scholarly journals Identification and Biochemical Characterization of a Novel Hormone-Sensitive Lipase Family Esterase Est19 from the Antarctic Bacterium Pseudomonas sp. E2-15

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1552
Author(s):  
Xiaoyu Liu ◽  
Mingyang Zhou ◽  
Shu Xing ◽  
Tao Wu ◽  
Hailun He ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Catalytic Triad ◽  
Hormone Sensitive Lipase ◽  
Substrate Affinity ◽  
Pseudomonas Sp ◽  
Antarctic Bacterium ◽  
Hormone Sensitive ◽  
The Antarctic

Esterases represent an important class of enzymes with a wide variety of industrial applications. A novel hormone-sensitive lipase (HSL) family esterase, Est19, from the Antarctic bacterium Pseudomonas sp. E2-15 is identified, cloned, and expressed. The enzyme possesses a GESAG motif containing an active serine (S) located within a highly conserved catalytic triad of Ser155, Asp253, and His282 residues. The catalytic efficiency (kcat/Km) of Est19 for the pNPC6 substrate is 148.68 s−1mM−1 at 40 °C. Replacing Glu154 juxtaposed to the critical catalytic serine with Asp (E154→D substitution) reduced the activity and catalytic efficiency of the enzyme two-fold, with little change in the substrate affinity. The wild-type enzyme retained near complete activity over a temperature range of 10–60 °C, while ~50% of its activity was retained at 0 °C. A phylogenetic analysis suggested that Est19 and its homologs may represent a new subfamily of HSL. The thermal stability and stereo-specificity suggest that the Est19 esterase may be useful for cold and chiral catalyses.

scholarly journals Domain-structure analysis of recombinant rat hormone-sensitive lipase

1996 ◽  
Vol 319 (2) ◽  
pp. 411-420 ◽  
Author(s):  
Torben ØSTERLUND ◽  
Birgitta DANIELSSON ◽  
Eva DEGERMAN ◽  
Juan Antonio CONTRERAS ◽  
Gudrun EDGREN ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Domain Structure ◽  
Expression System ◽  
Guanidine Hydrochloride ◽  
Catalytic Triad ◽  
Water Soluble ◽  
Hormone Sensitive Lipase ◽  
Structural Domain ◽  
Hydrolase Fold ◽  
Hormone Sensitive

Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the α/β-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and α/β-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains.

scholarly journals The first description of a hormone-sensitive lipase from a basidiomycete: Structural insights and biochemical characterization revealedBjerkandera adusta BaEstB as a novel esterase

2017 ◽  
Vol 6 (4) ◽  
pp. e00463 ◽  
Author(s):  
María del Rayo Sánchez-Carbente ◽  
Ramón Alberto Batista-García ◽  
Ayixón Sánchez-Reyes ◽  
Angela Escudero-Garcia ◽  
Catalina Morales-Herrera ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Hormone Sensitive Lipase ◽  
Novel Esterase ◽  
Hormone Sensitive ◽  
Structural Insights

scholarly journals Thermal Inactivation of a Cold-Active Esterase PMGL3 Isolated from the Permafrost Metagenomic Library

Biomolecules ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 880 ◽  
Author(s):  
M.V. Kryukova ◽  
L.E. Petrovskaya ◽  
E.A. Kryukova ◽  
G.Yu. Lomakina ◽  
S.A. Yakimov ◽  
...  
Keyword(s):  
Thermal Inactivation ◽  
Elevated Temperatures ◽  
Metagenomic Library ◽  
Catalytic Efficiency ◽  
Surface Hydrophobicity ◽  
Maximum Activity ◽  
Hormone Sensitive Lipase ◽  
Hydrophobic Residues ◽  
Improved Stability ◽  
Hormone Sensitive

