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[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]

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]

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]

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]

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]

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]

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]

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]

Highlights: [•] The CBM binding sites of cutinase–CBMCenAfusion protein was firstly modified. [•] The mutants of W68L and W68Y exhibited enhanced activity toward PET fiber. [•] The model of binding between the CBM and PET was proposed. [•] The mechanism of enhanced enzymatic activity toward PET upon mutation was explored. [ABSTRACT FROM AUTHOR]

Abstract: Solid state fermentation of lignocellulosic biomass by filamentous microorganisms to induced enzyme production has been recognized as an attractive and cost effective technology. The secretion profile of lignocellulolytic enzymes by thermostable filamentous Thermobifida fusca (T. fusca) in solid state fermentation of different lignocellulosic biomasses, such as corn stover, hay; saw dust; sugarcane bagasse; wood chips; and un-dried green plant were explored using label-free exponentially modified protein abundance index (emPAI) based quantitative proteomics. Comparative analyses of T. fusca secretion profiles between cellulose and the various lignocellulosic biomasses showed induced expression of cellulolytic proteins by cellulose, and expression of hemicellulose, pectin and lignin degrading enzymes were induced by lignocellulosic biomasses. The solid state fermentation by T. fusca on lignocellulosic biomasses also revealed increased expressions of various transport proteins and hypothetical proteins. The Bray–Curtis similarity indices, clustering, and multidimensional scaling plot explicated differential protein expressions by T. fusca on different lignocellulosic biomasses, indicating that protein secretion by T. fusca is reliant on substrate complexity. [Copyright &y& Elsevier]

Abstract: Thermobifida fusca is an aerobic, thermophilic, cellulose degrading bacterium identified in heated organic materials. This study applied iTRAQ quantitative proteomic analysis to the cellular and membrane proteomes of T. fusca grown in presence and absence of cellulose to elucidate the cellular processes induced by cellulose nutrient. Using an iTRAQ-based quantitative proteomic approach, 783 cytosolic and 181 membrane proteins expressed during cellulose hydrolysis were quantified with ≤1% false discovery rate. The comparative iTRAQ quantification revealed considerable induction in the expression levels and up-regulation of specific proteins in cellulosic medium than non-cellulosic medium. The regulated proteins in cellulosic medium were grouped under central carbohydrate metabolism such as glycolysis/gluconeogenesis, pentose phosphate pathways, citric acid cycle, starch, sugars, pyruvate, propanoate and butanoate metabolism; energy metabolism that includes oxidative phosphorylation, nitrogen, methane and sulfur metabolism; fatty acid metabolism, amino acid metabolic pathways, purine and pyrimidine metabolism, and main cellular genetic information processing functions like replication, transcription, translation, and cell wall synthesis; and environmental information processing (membrane transport and signal transduction). The results demonstrated cellulose induced several metabolic pathways during cellulose utilization. [Copyright &y& Elsevier]

Abstract: A gene (axe) encoding the AXE thermostable esterase in Thermobifida fusca NTU22 was cloned into a Yarrowia lipolytica P01g host strain. Recombinant expression resulted in extracellular esterase production at levels as high as 70.94U/ml in Hinton flask culture broth, approximately 140 times higher than observed in a Pichia pastoris expression system. After 72h of fermentation by the Y. lipolytica transformant in the fed-batch fermentor, the fermentation broth accumulated 41.11U/ml esterase activity. Rice bran, wheat bran, bagasse and corncob were used as hydrolysis substrates for the esterase, with corncob giving the best ferulic acid yield. The corncob was incubated with T. fusca xylanase (Tfx) for 12h and then with the AXE esterase for an additional 12h. Ferulic acid accumulated to 396μM in the culture broth, a higher concentration than with esterase alone or with Tfx and esterase together for 24h. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The gram-positive thermophilic actinomycete Thermobifida fusca KW3 secretes a highly hydrophobic carboxylesterase (TfCa) that is able to hydrolyze poly(ethylene terephthalate). TfCa was produced in the Escherichia coli strain BL21(DE3) as a fusion protein consisting of a pelB leader sequence to ensure periplasmic localization of the protein and a His6 tag for use in its purification. To enhance the recombinant enzyme yield, the tfca gene from T. fusca KW3 was successfully optimized for codon usage in E. coli. In addition, the gene expression induction conditions were optimized and the temperature for cell cultivation was lowered to reduce inclusion body formation. The optimized codons and expression conditions yielded 4500-fold higher TfCa activity than the wild-type strain. Using a pH-controlled bioreactor for cultivation, a TfCa protein concentration of 41.6mg/L was achieved. [Copyright &y& Elsevier]

Abstract: The thermostable truncated hemoglobin from the actinomyces Thermobifida fusca (Tf-trHb) displays a robust peroxidase activity, with optimum at acidic pH values, in experiments with the redox mediator ABTS. However, typical peroxidase substrates, such as phenolic or aromatic amine compounds, appear to be poor substrates for Tf-trHb. In turn, the protein is able to catalyze a unique dehydrogenation reaction of dibenzylbutanolides, suggested intermediates in the biosynthesis of podophyllotoxin, in the presence of hydrogen peroxide. Dibenzylbutanolides with a free 4″-hydroxyl group were thus converted into the corresponding 2,7″-dehydroderivatives thus setting up the basis for an efficient biotransformation of this important precursor. In particular, Tf-trHb mediated oxidation of trans-2-(4″-hydroxy-3″,5″-dimethoxybenzyl)-3-(3′,4′-methylenedioxy-7′β-hydroxybenzyl)butanolide 1 into the corresponding benzylidene-benzoyl-γ-butyrolactone 2 was obtained at high yield and with excellent selectivity. [Copyright &y& Elsevier]

