Willassen NP

References (6)

Title : Biochemical characterization and structural analysis of a new cold-active and salt-tolerant esterase from the marine bacterium Thalassospira sp - De Santi_2016_Extremophiles_20_323
Author(s) : De Santi C , Leiros HK , Di Scala A , de Pascale D , Altermark B , Willassen NP
Ref : Extremophiles , 20 :323 , 2016
Abstract : A gene encoding an esterase, ThaEst2349, was identified in the marine psychrophilic bacterium Thalassospira sp. GB04J01. The gene was cloned and overexpressed in E. coli as a His-tagged fusion protein. The recombinant enzyme showed optimal activity at 45 degrees C and the thermal stability displayed a retention of 75 % relative activity at 40 degrees C after 2 h. The optimal pH was 8.5 but the enzyme kept more than 75 % of its maximal activity between pH 8.0 and 9.5. ThaEst2349 also showed remarkable tolerance towards high concentrations of salt and it was active against short-chain p-nitrophenyl esters, displaying optimal activity with the acetate. The enzyme was tested for tolerance of organic solvents and the results are suggesting that it could function as an interesting candidate for biotechnological applications. The crystal structure of ThaEst2349 was determined to 1.69 A revealing an asymmetric unit containing two chains, which also is the biological unit. The structure has a characteristic cap domain and a catalytic triad comprising Ser158, His285 and Asp255. To explain the cold-active nature of the enzyme, we compared it against thermophilic counterparts. Our hypothesis is that a high methionine content, less hydrogen bonds and less ion pairs render the enzyme more flexible at low temperatures.
ESTHER : De Santi_2016_Extremophiles_20_323
PubMedSearch : De Santi_2016_Extremophiles_20_323
PubMedID: 27016194
Gene_locus related to this paper: 9prot-a0a023t3x2

Title : Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome - De Santi_2016_PLoS.One_11_e0159345
Author(s) : De Santi C , Willassen NP , Williamson A
Ref : PLoS ONE , 11 :e0159345 , 2016
Abstract : BACKGROUND: The glucuronoyl esterase enzymes of wood-degrading fungi (Carbohydrate Esterase family 15; CE15) form part of the hemicellulolytic and cellulolytic enzyme systems that break down plant biomass, and have possible applications in biotechnology. Homologous enzymes are predicted in the genomes of several bacteria, however these have been much less studied than their fungal counterparts. Here we describe the recombinant production and biochemical characterization of a bacterial CE15 enzyme denoted MZ0003, which was identified by in silico screening of a prokaryotic metagenome library derived from marine Arctic sediment. MZ0003 has high similarity to several uncharacterized gene products of polysaccharide-degrading bacterial species, and phylogenetic analysis indicates a deep evolutionary split between these CE15s and fungal homologs. RESULTS: MZ0003 appears to differ from previously-studied CE15s in some aspects. Some glucuronoyl esterase activity could be measured by qualitative thin-layer chromatography which confirms its assignment as a CE15, however MZ0003 can also hydrolyze a range of other esters, including p-nitrophenyl acetate, which is not acted upon by some fungal homologs. The structure of MZ0003 also appears to differ as it is predicted to have several large loop regions that are absent in previously studied CE15s, and a combination of homology-based modelling and site-directed mutagenesis indicate its catalytic residues deviate from the conserved Ser-His-Glu triad of many fungal CE15s. Taken together, these results indicate that potentially unexplored diversity exists among bacterial CE15s, and this may be accessed by investigation of the microbial metagenome. The combination of low activity on typical glucuronoyl esterase substrates, and the lack of glucuronic acid esters in the marine environment suggest that the physiological substrate of MZ0003 and its homologs is likely to be different from that of related fungal enzymes.
ESTHER : De Santi_2016_PLoS.One_11_e0159345
PubMedSearch : De Santi_2016_PLoS.One_11_e0159345
PubMedID: 27433797
Gene_locus related to this paper: unkp-CE15

Title : Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries - De Santi_2016_BMC.Biochem_17_1
Author(s) : De Santi C , Altermark B , Pierechod MM , Ambrosino L , de Pascale D , Willassen NP
Ref : BMC Biochem , 17 :1 , 2016
Abstract : BACKGROUND: The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields.
RESULTS: Here we present a novel esterase gene (lip3) identified by functional screening of three fosmid metagenomic libraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281 amino acids with similarity to class 3 lipases. The 3D modeling of Lip3 was generated by homology modeling on the basis of four lipases templates [PDB ID: 3O0D, 3NGM, 3G7N, 2QUB] to unravel structural features of this novel enzyme. The catalytic triad of Lip3 was predicted to be Asp207, His267 and the catalytic nucleophile Ser150 in a conserved pentapeptide (GXSXG). The 3D model highlighted the presence of a one-helix lid able to regulate the access of the substrate to the active site when the enzyme binds a hydrophobic interface. Moreover an analysis of the external surface of Lip3 model showed that the majority of the surface regions were hydrophobic (59.6 %) compared with homologous lipases (around 35 %) used as templates. The recombinant Lip3 esterase, expressed and purified from Escherichia coli, preferentially hydrolyzed short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate. Further characterization revealed a temperature optimum of 35 degrees C and a pH optimum of 8.0. Lip3 exhibits a broad temperature stability range and tolerates the presence of DTT, EDTA, PMSF, beta-mercaptoethanol and high concentrations of salt. The enzyme was also highly activated by NaCl.
CONCLUSIONS: The biochemical characterization and homology model reveals a novel esterase originating from the marine Arctic metagenomics libraries with features of a cold-active, relatively thermostable and highly halotolerant enzyme. Taken together, these results suggest that this esterase could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions and cheese ripening processes.
ESTHER : De Santi_2016_BMC.Biochem_17_1
PubMedSearch : De Santi_2016_BMC.Biochem_17_1
PubMedID: 26782782

