The estimated 5 × 10 30 prokaryotic cells inhabiting our planet sequester some 350-550 Petagrams ... more The estimated 5 × 10 30 prokaryotic cells inhabiting our planet sequester some 350-550 Petagrams (1 Pg = 1015 g) of carbon, 85-130 Pg of nitrogen, and 9-14 Pg of phosphorous, making them the largest reservoir of those nutrients on Earth (Whitman et al. 1998). However, reports suggest that only less than 1% of these microscopic organisms are cultivable (Torsvik et al. 1990; Sleator et al. 2008). Until recently with the development of metagenomic techniques, the knowledge of microbial diversity and their metabolic capabilities has been limited to this small fraction of cultivable organisms (Handelsman et al. 1998). While metagenomics has undoubtedly revolutionised the field of microbiology and biotechnology it has been generally acknowledged that the current approaches for metagenomic bio-prospecting / screening have limitations which hinder this approach to fully access the metabolic potentials and genetic variations contained in microbial genomes (Beloqui et al. 2008). In particular, the construction of metagenomic libraries and heterologous expression are amongst the major obstacles. The aim of this study was to develop an ultra-high throughput approach for screening enzyme activities using uncloned metagenomic DNA, thereby eliminating cloning steps, and employing in vitro heterologous expression. To achieve this, three widely used techniques: cell-free transcription-translation, in vitro compartmentalisation (IVC) and Fluorescence Activated Cell Sorting (FACS) were combined to develop this robust technique called metagenomic in vitro compartmentalisation (mIVC-FACS). Moreover, the E. coli commercial cell-free system was used in parallel to a novel, in-house Rhodococcus erythropolis based cell-free system. The versatility of this technique was tested by identifying novel beta-xylosidase encoding genes derived from a thermophilic compost metagenome. In addition, the efficiency of mIVC-FACS was compared to the traditional metagenomic approaches; function-based (clone library screening) and sequence-based (shotgun sequencing and PCR screening). List of abbreviation °C Degrees Celcius Amp Ampicillin Amp R Ampicillin resistance gene Bp Base pairs BLAST Basic local alignment search tool BSA Bovine serum albumin CAM Chloramphenicol cAMP cyclic Adenosine monophosphate CAZy Carbohydrate-Active Enzymes database CTAB Cetyl trimethylammonium bromide CFPS Cell-free protein synthesis C-terminus Carboxy terminus ddH 2 O Distilled deionised water dE Double emulsion dH 2 O Deionised water DNA Deoxyribonucleic acid dNTPs Deoxyribonucleotide triphosphates DTT Dithiothreitol EDTA Ethylenediamine tetraacetic acid EtOH Ethanol FACS Fluorescence activated cell sorter g Gram g Gravitational force gDNA Genomic DNA V Volts V i Initial velocity V max Maximum velocity vol Volume v/v Volume per volume w/v Weight per volume XylA 59 XylB 26 hypothetical protein [Elizabethkingia anophelis] 49 xylosidase [Elizabethkingia miricola] 49 xylosidase [Elizabethkingia genomo sp. 2] 49 hypothetical protein SCO7037 [Streptomyces coelicolor A3(2)] 65 XylA 113 XylB 53 hypothetical protein [Elizabethkingia anophelis] 113 xylosidase [Elizabethkingia miricola] 113 xylosidase [Elizabethkingia genomo sp. 2] 113
Background: There is a continued need for improved enzymes for industry. β-xylosidases are enzyme... more Background: There is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and although many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood. Results: Screening of a metagenomic library constructed of mDNA isolated from horse manure compost for β-xylosidase activity identified 26 positive hits. The fosmid clones were sequenced and bioinformatic analysis performed to identity putative β-xylosidases. Based on the novelty of its amino acid sequence and potential thermostability one enzyme (XylP81) was selected for expression and further characterization. XylP81 belongs to the family 39 β-xylosidases, a comparatively rarely found and characterized GH family. The enzyme displayed biochemical characteristics (K M-5.3 mM; V max-122 U/mg; k cat-107; T opt-50 °C; pH opt-6) comparable to previously characterized glycoside hydrolase family 39 (GH39) β-xylosidases and despite nucleotide identity to thermophilic species, the enzyme displayed only moderate thermostability with a half-life of 32 min at 60 °C. Apart from acting on substrates predicted for β-xylosidase (xylobiose and 4-nitrophenyl-β-D-xylopyranoside) the enzyme also displayed measurable α-Larabainofuranosidase, β-galactosidase and β-glucosidase activity. A remarkable feature of this enzyme is its ability to tolerate high concentrations of xylose with a K i of 1.33 M, a feature that is highly desirable for commercial applications. Conclusions: Here we describe a novel β-xylosidase from a poorly studied glycosyl hydrolase family (GH39) which despite having overall kinetic properties similar to other bacterial GH39 β-xylosidases, displays unusually high product tolerance. This trait is shared with only one other member of the GH39 family, the recently described β-xylosidases from Dictyoglomus thermophilum. This feature should allow its use as starting material for engineering of an enzyme that may prove useful to industry and should assist in the fundamental understanding of the mechanism by which glycosyl hydrolases evolve product tolerance.
