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.

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.

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.

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.

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.

A novel bifunctional aldehyde/alcohol dehydrogenase catalyzing reduction of acetyl-CoA to ethanol at temperatures up to 95 °C.

Hyperthermophilic Thermotoga spp. are excellent candidates for the biosynthesis of cellulosic ethanol producing strains because they can grow optimally at 80 °C with ability to degrade and utilize cellulosic biomass. In T. neapolitana (Tne), a putative iron-containing alcohol dehydrogenase was, for the first time, revealed to be a bifunctional aldehyde/alcohol dehydrogenase (Fe-AAdh) that catalyzed both reactions from acetyl-coenzyme A (ac-CoA) to acetaldehyde (ac-ald), and from ac-ald to ethanol, while the putative aldehyde dehydrogenase (Aldh) exhibited only CoA-independent activity that oxidizes ac-ald to acetic acid. The biochemical properties of Fe-AAdh were characterized, and bioinformatics were analyzed. Fe-AAdh exhibited the highest activities for the reductions of ac-CoA and acetaldehyde at 80-85 °C, pH 7.54, and had a 1-h half-life at about 92 °C. The Fe-AAdh gene is highly conserved in Thermotoga spp., Pyrococcus furiosus and Thermococcus kodakarensis, indicating the existence of a fermentation pathway from ac-CoA to ethanol via acetaldehyde as the intermediate in hyperthermophiles.

Efficient xylan-to-sugar biotransformation using an engineered xylanase in hyperthermic environment.

Hyperthermophilic xylanases play a critical role in bioconversion from xylan to sugar in the process of biomass utilization. The discovery of new or improvement of existing xylanases based on directed evolution is expected to be an effective approach to meet the increasing demand of thermostable xylanases. In this work, a xylanase B gene (CTN_0623) from Thermotoga neapolitana (Tne) was cloned and xylanase B (herein named TnexlnB) was solubly expressed in E. coli with a high amount using a heat shock vector pHsh. TnexlnB showed the highest endo-ß-1,4-xylan hydrolase activity at 75 °C and pH 6.0. Additionally, 1 mM Mg2+, Ba2+ and Ca2+ improved the activity of TnexlnB by 31%, 37%, and 53%, respectively. The optimal temperature reached 85 °C by site-directed mutation at the last three helices of TnexlnB. Km and Vmax towards birchwood xylan were determined for both wide type and the best mutant, as follow: 1.09 mg/mL, 191.76 U/mg and 0.29 mg/mL, 376.42 U/mg, respectively. Further characterization highlighted good thermal stability (>80% of enzymatic activity after 1 h at 90 °C) for the best mutant, which made this enzyme suitable for biomass degradation at high temperature.

Current status and future perspectives of solid waste management in Iran: a critical overview of Iranian metropolitan cities.

By growing urban population, Iran faces numerous environmental issues and solid waste management is on the top of these problems. Studies showed that a daily average of 700-1000 g of wastes are produced per person in Iran, in which organic waste accounts for a significant amount. On the other hand, hospital waste represents a part of the wastes, which need careful consideration from the environmental point of view. In the present study, the amount, composition, and management of urban and hospital wastes were evaluated in 7 Iranian metropolises, which account for about 30% of the population and produce about 35% of the country wastes. Based on prior surveys, landfill method is the current main method for waste management in these cities, which is generally not completely sanitary and therefore causes many environmental problems. The other common methods for waste management in these cities are composting of organic wastes, and the use of waste conversion methods to energy. However, the latter is ongoing only in Tehran which also includes some limitations. Therefore, the study also evaluated the future perspectives and feasibility of waste-to-energy conversion as a promising economic route for waste disposal.

Bio-Optimization of Chemical Parameters and Earthworm Biomass for Efficient Vermicomposting of Different Palm Oil Mill Waste Mixtures.

The present study reports mathematical modelling of palm oil mill effluent and palm-pressed fiber mixtures (0% to 100%) during vermicomposting process. The effects of different mixtures with respect to pH, C:N ratio and earthworms have been optimized using the modelling parameters. The results of analysis of variance have established effect of different mixtures of palm oil mill effluent plus palm press fiber and time, under selected physicochemical responses (pH, C:N ratio and earthworm numbers). Among all mixtures, 60% mixture was achieved optimal growth at pH 7.1 using 16.29 C:N ratio in 15 days of vermicomposting. The relationship between responses, time and different palm oil mill waste mixtures have been summarized in terms of regression models. The obtained results of mathematical modeling suggest that these findings have potential to serve a platform for further studies in terms of kinetic behavior and degradation of the biowastes via vermicomposting.

Current Scenario of the Tehran Municipal Solid Waste Handling Rules towards Green Technology.

This study aims to study the waste management process and recycling of municipal waste in Tehran. Currently, Kahrizak is the defined landfill area which collects the waste generated from 22 districts of Tehran. The organic wastes undergo to the windrow composting method in order to manage the partial of the waste generated in the city. Samples from the compost pile generated in Kahrizak were examined to evaluate its fertilizer value to be used further by the farmers. The results show that the obtained compost does not reach the acceptable quality to be used further in agriculture, due to lack of homogeneity, aeration and presence of heavy metals. Overall, it has been concluded that, due to the improper waste segregation and management prior to sending to landfill and presence of non-organic materials such as hazardous metals and medical wastes, causes difficulties in proper waste management, implementation and producing high quality compost. Based on this study the existence of stakeholders, society, economy and proper handling rules can effectively improve the waste management system in the country and leads to the sustainable green environment.

Food-grade gene transformation system constructed in Lactobacillus plantarum using a GlmS-encoding selection marker.

