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Background: Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. Methods and results: In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58 s−1 mM−1, 1.09 s−1 mM−1, and 0.062 s−1 mM−1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/β-hydrolase fold, which is consistent with other esterases. Conclusions: The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries. [ABSTRACT FROM AUTHOR]

Superoxide dismutases (SODs) are enzymes that catalyze the dismutation of the superoxide radical anion into O2 and H2O2 in a two-step reaction. They are ubiquitous to all forms of life and four different types of metal centers are detected, dividing this class of enzymes into Cu-/Zn-, Ni-, Mn-, and Fe-SODs. In this study, a superoxide dismutase from the thermophilic bacteria Thermobifida fusca (TfSOD) was cloned and expressed before the recombinant enzyme was characterized. The enzyme was found to be active for superoxide dismutation measured by inhibition of cytochrome c oxidation and the inhibition of the autoxidation of pyrogallol. Its pH-optimum was determined to be 7.5, while it has a broad temperature optimum ranging from 20 to 90 °C. Combined with the Tm that was found to be 78.5 ± 0.5 °C at pH 8.0, TfSOD can be defined as a thermostable enzyme. Moreover, the crystal structure of TfSOD was determined and refined to 1.25 Å resolution. With electron paramagnetic resonance spectroscopy, it was confirmed that iron is the metal co-factor of TfSOD. The cell potential (Em) for the TfSOD-Fe3+/TfSOD-Fe2+ redox couple was determined to be 287 mV. [ABSTRACT FROM AUTHOR]

[Display omitted] • Isolated A. terreus was used to hydrolyze food waste to a glucose rich feedstock. • The first-time food waste hydrolysate was used to produce recombinant cutinase. • Hydrolysate concentration was determined as the key player in cutinase production. • Successful surface degradation of commercial PET bottle by recombinant cutinase. Recently, there has been a significant surge in the utilization of single-use plastics. Cost-effective degradation of plastics is imperative to reduce the ecological burden. This study explores the overproduction and characterization of a PET (Polyethylene terephthalate) a degrading enzyme, cutinase, in the E. coli BL21 (DE3) expression system. Furthermore, this research delves into the utilization of mixed food waste (MFW) as a sustainable substrate for the production of Cutinase, replacing commercial glucose. The newly isolated strain, Aspergillus terreus , showed the highest glucoamylase activity of 76.10 ± 3.10 U/g. The enzymatic hydrolysis of mixed food waste resulted in 50 ± 0.4 g/L of glucose, used as feedstock for cutinase production. The crude enzyme activity was evaluated to be 11.64 U/mg, and a further 14.11-fold purification was achieved through IMAC. The purified recombinant enzyme showed its maximum activity at 70℃ and pH 7. The metal ion Na+ increased the enzyme activity by 26 % while Zn+2 significantly inhibited the same (47 % inhibition); other ions had little or no effect. Taguchi's optimization of five variables showed that the concentration of food waste hydrolysate has the maximum impact on enzyme production. The treatment of commercially available PET bottles using the purified enzyme showed characterizable surface degradation effects. This is the first time food waste hydrolysate was used as a sole carbon source to produce recombinant cutinase. This approach can potentially pave the way for sustainable biorefinery and waste management. [ABSTRACT FROM AUTHOR]

Plastic waste is the biggest global problem in present times due to its constant bioaccumulation in the environment. During the last year, 367 Mt of plastics were produced in the world, of which 28.6 Mt correspond to polyurethane waste. Polyurethanes can be found in products such as adhesives, preservatives, and foams, and are often difficult to recycle. The fragmentation of plastic waste in the environment generates microplastics causing a long-term effect on our ecosystem. In search of its solution via bioremediation using enzymes, in our present study cutinase enzyme has been chosen, as it appears to be a novel candidate due to the wide variety of substrates it hydrolyzes and its presence in different microorganisms. According to physiochemical characteristics, it was found that microbial cutinase enzymes are majorly made up of aliphatic amino acids. A higher aliphatic index (more than 80) indicates the great thermostability of the enzyme. Moreover, the enzyme is found to be hydrophilic and structurally stable. The negative GRAVY value of the enzyme confirmed its stable interaction with water. It was also observed that all chosen protein models had an average of 91.10 % amino acids in the favorable regions of the Ramachandran plot. The studied microbial cutinase enzymes from both fungi Humicola insolens and actinobacterium Thermobifida fusca successfully coupled with the polyurethane resin monomers. The main interactions were found in the catalytic triad with bonds close to the urethane bonds of the ligand, in addition to having an average binding energy of − 6 kJ/mol. The interaction between the cutinases with the PUR resin as a ligand was found to be evident from our study with stable binding energies, which makes microbial cutinases potential enzymes for polyurethane waste bioremediation processes.

The aerobic, thermophilic Actinobacterium, Thermobifida fusca has been proposed as an organism to be used for the efficient conversion of plant biomass to fatty acid-derived precursors of biofuels or biorenewable chemicals. Despite the potential of T. fusca to catabolize plant biomass, there is remarkably little data available concerning the natural ability of this organism to produce fatty acids. Therefore, we determined the fatty acids that T. fusca produces when it is grown on different carbon sources (i.e., glucose, cellobiose, cellulose and avicel) and at two different growth temperatures, namely at the optimal growth temperature of 50°C and at a suboptimal temperature of 37°C. These analyses establish that T. fusca produces a combination of linear and branched chain fatty acids (BCFAs), including iso-, anteiso-, and 10-methyl BCFAs that range between 14- and 18-carbons in length. Although different carbon sources and growth temperatures both quantitatively and qualitatively affect the fatty acid profiles produced by T. fusca, growth temperature is the greater modifier of these traits. Additionally, genome scanning enabled the identification of many of the fatty acid biosynthetic genes encoded by T. fusca.

