Thermophilic ß-mannanases from bacteria: production, resources, structural features and bioengineering strategies.

ß-mannanases are pivotal enzymes that cleave the mannan backbone to release short chain mannooligosaccharides, which have tremendous biotechnological applications including food/feed, prebiotics and biofuel production. Due to the high temperature conditions in many industrial applications, thermophilic mannanases seem to have great potential to overcome the thermal impediments. Thus, structural analysis of thermostable ß-mannanases is extremely important, as it could open up new avenues for genetic engineering, and protein engineering of these enzymes with enhanced properties and catalytic efficiencies. Under this scope, the present review provides a state-of-the-art discussion on the thermophilic ß-mannanases from bacterial origin, their production, engineering and structural characterization. It covers broad insights into various molecular biology techniques such as gene mutagenesis, heterologous gene expression, and protein engineering, that are employed to improve the catalytic efficiency and thermostability of bacterial mannanases for potential industrial applications. Further, the bottlenecks associated with mannanase production and process optimization are also discussed. Finally, future research related to bioengineering of mannanases with novel protein expression systems for commercial applications are also elaborated.

Changes in the microbiota during biological treatment of palm oil mill waste: A critical review.

Palm oil mill waste has a complex cellulosic structure, is rich in nutrients, and provides a habitat for diverse microbial communities. Current research focuses on how the microbiota and organic components interact during the degradation of this type of waste. Some recent studies have described the microbial communities present in different biodegradation processes of palm oil mill waste, identifying the dominant bacteria/fungi responsible for breaking down the cellulosic components. However, understanding the degradation process's mechanisms is vital to eliminating the need for further pretreatment of lignocellulosic compounds in the waste mixture and facilitating the commercialization of palm oil mill waste treatment technology. Thus, the present work aims to review microbial community dynamics via three biological treatment systems comprehensively: composting, vermicomposting, and dark fermentation, to understand how inspiration from nature can further enhance existing degradation processes. The information presented could be used as an umbrella to current research on biological treatment processes and specific research on the bioaugmentation of indigenous microbial consortia isolated during the biological degradation of palm oil mill waste.

Construction and characterization of the GFAT gene as a novel selection marker in Aspergillus nidulans.

Glutamine:fructose-6-phosphate aminotransferase (GFAT) catalyzes the formation of glucosamine-6-phosphate, and its gene is one of the genes essential for microbes. Using the GFAT-encoding gene can prevent the use of a drug-resistant gene as a selection marker in a bacterial system. Another unique property of the GFAT selection marker is that no particular compound is prohibited or required for creating a selective stress for a yeast. Filamentous fungi are major producers of industrial enzymes. However, there has been no report on the construction and application of the GFAT gene as a selection marker in filamentous fungi. To develop a new selection marker, the GFAT-encoding gene gfaA was deleted from the genome of the filamentous fungus Aspergillus nidulans, and the gfat gene of the straw mushroom Volvariella volvacea was used as the selection marker to mediate the transformation and overexpression of a thermostable bacterial laccase in A. nidulans. The GFAT-deficient strain A. nidulans ∆gfaA was not able to grow in the culture medium containing 0.5% yeast extract unless about 20 mM glucosamine was used to supplement to the medium. The gfat gene was amplified and inserted into the integration vector pAL5 and autonomous replication vector Prg3-AMA1-NotI for A. nidulans to generate the gfat vectors pALG and pAMAG, respectively. Using these gfat vectors, the laccase gene lcs from a hyperthermophilic bacterium was overexpressed intra- and extracellularly in A. nidulans ∆gfaA. Therefore, recombinant filamentous fungi can be constructed with gfat vectors, which can be maintained stably in host cells with the naturally occurred selective stress of a medium, forage, pulp, animal gut, wastewater, or soil.

Crystal structures of S-adenosylhomocysteine hydrolase from the thermophilic bacterium Thermotoga maritima.

