Diana C. Irwin

Processivity, Substrate Binding, and Mechanism of Cellulose Hydrolysis by Thermobifida fusca Cel9A

ABSTRACT Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(−1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(−2) to Glc(−4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(−2) to Glc(−4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.

Genetics and Properties of Cellulases

Cellulases are enzymes which degrade the insoluble, abundant polymer cellulose. In order to perform this task bacteria, fungi, plants and insects have developed a variety of different systems with multiple cellulases. In this review the similarities and differences of these enzymes are summarized based on the burgeoning information gained in recent years from amino acid sequences, three dimensional structures and biochemical experiments. The independent cellulases of aerobic organisms are contrasted with the cellulosomes of anaerobic organisms. The ability of different enzymes to synergize with each other is discussed along with the role of the different types of enzymes in cellulose degradation.

Regulation and characterization of Thermobifida fusca carbohydrate‐binding module proteins E7 and E8

E7, a single domain Family 33 cellulose binding module (CBM) protein, and E8, a non‐catalytic, three‐domain protein consisting of a Family 33 CBM, a FNIII domain, followed by a Family 2 CBM, were cloned, expressed, purified, and characterized. Western blots showed that E7 and E8 were induced and secreted when Thermobifida fusca was grown on cellobiose, Solka floc, switchgrass, or alfalfa as well as on β‐1,3 linked glucose molecules such as laminaribiose or pachyman. E8 bound well to α‐ and β‐chitin and bacterial microcrystalline cellulose (BMCC) at all pHs tested. E7 bound strongly to β‐chitin, less well to α‐chitin and more weakly to BMCC than E8. Filter paper binding assays showed that E7 was 28% bound, E8 was 39% bound, a purified CBM2 binding domain from Cel6B was 88% bound, and only 5% of the Cel5A catalytic domain was bound. A C‐terminal 6×His tag influenced binding of both E7 and E8 to these substrates. Filter paper activity assays showed enhanced activity of T. fusca cellulases when E7 or E8 was present. This effect was observed at very low concentrations of cellulases or at very long times into the reaction and was mainly independent of the type of cellulase and the number of cellulases in the mixture. E8, and to a lesser extent E7, significantly enhanced the activity of Serratia marscescens Chitinase C on β‐chitin. Biotechnol. Biotechnol. Bioeng. 2008;100: 1066–1077.

Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode

ABSTRACT We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockerin, and the resultant recombinant “cellulosomized” enzymes were incorporated into chimeric scaffolding proteins that contained cohesin(s) together with a CBM. The activities of the resultant designer cellulosomes were compared with an equivalent mixture of wild-type enzymes. In the present work, a fourth T. fusca cellulase, Cel5A, was equipped with a dockerin and intervening linker segments of different lengths to assess their contribution to the overall activity of simple one- and two-enzyme designer cellulosome complexes. The results demonstrated that cellulose binding played a major role in the degradation of crystalline cellulosic substrates. The combination of the converted Cel5A endoglucanase with the converted Cel48A exoglucanase also exhibited a measurable proximity effect for the most recalcitrant cellulosic substrate (Avicel). The length of the linker between the catalytic module and the dockerin had little, if any, effect on the activity. However, positioning of the dockerin on the opposite (C-terminal) side of the enzyme, consistent with the usual position of dockerins on most cellulosomal enzymes, resulted in an enhanced synergistic response. These results promote the development of more complex multienzyme designer cellulosomes, which may eventually be applied for improved degradation of plant cell wall biomass.

A Tomato Endo-β-1,4-glucanase, SlCel9C1, Represents a Distinct Subclass with a New Family of Carbohydrate Binding Modules (CBM49)

A critical structural feature of many microbial endo-β-1,4-glucanases (EGases, or cellulases) is a carbohydrate binding module (CBM), which is required for effective crystalline cellulose degradation. However, CBMs are absent from plant EGases that have been biochemically characterized to date, and accordingly, plant EGases are not generally thought to have the capacity to degrade crystalline cellulose. We report the biochemical characterization of a tomato EGase, Solanum lycopersicum Cel8 (SlCel9C1), with a distinct C-terminal noncatalytic module that represents a previously uncharacterized family of CBMs. In vitro binding studies demonstrated that this module indeed binds to crystalline cellulose and can similarly bind as part of a recombinant chimeric fusion protein containing an EGase catalytic domain from the bacterium Thermobifida fusca. Site-directed mutagenesis studies show that tryptophans 559 and 573 play a role in crystalline cellulose binding. The SlCel9C1 CBM, which represents a new CBM family (CBM49), is a defining feature of a new structural subclass (Class C) of plant EGases, with members present throughout the plant kingdom. In addition, the SlCel9C1 catalytic domain was shown to hydrolyze artificial cellulosic polymers, cellulose oligosaccharides, and a variety of plant cell wall polysaccharides.