Wastewater containing recalcitrant dyes causes environmental problems. A new superfamily of heme-containing peroxidases, dye-decolorizing peroxidases (DyPs), has been found to decolorize different kinds of dyes, especial anthraquinone dyes efficiently. However, the mechanism of dyes degradation by DyPs has not been fully understood and the toxicity of dye degradation intermediates by DyPs catalysis to microbes is unclear. In this study, a purified recombinant Thermobifida fusca DyP (TfuDyP) in E. coli BL21(DE3) was used to treat Reactive Blue 19 (RB19), an anthraquinone dye. The reaction intermediates analyzed by ultra performance liquid chromatography/mass spectroscopy (UPLC–MS) indicated the initial site of TfuDyP attack on RB19. In addition, it was found that both RB19 and its incomplete degradation products inhibited the growth of Bacillus subtilis. These findings provided a novel understanding of DyPs catalysis to anthraquinone dyes. [ABSTRACT FROM AUTHOR]

The rising production and consumption of plastics, coupled with their intrinsic robustness, has resulted in the accumulation of up to 200 million tons of plastic waste in landfills and the environment each year. Of all these synthetic polymers, polyethylene terephthalate (PET) is the most widely used in the world. As environmental microorganisms have begun evolving their own biological means to metabolize plastics, they potentially present a sustainable method to recycle PET and retain its material value for subsequent use through biodegradation. Here, we evaluate the feasibility of using multiple PET-degrading bacterial strains (Thermobifida fusca, Ideonella sakaiensis, Pseudomonas mendocina) concurrently to develop an industrial co-culture solution. The growth behavior of each strain was evaluated at temperatures ranging from 30-60 °C and in a Hipolypepton-based media. UV-treated PET pellets were then incubated in monoculture and co-culture cell suspensions for 2 days, and degradation was measured through a spectrophotometric assay. A 1:1 T. fusca/I. sakaiensis co-culture was found to achieve a higher biodegradation efficiency compared to I. sakaiensis monoculture but not compared to T. fusca monoculture, suggesting the need for further optimization of degradation conditions. Although our studies demonstrate the great potential of biodegradation as a solution for the plastic waste crisis, the direct application of microbes may not be suitable for industrial use when compared to alternative biodegradative methods. [ABSTRACT FROM AUTHOR]

Lignocelluloses are comprised of cellulose, hemicellulose, and lignins, which constitute plant biomass. Since peroxidases can degrade lignins, the authors examined peroxidase Tfu-1649, which is secreted from the thermophilic actinomycetes, Thermobifida fusca BCRC 19214. After cultivating for 48 h, the culture broth accumulated 43.66 U/mL of peroxidase activity. The treatment of four types of lignocellulolytic byproducts, i.e., bagasse, corncob, pin sawdust, and Zizania latifolia Turcz husk, with Tfu-1649 alone increased the total phenolic compounds, with limited reducing sugars, but treatment with xylanase, Tfu-11, and peroxidase Tfu-1649 showed synergistic effects. Hence, the co-operative degradation of lignocelluloses by both peroxidase and xylanase could contribute to biomass decomposition and further applications in the agricultural and environmental industries. [ABSTRACT FROM AUTHOR]

Effective artificial microbial consortia containing microorganisms with desired biological functions have the potential to optimize the lignocellulose-based bioindustry. Thermobifida fusca was a dominant actinobacterium in high-temperature corn stalk composts, but it was unable to grow alone in corn stalk solid medium. Interestingly, T. fusca showed good growth and secreted enzymes when cocultured with Thermomyces lanuginosus. T. lanuginosus grew firstly during the initial stage, whereas T. fusca dominated the system subsequently during cocultivation. The secretome indicated that T. lanuginosus mainly degraded xylan by expressing a GH11 xylanase (g4601.t1, GenBank AAB94633.1; with relative secretion of 4.95 ± 0.65%). T. fusca was induced by xylan mainly to secrete a xylanase from GH11 family (W8GGR4, GenBank AHK22788.1; with relative secretion of 8.71 ± 3.83%) which could rapidly degrade xylan to xylo-oligosaccharide (XOS) and xylose within 2 min, while high concentrations (>0.5%, w/v) of XOS or xylose suppressed the growth of T. fusca; which may be the reason why T. fusca unable to grow alone in corn stalk solid medium. However, T. lanuginosus could utilize the XOS and xylose produced by xylanases secreted by T. fusca. During the synergistic degradation of lignocellulose by T. lanuginosus and T. fusca, xylan was rapidly consumed by T. lanuginosus, the residual cellulose could specifically induced T. fusca to express a GH10 xylanase with a CBM2 domain (Q47KR6, GenBank AAZ56956.1; with relative secretion of 5.03 ± 1.33%) and 6 cellulases (2 exocellulases and 4 endocellulases). Moreover, T. lanuginosus increased the secretion of cellulases from T. fusca by 19–25%. The order of T. lanuginosus and T. fusca was consistent with the multilayered structures of lignocellulose and could be regulated by different concentrations of XOS and xylose. The novel synergism of T. lanuginosus and T. fusca gave a new sight for revealing more synergetic relationships in natural environments and exploring efficient microbial inoculants and enzyme cocktails for lignocellulose degradation.

