ABSTRACT
Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R-selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 Å. The structure exhibits an α/ß-hydrolase fold, very similar to the homologous, but S-selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser-His-Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)- and (S)-mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)-mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main-chain amide groups and an α-helix dipole very similar to α/ß-hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies.
Subject(s)
Aldehyde-Lyases/chemistry , Aldehyde-Lyases/metabolism , Arabidopsis/enzymology , Hevea/enzymology , Aldehyde-Lyases/genetics , Arabidopsis/chemistry , Arabidopsis/genetics , Arabidopsis/metabolism , Crystallography, X-Ray , Hevea/chemistry , Hevea/genetics , Hevea/metabolism , Models, Molecular , Point Mutation , Protein Binding , Protein Folding , Protein Structure, Secondary , Stereoisomerism , Substrate SpecificityABSTRACT
BACKGROUND: Microbial lipases represent the most important class of biocatalysts used for a wealth of applications in organic synthesis. An often applied reaction is the lipase-catalyzed transesterification of vinyl esters and alcohols resulting in the formation of acetaldehyde which is known to deactivate microbial lipases, presumably by structural changes caused by initial Schiff-base formation at solvent accessible lysine residues. Previous studies showed that several lipases were sensitive toward acetaldehyde deactivation whereas others were insensitive; however, a general explanation of the acetaldehyde-induced inactivation mechanism is missing. RESULTS: Based on five microbial lipases from Candida rugosa, Rhizopus oryzae, Pseudomonas fluorescens and Bacillus subtilis we demonstrate that the protonation state of lysine ε-amino groups is decisive for their sensitivity toward acetaldehyde. Analysis of the diverse modification products of Bacillus subtilis lipases in the presence of acetaldehyde revealed several stable products such as α,ß-unsaturated polyenals, which result from base and/or amino acid catalyzed aldol condensation of acetaldehyde. Our studies indicate that these products induce the formation of stable Michael-adducts at solvent-accessible amino acids and thus lead to enzyme deactivation. Further, our results indicate Schiff-base formation with acetaldehyde to be involved in crosslinking of lipase molecules. CONCLUSIONS: Differences in stability observed with various commercially available microbial lipases most probably result from different purification procedures carried out by the respective manufacturers. We observed that the pH of the buffer used prior to lyophilization of the enzyme sample is of utmost importance. The mechanism of acetaldehyde-induced deactivation of microbial lipases involves the generation of α,ß-unsaturated polyenals from acetaldehyde which subsequently form stable Michael-adducts with the enzymes. Lyophilization of the enzymes from buffer at pH 6.0 can provide an easy and effective way to stabilize lipases toward inactivation by acetaldehyde.
Subject(s)
Acetaldehyde/chemistry , Bacteria/enzymology , Bacterial Proteins/chemistry , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Fungi/enzymology , Lipase/chemistry , Acetaldehyde/metabolism , Bacteria/chemistry , Bacteria/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Enzyme Activation , Fungal Proteins/genetics , Fungi/chemistry , Fungi/genetics , Kinetics , Lipase/genetics , Lipase/metabolismABSTRACT
Bacillus subtilis and Bacillus licheniformis are widely used for the large-scale industrial production of proteins. These strains can efficiently secrete proteins into the culture medium using the general secretion (Sec) pathway. A characteristic feature of all secreted proteins is their N-terminal signal peptides, which are recognized by the secretion machinery. Here, we have studied the production of an industrially important secreted protease, namely, subtilisin BPN' from Bacillus amyloliquefaciens. One hundred seventy-three signal peptides originating from B. subtilis and 220 signal peptides from the B. licheniformis type strain were fused to this secretion target and expressed in B. subtilis, and the resulting library was analyzed by high-throughput screening for extracellular proteolytic activity. We have identified a number of signal peptides originating from both organisms which produced significantly increased yield of the secreted protease. Interestingly, we observed that levels of extracellular protease were improved not only in B. subtilis, which was used as the screening host, but also in two different B. licheniformis strains. To date, it is impossible to predict which signal peptide will result in better secretion and thus an improved yield of a given extracellular target protein. Our data show that screening a library consisting of homologous and heterologous signal peptides fused to a target protein can identify more-effective signal peptides, resulting in improved protein export not only in the original screening host but also in different production strains.
