scholarly journals COMBINATION TEST OF CHINESE LEAF EXTRACT (Leucaena leucocephala folium) AND ALOE VERA INHIBITING GROWTH Escherichia coli

2020 ◽  
Vol 2 (2) ◽  
pp. 60-67
Author(s):  
Putra Rahmadea Utami ◽  
Chairani ◽  
Hendra Yudha
Keyword(s):  
Escherichia Coli ◽  
Leucaena Leucocephala ◽  
Aloe Vera ◽  
Ethanol Extract ◽  
Laboratory Research ◽  
The Public ◽  
E Coli ◽  
Experimental Laboratory ◽  
Medicinal Properties

Chinese petai (Leucaena leucocephala folium) and aloe vera (Aloe vera L.) have medicinal properties among the plants.  The objective of this study was to determine the number of inhibitory zones produced by the ethanol extract of Chinese petai and aloe vera on the growth of Escherichia coli. The research method was In-vitro Experimental Laboratory research design with the Kirby Bauer method. The samples used was Chinese petai and aloe vera with pure strains of E. coli. One Way ANOVA was used to compare the differences in inhibition of Chinese petai and aloe vera on the growth of E. coli. The results of a combination of Chinese petai and aloe vera extract test showed that there were significant differences in the concentration of 25 g, 50 g, 75 g, and 100 g (p <0.05). The ethanol extract of Chinese petai and aloe vera can inhibit the growth of E. coli. From the results of this study found that there was an interaction on the combination of ethanol extract of Chinese petai and aloe vera inhibiting the growth of E. coli with the most effective concentration being 100 g/mL. This study can find out the benefits of petai cina and aloe vera also the public will know the benefits and efficacy of Chinese petai and aloe vera leaves in medicine.

scholarly journals Isolasi dan identifikasi Escherichia coli dari Sumber Air Minum Kandang Broiler serta Uji Aktivitas Antibakteri Lidah Buaya

2020 ◽  
Vol 10 (2) ◽  
pp. 106
Author(s):  
Dian Meididewi Nuraini ◽  
Morsid Andityas ◽  
Adi Paramarta ◽  
Nur Rohman Najib ◽  
Agustina Dwi Wijayanti
Keyword(s):  
Escherichia Coli ◽  
Methylene Blue ◽  
Drinking Water ◽  
Inhibitory Activity ◽  
Aloe Vera ◽  
Ethanol Extract ◽  
Inhibition Zone ◽  
Inhibition Test ◽  
Aloe Barbadensis ◽  
E Coli

