Bacterial non-specific nucleases of the phospholipase D superfamily and their biotechnological potential.

Bacterial non-specific nucleases are ubiquitously distributed and involved in numerous intra- and extracellular processes. Although all nucleases share the basic chemistry for the hydrolysis of phosphodiester bonds in nucleic acid molecules, the catalysis comprises diverse modes of action, which offers great potential for versatile biotechnological applications. A major criterium for their differentiation is substrate specificity. Specific endonucleases are widely used as restriction enzymes in molecular biology approaches, whereas the main applications of non-specific nucleases (NSNs) are the removal of nucleic acids from crude extracts in industrial downstream processing and the prevention of cell clumping in microfabricated channels. In nature, the predominant role of NSNs is the acquisition of nutrient sources such as nucleotides and phosphates. The number of extensively characterized NSNs and available structures is limited. Moreover, their applicability is mostly challenged by the presence of metal chelators that impede the hydrolysis of nucleic acids in a metal ion-dependent manner. However, a few metal ion-independent NSNs that tolerate the presence of metal chelators have been characterized in recent years with none being commercially available to date. The classification and biotechnological potential of bacterial NSNs with a special focus on metal ion-independent nucleases are presented and discussed.Key Points • Bacterial phospholipases (PLD-family) exhibit nucleolytic activity. • Bacterial nucleases of the PLD-family are metal ion-independent. • NSNs can be used in downstream processing approaches.

Characterization of Single Amino Acid Variations in an EDTA-Tolerating Non-specific Nuclease from the Ice-Nucleating Bacterium Pseudomonas syringae.

Non-specific nuclease (NSN) can be applied in industrial downstream processing to remove nucleic acids from crude protein extracts or in cell-sorting systems to degrade nucleic acids derived from lysed cells. PsNuc from the ice-nucleating bacterium Pseudomonas syringae has the ability to decompose double- and single-stranded DNA in linear or circular form and RNA. It is not affected by the presence of metal-ion chelators such as EDTA and tolerates several protease inhibitors and reducing agents. A multiple sequence alignment of PsNuc with closely related enzymes (97-99% identity on the protein level) within the family Pseudomonaceae revealed the presence of only six amino acid residues that are variable in putative NSN from different members of the genus Pseudomonas. Single amino acid variants were produced in recombinant form in Escherichia coli, purified, and characterized. They showed similar activity compared to PsNuc, but a single variant even displayed an improved performance with an activity of > 20,000 U/mg at 35 °C, while amino acid residues S148 and V161 were found to be essential for enzymatic functionality. These results suggest that homologous nucleases from Pseudomonaceae display high activity levels in a metal-ion-independent manner and are therefore of interest for applications in biotechnology.

Comparative analysis of two non-specific nucleases of the phospholipase D family from the plant pathogen competitor bacterium Pantoea agglomerans.

Bacterial non-specific nucleases of the phospholipase D family are widely distributed among the members of the Enterobacteriaceae. Each genome mainly contains a single copy of a gene encoding a phospholipase D family protein. However, two distantly related isozymes (< 40% identity at the protein level) were identified by BLAST-analyses in the plant pathogenic competitor enterobacterium Pantoea agglomerans. The two nucleases PaNuc-1 and PaNuc-2 were produced in Escherichia coli. Identical gene constructs and expression conditions resulted in the production of PaNuc-1 in soluble form, while PaNuc-2 remained insoluble in inclusion bodies. PaNuc-2 was refolded and both proteins were purified by a combination of affinity and ion exchange chromatography. Proteolytic removal of the HIS-tag allowed the characterization of pure and mature tag-less proteins. Enzymatic properties of both isozymes revealed that they are non-specific nucleases, displaying activities against RNA, single- and double-stranded genomic DNA as well as circular plasmids. However, their biochemical activity profiles were clearly different, with PaNuc-1 being optimally active at 70 °C and pH 7.0, while PaNuc-2 was most active at 45 °C and pH 7.0. The enzymes retained > 90% nuclease activity at EDTA concentrations of 4 mM (PaNuc-2) and 20 mM (PaNuc-1), respectively. Different enzymatic properties suggest that the roles of PaNuc-1 and PaNuc-2 differ in the cell and might be the result of functional diversification after an ancient gene duplication event took place. The fact that both enzymes could be easily produced in recombinant form and their tolerance against metal ion chelators in combination with a broad substrate promiscuity might pave the way to versatile biotechnological applications.