Magnetosomes, produced by magnetotactic bacteria (MTB), are the most attractive alternative sourc... more Magnetosomes, produced by magnetotactic bacteria (MTB), are the most attractive alternative source of non-toxic biocompatible magnetic nanoparticles (MNPs). A magnetosome contains Fe 2 O 4 magnetite with properties superior to MNPs synthesized by the traditional chemical route. However, synthesis of magnetosomes on large scale has not been achieved yet because magnetotactic bacteria are fastidious to grow. In addition, magnetosomes are generally "soft" magnetic materials which can only be used for some applications, while other applications require "hard" magnetic materials. Here at the Institute of Microbial Biotechnology and Metagenomic (IMBM), a study is being conducted on cloning and expression of the magnetosome gene island (MIA), the genetic machinery for magnetosome formation, in an easy to culture E. coli strain. The magnetic properties of the magnetosome can be manipulated by doping with divalent metals such as Ni 2+ or Co 2+ for a variety of applications. The specific objective of this study was to genetically engineer E. coli strains which accumulate intracellular Ni 2+ or Co 2+ in order to manipulate the magnetic properties of the magnetosomes. Three E. coli mutants and a wild type strain were transformed with high affinity Ni 2+ or Co 2+ uptake genes and evaluated for intracellular accumulation at different medium concentrations of NiCl 2 or CoCl 2. Cellular iron and magnesium were also evaluated because iron is the major component of the magnetosome and magnesium is important for cell growth. The wild type strain, EPI 300 habouring Ni 2+ uptake permease the hoxN gene or Co 2+ uptake ABC type transporter cbiKMQO operon was found to accumulate the most intracellular Ni 2+ or Co 2+ in medium conditions most likely to induce magnetosome formation and magnetite manipulation. This strain can be used to co-express the MIA and v Ni 2+ or Co 2+ uptake gene for mass production of magnetosome with altered magnetic properties.
Additional file 1: Figure S1. SDS-PAGE analysis XylP81 expression. A) Lane M: ColorPlus prestaine... more Additional file 1: Figure S1. SDS-PAGE analysis XylP81 expression. A) Lane M: ColorPlus prestained protein ladder, broad range (10-230 kDa); lane 1: E. coli-pET21a no insert uninduced; lane 2: E. coli-pETP81 uninduced soluble fraction; lane 3: E. coli-pETP81 induced soluble fraction; lane 4: E. coli-pET21a no insert induced; lane 5: E. coli-pETP81 uninduced insoluble fraction; lane 6: E. coli-pETP81 induced insoluble fraction. B) Purified XylP81 following metal affinity chromatography purification. lane M: ColorPlus prestained protein ladder. Figure S2. Unrooted maximum likelihood phylogenetic tree of characterized GH39 amino acid sequences including XylP81, excluding unchracterized and sequences closely related to XylP81. Figure S3. A) Alignment of the structures for TsXynB (magenta) and GsXynB1 (blue) with a model of XylP81(light green) B) Alignment of all published GH39 β-Xylosidase structures with a model of XylP81 based on the Xanthomonas axonopodis pv. citri structure (6uqj). ...
