Teichoic Acids in the Cell Walls of Microbispora mesophila Ac-1953T and Thermobifida fusca Ac-1952T

The cell walls of Microbispora mesophila strain Ac-1953^T (the family Streptosporangiaceae) and Thermobifida fusca Ac-1952^T (the family Nocardiopsaceae) were found to contain teichoic acids of a poly(glycerol phosphate) nature. The teichoic acid of M. mesophila (formerly Thermomonospora mesophila) represents a 1,3-poly(glycerol phosphate) containing 5% of substituent 2-acetamido-2-deoxy--D-galactosaminyl residues. Teichoic acid of such a kind was found in actinomycetes for the first time. The cell wall of T. fusca (formerly Thermonospora fusca) contains two teichoic acids, namely, unsubstituted 1,3-poly(glycerol phosphate) and -glucosylated 1,3-poly(glycerol phosphate).     

Teichoic acids from the cell wall of Nocardiopsis prasina BKM Ac-1880T

The cell wall of Nocardiopsis prasina VKM Ac-1880T was found to contain two structurally different teichoic acids: unsubstituted 3,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate), substituted at position 2 by 10% with alpha-N-acetylglucosamine and by 5% with O-acetyl groups. The structure of the polymers was studied by chemical analysis and NMR spectroscopy. The results obtained correlate well with 16S rRNA sequence data and confirm the species-specificity of teichoic acids in the genus Nocardiopsis.     

Biochemical characterization of the cutinases from Thermobifida fusca

Thermobifida fusca produces two cutinases which share 93% identity in amino acid sequence. In the present study, we investigated the detailed biochemical properties of T. fusca cutinases for the first time. For a better comparison between bacterial and fungal cutinases, recombinant Fusarium solani pisi cutinase was subjected to the similar analysis. The results showed that both bacterial and fungal cutinases are monomeric proteins in solution. The bacterial cutinases exhibited a broad substrate specificity against plant cutin, synthetic polyesters, insoluble triglycerides, and soluble esters. In addition, the two isoenzymes of T. fusca and the F. solani pisi cutinase are similar in substrate kinetics, the lack of interfacial activation, and metal ion requirements. However, the T. fusca cutinases showed higher stability in the presence of surfactants and organic solvents. Considering the versatile hydrolytic activity, good tolerance to surfactants, superior stability in organic solvents, and thermostability demonstrated by T. fusca cutinases, they may have promising applications in related industries.     

Purification and characterization of Thermobifida fusca xylanase 10B

Thermobifida fusca grows well on cellulose and xylan, and produces a number of cellulases and xylanases. The gene encoding a previously unstudied endoxylanase, xyl10B, was overexpressed in E. coli, and the protein was purified and characterized. Mature Xyl10B is a 43-kDa glycohydrolase with a short basic domain at the C-terminus. It has moderate thermostability, maintaining 50% of its activity after incubation for 16 h at 62 degrees C, and is most active between pH 5 and 8. Xyl10B is produced by growth of T. fusca on xylan or Solka Floc but not on pure cellulose. Mass spectroscopic analysis showed that Xyl10B produces xylobiose as the major product from birchwood and oat spelts xylan and that its hydrolysis products differ from those of T. fusca Xyl11A. Xyl10B hydrolyzes various p-nitrophenyl-sugars, including p-nitrophenyl alpha-D-arabinofuranoside, p-nitrophenyl-beta-D-xylobioside, p-nitrophenyl-beta-D-xyloside, and p-nitrophenyl-beta-D-cellobioside. Xyl11A has higher activity on xylan substrates, but Xyl10B produced more reducing sugars from corn fiber than did Xyl11A.     