A non-specific nucleolytic enzyme and its application potential in EDTA-containing buffer solutions.

OBJECTIVES: Metal-ion independent non-specific nucleases are of high potential for applications in EDTA-containing bioprocessing workflows. RESULTS: A novel extracellular non-specific nuclease EcNuc from the enterobacterium Escherichia coli has been identified. The recombinant gene was expressed and the protein was purified. Maximum activity of the enzyme was detected at 41.7 °C and at an acidic pH of 5.8. EcNuc tolerates EDTA in the reaction buffer at concentrations of up to 20 mM and the activity is not impaired by high concentrations of mono- and divalent metal ions in the absence of EDTA. The viscosity of crude protein extracts after cell lysis in EDTA-containing buffers is reduced when supplemented with EcNuc. CONCLUSION: Proof-of-concept has been demonstrated that a metal-ion independent non-specific nuclease can be applied for removal of nucleic acids in EDTA-containing buffers for the subsequent purification of proteins from crude extracts.

Facing the challenge of sustainable bioenergy production: Could halophytes be part of the solution?

Due to steadily growing population and economic transitions in the more populous countries, renewable sources of energy are needed more than ever. Plant biomass as a raw source of bioenergy and biofuel products may meet the demand for sustainable energy; however, such plants typically compete with food crops, which should not be wasted for producing energy and chemicals. Second-generation or advanced biofuels that are based on renewable and non-edible biomass resources are processed to produce cellulosic ethanol, which could be further used for producing energy, but also bio-based chemicals including higher alcohols, organic acids, and bulk chemicals. Halophytes do not compete with conventional crops for arable areas and freshwater resources, since they grow naturally in saline ecosystems, mostly in semi-arid and arid areas. Using halophytes for biofuel production may provide a mid-term economically feasible and environmentally sustainable solution to producing bioenergy, contributing, at the same time, to making saline areas - which have been considered unproductive for a long time - more valuable. This review emphasises on halophyte definition, global distribution, and environmental requirements. It also examines their enzymatic valorization, focusing on salt-tolerant enzymes from halophilic microbial species that may be deployed with greater advantage compared to their conventional mesophilic counterparts for faster degradation of halophyte biomass.

Complete genome sequence of Thermus brockianus GE-1 reveals key enzymes of xylan/xylose metabolism.

Thermus brockianus strain GE-1 is a thermophilic, Gram-negative, rod-shaped and non-motile bacterium that was isolated from the Geysir geothermal area, Iceland. Like other thermophiles, Thermus species are often used as model organisms to understand the mechanism of action of extremozymes, especially focusing on their heat-activity and thermostability. Genome-specific features of T. brockianus GE-1 and their properties further help to explain processes of the adaption of extremophiles at elevated temperatures. Here we analyze the first whole genome sequence of T. brockianus strain GE-1. Insights of the genome sequence and the methodologies that were applied during de novo assembly and annotation are given in detail. The finished genome shows a phred quality value of QV50. The complete genome size is 2.38 Mb, comprising the chromosome (2,035,182 bp), the megaplasmid pTB1 (342,792 bp) and the smaller plasmid pTB2 (10,299 bp). Gene prediction revealed 2,511 genes in total, including 2,458 protein-encoding genes, 53 RNA and 66 pseudo genes. A unique genomic region on megaplasmid pTB1 was identified encoding key enzymes for xylan depolymerization and xylose metabolism. This is in agreement with the growth experiments in which xylan is utilized as sole source of carbon. Accordingly, we identified sequences encoding the xylanase Xyn10, an endoglucanase, the membrane ABC sugar transporter XylH, the xylose-binding protein XylF, the xylose isomerase XylA catalyzing the first step of xylose metabolism and the xylulokinase XylB, responsible for the second step of xylose metabolism. Our data indicate that an ancestor of T. brockianus obtained the ability to use xylose as alternative carbon source by horizontal gene transfer.

