Novel Penicillium cellulases for total hydrolysis of lignocellulosics

https://doi.org/10.1016/j.enzmictec.2013.03.003Get rights and content

Highlights

  • Two novel Penicillium strains (TUB F-2220 and TUB F-2378) with high cellulase production were discovered.

  • Enzyme mixtures of both Penicillium strains hydrolyzed efficiently of crystalline cellulose, were similar in hydrolysis of wheat straw but differed in hydrolysis of spruce.

  • Spruce lignin inhibited especially the F-2378 enzyme mixture in temperature dependent manner.

  • The main proteins in the TUB F-2220 (Penicillium pulvillorum) cultures were GH7 and GH6 CBHs (PpCBH1 and PpCBH2).

  • Adsorption of PpCBH1 on spruce lignin was pH and temperature dependent.

Abstract

The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and β-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35 °C while at 45 °C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases.

Introduction

Decreasing resources of fossil carbon sources and the detrimental effects of their usage on natural environments has generated growing demand to utilize plant biomass for fuel and chemical production [1], [2]. The first generation bioethanol plants rely on starch-based processes, however the plant cell wall components generally referred as lignocelluloses are well recognized as important raw materials for future biorefineries [3], [4]. Raw materials used by these plants range from dedicated energy plants and trees to agro- and forestry residues. Major components in the lignocelluloses are cellulose, hemicelluloses and lignin. Natural carbon recycling of the polymers relies on lignocellulose degrading microorganisms which produce a wide variety of glycosidic hydrolases (GH) [5], [6].

Most of the currently used commercial GH mixtures originate from Trichoderma and Aspergillus species, where T. reesei is the most exploited source of cellulases and hemicellulases for industrial purposes. Novel, more efficient or alternative enzymes are however under continuous search in order to improve the economical efficiency of the enzymatic hydrolysis aiming at lowering the required protein loadings, improved stability and recycling of the enzyme. Cellulase systems of Penicillium strains have shown to have potential in enhanced saccharification of lignocelluloses in the last 10 years [7], [8]. They include cellulases from Penicillium brasilianum [9], [10], Penicillium echinulatum [11], [12], Penicillium decumbens [13], Penicillium funiculosum [14], [15], Penicillium janthinellum [16], Penicillium pinophilum [17], [18], [19], and Penicillium purpurogenum [20]. Some cellulase complexes of Penicillium origin proved to be competitive with T. reesei cellulases in the hydrolysis of pure cellulose and different lignocellulosics [7], [12], [14], [21].

Lignin causes problems in industrial lignocellulose utilization by restricting the access of GHs to the carbohydrates and on the other hand causing the irreversible adsorption of the GHs on lignin surfaces [22], [23], [24], [25]. Inhibition is not only dependent on lignin content but also on lignin sources (structures) [26]. Recent studies have shown that soft wood lignin is the more inhibitory to enzymes than hardwood or grass lignins [24]. Weak lignin-binding enzymes for the hydrolysis of lignocellulosics are of importance in order to make the enzymatic hydrolysis of plant biomass more economical [27].

The hydrolytic potential of enzymes derived from two novel Penicillium sp. (TUB F-2220 and TUB F-2378) was elucidated in the present work. The strains were cultivated on several (ligno)cellulosic carbon sources and the secreted protein mixtures were assayed for cellulase and hemicellulase activities and evaluated in the hydrolysis of (ligno)celluloses and for lignin sensitivity in comparison to the enzyme mixtures from well-known T. reesei strains. The major cellulases in the TUB F-2220 cultures were partially purified and identified and characterized including evaluation of adsorption to cellulose and lignin. Coding sequence for the major GH7 enzyme of TUB-F-2220 cellulase mixture was obtained using PCR cloning.

Section snippets

Preparation and analyses of pretreated lignocelluloses and EnzHR lignins

Natural wheat straw was obtained from local sources in Hungary and milled in a coffee grinder to powder (<0.3 mm particle size) before use. Hydrothermally pre-treated wheat straw samples were obtained from Biogold OU (Tallinn, Estonia). High pressure steam was used at 200 °C for 20 min without any chemical additive as a pre-treatment method in Biogold. Steam pre-treated spruce (SO2 catalyzed pre-treatment) was provided by SEKAB E-Technology (Örnsköldsvik, Sweden). Only the solid fractions of the

Comparison of (hemi)cellulase activities in culture supernatants of the Penicillium species and T. reesei

Total cellulase and xylanase activities in the culture supernatants of the two Penicillium strains (TUB F-2220 (P. pulvillorum) and TUB F-2378 (P. cf simplicissimum)) and T. reesei RUT C30 during cultivation on cellulose (Avicel) are shown in Fig. 1. Maximal volumetric cellulase and xylanase activities in the cultures were obtained within 4–6 days of cultivation. The total volumetric cellulase activities (FPU) in the cultures of the two novel Penicillium strains were at a similar level as T.

Discussion

Two novel lignocellulolytic Penicillium strains, viz. P. pulvillorum TUB F-2220 and P. cf simplicissimum TUB F-2378 were tested in comparison to the well-known cellulase and hemicellulase producer T. reesei RUT C30 and QM 6a for the production and hydrolytic efficiency of cellulolytic and hemicellulolytic enzymes. Both novel wild-type Penicillium strains secreted approximately equal total volumetric cellulase (FPase) activities as the T. reesei RUT C30 despite of their lower level of total

Conclusions

Evaluation of the cellulase and hemicellulase systems of two novel lignocellulolytic Penicillium strains indicated that both strains secrete high level of cellulase activities. Specific cellulase activities for the wild-type P. pulvillorum TUB F-2220 strain are approx. two times higher as compared to T. reesei RUT C30. This attribute can make isolate F-2220 to an attractive biological starting material for strain improvement. The enzyme mixture secreted by P. pulvillorum (TUB F-2220) has

Acknowledgements

The experiments were carried out in the framework of the research project “Targeted DISCOvery of novel cellulases and hemicellulases and their reaction mechanisms for hydrolysis of lignocellulosic biomass” funded by the European Commission (http://www.disco-project.eu/index.htm, FP7-KBBE-2007-3.2-01). ROAL Ltd (Rajamäki, Finland), Inbicon A/S (Kalundborg, Denmark) and Prof. G. Zacchi (Department of Chemical Engineering, Lund University, Sweden) are acknowledged for provision of the materials.

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