产几丁质酶褐色喜热裂孢菌的分离鉴定及产酶特性初步研究

采用选择性培养基从土壤中分离到1株产几丁质酶的微生物菌株YX,经形态和分子鉴定为褐色喜热裂孢菌(Thermobifida fusca)。进一步在摇瓶中比较了T.fusca YX在纤维二糖、几丁质、或羧甲基纤维素钠为碳源的培养基中的产酶特性,YX菌株在5 L发酵罐中以几丁质为碳源的培养基发酵到22 h左右时发酵液几丁质酶活即可达到1.7 U/m L。本文首次报道褐色喜热裂孢菌能够产生几丁质酶,具有潜在的应用价值。     

Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP⁺, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs.     

Purification and properties of an acetylxylan esterase from Thermobifida fusca

An acetylxylan esterase from Thermobifida fusca NTU22 was purified 51-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified enzyme was 14.4%. The purified enzyme gave an apparent single protein band on an SDS-PAGE. The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B was found to be 30 and 28kDa, respectively, indicating that the acetylxylan esterase from T. fusca NTU22 is a monomer. The pI value of the purified enzyme was estimated to be 6.55 by isoelectric focusing gel electrophoresis. The N-terminal amino acid sequence of the purified esterase was ANPYERGP. The optimum pH and temperature for the purified enzyme were 8.0 and 80°C, respectively. The Zn(2+), Hg(2+), PMSF and DIPF inhibited the enzyme activity. The K(m) value for p-nitrophenyl acetate and acetylxylan were 1.86μM and 0.15%, respectively. Co-operative enzymatic degradation of oat-spelt xylan by purified acetylxylan esterase and xylanase significantly increased the acetic acid liberation compared to the acetylxylan esterase action alone.     

Laboratory evolution and multi-platform genome re-sequencing of the cellulolytic actinobacterium Thermobifida fusca

Biological utilization of cellulose is a complex process involving the coordinated expression of different cellulases, often in a synergistic manner. One possible means of inducing an organism-level change in cellulase activity is to use laboratory adaptive evolution. In this study, evolved strains of the cellulolytic actinobacterium, Thermobifida fusca, were generated for two different scenarios: continuous exposure to cellobiose (strain muC) or alternating exposure to cellobiose and glucose (strain muS). These environmental conditions produced a phenotype specialized for growth on cellobiose (muC) and an adaptable, generalist phenotype (muS). Characterization of cellular phenotypes and whole genome re-sequencing were conducted for both the muC and muS strains. Phenotypically, the muC strain showed decreased cell yield over the course of evolution concurrent with decreased cellulase activity, increased intracellular ATP concentrations, and higher end-product secretions. The muS strain increased its cell yield for growth on glucose and exhibited a more generalist phenotype with higher cellulase activity and growth capabilities on different substrates. Whole genome re-sequencing identified 48 errors in the reference genome and 18 and 14 point mutations in the muC and muS strains, respectively. Among these mutations, the site mutation of Tfu_1867 was found to contribute the specialist phenotype and the site mutation of Tfu_0423 was found to contribute the generalist phenotype. By conducting and characterizing evolution experiments on Thermobifida fusca, we were able to show that evolutionary changes balance ATP energetic considerations with cellulase activity. Increased cellulase activity is achieved in stress environments (switching carbon sources), otherwise cellulase activity is minimized to conserve ATP.     

A teichoic acid of Nocardioides albus VKM Ac-805T cell walls

A teichoic acid of Nocardioides albus VKM Ac-805T cell walls, a typical species of the genus Nocardioides, contains a poly(glycosylglycerol phosphate). The repeating unit of the polymer has the structure: [figure]. These units are in phosphodiester linkage at C-3 of glycerol and C-3 of beta-D-galactopyranose. beta-D-Galactopyranosyl residues are substituted at C-4 by beta-D-glucopyranose carrying a 4,6-pyruvate ketal group in S-configuration. The presence of pyruvic acid in the majority of repeating units increases the anionic properties of the polymer in comparison with most other common teichoic acids. This is the first report of the occurrence of a beta-D-galactofuranosyl residue in teichoic acids; it probably acts as a terminator of an extending chain of the polymer. The ratio of beta-D-galactopyranosyl to beta-D-galactofuranosyl units is 7:1. The polymer structure was determined by NMR spectroscopy. This type of teichoic acid structure has not been reported previously.     