Effective artificial microbial consortia containing microorganisms with desired biological functions have the potential to optimize the lignocellulose-based bioindustry. Thermobifida fusca was a dominant actinobacterium in high-temperature corn stalk composts, but it was unable to grow alone in corn stalk solid medium. Interestingly, T. fusca showed good growth and secreted enzymes when cocultured with Thermomyces lanuginosus. T. lanuginosus grew firstly during the initial stage, whereas T. fusca dominated the system subsequently during cocultivation. The secretome indicated that T. lanuginosus mainly degraded xylan by expressing a GH11 xylanase (g4601.t1, GenBank AAB94633.1; with relative secretion of 4.95 ± 0.65%). T. fusca was induced by xylan mainly to secrete a xylanase from GH11 family (W8GGR4, GenBank AHK22788.1; with relative secretion of 8.71 ± 3.83%) which could rapidly degrade xylan to xylo-oligosaccharide (XOS) and xylose within 2 min, while high concentrations (>0.5%, w/v) of XOS or xylose suppressed the growth of T. fusca ; which may be the reason why T. fusca unable to grow alone in corn stalk solid medium. However, T. lanuginosus could utilize the XOS and xylose produced by xylanases secreted by T. fusca. During the synergistic degradation of lignocellulose by T. lanuginosus and T. fusca , xylan was rapidly consumed by T. lanuginosus , the residual cellulose could specifically induced T. fusca to express a GH10 xylanase with a CBM2 domain (Q47KR6, GenBank AAZ56956.1; with relative secretion of 5.03 ± 1.33%) and 6 cellulases (2 exocellulases and 4 endocellulases). Moreover, T. lanuginosus increased the secretion of cellulases from T. fusca by 19–25%. The order of T. lanuginosus and T. fusca was consistent with the multilayered structures of lignocellulose and could be regulated by different concentrations of XOS and xylose. The novel synergism of T. lanuginosus and T. fusca gave a new sight for revealing more synergetic relationships in natural environments and exploring efficient microbial inoculants and enzyme cocktails for lignocellulose degradation. [ABSTRACT FROM AUTHOR]

The ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase‐coupled NADH‐dependent ferredoxin reductase (FNR) family, catalyses electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a putative three‐component cytochrome P450 monooxygenase system in T. fusca comprising CYP222A1 and the [3Fe–4S]‐cluster ferredoxin Fdx8 as well as FdR9. Here, FdR9 was overexpressed and purified and its crystal structure was determined at 1.9 Å resolution. The overall structure of FdR9 is similar to those of other members of the FNR family and is composed of an FAD‐binding domain, an NAD‐binding domain and a C‐terminal domain. Activity measurements with FdR9 confirmed a strong preference for NADH as the cofactor. Comparison of the FAD‐ and NAD‐binding domains of FdR9 with those of other ferredoxin reductases revealed the presence of conserved sequence motifs in the FAD‐binding domain as well as several highly conserved residues involved in FAD and NAD cofactor binding. Moreover, the NAD‐binding site of FdR9 contains a modified Rossmann‐fold motif, GxSxxS, instead of the classical GxGxxG motif. [ABSTRACT FROM AUTHOR]

Abstract Background Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The point mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity. Results In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination. Conclusions The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized.

Cel48A of Thermobifida fusca (TfCel48A) is a processive exocellulase that contains an active site tunnel and digests lignocellulosic biomass via synergistic interactions between different cellulases. Cel48A possesses a number of aromatic amino acids lining the tunnel entrance, which are highly conserved across a diverse number of microbial species and appear to play a role in the selection and threading of individual strands of cellulose from highly recalcitrant substrates. In this study, we sought to further elucidate the roles of these tunnel entrance aromatic amino acids by creating a series of double mutants and examining their effect on TfCel48A activity, processivity, and synergistic interactions with the well‐studied processive endocellulase TfCel9A. Our results provide further insight concerning the mechanism of Cel48A kinetics with soluble and insoluble substrates and could play an influential role in the application of Cel48A and other exocellulases for industrial purposes. [ABSTRACT FROM AUTHOR]

Carotenoids are important colored components of food with several essential health benefits. Because of their characteristic fruity aroma, the cleavage products of carotenoids are being used as flavouring agents in the food industry. Enzymes are needed to get cleavage products of carotenoids in order to avoid the very laborious and costly extraction processes of particular aroma compounds, such as α- and β-ionone, directly from their plant sources. So, for an easy and cost effective access to flavouring agents, the role of enzymes is undeniably crucial. The present work was therefore undertaken to isolate an industrially important recombinant peroxidase enzyme to obtain the flavouring agents. Thermobifida fusca (accession number KM677184), isolated from manure piles, was found to effectively degrade β-carotene. The gene encoding T. fusca extracellular heme-containing peroxidase (TfuDyP) was amplified from genomic DNA of T. fusca and was cloned in vector pET24b. TfuDyP was expressed heterologously in E. coli (BL21) and a recombinant enzyme was used for the transformation of β-carotene into volatile products which were identified by the GC-MS (Gas Chromatography-Mass Spectrophotometry). The enzyme was found effective as various volatile degradation products of β-carotene, such as β-ionone, β-cyclocitral, β-ionone-5,6-epoxide and dihydroactinidiolide were identified. With these results, we are inclined to conclude that the extracellular peroxidase enzyme obtained from T. fusca IP1 has the potential to be used in the food industry. In spite of the various reports on the subject, the use of this particular bacterial strain in the enzyme production is being reported for the first time in the present work. [ABSTRACT FROM AUTHOR]

Abstract Adipic acid is a platform chemical, and is the most important commercial dicarboxylic acid. It has been targeted for biochemical conversion as an alternative to present chemical production routes. From the perspective of bioeconomy, several kinds of raw material are of interest including the sugar platform (derived from starch, cellulose or hemicellulose), the lignin platform (aromatics) and the fatty acid platform (lipid derived). Two main biochemical-based production schemes may be employed: (i) direct fermentation to adipic acid, or (ii) fermentation to muconic or glucaric acid, followed by chemical hydrogenation (indirect fermentation). This review presents a comprehensive description of the metabolic pathways that could be constructed and analyzes their respective theoretical yields and metabolic constraints. The experimental yields and titers obtained so far are low, with the exception of processes based on palm oil and glycerol, which have been reported to yield up to 50 g and 68 g adipic acid/L, respectively. The challenges that remain to be addressed in order to achieve industrially relevant production levels include solving redox constraints, and identifying and/or engineering enzymes for parts of the metabolic pathways that have yet to be metabolically demonstrated. This review provides new insights into ways in which metabolic pathways can be constructed to achieve efficient adipic acid production. The production host provides the chassis to be engineered via an appropriate metabolic pathway, and should also have properties suitable for the industrial production of adipic acid. An acidic process pH is attractive to reduce the cost of downstream processing. The production host should exhibit high tolerance to complex raw material streams and high adipic acid concentrations at acidic pH. [ABSTRACT FROM AUTHOR]