S-adenosylhomocysteine (SAH) hydrolase catalyzes the reversible hydrolysis of SAH into adenosine and homocysteine by using NAD(+) as a cofactor. The enzyme from Thermotoga maritima (tmSAHH) has great potentials in industrial applications because of its hyperthermophilic properties. Here, two crystal structures of tmSAHH in complex with NAD(+) show both open and closed conformations despite the absence of bound substrate. Each subunit of the tetrameric enzyme is composed of three domains, namely the catalytic domain, the NAD(+)-binding domain and the C-terminal domain. The NAD(+) binding mode is clearly observed and a substrate analogue can also be modeled into the active site, where two cysteine residues in mesophilic enzymes are replaced by serine and threonine in tmSAHH. Notably, the C-terminal domain of tmSAHH lacks the second loop region of mesophilic SAHH, which is important in NAD(+) binding, and thus exposes the bound cofactor to the solvent. The difference explains the higher NAD(+) requirement of tmSAHH because of the reduced affinity. Furthermore, the feature of missing loop is consistently observed in thermophilic bacterial and archaeal SAHHs, and may be related to their thermostability.

Binding mode of the oxidized α-anomer of NAD+ to RSP, a Rex-family repressor.

The Rex-family repressors sense redox levels by alternative binding to NADH or NAD(+). RSP is the homologue of Rex in Thermoanaerobacter ethanolicus JW200(T) and regulates ethanol fermentation in this obligate anaerobe. The dimeric repressor binds to DNA by an open conformation. The crystal structure of RSP/α-NAD(+) complex shows a different set of ligand interactions mainly due to the unique configuration of the nicotinamide moiety. The positively charged ring is covered by the Tyr102 side chain and interacts with a sulfate ion adjacent to the N-terminus of helix α8. Consequently, the RSP dimer may be locked in a closed conformation that does not bind to DNA. However, α-NAD(+) does not show a higher affinity to RSP than ß-NAD(+). It has to be improved for possible use as an effector in modulating the repressor.

Distinct structural features of Rex-family repressors to sense redox levels in anaerobes and aerobes.

The Rex-family repressors sense redox levels by alternative binding to NADH or NAD(+). Unlike other Rex proteins that regulate aerobic respiration, RSP controls ethanol fermentation in the obligate anaerobe Thermoanaerobacter ethanolicus JW200(T). It is also found in other anaerobic microorganisms. Here we present the crystal structures of apo-RSP, RSP/NADH and RSP/NAD(+)/DNA, which are the first structures of Rex-family members from an obligate anaerobe. RSP functions as a homodimer. It assumes an open conformation when bound to the operator DNA and a closed conformation when not DNA-bound. The DNA binds to the N-terminal winged-helix domain and the dinucleotide, either reduced or oxidized, binds to the C-terminal Rossmann-fold domain. The two distinct orientations of nicotinamide ring, anti in NADH and syn in NAD(+), give rise to two sets of protein-ligand interactions. Consequently, NADH binding makes RSP into a closed conformation, which does not bind to DNA. Both the conserved residues and the DNA specificity of RSP show a number of variations from those of the aerobic Rex, reflecting different structural bases for redox-sensing by the anaerobic and aerobic Rex-family members.

Identification and refinement of two strong constitutive promoters for gene expression system of Schizosaccharomyces pombe.

Fission yeast Schizosaccharomyces pombe shares various important properties with higher eukaryotes and is now considered a useful host for elevated production of mammalian proteins for medicinal applications. The full-length nmt1 promoter has been widely used as a strong promoter in S. pombe expression system. In the present study, the promoters of the eno101 and gpd3 genes in S. pombe were identified as strong constitutive promoters. For convenient applications in the plasmids of S. pombe, these promoters were refined to 276-bp eno and 273-bp gpd promoters by deleting undesired sequences and examining the expression of reporter genes including lacZ and xynA. Both the refined eno and gpd promoters provided approximately 1.5-fold higher expression of LacZ than nmt1 promoter. Furthermore, gene expression under the control of the eno or gpd promoter was not repressed by the components of YES medium while nmt1 promoter was inhibited by thiamine in yeast extract. Therefore, both eno and gpd promoters offer opportunities for efficient production of recombinant proteins by S. pombe in high cell-density fermentation.

