Metabolic engineering ofLactococcus lactisinfluence of the overproduction of lipase enzyme

2013 ◽  
Vol 80 (4) ◽  
pp. 490-495 ◽  
Author(s):  
Mohammad Raftari ◽  
Sobhan Ghafourian ◽  
Fatimah Abu Bakar
Keyword(s):  
Western Blotting ◽  
Milk Fat ◽  
Burkholderia Cepacia ◽  
Nile Blue ◽  
Dairy Industry ◽  
Nucleotide Sequencing ◽  
Protein Assay ◽  
Recombinant Lipase ◽  
Fatty Acid Chain ◽  
Sds Page

The dairy industry uses lipase extensively for hydrolysis of milk fat. Lipase is used in the modification of the fatty acid chain length, to enhance the flavours of various chesses. Therefore finding the unlimited source of lipase is a concern of dairy industry. Due to the importance of lipase, this study was an attempt to express the lipase fromBurkholderia cepaciainLactococcus lactis.To achieve this, a gene associated with lipase transport was amplified and subcloned in inducible pNZ8148 vector, and subsequently transformed intoLc. lactisNZ9000. The enzyme assay as well as SDS-PAGE and western blotting were carried out to analysis the recombinant lipase expression. Nucleotide sequencing of the DNA insert from the clone revealed that the lipase activity corresponded to an open reading frame consisting of 1092 bp coding for a 37·5-kDa size protein. Blue colour colonies on nile blue sulphate agar and sharp band on 37·5-kD size on SDS-PAGE and western blotting results confirm the successful expression of lipase byLc. lactis. The protein assay also showed high expression, approximately 152·2 μg/ml.h, of lipase by recombinantLc. lactis.The results indicate thatLc. lactishas high potential to overproduce the recombinant lipase which can be used commercially for industrially purposes.

Overexpression of Recombinant Lipase from Burkholderia Cepacia in Escherichia Coli

2012 ◽  
Vol 10 (3) ◽  
pp. 365-369 ◽  
Author(s):  
M. Raftari ◽  
S. Ghafourian ◽  
N. Sadeghifard ◽  
F. Abu Bakar ◽  
N. Saari ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Lipase Activity ◽  
Burkholderia Cepacia ◽  
Nucleotide Sequencing ◽  
Blue Color ◽  
Lipase Gene ◽  
E Coli ◽  
Recombinant Lipase ◽  
Sds Page

This study attempts to clone and express the extracellular lipase from Burkholderia cepacia in Escherichia coli using pET system as well as to determine the enzyme activity of recombinant lipase. The extracted DNA from B. cepacia was used as a template for amplifying lipase gene, and then the lipase gene was subcloned into pET-32a and subsequently transformed into E. coli BL21. Media assay and SDS-PAGE were carried out to analyse the results. Nucleotide sequencing of the DNA insert from the clone revealed that the lipase activity corresponded to an open reading frame consisting of 1092 bp coding for a 37.5-kDa protein. The successful expression of lipase was confirmed by obtaining blue color colonies on Nile Blue Sulphate Agar and big band at 37.5-kD size on SDS-PAGE. The enzyme activity assay also showed the high lipase activity around 590 μg lipase ml−1 culture 30 min−1 of recombinant E. coli BL21. The specific lipolytic activity of the recombinant lipase was 185 U/mL which is around 35-fold higher than the native baseline. The findings suggest that the crude recombinant lipase has potential application in digestion of lipids and fatty acids. In conclusion, the results of the current study showed a lipase gene encoding an enzyme with non-specific hydrolysis activity, which could be applied as lipase biosensor for digestion of lipids in food and medicine as well as oil-contamination treatment.

scholarly journals Nitrotyrosine Density of Rabbit Urinary Bladder Muscle and Mucosa Measured via Western Blotting and 96-Well Plate Analysis

ISRN Urology ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-5
Author(s):  
Brittany Fitzpatrick ◽  
Catherine Schuler ◽  
Robert E. Leggett ◽  
Robert M. Levin
Keyword(s):  
Quantitative Analysis ◽  
Western Blotting ◽  
Optical Density ◽  
Tissue Concentration ◽  
Detrusor Muscle ◽  
Pvdf Membrane ◽  
Sds Page ◽  
Wash Buffer ◽  
Rabbit Bladder ◽  
Plate Analysis