Cellulases are a set of lignocellulolytic enzymes, capable of producing eco-friendly low-cost renewable bioethanol. However, low stability and hydrolytic activity limit their wide-scale applicability at the industrial scale. In this work, we report the domain engineering of endoglucanase (cel6A) of Thermobifida fusca to improve their catalytic activity and thermal stability. Later, enzymatic activity and thermostability of the most efficient variant named as cel6A.CBC was analyzed by molecular dynamics simulations. This variant demonstrated profound activity against soluble and insoluble cellulosic substrates like filter paper, alkali-treated bagasse, regenerated amorphous cellulose (RAC), and bacterial microcrystalline cellulose. The variant cel6A.CBC showed the highest catalysis of carboxymethyl cellulose (CMC) and other related insoluble substrates at a pH of 6.0 and a temperature of 60 °C. Furthermore, a sound rationale was observed between experimental findings and molecular modeling of cel6A.CBC which revealed thermostability of cel6A.CBC at 26.85, 60.85, and 74.85 °C as well as structural flexibility at 126.85 °C. Therefore, a thermostable derivative of cel6A engineered in the present work has enhanced biological performance and can be a useful construct for the mass production of bioethanol from plant biomass.

Thermobifida fusca is of biotechnological interest due to its ability to produce an array of plant cell wall hydrolytic enzymes. Nonetheless, only one T. fusca bacteriophage with genome information has been reported to date. This study was aimed at discovering more relevant bacteriophages to expand the existing knowledge of phage diversity for this host species. With this end in view, a thermostable T. fusca bacteriophage P318, which belongs to the Siphoviridae family, was isolated and characterized. P318 has a double-stranded DNA genome of 48,045 base pairs with 3′-extended COS ends, on which 52 putative ORFs are organized into clusters responsible for the order of genome replication, virion morphogenesis, and the regulation of the lytic/lysogenic cycle. In comparison with T. fusca and the previously discovered bacteriophage P1312, P318 has a much lower G+C content in its genome except at the region encompassing ORF42, which produced a protein with unknown function. P1312 and P318 share very few similarities in their genomes except for the regions encompassing ORF42 of P318 and ORF51 of P1312 that are homologous. Thus, acquisition of ORF42 by lateral gene transfer might be an important step in the evolution of P318.

Thermobifida fusca produces a firmly attached biofilm on nutritive and non-nutritive surfaces, such as cellulose, glass, plastic, metal and Teflon®. The ability to bind to surfaces has been suggested as a competitive advantage for microbes in soil environments. Results of previous investigations indicated that a Gram-positive cellulolytic soil bacteria, Cellulomonas uda, a facultative aerobe, specifically adhered to nutritive surfaces forming biofilms, but cells did not colonize non-nutritive surfaces. Cell surface hydrophobicity has been implicated in the interactions between bacteria and the adhesion to surfaces. It was recently described that the cellulolytic actinomycete T. fusca cells hydrophobicity was measured and compared to the cellulolytic soil bacteria C. uda. Also, T. fusca biofilm formation on non-nutritive surface, such as polyvinyl chloride, was examined by testing various culture ingredients to determine a possible trigger mechanism for biofilm formation. Experimental results showed that partitioning of bacterial cells to various hydrocarbons was higher in T. fusca cells than in C. uda. The results of this study suggest that the attachment to multiple surfaces by T. fusca could depend on nutrient availability, pH, salt concentrations, and the higher hydrophobic nature of bacterial cells. Possibly, these characteristics may confer T. fusca a selective advantage to compete and survive among the many environments it thrives.

CelR is a transcriptional regulator that controls the expression of cellulases catalyzing cellulose hydrolysis. However, the structural mechanism of its regulation has remained unclear. Here, we report the first structure of CelR, in this case with cellobiose bound. CelR consists of a DNA‐binding domain (DBD) and a regulatory domain (RD), and homodimerizes with each monomer bound to cellobiose. A hinge region (HR) in CelR connects the DBD with the RD, and Leu59 in the HR acts as a ‘leucine lever’ that transduces a transcriptional activation signal. Furthermore, an α4 helix mediates the ligand‐binding signal for transcriptional activation. Tyr84 and Gln301 can potentially alter the ligand specificity of CelR. This study provides a pivotal step toward understanding transcription of the cellulases. [ABSTRACT FROM AUTHOR]

Locust bean gum (LBG) galactomannan has been claimed to have applications in the biopharmaceutical field. However, the effects of LBG galactomannan on immunomodulatory aspects are not yet clear. The purpose of this study was to over-express thermostable β- d -mannanase from the thermophilic actinomycete Thermobifida fusca BCRC 19214 using a Pichia pastoris expression system. The maximum intracellular β- d -mannanase activity obtained from the cell-free extract was approximately 40.0 U/mL after 72 h of cultivating a P. pastoris transformant (pPICZ- man ) induced with methanol. Hydrolysis of native LBG galactomannan with 8 U/mL β- d -mannanase for 24 h significantly decreased the weight-average molecular weight of LBG galactomannan from 5,580,010 to 3188. Native and hydrolyzed LBG galactomannan in a range of 0–0.2% did not trigger significant cytotoxicity after 24 h of treatment compared with the control. The native LBG galactomannan stimulated RAW 264.7 cells to produce cytokine TNF-α dose-dependently, but there was no significant IL-1β or nitric oxide production. The native LBG galactomannan also stimulated β-hexosaminidase secretion in RBL-2H3 cells. After the native LBG galactomannan was hydrolyzed with β- d -mannanase, all of the immunological properties disappeared. These results suggest the possible immunomodulatory effects of native LBG galactomannan. [ABSTRACT FROM AUTHOR]

