Cowan DA

References (7)

Title : Structural characterization and directed evolution of a novel acetyl xylan esterase reveals thermostability determinants of the Carbohydrate Esterase 7 family - Adesioye_2018_Appl.Environ.Microbiol_84_e02695
Author(s) : Adesioye FA , Makhalanyane TP , Vikram S , Sewell BT , Schubert WD , Cowan DA
Ref : Applied Environmental Microbiology , 84 :e02695 , 2018
Abstract : A hot desert hypolith metagenomic DNA sequence dataset was screened in-silico for genes annotated as acetyl xylan esterases (AcXEs). One of the genes identified encoded a approximately 36 kDa protein (Axe1NaM1). The synthesised gene was cloned, expressed and the resulting protein, purified. NaM1 was optimally active at pH 8.5 and 30 degrees C, and functionally stable at salt concentrations up to 5 M. The specific activity and catalytic efficiency were 488.9 Umg(-1) and 3.26x10(6) M(-1)s(-1), respectively. The crystal structure of wild type NaM1 was solved at a resolution of 2.03 A and a comparison with the structures and models of more thermostable carbohydrate esterase (CE) 7 family enzymes and variants of NaM1 from a directed evolution experiment, respectively, suggest that reduced side chain volume of protein core residues is relevant to the thermal stability of NaM1. Surprisingly, a single point mutation (N96S), not only resulted in a simultaneous improvement in thermal stability and catalytic efficiency, but also increased the acyl moiety substrate range of NaM1.IMPORTANCE Acetyl xylan esterases (AcXEs) belong to nine carbohydrate esterase families (CE 1-7, 12 and 16), of which CE7 enzymes possess a unique and narrow specificity for acetylated substrates. All structurally characterised members of this family are moderately to highly thermostable. The crystal structure of a novel, mesophilic CE7 AcXE (Axe1NaM1), from a soil metagenome, provides a basis for comparisons with thermostable CE7 enzymes. Using error-prone polymerase chain reaction (PCR) and site-directed mutagenesis, we enhanced both the stability and activity of the mesophilic AcXE. With comparative structural analyses, we have also identified possible thermal stability determinants. These are valuable for understanding the thermal stability of enzymes within this family and as a guide for future protein engineering of CE7 and other alpha/beta hydrolase enzymes.
ESTHER : Adesioye_2018_Appl.Environ.Microbiol_84_e02695
PubMedSearch : Adesioye_2018_Appl.Environ.Microbiol_84_e02695
PubMedID: 29453256
Gene_locus related to this paper: 9zzzz-Axe1NaM1 , 9zzzz-Axe1NaM2

Title : Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases - Adesioye_2016_Enzyme.Microb.Technol_93-94_79
Author(s) : Adesioye FA , Makhalanyane TP , Biely P , Cowan DA
Ref : Enzyme Microb Technol , 93-94 :79 , 2016
Abstract : Acetyl xylan esterases (AcXEs), also termed xylan deacetylases, are broad specificity Carbohydrate-Active Enzymes (CAZymes) that hydrolyse ester bonds to liberate acetic acid from acetylated hemicellulose (typically polymeric xylan and xylooligosaccharides). They belong to eight families within the Carbohydrate Esterase (CE) class of the CAZy database. AcXE classification is largely based on sequence-dependent phylogenetic relationships, supported in some instances with substrate specificity data. However, some sequence-based predictions of AcXE-encoding gene identity have proved to be functionally incorrect. Such ambiguities can lead to mis-assignment of genes and enzymes during sequence data-mining, reinforcing the necessity for the experimental confirmation of the functional properties of putative AcXE-encoding gene products. Although one-third of all characterized CEs within CAZy families 1-7 and 16 are AcXEs, there is a need to expand the sequence database in order to strengthen the link between AcXE gene sequence and specificity. Currently, most AcXEs are derived from a limited range of (mostly microbial) sources and have been identified via culture-based bioprospecting methods, restricting current knowledge of AcXEs to data from relatively few microbial species. More recently, the successful identification of AcXEs via genome and metagenome mining has emphasised the huge potential of culture-independent bioprospecting strategies. We note, however, that the functional metagenomics approach is still hampered by screening bottlenecks. The most relevant recent reviews of AcXEs have focused primarily on the biochemical and functional properties of these enzymes. In this review, we focus on AcXE phylogeny, classification and the future of metagenomic bioprospecting for novel AcXEs.
ESTHER : Adesioye_2016_Enzyme.Microb.Technol_93-94_79
PubMedSearch : Adesioye_2016_Enzyme.Microb.Technol_93-94_79
PubMedID: 27702488