Abstract Colibacillosis is one of the most problematic issues in the boiler industry. However, the antibiotic overuse has induced Escherichia coli resistance so that other alternative to reduce colibacillosis is needed. One of the alternatives is using aloe vera (Aloe barbadensis Miller), which has been widely used as an antibacterial agent. This study aims to isolate and identify E. coli from the broiler drinking water source and test the aloe vera antibacterial activity against it. Escherichia coli were isolated from well in three broiler farms in Moyudan District, Sleman, Yogyakarta that previously had colibacillosis. Escherichia coli were isolated using eosin methylene blue (EMB) agar and the metallic sheen colony was tested to confirm the biochemist reaction. The pure isolate of E. coli was used in the aloe vera inhibition test using Muller Hinton agar (MHA) by a Well Diffusion method. Aloe vera was processed using aquades and ethanol 70%. The aquades infusion was diluted into 12.5%, 25%, 50%, 75%, and 100% and the extract ethanol 70% was diluted into 10%, 12.5, %, 25%, 40%, and 50%. The bacterial identification showed that one of three samples contained E. coli which was then used for inhibition test. The result showed no inhibition zone in the aquades infusion while ethanol extract showed an inhibition zone in concentration 25%, 40%, and 50% of aloe vera extract with a diameter 19.5 mm, 24 mm, and 25 mm. It can be concluded that aloe vera ethanol extract has inhibitory activity against E. coli in poultry drinking water with a minimum concentration of 25%.  Keywords: Aloe vera; Broiler drinking water; Escherichia coli; Inhibitory activity   Abstrak  Colibacilosis masih menjadi permasalahan dalam industri broiler. Penggunaan antibiotik berlebihan telah menyebabkan resistensi sehingga perlu alternatif lain. Salah satu alternatif adalah menggunakan bahan alami seperti adalah lidah buaya (Aloe barbadensis Miller) yang memilliki aktivitas antibakteri. Penelitian ini bertujuan untuk mengisolasi dan mengidentifikasi E. coli dari sumber air minum di kandang broiler serta menguji aktivitas inhibisi lidah buaya terhadap bakteri tersebut. Air yang digunakan sebagai sumber E. coli berasal dari sumur di tiga peternakan broiler di Kecamatan Moyudan, Sleman, Yogyakarta yang memiliki riwayat infeksi colibacilosis. Bakteri E. coli diisolasi menggunakan media eosin methylene blue (EMB) dan diuji sifat biokimia untuk mengkonfirmasi sifat bakteri E. coli. Isolat murni E. coli digunakan pada uji daya hambat bakteri dengan metode difusi sumuran menggunakan media Muller Hinton Agar (MHA). Lidah buaya diproses menggunakan aquades dan ethanol 70%. Infusa aquades diencerkan menjadi konsentrasi 12,5%, 25%, 50%, 75%, dan 100% dan ekstrak ethanol 70% diencerkan menjadi 10%, 12., %, 25%, 40%, dan 50%.  Hasil isolasi menunjukan bahwa satu sumber air dari sumur di Desa Kolowenang mengandung E. coli yang kemudian digunakan pada pengujian daya hambat. Hasil pengujian menunjukan tidak ada daya hambat yang terbentuk pada infusa aquades sedangkan ekstrak etanol lidah buaya 25%, 40%, dan 50% menunjukan adanya zona hambat sebesar 19,5 mm, 24 mm, dan 25 mm berturut-turut. Ekstrak etanol lidah buaya pada penelitian ini memiliki kemampuan menghambat pertumbuhan E. coli yang bersumber dari air minum broiler dengan konsentrasi terendah 25%. Kata kunci: Air minum broiler; Escherichia coli; Lidah buaya; Daya hambat bakteri

scholarly journals Pengaruh Uji Antibakteri Ekstrak Rimpang Jahe Terhadap Pertumbuhan Staphylococcus Aureus Dan Escherichia Coli Secara In Vitro

2020 ◽  
Vol 1 (2) ◽  
pp. 71-80
Author(s):  
Siti Zamilatul Azkiyah
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Bacterial Growth ◽  
Ethanol Extract ◽  
Inhibition Zone ◽  
E Coli ◽  
Randomized Design ◽  
Ginger Rhizome ◽  
Completely Randomized Design

The problem of resistance is increasing along with the need for antimicrobials as an alternative in overcoming resistance problems. One solution is to utilize the content of secondary metabolite compounds in rhizome plants such as ginger. The content of compounds that are usually the most dominant in ginger such as essential oils, flavonoids, terpenoids and phenols. This study aims to determine the effect of ginger rhizome extract as an antibacterial on the growth of Staphylococcus aureus and Escherichia coli in vitro. This study was a laboratory experimental study with a completely randomized design (CRD) consisting of 7 treatments with 3 replications. The results showed that the ethanol extract of ginger rhizome had antibacterial activity against S. aureus and E. coli. At concentrations of 20%, 40% and 80% which are more effective in inhibiting the growth of these two bacteria. The higher the extract concentration level, the larger the diameter of the inhibition zone from bacterial growth. This inhibition of bacterial growth is thought to have an effect on the content of the ginger root.   Keywords: Ginger rhizome, Staphylococcus aureus, Escherichia coli, antibacterial ABSTRAK   Permasalahan tentang resistensi semakin meningkat seiring dengan kebutuhan antimikroba sebagai alternatif dalam mengatasi masalah resistensi. Salah satu solusinya yaitu dengan memanfaatkan kandungan dari senyawa metabolit sekunder pada tanaman rimpang-rimpangan seperti rimpang jahe. Kandungan senyawa yang biasanya paling dominan pada jahe seperti minyak atsiri, flavonoid, terpenoid dan fenol. Penelitian ini bertujuan untuk mengetahui pengaruh dari ekstrak rimpang jahe sebagai antbakteri terhadap pertumbuhan Staphylococcus aureus dan Escherichia coli secara in vitro. Penelitian ini termasuk penelitian eksperimental laboratorium dengan rancangan acak lengkap (RAL) yang terdiri dari 7 perlakuan dengan 3 kali ulangan. Hasil penelitian diperoleh bahwa ekstrak etanol rimpang jahe memiliki aktivitas antibakteri terhadap S. Aureus dan E. coli. Pada konsenrasi 20%, 40% dan 80% yang lebih efktif dalam menghambat pertumbuhan kedua bakteri tersebut. Semakin tinggi tingkatan konsentrasi ekstrak maka diameter zona hambat dari pertumbuhan bakteri juga semakin besar. Terhambatnya pertumbuhan bakteri ini diduga adanya pengaruh dari kandungan pada rimpang jahe.   Kata Kunci: Rimpang Jahe, Staphylococcus aureus, Escherichia coli, antibakteri

