Papers by Jerry Ståhlberg
Biotechnology for biofuels, 2018
The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conv... more The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate. The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. The catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than eit...
Journal of Biotechnology
Despite recent progress, saccharification of lignocellulosic biomass is still a major cost driver... more Despite recent progress, saccharification of lignocellulosic biomass is still a major cost driver in biorefining. In this study, we present the development of minimal enzyme cocktails for hydrolysis of Norway spruce and sugarcane bagasse, which were pretreated using the so-called BALI™ process, which is based on sulfite pulping technology. Minimal enzyme cocktails were composed using several glycoside hydrolases purified from the industrially relevant filamentous fungus Trichoderma reesei and a purified commercial β-glucosidase from Aspergillus niger. The contribution of in-house expressed lytic polysaccharide monooxygenases (LPMOs) was also tested, since oxidative cleavage of cellulose by such LPMOs is known to be beneficial for conversion efficiency. We show that the optimized cocktails permit efficient saccharification at reasonable enzyme loadings and that the effect of the LPMOs is substrate-dependent. Using a cocktail comprising only four enzymes, glucan conversion for Norway spruce reached >80% at enzyme loadings of 8mg/g glucan, whereas almost 100% conversion was achieved at 16mg/g.
Described herein are variants of H. jecorina CBH I, a Cel7 enzyme. The present invention provides... more Described herein are variants of H. jecorina CBH I, a Cel7 enzyme. The present invention provides novel cellobiohydrolases that have improved thermostability and reversibility.
Chemical reviews, Jan 11, 2015
European journal of biochemistry / FEBS, Jan 5, 1988
From the culture filtrate of Trichoderma reesei we have isolated a novel endoglucanase (38 kDa) w... more From the culture filtrate of Trichoderma reesei we have isolated a novel endoglucanase (38 kDa) which was shown to be identical to endoglucanase III (E III, 50 kDa), but lacking the first 61 N-terminal amino acids. This core protein, designated E III core, is fully active against soluble substrates, such as carboxymethylcellulose, whereas both activity against and adsorption to microcrystalline cellulose (Avicel) is markedly decreased. Sedimentation velocity experiments revealed that the intact E III enzyme has much higher asymmetry than the E III core protein, suggesting that the N-terminal region split off constitutes a protruding part of the native enzyme. These results lead to the proposal that native E III consists of two functionally separated domains: a catalytically active core and a protruding N-terminal domain which acts in the binding to insoluble cellulose. The N-terminal peptide missing in E III core corresponds to the heavily glycosylated common structural element foun...
ACS Symposium Series, 2004
... 23. van Tilbeurgh, H.; Tomme, P.; Claeyssens, M.; Bhikhabhai, R.; Pettersson, G. FEBS Lett. 1... more ... 23. van Tilbeurgh, H.; Tomme, P.; Claeyssens, M.; Bhikhabhai, R.; Pettersson, G. FEBS Lett. 1986, 204, 223-227. 24. Tomme, P.; van Tilbeurgh, H.; Pettersson, G.; Van Damme, J.; Vandekerckhove, J.; Knowles, JKC; Teeri, TT; Claeyssens, M. Eur. J. Biochem. ...
Acta Crystallographica Section D Biological Crystallography, 2014
Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) play a key role in biomass recycling... more Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) play a key role in biomass recycling in nature. They are typically the most abundant enzymes expressed by potent cellulolytic fungi, and are also responsible for the majority of hydrolytic potential in enzyme cocktails for industrial processing of plant biomass. The thermostability of the enzyme is an important parameter for industrial utilization. In this study, Cel7 enzymes from different fungi were expressed in a fungal host and assayed for thermostability, includingHypocrea jecorinaCel7A as a reference. The most stable of the homologues,Humicola griseavar.thermoideaCel7A, exhibits a 10°C higher melting temperature (Tmof 72.5°C) and showed a 4–5 times higher initial hydrolysis rate thanH. jecorinaCel7A on phosphoric acid-swollen cellulose and showed the best performance of the tested enzymes on pretreated corn stover at elevated temperature (65°C, 24 h). The enzyme shares 57% sequence identity withH. jecorinaCel7A and c...
