High-level soluble expression and enzymatic characterization of Burkholderia sp. lipase with steryl ester hydrolysis activity in E. coli

Abstract Background Burkholderia cepacia lipase is an important industrial biocatalyst for biodiesel production and chiral pharmaceutical synthesis. Heterologous soluble expression of lipase lipA gene from B. cepacia in Escherichia coli highly depends on co-expression of its cognate foldase gene, lipB. However, the interaction between recombinant lipase LipA and chaperonin LipB is rather complicated and confusing. In this research, various systems of lipA/lipB co-expression combinations are investigated to obtain high-level soluble expression of lipA, respectively. Results The best co-expression combination system for lipA and lipB is E. coli Origami 2 (DE3)/pETDuet-lipB(MCS1)/lipA(MCS2). The soluble expression level of lipA is 100.4 U/OD600 towards 4-nitrophenyl laurate hydrolysis. The recombinant LipA can be rapidly isolated from cell-free supernatant of recombinant E. coli lysate using HisTrap HP affinity chromatography column, and the lipA/LipB complex is obtained. Enzymatic characterization analysis shows that the purified LipA is a mesothermal and alkaline enzyme. LipA displays preference for medium-chain-length acyl groups (C10-C12) and sn-1,3 regioselectivity. Besides triacylglycerol hydrolase activity (EC. 3.1.1.3), LipA also displays steryl ester hydrolase activity (EC. 3.1.1.13). The specific activity of LipA towards 4-nitrophenyl decanoate and cholesterol linoleate are 638.9 U/mg and 1111.5 mU/mg, respectively. Conclusions Host strain E. coli Origami 2 (DE3), lipB locus at MCS1 on the dual expression cassette plasmid pETDuet, and low-temperature induction contribute to the soluble expression of lipA. Recombinant LipA displays both triacylglycerol hydrolase activity and steryl ester hydrolase activity.

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Constitutive High Level Expression of an Endoxylanase Gene from the Newly IsolatedBacillus subtilisAQ1 inEscherichia coli

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/980567 ◽

2010 ◽

Vol 2010 ◽

pp. 1-12 ◽

Cited By ~ 7

Author(s):

Is Helianti ◽

Niknik Nurhayati ◽

Maria Ulfah ◽

Budiasih Wahyuntari ◽

Siswa Setyahadi

Keyword(s):

Specific Activity ◽

Biochemical Properties ◽

Extracellular Expression ◽

High Level Expression ◽

Periplasmic Fraction ◽

16S Rdna Sequence ◽

E Coli ◽

Encoding Gene ◽

High Level ◽

Very High

A xylanolytic bacterium was isolated from the sediment of an aquarium. Based on the 16S rDNA sequence as well as morphological and biochemical properties the isolate was identified and denoted asBacillus subtilis(B. subtilis) AQ1 strain. An endoxylanase-encoding gene along with its indigenous promoter was PCR amplified and after cloning expressed inE. coli. InE. colithe recombinant enzyme was found in the extracellular, in the cytoplasmic, and in the periplasmic fraction. The specific activity of the extracellular AQ1 recombinant endoxylanase after 24-hour fermentation was very high, namely,2173.6 ± 51.4and2745.3 ± 11 U/mg in LB and LB-xylan medium, respectively. This activity was clearly exceeding that of the nativeB. subtilisAQ1 endoxylanase and that of 95% homologous recombinant one fromB. subtilisDB104. The result shows that the original AQ1 endoxylanase promoter and the signal peptide gave a very high constitutive extracellular expression inE. coliand hence made the production inE. colifeasible.

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High-level soluble expression of bioactive porcine myostatin propeptide in E. coli

Applied Microbiology and Biotechnology ◽

10.1007/s00253-013-5134-0 ◽

2013 ◽

Vol 97 (19) ◽

pp. 8517-8527 ◽

Cited By ~ 5

Author(s):

Wing Yeung Haq ◽

Sang Kee Kang ◽

Sang Beum Lee ◽

Hee Chul Kang ◽

Yun Jaie Choi ◽

...

Keyword(s):

Soluble Expression ◽

E Coli ◽

High Level

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Inhibitors of sterol synthesis. 15-Oxygenated steryl ester hydrolase activity of rat liver at neutral and acid pH

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism ◽

10.1016/0005-2760(89)90138-0 ◽

1989 ◽

Vol 1001 (2) ◽

pp. 127-133 ◽

Cited By ~ 2

Author(s):

Thomas W. Stephens ◽

George J. Schroepfer

Keyword(s):

Rat Liver ◽

Steryl Ester ◽

Hydrolase Activity ◽

Sterol Synthesis ◽

Acid Ph ◽

Ester Hydrolase

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High level of soluble expression and purification of catalytically active native UDP-galactose 4-epimerase of <i>Aeromonas hydrophila</i> in <i>E. coli</i>

Advances in Bioscience and Biotechnology ◽

10.4236/abb.2011.26058 ◽

2011 ◽

Vol 02 (06) ◽

pp. 397-403

Author(s):

Keshav Gopal ◽

Shivani Agarwal ◽

Aparna Dixit

Keyword(s):

Aeromonas Hydrophila ◽

Soluble Expression ◽

Expression And Purification ◽

E Coli ◽

Catalytically Active ◽

High Level

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High-level soluble expression of isopenicillin N synthase isozymes in E. coli

Tetrahedron ◽

10.1016/s0040-4020(01)86491-7 ◽

1991 ◽

Vol 47 (31) ◽

pp. 5991-6002 ◽

Cited By ~ 17

Author(s):

J.E. Baldwin ◽

J.M. Blackburn ◽

J.D. Sutherland ◽

M.C. Wright

Keyword(s):