Title : High quality draft genome sequence of Streptomyces sp. strain AW19M42 isolated from a sea squirt in Northern Norway - Bjerga_2014_Stand.Genomic.Sci_9_676
Author(s) : Bjerga GE , Hjerde E , De Santi C , Williamson AK , Smalas AO , Willassen NP , Altermark B
Ref : Stand Genomic Sci , 9 :676 , 2014
Abstract : Here we report the 8 Mb high quality draft genome of Streptomyces sp. strain AW19M42, together with specific properties of the organism and the generation, annotation and analysis of its genome sequence. The genome encodes 7,727 putative open reading frames, of which 6,400 could be assigned with COG categories. Also, 62 tRNA genes and 8 rRNA operons were identified. The genome harbors several gene clusters involved in the production of secondary metabolites. Functional screening of the isolate was positive for several enzymatic activities, and some candidate genes coding for those activities are listed in this report. We find that this isolate shows biotechnological potential and is an interesting target for bioprospecting.
ESTHER : Bjerga_2014_Stand.Genomic.Sci_9_676
PubMedSearch : Bjerga_2014_Stand.Genomic.Sci_9_676
PubMedID: 25197453

Title : A new alkaliphilic cold-active esterase from the psychrophilic marine bacterium Rhodococcus sp.: functional and structural studies and biotechnological potential - De Santi_2014_Appl.Biochem.Biotechnol_172_3054
Author(s) : De Santi C , Tedesco P , Ambrosino L , Altermark B , Willassen NP , de Pascale D
Ref : Appl Biochem Biotechnol , 172 :3054 , 2014
Abstract : The special features of cold-adapted lipolytic biocatalysts have made their use possible in several industrial applications. In fact, cold-active enzymes are known to be able to catalyze reactions at low temperatures, avoiding side reactions taking place at higher temperatures and preserving the integrity of products. A lipolytic gene was isolated from the Arctic marine bacterium Rhodococcus sp. AW25M09 and expressed in Escherichia coli as inclusion bodies. The recombinant enzyme (hereafter called RhLip) showed interesting cold-active esterase activity. The refolded purified enzyme displayed optimal activity at 30 degrees C and was cold-active with retention of 50% activity at 10 degrees C. It is worth noting that the optimal pH was 11, and the low relative activity below pH 10 revealed that RhLip was an alkaliphilic esterase. The enzyme was active toward short-chain p-nitrophenyl esters (C2-C6), displaying optimal activity with the butyrate (C4) ester. In addition, the enzyme revealed a good organic solvent and salt tolerance. These features make this an interesting enzyme for exploitation in some industrial applications.
ESTHER : De Santi_2014_Appl.Biochem.Biotechnol_172_3054
PubMedSearch : De Santi_2014_Appl.Biochem.Biotechnol_172_3054
PubMedID: 24488777

Title : The genome sequence of the fish pathogen Aliivibrio salmonicida strain LFI1238 shows extensive evidence of gene decay - Hjerde_2008_BMC.Genomics_9_616
Author(s) : Hjerde E , Lorentzen MS , Holden MT , Seeger K , Paulsen S , Bason N , Churcher C , Harris D , Norbertczak H , Quail MA , Sanders S , Thurston S , Parkhill J , Willassen NP , Thomson NR
Ref : BMC Genomics , 9 :616 , 2008
Abstract : BACKGROUND: The fish pathogen Aliivibrio salmonicida is the causative agent of cold-water vibriosis in marine aquaculture. The Gram-negative bacterium causes tissue degradation, hemolysis and sepsis in vivo.
RESULTS: In total, 4 286 protein coding sequences were identified, and the 4.6 Mb genome of A. salmonicida has a six partite architecture with two chromosomes and four plasmids. Sequence analysis revealed a highly fragmented genome structure caused by the insertion of an extensive number of insertion sequence (IS) elements. The IS elements can be related to important evolutionary events such as gene acquisition, gene loss and chromosomal rearrangements. New A. salmonicida functional capabilities that may have been aquired through horizontal DNA transfer include genes involved in iron-acquisition, and protein secretion and play potential roles in pathogenicity. On the other hand, the degeneration of 370 genes and consequent loss of specific functions suggest that A. salmonicida has a reduced metabolic and physiological capacity in comparison to related Vibrionaceae species. CONCLUSION: Most prominent is the loss of several genes involved in the utilisation of the polysaccharide chitin. In particular, the disruption of three extracellular chitinases responsible for enzymatic breakdown of chitin makes A. salmonicida unable to grow on the polymer form of chitin. These, and other losses could restrict the variety of carrier organisms A. salmonicida can attach to, and associate with. Gene acquisition and gene loss may be related to the emergence of A. salmonicida as a fish pathogen.
ESTHER : Hjerde_2008_BMC.Genomics_9_616
PubMedSearch : Hjerde_2008_BMC.Genomics_9_616
PubMedID: 19099551
Gene_locus related to this paper: alisl-b6elz2 , alisl-b6ek45