FEBS Journal, 2005
Cellulose is the most abundant polymer on earth. It has been estimated that as much as 15% of all... more Cellulose is the most abundant polymer on earth. It has been estimated that as much as 15% of all atmospheric carbon dioxide is fixed yearly, resulting in vast quantities of plant biomass, mostly as a complex mixture of cellulose and lignin [1]. The recycling of this carbon is critically dependent on the action of microbial organisms, primarily fungi and bacteria. An understanding of the processes at work is obviously of enormous environmental importance. The enzymes involved are also useful for applications that include, among others, their use in commercial laundry powders, as well as in the de-inking of recycled paper and the synthesis of fine chemicals. Cellulases, the enzymes that hydrolyse cellulose, have been broadly characterized as cellobiohydrolases (1,4-b-d-glucan cellobiohydrolase, EC 3.2.1.91) and
FEBS Journal, 2009
Glucan architecture is determined by the pattern of linkages connecting the glucose units. Branch... more Glucan architecture is determined by the pattern of linkages connecting the glucose units. Branching, substituents and the degree of polymerization further distingiuish different types of glucans [1,2]. Enzymes such as laminarinase 16A (Lam16A) from the whiterot fungus Phanerochaete chrysosporium exploit such differences [3] for binding and hydrolysis of glucans such as laminarin and lichenin (lichenan). b-1,3-glucans are omnipresent in the natural habitat of P. chrysosporium, which comprises fallen trees and forest litter. b-1,3-glucans are a prominent component of fungal cell walls and are produced to varying degrees by plants (as callose) in response to tissue damage. Furthermore, fungi and bacteria are often able to produce characteristic b-glucan exopolysaccharides, and P. chrysosporium itself can produce extracellular b-1,3-glucan that forms a gel-like sheath at its hyphae [4-7]. The usefulness of discriminating self from non-self in such an environment would suggest that P. chrysosporium has different glycoside hydrolases
FEBS Journal, 2013
Methylumbelliferyl-b-cellobioside (MUF-G2) is a convenient fluorogenic substrate for certain b-gl... more Methylumbelliferyl-b-cellobioside (MUF-G2) is a convenient fluorogenic substrate for certain b-glycoside hydrolases (GH). However, hydrolysis of the aglycone is poor with GH family 6 enzymes (GH6), despite strong binding. Prediction of the orientation of the aglycone of MUF-G2 in the +1 subsite of Hypocrea jecorina Cel6A by automated docking suggested umbelliferyl modifications at C4 and C6 for improved recognition. Four modified umbelliferyl-b-cellobiosides [6-chloro-4-methyl-(ClMUF); 6-chloro-4-trifluoromethyl-(ClF3MUF); 4-phenyl-(PhUF); 6-chloro-4phenyl-(ClPhUF)] were synthesized and tested with GH6, GH7, GH9, GH5 and GH45 cellulases. Indeed the rate of aglycone release by H. jecorina Cel6A was 10-150 times higher than with MUF-G2, although it was still three orders of magnitude lower than with H. jecorina Cel7B. The 4-phenyl substitution drastically reduced the fluorescence intensity of the free aglycone, while ClMUF-G2 could be used for determination of k cat and K M for H. jecorina Cel6A and Thermobifida fusca Cel6A. Crystal structures of H. jecorina Cel6A D221A mutant soaked with the MUF-, ClMUF-and ClPhUF-b-cellobioside substrates show that the modifications turned the umbelliferyl group 'upside down', with the glycosidic bond better positioned for protonation than with MUF-G2. Database Structural data have been submitted to the Protein Data Bank under accession numbers pdb 4AU0, 4AX7, 4AX6 Structured digital abstract • http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260296 • Cel6A and Cel6A bind by x-ray crystallography (View Interaction: 1, 2
European Journal of Biochemistry, 1999
A 28-kDa endoglucanase was isolated from the culture filtrate of Phanerochaete chrysosporium stra... more A 28-kDa endoglucanase was isolated from the culture filtrate of Phanerochaete chrysosporium strain K3 and named EG 28. It degrades carboxymethylated cellulose and amorphous cellulose, and to a lesser degree xylan and mannan but not microcrystalline cellulose (Avicel). EG 28 is unusual among cellulases from aerobic fungi, in that it appears to lack a cellulose-binding domain and does not bind to crystalline cellulose. The enzyme is efficient at releasing short fibres from filter paper and mechanical pulp, and acts synergistically with cellobiohydrolases. Its mode of degrading filter paper appears to be different to that of endoglucanase I from Trichoderma reesei. Furthermore, EG 28 releases colour from stained cellulose beads faster than any other enzyme tested. Peptide mapping suggests that it is not a fragment of another known endoglucanases from P. chrysosporium and peptide sequences indicate that it belongs to family 12 of the glycosyl hydrolases. EG 28 is glycosylated. The biological function of the enzyme is discussed, and it is hypothesized that it is homologous to EG III in Trichoderma reesei and the role of the enzyme is to make the cellulose in wood more accessible to other cellulases.
