Enhanced Biocatalytic Activity of Recombinant Lipase Immobilized on Gold Nanoparticles

Background: Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications. Objectives: With the better understanding and progressive needs for improving its activity in accordance with the growing market demand, we aimed in this study to improve the recombinant production and biocatalytic activity of lipases via surface conjugation on gold nanoparticles. Methods: The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14 and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were immobilized through surface conjugation with gold nanoparticles and enzymatic activity was colorimetrically quantified. Results: Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively. However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs), the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively. The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf, respectively. Conclusion: The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased relative to the free recombinant enzyme where immobilization had made the enzyme attain its optimum performance.

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High Expression of Human Amelogenin in E. Coli

Advances in Dental Research ◽

10.1177/08959374960100021201 ◽

1996 ◽

Vol 10 (2) ◽

pp. 187-194 ◽

Cited By ~ 6

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.

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Overexpression of Recombinant Lipase from Burkholderia Cepacia in Escherichia Coli

European Journal of Inflammation ◽

10.1177/1721727x1201000312 ◽

2012 ◽

Vol 10 (3) ◽

pp. 365-369 ◽

Cited By ~ 1

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.

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Enhanced Extracellular Synthesis of Gold Nanoparticles by Soluble Extracts from Escherichia coli Transformed with Rhizobium tropici Phytochelatin Synthase Gene

Metals ◽

10.3390/met11030472 ◽

2021 ◽

Vol 11 (3) ◽

pp. 472

Author(s):

Qunying Yuan ◽

Manjula Bomma ◽

Zhigang Xiao

Keyword(s):

Gold Nanoparticles ◽

Gold Nanoparticle ◽

Synthesis Method ◽

Nanoparticle Synthesis ◽

Morphological Properties ◽

Phytochelatin Synthase ◽

Rhizobium Tropici ◽

Whole Cells ◽

E Coli ◽

Recombinant Cells

Phytochelatins, the enzymatic products of phytochelatin synthase, play a principal role in protecting the plants from heavy metal and metalloid toxicity due to their ability to scavenge metal ions. In the present study, we investigated the capacity of soluble intracellular extracts from E. coli cells expressing R. tropici phytochelatin synthase to synthesize gold nanoparticle. We discovered that the reaction mediated by soluble extracts from the recombinant E. coli cells had a higher yield of gold nanoparticles, compared to that from the control cells. The compositional and morphological properties of the gold nanoparticles synthesized by the intracellular extracts from recombinant cells and control cells were similar. In addition, this extracellular nanoparticle synthesis method produced purer gold nanoparticles, avoiding the isolation of nanoparticles from cellular debris when whole cells are used to synthesize nanoparticles. Our results suggested that phytochelatins can improve the efficiency of gold nanoparticle synthesis mediated by bacterial soluble intracellular extracts, and the potential of extracellular nanoparticle synthesis platform for the production of nanoparticles in large quantity and pure form is worth further investigation.

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Multifunctional Activities of Gold Nanoparticles Biosynthesized Using Bacteria Isolated from Mining Areas

Applied Sciences ◽

10.3390/app11083670 ◽

2021 ◽

Vol 11 (8) ◽

pp. 3670

Author(s):

Chih-Yu Chen ◽

Yung-Chu Chang ◽

Teh-Hua Tsai ◽

Man-Hai Liu ◽

Ying-Chien Chung

Keyword(s):

Gold Nanoparticles ◽

Mining Area ◽

Synthesis Process ◽

Average Particle ◽

Bifidobacterium Lactis ◽

E Coli ◽

Mining Areas ◽

Skin Cells ◽

Study Results ◽

Antityrosinase Activity

Research on gold nanoparticles (AuNPs) has often focused on their physical, chemical, and crystalline characteristics. Commercial AuNPs have been applied in the diverse fields of biomedicine, catalysis, photovoltaics, and sensing. In this study, we explored the various activities of AuNPs to widen their applicability. This paper presents a simple and rapid synthesis process of AuNPs with bacteria isolated from a gold mining area. We also investigated the optimization of reaction parameters for AuNP synthesis. The study results revealed that among the isolated strains, Bifidobacterium lactis and Escherichia coli demonstrated the highest capabilities of AuNP synthesis. The optimal pH values for AuNP synthesis by B. lactis (BLAuNPs) and E. coli (ECAuNPs) were 5.0 for 72 h of incubation and 8.0 for 24 h of incubation. The average particle sizes of ECAuNPs and BLAuNPs were 4.2 and 5.6 nm, respectively. Furthermore, these biogenic AuNPs were found to be stable with no aggregation after 3 months of storage. BLAuNPs and ECAuNPs exhibited high levels of antimicrobial, antioxidant, photocatalytic, and antityrosinase activity. Moreover, they were noncytotoxic to skin cells even at 100% melanin inhibitory concentrations. Considering the demonstrated multifunctional activities of AuNPs, BLAuNPs and ECAuNPs have promising potential for commercialization.