Cellulase activity on insoluble cellulose substrates declines as the substrate is modified. The role of structural changes that result in substrate recalcitrance, such as changes to cellulose crystallinity, requires further investigation. Crystallinity of cellulose samples with varying extents of digestion can only be compared meaningfully using a high throughput - Fourier transform infrared spectroscopy (HTS-FTIR) technique when the many variables involved are carefully controlled. Hence, changes to the HTS-FTIR sample preparation methods previously described in literature were necessary in order to obtain clean raw spectra and reliable measures of cellulose crystallinity. The sample preparation methods of residual cellulose after digestion by individual cellulases and a complex cellulase mixture from T. fusca were improved to remove extraneous overlapping signals, provide accurate extent of digestion, and correct errors caused by varying concentrations. These improved preparation methods enabled measurement of crystallinity index values of residual cellulose which did not show a correlation between cellulose crystallinity and the decline in cellulase activity. [ABSTRACT FROM AUTHOR]

Background: Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The point mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity. Results: In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination. Conclusions: The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized. [ABSTRACT FROM AUTHOR]

A codon optimized synthetic alkaline thermostable Thermobifida fusca β-mannanase ManB ( KJ806638 ) was expressed in Pichia pastoris and used in ramie degumming. To improve the expression level, a multi-copy secretion expression vector pAOhr was constructed to introduce the ManB gene into Pichia pastoris GS115. The highest secretion yield was obtained from a transformant strain containing six copies of ManB gene. The size of ManB protein was 34 kDa in SDS-PAGE and the secreted protein was the main protein in the culture broth. The optimal activity region of ManB was at pH 7–9 and the enzyme was quite stable at pH 6–10. At pH 9, the specific activity of ManB was 493.8 IU/mg and the optimum temperature was 70–75 °C. ManB appeared to be inhibited by Tris buffer. Molecular docking showed that Tris molecule can bind to the enzyme active site. ManB exhibited high activity for locust bean gum, whereas it showed in practice no activity for CMC-Na. Ramie degumming was performed with combined treatment by ManB and Bacillus sp. HG-28 expressing pectinase and xylanase. The obtained results demonstrated that the combination treatment with additional mannanase enzyme was more efficient in removing the gums than the treatment merely by the bacterial strain. [ABSTRACT FROM AUTHOR]

Cutinase as a promising biocatalyst has been intensively studied and applied in processes targeted for industrial scale. In this work, the cutinase gene tfu from Thermobifida fusca was artificially synthesized according to codon usage bias of Saccharomyces cerevisiae and investigated in Saccharomyces cerevisiae. Using the α-factor signal peptide, the T. fusca cutinase was successfully overexpressed and secreted with the GAL1 expression system. To increase the cutinase level and overcome some of the drawbacks of induction, four different strong promoters ( ADH1, HXT1, TEF1, and TDH3) were comparatively evaluated for cutinase production. By comparison, promoter TEF1 exhibited an outstanding property and significantly increased the expression level. By fed-batch fermentation with a constant feeding approach, the activity of cutinase was increased to 29.7 U/ml. The result will contribute to apply constitutive promoter TEF1 as a tool for targeted cutinase production in S. cerevisiae cell factory. [ABSTRACT FROM AUTHOR]

A significant problem in the oxidative breakdown of lignin is the tendency of phenolic radical fragments to re-polymerise to form higher molecular weight species. In this paper we identify an extracellular flavin-dependent dehydrolipoamide dehydrogenase from Thermobifida fusca that prevents oxidative dimerization of a dimeric lignin model compound, which could be used as an accessory enzyme for lignin depolymerisation. [ABSTRACT FROM AUTHOR]

The heterologous overexpression level of the bacterial dye decolorizing peroxidase Tfu DyP in Escherichia coli was increased sixty fold to approximately 200 mg of purified enzyme per liter culture broth by fusing the enzyme to the small ubiquitin-related modifier protein (SUMO). The highly overexpressed SUMO- Tfu DyP was, however, almost inactive. Analysis of the enzyme by UV–vis absorption spectroscopy and high-resolution mass spectrometry showed that a large fraction of the highly overexpressed enzyme contained the iron deficient heme precursor protoporphyrin IX (PPIX) instead of heme. Here we show that the activity of the enzyme was dependent on the expression level of the protein. [ABSTRACT FROM AUTHOR]

Dye-decolorizing peroxidases (DyPs) represent a new class of oxidative enzymes for which the natural substrates are largely unknown. To explore the biocatalytic potential of a DyP, we have studied the substrate acceptance profile of a robust DyP peroxidase, a DyP from Thermobifida fusca ( Tfu DyP). While previous work established that this bacterial peroxidase is able to act on a few reactive dyes and aromatic sulfides, this work significantly expands its substrate scope towards lignin related compounds, flavors, and various dyes. [ABSTRACT FROM AUTHOR]

The aim of this research was to evaluate the production of cellulases from locally isolated bacteria, Thermobifida fusca, using thermal and chemical treated oil palm empty fruit bunch (OPEFB) as substrate in liquid-state fermentation (LSF). T. fusca was successfully isolated and was a dominant cellulase producer in OPEFB composting at the thermophilic stage. Analysis of the surface morphology of OPEFB samples using Scanning Electron Microscopy (SEM) showed that the most significant changes after the combination of thermal and chemical pretreatment was the removal of silica bodies, and this observation was supported by X-ray Diffraction analysis (XRD), Fourier Transform Infrared (FTIR), and Thermogravimetric analysis (TG) showing changes on the hemicelluloses, cellulose, and lignin structures throughout the pretreatment process. As a result of the pretreatment, higher cellulase production by T. fusca was obtained. The highest activity for CMCase, FPase, and β-glucosidase using optimally treated OPEFB were 0.24 U/mL, 0.34 U/mL, and 0.04 U/mL, respectively. Therefore, it can be suggested that the combination of chemical and thermal pretreatments enhances the degradation of OPEFB for subsequent use as fermentation substrate, contributing to a higher cellulases yield by T. fusca.

