Enzymes as Biocatalysts for Lipid-based Bioproducts Processing - Food and Industrial Bioproducts and Bioprocessing - page 338

usually performed to obtain lysophospholipids (LPL). LPL possess superior emulsification
properties and are often used in the food, pharmaceutical, agrochemicals and personal care
industries (Fujita and Suzuki, 1990). Enzymatic hydrolysis of PL to form LPL can be
conducted using phospholipase A 1 (Kim et al ., 1997 ), phospholipase A 2 (Yesair, 1998 ) or
lipase (Kim and Kim, 2000; Virto and Adlercreutz, 2000). In fact, enzymatic hydrolysis of
phospholipids has been carried out industrially (Sas and Peys, 2000; Schmitt et al ., 2005 ;
Schmitt and Heirman, 2007). Hydrolytic reaction requires the presence of water in the
reaction mixture. As PLs have limited solubility in water, the reaction is inefficient. Haas
and co-workers (1993) conducted enzymatic hydrolysis of phosphatidylcholine (PC) in the
presence of organic solvents. Hydrophobic solvents were found to increase solubility of PLs
significantly, which subsequently enhanced the reaction efficiency and yield of LPL. MAG
and fatty acids can be added to the reaction mixtures to form a microemulsion system,
which produces a higher yield of LPL and minimizes formation of by-product.
Enzymatic alcoholysis of PLs can be used to produce LPLs and fatty acid esters simulta-
neously. The main factors affecting enzymatic alcoholysis of PLs are the type of lipase and
chain length of the alcohol. For example, immobilized RML exhibited significant preference
for short chain alcohols like ethanol, whereas lipase TLL preferred long chain alcohol
(Sarney et al ., 1994). Sarney and co-workers (1994) reported that addition of a small amount
of water to the reaction mixture increased the LPLs yield significantly.
Enzymatic transesterification of PLs is used to synthesize structured phospholipids. For
example, microbial lipases from Aspergillus , Candida , RML and TLL have been used for
transesterification of PLs with fatty acids of different chain lengths (Peng et al ., 2002 ; Hara
et al ., 2002; Doig and Diks, 2003). The type of acyl donor affects enzymatic transesterification
of PLs significantly. Generally, FFA is a more efficient acyl donor in comparison to esters.
Although the reaction kinetics is slightly slower when using FFA as acyl donor, the yield of
structured PLs is higher because of the inhibition of hydrolytic reaction. Another important
factor is the use of solvent, especially hydrophobic solvent, to improve miscibility of
substrates, which subsequently enhances mass transfer and overall reaction rate.
Enzymatic transphosphatidylation of PLs can be conducted using PLC and PLD. PLC
specifically hydrolyzes the C-O-P bond in phospholipids yielding sn-1,2 DAG and the
phosphate residue bearing the corresponding head group (Durban and Bornscheuer, 2007).
DAG produced from enzymatic transphosphatidylation possesses the enantiomeric structure
of the natural product, which can be used directly for synthesis of stereospecific compounds.
This is not possible when lipases are used for DAG production (Anthonsen et al ., 1999 ).
For example, PLC from Bacillus cereus has been used to produce biologically active
sphingosines, ceramides, sphingosine 1-phosphate (Morigaki et al ., 1998 ), 1-lauroyl-rac-
glycerophosphate (Virto and Adlercreutz, 2000) and cyclic five-membered phosphodiesters
(Bruzik et al ., 1996). PLD is an expensive biocatalyst and is used primarily for production
of high value pharmaceutical products. Parameters considered important in enzymatic
transphosphatidylation include the nature of acceptor alcohol and reaction media. Primary
alcohols can be easily incorporated to PLs with PLD, especially PLD from streptomyces
species (Hosokawa et al ., 2000 ; Yaqoob et al ., 2001 ; Iwasaki et al ., 2003 ). Small water-
soluble alcohols, such as ethanol and glycerol, are also easier to incorporate into PLs.
Selection of a suitable reaction medium capable of dissolving substrates that differ in terms
of polarity is crucial for the efficiency of enzymatic transphosphatidylation. A biphasic
solvent system consisting of ethyl acetate and diethyl ether is commonly used for transpho-
sphatidylation reactions. In some cases, benzene or a mixture of hexane and octanol has
been used as well.