Heterologous expression and biochemical characterization of glucose isomerase from Thermobifida fusca

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 pI 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 _m and K _cat values of the enzyme are 197 mM and 1,688 min^?1, 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.     

Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli

The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His₆ tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L⁻¹ could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process.     

Mutational Bias and Gene Expression Level Shape Codon Usage in Thermobifida fusca YX

In this study major factors shaping codon and amino acid usage variation in Thermobifida fusca YX are reported. It is a major degrader of plant cell walls. It produces spores that can be allergenic and has been associated with a condition called farmers lung. For comparison, two other closely related Actinobacteria, S. coelicolor and N. farcinica were considered. Correspondence analysis on RSCU (Relative Synonymous Codon Usage) showed significant correlation between the major trend of codon usage variation and gene expression level assessed by the "Codon Adaptation Index" (CAI) values. The result was further confirmed from distribution of genes along the first axis. In addition, N_{c} (effective number of codons) plot, SCUO (synonymous codon usage order) plot and correlation analyses showed that base composition and mutational bias have a dominant role in codon usage variation. Furthermore, gene expression level, hydrophobicity and aromaticity have played a significant role in the source of variations for amino acid usage. In addition, codon preference for genes at higher expression level was found to be similar among three different genera. Notably, 14 codons optimally used by Thermobifida fusca YX and its comparative study with S. coelicolor and N. farcinica might provide some useful information for their further study of molecular evolution and genetic engineering.     

Interaction between the CBM of Cel9A from Thermobifida fusca and cellulose fibers

Molecular docking and molecular dynamics (MD) simulations were used to investigate the binding of a cellodextrin chain in a crystal-like conformation to the carbohydrate-binding module (CBM) of Cel9A from Thermobifida fusca. The fiber was found to bind to the CBM in a single and well-defined configuration in-line with the catalytic cleft, supporting the hypothesis that this CBM plays a role in the catalysis by feeding the catalytic domain (CD) with a polysaccharide chain. The results also expand the current known list of residues involved in the binding. The polysaccharide-protein attachment is shown to be mediated by five amine/amide-containing residues. E478 and E559 were found not to interact directly with the sugar chain; instead they seem to be responsible to stabilize the binding motif via hydrogen bonds.     

Characterization of Thermobifida fusca Cutinase-Carbohydrate-Binding Module Fusion Proteins and Their Potential Application in Bioscouring