Soluble Expression ◽

E Coli ◽

High Level ◽

Isopenicillin N

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High-Level Formation of Active Pseudomonas cepaciaLipase after Heterologous Expression of the Encoding Gene and Its Modified Chaperone in Escherichia coli and Rapid In Vitro Refolding

Applied and Environmental Microbiology ◽

10.1128/aem.65.2.787-794.1999 ◽

1999 ◽

Vol 65 (2) ◽

pp. 787-794 ◽

Cited By ~ 57

Author(s):

Dinh Thi Quyen ◽

Claudia Schmidt-Dannert ◽

Rolf D. Schmid

Keyword(s):

Escherichia Coli ◽

Burkholderia Cepacia ◽

Specific Activity ◽

Gc Content ◽

Pseudomonas Cepacia ◽

Membrane Anchor ◽

E Coli ◽

Highly Active ◽

High Level

ABSTRACT The lipase from Pseudomonas cepacia ATCC 21808 (recently reclassified as Burkholderia cepacia) is widely used by organic chemists for enantioselective synthesis and is manufactured from recombinant P. cepacia harboring on a plasmid the clustered genes for lipase and its chaperone. High levels of expression of inactive lipase (40%) in Escherichia coli were achieved with pCYTEXP1 under the control of the strong, temperature-inducible λPRL promoter. However, no overexpression of the lipase chaperone was achieved in E. coli. Thus, chemical refolding of inactive lipase in the absence of its chaperone yielded only 25 U/mg, compared to 3,470 U of the purified lipase secreted by recombinant P. cepacia per mg. Sequence analysis of the chaperone revealed a high GC content (>90%) in the 5′ region of the gene and the presence of a putative membrane anchor at the N terminus. Hence, the 5′ region of the gene was replaced by a synthetic fragment, and the putative membrane anchor was removed by deletion of the first 34 or 70 N-terminal amino acids. Only truncation of the gene led to overexpression of the chaperone (up to 60%) in E. coli. With this chaperone, it was possible to obtain for the first time in a simple refolding procedure a highly active Pseudomonas lipase (classes I and II) expressed inE. coli with a specific activity of up to 4,850 U/mg and a yield of 314,000 U/g of E. coli wet cells.

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Toxic Effect of 2-ethyl (bicyclo[2.2.1] heptane) on Bacterial Cells

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-6-67-72 ◽

2019 ◽

Vol 35 (6) ◽

pp. 67-72 ◽

Cited By ~ 1

Author(s):

I.V. Manukhov ◽

L.S. Yaguzhinsky ◽

M.V. Bermeshev ◽

M.A. Zisman ◽

V.G. Pevgov ◽

...

Keyword(s):

Toxic Effect ◽

Atmospheric Oxygen ◽

Inducible Promoter ◽

Oxygen Species ◽

Bacterial Cells ◽

Unique Identifier ◽

E Coli ◽

Lux Biosensors ◽

High Level ◽

Ministry Of Higher Education

Toxic effect of 2-ethylnorbornane (2-ethyl(bicyclo[2.2.1]heptane) (EBH)) on bacteria has been studied using the E. coli pRecA-lux and E. coli pKatG- lux cells as lux-biosensors. It was shown that the addition of EBH to the incubation medium leads to death and growth retardation, high level oxidative stress and DNA damage in E. coli cells. It is assumed that the oxidation of EBH with atmospheric oxygen causes the formation of reactive oxygen species in the medium, which makes a major contribution to the toxicity of this substance. biosensor, luciferase, bioluminescence, inducible promoter, PrecA, PkatG The authors are grateful to Stanislav Filippovich Chalkin for the development of interdisciplinary ties in the scientific community. The work was financially supported by the Ministry of Higher Education and Science of Russia (Project Unique Identifier RFMEFI60417X0181, Agreement No. 14.604.21.0181 of 26.09.2017).

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Soluble Expression and Efficient Purification of Recombinant Class I Hydrophobin DewA

International Journal of Molecular Sciences ◽

10.3390/ijms22157843 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7843

Author(s):

Sang-Oh Ahn ◽

Ho-Dong Lim ◽

Sung-Hwan You ◽

Dae-Eun Cheong ◽

Geun-Joong Kim

Keyword(s):

Tertiary Structure ◽

Signal Sequence ◽

Soluble Expression ◽

Class I ◽

Soluble Form ◽

Two Phase ◽

E Coli ◽

Small Proteins ◽

Industrial Use ◽

Shed Light

Hydrophobins are small proteins (<20 kDa) with an amphipathic tertiary structure that are secreted by various filamentous fungi. Their amphipathic properties provide surfactant-like activity, leading to the formation of robust amphipathic layers at hydrophilic–hydrophobic interfaces, which make them useful for a wide variety of industrial fields spanning protein immobilization to surface functionalization. However, the industrial use of recombinant hydrophobins has been hampered due to low yield from inclusion bodies owing to the complicated process, including an auxiliary refolding step. Herein, we report the soluble expression of a recombinant class I hydrophobin DewA originating from Aspergillus nidulans, and its efficient purification from recombinant Escherichia coli. Soluble expression of the recombinant hydrophobin DewA was achieved by a tagging strategy using a systematically designed expression tag (ramp tag) that was fused to the N-terminus of DewA lacking the innate signal sequence. Highly expressed recombinant hydrophobin DewA in a soluble form was efficiently purified by a modified aqueous two-phase separation technique using isopropyl alcohol. Our approach for expression and purification of the recombinant hydrophobin DewA in E. coli shed light on the industrial production of hydrophobins from prokaryotic hosts.

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