Food-grade gene expression systems in lactic acid bacteria enable production of functional proteins or product testing without antibiotic requirement. Here, we expanded the available selection markers by developing a novel food-grade genetic transformation system for Lactobacillus plantarum WCFS1 using the glucosamine-6-phosphate synthase gene (glmS1). A glmS-vector pSIPH497 was constructed by replacing the erythromycin resistance gene (erm) with L. plantarum glmS1 under control of the PldhL promoter from WCFS1. The selection efficiency and stability of the glmS-vector were shown to be comparable to those of the erm-based plasmid. Moreover, using mCherry expression as a reporter gene, we showed the feasibility of the system for producing foreign proteins. This food-grade host/vector system will provide an effective and safe technique for the application of lactic acid bacteria in the food and medical industries. Furthermore, this study provides a useful strategy for developing food-grade selection markers in other host/vector systems.

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.

Enhancement of Spirulina biomass production and cadmium biosorption using combined static magnetic field.

The effect of static magnetic field (SMF) on Spirulina platensis growth and its influence on cadmium ions (Cd2+) removal efficiency were studied. Application of 6 h day-1 SMF resulted in the highest significant biomass productivity of 0.198 g L-1 day-1. However, 10 and 15 mg L-1 of Cd2+ resulted in significant reduction in biomass productivity by 8.8 and 12.5%, respectively, below the control. Combined SMF showed 30.1% significant increase in biomass productivity over the control. On the other hand, increase of initial Cd2+ concentration resulted in significant reduction of Cd2+ removal efficiency, representing 79.7% and 61.5% at 10 and 15 mg L-1, respectively, after 16 days. Interestingly, application of SMF for 6 h day-1 enhanced Cd2+ removal efficiency counted by 91.4% and 82.3% after 20 days for cultures with initial Cd2+ concentration of 10 and 15 mg L-1, representing increase by 6.3 and 25.3%, respectively, over the SMF-untreated cultures.

Development and Use of a Novel Random Mutagenesis Method: In Situ Error-Prone PCR (is-epPCR).

Directed evolution methods are increasingly needed to improve gene and protein properties. Error-prone PCR is the most efficient method to introduce random mutations by reducing the fidelity of the DNA polymerase. However, a highly efficient process is required for constructing and screening a diverse mutagenesis library since a large pool of transformants is needed to generate a desired mutant. We developed a method called in situ error-prone PCR (is-epPCR) to improve the efficiency of constructing a mutation library for directed evolution. This method offers the following advantages: (1) closed-circular PCR products can be directly transformed into competent E. coli cells and easily selected by using an alternative antibiotic; (2) a mutant library can be created and screened by one-step error-prone amplification of a variable DNA region in an expression plasmid; and (3) accumulation of desired mutations in one sequence can be obtained by multiple rounds of is-epPCR. Is-epPCR offers a novel, convenient, and efficient approach for improving genes and proteins through directed evolution.

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.

[An expression T-vector and its application at low temperatures].

In modern biology and biotechnology research, recombinant gene expression has been the most popular method to obtain the target protein. In recent years, many foreign genes have been efficiently expressed in Escherichia coli. However, proteins encoded by animal, plant or mesophilic microbial genes often lose activities or become denatured within a few hours at regular growth temperatures for E. coli; some other target proteins are toxic to host cells and therefore difficult to be over-expressed. The new T-vector, pEXC-T, was constructed by combining TA cloning and cold-shock induction to obtain high expression levels with low costs. This paper reports the construction of pEXC-T and optimization of induction techniques for gene expression. Two instable proteins were tested and successfully expressed in soluble form by using pEXC vector. The development of pEXC-T offers a convenient technique for the preparations of recombinant proteins to be used in structure/function studies, or as diagnostic markers and medicinal proteins.

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.

Crystallization and preliminary X-ray diffraction analysis of the S-adenosylhomocysteine hydrolase (SAHH) from Thermotoga maritima.

S-Adenosylhomocysteine hydrolase (SAHH) catalyzes the reversible conversion of S-adenosylhomocysteine into adenosine and homocysteine. The SAHH from Thermotoga maritima (TmSAHH) was expressed in Escherichia coli and the recombinant protein was purified and crystallized. TmSAHH crystals belonging to space group C2, with unit-cell parameters a=106.3, b=112.0, c=164.9 Å, ß=103.5°, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.85 Šresolution. Initial phase determination by molecular replacement clearly indicated that the crystal contains one homotetramer per asymmetric unit. Further refinement of the crystal structure is in progress.

[Expression, purification and characterization of a thermostable lactate dehydrogenase from Thermotoga maritima].

The gene encoding thermostable lactate dehydrogenase (Tm-LDH) was cloned into the plasmid pHsh from Thermotoga maritima, and expressed in Escherichia coli JM 109. The recombinant protein was purified to homogeneity by a simple step, heat treatment. The recombinant enzyme had a molecular mass of 33 kDa. The optimal temperature and pH of Tm-LDH were observed 95 degrees C and 7.0. The purified enzyme had a half-life of 2 h at 90 degrees C, and exhibited better stability over a pH range from 5.5 to 8.0. The K(m) and V(max) values were 1.7 mmol/L, 3.8 x 10(4) U/mg of protein for pyruvate, and 7.2 mmol/L and 1.1 x 10(5) U/mg for NADH, respectively. The expression of Tm-LDH in T7 system could not obtain high efficiency, but it has been soluble over-expression in pHsh system and reached 340 mg/L. The superior stability and productivity of Tm-LDH will lay the foundation of its industrial-scale fermentation and application in the NAD regeneration.