Parallel N- and C-Terminal Truncations Facilitate Purification and Analysis of a 155-kDa Cold-Adapted Type-I Pullulanase.

The cold-adapted pullulanase Pul13A is an industrial useful amylolytic enzyme, but its low solubility is the major bottleneck to produce the protein in recombinant form. In a previous approach, a complex and time-consuming purification strategy including a step-wise dialysis procedure using decreasing concentrations of urea to renature the insoluble protein from inclusion bodies had been established. In this study, a truncation strategy was developed to facilitate the purification and handling of the type-I pullulanase. Pul13A has a size of 155-kDa with a multidomain architecture that is composed of the following predicted modules: CBM41/E-set/Amy-Pul/DUF3372/E-set/E-set/E-set, with CBM and E-set domains being putative carbohydrate-binding modules, Amy-Pul is the catalytic region and DUF is a domain of unknown function. Consecutive N- and C-terminal deletions of domains were applied to construct minimized enzyme variants retaining pullulanase activity and exhibiting improved renaturation efficiencies. A total of seven truncation constructs were generated and tested, which still led to the production of inclusion bodies. However, the parallel deletion of the exterior CBM41 and E-set domain enabled the direct refolding of active enzymes during one-step dialysis in urea-free buffer. Catalytic properties of truncation construct Pul13A-N1/C1 were not impaired indicating that this enzyme variant may be superior for industrial applications over the full-length pullulanase.

Is it possible to optimize the protein production yield by the generation of homomultimeric fusion enzymes?

BACKGROUND: The supply of industrially relevant biocatalysts demands an easy and efficient protein production in high yield. In a conventional approach, a recombinant protein is produced in a heterologous host enabling the manipulation of multiple parameters including expression plasmids, growth conditions and regulation of protein biosynthesis. In this study, the generation of homomultimeric fusion genes is tested as an additional parameter to increase the production yield of a heat-stable cellulase. FINDINGS: The LE (LguI/Eco81I)-cloning strategy was used to generate a set of plasmids containing a single copy or two to four repetitions of the endoglucanase-encoding gene cel5A from the thermophilic anaerobe Fervidobacterium gondwanense. Serial up-scaling of shaking flask volumes from 50 to 500 mL were used to determine the production yield of active cellulolytic enzyme Cel5A in recombinant form in Escherichia coli. Monitoring the cellular wet weight and total protein proved that the bacterial growth rate is not depending on the production of fusion enzymes, however activity assays in combination with Western blotting analyses indicated instability effects of large homomultimeric fusion enzymes. CONCLUSION: The production yield of fusion cellulases is constant with increasing molecular weights, but improved activities were not observed for recombinant Cel5A homomultimers. This strategy may serve as a starting point for further studies to generate more stable fusion proteins with improved catalytic activities and higher protein yield in the future.

Influence of Linker Length Variations on the Biomass-Degrading Performance of Heat-Active Enzyme Chimeras.

Plant cell walls are composed of complex polysaccharides such as cellulose and hemicellulose. In order to efficiently hydrolyze cellulose, the synergistic action of several cellulases is required. Some anaerobic cellulolytic bacteria form multienzyme complexes, namely cellulosomes, while other microorganisms produce a portfolio of diverse enzymes that work in synergistic fashion. Molecular biological methods can mimic such effects through the generation of artificial bi- or multifunctional fusion enzymes. Endoglucanase and ß-glucosidase from extremely thermophilic anaerobic bacteria Fervidobacterium gondwanense and Fervidobacterium islandicum, respectively, were fused end-to-end in an approach to optimize polysaccharide degradation. Both enzymes are optimally active at 90 °C and pH 6.0-7.0 representing excellent candidates for fusion experiments. The direct linkage of both enzymes led to an increased activity toward the substrate specific for ß-glucosidase, but to a decreased activity of endoglucanase. However, these enzyme chimeras were superior over 1:1 mixtures of individual enzymes, because combined activities resulted in a higher final product yield. Therefore, such fusion enzymes exhibit promising features for application in industrial bioethanol production processes.

Fast and reliable production, purification and characterization of heat-stable, bifunctional enzyme chimeras.