scholarly journals Study of the Effect of Carob (Ceratoniasiliqua L.) Extract Activity as Antibiotic from UTI

2018 ◽  
Vol 8 (1) ◽  
pp. 17-25
Author(s):  
Iman Fadhil Abdul-Husin
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Plant Extracts ◽  
Ethanol Extract ◽  
Species Level ◽  
Bacterial Cells ◽  
E Coli ◽  
In Vitro Antibacterial Activity ◽  
Resistant Strains

Escherichia coli  bacterial cells have been collected and selected from(30) patients (most found strain) in urine samples 25 (83.3 %) suffering from infection of urinary tract laid down in Hashimiyaha teaching hospital, Babylon during a period from Novembertwo thousand sixty to February two thousand seventy. The isolated strain  diagnosis is confirmed withVitek2 system apparatus which performed to identify species level ofEscherichia coli  isolates.To evaluate the antimicrobialaction of the ethanol extract of  Carob (CeratoniasiliquaL.) podsonlyas well as in mixture with certain drugs (64µg /ml ampicillin, 32µg /ml gentamicin, 128µg /ml amikacin,8µg /ml clindamycin.) as the wide usageantibiotics in the treatment of UTI bacterial infectionswhich has led to the emergence and spread of resistant strains. Many studies showed that the efficacy of antimicrobials can be improved by combining them with crude plant extracts. The antimicrobial activity of the ethanol extract of pods of Carob(CeratoniasiliquaL.)alone as well as in mixture with some  standard antimicrobials has been evaluated using well diffusion methodwhich demonstrates an in-vitro antibacterial activity of the tested extracts against E. Coli bacteria.  A combination of the tested extracts( concentration 100%,50%) with antibacterial has increased that activity of the tested antimicrobials.The results revealed the importance of  Carob plant extracts when associated with antibiotics to regulatorresistance E. Coli bacteria that developed as adanger to human health

scholarly journals Uji daya hambat ekstrak daun lidah mertua (Sansevieriae trifasciata folium) terhadap pertumbuhan bakteri Escherichia coli dan Streptococcus sp

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
Brily Lombogia ◽  
Fona Budiarso ◽  
Widdhi Bodhi
Keyword(s):  
Escherichia Coli ◽  
Laboratory Study ◽  
Leaf Extract ◽  
Ethanol Extract ◽  
Inhibition Zone ◽  
Antibacterial Activities ◽  
Antibacterial Effects ◽  
E Coli ◽  
Experimental Laboratory ◽  
Inhibition Zones