The substrate/product-binding modes of a novel GH120 ß-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-YS485.

Xylan-1,4-ß-xylosidase (ß-xylosidase) hydrolyses xylo-oligomers at their non-reducing ends into individual xylose units. Recently, XylC, a ß-xylosidase from Thermoanaerobacterium saccharolyticum JW/SL-YS485, was found to be structurally different from corresponding glycosyl hydrolases in the CAZy database (http://www.cazy.org/), and was subsequently classified as the first member of a novel family of glycoside hydrolases (GH120). In the present paper, we report three crystal structures of XylC in complex with Tris, xylobiose and xylose at 1.48-2.05 Å (1 Å=0.1 nm) resolution. XylC assembles into a tetramer, and each monomer comprises two distinct domains. The core domain is a right-handed parallel ß-helix (residues 1-75 and 201-638) and the flanking region (residues 76-200) folds into a ß-sandwich domain. The enzyme contains an open carbohydrate-binding cleft, allowing accommodation of longer xylo-oligosaccharides. On the basis of the crystal structures and in agreement with previous kinetic data, we propose that XylC cleaves the glycosidic bond by the retaining mechanism using two acidic residues Asp382 (nucleophile) and Glu405 (general acid/base). In addition to the active site, nine other xylose-binding sites were consistently observed in each of the four monomers, providing a possible reason for the high tolerance of product inhibition.

Genome sequences of Pseudomonas fragi strains A22 and B25.

Pseudomonas fragi A22 is a novel isolate that produces bead-like particles (A22B) in its cell wall. To explore the genetic basis for the formation of A22B, P. fragi A22 and the type strain of the species, P. fragi B25, were subjected to genome sequence analysis. Here, we report the draft genome sequences and automatic annotation of both strains. These data offer a solid base for related studies of P. fragi, including comparative genomics, proteomics, and gene mining.

Crystallization and preliminary X-ray diffraction analysis of a novel GH120 ß-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-YS485.

Xylosidases hydrolyze xylopolymers at the nonreducing end to free xylose units. The ß-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-YS485 was expressed in Escherichia coli and the recombinant protein was purified and crystallized. A BLASTP search with the XylC protein sequence showed that no similar structure had previously been solved. XylC was classified as a member of the new glycoside hydrolase family GH120 according to the CAZy website (http://www.cazy.org/). Crystals belonging to the monoclinic space group P2(1), with unit-cell parameters a = 88.36, b = 202.20, c = 99.87 Å, ß = 99.04°, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.2 Šresolution. Structure determination using MIR and MAD methods is in progress.

Biobleaching of wheat straw pulp with recombinant laccase from the hyperthermophilic Thermus thermophilus.

The recombinant laccase from Thermus thermophilus was applied to the biobleaching of wheat straw pulp. The best bleaching effect was when the pulp was treated with 3 U laccase g(-1) dry pulp at 90°C, pH 4.5, 8% consistency for 1.5 h. Under these conditions, the pulp brightness was increased by 3.3% ISO, and the pulp kappa number was decreased by 5.6 U. Enzymatic treatment improved the bleachability of wheat straw pulp but caused no damage to the pulp fibers. The use of enzyme-treated pulp saved 25% H(2)O(2) consumption in subsequent peroxide bleaching without decreasing the final brightness. Pulp biobleaching in the presence of 5 mM ABTS further increased the pulp brightness by 1.5% ISO. This is the first report on the application of laccase from T. thermophilus in the pulp and paper sector.

Modifying Thermostability and Reusability of Hyperthermophilic Mannanase by Immobilization on Glutaraldehyde Cross-Linked Chitosan Beads.