A gene encoding the thermostable acetylxylan esterase (AXE) in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into the Pichia pastoris X-33 host strain using the vector pGAPZαA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular AXE production, as high as 526 U/mL in the Hinton flask culture broth. The purified enzyme showed a single band at about 28 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H; this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60 °C for three hours. The optimal pH and temperature of the purified enzyme were 8.0 and 60 °C, respectively. The properties of the purified AXE from the P. pastoris transformant are similar to those of the AXE from an E. coli transformant.

In recent years, Poly(butylene adipate-co-terephthalate) (PBAT) films were wildly used due to its biodegradable properties. However, there are few reports of strains that can high efficiently degrade PBAT. Thermobifida fusca FXJ-1, a thermophilic actinomycete, was screened and identified from compost. FXJ-1 can efficiently degrade PBAT at 55 °C in MSM medium. The degradation rates of the pure PBAT film (PF), PBAT film used for mulching on agricultural fields (PAF), and PBAT-PLA-ST film (PPSF) were 82.87 ± 1.01%, 87.83 ± 2.00% and 52.53 ± 0.54%, respectively, after nine days of incubation in MSM medium. Cracking areas were monitored uniformly distributed on the surfaces of three kinds of PBAT-based films after treatment with FXJ-1 using scanning electron microscopy. The LC–MS results showed that PBAT might be degraded into adipic acid, terephthalic acid, butylene adipate, butylene terephthalate and butylene adipate-co-terephthalate, and these products are involved in the cleavage of ester bonds. We also found that amylase produced by FXJ-1 played an important role in the degradation of PPSF. FXJ-1 also showed an efficient PBAT-based films degradation ability in simulating compost environment, which implied its potential application in PBAT and starch-based film degradation by industrial composting. [Display omitted] • Thermobifida fusca FXJ-1 can high efficiently degrade PBAT films to 87.83% in 9 days. • PBAT was degraded into five kinds of oligomers and monomers by FXJ-1. • FXJ-1 can also degrade PPSF film by catalyzing starch in it. • FXJ-1 can directly degrade PBAT films in compost environment. [ABSTRACT FROM AUTHOR]

Thermobifida fusca is a moderately thermophilic and cellulolytic actinobacterium. It is of particular interest due to its ability to not only produce a variety of biotechnologically relevant enzymes but also serve as an alternative host for metabolic engineering for the production of valuable chemicals from lignocellulosic agricultural wastes. No bacteriophage that infects T. fusca has been reported, despite its potential impacts on the utilization of T. fusca. In this study, an extremely thermostable bacteriophage P1312 that infects T. fusca was isolated from manure compost. Electron microscopy showed that P1312 has an icosahedral head and a long flexible non-contractile tail, a characteristic of the family Siphoviridae. P1312 has a double-stranded DNA genome of 60,284 bp with 93 potential ORFs. 31 ORFs encode proteins having putative biological functions. The genes involved in phage particle formation cluster together in a region of approximately 16 kb, followed by a segment containing genes presumably for DNA degradation/modification and cell wall disruption. The genes required for DNA replication and transcriptional control are dispersed within the rest of the genome. Phylogenetic analysis of large terminase subunit suggests that P1312 is a headful packaging phage containing a chromosome with circularly permuted direct terminal repeats.

Agricultural lignocelluloses could represent great resources for the production of biofuels and renewable chemicals if the recalcitrant lignin polymer could be efficiently removed without negatively impacting the environment. Recently, a monocopper polyphenol oxidase (Tfu1114) from Thermobifida fusca was found capable of assisting the bacterium’s own xylanase/cellulase to release reducing sugars from sugarcane bagasse. This study demonstrates that molecular oxygen was reduced to hydrogen peroxide by Tfu1114, coupled to the oxidation of 2,6-dimethoxyphenol, alkaline lignin, or sugarcane bagasse. Tfu1114 was able to reduce the total phenolic content of alkaline lignin and shift the molecular weight distribution to species with smaller sizes. Dilignol subunits such as syringaresinol and simulanol were generated in the hydrolysate of bagasse treated with Tfu1114. FTIR analysis suggests that Tfu1114 removed some C C and/or C O bonds adjacent to aromatic rings of lignin. From an applied perspective, Tfu1114 and cellulases acted in concert to release reducing sugars that were as usable as glucose for the cultivation of Escherichia coli , suggesting that toxins such as furfural and furan were absent, or at ignorable concentrations, in the hydrolysate. Tfu1114, therefore, represents a new type of lignin-degrading enzyme with potential to provide an eco-friendly way to modify the lignin in lignocellulose. [ABSTRACT FROM AUTHOR]

Short-chain aliphatic esters are commonly used as fruit flavorings in the food industry. In this study, Thermobifida fusca ( T. fusca ) cutinase was used for the synthesis of aliphatic esters, and the maximum yield of ethyl caproate reached 99.2% at a cutinase concentration of 50 U/ml, 40 °C, and water content of 0.5%, representing the highest ester yield to date. The cutinase-catalyzed esterification displayed strong tolerance for water content (up to 8%) and acid concentration (up to 0.8 M). At substrate concentrations ⩽0.8 M, the ester yield remained above 80%. Moreover, ester yields of more than 98% and 95% were achieved for acids of C3–C8 and alcohols of C1–C6, respectively, indicating extensive chain length selectivity of the cutinase. These results demonstrate the superior ability of T. fusca cutinase to catalyze the synthesis of short-chain esters. This study provides the basis for industrial production of short-chain esters using T. fusca cutinase. [ABSTRACT FROM AUTHOR]