The estimated 5 × 10 30 prokaryotic cells inhabiting our planet sequester some 350-550 Petagrams ... more The estimated 5 × 10 30 prokaryotic cells inhabiting our planet sequester some 350-550 Petagrams (1 Pg = 1015 g) of carbon, 85-130 Pg of nitrogen, and 9-14 Pg of phosphorous, making them the largest reservoir of those nutrients on Earth (Whitman et al. 1998). However, reports suggest that only less than 1% of these microscopic organisms are cultivable (Torsvik et al. 1990; Sleator et al. 2008). Until recently with the development of metagenomic techniques, the knowledge of microbial diversity and their metabolic capabilities has been limited to this small fraction of cultivable organisms (Handelsman et al. 1998). While metagenomics has undoubtedly revolutionised the field of microbiology and biotechnology it has been generally acknowledged that the current approaches for metagenomic bio-prospecting / screening have limitations which hinder this approach to fully access the metabolic potentials and genetic variations contained in microbial genomes (Beloqui et al. 2008). In particular, the construction of metagenomic libraries and heterologous expression are amongst the major obstacles. The aim of this study was to develop an ultra-high throughput approach for screening enzyme activities using uncloned metagenomic DNA, thereby eliminating cloning steps, and employing in vitro heterologous expression. To achieve this, three widely used techniques: cell-free transcription-translation, in vitro compartmentalisation (IVC) and Fluorescence Activated Cell Sorting (FACS) were combined to develop this robust technique called metagenomic in vitro compartmentalisation (mIVC-FACS). Moreover, the E. coli commercial cell-free system was used in parallel to a novel, in-house Rhodococcus erythropolis based cell-free system. The versatility of this technique was tested by identifying novel beta-xylosidase encoding genes derived from a thermophilic compost metagenome. In addition, the efficiency of mIVC-FACS was compared to the traditional metagenomic approaches; function-based (clone library screening) and sequence-based (shotgun sequencing and PCR screening). List of abbreviation °C Degrees Celcius Amp Ampicillin Amp R Ampicillin resistance gene Bp Base pairs BLAST Basic local alignment search tool BSA Bovine serum albumin CAM Chloramphenicol cAMP cyclic Adenosine monophosphate CAZy Carbohydrate-Active Enzymes database CTAB Cetyl trimethylammonium bromide CFPS Cell-free protein synthesis C-terminus Carboxy terminus ddH 2 O Distilled deionised water dE Double emulsion dH 2 O Deionised water DNA Deoxyribonucleic acid dNTPs Deoxyribonucleotide triphosphates DTT Dithiothreitol EDTA Ethylenediamine tetraacetic acid EtOH Ethanol FACS Fluorescence activated cell sorter g Gram g Gravitational force gDNA Genomic DNA V Volts V i Initial velocity V max Maximum velocity vol Volume v/v Volume per volume w/v Weight per volume XylA 59 XylB 26 hypothetical protein [Elizabethkingia anophelis] 49 xylosidase [Elizabethkingia miricola] 49 xylosidase [Elizabethkingia genomo sp. 2] 49 hypothetical protein SCO7037 [Streptomyces coelicolor A3(2)] 65 XylA 113 XylB 53 hypothetical protein [Elizabethkingia anophelis] 113 xylosidase [Elizabethkingia miricola] 113 xylosidase [Elizabethkingia genomo sp. 2] 113