In the current study, the purified ß-mannanase (Man/Cel5B) from Thermotoga maritima was immobilized on glutaraldehyde cross-linked chitosan beads. The immobilization of Man/Cel5B on chitosan beads was confirmed by Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. After immobilization, the protein loading efficiency and immobilization yield were found to be 73.3% and 71.8%, respectively. The optimum pH for both free and immobilized enzymes was found to be pH 5.5. However, the optimum temperature of immobilized Man/Cel5B increased by 10 °C, from 85 °C (free Man/Cel5B) to 95 °C (Immobilized). The half-life of free and immobilized enzymes was found to be 7 h and 9 h, respectively, at 85 °C owing to the higher thermostability of immobilized Man/Cel5B. The increase in thermostability was also demonstrated by an increase in the energy of deactivation (209 kJmol-1) for immobilized enzyme compared to its native form (92 kJmol-1), at 85 °C. Furthermore, the immobilized Man/Cel5B displayed good operational stability as it retained 54% of its original activity after 15 repeated catalytic reactions concerning its free form.

Characterization of Thermotoga neapolitana Alcohol Dehydrogenases in the Ethanol Fermentation Pathway.

Hyperthermophilic Thermotoga spp. are candidates for cellulosic ethanol fermentation. A bifunctional iron-acetaldehyde/alcohol dehydrogenase (Fe-AAdh) has been revealed to catalyze the acetyl-CoA (Ac-CoA) reduction to form ethanol via an acetaldehyde intermediate in Thermotoga neapolitana (T. neapolitana). In this organism, there are three additional alcohol dehydrogenases, Zn-Adh, Fe-Adh1, and Fe-Adh2, encoded by genes CTN_0257, CTN_1655, and CTN_1756, respectively. This paper reports the properties and functions of these enzymes in the fermentation pathway from Ac-CoA to ethanol. It was determined that Zn-Adh only exhibited activity when oxidizing ethanol to acetaldehyde, and no detectable activity for the reaction from acetaldehyde to ethanol. Fe-Adh1 had specific activities of approximately 0.7 and 0.4 U/mg for the forward and reverse reactions between acetaldehyde and ethanol at a pHopt of 8.5 and Topt of 95 °C. Catalyzing the reduction of acetaldehyde to produce ethanol, Fe-Adh2 exhibited the highest activity of approximately 3 U/mg at a pHopt of 7.0 and Topt of 85 °C, which were close to the optimal growth conditions. These results indicate that Fe-Adh2 and Zn-Adh are the main enzymes that catalyze ethanol formation and consumption in the hyperthermophilic bacterium, respectively.

Transcription factor AtMYB44 regulates induced expression of the ETHYLENE INSENSITIVE2 gene in Arabidopsis responding to a harpin protein.

AtMYB44 is a transcription factor that functions in association with the ethylene-signaling pathway in Arabidopsis thaliana. The pathway depends on ETHYLENE INSENSITIVE2 (EIN2), an essential component of ethylene signaling, to regulate defense responses in the plant following treatment with HrpN(Ea), a harpin protein from a bacterial plant pathogen. Here, we show that AtMYB44 regulates induced expression of the EIN2 gene in HrpN(Ea)-treated Arabidopsis plants. A HrpN(Ea) and ethylene-responsive fragment of the AtMYB44 promoter is sufficient to support coordinate expression of AtMYB44 and EIN2 in specific transgenic Arabidopsis. In the plant, the AtMYB44 protein localizes to nuclei and binds the EIN2 promoter; the HrpN(Ea) treatment promotes AtMYB44 production, binding activity, and transcription of AtMYB44 and EIN2. AtMYB44 overexpression results in increased production of the AtMYB44 protein and the occurrence of AtMYB44-EIN2 interaction under all genetic backgrounds of wild-type Arabidopsis and the etr1-1, ein2-1, ein3-1, and ein5-1 mutants, which have defects in the ethylene receptor ETR1 and the signal regulators EIN2, EIN3, and EIN5. However, AtMYB44 overexpression leads to enhanced EIN2 expression only under backgrounds of wild type, ein3-1, and ein5-1 but not etr1-1 and ein2-1, suggesting that ethylene perception is necessary to the regulation of EIN2 transcription by AtMYB44.