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On-column Refolding of an Insoluble His6-tagged Recombinant EC-SOD Overexpressed in Escherichia coli

Acta Biochimica et Biophysica Sinica ◽

10.1111/j.1745-7270.2005.00035.x ◽

2005 ◽

Vol 37 (4) ◽

pp. 265-269 ◽

Cited By ~ 11

Author(s):

Xi-Qiang Zhu ◽

Su-Xia Li ◽

Hua-Jun He ◽

Qin-Sheng Yuan

Keyword(s):

Escherichia Coli ◽

Inclusion Bodies ◽

Mass Ratio ◽

Chain Reaction ◽

Expression Plasmid ◽

Protein Subunits ◽

E Coli ◽

Metal Affinity ◽

Polymerase Chain ◽

Escherichia Coli Expression

Abstract The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile. Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.

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Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

Pesquisa Agropecuária Brasileira ◽

10.1590/s0100-204x2008001000020 ◽

2008 ◽

Vol 43 (10) ◽

pp. 1405-1411 ◽

Cited By ~ 6

Author(s):

Paula Radaelli ◽

Thor Vinícius Martins Fajardo ◽

Osmar Nickel ◽

Marcelo Eiras ◽

Gilvan Pio-Ribeiro

Keyword(s):

Virus Disease ◽

Disease Diagnosis ◽

Coat Proteins ◽

Grapevine Virus ◽

Western Blots ◽

E Coli ◽

Sds Page ◽

Grapevine Virus B ◽

Polyclonal Antisera ◽

Infected Grapevine

The objective of this work was to produce and characterize specific antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) and Grapevine virus B (GVB), developed from expressed coat proteins (CPs) in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP) genes were RT-PCR-amplified, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purified, and their identities were confirmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A405) of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.

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Biosynthesis, purification, characterization and immobilization of laccase from Lithothelium sp.

Chemija ◽

10.6001/chemija.v31i3.4292 ◽

2020 ◽

Vol 31 (3) ◽

Author(s):

Ingrida Radveikienė ◽

Ingrida Pilotaitė ◽

Rimgailė Dainytė ◽

Regina Vidžiūnaitė

Keyword(s):

Residual Activity ◽

Catalytic Efficiency ◽

Hydrophobic Interaction Chromatography ◽

Metal Salts ◽

Biotechnological Applications ◽

Affinity Constants ◽

Sds Page ◽

Wide Range ◽

Optimum Ph ◽

Sulphate Salts

Novel fungal laccase isoenzymes (namely L95-1 and L95-2) produced by the Ascomycete Lithothelium sp. isolated from the forest soil were purified. However, only one of them was characterized, because the other isoenzyme lost its activity during purification. Extracellular L95-1 laccase was purified 30-fold using ion-exchange and hydrophobic interaction chromatography, with an overall yield of 88%. The molecular mass of purified L95-1 was estimated to be 85 kDa by SDS-PAGE analysis. L95-1 laccase was stable at temperature 4–22°C and pH 6.0–6.5. The substrate specificity of L95-1 laccase was examined with various compounds. Determined affinity constants (KM) varied in a wide range of 3.7–2020.0 µM, whereas catalytic efficiency constants (kcat/KM) covered a range of 0.008–1.9 µM–1 s–1. The optimum pH for most substrates varied in a range from pH 5.0 to 6.0. Sodium azide and fluoride strongly inhibited L95-1 activity, whereas sulphate salts inhibited weakly. The laccase was immobilized on the Fe3O4 nanoparticles and characterized. Residual activity remained at 20% after ten cycles of ABTS oxidation reaction. The immobilized laccase showed higher tolerance to various metal salts. The properties of L95-1 laccase make it potentially useful in the biotechnological applications.

