Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Gene cloning, expression, and characterization of the Bacillus amyloliquefaciens PS35 lipase.

ABSTRACT: Lipases are enzymes of immense industrial relevance, and, therefore, are being intensely investigated. In an attempt to characterize lipases at molecular level from novel sources, a lipase gene from Bacillus amyloliquefaciens PS35 was cloned, heterologously expressed in Escherichia coli DH5? cells and sequenced. It showed up to 98% homology with other lipase sequences in the NCBI database. The recombinant enzyme was then purified from E. coli culture, resulting in a 19.41-fold purification with 9.7% yield. It displayed a preference for long-chain para-nitrophenyl esters, a characteristic that is typical of true lipases. Its optimum pH and temperature were determined to be 8.0 and 40 °C, respectively. The half-lives were 2.0, 1.0 and 0.5 h at 50 °C, 60 °C and 70 °C, respectively. The metal ions K+ and Fe3+ enhanced the enzyme activity. The enzyme displayed substantial residual activity in the presence of various tested chemical modifiers, and interestingly, the organic solvents, such as n-hexane and toluene, also favored the enzyme activity. Thus, this study involves characterization of B. amyloliquefaciens lipase at molecular level. The key outcomes are novelty of the bacterial source and purification of the enzyme with desirable properties for industrial applications.

SUBMITTER: Kanmani P

PROVIDER: S-EPMC4704641 | biostudies-literature | 2015 Oct-Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Cloning, expression and characterization of a lipase encoding gene from human oral metagenome.

Project description:The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6-7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes.

| S-EPMC4039722 | biostudies-literature

Cloning of a Family 11 Xylanase Gene from Bacillus amyloliquefaciens CH51 Isolated from Cheonggukjang.

Project description:Bacillus amyloliquefaciens CH51, an isolate from cheonggukjang, Korean fermented soyfood, secretes several enzymes into culture medium. A gene encoding 19 kDa xylanase was cloned by PCR. Sequencing showed that the gene encoded a glycohydrolase family 11 xylanase and named xynA. xynAHis, xynA with additional codons for his-tag, was overexpressed in Escherichia coli BL21(DE3) using pET-26b(+). XynAHis was purified using HisTrap affinity column. Km and Vmax of XynAHis were 0.363 mg/ml and 701.1 μmol/min/mg, respectively with birchwood xylan as a substrate. The optimum pH and temperature were pH 4 and 25 °C, respectively. When xynA was introduced into Bacillus subtilis WB600, active XynA was secreted into culture medium.

| S-EPMC3516658 | biostudies-literature

Cloning, expression, purification and characterization of lipase from Bacillus licheniformis, isolated from hot spring of Himachal Pradesh, India.

Project description:In the present investigation, a gene encoding extracellular lipase was cloned from a Bacillus licheniformis. The recombinant protein containing His-tag was expressed as inclusion bodies in Esherichia coli BL21DE3 cells, using pET-23a as expression vector. Expressed protein purified from the inclusion bodies demonstrated ~22 kDa protein band on 12 % SDS-PAGE. It exhibited specific activity of 0.49 U mg-1 and % yield of 8.58. Interestingly, the lipase displayed activity at wide range of pH and temperature, i.e., 9.0-14.0 pH and 30-80 °C, respectively. It further demonstrated ~100 % enzyme activity in presence of various organic solvents. Enzyme activity was strongly inhibited in the presence of ?-ME. Additionally, the serine and histidine modifiers also inhibited the enzyme activities strongly at all concentrations that suggest their role in the catalytic center. Enzyme could retain its activity in presence of various detergents (Triton X-100, Tween 20, Tween 40, SDS). Sequence and structural analysis employing in silico tools revealed that the lipase contained two highly conserved sequences consisting of ITITGCGNDL and NLYNP, arranged as parallel ?-sheet in the core of the 3D structure. The function of these conserve sequences have not fully understood.

| S-EPMC4746201 | biostudies-literature

Transesterification Synthesis of Chloramphenicol Esters with the Lipase from Bacillus amyloliquefaciens.