The mechanism for regulating ethanol fermentation by redox levels in Thermoanaerobacter ethanolicus.

Anaerobes can obtain the entire cell's ATP by glycolysis and remove resulting reducing power by fermentation. There is a delicate balance in redox status to obtain a maximal growth of these cells, and the conditions to change redox fluxes can induce kinds of changes in metabolism. The fundamental knowledge on sensing redox status and coupling redox signals with fermentation pathways is essential for the metabolic engineering to control redox fluxes at the molecular level. A redox sensing protein (RSP) was isolated by DNA affinity chromatography, and corresponding gene was mined from genomic sequences of Thermoanaerobacter spp. The RSP shares up to 41% identity with the regulatory proteins which sense NADH and control the expression of NADH dehydrogenase in aerobic microorganisms. The operator sites for RSP were located in all the operons for ethanol fermentation rather than in that of NADH dehydrogenase. The typical operator was identified as a palindromic sequence, -ATTGTTANNNNNNTAACAAT-. NADH caused a transition of RSP from an α-helix rich to ß-sheet rich conformation. In an in vitro transcription system of T. ethanolicus, RSP repressed the transcription of an alcohol dehydrogenase, whereas the repression was reversed by adding NADH. Base substitutes in the repeats of the palindrome reduced the affinity between RSP and the operator, and thus delicate regulation could be achieved. This study reveals for the first time a repressor/operator system that couples a redox signal with a fermentation pathway, and the results presented here provide valuable insights for the design of metabolic engineering.

Characterization of a novel beta-xylosidase, XylC, from Thermoanaerobacterium saccharolyticum JW/SL-YS485.

The 1,914-bp open reading frame of xylC from Thermoanaerobacterium saccharolyticum JW/SL-YS485 encodes a calculated 73-kDa ß-xylosidase, XylC, different from any glycosyl hydrolase in the database and representing a novel glycohydrolase family. Hydrolysis occurred under retention of the anomeric configuration, and transglycosylation occurred in the presence of alcohols as acceptors. With the use of vector pHsh, expression of XylC, the third ß-xylosidase in this bacterium, increased approximately 4-fold when a loop within the translational initiation region in the mRNA was removed by site-directed mutagenesis. The increased expression of xylC(m) is due to removal of a stem-loop structure without a change of the amino acid sequence of the heterologously expressed enzyme (XylC(rec)). When gel filtration was applied, purified XylC had molecular masses of 210 kDa and 265 kDa using native gradient gel electrophoresis. The protein consisted of 78-kDa subunits based on SDS gel electrophoresis and contained 6% carbohydrates. XylC and XylC(rec) exhibited maximum activity at 65°C and pH(65°C) 6.0, a 1-h half-life at 67°C, a K(m) for p-nitrophenyl-ß-D-xyloside of 28 mM, and a V(max) of 276 U/mg and retained 70% activity in the presence of 200 mM xylose, suggesting potential for industrial applications.

Enhanced soluble expression of a thermostable cellulase from Clostridium thermocellum in Escherichia coli.

In this study, the cellulase gene celD from Clostridium thermocellum was cloned into expression vectors pET-20b(+) and pHsh. While high expression can be achieved by means of both these expression systems, only the pHsh expression system gives soluble proteins. By weakening the mRNA secondary structure and replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of CelD was increased from 4.1 ± 0.3 to 6.4 ± 0.4 U ml(-1) in LB medium. Recombinant CelD was purified by heat treatment followed by Ni-NTA affinity. The purified CelD exhibited the highest activity at pH 5.4 and 60°C, and retained more than 50% activity after incubation at 70°C for 1 h. The cellulase activity of CelD was significantly enhanced by Ca(2+) but inhibited by EDTA. The favorable properties of CelD offer the potential for genetic modification of strains for biomass degradation. Presently, one of the major bottlenecks for industrial cellulase users is the high cost of enzyme production. The high level expression of soluble enzymes from the pHsh expression system offers a novel approach for the production of cellulases to be used in various agro-industrial processes such as chemical, food and textile.

