Comparison of Two Methods for Purification of Enterocin B, a Bacteriocin Produced by Enterococcus faecium W3

Date

2015-11-17

Author

Dundar, Halil
ATAKAY, MEHMET
Celikbicak, Omuer
SALİH, BEKİR
Bozoglu, Faruk

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

118
views

0
downloads

This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography, and sequential reverse-phase high-performance liquid chromatography. The latter approach exploited a pH-mediated cell adsorption-desorption method to extract cell-bound bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide were confirmed again by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) analysis. Although both approaches were satisfactory to obtain a sufficient amount of enterocin B for use in MS and amino acid sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B.

Subject Keywords

Biotechnology, Biochemistry, General Medicine

URI

https://hdl.handle.net/11511/67964

Journal

PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY

DOI

https://doi.org/10.1080/10826068.2014.958165

Collections

Department of Biology, Article

Suggestions

OpenMETU
Core

Comparison of benzaldehyde lyase production capacity in recombinant Escherichia coli and recombinant Bacillus species Kaya, Hande; Çalık, Pınar; Department of Chemical Engineering (2006) In this study, the benzaldehyde lyase (BAL, EC 4.1.2.38) production in E. coli BL21 (DE3) pLySs as intracellular and in Bacillus species as extracellular were investigated, and comparison of the production capacity of the enzyme in the developed recombinant microorganisms were compared. For this purpose, firstly, PCR amplified bal gene was cloned into pRSETA vector which is under the control of strong T7 promoter and expressed in E. coli BL21 (DE3) pLysS strain. With developed recombinant E. coli BL21 (DE3)...
Analysis of acyl CoA ester intermediates of the mevalonate pathway in Saccharomyces cerevisiae. Şeker, Tamay; Nielsen, J (Springer Science and Business Media LLC, 2005-04-01) The mevalonate pathway plays an important role in providing the cell with a number of essential precursors for the synthesis of biomass constituents. With respect to their chemical structure, the metabolites of this pathway can be divided into two groups: acyl esters [acetoacetyl CoA, acetyl CoA, hydroxymethylglutaryl (HMG) CoA] and phosphorylated metabolites (isopentenyl pyrophosphate, dimethylallyl pyrophosphate, geranyl pyrophosphate, farnesyl pyrophosphate). In this study, we developed a method for the ...
Determination and metabolic engineering of rate limiting reactions in aromatic amino acid pathway in Bacillus subtilis for L-phenylaianine production Guzide, Calik; Yasemin, Demirci; Pinar, Calik; Ozdamar, Tuncer H. (2007-09-01) Rate limiting reactions in the aromatic-group amino acid pathway (AAAP) in Bacillus subtilis for l-phenylalanine (Phe) production were determined as reported elsewhere (Özçelik-Şenver et al., 2004). The biochemical reactions in the AAAP start with the reaction using phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) synthesised in the glycolysis pathway and the PPP, respectively, leading to the formation of Phe via chorismate by branching at prephenate. The branch-point metabolites E4P supplied in vi...
PRODUCTION OF THE LEPIDOPTERA-SPECIFIC CRYSTAL PROTEIN BY A LOCAL ISOLATE OF BACILLUS-THURINGIENSIS AFKHAMI, P; Özcengiz, Gülay; ALAEDDINOGLU, NG (Springer Science and Business Media LLC, 1993-12-01) A local isolate of Bacillus thuringiensis (B.t.81) produced crystal protein was identified as a cry I gene of Class I. The synthesis and assembly of crystal complements were investigated at intervals throughout the growth cycle. Incubation temperature had a marked effect on toxin synthesis; production being the highest at 25-degrees-C and the lowest at 42-degrees-C. The mutants of B. t. 81, unable to synthesize crystal protein complements, have also been described.
Determination of metabolic bottlenecks using reaction engineering principles in serine alkaline protease production by recombinant bacillus sp. Telli, İlkin Ece; Çalık, Pınar; Department of Chemical Engineering (2004) In this study, firstly, bioprocess characteristics for Serine Alkaline Protease (SAP) production, using recombinant Bacillus subtilis carrying pHV1431::subC, were examined. The cell concentration, substrate concentration, SAP activity and SAP synthesis rate profiles demonstrated that the system reaches to a steady state in terms of cell growth and SAP synthesis between t=15-25 h, therefore, this time interval is appropriate to employ both metabolic flux analysis and metabolic control analysis, which apply s...

Citation Formats

H. Dundar, M. ATAKAY, O. Celikbicak, B. SALİH, and F. Bozoglu, “Comparison of Two Methods for Purification of Enterocin B, a Bacteriocin Produced by Enterococcus faecium W3,” PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY, pp. 796–809, 2015, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/67964.