Subject(s)
Bacillus/metabolism , Bacterial Proteins/metabolism , Protein Sorting Signals , Subtilisins/metabolism , Bacterial Proteins/genetics , Biotechnology/methods , Industrial Microbiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Subtilisins/geneticsABSTRACT
A whole-cell catalyst using Escherichia coli BL21(DE3) as a host, co-expressing glycerol dehydrogenase (GlyDH) from Gluconobacter oxydans and glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration, has been successfully constructed and used for the reduction of aliphatic aldehydes, such as hexanal or glyceraldehyde to the corresponding alcohols. This catalyst was characterized in terms of growth conditions, temperature and pH dependency, and regarding the influence of external cofactor and permeabilization. In the case of external cofactor addition we found a 4.6-fold increase in reaction rate caused by the addition of 1 mM NADP(+). Due to the fact that pH and temperature are also factors which may affect the reaction rate, their effect on the whole-cell catalyst was studied as well. Comparative studies between the whole-cell catalyst and the cell-free system were investigated. Furthermore, the successful application of the whole-cell catalyst in repetitive batch conversions could be demonstrated in the present study. Since the GlyDH was recently characterized and successfully applied in the kinetic resolution of racemic glyceraldehyde, we were now able to transfer and establish the process to a whole-cell system, which facilitated the access to L-glyceraldehyde in high enantioselectivity at 54% conversion. All in all, the whole-cell catalyst shows several advantages over the cell-free system like a higher thermal, a similar operational stability and the ability to recycle the catalyst without any loss-of-activity. The results obtained making the described whole-cell catalyst an improved catalyst for a more efficient production of enantiopure L-glyceraldehyde.
Subject(s)
Bacillus subtilis/enzymology , Escherichia coli/metabolism , Gluconobacter oxydans/enzymology , Glucose 1-Dehydrogenase/metabolism , Glyceraldehyde/metabolism , Sugar Alcohol Dehydrogenases/metabolism , Biotransformation , Escherichia coli/genetics , Glucose 1-Dehydrogenase/genetics , Hydrogen-Ion Concentration , Kinetics , NADP/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sugar Alcohol Dehydrogenases/genetics , TemperatureABSTRACT
Due to the lack of an outer membrane, Gram-positive bacteria (e.g., Bacillus species) are considered as promising host organisms for the secretory production of biotechnologically relevant heterologous proteins. However, the yields of the desired target proteins were often reported to be disappointingly low. Here, we used saturation mutagenesis of the positively charged N-domain (positions 2-7) of the signal peptide of the Bacillus subtilis alpha-amylase (AmyE) as a novel approach for the improvement of the secretion of a heterologous model protein, cutinase from Fusarium solani pisi, by the general secretory pathway of B. subtilis. Automated high-throughput screening of the resulting signal peptide libraries allowed for the identification of four single point mutations that resulted in significantly increased cutinase amounts, three of which surprisingly reduced the net charge of the N-domain from +3 to +2. Characterization of the effects of the identified mutations on protein synthesis and export kinetics by pulse-chase analyses indicates that an optimal balance between biosynthesis and the flow of the target protein through all stages of the B. subtilis secretion pathway is of crucial importance with respect to yield and quality of secreted heterologous proteins.
Subject(s)
Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/metabolism , Protein Sorting Signals/genetics , Recombinant Fusion Proteins/metabolism , alpha-Amylases/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biotechnology , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fusarium/enzymology , Fusarium/genetics , Mutagenesis , Mutant Proteins/metabolism , Peptide Library , Point Mutation , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , alpha-Amylases/chemistry , alpha-Amylases/metabolismABSTRACT
Fluorescent reporter proteins such as green fluorescent protein are valuable noninvasive molecular tools for in vivo real-time imaging of living specimens. However, their use is generally restricted to aerobic systems, as the formation of their chromophores strictly requires oxygen. Starting with blue-light photoreceptors from Bacillus subtilis and Pseudomonas putida that contain light-oxygen-voltage-sensing domains, we engineered flavin mononucleotide-based fluorescent proteins that can be used as fluorescent reporters in both aerobic and anaerobic biological systems.