Abstract: Mother in law’s tongue plant has some active compounds inter alia saponin, polyphenol, and flavonoid that have antibacterial effects. This study aimed to identify whether the antibacterial effects of mother in law’s tongue leaf (Sansevieria Trifasciata) towards the growth of Escherichia coli and Streptococcus sp. This was an experimental laboratory study. The concentrations of mother in law’s tongue leaf extract were tested with well methods, as follows: 5%, 10%, 20%, and 40%. The results showed that this extract at concentration of 5%, 10%, 20%, and 40% could inhibit the growth of E. coli with the average diameters of inhibition zones as follows: 7.8 mm, 13 mm, 14.5 mm, and 17.3 mm meanwhile of Streptococcus sp. with the average diameters of inhibition zones, as follows: 4.6 mm, 9.6 mm, 13 mm, and 15.3 mm. Conclusion: Ethanol extract of mother in law’s tongue leaves (Sansevieria Trifasciata) has antibacterial activities against the growth of E. coli and Streptococcus sp. The higher the concentration is, the broader the inhibition zone is.Keywords: Sansevieriae trifasciata folium, inhibition zone, E. coli, Streptococcus sp. Abstrak: Tanaman Lidah Mertua (Sansevieria Trifasciata) memiliki senyawa aktif yaitu Saponin, Polifenol, dan Flavonoid yang mampu bekerja sebagai antibakteri. Penelitian ini bertujuan untuk mengetahui ada tidaknya daya hambat ekstrak daun lidah mertua (Sansevieria trifasciata) terhadap pertumbuhan bakteri E. coli, dan Streptococcus sp. Jenis penelitian ini eksperimental laboratorik. Kadar ekstrak etanol daun lidah mertua (Sansevieria trifasciata) yang diujikan dengan metode sumuran yaitu 5%, 10%, 20%, dan 40%. Ekstrak etanol daun lidah mertua (Sansevieria trifasciata) dengan konsentrasi 5%, 10%, 20%, dan 40% dapat menghambat pertumbuhan bakteri E. coli dengan rerata diameter zona hambat masing-masing yaitu 7,8 mm, 13 mm, 14,5 mm, dan 17,3 mm sedangkan Streptococcus sp. dengan masing-masing rerata diameter zona hambat yaitu 4,6 mm, 9,6 mm, 13 mm, dan 15,3 mm. Simpulan: Ekstrak etanol daun lidah mertua (Sansevieria trifasciata) mempunyai aktifitas antimikroba terhadap pertumbuhan bakteri E. coli dan Streptococcus sp, dimana makin tinggi konsentrasi ekstrak daun lidah mertua, makin luas zona jernih pada media kultur bakteri E. coli dan Streptococcus sp. Kata kunci: Sansevieriae trifasciata folium, daya hambat, E. coli, Streptococcus sp.

Substituted 4-Formyl-2H-chromen-2-ones: Their Reaction with N-(2,3,4,6-Tetra-Oacetyl- β-D-galactopyranosyl)thiosemicarbazide, Antibacterial and Antifungal Activity of Their Thiosemicarbazone Products

2020 ◽  
Vol 24 (19) ◽  
pp. 2272-2282
Author(s):  
Vu Ngoc Toan ◽  
Nguyen Minh Tri ◽  
Nguyen Dinh Thanh
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Selenium Dioxide ◽  
Antibacterial And Antifungal Activity ◽  
E Coli ◽  
Antibacterial And Antifungal Activities ◽  
Alkyl Ethers ◽  
Antibacterial And Antifungal

Several 6- and 7-alkoxy-2-oxo-2H-chromene-4-carbaldehydes were prepared from corresponding alkyl ethers of 6- and 7-hydroxy-4-methyl-2-oxo-2H-chromen-2-ones by oxidation using selenium dioxide. 6- and 7-Alkoxy-4-methyl-2H-chromenes were obtained with yields of 57-85%. Corresponding 4-carbaldehyde derivatives were prepared with yields of 41-67%. Thiosemicarbazones of these aldehydes with D-galactose moiety were synthesized by reaction of these aldehydes with N-(2,3,4,6-tetra-O-acetyl-β-Dgalactopyranosyl) thiosemicarbazide with yields of 62-74%. These thiosemicarbazones were screened for their antibacterial and antifungal activities in vitro against bacteria, such as Staphylococcus aureus, Escherichia coli, and fungi, such as Aspergillus niger, Candida albicans. Several compounds exhibited strong inhibitory activity with MIC values of 0.78- 1.56 μM, including 8a (against S. aureus, E. coli, and C. albicans), 8d (against E. coli and A. niger), 9a (against S. aureus), and 9c (against S. aureus and C. albicans).