Degradation of complex plant biomass demands a fine-regulated portfolio of glycoside hydrolases. The LE (LguI/Eco81I)-cloning approach was used to produce two enzyme chimeras CB and BC composed of an endoglucanase Cel5A (C) from the extreme thermophilic bacterium Fervidobacterium gondwanense and an archaeal ß-glucosidase Bgl1 (B) derived from a hydrothermal spring metagenome. Recombinant chimeras and parental enzymes were produced in Escherichia coli and purified using a two-step affinity chromatography approach. Enzymatic properties revealed that both chimeras closely resemble the parental enzymes and physical mixtures, but Cel5A displayed lower temperature tolerance at 100°C when fused to Bgl1 independent of the conformational order. Moreover, the determination of enzymatic performances resulted in the detection of additive effects in case of BC fusion chimera. Kinetic measurements in combination with HPLC-mediated product analyses and site-directed mutation constructs indicated that Cel5A was strongly impaired when fused at the N-terminus, while activity was reduced to a slighter extend as C-terminal fusion partner. In contrast to these results, catalytic activity of Bgl1 at the N-terminus was improved 1.2-fold, effectively counteracting the slightly reduced activity of Cel5A by converting cellobiose into glucose. In addition, cellobiose exhibited inhibitory effects on Cel5A, resulting in a higher yield of cellobiose and glucose by application of an enzyme mixture (53.1%) compared to cellobiose produced from endoglucanase alone (10.9%). However, the overall release of cellobiose and glucose was even increased by catalytic action of BC (59.2%). These results indicate possible advantages of easily produced bifunctional fusion enzymes for the improved conversion of complex polysaccharide plant materials.

A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein.

Extein amino acid residues around the splice site junctions affect the functionality of inteins. To identify an optimal sequence context for efficient protein splicing of an intein from the thermoacidophilic archaeon Picrophilus torridus, single extein amino acid residues at the splice site junctions were continuously deleted. The construction of a set of different truncated extein variants showed that this intein tolerates multiple amino acid variations near the excision sites and exhibits full activity when -1 and +1 extein amino acid residues are conserved in an artificial GST-intein-HIS fusion construct. Moreover, splicing of the recombinant intein took place at temperatures between 4 and 42 °C with high efficiency, when produced in Escherichia coli. Therefore, structural model predictions were used to identify optimal insertion sites for the intein to be embedded within a hemicellulase from the psychrophilic bacterium Pseudoalteromonas arctica. The P. torridus intein inserted before amino acid residue Thr75 of the reporter enzyme retained catalytic activity. Moreover, the catalytic activity of the xylan-degrading hydrolase could be easily monitored in routine plate assays and in liquid test measurements at room temperature when produced in recombinant form in E. coli. This tool allows the indirect detection of the intein's catalytic activity to be used in screenings.

Exploration of extremophiles for high temperature biotechnological processes.

Industrial processes often take place under harsh conditions that are hostile to microorganisms and their biocatalysts. Microorganisms surviving at temperatures above 60°C represent a chest of biotechnological treasures for high-temperature bioprocesses by producing a large portfolio of biocatalysts (thermozymes). Due to the unique requirements to cultivate thermophilic (60-80°C) and hyperthermophilic (80-110°C) Bacteria and Archaea, less than 5% are cultivable in the laboratory. Therefore, other approaches including sequence-based screenings and metagenomics have been successful in providing novel thermozymes. In particular, polysaccharide-degrading enzymes (amylolytic enzymes, hemicellulases, cellulases, pectinases and chitinases), lipolytic enzymes and proteases from thermophiles have attracted interest due to their potential for versatile applications in pharmaceutical, chemical, food, textile, paper, leather and feed industries as well as in biorefineries.

Generating bifunctional fusion enzymes composed of heat-active endoglucanase (Cel5A) and endoxylanase (XylT).