Title : The Geobacillus Pan-Genome: Implications for the Evolution of the Genus - Bezuidt_2016_Front.Microbiol_7_723
Author(s) : Bezuidt OK , Pierneef R , Gomri AM , Adesioye F , Makhalanyane TP , Kharroub K , Cowan DA
Ref : Front Microbiol , 7 :723 , 2016
Abstract : The genus Geobacillus is comprised of a diverse group of spore-forming Gram-positive thermophilic bacterial species and is well known for both its ecological diversity and as a source of novel thermostable enzymes. Although the mechanisms underlying the thermophilicity of the organism and the thermostability of its macromolecules are reasonably well understood, relatively little is known of the evolutionary mechanisms, which underlie the structural and functional properties of members of this genus. In this study, we have compared 29 Geobacillus genomes, with a specific focus on the elements, which comprise the conserved core and flexible genomes. Based on comparisons of conserved core and flexible genomes, we present evidence of habitat delineation with specific Geobacillus genomes linked to specific niches. Our analysis revealed that Geobacillus and Anoxybacillus share a high proportion of genes. Moreover, the results strongly suggest that horizontal gene transfer is a major factor deriving the evolution of Geobacillus from Bacillus, with genetic contributions from other phylogenetically distant taxa.
ESTHER : Bezuidt_2016_Front.Microbiol_7_723
PubMedSearch : Bezuidt_2016_Front.Microbiol_7_723
PubMedID: 27252683

Title : Draft Genome Sequence of Williamsia sp. Strain D3, Isolated From the Darwin Mountains, Antarctica - Guerrero_2014_Genome.Announc_2_e01230
Author(s) : Guerrero LD , Makhalanyane TP , Aislabie JM , Cowan DA
Ref : Genome Announc , 2 : , 2014
Abstract : Actinobacteria are the dominant taxa in Antarctic desert soils. Here, we describe the first draft genome of a member of the genus Williamsia (strain D3) isolated from Antarctic soil. The genome of this psychrotolerant bacterium may help to elucidate crucial survival mechanisms for organisms inhabiting cold desert soil systems.
ESTHER : Guerrero_2014_Genome.Announc_2_e01230
PubMedSearch : Guerrero_2014_Genome.Announc_2_e01230
PubMedID: 24459282
Gene_locus related to this paper: 9acto-v8cz58 , 9acto-v8cun2 , 9acto-v8d0d9 , 9actn-v8ctk1 , 9actn-v8d1p4

Title : Draft Genome Sequence of the Antarctic Polyextremophile Nesterenkonia sp. Strain AN1 - Aliyu_2014_Genome.Announc_2_e00197
Author(s) : Aliyu H , De Maayer P , Rees J , Tuffin M , Cowan DA
Ref : Genome Announc , 2 : , 2014
Abstract : Nesterenkonia sp. strain AN1 was isolated from Antarctic soil and is a polyextremophile, being tolerant of low temperatures, high salt concentrations, and high alkalinity. Here we report the draft genome sequence of this strain.
ESTHER : Aliyu_2014_Genome.Announc_2_e00197
PubMedSearch : Aliyu_2014_Genome.Announc_2_e00197
PubMedID: 24675854
Gene_locus related to this paper: 9micc-a0a010ngc6