Subject(s)
Genes, Reporter , Luminescent Proteins/metabolism , Oxygen/metabolism , Anaerobiosis , Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Flavin Mononucleotide/metabolism , Fluorescence , Protein Structure, Tertiary , Pseudomonas putida/metabolism , Rhodobacter capsulatus/metabolismABSTRACT
Plants and fungi respond to environmental light stimuli via the action of different photoreceptor modules. One such class, responding to the blue region of light, is constituted by photoreceptors containing so-called light-oxygen-voltage (LOV) domains as sensor modules. Four major LOV families are currently identified in eukaryotes: (i) the plant phototropins, regulating various physiological effects such as phototropism, chloroplast relocation, and stomatal opening; (ii) the aureochromes, mediating photomorphogenesis in photosynthetic stramenopile algae; (iii) the plant circadian photoreceptors of the zeitlupe (ZTL)/adagio (ADO)/flavin-binding Kelch repeat F-box protein 1 (FKF1) family; and (iv) the fungal circadian photoreceptors white-collar 1 (WC-1). Blue-light-sensitive LOV signaling modules are also widespread throughout the prokaryotic world, and physiological responses mediated by bacterial LOV photoreceptors were recently reported. Thus, the question arises as to the evolutionary relationship between the pro- and eukaryotic LOV photoreceptor systems. We used Bayesian and maximum-likelihood tree reconstruction methods to infer evolutionary scenarios that might have led to the widespread appearance of LOV domains among the pro- and eukaryotes. The phylogenetic study presented here suggests a bacterial origin for the LOV domains of the four major eukaryotic LOV photoreceptor families, whereas the LOV sensor domains were most likely recruited from the bacteria in the course of plastid and mitochondrial endosymbiosis.
Subject(s)
Bacteria/classification , Fungi/classification , Phylogeny , Plants/classification , Algal Proteins/genetics , Arabidopsis Proteins/classification , Arabidopsis Proteins/genetics , Bacterial Proteins/genetics , Bayes Theorem , DNA-Binding Proteins/classification , DNA-Binding Proteins/genetics , Fungal Proteins/classification , Fungal Proteins/genetics , Likelihood Functions , Phototropins/classification , Phototropins/genetics , Plant Proteins/genetics , Transcription Factors/classification , Transcription Factors/geneticsABSTRACT
The acetic acid bacterium Gluconobacter oxydans has a high potential for oxidoreductases with a variety of different catalytic abilities. One putative oxidoreductase gene codes for an enzyme with a high similarity to the NADP+-dependent glycerol dehydrogenase (GlyDH) from Hypocrea jecorina. Due to this homology, the GlyDH (Gox1615) has been cloned, over-expressed in Escherichia coli, purified and characterised. Gox1615 shows an apparent native molecular mass of 39 kDa, which corresponds well to the mass of 37.213 kDa calculated from the primary structure. From HPLC measurements, a monomeric structure can be deduced. Kinetic parameters and the dependence of the activity on temperature and pH were determined. The enzyme shows a broad substrate spectrum in the reduction of different aliphatic, branched and aromatic aldehydes. Additionally, the enzyme has been shown to oxidize a variety of different alcohols. The highest activities were observed for the conversion of D-glyceraldehyde in the reductive and L-arabitol in the oxidative direction. Since high enantioselectivities were observed for the reduction of glyceraldehyde, the kinetic resolution of glyceraldehyde was investigated and found to yield enantiopure L-glyceraldehyde on preparative scale.
Subject(s)
Gluconobacter oxydans/enzymology , Glyceraldehyde/metabolism , Sugar Alcohol Dehydrogenases/metabolism , Amino Acid Sequence , Animals , Molecular Sequence Data , NADP/metabolism , Oxidation-Reduction , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Substrate Specificity , Sugar Alcohol Dehydrogenases/genetics , Sugar Alcohol Dehydrogenases/isolation & purificationABSTRACT
Efficient protein secretion is very important in biotechnology as it provides active and stable enzymes, which are an essential prerequisite for successful biocatalysis. Therefore, optimizing enzyme-producing bacterial strains is a major challenge in the field of biotechnology and protein production. In this study, the Gram-positive model bacterium Bacillus subtilis was optimized for heterologous protein secretion using a novel approach. Two lipolytic enzymes, cutinase from Fusarium solani pisi and a cytoplasmatic esterase of metagenomic origin, were chosen as reporters for heterologous protein secretion. In a systematic screening approach, all naturally occurring (non-lipoprotein) Sec-type signal peptides (SPs) from B. subtilis were characterized for their potential in heterologous protein secretion. Surprisingly, optimal SPs in cutinase secretion were inefficient in esterase secretion and vice versa, indicating the importance of an optimal fit between the SP and the respective mature part of the desired secretion target proteins. These results highlight the need for individually optimal signal peptides for every heterologous secretion target. Therefore, the SP library generated in this study represents a powerful tool for secretion optimization in Gram-positive expression hosts.