Purpose. Nitrotyrosine was quantitated in rabbit bladder muscle and mucosa using two analytical systems: Western blotting analyses and a 96-well plate quantitative analysis kit. Materials and Methods. Rabbit bladder muscle and mucosa were obtained from control rabbits. For the Western analysis, the samples were loaded into a SDS page gel and then transferred to a PVDF membrane. The optical density was measured using a Kodak Scanner. Using the 96-well plate, the samples and standards were loaded, incubated with primary and secondary antibody, washed and vacuumed with 10x wash buffer three times between each incubation period. Stop buffer was added to the plate and the results were quantified via the plate reader. Results. For both muscle and mucosa tissue, the optical density readings were linear with tissue concentration; the concentration of nitrotyrosine in the mucosa was significantly higher than in the muscle. However, whereas the Western blot analysis is based on relative optical densities, the 96-well plate kit provides a truly quantitative analysis. Discussion. Mucosa tissue displayed a higher density of nitrotyrosine than did detrusor muscle tissue. This may well be due to the significantly higher metabolic activity of the mucosa compared to the muscle.

scholarly journals Generating and characterizing the anti-human CD45 monoclonal antibody

2020 ◽  
Vol 23 (3) ◽  
pp. 665-672
Author(s):  
Giang Huong Ta ◽  
Huy Quoc Nguyen ◽  
Quan Dang Nguyen
Keyword(s):  
Monoclonal Antibody ◽  
Western Blotting ◽  
Jurkat Cells ◽  
Spleen Cells ◽  
Myeloma Cells ◽  
E Coli ◽  
Common Marker ◽  
Sds Page ◽  
Hybridoma Technology ◽  
Mab Production

Introduction: CD45 is a common marker of leukocytes. Anti-human CD45 monoclonal antibody (MAb) has been used widely in diagnosing and monitoring hematologic diseases. The aim of this study was to generate an anti-human CD45 MAb, which can be used in research and diagnosis. Methods: Recombinant human CD45RO antigen was expressed from E. coli BL21 (DE3), purified and analyzed by SDS-PAGE and Western blotting. The purified CD45RO antigen was used to immunize Balb/c mice. Spleen cells from immunized mouse were collected and fused with P3X63Ag8.653 myeloma cells to form hybridoma. Anti-CD45 antibody-secreting capacity of hybridoma clones was evaluated by ELISA assay. Anti-CD45 MAb from the culture supernatant of the chosen hybridoma clone was purified by affinity chromatography. The MAb was characterized the biochemical characteristics and biological activity. Results: Recombinant human CD45RO antigen was expressed and purified from E.coli BL21 (DE3). Injection of purified CD45RO antigen provoked the immune response in Balb/c mice. Hybridoma clones were generated successfully by the fusion of spleen cells from the selected immunized-mouse and myeloma cells. Among these hybridoma clones, one with the highest yield of MAb production was identified. The isotype of the anti-CD45 MAb created in this work is IgG2b, while its the light chain is kappa (k) type. The affinity of this MAb with CD45RO antigen is high with Kd value at the picomolar level. The anti-CD45 MAb can interact with CD45 naturally expressed on the surface of Jurkat cells in Western blotting and fluorescent immuno-staining assay. Conclusion: We have developed successfully an anti-human CD45 MAb using hybridoma technology, which can recognize CD45 in ELISA, Western blotting, and fluorescent immuno-staining analysis. Although further investigations are necessary, obviously, our anti-human CD45 MAb is potential for research and diagnosis applications.

Genetic and phenotypic trends in production traits in the United Kingdom (UK) dairy herd

1998 ◽  
Vol 1998 ◽  
pp. 191-191
Author(s):  
C. M. Lindberg ◽  
G. J. T. Swanson ◽  
R. A. Mrode
Keyword(s):  
Animal Model ◽  
United Kingdom ◽  
Milk Fat ◽  
Dairy Industry ◽  
Dairy Herd ◽  
Production Traits ◽  
Individual Animal ◽  
Know How ◽  
The United Kingdom ◽  
Breeding Schemes

It is important for the dairy industry to be aware of the consequences of past selection policies. This can provide guidance on how to improve or change current breeding schemes. In addition it is important to know how much of current progress is due to breeding and how much to management. The objective of the study was to analyse genetic and phenotypic trends for the production traits (milk, fat and protein) using the results from the latest UK Individual Animal Model evaluations.

scholarly journals Vírus do mosaico severo do caupi-CPSMV como molécula carreadora para a p28 do vírus da artrite-encefalite caprina-CAEV

