Enzymatic synthesis and modification of
structured phospholipids:
recent advances in enzyme preparation and
biocatalytic processes
Saarbrücken, 16th of November 2016
Shinji Hama & Chiaki Ogino & Akihiko Kondo
> 
Overview
Overview
1.  Introduction
2.  Enzymes involved in PL modification
3.  Enzyme preparation for PL modification
4.  Enzymatic modification of PLs
5.  Conclusion
07.12.16
Page
2
> 
Introduction
Introduktion
Phospholipids (PL)
à  head: glycerol phosphate
tail: fatty acids
à  chemical and physical properties depend on their
molecular structure
Quelle:https://
www.boundless.com/
à  used in applications including
food, cosmetics, pharmaceuticals, plastics, paints, and pesticides
à  function:
emulsifiers
stabilizers
antioxidants
07.12.16
Page
3
> 
Introduction
Natural PLs extracted from biological materials
à  compositional variety of natural PLs is limited
à  difficult: separating specific PLs that rarely exist in nature
Modification of PL
Lecithin (major fraction: Phosphatidylcholine)
à  obtained from biological materials:
soybean
egg yolk (i.e., lecithin)
à  oil processing industry
07.12.16
Page
4
> 
Enzymes involved in PL modification
Enzymes involved in PL modification
Enzyme sources: microbial phospholipases and lipases
Phospholipase
à hydrolyze PLs at different ester bonds
Phospholipase A1 (PLA1) and A2 (PLA2)
Phospholipase C (PLC)
, Lipase
sn-1
Phospholipase D (PLD)
Phospholipase B (PLB)
Lysophospholipase
sn-2
Lipase
à modifying the sn-1 position of PLs
07.12.16
Page
5
> 
Enzyme preparation for PL modification
Enzyme preparation for PL modification
Overexpression of phospholipases for extracellular enzyme production
à  secretory enzymes dispersed in the liquid phase
à  mixture of lecithin and solvents
Immobilized enzyme preparations
à  immobilized on solid supports
à  cell bound enzymes as whole-cell biocatalyst
no purification or immobilization process
07.12.16
Page
6
> 
Enzyme preparation for PL modification
Immobilized enzyme preparations
Rhizopus oryzae
à 
culture of a filamentous fungus
à 
immobilized within reticulated particles:
change in their morphology à higher amount of membrane-bound lipase
ROL34
à 
localized in the cell wall and secreted extracellularly
à 
N28 plays role in the translocation of proteins
ROL31
07.12.16
à 
bound to the cell membrane
à 
high transesterification activity
à 
potential use for PL modification
Page
7
> 
07.12.16
Enzyme preparation for PL modification
Page
8
> 
Enzyme preparation for PL modification
Whole-cell biocatalysts
à  genetically engineered:
yeasts and bacteria
à  selecting the enzymeencoding genes and microbial
hosts
à  heterologous enzymes fused
to cell-wall-localized anchor
proteins
07.12.16
Page
9
> 
Enzymatic modification of PLs
Enzymatic modification of PLs
Hydrolysis for production of lysophospholipids (LPLs)
à 
hydrolysis of lecithin
à 
PLA1, PLA2, and lipases
à 
improve the emulsification properties, heat stability, and viscosity of natural PLs
Transesterification (acidolysis) for exchanging acyl groups in PLs
à 
substrate: lecithin und PC
à 
PLA1, PLA2, and lipases
à 
replaces the original fatty acids with exogenous ones
Transphosphatidylation for exchanging polar head groups in PLs
07.12.16
à 
substrate: lecithin und PC
à 
PLD
à 
exchange of a choline head group
Page
10
> 
Conclusion
Conclusion
à 
PL produced by enzyme-catalyzed reactions
à 
phospholipases and lipases have different specificities
à 
structured PLs improve the chemical and physical properties of natural PLs (required in
industries)
à 
Lecithin
source for natural PLs
plant oils a viable source for lecithin production
07.12.16
à
sophisticated designs of PLs à cannot be obtained by chemical catalysis
à 
genetically engineered microorganisms are more efficiently than enzymes from mammals
à 
Products: high price and low availability (pharmaceutical products)
Page
11
> 
Conclusion
References
Enzymatic synthesis and modification of structured phospholipids: recent advances in enzyme
preparation and biocatalytic processes
Shinji Hama & Chiaki Ogino & Akihiko Kondo
Advancing oleaginous microorganisms to produce lipid via metabolic engineering technology
Liang M-H, Jiang J-G (2013)
Whole-cell biocatalysts for biodiesel fuel production
H. Fukuda, S. Hama, S. Tamalampudi and H. Noda
https://www.boundless.com/biology/textbooks/boundless-biology-textbook/biologicalmacromolecules-3/lipids-55/phospholipids-300-11433/
https://upload.wikimedia.org/wikipedia/commons/3/3d/Phospholipase.jpg
07.12.16
Page
12
UNIVERSITÄT
DES
SAARLANDES
Vielen Dank für Ihre Aufmerksamkeit.