PMGL3 is a cold-adapted esterase which was recently isolated from the permafrost metagenomic library. It exhibits maximum activity at 30 °C and low stability at elevated temperatures (40 °C and higher). Sequence alignment has revealed that PMGL3 is a member of the hormone-sensitive lipase (HSL) family. In this work, we demonstrated that incubation at 40 °C led to the inactivation of the enzyme (t1/2 = 36 min), which was accompanied by the formation of tetramers and higher molecular weight aggregates. In order to increase the thermal stability of PMGL3, its two cysteines Cys49 and Cys207 were substituted by the hydrophobic residues, which are found at the corresponding positions of thermostable esterases from the HSL family. One of the obtained mutants, C207F, possessed improved stability at 40 °C (t1/2 = 169 min) and increased surface hydrophobicity, whereas C49V was less stable in comparison with the wild type PMGL3. Both mutants exhibited reduced values of Vmax and kcat, while C207F demonstrated increased affinity to the substrate, and improved catalytic efficiency.

scholarly journals Peroxidase from Coleus Forskohlii: Purification and Biochemical Characterization

2020 ◽  
Vol 5 (1) ◽  
pp. 9-20
Author(s):  
Yaaser Q. Almulaiky ◽  
Yaaser Q. Almulaiky
Keyword(s):  
Ion Exchange ◽  
Peroxidase Activity ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Gel Filtration ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Coleus Forskohlii ◽  
Sds Page ◽  
Optimum Ph

In this study, a peroxidase from new source was purified using ion exchange and gel filtration techniques. The recovery for peroxidase activity was 19% with 11-fold purification and specific activity of 749 unit/mg protein. Purified peroxidase demonstrated a molecular mass of 39 kDa using gel filtration and was confirmed as a single band on SDS-PAGE. The purified peroxidase revealed a broad optimum pH activity at 6.0-6.5 and 50°C temperature. The kinetic parameters for purified peroxidase toward H2O2 and guaiacol as substrates were found to be Km = 3.355, 5.395 mM, Kcat = 99.52, 79.56 s-1 and Vmax =1.531, 1.242 µmole ml-1 min-1, respectively. The catalytic efficiency (kcat/Km) of the purified peroxidase was 14.75 and 29.66 s−1 mM−1 for guaiacol and H2O2, respectively. Peroxidase activity was observed to be enhanced by Cu2+, Co2+, Ni2+ and inhibited in the presence of Sn2+, Al3+, Hg2+, NaN3, EDTA and urea. Characterization showed that peroxidase purified from C. forskohlii has the ability to be used for food industrial applications.

scholarly journals Kinetic and structural optimization to catalysis at low temperatures in a psychrophilic cellulase from the Antarctic bacterium Pseudoalteromonas haloplanktis

2004 ◽  
Vol 384 (2) ◽  
pp. 247-253 ◽  
Author(s):  
Geneviève GARSOUX ◽  
Josette LAMOTTE ◽  
Charles GERDAY ◽  
Georges FELLER
Keyword(s):  
Active Site ◽  
Low Temperatures ◽  
Catalytic Domain ◽  
Homology Modelling ◽  
Temperature Adaptation ◽  
Substrate Affinity ◽  
Structural Factors ◽  
Pseudoalteromonas Haloplanktis ◽  
Antarctic Bacterium ◽  
The Antarctic

The cold-adapted cellulase CelG has been purified from the culture supernatant of the Antarctic bacterium Pseudoalteromonas haloplanktis and the gene coding for this enzyme has been cloned, sequenced and expressed in Escherichia coli. This cellulase is composed of three structurally and functionally distinct regions: an N-terminal catalytic domain belonging to glycosidase family 5 and a C-terminal cellulose-binding domain belonging to carbohydrate-binding module family 5. The linker of 107 residues connecting both domains is one of the longest found in cellulases, and optimizes substrate accessibility to the catalytic domain by drastically increasing the surface of cellulose available to a bound enzyme molecule. The psychrophilic enzyme is closely related to the cellulase Cel5 from Erwinia chrysanthemi. Both kcat and kcat/Km values at 4 °C for the psychrophilic cellulase are similar to the values for Cel5 at 30–35 °C, suggesting temperature adaptation of the kinetic parameters. The thermodynamic parameters of activation of CelG suggest a heat-labile, relatively disordered active site with low substrate affinity, in agreement with the experimental data. The structure of CelG has been constructed by homology modelling with a molecule of cellotetraose docked into the active site. No structural alteration related to cold-activity can be found in the catalytic cleft, whereas several structural factors in the overall structure can explain the weak thermal stability, suggesting that the loss of stability provides the required active-site mobility at low temperatures.