Subject(s)
Bacillus subtilis/metabolism , Protein Sorting Signals/physiology , Recombinant Proteins/biosynthesis , Amino Acid Sequence , Bacillus subtilis/genetics , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Base Sequence , Biological Transport, Active , Biotechnology , Carboxylic Ester Hydrolases/biosynthesis , Carboxylic Ester Hydrolases/genetics , DNA, Bacterial/genetics , Escherichia coli/genetics , Esterases/biosynthesis , Esterases/genetics , Fusarium/enzymology , Fusarium/genetics , Gram-Positive Bacteria/genetics , Gram-Positive Bacteria/metabolism , Kinetics , Molecular Sequence Data , Peptide Library , Plasmids/genetics , Protein Processing, Post-Translational , Protein Sorting Signals/genetics , Recombinant Proteins/geneticsABSTRACT
A high-throughput screening assay for hydroxynitrile lyase activity accepting a wide range of HNL-substrates is presented, which is useful either for enzyme fingerprinting or screening of huge variant libraries generated in metagenome or directed evolution approaches.
Subject(s)
Aldehyde-Lyases , Directed Molecular Evolution/methods , Nitriles/chemistry , Plant Proteins , Aldehyde-Lyases/chemistry , Aldehyde-Lyases/genetics , Aldehyde-Lyases/metabolism , Catalysis , Cloning, Molecular , Escherichia coli/genetics , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Conformation , Spectrophotometry , Substrate SpecificityABSTRACT
Chiral compounds can be produced efficiently by using biocatalysts. However, wild-type enzymes often do not meet the requirements of a production process, making optimization by rational design or directed evolution necessary. Here, we studied the lipase-catalyzed hydrolysis of the model substrate 1-(2-naphthyl)ethyl acetate both theoretically and experimentally. We found that a computational equivalent of alanine scanning mutagenesis based on QM/MM methodology can be applied to identify amino acid positions important for the activity of the enzyme. The theoretical results are consistent with concomitant experimental work using complete saturation mutagenesis and high-throughput screening of the target biocatalyst, a lipase from Bacillus subtilis. Both QM/MM-based calculations and molecular biology experiments identify histidine 76 as a residue that strongly affects the catalytic activity. The experiments demonstrate its important influence on enantioselectivity.
Subject(s)
Computational Biology/methods , Directed Molecular Evolution/methods , Gene Library , Amino Acid Substitution/genetics , Bacillus subtilis/chemistry , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Hydrolysis , Lipase/chemistry , Lipase/genetics , Models, Chemical , Mutagenesis , Protein Structure, Secondary , Substrate SpecificityABSTRACT
Several primer prediction programs have been developed for a variety of applications. However none of these tools allows the prediction of a large set of primers for whole gene site-directed mutagenesis experiments using the megaprimer method. We report a novel primer prediction tool (insilico.mutagenesis), accessible at www.insilico.uni-duesseldorf.de, developed for the application to high-throughput mutagenesis used in directed evolution or structure-function dependency projects, which involve the subsequent mutagenesis of a large number of amino acid positions (e.g., in whole gene saturation or gene scanning mutagenesis experiments). Furthermore, the program is suitable for all site-directed (saturation) mutagenesis approaches, such as saturation mutagenesis of promoter sequences and other types of untranslated intergenic regions. In anticipation of downstream cloning steps, the primer design tool also includes a restriction site control feature alerting the user if unwanted restriction sites have been introduced within the mutagenesis primer. The use of our tool promises to speed up the process of site-directed mutagenesis, as it instantly allows predicting a large set of primers.
Subject(s)
Computational Biology/methods , Evolution, Molecular , Genetic Techniques , Sequence Analysis, DNA/methods , Algorithms , Bacillus subtilis/genetics , Base Sequence , Cloning, Molecular , DNA Primers/chemistry , Internet , Models, Biological , Molecular Sequence Data , Mutagenesis , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Software , Structure-Activity RelationshipABSTRACT
Directed evolution methods are now widely used for the optimization of diverse enzyme properties, which include biotechnologically relevant characteristics like stability, regioselectivity and, in particular, enantioselectivity. In principle, three different approaches are followed to optimize enantioselective reactions: the development of whole-cell biocatalysts through the creation of designer organisms; the optimization of enzymes with existing enantioselectivity for process conditions; and the evolution of novel enantioselective biocatalysts starting from non-selective wild-type enzymes.