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Recombinant Production of Biliverdin IXβ and δ Isomers in the T7 Promoter Compatible Escherichia coli Nissle

Frontiers in Microbiology ◽

10.3389/fmicb.2021.787609 ◽

2021 ◽

Vol 12 ◽

Author(s):

Elizabeth A. Robinson ◽

Nicole Frankenberg-Dinkel ◽

Fengtian Xue ◽

Angela Wilks

Keyword(s):

Heme Oxygenase ◽

Chemical Oxidation ◽

Heme Biosynthesis ◽

T7 Promoter ◽

E Coli ◽

Recombinant Production ◽

Alternative Approaches ◽

Biliverdin Ix ◽

Scalable Production ◽

Escherichia Coli Nissle

The ability to obtain purified biliverdin IX (BVIX) isomers other than the commercially available BVIXα is limited due to the low yields obtained by the chemical coupled oxidation of heme. Chemical oxidation requires toxic chemicals, has very poor BVIX yields (<0.05%), and is not conducive to scalable production. Alternative approaches utilizing recombinant E. coli BL21 expressing a cyanobacterial heme oxygenase have been employed for the production BVIXα, but yields are limited by the rate of endogenous heme biosynthesis. Furthermore, the emerging roles of BVIXβ and BVIXδ in biology and their lack of commercial availability has led to a need for an efficient and scalable method with the flexibility to produce all three physiologically relevant BVIX isomers. Herein, we have taken advantage of an optimized non-pathogenic E. coli Nissle (EcN(T7)) strain that encodes an endogenous heme transporter and an integrated T7 polymerase gene. Protein production of the Pseudomonas aeruginosa BVIXβ and BVIXδ selective heme oxygenase (HemO) or its BVIXα producing mutant (HemOα) in the EcN(T7) strain provides a scalable method to obtain all three isomers, that is not limited by the rate of endogenous heme biosynthesis, due to the natural ability of EcN(T7) to transport extracellular heme. Additionally, we have optimized our previous LC-MS/MS protocol for semi-preparative separation and validation of the BVIX isomers. Utilizing this new methodology for scalable production and separation we have increased the yields of the BVIXβ and -δ isomers >300-fold when compared to the chemical oxidation of heme.

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Green Synthesis of Gold Nanoparticles from Leaf Extracts of Ginkgo biloba L. and its Antibiotic Potential Against Escherichia coli and Bacillus subtilis

Proceedings International ◽

10.33263/proceedings21.029029 ◽

2020 ◽

Vol 2 (1) ◽

pp. 29

Keyword(s):

Gold Nanoparticles ◽

Bacillus Subtilis ◽

Aqueous Extract ◽

Ginkgo Biloba ◽

Bacterial Pathogens ◽

Sem Analysis ◽

Leaf Extracts ◽

Living Fossil ◽

E Coli ◽

Global Environmental

Based on the global environmental pollution problems, the main focus of every nano-research is to produce the nanomaterial in a green and eco-friendly way without any interference of chemical synthesis. By the way, the present study was intended to use an aqueous extract of the living fossil plant viz., Ginkgo biloba L., to synthesize the gold nanoparticles and evaluate their antibiotic activity against bacterial pathogens. The gold nanoparticles (AuNps) were successfully synthesized by mixing the Ginkgo biloba aqueous extract and the auric chloride solution for approximately 24 hours. The UV-Vis spectra of Gold nanoparticles (AuNps) showed the maximum absorption peak at 520nm. The SEM analysis also showed the gold nanoparticles synthesized from Ginkgo biloba were spherical with particle size ranging from 40 to 60nm. During our study, the gold nanoparticles exhibited significant antimicrobial activity against bacterial pathogens, i.e., E. coli and Bacillus subtilis. The later bacterium was found to be more susceptible to the nanoparticles as well as the extracts of G. biloba in comparison to the former bacterium.

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CONSTRUCTION, EXPRESSION AND PURIFICATION OF RECOMBINANT PRE-MATURE PEPTIDE OF PLANTARICIN F FROM Lactobacillus plantarum S34 IN Escherichia coli

Indonesian Journal of Agricultural Science ◽

10.21082/ijas.v16n1.2015.31-38 ◽

2015 ◽

Vol 16 (1) ◽

pp. 31

Author(s):

Kusdianawati Kusdianawati ◽

Apon Zaenal Mustopa ◽

Suharsono Suharsono ◽

Bugi Ratno Budiarto ◽

Fatimah Fatimah ◽

...

Keyword(s):

Escherichia Coli ◽

Lactobacillus Plantarum ◽

Proteolytic Enzymes ◽

Leader Peptide ◽

Human Consumption ◽

Mature Peptide ◽

Expression And Purification ◽

Food Preservative ◽

E Coli ◽

Sds Page

Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from Lactobacillus plantarum S34 in Escherichia coli. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. plantarum S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3) pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. plantarum S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His)6tag had successfully been expressed in E. coli BL21 (DE3) pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1). Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F’s leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His)6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.

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