In Vitro Antibacterial activity of ethanolic extract of Myrsine africana against laboratory Strains of Pathogenic Organisms

2016 ◽  
Vol 5 (04) ◽  
pp. 4512
Author(s):  
Jackie K. Obey ◽  
Anthoney Swamy T* ◽  
Lasiti Timothy ◽  
Makani Rachel
Keyword(s):  
Antibacterial Activity ◽  
Minimum Inhibitory Concentration ◽  
Inhibitory Concentration ◽  
Ethanolic Extract ◽  
Ethanol Extract ◽  
E Coli ◽  
Zones Of Inhibition ◽  
In Vitro Antibacterial Activity ◽  
Myrsine Africana

The determination of the antibacterial activity (zone of inhibition) and minimum inhibitory concentration of medicinal plants a crucial step in drug development. In this study, the antibacterial activity and minimum inhibitory concentration of the ethanol extract of Myrsine africana were determined for Escherichia coli, Bacillus cereus, Staphylococcus epidermidis and Streptococcus pneumoniae. The zones of inhibition (mm±S.E) of 500mg/ml of M. africana ethanol extract were 22.00± 0.00 for E. coli,20.33 ±0.33 for B. cereus,25.00± 0.00 for S. epidermidis and 18. 17±0.17 for S. pneumoniae. The minimum inhibitory concentration(MIC) is the minimum dose required to inhibit growth a microorganism. Upon further double dilution of the 500mg/ml of M. africana extract, MIC was obtained for each organism. The MIC for E. coli, B. cereus, S. epidermidis and S. pneumoniae were 7.81mg/ml, 7.81mg/ml, 15.63mg/ml and 15.63mg/ml respectively. Crude extracts are considered active when they inhibit microorganisms with zones of inhibition of 8mm and above. Therefore, this study has shown that the ethanol extract of M. africana can control the growth of the four organisms tested.

scholarly journals In vitro activity of antimicrobial peptide CDP-B11 alone and in combination with colistin against colistin-resistant and multidrug-resistant Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kaitlin S. Witherell ◽  
Jason Price ◽  
Ashok D. Bandaranayake ◽  
James Olson ◽  
Douglas R. Call
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptide ◽  
Membrane Integrity ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Multidrug Resistant Bacteria ◽  
E Coli ◽  
Minimal Media

AbstractMultidrug-resistant bacteria are a growing global concern, and with increasingly prevalent resistance to last line antibiotics such as colistin, it is imperative that alternative treatment options are identified. Herein we investigated the mechanism of action of a novel antimicrobial peptide (CDP-B11) and its effectiveness against multidrug-resistant bacteria including Escherichia coli #0346, which harbors multiple antibiotic-resistance genes, including mobilized colistin resistance gene (mcr-1). Bacterial membrane potential and membrane integrity assays, measured by flow cytometry, were used to test membrane disruption. Bacterial growth inhibition assays and time to kill assays measured the effectiveness of CDP-B11 alone and in combination with colistin against E. coli #0346 and other bacteria. Hemolysis assays were used to quantify the hemolytic effects of CDP-B11 alone and in combination with colistin. Findings show CDP-B11 disrupts the outer membrane of E. coli #0346. CDP-B11 with colistin inhibits the growth of E. coli #0346 at ≥ 10× lower colistin concentrations compared to colistin alone in Mueller–Hinton media and M9 media. Growth is significantly inhibited in other clinically relevant strains, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In rich media and minimal media, the drug combination kills bacteria at a lower colistin concentration (1.25 μg/mL) compared to colistin alone (2.5 μg/mL). In minimal media, the combination is bactericidal with killing accelerated by up to 2 h compared to colistin alone. Importantly, no significant red blood hemolysis is evident for CDP-B11 alone or in combination with colistin. The characteristics of CDP-B11 presented here indicate that it can be used as a potential monotherapy or as combination therapy with colistin for the treatment of multidrug-resistant infections, including colistin-resistant infections.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Christopher W. Lennon ◽  
Kimberly C. Lemmer ◽  
Jessica L. Irons ◽  
Max I. Sellman ◽  
Timothy J. Donohue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Rhodobacter Sphaeroides ◽  
Fatty Acid Content ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Globular Domain ◽  
Content Type ◽  
E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

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