Cutinase from Thermobifida fusca is thermally stable and has potential application in the bioscouring of cotton in the textile industry. In the present study, the carbohydrate-binding modules (CBMs) from T. fusca cellulase Cel6A (CBM_Cel6A) and Cellulomonas fimi cellulase CenA (CBM_CenA) were fused, separately, to the carboxyl terminus of T. fusca cutinase. Both fusion enzymes, cutinase-CBM_Cel6A and cutinase-CBM_CenA, were expressed in Escherichia coli and purified to homogeneity. Enzyme characterization showed that both displayed similar catalytic properties and pH stabilities in response to T. fusca cutinase. In addition, both fusion proteins displayed an activity half-life of 53 h at their optimal temperature of 50°C. Compared to T. fusca cutinase, in the absence of pectinase, the binding activity on cotton fiber was enhanced by 2% for cutinase-CBM_Cel6A and by 28% for cutinase-CBM_CenA, whereas in the presence of pectinase, the binding activity was enhanced by 40% for the former and 45% for the latter. Notably, a dramatic increase of up to 3-fold was observed in the amount of released fatty acids from cotton fiber by both cutinase-CBM fusion proteins when acting in concert with pectinase. This is the first report of improving the scouring efficiency of cutinase by fusing it with CBM. The improvement in activity and the strong synergistic effect between the fusion proteins and pectinase suggest that they may have better applications in textile bioscouring than the native cutinase.Cotton fiber has a multilayered structure, with its outermost surface being the cuticle that is cross-linked to the primary cell wall of cotton fiber by esterified pectin substances. The major component of the cuticle is cutin, an insoluble polyester composed mainly of saturated C₁₆ and C₁₈ hydroxy and epoxy fatty acids (14, 16, 27, 38). During the process of scouring in the textile industry, the cuticle layer has to be removed in order to improve the wettability of cotton fiber, which then facilitates uniform dyeing and finishing. Traditionally, this process is performed by hot hydrolysis in alkaline medium, which not only consumes large quantities of water and energy but also causes severe pollution and fiber damage (20, 21, 33). Therefore, environment-friendly scouring methods based on biocatalysts have been actively sought (2, 30, 36).Cutinase is a multifunctional esterase capable of degrading the cutin component of the cuticle. Earlier reports showed that the fungal cutinase from Fusarium solani pisi has potential use for cotton cuticle degradation and exhibits a good synergistic effect with pectinase, an enzyme utilized to degrade pectin, in the scouring of cotton fiber (1, 7, 8, 14). Moreover, site-directed mutagenesis has been performed to replace the specific amino acid residues near the active site of cutinase (3) to improve its hydrolytic activity toward polyesters. More recently, a cutinase from the thermophilic bacterium Thermobifida fusca has been identified and overexpressed in Escherichia coli in our laboratory (10). The good thermal stability and alkali resistance of this recombinant T. fusca cutinase make it potentially more amenable to textile bioscouring (10).To further improve the applicability and/or catalytic efficiency of T. fusca cutinase, the present study attempts to engineer a novel cutin-degrading enzyme, based on analysis of the surface structure of cotton fiber. It has been observed that, in addition to cutin, pectin, proteins and other components, there is also a large amount of cellulose on the surface layer of cotton fiber (23). Thus, it is tempting to hypothesize that if the enzyme can be engineered to specifically bind to cellulose through a “gain of function” modification, its concentration on the surface of cotton fiber could increase significantly. Subsequently, its catalytic efficiency for cutin breakdown could be improved due to a proximity effect. In order to design such an enzyme, a fusion protein strategy in which a cellulose-binding protein/module will be attached to cutinase is considered.It is well known that cellulase is capable of binding specifically to cellulose (25, 31). This enzyme has two separate modules: a catalytic module and a carbohydrate-binding module (CBM) (11). The two modules are discrete structural and functional units usually connected by a flexible linker (5, 17, 28). CBM has high specific capacities for cellulose binding. Previously, it has been reported that CBM is able to be fused to a chosen target protein by genetic manipulation (36), resulting in enhanced binding of this fusion protein to cellulose (6, 29). For example, fusion proteins were constructed by fusing CBM to β-glucose nucleotide enzyme (GUS) (13) or β-glycosidase (BglA) (19), which facilitates biochemical analysis of scouring efficiency for cotton fabrics.In the present study, the CBM from T. fusca cellulase Cel6A (CBM_Cel6A) and the CBM from Cellulomonas fimi cellulase CenA (CBM_CenA) were fused, separately, to the carboxyl terminus of T. fusca cutinase. The resulting fusion enzymes were compared to the native cutinase in terms of their biochemical properties, as well as the catalytic efficiency in cutin breakdown on cotton fiber. This is the first report of improving the scouring efficiency of cutinase by fusing it with CBM.     