Magnetosomes, produced by magnetotactic bacteria (MTB), are the most attractive alternative sourc... more Magnetosomes, produced by magnetotactic bacteria (MTB), are the most attractive alternative source of non-toxic biocompatible magnetic nanoparticles (MNPs). A magnetosome contains Fe 2 O 4 magnetite with properties superior to MNPs synthesized by the traditional chemical route. However, synthesis of magnetosomes on large scale has not been achieved yet because magnetotactic bacteria are fastidious to grow. In addition, magnetosomes are generally "soft" magnetic materials which can only be used for some applications, while other applications require "hard" magnetic materials. Here at the Institute of Microbial Biotechnology and Metagenomic (IMBM), a study is being conducted on cloning and expression of the magnetosome gene island (MIA), the genetic machinery for magnetosome formation, in an easy to culture E. coli strain. The magnetic properties of the magnetosome can be manipulated by doping with divalent metals such as Ni 2+ or Co 2+ for a variety of applications. The specific objective of this study was to genetically engineer E. coli strains which accumulate intracellular Ni 2+ or Co 2+ in order to manipulate the magnetic properties of the magnetosomes. Three E. coli mutants and a wild type strain were transformed with high affinity Ni 2+ or Co 2+ uptake genes and evaluated for intracellular accumulation at different medium concentrations of NiCl 2 or CoCl 2. Cellular iron and magnesium were also evaluated because iron is the major component of the magnetosome and magnesium is important for cell growth. The wild type strain, EPI 300 habouring Ni 2+ uptake permease the hoxN gene or Co 2+ uptake ABC type transporter cbiKMQO operon was found to accumulate the most intracellular Ni 2+ or Co 2+ in medium conditions most likely to induce magnetosome formation and magnetite manipulation. This strain can be used to co-express the MIA and v Ni 2+ or Co 2+ uptake gene for mass production of magnetosome with altered magnetic properties.
Background There is a continued need for improved enzymes for industry. β-xylosidases are enzymes... more Background There is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and although many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood. Results Screening of a metagenomic library constructed of mDNA isolated from horse manure compost for β-xylosidase activity identified 26 positive hits. The fosmid clones were sequenced and bioinformatic analysis performed to identity putative β-xylosidases. Based on the novelty of its amino acid sequence and potential thermostability one enzyme (XylP81) was selected for expression and further characterization. XylP81 belongs to the family 39 β-xylosidases, a comparatively rarely found and characterized GH family. The enzyme displayed biochemical characteristics (K M —5.3 mM; V max —122 U/mg; k cat —107; T opt —50 °C...
The estimated 5 × 10 30 prokaryotic cells inhabiting our planet sequester some 350-550 Petagrams ... more The estimated 5 × 10 30 prokaryotic cells inhabiting our planet sequester some 350-550 Petagrams (1 Pg = 1015 g) of carbon, 85-130 Pg of nitrogen, and 9-14 Pg of phosphorous, making them the largest reservoir of those nutrients on Earth (Whitman et al. 1998). However, reports suggest that only less than 1% of these microscopic organisms are cultivable (Torsvik et al. 1990; Sleator et al. 2008). Until recently with the development of metagenomic techniques, the knowledge of microbial diversity and their metabolic capabilities has been limited to this small fraction of cultivable organisms (Handelsman et al. 1998). While metagenomics has undoubtedly revolutionised the field of microbiology and biotechnology it has been generally acknowledged that the current approaches for metagenomic bio-prospecting / screening have limitations which hinder this approach to fully access the metabolic potentials and genetic variations contained in microbial genomes (Beloqui et al. 2008). In particular, the construction of metagenomic libraries and heterologous expression are amongst the major obstacles. The aim of this study was to develop an ultra-high throughput approach for screening enzyme activities using uncloned metagenomic DNA, thereby eliminating cloning steps, and employing in vitro heterologous expression. To achieve this, three widely used techniques: cell-free transcription-translation, in vitro compartmentalisation (IVC) and