2005 ◽  
Vol 35 (6) ◽  
pp. 1363-1367 ◽  
Author(s):  
Francisco Jarbas Santos de Sousa ◽  
Marcelo Róseo de Oliveira ◽  
Ney de Carvalho Almeida ◽  
Marlos Gomes Martins ◽  
Maria Erivalda Farias de Aragão ◽  
...  
Keyword(s):  
Western Blotting ◽  
Sds Page

O vírus da Artrite-encefalite caprina (CAEV) pertence à família Retroviridae, gênero Lentivirus. O CAEV infecta caprinos do mundo inteiro causando artrite, encefalite, mamite, pneumonia e emagrecimento progressivo. Este trabalho mostra a formação de uma quimera construída através da mistura da p28 do CAEV com glutaraldeído e CPSMV, purificada por meio de cromatografia em biogel e sephadex G-150. As cromatografias foram monitoradas através de leituras em espectrofotômetro no comprimento de onda de 280nm, dos líquidos coletados nos tubos. Os picos contendo a quimera foram coletados e submetidos à eletroforese (SDS-PAGE), sendo assim evidenciada a banda correspondente à mesma. Grupos de camundongos swiss foram imunizados com o vírus quimérico (CPSMV + p28), com o vírus CPSMV purificado e com a proteína p28 do CAEV, utilizando o adjuvante de Freund incompleto. Os anticorpos específicos produzidos contra o CPSMV e p28 reconheceram a proteína quimérica em Western Blotting e em teste de ELISA. Os anticorpos contra o vírus quimérico apresentaram títulos mais elevados do que os anticorpos produzidos contra a p28, demonstrando que o vírus quimérico apresenta maior imunogenicidade do que a proteína p28 sozinha. Os resultados mostraram que o acoplamento covalente entre o CPSMV e a p28 do CAEV foi obtido com sucesso, originando uma molécula estável não comprometendo a estrutura do capsídeo do CPSMV. Desta forma, sugere-se que o CPSMV possa ser utilizado como molécula carreadora na produção de vacinas para vírus que infectam animais.

scholarly journals Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

1990 ◽  
Vol 32 (5) ◽  
pp. 379-383 ◽  
Author(s):  
Anna Maria Simonsen Stolf ◽  
Eufrosina Setsu Umezawa ◽  
Bianca Zingales
Keyword(s):  
Trypanosoma Cruzi ◽  
Western Blot ◽  
Chagas Disease ◽  
Western Blotting ◽  
Specific Antibodies ◽  
Internal Parasite ◽  
Sds Page ◽  
Blotting Technique ◽  
Simultaneous Identification ◽  
Acute Chagas Disease

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

High Expression of Human Amelogenin in E. Coli

1996 ◽  
Vol 10 (2) ◽  
pp. 187-194 ◽  
Author(s):  
D. Deutsch ◽  
E. Chityat ◽  
M. Hekmati ◽  
A. Palmon ◽  
Y. Farkash ◽  
...  
Keyword(s):  
Amino Acid ◽  
Western Blotting ◽  
Rt Pcr ◽  
Amino Acid Sequencing ◽  
Terminal Amino Acid ◽  
Expression Plasmid ◽  
Over Expression ◽  
E Coli ◽  
Sds Page ◽  
Terminal Amino

A human cDNA, encoding for the 175-aminoacid human amelogenin, was prepared by RT PCR from tooth bud mRNA and sub-cloned into pGEX-KG expression plasmid for over-expression in E. coli. The expressed protein was characterized by SDS-PAGE, Western blotting, and N-terminal amino acid sequencing.

Setting targets for the Irish dairy industry

2020 ◽  
Vol 60 (1) ◽  
pp. 159
Author(s):  
Laurence Shalloo ◽  
Liam Hanrahan
Keyword(s):  
Milk Fat ◽  
Dairy Industry ◽  
Sustainability Indicators ◽  
Farm Level ◽  
Significant Potential ◽  
Grass Growth ◽  
Farm Business ◽  
Milk Solids ◽  
On Farm ◽  
Efficiency And Productivity

A resilient dairy business will be sustainable across all of the sustainability indicators, survive milk-price drops and be very profitable when milk price is high. The term resilient means able to ‘recover, respond, deal or withstand’ different internal and external challenges that may manifest themselves within the farm business from time to time. There is significant potential to increase efficiency and productivity at farm level when compared with the average farm nationally. The focus at a farm level must be about increasing grass growth and utilisation and converting that feed to milk solids (kg of milk fat and protein) sales at as low a cost as possible. Increasing labour efficiency by operating more streamlined work practices, using contractors and contract rearing of heifers will have a major impact on farm labour requirements.

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