acid proteases and a Kex2-like dibasic endopeptidase were reported [8], [15], [16].

In the present study, the full length Cel7B was expressed by a constitutive promoter in T. reesei QM9414 under glucose repressing conditions. Observed degradation of the full length Cel7B inspired an investigation to possibly identify the source of the protease activity resulting in degradation of the target protein. The objective of the present work was to identify a major extracellular protease and characterize it in order to establish a possible relation to the target protein degradation. As a result a previously uncharacterized protease was identified, purified and analyzed by mass spectrometry (MS). Database searches of MS spectra from a tryptic digest of the purified protein suggested that it is a trypsin-like serine protease. Trypsin-like activity was also confirmed by protease assays and the conditions for optimal activity for the substrates azocasein and BApNA, as well as the kinetic parameters for BApNA were determined. Furthermore, degradation of full length Cel7B was tested.

Section snippets

Expression of full-length Cel7B enzyme

Genetically modified T. reesei strain QM9414 was used to produce full-length Cel7B. The target protein was expressed under the constitutive promoter of the glyceraldehyde phosphate dehydrogenase gene (gpdA) from Aspergillus nidulans as described previously [17]. Endogenous production of cellulases was expected to be repressed at the high glucose concentration applied in the cultivations, thereby allowing a relatively pure Cel7B to be produced [2], [18], [19].

Cultivation conditions

The stock culture of parent and the

Cultivation of genetically modified T. reesei QM9414 in laboratory fermenter

In a previous work, Cel7B and Cel7B core proteins were produced using recombinants of T. reesei QM9414 (hereafter referred to as TrCel7B and TrCel7Bcore recombinants) cultivated in shake flask on glucose containing medium [31]. The supernatant was used in enzymatic treatment of secondary fibers. Positive results were achieved with respect to drainage without causing significant deterioration in strength properties of paper.

In the present work, in an attempt to increase the Cel7B yield, the

Discussion

In the present work a recombinant of T. reesei QM9414 was used to produce Cel7B. As previously discussed [4], [34], the glucose repression exerted on the expression of the other cellulolytic enzymes in T. reesei provides the basis for an attractive option to produce specific cellulolytic enzymes in a highly efficient secreting host organism. However, good yield depends on (a) strict glucose repression of all other cellulase enzymes in T. reesei, (b) efficient secretion, and (c) no proteolytic

Acknowledgements

Marie Curie Fellowship Association (Marie Curie training site, QCIM), European Cooperation in the field of Scientific and Technical Research (Short Term Scientific Mission, COST Action E23), the National Research Fund of Hungary (OTKA TS049849) and the Swedish Research Council (VR) are acknowledged for the financial support.

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