Fluorescence Activated Cell Sorting (FACS) were combined to develop this robust technique called metagenomic in vitro compartmentalisation (mIVC-FACS). Moreover, the E. coli commercial cell-free system was used in parallel to a novel, in-house Rhodococcus erythropolis based cell-free system. The versatility of this technique was tested by identifying novel beta-xylosidase encoding genes derived from a thermophilic compost metagenome. In addition, the efficiency of mIVC-FACS was compared to the traditional metagenomic approaches; function-based (clone library screening) and sequence-based (shotgun sequencing and PCR screening). List of abbreviation °C Degrees Celcius Amp Ampicillin Amp R Ampicillin resistance gene Bp Base pairs BLAST Basic local alignment search tool BSA Bovine serum albumin CAM Chloramphenicol cAMP cyclic Adenosine monophosphate CAZy Carbohydrate-Active Enzymes database CTAB Cetyl trimethylammonium bromide CFPS Cell-free protein synthesis C-terminus Carboxy terminus ddH 2 O Distilled deionised water dE Double emulsion dH 2 O Deionised water DNA Deoxyribonucleic acid dNTPs Deoxyribonucleotide triphosphates DTT Dithiothreitol EDTA Ethylenediamine tetraacetic acid EtOH Ethanol FACS Fluorescence activated cell sorter g Gram g Gravitational force gDNA Genomic DNA V Volts V i Initial velocity V max Maximum velocity vol Volume v/v Volume per volume w/v Weight per volume XylA 59 XylB 26 hypothetical protein [Elizabethkingia anophelis] 49 xylosidase [Elizabethkingia miricola] 49 xylosidase [Elizabethkingia genomo sp. 2] 49 hypothetical protein SCO7037 [Streptomyces coelicolor A3(2)] 65 XylA 113 XylB 53 hypothetical protein [Elizabethkingia anophelis] 113 xylosidase [Elizabethkingia miricola] 113 xylosidase [Elizabethkingia genomo sp. 2] 113
Background: There is a continued need for improved enzymes for industry. β-xylosidases are enzyme... more Background: There is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and although many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood. Results: Screening of a metagenomic library constructed of mDNA isolated from horse manure compost for β-xylosidase activity identified 26 positive hits. The fosmid clones were sequenced and bioinformatic analysis performed to identity putative β-xylosidases. Based on the novelty of its amino acid sequence and potential thermostability one enzyme (XylP81) was selected for expression and further characterization. XylP81 belongs to the family 39 β-xylosidases, a comparatively rarely found and characterized GH family. The enzyme displayed biochemical characteristics (K M-5.3 mM; V max-122 U/mg; k cat-107; T opt-50 °C; pH opt-6) comparable to previously characterized glycoside hydrolase family 39 (GH39) β-xylosidases and despite nucleotide identity to thermophilic species, the enzyme displayed only moderate thermostability with a half-life of 32 min at 60 °C. Apart from acting on substrates predicted for β-xylosidase (xylobiose and 4-nitrophenyl-β-D-xylopyranoside) the enzyme also displayed measurable α-Larabainofuranosidase, β-galactosidase and β-glucosidase activity. A remarkable feature of this enzyme is its ability to tolerate high concentrations of xylose with a K i of 1.33 M, a feature that is highly desirable for commercial applications. Conclusions: Here we describe a novel β-xylosidase from a poorly studied glycosyl hydrolase family (GH39) which despite having overall kinetic properties similar to other bacterial GH39 β-xylosidases, displays unusually high product tolerance. This trait is shared with only one other member of the GH39 family, the recently described β-xylosidases from Dictyoglomus thermophilum. This feature should allow its use as starting material for engineering of an enzyme that may prove useful to industry and should assist in the fundamental understanding of the mechanism by which glycosyl hydrolases evolve product tolerance.
