Novel bacteriocin design

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Novel bacteriocin design
by
Burcu Kaplan
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Bacteriocins
Proteinaceous subtances that kill closely related
bacteria
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Mode of action
Enzyme activity modulation
Inhibition of outgrowth of spores
Anion carrier activity to the formation of pores
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Lacticin 3147
Production and immunity encoded on a 60.2 kb
conjugative plasmid,pMCR01
No inhibitory effect on G(-) bacteria
Active at neutral pH and after spraydrying
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Problem
Inhibition of G(-) spoilage / pathogen bacteria
by use of these natural antimicrobial substances
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Bacteriocins
colicins

• Class I the lantibiotic family
• Class II small non-modified peptides
• Class III large heat-labile proteins

colicins 5, 10, A, B, E1, Ia, Ib, K, and N
form cytoplasmic pores which result in a
reduction of electrochemical membrane potential.
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Bacteriocins
Propose
Pore forming domains of various colicins are
identified

• Class I the lantibiotic family
• Class II small non-modified peptides
• Class III large heat-labile proteins

G() bacteriocins can not pass from the OM of G(-)
Insertion of this domain into pMRC01 may result
in a novel bacteriocin
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Bacteriocins
Experimental
Gene fusion

• Class I the lantibiotic family
• Class II small non-modified peptides
• Class III large heat-labile proteins

Protein expression
Protein structure analysis
Protein function analysis
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Bacteriocins
Experimental
Gene fusion

• Class I the lantibiotic family
• Class II small non-modified peptides
• Class III large heat-labile proteins

Protein expression
Protein structure analysis
Protein function analysis
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Expected result
Novel bacteriocin
Inhibitory to both G(-) and G() bacteria
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Lacticin 3147

• Two subunits
• LNTA1
• LNTA2

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Experimental
Delete immunity gene
Express this protein in E.coli
Structure-function studies
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Applications

• fermentation

• Bioprocessing

• Agriculture

• food

• medicine

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NISIN

• first identified bacteriocin

• inhibitiory effect on some LAB

• first marketed in England in 1953

• FAO/WHO Codex Committee on milk
• and milk products accepted nisin as a food
  additive in 1969.

• added to the European food additive list as E234
• (EEC, 1983).

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Biopreservative Effect

• antimicrobial compounds and proteinaceous
  substances which can inhibit or reduce
  undesirable flora in food products
• Microorganisms can also produce a range of
  anti-microbial peptides and proteins which are
  collectively referred to as bacteriocins.

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LAB

• a variety of dairy, vegetable
• and meat fermentations.

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