Native konjac glucomannan was used as the substrate for thermophilic actinomycetes, Thermobifida fusca BCRC19214, to produce β-mannanase. The β-mannanase was purified and five internal amino acid sequences were determined by LC-MS/MS. These sequences had high homology with the β-mannanase from T. fusca YX. The tfm gene which encoded the β-mannanase was cloned, sequenced and heterologous expressed in Yarrowia lipolytica P01g expression system. Recombinant heterologous expression resulted in extracellular β-mannanase production at levels as high as 3.16 U/ml in the culture broth within 48 h cultivation. The recombinant β-mannanase from Y. lipolytica transformant had superior thermal property. The optimal temperature of the recombinant β-mannanase from Y. lipolytica transformant (pYLSC1- tfm ) was 80 °C. When native konjac glucomannan was incubated with the recombinant β-mannanase from Y. lipolytica transformant (pYLSC1- tfm ) at 50 °C, there was a fast decrease of viscosity happen during the initial phase of reaction. This viscosity reduction was accompanied by an increase of reducing sugars. The surface of konjac glucomannan film became smooth. After 24 h of treatment, the DPw of native konjac glucomannan decreased from 6,435,139 to 3089. [ABSTRACT FROM AUTHOR]

Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and overexpressed in Escherichia coli with a yield of 400 mg/L. Heat treatment of disrupted cells at 60 °C for 1 h resulted in enzyme preparation of high purity; hence, no chromatography steps are needed for large-scale production. Except for catalyzing the dismutation of hydrogen peroxide, TfuCat was also found to catalyze oxidations of phenolic compounds. The catalase activity was comparable to other described catalases while peroxidase activity was quite remarkable with a k of nearly 1000 s for catechol. Site directed mutagenesis was used to alter the ratio of peroxidase/catalase activity. Resistance to inhibition by classic catalase inhibitors and an apparent melting temperature of 74 °C classifies this enzyme as a robust biocatalyst. As such, it could compete with other commercially available catalases while the relatively high peroxidase activity also offers new biocatalytic possibilities. [ABSTRACT FROM AUTHOR]

The sequence and structure of mRNA plays an important role in solubility and expression of the translated protein. To divulge the role of mRNA secondary structure and its thermodynamics in the expression level of the recombinant endoglucanase in Escherichia coli, 5′-end of the mRNA was thermodynamically optimized. Molecular engineering was done by introducing two silent synonymous mutations at positions +5 (UCU with UCC) and +7 (UUC with UUU) of the 5′-end of mRNA to relieve hybridization with ribosomal binding site. Two variants of glycoside hydrolase family six endoglucanase, wild type ( cel6A.wt) and mutant ( cel6A.mut) from Thermobifida fusca were expressed and characterized in E. coli using T7 promoter-based expression vector; pET22b(+). Enhanced expression level of engineered construct (Cel6A. mut) with ∆G = −2.7 kcal molwas observed. It showed up to ~45 % higher expression as compared to the wild type construct (Cel6A. wt) having ∆G = −7.8 kcal mol and ~25 % expression to the total cell proteins. Heterologous protein was purified by heating the recombinant E. coli BL21 (DE3) CodonPlus at 60 °C. The optimum pH for enzyme activity was six and optimum temperature was 60 °C. Maximum activity was observed 4.5 Umg on CMC. Hydrolytic activity was also observed on insoluble substrates, i.e. RAC (2.8 Umg), alkali treated bagass (1.7 Umg), filter paper (1.2 Umg) and BMCC (0.3 Umg). Metal ions affect endoglucanase activity in different ways. Only Fe exhibited 20.8 % stimulatory effects on enzyme activity. Enzyme activity was profoundly inhibited by Hg2 (91.8 %). [ABSTRACT FROM AUTHOR]

Thermobifida fusca is an aerobic, moderately thermophilic, filamentous soil bacterium. Iron is an essential metal involved in the vital metabolic functions in microorganisms. Thermobifida fusca was found to survive in the iron-deficiency condition and produce siderophores which synthesized to acquire iron. The iron transport was regulated by DtxR, a prototypic member of a superfamily of transition metal ion-activated transcriptional regulators. Tfu 0249 was found to be working as DtxR in T. fusca. The promoter regions of the three gene clusters related to the iron transport bound and regulated by Tfu 0249 were identified by bioinformatics analysis. The gel shift assays confirmed that Tfu 0249 bound the binding sites firmly on the upstream of three gene clusters. By comparing the binding sites of three clusters and gel shift assay, TWAGGTWAGSCTWACCTWA was found to be recognized and bound by Tfu 0249 to control iron transport in T. fusca. [ABSTRACT FROM AUTHOR]

A comparative analysis of the structural and functional aspects along with equilibrium unfolding of two homologous cutinases, Cut1 and Cut2, from Thermobifida fusca was carried out. The CD and fluorescence profile at different pH in the range of 6 to 9 showed no structural variations for both cutinases, indicating their stability to a wide range of pH. Tryptophan quenching studies suggested that all the four Trp residues in the protein are in inaccessible hydrophobic pockets. Further, near-UV CD analysis of tertiary structure revealed a dissimilar distribution of aromatic amino acid on the surface of these two enzymes. Denaturation profiles obtained in aqueous solutions of the guanidine hydrochloride revealed different tolerance levels for unfolding of the two cutinases, with Cut2 showing higher resistivity to unfolding in comparison to Cut1. Both cutinases retained all the structural parameters even in the presence of 8 M urea, indicating the protein to be highly resistant to urea-induced unfolding. Structural study by homology modeling revealed a high resemblance of secondary structure between the two cutinases; however, their tertiary structure, hydrophobicity, and surface electrostatic properties were very different, which contributed to the difference in the structural stability of these two cutinases. [ABSTRACT FROM AUTHOR]