Title : A novel recombinant ethyl ferulate esterase from Burkholderia multivorans - Rashamuse_2007_J.Appl.Microbiol_103_1610
Author(s) : Rashamuse KJ , Burton SG , Cowan DA
Ref : J Appl Microbiol , 103 :1610 , 2007
Abstract : AIMS: Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity. METHODS AND
RESULTS: A micro-organism with ethyl ferulate hydrolysing (EFH) activity was isolated by culture enrichment techniques. Detailed molecular identification based on species-specific primers and two conserved genes (16S rRNA and recA) led to the identification of the isolate as Burkholderia multivorans UWC10. A gene (designated estEFH5) encoding an EFH enzyme was cloned and its nucleotide sequence determined. Translational analysis revealed that estEFH5 encoded a polypeptide of 326 amino acids with an estimated molecular weight of 34.83 kDa. The EstEFH5 primary structure showed a typical serine hydrolase motif (G-H-S-L-G). The estEFH5 gene was over-expressed in Escherichia coli in an insoluble form. Following urea denaturation and in vitro refolding, the enzyme was purified using one-step His Select Nickel chromatographic column. CONCLUSION: Purified EstEFH5 showed a preference for short-chain rho-nitrophenyl esters (C2 and C3) a typical feature for carboxylesterase. Furthermore, the recombinant enzyme also retained the activity against ethyl ferulate (EF). SIGNIFICANCE AND IMPACT OF THE STUDY: A biocatalytic process for the production of FA from EF as a model substrate was demonstrated. This is the first report that describes the cloning and expression of a gene encoding FA esterase activity from the genus Burkholderia.
ESTHER : Rashamuse_2007_J.Appl.Microbiol_103_1610
PubMedSearch : Rashamuse_2007_J.Appl.Microbiol_103_1610
PubMedID: 17953572

Title : Purification and partial characterization of a novel thermophilic carboxylesterase with high mesophilic specific activity - Wood_1995_Enzyme.Microb.Technol_17_816
Author(s) : Wood AN , Fernandez-Lafuente R , Cowan DA
Ref : Enzyme Microb Technol , 17 :816 , 1995
Abstract : An esterase activity obtained from a strain of Bacillus stearothermophilus was purified 5,133-fold to electrophoretic homogeneity with 26% recovery. The purified esterase had a specific activity of 2,032 mumol min-1 mg-1 based on the hydrolysis of p-nitrophenyl caproate at pH 7.0 and 30 degrees C. The apparent molecular mass was 50,000 +/- 2,000 daltons from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45,000 +/- 3,000 daltons from gel filtration. Native polyacrylamide gels stained for esterase activity showed three bands. The isoelectric points were estimated to be 5.7, 5.8, and 6.0. Forty amino acid residues were sequenced at the N-terminus. The sequence showed no degeneracy, suggesting that the three esterases are functionally identical carboxylesterases differing by a limited number of amino acids. The enzyme showed maximum activity at pH 7.0 and was very stable at pH 6.0-8.9 with optimum stability at pH 6.0. At this pH and 60 degrees C the half-life was 170 h. Esterase activity was totally inhibited by phenylmethanesulfonyl fluoride, parahydroxymercuribenzoate, eserine, and tosyl-L-phenylalanine, but not by ethylendiaminetetra acetic acid. The esterase obeyed Michaelis-Menten kinetics in the hydrolysis of p-nitrophenyl esters, but both Vmax and KM were protein concentration-dependent. The esterase was able to hydrolyse a number of p-nitrophenyl derivatives (amino acid derivatives and aliphatic acids with different chain lengths).
ESTHER : Wood_1995_Enzyme.Microb.Technol_17_816
PubMedSearch : Wood_1995_Enzyme.Microb.Technol_17_816
PubMedID: 7576531
Gene_locus related to this paper: geost-est50