Processivity,Synergism, and Substrate Specificity of Thermobifida fusca Cel6B

A relationship between processivity and synergism has not been reported for cellulases, although both characteristics are very important for hydrolysis of insoluble substrates. Mutation of two residues located in the active site tunnel of Thermobifida fusca exocellulase Cel6B increased processivity on filter paper. Surprisingly, mixtures of the Cel6B mutant enzymes and T. fusca endocellulase Cel5A did not show increased synergism or processivity, and the mutant enzyme which had the highest processivity gave the poorest synergism. This study suggests that improving exocellulase processivity might be not an effective strategy for producing improved cellulase mixtures for biomass conversion. The inverse relationship between the activities of many of the mutant enzymes with bacterial microcrystalline cellulose and their activities with carboxymethyl cellulose indicated that there are differences in the mechanisms of hydrolysis for these substrates, supporting the possibility of engineering Cel6B to target selected substrates.Cellulose is a linear homopolymer of β-1,4-linked anhydrous glucosyl residues with a degree of polymerization (DP) of up to 15,000 (5). Adjacent glucose residues in cellulose are oriented at an angle of 180° to each other, making cellobiose the basic unit of cellulose structure (5). The β-1,4-glycosidic bonds of cellulose are enzymatically hydrolyzed by three classes of cellulases. Endocellulases (EC 3.2.1.4) cleave cellulose chains internally, generating products of variable length with new chain ends, while exocellulases, also called cellobiohydrolases (EC 3.2.1.91), act from one end of a cellulose chain and processively cleave off cellobiose as the main product. The third class is the processive endocellulases, which can be produced by bacteria (2, 20).Processivity and synergism are important properties of cellulases, particularly for hydrolysis of crystalline substrates. Processivity indicates how far a cellulase molecule proceeds and hydrolyzes a substrate chain before there is dissociation. Processivity can be measured indirectly by determining the ratio of soluble products to insoluble products in filter paper assays (14, 19, 39). Although this approach might not discriminate exocellulases from highly processive endocellulases (12), it is very helpful for comparing mutants of the same enzyme (19). The processivity of some glycoside hydrolases also can be determined from the ratio of dimers to monomers in the hydrolysate (13).Four types of synergism have been demonstrated in cellulase systems: synergism between endocellulases and exocellulases, synergism between reducing- and nonreducing-end-directed exocellulases, synergism between processive endocellulases and endo- or exocellulases, and synergism between β-glucosidases and other cellulases (3). Synergism is dependent on a number of factors, including the physicochemical properties of the substrate and the ratio of the individual enzymes (10).Great effort has been focused on improving enzymatic hydrolysis of cellulases in biomass (24). However, studying biomass is difficult due to its complexity; instead, nearly pure cellulose, amorphous cellulose, or carboxymethyl cellulose (CMC) are commonly used as substrates (22).Random mutagenesis approaches and rational protein design have been used to study cellulose hydrolysis (18), to improve the activity of catalytic domains and carbohydrate-binding modules (19), and to thermostabilize cellulases (9). Increased knowledge of cellulase structures and improvements in modeling software (1) have facilitated rational protein design. The structures of five glycoside hydrolase family 6 cellulases from four microorganisms, Trichoderma reesei (23), Thermobifida fusca (26), Humicola insolens (6, 29), and Mycobacterium tuberculosis (30), have been determined. Structural analysis showed that the active sites of the exocellulases are enclosed by two long loops forming a tunnel, while the endocellulases have an open active site groove. Movement of one of these loops is important for enzymatic activity (6, 35, 37).In nature, as well as for industrial applications, mixtures of cellulase are required; therefore, a better strategy for designing individual enzymes to improve the activity of mixtures is critical. In this study, we used Cel6B, a nonreducing-end-directed, inverting exocellulase from Thermobifida fusca, a thermophilic soil bacterium, as a model cellulase to investigate the impact of improved exocellulases in mixtures with endocellulases since T. fusca Cel6B is important for achieving the maximum activity of synergistic mixtures (35). Cel6B activity is similar to that of the fungal T. reesei exocellulase Cel6A, but Cel6B has higher thermostability and a much broader pH optimum (36). Six noncatalytic residues in the active site tunnel of T. fusca exocellulase Cel6B were mutated to obtain insight into the role of these residues in processivity and substrate specificity. Two mutant enzymes that showed higher activity with filter paper and processivity were investigated further for production of oligosaccharides and synergism to analyze the relationship between processivity and synergism.