Abstract: Changes in organic matter degradation and microbial communities during thermophilic composting were compared using two different types of anaerobic sludge, one from mesophilic methane fermentation, containing a high concentration of proteins (S-sludge), and the other from thermophilic methane fermentation, containing high concentrations of lipids and fibers (K-sludge). The difference in the organic matter degradation rate corresponded to the difference in the organic matter constituents; the CO2 evolution rate was greater in the composting of S-sludge than of K-sludge; moreover, the NH3 evolution resulting from the protein degradation was especially higher in the composting of S-sludge. Then the differences in the microbial communities that contributed to each composting were determined by the PCR-DGGE method. Ureibacillus sp., which is known as a degrader with high organic matter degradation activity, was observed during the composting of S-sludge, whereas Thermobifida fusca, which is a well known thermophilic actinomycete that produces enzymes for lignocellulose degradation, were observed during the composting of K-sludge. [Copyright &y& Elsevier]

Abstract: 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 Zn2+, Hg2+, 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. [Copyright &y& Elsevier]

Abstract: A gene (xyl11) encoding xylanase from Thermobifida fusca NTU22 was cloned, sequenced and expressed in Escherichia coli. The gene consists of 1014 base pairs and encodes a protein of 338 amino acids. The gene was then cloned into and secretively expressed in Pichia pastoris under the control of the AOX1 promoter. The xylanase productivity of the P. pastoris transformant (pPICαXYL) was about 67-fold higher than that of T. fusca NTU22 cultured in a 5-liter fermentor. The purified xylanase showed a single band at about 36kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H. The optimal pH and temperature of the purified xylanase were 7.0 and 70°C, respectively. About 70% of original activity remained after heat treatment at 70°C for 3h. [Copyright &y& Elsevier]

A maltotriose-producing α-amylase from Thermobifida fusca NTU22 was purified 7.2-fold as measured by specific activity from crude culture filtrate by ammonium sulfate fractionation, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified enzyme was 22%. The purified enzyme gave an apparent single protein band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B to be 64 and 60 kDa, respectively, indicated that the maltotriose-producing α-amylase from T. fusca NTU22 is a monomer. The optimum pH and temperature for the purified enzyme were 7.0 and 60 °C, respectively. About 70% of the original activity still remained after treatment at 60 °C for 3 h. The enzyme activity was completely inhibited by 1 mM Hg2+. The Km value for soluble starch was 0.88 mg/ml. The purified enzyme exhibited a high level of activity with raw sago starch. Maltotriose was formed as the major enzymatic product from both soluble starch and raw sago starch. [Copyright &y& Elsevier]

Ten cysteine mutations in the Thermobifida fusca cellulase Cel6A and Cel6B catalytic domains were created at different positions by site-directed mutagenesis for the purpose of testing their effects on thermostability and catalytic activity. The level of in vivo expression of these mutant proteins was significantly reduced by many of these mutations. There were positional effects of the mutations, which are probably due to the formation of incorrect disulfide bonds. These results increase our understanding of the role of cysteine residues in protein folding, and also help guide the choice of sites for other mutations in these enzymes. [Copyright &y& Elsevier]

The binding of the catalytic domain of Thermobifida fusca endoglucanase (EC 3.2.1.4) CDCel5A and exoglucanases (EC 3.2.1.91) CDCel6B and CDCel48A to BMCC was studied. At 5 °C, the binding of these CDs to BMCC is rapid with maximum binding occurring just after CD loading. An observed desorption of the bound CDs with time was attributed to the loss of binding sites due to hydrolysis of the easily hydrolysable BMCC. This conclusion was supported by prehydrolysis experiments where the easily hydrolysable BMCC fraction was removed, and binding to the recalcitrant fraction was observed. More of the CDs were bound to the recalcitrant fraction. This was especially true for the exoglucanases, CDCel6B and CDCel48A, where 81–76% of the total CD binding was to the recalcitrant BMCC fraction. The binding to the easily hydrolysable fraction for the endo CDCel5A was 1.5–4 times higher than that of the exo CDCel6B and CDCel48A at the same enzyme concentration. Although CDCel5A saturated the easily hydrolysable fraction of BMCC first, the ratio of the bound to the recalcitrant substrate was constant for CDCel6B and CDCel48A as 81 and 76%, respectively. As the reaction temperature was increased to 50 °C, CDCel5A and CDCel48A exhibited a rapid increase in the rate and extent of binding, while CDCel6B exhibited a decrease in the extent of binding. [Copyright &y& Elsevier]

A versatile system for expression and purification of cellulase Cel6B from Thermobifida fusca in Escherichia coli was developed. A double mutation, N233C-D506C, of Cel6B was created to try to increase its thermostability by forming an extra-disulfide bond joining two loops of Cel6B that cover the active site cleft. The predicted mutant enzyme was slightly more thermostable with no CD spectroscopic changes, but a disulfide bond did form and there was a significant decrease in enzyme production. [Copyright &y& Elsevier]

Abstract: cis-Prenyltransferase catalyzes the synthesis of Z,E-mixed prenyl diphosphates by a condensation of isopentenyl diphosphate to an allylic diphosphate. A novel gene encoding a cis-prenyltransferase is cloned from Thermobifida fusca. It showed a unique substrate specificity accepting dimethylallyl diphosphate as a shortest allylic substrate, and synthesizes polyprenyl products up to C70. [Copyright &y& Elsevier]