Structure, 2003
and Molecular Biology nism proceeds by inversion or retention of the anomeric carbon. Biomedical ... more and Molecular Biology nism proceeds by inversion or retention of the anomeric carbon. Biomedical Centre University of Uppsala Dextranase enzymes are found in two glycoside hydrolase families (GH), 49 and 66 (Coutinho and Henris-Box 596 SE-751 24 Uppsala sat, 1999), with no sequence similarity between the two families. The bacterial dextranases from the Streptococ-Sweden 2 Department of Chemistry cus species have been classified into family 66. In family 49, both bacterial dextranases from the Arthrobacter Swedish University of Agriculture Sciences Box 7015 species and fungal dextranases from Penicillium species are found. Dextran 1,6-␣-isomaltotriosidase from SE-750 07 Uppsala Sweden Brevibacterium fuscum var. dextranlyticum and isopullulanse from Aspergillus niger belong to the same family 3 Department of Molecular Biology Biomedical Centre 49. It has recently been predicted (Rigden and Franco, 2002) that GH families 49, 55, and 87 share a common Swedish University of Agriculture Sciences Box 590 evolutionary ancestor with families 28 and 82. P. minioluteum dextranase is a 67 kDa glycoprotein SE-751 24 Uppsala Sweden with an optimal activity at pH 5 and an isoelectric point of 3.88 (Raices et al., 1991). The dex gene encoding the enzyme has been cloned and sequenced by Garcia et al. (1996) (GenBank accession number L41562) and ex-Summary pressed in Pichia pastoris at a level of 3.2 g/l (Roca et al., 1996). Since the P. minioluteum dextranase belongs Dextranase catalyzes the hydrolysis of the ␣-1,6-glyto GH 49, we propose the name Dex49A for the enzyme cosidic linkage in dextran polymers. The structure of (Henrissat et al., 1998). To facilitate the crystallographic dextranase, Dex49A, from Penicillium minioluteum studies of the protein, we used a glycosylation-free muwas solved in the apo-enzyme and product-bound tant, N5A/N537A/N540A, of Dex49A in this work. In all forms. The main domain of the enzyme is a rightthree potential glycosylation sites, the asparagines were handed parallel  helix, which is connected to a  replaced by alanines (Larsson et al., unpublished data). sandwich domain at the N terminus. In the structure We have previously described the preparation and crysof the product complex, isomaltose was found to bind tallization of selenomethionine-labeled dextranase (Larsin a crevice on the surface of the enzyme. The glycoson et al., 2002). In the current publication, we present sidic oxygen of the glucose unit in subsite ϩ1 forms the three-dimensional structure of the apo-enzyme at a hydrogen bond to the suggested catalytic acid, 1.8 Å resolution and the structure of a product complex Asp395. By NMR spectroscopy the reaction course at 1.65 Å resolution. We also show by NMR that Dex49A was shown to occur with net inversion at the anomeric is an inverting enzyme, which, together with the struccarbon, implying a single displacement mechanism. ture, allows us to define details of the enzyme mecha-Both Asp376 and Asp396 are suitably positioned to nism and its relationship to other GH families. activate the water molecule that performs the nucleophilic attack. A new clan that links glycoside hydrolase families 28 and 49 is suggested. Results and Discussion
Structure, 1999
Background: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The cataly... more Background: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. Results: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-β-cellobiosyl-4-thio-β-cellobioside (Glc) 2-S-(Glc) 2 , which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl β-D-glucopyranosyl-β(1,4)-D-xylopyranoside (IBXG). Conclusions: The (Glc) 2-S-(Glc) 2 ligand binds in the-2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the-1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the-2 to +1 sites, with the xylosyl unit in the-1 site where it adopts the energetically favourable chair conformation. The-1 site glucosyl of the (Glc) 2-S-(Glc) 2 ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.
Progress in Biophysics and Molecular Biology, 2005
In this review we will describe how we have gathered structural and biochemical information from ... more In this review we will describe how we have gathered structural and biochemical information from several homologous cellulases from one class of glycoside hydrolases (GH family 12), and used this information within the framework of a protein-engineering program for the design of new variants of these enzymes. These variants have been characterized to identify some of the positions and the types of mutations in the enzymes that are responsible for some of the biochemical differences in thermal stability and activity between the homologous enzymes. In this process we have solved the three-dimensional structure of four of these homologous GH 12 cellulases: Three fungal enzymes, Humicola grisea Cel12A, Hypocrea jecorina Cel12A and Hypocrea schweinitzii Cel12A, and one bacterial, Streptomyces sp. 11AG8 Cel12A. We have also determined the three-dimensional structures of the two most stable H. jecorina Cel12A variants. In addition, four ligand-complex structures of the wild-type H. grisea Cel12A enzyme have been solved and have made it possible to characterize some of the interactions between substrate and enzyme. The structural and biochemical studies of these related GH 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can serve as a structural toolbox for the design of Cel12A enzymes with specific properties and features suited to existing or new applications.
PLoS ONE, 2011
Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase ... more Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61), some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33), this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency.
New Phytologist, 2012
Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and n... more Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and nutrient cycling in forest ecosystems. An opportunity to assess the trade-off between these strategies is provided by the forest pathogen and wood decayer Heterobasidion annosum sensu lato. We report the annotated genome sequence and transcript profiling, as well as the quantitative trait loci mapping, of one member of the species complex: H. irregulare. Quantitative trait loci critical for pathogenicity, and rich in transposable elements, orphan and secreted genes, were identified. A wide range of cellulose-degrading enzymes are expressed during wood decay. By contrast, pathogenic interaction between H. irregulare and pine engages fewer carbohydrate-active enzymes, but involves an increase in pectinolytic enzymes, transcription modules for oxidative stress and secondary metabolite production. Our results show a trade-off in terms of constrained carbohydrate decomposition and membrane transport capacity during interaction with living hosts. Our findings establish that saprotrophic wood decay and necrotrophic parasitism involve two distinct, yet overlapping, processes.
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Papers by Jerry Ståhlberg