Bifunctional enzyme constructs were generated comprising two genes encoding heat-active endoglucanase (cel5A) and endoxylanase (xylT). The fused proteins Cel5A-XylT and XylT-Cel5A were active on both ß-glucan and beechwood xylan. An improvement in endoglucanase and endoxylanase catalytic activities was observed. The specific activity of the fusion towards xylan was significantly raised when compared to XylT. The fusion constructs were active from 40 to 100 °C for endoglucanase and from 40 to 90 °C for endoxylanase, but the temperature optima were lowered from 90 to 80 °C for the endoglucanase and from 80 to 70 °C for the endoxylanase. XylT in the construct XylT-Cel5A was less stable at higher temperatures compared to Cel5A-XylT. Due to the enzymatic performance, these fusion enzymes are attractive candidates for applications in biorefineries based on plant waste.

Bringing functions together with fusion enzymes--from nature's inventions to biotechnological applications.

It is a mammoth task to develop a modular protein toolbox enabling the production of posttranslational organized multifunctional enzymes that catalyze reactions in complex pathways. However, nature has always guided scientists to mimic evolutionary inventions in the laboratory and, nowadays, versatile methods have been established to experimentally connect enzymatic activities with multiple advantages. Among the oldest known natural examples is the linkage of two or more juxtaposed proteins catalyzing consecutive, non-consecutive, or opposing reactions by a native peptide bond. There are multiple reasons for the artificial construction of such fusion enzymes including improved catalytic activities, enabled substrate channelling by proximity of biocatalysts, higher stabilities, and cheaper production processes. To produce fused proteins, it is either possible to genetically fuse coding open reading frames or to connect proteins in a posttranslational process. Molecular biology techniques that have been established for the production of end-to-end or insertional fusions include overlap extension polymerase chain reaction, cloning, and recombination approaches. Depending on their flexibility and applicability, these methods offer various advantages to produce fusion genes in high throughput, different orientations, and including linker sequences to maximize the flexibility and performance of fusion partners. In this review, practical techniques to fuse genes are highlighted, enzymatic parameters to choose adequate enzymes for fusion approaches are summarized, and examples with biotechnological relevance are presented including a focus on plant biomass-degrading glycosyl hydrolases.

Purification and characterization of a cold-adapted pullulanase from a psychrophilic bacterial isolate.

There is a considerable potential of cold-active biocatalysts for versatile industrial applications. A psychrophilic bacterial strain, Shewanella arctica 40-3, has been isolated from arctic sea ice and was shown to exhibit pullulan-degrading activity. Purification of a monomeric, 150-kDa pullulanase was achieved using a five-step purification approach. The native enzyme was purified 50.0-fold to a final specific activity of 3.0 U/mg. The enzyme was active at a broad range of temperature (10-50 °C) and pH (5-9). Optimal activity was determined at 45 °C and pH 7. The presence of various metal ions is tolerated by the pullulanase, while detergents resulted in decreased activity. Complete conversion of pullulan to maltotriose as the sole product and N-terminal amino acid sequence indicated that the enzyme is a type-I pullulanase and belongs to rarely characterized pullulan-degrading enzymes from psychrophiles.

Design and establishment of a vector system that enables production of multifusion proteins and easy purification by a two-step affinity chromatography approach.

The LE (LguI/Eco81I)-cloning procedure allows a step-wise, directional fusion of multiple DNA-fragments into a vector by utilizing two restriction enzymes generating identical non-palindromic overhangs. This strategy was applied to produce heat-stable cellulase-fusion proteins containing up to five single moieties. Terminal affinity tags enable efficient purification using a simple two-step approach.

Extremozymes--biocatalysts with unique properties from extremophilic microorganisms.

Extremozymes are enzymes derived from extremophilic microorganisms that are able to withstand harsh conditions in industrial processes that were long thought to be destructive to proteins. Heat-stable and solvent-tolerant biocatalysts are valuable tools for processes in which for example hardly decomposable polymers need to be liquefied and degraded, while cold-active enzymes are of relevance for food and detergent industries. Extremophilic microorganisms are a rich source of naturally tailored enzymes, which are more superior over their mesophilic counterparts for applications at extreme conditions. Especially lignocellulolytic, amylolytic, and other biomass processing extremozymes with unique properties are widely distributed in thermophilic prokaryotes and are of high potential for versatile industrial processes.

The filamentous ascomycete Sordaria macrospora can survive in ambient air without carbonic anhydrases.