Subject(s)
Catalysis , Directed Molecular Evolution/methods , Enzymes/biosynthesis , Enzymes/genetics , Models, Genetic , Protein Engineering/methods , Sequence Analysis, Protein/methods , Algorithms , Biotechnology/methods , Drug Design , Enzymes/chemistry , Evolution, Molecular , Gene Expression Profiling/methods , Isomerism , Mutagenesis, Site-Directed/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistryABSTRACT
Lipases constitute the most important group of biocatalysts for biotechnological applications. The high-level production of microbial lipases requires not only the efficient overexpression of the corresponding genes but also a detailed understanding of the molecular mechanisms governing their folding and secretion. The optimisation of industrially relevant lipase properties can be achieved by directed evolution. Furthermore, novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agrochemicals, and flavour compounds.
Subject(s)
Bacteria/genetics , Bacteria/metabolism , Biotechnology/methods , Biotechnology/trends , Lipase/biosynthesis , Lipase/chemistry , Agrochemicals/chemical synthesis , Bacteria/classification , Biopolymers/biosynthesis , Catalysis , Cosmetics/chemical synthesis , Directed Molecular Evolution/methods , Directed Molecular Evolution/trends , Flavoring Agents/chemical synthesis , Fossil Fuels , Gene Expression Regulation, Bacterial , Lipase/chemical synthesis , Lipase/classification , Models, Biological , Models, Chemical , Pharmaceutical Preparations/chemical synthesis , Plant Oils/chemistry , Quality Control , Species Specificity , Technology, PharmaceuticalABSTRACT
Bacillus subtilis secretes two lipases LipA and LipB into the culture medium. Both enzyme genes were differentially expressed depending on the growth conditions as determined by activity assays and Western blotting in B. subtilis mutant strains lipA, lipB, and the corresponding lipA/lipB double mutant. In minimal medium, LipA was produced at wild-type level in a lipB mutant, however, no LipB protein was detected in a lipA mutant. Interestingly, LipA was produced and secreted at wild-type level in rich medium, but the enzyme remained enzymatically inactive, presumably being caused by a shift of the growth medium to acid pH. Furthermore, expression of the lipase genes was studied using transcriptional fusions with the lacZ reporter gene. The expression of lipA was repressed by high amino acid concentrations, whereas the lipB gene expression remained unaffected.
Subject(s)
Amino Acids/metabolism , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Gene Expression Regulation, Bacterial , Lipase/genetics , Lipase/metabolism , Blotting, Western , Caprylates/metabolism , Electrophoresis, Polyacrylamide Gel , Gene Deletion , Genes, Reporter , Hydrogen-Ion Concentration , beta-Galactosidase/metabolismABSTRACT
Ursodeoxycholic acid, a secondary bile acid, is used as a drug for the treatment of various liver diseases, the optimal dose comprises the range of 8-10mg/kg/day. For industrial syntheses, the structural complexity of this bile acid requires the use of an appropriate starting material as well as the application of regio- and enantio-selective enzymes for its derivatization. Most strategies for the synthesis start from cholic acid or chenodeoxycholic acid. The latter requires the conversion of the hydroxyl group at C-7 from α- into ß-position in order to obtain ursodeoxycholic acid. Cholic acid on the other hand does not only require the same epimerization reaction at C-7 but the removal of the hydroxyl group at C-12 as well. There are several bacterial regio- and enantio-selective hydroxysteroid dehydrogenases (HSDHs) to carry out the desired reactions, for example 7α-HSDHs from strains of Clostridium, Bacteroides or Xanthomonas, 7ß-HSDHs from Clostridium, Collinsella, or Ruminococcus, or 12α-HSDH from Clostridium or from Eggerthella. However, all these bioconversion reactions need additional steps for the regeneration of the coenzymes. Selected multi-step reaction systems for the synthesis of ursodeoxycholic acid are presented in this review.