Background Thermobifida fusca is a cellulolytic bacterium with potential to be used as a platform organism for sustainable industrial production of biofuels, pharmaceutical ingredients and other bioprocesses due to its capability of potential to convert plant biomass to value-added chemicals. To best develop T. fusca as a bioprocess organism, it is important to understand its native cellular processes. In the current study, we characterize the metabolic network of T. fusca through reconstruction of a genome-scale metabolic model and proteomics data. The overall goal of this study was to use multiple metabolic models generated by different methods and comparison to experimental data to gain a high-confidence understanding of the T. fusca metabolic network. Results We report the generation of three versions of a metabolic model of Thermobifida fusca sp. XY developed using three different approaches (automated, semi-automated, and proteomicsderived). The model closest to in vivo growth was the proteomics-derived model that consists of 975 reactions involving 1382 metabolites and account for 316 EC numbers (296 genes). The model was optimized for biomass production with the optimal flux of 0.48 doublings per hour when grown on cellobiose with a substrate uptake rate of 0.25mmole/h. In vivo activity of the DXP pathway for terpenoid biosynthesis was also confirmed using real-time PCR. Conclusions iTfu296 provides a platform to understand and explore the metabolic capabilities of the actinomycete T. fusca for the potential use in bioprocess industries for the production of biofuel and pharmaceutical ingredients. By comparing different model reconstruction methods, the use of high-throughput proteomics data as a starting point proved to be the most accurate to in vivo growth. [ABSTRACT FROM AUTHOR]

The process of bioethanol production from biomass comprises pretreatments and enzyme-mediated hydrolysis to convert lignocellulose into fermentable sugars. Because of the recalcitrant character of cellulose, the enzymatic hydrolysis is considered the major challenge in this process to be economically competitive. These technical difficulties highlight the need for the discovery of new enzymes to optimize and lower the cost of current technologies. Microorganisms have developed efficient systems for cellulose degradation. Among cellulolytic microbes, Thermobifida fusca possesses great physiological and cellulolytic characteristics (thermostability, high activity and tolerance to a broad pH range) making it an interesting organism to be studied from an applied perspective. In this review we describe the main enzymes/proteins produced by T.fusca (cellulases, xylanases, mannanase, manosidase, CBM33 and CelR), the effect of substrate on T. fusca proteome, enzyme improvement approaches, synergism between enzymes/proteins and artificial cellulosomes. [ABSTRACT FROM AUTHOR]

Abstract: Background: Understanding the molecular mechanism through which proteins are functional at extreme high and low temperatures is one of the key issues in structural biology. To investigate this phenomenon, we have focused on two instructive truncated hemoglobins from Thermobifida fusca (Tf-trHbO) and Mycobacterium tuberculosis (Mt-trHbO); although the two proteins are structurally nearly identical, only the former is stable at high temperatures. Methods: We used molecular dynamics simulations at different temperatures as well as thermal melting profile measurements of both wild type proteins and two mutants designed to interchange the amino acid residue, either Pro or Gly, at E3 position. Results: The results show that the presence of a Pro at the E3 position is able to increase (by 8°) or decrease (by 4°) the melting temperature of Mt-trHbO and Tf-trHbO, respectively. We observed that the ProE3 alters the structure of the CD loop, making it more flexible. Conclusions: This gain in flexibility allows the protein to concentrate its fluctuations in this single loop and avoid unfolding. The alternate conformations of the CD loop also favor the formation of more salt-bridge interactions, together augmenting the protein's thermostability. General significance: These results indicate a clear structural and dynamical role of a key residue for thermal stability in truncated hemoglobins. [Copyright &y& Elsevier]

Glucose isomerase (GIase) catalyzes the isomerization of d-glucose to d-fructose. The GIase from Thermobifida fusca WSH03-11 was expressed in Escherichia coli BL21(DE3), and the purified enzyme took the form of a tetramer in solution and displayed a p I value of 5.05. The temperature optimum of GIase was 80 °C and its half life was about 2 h at 80 °C or 15 h at 70 °C. The pH optimum of GIase was 10 and the enzyme retained 95 % activity over the pH range of 5-10 after incubating at 4 °C for 24 h. Kinetic studies showed that the K and K values of the enzyme are 197 mM and 1,688 min, respectively. The maximum conversion yield of glucose (45 %, w/v) to fructose of the enzyme was 53 % at pH 7.5 and 70 °C. The present study provides the basis for the industrial application of recombinant T. fusca GIase in the production of high fructose syrup. [ABSTRACT FROM AUTHOR]

ABSTRACT Lignocellulosic biomass is a potential source of sustainable transportation fuels, but efficient enzymatic saccharification of cellulose is a key challenge in its utilization. Cellulases from the glycoside hydrolase (GH) family 48 constitute an important component of bacterial biomass degrading systems and structures of three enzymes from this family have been previously published. We report a new crystal structure of TfCel48A, a reducing end directed exocellulase from Thermobifida fusca, which shows that this enzyme shares important structural features with the other members of the GH48 family. The active site tunnel entrance of the known GH48 exocellulases is enriched in aromatic residues, which are known to interact well with anhydroglucose units of cellulose. We carried out site-directed mutagenesis studies of these aromatic residues (Y97, F195, Y213, and W313) along with two non-aromatic residues (N212 and S311) also located around the tunnel entrance and a W315 residue inside the active site tunnel. Only the aromatic residues located around the tunnel entrance appear to be important for the ability of TfCel48A to access individual cellulose chains on bacterial cellulose (BC), a crystalline substrate. Both Trp residues were found to be important for the processivity of TfCel48A on BC and phosphoric acid swollen cellulose (PASC), but only W313 appears to play a role in the ability of the enzyme to access individual cellulose chains in BC. When acting on BC, reduced processivity was found to correlate with reduced enzyme activity. The reverse, however, is true when PASC is the substrate. Presumably, higher density of available cellulose chain ends and the amorphous nature of PASC explain the increased initial activity of mutants with lower processivity. Biotechnol. Bioeng. 2014;111: 664-673. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]