The rapid interconversion of carbon dioxide and bicarbonate (hydrogen carbonate) is catalysed by metalloenzymes termed carbonic anhydrases (CAs). CAs have been identified in all three domains of life and can be divided into five evolutionarily unrelated classes (α, ß, γ, δ and ζ) that do not share significant sequence similarities. The function of the mammalian, prokaryotic and plant α-CAs has been intensively studied but the function of CAs in filamentous ascomycetes is mostly unknown. The filamentous ascomycete Sordaria macrospora codes for four CAs, three of the ß-class and one of the α-class. Here, we present a functional analysis of CAS4, the S. macrospora α-class CA. The CAS4 protein was post-translationally glycosylated and secreted. The knockout strain Δcas4 had a significantly reduced rate of ascospore germination. To determine the cas genes required for S. macrospora growth under ambient air conditions, we constructed double and triple mutations of the four cas genes in all possible combinations and a quadruple mutant. Vegetative growth rate of the quadruple mutant lacking all cas genes was drastically reduced compared to the wild type and invaded the agar under normal air conditions. Likewise the fruiting bodies were embedded in the agar and completely devoid of mature ascospores.

Characterization of a heat-active archaeal ß-glucosidase from a hydrothermal spring metagenome.

Thermostable enzymes are required for application in a wide range of harsh industrial processes. High stability and activity at elevated temperatures, as well as high tolerances toward various reagents and solvents, are needed. In this work, a glycoside hydrolase family 1 ß-glucosidase (Bgl1) of archaeal origin was isolated from a hydrothermal spring metagenome. The enzyme showed a broad substrate spectrum with activity toward cellobiose, cellotriose and lactose. Compared to most enzymes, extremely high specific activity with 3195U/mg was observed at 90°C and pH 6.5. Bgl1 was completely stable at pH 4.5-9.5 for 48 h at 4 °C. More than 40% of activity was measured at 105 °C. A thermal activation was observed at 90 °C after 30 min. Enzyme stability was enhanced (5- and 7-fold) after applying pressure of 100 and 200 bar at 90 °C for 2h, respectively. The affinity of the ß-glucosidase to its substrate was significantly increased in the presence of AlCl3. The K(i) value for glucose was 150 mM. These distinctive characteristics distinguish Bgl1 from other enzymes described so far and make this enzyme suitable for application in numerous processes that run at high temperatures.

Crystal structures of two tetrameric ß-carbonic anhydrases from the filamentous ascomycete Sordaria macrospora.

Carbonic anhydrases (CAs) are metalloenzymes catalyzing the reversible hydration of carbon dioxide to bicarbonate (hydrogen carbonate) and protons. CAs have been identified in archaea, bacteria and eukaryotes and can be classified into five groups (α, ß, γ, δ, ζ) that are unrelated in sequence and structure. The fungal ß-class has only recently attracted attention. In the present study, we investigated the structure and function of the plant-like ß-CA proteins CAS1 and CAS2 from the filamentous ascomycete Sordaria macrospora. We demonstrated that both proteins can substitute for the Saccharomyces cerevisiae ß-CA Nce103 and exhibit an in vitro CO2 hydration activity (kcat /Km of CAS1: 1.30 × 10(6) m(-1) ·s(-1) ; CAS2: 1.21 × 10(6 ) m(-1) ·s(-1) ). To further investigate the structural properties of CAS1 and CAS2, we determined their crystal structures to a resolution of 2.7 Å and 1.8 Å, respectively. The oligomeric state of both proteins is tetrameric. With the exception of the active site composition, no further major differences have been found. In both enzymes, the Zn(2) (+) -ion is tetrahedrally coordinated; in CAS1 by Cys45, His101 and Cys104 and a water molecule and in CAS2 by the side chains of four residues (Cys56, His112, Cys115 and Asp58). Both CAs are only weakly inhibited by anions, making them good candidates for industrial applications. STRUCTURED DIGITAL ABSTRACT: CAS1 and CAS2 bind by x-ray crystallography (View interaction) DATABASE: Structural data have been deposited in the Protein Data Bank database under accession numbers 4O1J for CAS1 and 4O1K for CAS2.