Factorial optimization of a six‐cellulase mixture

A factorial experimental design approach was used to optimize mixtures of six cellulases (five Thermomonospora fusca cellulases and plus/minus Trichoderma reesei CBHI along with beta-glucosidase) so as to maximize the glucose produced from filter paper. Optimized mixture A and mixture B produced glucose at 25 and 8.3 μmol glucose/μmol enzyme/min, respectively, which are 8 and 1.5 times higher than the sum of the activity of the individual cellulases. In both mixtures, the glucose yield depended on the ratio and the cellulases used. Most enzymes showed synergistic interactions that increased the glucose yield. The yield of glucose with the optimum mixtures depended on the total enzyme concentration. Copyright 1998 John Wiley & Sons, Inc.

Thermobifida fusca family-6 cellulases as potential designer cellulosome components

During the course of our studies on the structure–function relationship of cellulosomes, we were interested in converting the free cellulase system of the aerobic bacterium, Thermobifida fusca, to a cellulosomal system. For this purpose, the cellulose-binding modules (CBM) of two T. fusca family-6 cellulases, endoglucanase Cel6A and exoglucanase Cel6B, were replaced by divergent dockerin modules. Thus far, family-6 cellulases have not been shown to be members of natural cellulosome systems. The resultant chimaeric proteins, 6A-c and t-6B, respectively, were purified and found to interact specifically and stoichiometrically with their corresponding cohesin modules, indicating their suitability for use as components in ‘designer cellulosomes’. Both chimaeric enzymes showed somewhat decreased but measurable levels of activity on carboxymethyl cellulose, consistent with the known endo- and exo-glucanase character of the parent enzymes. The activity of 6A-c on phosphoric acid swollen cellulose was also consistent with that of the wild-type endoglucanase Cel6A. The startling finding of the present research was the extent of degradation of this substrate by the chimaeric enzyme t-6B. Wild-type exoglucanase Cel6B exhibited very low activity on this substrate, while the specific activity of t-6B was 14-fold higher than the parent enzyme.

The impact of initial particle size on the fragmentation of cellulose by the cellulase of thermomonospora fusca

This paper explores the impact of initial particle size on the rate and extent of cellulose fragmentation. Microcrystalline cellulose Avicel PH 102 was sieved with a set of US standard sieves to obtain six monodisperse, particle size ranges. Three of these size ranges (74 μm < x < 105 μm, 46 μm < x 63 μm < x 46 μm) were made into 5% suspensions and hydrolyzed with 0·1 and 0·25 mg ml−1Thermomonospora fusca crude cellulase. Samples were removed very 20 min during hydrolysis and analyzed for reducing sugar, free enzyme and particle size distribution. The results showed that initial particle size had no impact on the rate and extent of reducing sugar production or on enzyme binding. However, the rate and extent of cellulose fragmentation was strongly affected by initial particle size. The change in the volume fraction distribution with time was also influenced by initial particle size, with larger particles giving rise to a bimodal distribution and smaller particles to a skewed distribution. The changes in the volume fraction distributions with time suggested fragmentation to be the dominant reaction for larger particles and erosion to be the dominant reaction for smaller particles. These experiments show that fragmentation is not the rate limiting step in the enzymatic hydrolysis of cellulose.

Increased Crystalline Cellulose Activity via Combinations of Amino Acid Changes in the Family 9 Catalytic Domain and Family 3c Cellulose Binding Module of Thermobifida fusca Cel9A

ABSTRACT Amino acid modifications of the Thermobifida fusca Cel9A-68 catalytic domain or carbohydrate binding module 3c (CBM3c) were combined to create enzymes with changed amino acids in both domains. Bacterial crystalline cellulose (BC) and swollen cellulose (SWC) assays of the expressed and purified enzymes showed that three combinations resulted in 150% and 200% increased activity, respectively, and also increased synergistic activity with other cellulases. Several other combinations resulted in drastically lowered activity, giving insight into the need for a balance between the binding in the catalytic cleft on either side of the cleavage site, as well as coordination between binding affinity for the catalytic domain and CBM3c. The same combinations of amino acid variants in the whole enzyme, Cel9A-90, did not increase BC or SWC activity but did have higher filter paper (FP) activity at 12% digestion.

Binding reversibility and surface exchange of Thermomonospora fusca E3 and E5 and Trichoderma reesei CBHI

Abstract The sorption of Thermomonospora fusca E 3 and E 5 and Trichoderma reesei CBHI cellulases on bacterial microcrystalline cellulose (BMCC) was investigated to measure binding reversibility and surface exchange. The adsorption and desorption curves for CBHI were comparable; this suggests a totally reversible binding process. By contrast, the desorption isotherms of E 3 and E 5 did not retrace their respective adsorption curves; instead, hysteresis loops were formed. The calculated percent reversibilities for E 3 , E 5 , and CBHI were 73 ± 7%, 80 ± 7%, and 95 ± 7%, respectively. Surface exchange studies indicated that the adsorbed and free cellulases were exchanging at the cellulose surface. Despite this confirmation of molecular exchange, the extent of exchange did not reach the level that would be predicted if the preadsorbed cellulase were to equally redistribute between the free and bound states. The percent of exchange for E 3 and E 5 decreased with increasing initial total cellulase concentration (E t ) while that for CBHI showed no dependence on E t .