scholarly journals Species-specific alternative splicing generates a catalytically inactive form of human hormone-sensitive lipase

1997 ◽  
Vol 328 (1) ◽  
pp. 137-143 ◽  
Author(s):  
Henrik LAURELL ◽  
Jacques GROBER ◽  
Cécile VINDIS ◽  
Thierry LACOMBE ◽  
Michèle DAUZATS ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Alternative Splicing ◽  
Cell Lines ◽  
Catalytic Triad ◽  
Hormone Sensitive Lipase ◽  
Human Adipocytes ◽  
Brown Adipose ◽  
Specific Alternative ◽  
Hormone Sensitive ◽  
Species Specific

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The enzyme is also expressed in steroidogenic tissues, mammary gland, muscle tissues and macrophages. A novel HSL mRNA termed hHSL-S, 228 bp shorter than the full-length HSL mRNA, was detected in human adipocytes. hHSL-S mRNA results from the in-frame skipping of exon 6, which encodes the serine residue of the catalytic triad. The corresponding 80 kDa protein was identified in human adipocytes after immunoprecipitation. The truncated protein expressed in COS cells showed neither lipase nor esterase activity but was phosphorylated by cAMP-dependent protein kinase. hHSL-S mRNA was found in all human tissues expressing HSL, except brown adipose tissue from newborns. It represented approx. 20% of total HSL transcripts in human subcutaneous adipocytes. No alternative splicing was detected in other mammals. Human and mouse three-exon HSL minigenes transfected into primate and rodent cell lines reproduced the splicing pattern of the endogenous HSL genes. Analysis of hybrid human/mouse minigenes transfected into human cell lines showed that cis-acting elements responsible for the skipping of human exon 6 were restricted to a 247 bp region including exon 6 and the first 19 nt of intron 6. Moreover, divergence in exonic splicing elements between mouse and human was shown to be critical for the species-specific alternative splicing.

scholarly journals Comparative Analysis and Biochemical Characterization of Two Endo-β-1,3-Glucanases from the Thermophilic Bacterium Fervidobacterium sp.

Catalysts ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 830 ◽  
Author(s):  
Burkhardt ◽  
Schäfers ◽  
Claren ◽  
Schirrmacher ◽  
Antranikian
Keyword(s):  
Elevated Temperatures ◽  
Biochemical Characterization ◽  
Hot Spring ◽  
Biomass Conversion ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Thermophilic Bacterium ◽  
Metagenomic Sample ◽  
Wide Range ◽  
Active Site Cleft

Laminarinases exhibit potential in a wide range of industrial applications including the production of biofuels and pharmaceuticals. In this study, we present the genetic and biochemical characteristics of FLamA and FLamB, two laminarinases derived from a metagenomic sample from a hot spring in the Azores. Sequence comparison revealed that both genes had high similarities to genes from Fervidobacterium nodosum Rt17-B1. The two proteins showed sequence similarities of 62% to each other and belong to the glycoside hydrolase (GH) family 16. For biochemical characterization, both laminarinases were heterologously produced in Escherichia coli and purified to homogeneity. FLamA and FLamB exhibited similar properties and both showed highest activity towards laminarin at 90 °C and pH 6.5. The two enzymes were thermostable but differed in their half-life at 80 °C with 5 h and 1 h for FLamA and FLamB, respectively. In contrast to other laminarinases, both enzymes prefer β-1,3-glucans and mixed-linked glucans as substrates. However, FLamA and FLamB differ in their catalytic efficiency towards laminarin. Structure predictions were made and showed minor differences particularly in a kink adjacent to the active site cleft. The high specific activities and resistance to elevated temperatures and various additives make both enzymes suitable candidates for application in biomass conversion.

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