Subject(s)
Bile Acids and Salts/biosynthesis , Hydroxysteroid Dehydrogenases/biosynthesis , Ursodeoxycholic Acid/biosynthesis , Actinobacteria/enzymology , Bile Acids and Salts/chemistry , Cholic Acid/chemistry , Clostridium/enzymology , Escherichia coli , Humans , Hydroxysteroid Dehydrogenases/chemistry , Ursodeoxycholic Acid/chemistry , Ursodeoxycholic Acid/therapeutic useABSTRACT
Usage of the enhanced green fluorescent protein (eGFP) in living mammalian cells is limited to aerobic conditions due to requirement of oxygen during chromophore formation. Since many diseases or disease models are associated with acute or chronic hypoxia, eGFP-labeling of structures of interest in experimental studies might be unreliable leading to biased results. Thus, a chromophore yielding a stable fluorescence under hypoxic conditions is desirable. The fluorescence of flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) does not require molecular oxygen. Recently, the advantages of FbFPs for several bacterial strains and yeasts were described, specifically, their usage as a real time fluorescence marker in bacterial expression studies and their ability of chromophore formation under anaerobic conditions. Our objective was to verify if FbFPs also function in mammalian cells in order to potentially broaden the repertoire of chromophores with ones that can reliably be used in mammalian studies under hypoxic conditions. In the present study, we demonstrate for the first time, that FbFPs can be expressed in different mammalian cells, among them murine neural stem cells during proliferative and differentiated stages. Fluorescence intensities were comparable to eGFP. In contrast to eGFP, the FbFP fluorescence did not decrease when cells were exposed to defined hypoxic conditions neither in proliferating nor in differentiated cells. Thus, FbFPs can be regarded as an alternative to eGFP in studies that target cellular structures which are exposed to hypoxic conditions.
Subject(s)
Flavin Mononucleotide/metabolism , Green Fluorescent Proteins/metabolism , Mammals/metabolism , Oxygen/metabolism , Animals , CHO Cells , Cell Hypoxia , Cell Line, Tumor , Cell Size , Cricetinae , Gene Expression , HEK293 Cells , Humans , Luminescent Proteins/metabolism , Mice , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Transfection , Transgenes/geneticsABSTRACT
A wide variety of random- and site-directed mutagenesis techniques have been developed to investigate the structure-function relationship in proteins and intergenic regions like promoter sequences. Similar techniques can be employed to optimize protein properties like enantioselectivity, substrate specificity, and stability in a directed evolution approach. Due to the tremendous genetic diversity that is created by common random-mutagenesis methods, directed evolution techniques usually require the time-consuming and cumbersome screening of large numbers of variants. A gene-scanning saturation-mutagenesis approach represents one efficient way to limit the screening effort by reducing the created genetic diversity. In structure/function studies often a similar method, e.g., alanine- or arginine-scanning mutagenesis, is used to probe the role of specific amino acids in a protein. Here, we present a standardized mutagenesis strategy that can speed up the process of scanning whole proteins for structure/function studies and, furthermore, allows for the fast and efficient generation of gene-scanning saturation-mutagenesis libraries to be used in the directed evolution of enzyme functions and properties. The described method uses automated computer-assisted oligonucleotide design, and a two-step PCR-mutagenesis protocol to amplify site-specifically mutated circular plasmids that can be directly transformed in Escherichia coli expression strains.
Subject(s)
Mutagenesis , Plasmids , Polymerase Chain Reaction/methods , DNA Primers , Directed Molecular Evolution , Escherichia coli/geneticsABSTRACT
Hydroxynitrile lyases (HNLs) are applied in technical processes for the synthesis of chiral cyanohydrins. Here we describe the thorough characterization of the recently discovered R-hydroxynitrile lyase from Arabidopsis thaliana and its S-selective counterpart from Manihot esculenta (MeHNL) concerning their properties relevant for technical applications. The results are compared to available data of the structurally related S-HNL from Hevea brasiliensis (HbHNL), which is frequently applied in technical processes. Whereas substrate ranges are highly similar for all three enzymes, the stability of MeHNL with respect to higher temperature and low pH-values is superior to the other HNLs with alpha/beta-hydrolase fold. This enhanced stability is supposed to be due to the ability of MeHNL to form tetramers in solution, while HbHNL and AtHNL are dimers. The different inactivation pathways, deduced by means of circular dichroism, tryptophan fluorescence and static light scattering further support these results. Our data suggest different possibilities to stabilize MeHNL and AtHNL for technical applications: whereas the application of crude cell extracts is appropriate for MeHNL, AtHNL is stabilized by addition of polyols. In addition, the molecular reason for the inhibition of MeHNL and HbHNL by acetate could be elucidated, whereas no such inhibition was observed with AtHNL.