(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Proteobacteria: NE > Gammaproteobacteria: NE > Pseudomonadales: NE > Moraxellaceae: NE > Moraxella: NE > Moraxella sp.: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MPILPVPALNALLTKTIKTIKTGAAKNAHQHHVLHHTLKGLDNLPAPVLE RINRRLKASTAEQYPLADAHLRLILAISNKLKRPLAIDKLPKLRQKFGTD AVSLQAPSVWQQNADASGSTENAVSWQDKTIANADGGDMTVRCYQKSTQN SERKSTDEAAMLFFHGGGFCIGDIDTHHEFCHTVCAQTGWAVVSVDYRMA PEYPAPTALKDCLAAYAWLAEHSQSLGASPSRIVLSGDSAGGCLAALVAQ QVIKPIDALWQDNNQAPAADKKVNDTFKNSLADLPRPLAQLPLYPVTDYE AEYPSWELYGEGLLLDHNDAEVFNSAYTQHSGLPQSHPLISVMHGDNTQL CPSYIVVAELDILRDEGLAYAELLQKEGVQVQTYTVLGAPHGFINLMSVH QGLGNQTTYIINEFACLVQNLLTSEGDKPNLRA
References
Title: Native expression and purification of hormone-sensitive lipase from Psychrobacter sp. TA144 enhances protein stability and activity Ascione G, de Pascale D, De Santi C, Pedone C, Dathan NA, Monti SM Ref: Biochemical & Biophysical Research Communications, 420:542, 2012 : PubMed
Psychrobacter, a micro-organism originally isolated from Antarctic sea water, expresses an extremely active hormone-sensitive lipase (HSL) which catalyzes the hydrolysis of fatty acid esters at very low temperature and is therefore of great potential industrial and pharmaceutical interest. An insoluble form of the entire enzyme has previously been cloned and expressed in Escherichia coli, subsequently refolded and shown to be active, whilst a shorter but completely inactive version, lacking the N-terminal 98 amino acids has been expressed in soluble form. In this study the entire enzyme has been expressed as a fully soluble protein in E. coli in the presence of either the osmolyte trehalose, plus high salt concentration, or the membrane fluidizer benzyl alcohol. Trehalose promotes protein mono-dispersion by increasing the viscosity of the growth medium for bacterial cells, thereby helping circumvent protein aggregation, whilst the heat-shock inducer benzyl alcohol stimulates the production of a network of endogenous chaperones which actively prevent protein misfolding, whilst also converting recombinant aggregates to native, correctly folded proteins. The resultant recombinant protein proved to be more stable than its previously expressed counterpart, as shown by CD and enzymatic activity data which proved the enzyme to be more active at a higher temperature than its refolded counterpart. By light scattering analysis it was shown that the newly expressed protein was monomeric. The stability of the full length native protein will help in understanding the structure of PsyHSL and the role of its regulatory N-terminal for eventual application in a myriad of biotechnological processes.
Cold-adapted esterases and lipases have been found to be dominant activities throughout the cold marine environment, indicating their importance in bacterial degradation of the organic matter. lip2 Gene from Psychrobacter sp. TA144, a micro-organism isolated from the Antarctic sea water, was cloned and over-expressed in Escherichia coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from which it was recovered in active form, purified to homogeneity and deeply characterised. Temperature dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 degrees C at pH 8.0. The enzyme resulted to be active on pNP-esters of fatty acids with acyl chain length from C(2) to C(12) and the preferred substrate was pNP-pentanoate showing a k(cat) = 26.2 +/- 0.1 s(-1), K(M) = 0.122 +/- 0.006 mM and a k(cat)/K(M) = 215 +/- 11 mM(-1) s(-1). The enzyme was strongly inhibited by Hg(2+), Zn(2+), Cu(2+), Fe(3+), Mn(2+) ions and it resulted to be activated in presence of methanol and acetonitrile, with calculated C(50) values of 1.98 M and 0.92 M, respectively. The region surrounding PsyHSL catalytic site showed an unexpected homology with the human HSL. Further, both enzymes are characterised by the presence of an extra N-terminal domain, which role in the human protein has been related to regulative function. To clarify the function of PsyHSL N-terminal domain, a 97 amino acids deleted version of the enzyme was produced in E. coli in soluble form, purified and characterised. This mutant was inactive towards all tested substrates, indicating the involvement of this region in the catalytic process.
Title: Nucleotide sequence of the lipase gene lip2 from the antarctic psychrotroph Moraxella TA144 and site-specific mutagenesis of the conserved serine and histidine residues Feller G, Thiry M, Gerday C Ref: DNA & Cell Biology, 10:381, 1991 : PubMed
The lip2 gene from the antarctic psychotroph Moraxella TA144 was sequenced. The primary structure of the Lip2 preprotein deduced from the nucleotide sequence is composed of 433 amino acids with a predicted Mr of 47,222. This enzyme contains a Ser-centered consensus sequence and a conserved His-Gly dipeptide found in most lipase amino-terminal domains. These sequences are involved in the lipase active site conformation since substitution of the conserved Ser or His residues by Ala and Gln, respectively, results in the loss of both lipase and esterase activities. Structural factors that would allow proper enzyme flexibility at low temperatures are discussed. It is suggested that only subtle changes in the primary structure of these psychrotrophic enzymes can account for their ability to catalyze lipolysis at temperatures close to 0 degrees C.
Moraxella L1 gene for lipase 1 (EC 3.1.1.3) PET8 MoPE