Cellobiose binding

Abstract: Enzymes that degrade cellulose into glucose are one of the most expensive components of processes for converting cellulosic biomass to fuels and chemicals. Cellulase enzyme Cel7A is the most abundant enzyme naturally employed by fungi to depolymerize cellulose, and like other cellulases is inhibited by its product, cellobiose. There is thus great economic incentive for minimizing the detrimental effects of product inhibition on Cel7A. In this work, we experimentally generated 10 previously proposed site-directed mutant Cel7A enzymes expected to have reduced cellobiose binding energies (the majority of mutations were to alanine). We then tested their resilience to cellobiose as well as their hydrolytic activities on microcrystalline cellulose. Although every mutation tested conferred reduced product inhibition (and abolished it for some), our results confirm a trade-off between Cel7A tolerance to cellobiose and enzymatic activity: Reduced product inhibition was accompanied by lower overall enzymatic activity on crystalline cellulose for the mutants tested. The tempering effect of mutations on inhibition was nearly constant despite relatively large differences in activities of the mutants. Our work identifies an amino acid in the Cel7A product binding site of interest for further mutational studies, and highlights both the challenge and the opportunity of enzyme engineering toward improving product tolerance in Cel7A.

Abstract: To gain further insight into the difference in substrate specificity between endoglucanase and cellobiohydrolase, the intrinsic fluorescence properties of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) from Trichoderma pseudokiningii S-38 were investigated. The results for the spectral characteristics, ligand binding and fluorescence quenching suggest that the fluorescence of two enzymes comes from tryptophan residues, and that tryptophan residue(s) may be involved in the function of the two enzymes. The results also suggest that the binding tryptophan in EG I may be more exposed to solvent than that in CBH I. This interpretation is supported by the observations that the effects of pH upon the fluorescence of EG I are greater than that of CBH I; spectral shifts are different in EG I and CBH I under various conditions, and fluorescence lifetime changes caused by cellobiose binding are larger for EG I than for CBH I.

Abstract: Cellobiohydrolase Cel7A (Hje_Cel7A) from the ascomycete fungus Hypocrea jecorina is the major component of enzyme cocktails for degradation of plant biomass to soluble sugars for conversion to biofuels. It’s active site is enclosed in a cellulose-binding tunnel and it is able to processively cleave off several cellobiose units from the end of a cellulose chain before being released. However, Cel7A is sensitive to product inhibition due to strong binding of cellobiose in subsites +1 and +2 in the active site. Prior to this study, variants of Hje Cel7A, aimed at both stronger and weaker cellobiose binding, have been designed and expressed. Large increase of kcat and KM and reduced cellobiose inhibition were found on soluble substrates. The purpose of the present study is to complement the previous results with evaluation of the performance of the Cel7A variants on “real” insoluble cellulosic substrates, and also compare with the corresponding major enzymes from two basidiomycete fungi, Phanerochaete chrysosporium Cel7D (Pch Cel7D) and Heterobasidion irregulare Cel7A (Hir Cel7A). Activity was measured at 40°C, pH 5.0, on Avicel cellulose (5g/l) incubated for 2 hours, untreated spruce saw dust powder (30 g/l) incubated for 20 hours, and industrially pretreated spruce material (50 g/l) incubated for 20 hours with 50 mg/l of enzyme, either Cel7 enzymes alone, or together with a commercial cellulase enzyme cocktail, or mixed with the same cocktail where the Hje Cel7A wildtype enzyme had been selectively removed. The differences in activity were less dramatic than previously observed with soluble substrates. On Avicel, the AAA variant alone showed slightly higher activity, but in all other cases Hje Cel7A WT was most active, closely followed by the 2CC variant. On untreated spruce, the 2CC variant gave most reducing sugar when added to Cel7A-free Accellerase and Accellerase 1500. Interestingly, Hir Cel7A was much more active than the others when acting alone on untreated spruce. However, it was not analysed which sugars were actually released. No conclusions could be drawn with thermo-chemically pretreated spruce, because the amount of soluble sugar was rather high already before adding the enzymes, only small amounts of sugars were released by the enzymes, and the variation was high between samples. The possible presence of enzyme inhibitors need to be further investigated. Overall, the results indicate that a more open active site and weaker cellobiose binding is not beneficial for the enzyme performance on insoluble lignocellulose. Rather it is an advantage with a more closed tunnel.

Abstract: Streptomyces reticuli has an inducible ATP-dependent uptake system specific for cellobiose and cellotriose. By reversed genetics a gene cluster encoding components of a binding protein-dependent cellobiose and cellotriose ABC transporter was cloned and sequenced. The deduced gene products comprise a regulatory protein (CebR), a cellobiose binding lipoprotein (CebE), two integral membrane proteins (CebF and CebG), and the NH2-terminal part of an intracellular beta-glucosidase (BglC). The gene for the ATP binding protein MsiK is not linked to the ceb operon. We have shown earlier that MsiK is part of two different ABC transport systems, one for maltose and one for cellobiose and cellotriose, in S. reticuli and Streptomyces lividans. Transcription of polycistronic cebEFG and bglC mRNAs is induced by cellobiose, whereas the cebR gene is transcribed independently. Immunological experiments showed that CebE is synthesized during growth with cellobiose and that MsiK is produced in the presence of several sugars at high or moderate levels. The described ABC transporter is the first one of its kind and is the only specific cellobiose/cellotriose uptake system of S. reticuli, since insertional inactivation of the cebE gene prevents high-affinity uptake of cellobiose.