Project description:This work presents a synthetic route to produce chloramphenicol esters by taking advantage the high enantio- and regio-selectivity of lipases. A series of chloramphenicol esters were synthesized using chloramphenicol, acyl donors of different carbon chain length and lipase LipBA (lipase cloned from Bacillus amyloliquefaciens). Among acyl donors with different carbon chain lengths, vinyl propionate was found to be the best. The influences of different organic solvents, reaction temperature, reaction time, enzyme loading and water content on the synthesis of the chloramphenicol esters were studied. The synthesis of chloramphenicol propionate (0.25 M) with 4.0 g L-1 of LipBA loading gave a conversion of ~98% and a purity of ~99% within 8 h at 50 °C in 1,4-dioxane as solvent. The optimum mole ratio of vinyl propionate to chloramphenicol was increased to 5:1. This is the first report of B. amyloliquefaciens lipase being used in chloramphenicol ester synthesis and a detailed study of the synthesis of chloramphenicol propionate using this reaction. The high enzyme activity and selectivity make lipase LipBA an attractive catalyst for green chemical synthesis of molecules with complex structures.

| S-EPMC6151736 | biostudies-literature

Cloning and Expression of a Subfamily 1.4 Lipase from Bacillus licheniformis IBRL-CHS2.

Project description:We report on the cloning of the lipase gene from Bacillus licheniformis IBRL-CHS2 and the expression of the recombinant lipase. DNA sequencing analysis of the cloned lipase gene showed that it shares 99% identity with the lipase gene from B. licheniformis ATCC 14580 and belongs to subfamily 1.4 of true lipases based on amino acid sequence alignment of various Bacillus lipases. The 612 bp lipase gene was then cloned into the pET-15b(+) expression vector and the construct was transformed into E. coli BL21 (DE3) for bulk expression of the lipase. Expression was analysed by SDS-PAGE where the lipase was found to have a molecular weight of about 23 kDa.

| S-EPMC5131670 | biostudies-literature

Gene Cloning, Heterologous Expression and Biochemical Characterization of A Novel Extracellular Lipase from Rhizopus Oryzae KU45.

Project description:BackgroundLipases secreted from various Rhizopus oryzae strains were previously expressed in Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae and was shown to have distinct activities in response to different temperatures, metal ions, organic solvents, and specific substrates. However, until now, no other research biochemically characterized the functions of extracellular pro-lipase in a novel Rhizopus oryzae KU45.ObjectivesCharacterization of a novel extracellular lipase from fungus R. oryzae KU45 after heterologous expression in E. coli BL21 (DE3) strain.Materials and methodsAn extracellular lipase producing fungus was isolated from a soil sample and identified as a strain of R. oryzae by partial 18S rRNA gene sequencing. It was named as R. oryzae KU45. The lipase gene of KU45 was cloned into pET-28a expression vector and expressed in E. coli as inclusion bodies. The recombinant lipase was purified, refolded and characterized.ResultsThe lipase exhibited maximum activity at 45ºC, at slightly alkaline pH. It showed a broad substrate specificity acting on p-nitrophenyl esters with C8-C16 acyl groups as substrates and, many of the organic solvents and metal ions tested did not have any adverse effects on the enzyme activity.ConclusionsHigh stability, broad substrate specificity and activity at mesophilic temperatures in the presence of organic solvents, and metal ions make the extracellular lipase of KU45 a candidate for various biotechnological applications.

| S-EPMC7856406 | biostudies-literature

A novel cold-adapted lipase from Sorangium cellulosum strain So0157-2: gene cloning, expression, and enzymatic characterization.

Project description:Genome sequencing of cellulolytic myxobacterium Sorangium cellulosum reveals many open-reading frames (ORFs) encoding various degradation enzymes with low sequence similarity to those reported, but none of them has been characterized. In this paper, a predicted lipase gene (lipA) was cloned from S. cellulosum strain So0157-2 and characterized. lipA is 981-bp in size, encoding a polypeptide of 326 amino acids that contains the pentapeptide (GHSMG) and catalytic triad residues (Ser114, Asp250 and His284). Searching in the GenBank database shows that the LipA protein has only the 30% maximal identity to a human monoglyceride lipase. The novel lipA gene was expressed in Escherichia coli BL21 and the recombinant protein (r-LipA) was purified using Ni-NTA affinity chromatography. The enzyme hydrolyzed the p-nitrophenyl (pNP) esters of short or medium chain fatty acids (?C(10)), and the maximal activity was on pNP acetate. The r- LipA is a cold-adapted lipase, with high enzymatic activity in a wide range of temperature and pH values. At 4 °C and 30 °C, the K(m) values of r-LipA on pNP acetate are 0.037 ± 0.001 and 0.174 ± 0.006 mM, respectively. Higher pH and temperature conditions promoted hydrolytic activity toward the pNP esters with longer chain fatty acids. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents. The results suggest that the r-LipA protein has some new characteristics potentially promising for industrial applications and S. cellulosum is an intriguing resource for lipase screening.

| S-EPMC3211009 | biostudies-literature

Characterization of lpaH2 gene corresponding to lipopeptide synthesis in Bacillus amyloliquefaciens HAB-2.