Magnetosomes, produced by magnetotactic bacteria (MTB), are the most attractive alternative sourc... more Magnetosomes, produced by magnetotactic bacteria (MTB), are the most attractive alternative source of non-toxic biocompatible magnetic nanoparticles (MNPs). A magnetosome contains Fe 2 O 4 magnetite with properties superior to MNPs synthesized by the traditional chemical route. However, synthesis of magnetosomes on large scale has not been achieved yet because magnetotactic bacteria are fastidious to grow. In addition, magnetosomes are generally "soft" magnetic materials which can only be used for some applications, while other applications require "hard" magnetic materials. Here at the Institute of Microbial Biotechnology and Metagenomic (IMBM), a study is being conducted on cloning and expression of the magnetosome gene island (MIA), the genetic machinery for magnetosome formation, in an easy to culture E. coli strain. The magnetic properties of the magnetosome can be manipulated by doping with divalent metals such as Ni 2+ or Co 2+ for a variety of applications. The specific objective of this study was to genetically engineer E. coli strains which accumulate intracellular Ni 2+ or Co 2+ in order to manipulate the magnetic properties of the magnetosomes. Three E. coli mutants and a wild type strain were transformed with high affinity Ni 2+ or Co 2+ uptake genes and evaluated for intracellular accumulation at different medium concentrations of NiCl 2 or CoCl 2. Cellular iron and magnesium were also evaluated because iron is the major component of the magnetosome and magnesium is important for cell growth. The wild type strain, EPI 300 habouring Ni 2+ uptake permease the hoxN gene or Co 2+ uptake ABC type transporter cbiKMQO operon was found to accumulate the most intracellular Ni 2+ or Co 2+ in medium conditions most likely to induce magnetosome formation and magnetite manipulation. This strain can be used to co-express the MIA and v Ni 2+ or Co 2+ uptake gene for mass production of magnetosome with altered magnetic properties.
Additional file 1: Figure S1. SDS-PAGE analysis XylP81 expression. A) Lane M: ColorPlus prestaine... more Additional file 1: Figure S1. SDS-PAGE analysis XylP81 expression. A) Lane M: ColorPlus prestained protein ladder, broad range (10-230 kDa); lane 1: E. coli-pET21a no insert uninduced; lane 2: E. coli-pETP81 uninduced soluble fraction; lane 3: E. coli-pETP81 induced soluble fraction; lane 4: E. coli-pET21a no insert induced; lane 5: E. coli-pETP81 uninduced insoluble fraction; lane 6: E. coli-pETP81 induced insoluble fraction. B) Purified XylP81 following metal affinity chromatography purification. lane M: ColorPlus prestained protein ladder. Figure S2. Unrooted maximum likelihood phylogenetic tree of characterized GH39 amino acid sequences including XylP81, excluding unchracterized and sequences closely related to XylP81. Figure S3. A) Alignment of the structures for TsXynB (magenta) and GsXynB1 (blue) with a model of XylP81(light green) B) Alignment of all published GH39 β-Xylosidase structures with a model of XylP81 based on the Xanthomonas axonopodis pv. citri structure (6uqj). ...
The estimated 5 × 10 30 prokaryotic cells inhabiting our planet sequester some 350-550 Petagrams ... more The estimated 5 × 10 30 prokaryotic cells inhabiting our planet sequester some 350-550 Petagrams (1 Pg = 1015 g) of carbon, 85-130 Pg of nitrogen, and 9-14 Pg of phosphorous, making them the largest reservoir of those nutrients on Earth (Whitman et al. 1998). However, reports suggest that only less than 1% of these microscopic organisms are cultivable (Torsvik et al. 1990; Sleator et al. 2008). Until recently with the development of metagenomic techniques, the knowledge of microbial diversity and their metabolic capabilities has been limited to this small fraction of cultivable organisms (Handelsman et al. 1998). While metagenomics has undoubtedly revolutionised the field of microbiology and biotechnology it has been generally acknowledged that the current approaches for metagenomic bio-prospecting / screening have limitations which hinder this approach to fully access the metabolic potentials and genetic variations contained in microbial genomes (Beloqui et al. 2008). In particular, the construction of metagenomic libraries and heterologous expression are amongst the major obstacles. The aim of this study was to develop an ultra-high throughput approach for screening enzyme activities using uncloned metagenomic DNA, thereby eliminating cloning steps, and employing in vitro heterologous expression. To achieve this, three widely used techniques: cell-free transcription-translation, in vitro compartmentalisation (IVC) and Fluorescence Activated Cell