Sophorolipids Characteristics, Production, and Applications - Biosurfactants: Research Trends and Applications - page 109

further β-oxidation (van Bogaert et al., 2007, 2011). While in the absence of the
hydrophobic substrate in the medium, the fatty acids are formed de novo starting
from acetyl-CoA derived from glycolysis (van Bogaert et al., 2007).
In Figure 4.2, steps 1-3 are involved in sophorolipid production on lipophilic
substrates other than fatty acids or esters thereof such as alkenes. While using
the n-alkane as a substrate for the production sophorolipid, the cytochrome P450
monooxygenase-mediated oxidation of the n -alkenes to alcohol is the primary
step, which is further converted into a fatty acid by successive oxidation mediated
by alcohol- and aldehyde-dehydrogenase. Two genes of cytochrome P450 mono-
oxygenase from C. apicola (European Molecular Biology Laboratory/GenBank
accession numbers X76225 and X87640) have been reported by Lottermoser
et al. (1996) and classified into the CYP52 family using amino acid similarity
analysis. The cytochrome P450 enzymes of yeasts are capable of hydroxylat-
ing alkanes and/or fatty acids (Nelson, 1999; Franzetti et al., 2010; Kang et al.,
2010). However, there is not so much literature available, which verify whether
the mentioned gene products were involved in sophorolipid production or alkane
assimilation and if they were expressed at all (van Bogaert et al., 2007). From
C. bombicola ATCC 22214, five different cytochrome P450 monooxygenase genes
has been identified which belongs to CYP52 family. One of them exposes very
high similarity (91% AA identity) to the CYP52E2 gene of C. apicola , whereas
the others probably belong to one or more new CYP52 subfamilies (van Bogaert
et al. 2007). Alternatively, extracellular lipases convert any fatty acid esters into
free fatty acids, which are utilized as a substrate. In the absence of lipophilic
substrate, the de novo synthesis of fatty acids occurs to form sophorolipid using
acetyl-CoA, derived from the glycolytic pathway. These fatty acids convert into
sophorolipid after a series of reactions, which is initiated by hydroxylation of fatty
acids in the presence of molecular oxygen and nicotinamide adenine dinucleotide
phosphate—reduced form—at the ω or ω−1 position, again through the action of
cytochrome P450 monooxygenase. Two glucose units are added in the resulting
hydroxy fatty acid by two different glucosyltransferases. Glucose is glycosidically
coupled (position C1) to the hydroxyl group of the fatty acid through the action of
a specific glycosyltransferase I. Experiments with 13 C-labeled glucose pointed out
that the bulk of the added glucose first passed through glycolysis, in this way sup-
plementing trioses for the gluconeogenesis of glucose for sophorolipid synthesis
(Hommel et al., 1987, 1994). The transferase reaction requires nucleotide-activated
glucose (uridine diphosphate-glucose) as glucosyl donor (Breithaupt and Light,
1982). In a subsequent step, a second glucose is glycosidically coupled to the C2′-
position of the first glucose moiety by glycosyltransferase II. Both glycosyltrans-
ferases are involved in sophorolipid synthesis. The two enzyme activities could
however not be separated, and highly purified samples exhibit a single major band
of 52 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (Esders
and Light, 1972; Breithaupt and Light, 1982). Therefore, it remains open for dis-
cussion whether the consecutive glucose transfers are carried out by two different
(but copurified) enzymes or by one and the same (multi)enzyme. It is supposed that
sophorolipid synthesis in C. bombicola involves analogous enzymes. The sopho-
rolipids obtained after the action of glucosyltransferase II are as such detected