Project description:BACKGROUND:Bacillus spp. have prominent ability to suppress plant pathogens and corresponding diseases. Previous analyses of Bacillus spp. revealed numerous gene clusters involved in nonribosomal synthesis of cyclic lipopeptides with distinct antimicrobial action. The 4'-phosphopantetheinyl transferase (PPTase) encoded by sfp gene is a key factor in lipopeptide synthesis in Bacillus spp. In previous study, B. amyloliquefaciens strain HAB-2 was found to inhibit a broad range of plant pathogens, which was attributed to its secondary metabolite lipopeptide. RESULTS:A sfp homologue lpaH2 which encoded phosphopantetheinyl transferase but shared 71% sequence similarity was detected in strain HAB-2. Disruption of lpaH2 gene resulted in losing the ability of strain HAB-2 to produce lipopeptide, as well as antifungal and hemolytic activities. When lpaH2 replaced sfp gene of B. subtilis strain 168, a non-lipopeptide producer, the genetically engineered strain 168 could produced lipopeptides and recovered antifungal activity. Quantitative PCR assays indicated that, the expression level of lpaH2 in B. subtilis 168 strain decrease to 0.27-fold compared that of the wild type B. amyloliquefaciens strain HAB-2. CONCLUSION:Few studies have reported about lpa gene which can replace sfp gene in the different species. Taken together, our study showed for the first time that lpaH2 from B. amyloliquefaciens could replace sfp gene.

| S-EPMC5716053 | biostudies-literature

Gene cloning, expression, purification and characterization of a sn-1,3 extracellular lipase from <i>Aspergillus niger</i> GZUF36.

Project description:Sn-1,3 extracellular Aspergillus niger GZUF36 lipase (EXANL1) has wide application potential in the food industry. However, the A. niger strain has defects such as easy degradation and instability in the expression of sn-1,3 lipase. To obtain a stable expression of this lipase and its subsequent enzymatic properties, the gene encoding EXANL1 was cloned and expressed in Escherichia coli BL21 (DE3) cells using pET-28a as the expression vector. The temperature-induced conditions were optimized, and we successfully achieved its active expression in E. coli. These conditions significantly influenced the active expression of EXANL1 (P < 0.05), and the highest enzyme activity of the supernatant of lysis cells expressed at 20 °C was at 7.02 ± 0.05 U/mL. The expressed recombinant EXANL1 was purified using Ni-NTA, showing an estimated relative molecular mass of 35 kDa. The recombinant EXANL1 exhibited maximum activity at 35 °C and pH 4.0, with a wide acid pH range. Thin-layer chromatography analysis showed that the enzyme displayed sn-1,3 positional selectivity toward triolein. The recombinant EXANL1 could maintain its relative activities (> 80%) after 24 h of incubation at pH 3-10, suggesting its suitability for a wide range of industrial applications. After comparing these properties with those of the other A. niger lipases, we found that some key amino acids may play a decisive role in enzymology. This work laid a foundation for the stable expression of the EXANL1 gene and its potential industrial application.

| S-EPMC7270473 | biostudies-literature

Cloning and expression of Aspergillus tamarii FS132 lipase gene in Pichia pastoris.

Project description:A lipase gene (atl) was cloned from Aspergillus tamarii FS132 for the first time. The gene was found to have an open reading frame of 1024 base pairs (bp), and the coding region of the gene contained two introns (51 bp and 52 bp). Multi-alignment analysis of the deduced amino acid sequence indicated high homology between the enzyme and mono-and diacylglycerol lipases from fungi Aspergillus. The recombinant lipase was expressed in Pichia pastoris GS115 cells. The recombinant lipase was found to have a molecular mass of 36.7 kDa, and it exhibited lipase activity of 20 U/mL in culture supernatant when tributyrin was used as the substrate.

| S-EPMC2904922 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data