Sorting (FACS) were combined to develop this robust technique called metagenomic in vitro compartmentalisation (mIVC-FACS). Moreover, the E. coli commercial cell-free system was used in parallel to a novel, in-house Rhodococcus erythropolis based cell-free system. The versatility of this technique was tested by identifying novel beta-xylosidase encoding genes derived from a thermophilic compost metagenome. In addition, the efficiency of mIVC-FACS was compared to the traditional metagenomic approaches; function-based (clone library screening) and sequence-based (shotgun sequencing and PCR screening). List of abbreviation °C Degrees Celcius Amp Ampicillin Amp R Ampicillin resistance gene Bp Base pairs BLAST Basic local alignment search tool BSA Bovine serum albumin CAM Chloramphenicol cAMP cyclic Adenosine monophosphate CAZy Carbohydrate-Active Enzymes database CTAB Cetyl trimethylammonium bromide CFPS Cell-free protein synthesis C-terminus Carboxy terminus ddH 2 O Distilled deionised water dE Double emulsion dH 2 O Deionised water DNA Deoxyribonucleic acid dNTPs Deoxyribonucleotide triphosphates DTT Dithiothreitol EDTA Ethylenediamine tetraacetic acid EtOH Ethanol FACS Fluorescence activated cell sorter g Gram g Gravitational force gDNA Genomic DNA V Volts V i Initial velocity V max Maximum velocity vol Volume v/v Volume per volume w/v Weight per volume XylA 59 XylB 26 hypothetical protein [Elizabethkingia anophelis] 49 xylosidase [Elizabethkingia miricola] 49 xylosidase [Elizabethkingia genomo sp. 2] 49 hypothetical protein SCO7037 [Streptomyces coelicolor A3(2)] 65 XylA 113 XylB 53 hypothetical protein [Elizabethkingia anophelis] 113 xylosidase [Elizabethkingia miricola] 113 xylosidase [Elizabethkingia genomo sp. 2] 113
Magnetosomes, produced by magnetotactic bacteria (MTB), are the most attractive alternative sourc... more Magnetosomes, produced by magnetotactic bacteria (MTB), are the most attractive alternative source of non-toxic biocompatible magnetic nanoparticles (MNPs). A magnetosome contains Fe 2 O 4 magnetite with properties superior to MNPs synthesized by the traditional chemical route. However, synthesis of magnetosomes on large scale has not been achieved yet because magnetotactic bacteria are fastidious to grow. In addition, magnetosomes are generally "soft" magnetic materials which can only be used for some applications, while other applications require "hard" magnetic materials. Here at the Institute of Microbial Biotechnology and Metagenomic (IMBM), a study is being conducted on cloning and expression of the magnetosome gene island (MIA), the genetic machinery for magnetosome formation, in an easy to culture E. coli strain. The magnetic properties of the magnetosome can be manipulated by doping with divalent metals such as Ni 2+ or Co 2+ for a variety of applications. The specific objective of this study was to genetically engineer E. coli strains which accumulate intracellular Ni 2+ or Co 2+ in order to manipulate the magnetic properties of the magnetosomes. Three E. coli mutants and a wild type strain were transformed with high affinity Ni 2+ or Co 2+ uptake genes and evaluated for intracellular accumulation at different medium concentrations of NiCl 2 or CoCl 2. Cellular iron and magnesium were also evaluated because iron is the major component of the magnetosome and magnesium is important for cell growth. The wild type strain, EPI 300 habouring Ni 2+ uptake permease the hoxN gene or Co 2+ uptake ABC type transporter cbiKMQO operon was found to accumulate the most intracellular Ni 2+ or Co 2+ in medium conditions most likely to induce magnetosome formation and magnetite manipulation. This strain can be used to co-express the MIA and v Ni 2+ or Co 2+ uptake gene for mass production of magnetosome with altered magnetic properties.
Background There is a continued need for improved enzymes for industry. β-xylosidases are enzymes... more Background There is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and although many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood. Results Screening of a metagenomic library constructed of mDNA isolated from horse manure compost for β-xylosidase activity identified 26 positive hits. The fosmid clones were sequenced and bioinformatic analysis performed to identity putative β-xylosidases. Based on the novelty of its amino acid sequence and potential thermostability one enzyme (XylP81) was selected for expression and further characterization. XylP81 belongs to the family 39 β-xylosidases, a comparatively rarely found and characterized GH family. The enzyme displayed biochemical characteristics (K M —5.3 mM; V max —122 U/mg; k cat —107; T opt —50 °C...
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