Cloning, expression and characterization of glycoside hydrolase family 11 endoxylanase from Bacillus pumilus ARA.

An endoxylanase gene, xynA, was cloned from Bacillus pumilus ARA and expressed in Escherichia coli. The open reading frame of the xynA gene was 687 bp encoding a signal peptide and a mature xylanase with a molecular mass of 23 kDa. The enzyme was categorized as a glycosyl hydrolase family 11 member based on the sequence analysis of the putative catalytic domain. The recombinant XynA (Bpu XynA) was purified to homogeneity by Ni-NTA and ion exchange chromatography on DEAE-Sepharose FF. The enzyme exhibited highest activity at pH 6.6 and 50°C. The purified Bpu XynA was stable for at least 2 h at 45°C, and retained over 50% residual activity after being incubated at 60°C for 1 h. The activity of the xylanase was not significantly affected by metal ions and EDTA. The K ( m ) and K ( cat ) /K ( m ) of Bpu XynA for oat-spelt xylan were 5.53 mg/ml and 10.14 ml/mg s at 50°C and pH 6.6. The main product of hydrolysis by Bpu XynA was xylooligosaccharide. The results revealed that the consumption of grass xylan by B. pumilus ARA depended on the synergistic reactions of Bpu XynA and Bpu arabinosidase, and that a typical GH11 xylanase e.g. Tla XynA had capability to remove the side chain of xylan. The properties Bpu XynA make it promising for application in the production of Bifidobacterium growth-promoting factors and in feed industry.

Cloning and analysis of the xylAB operon and characterization of xylose isomerase from Thermoanaerobacter ethanolicus.

Three genes, xylA-like, xylA and xylB, were cloned and sequenced from the chromosome of Thermoanaerobacter ethanolicus JW200. xylA and xylB share an operon and encode xylose isomerase and xylulokinase, respectively. The xylA-like gene locates upstream of xylAB operon and encodes a hypothetical protein that lacks xylose isomerase activity. The xylose isomerase was expressed in Escherichia coli and purified by heat treatment and an ion-exchange chromatography. The enzyme had highest activity at 85°C and pH 7.0, and a half-life for 1 h at 85°C. The K (m) and V (max) values for xylose were 11 mM and 25 U/mg, respectively. The high level of expression, easy purification, and thermostability of the XylA from T. ethanolicus JW200 suggests industrial usefulness.

Man/Cel5B, a Bifunctional Enzyme Having the Highest Mannanase Activity in the Hyperthermic Environment.

Thermotoga maritima (Tma) contains genes encoding various hyperthermophilic enzymes with great potential for industrial applications. The gene TM1752 in Tma genome has been annotated as cellulase gene encoding protein Cel5B. In this work, the gene TM1752 was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. Interestingly, the purified enzyme exhibited specific activities of 416 and 215 U/mg on substrates galactomannan and carboxy methyl cellulose, which is the highest among thermophilic mannanases. However, the putative enzyme did not show sequence homology with any of the previously reported mannanases; therefore, the enzyme Cel5B was identified as bifunctional mannanase and cellulase and renamed as Man/Cel5B. Man/Cel5B exhibited maximum activity at 85°C and pH 5.5. This enzyme retained more than 50% activity after 5 h of incubation at 85°C, and retained up to 80% activity after incubated for 1 h at pH 5-8. The K m and V max of Man/Cel5B were observed to be 4.5 mg/mL galactomannan and 769 U/mg, respectively. Thin layer chromatography depicted that locust bean gum could be efficiently degraded to mannobiose, mannotriose, and mannooligosaccharides by Man/Cel5B. These characteristics suggest that Man/Cel5B has attractive applications for future food, feed, and biofuel industries.