Safety evaluation of certain food additives - ipcs inchem

108 PHOSPHOLIPASE C EXPRESSED IN PICHIA PASTORIS
cholinephosphohydrolase, and its Enzyme Commission (EC) number is 3.1.4.3.
Phospholipase C catalyses the hydrolysis of phosphodiester bonds at the sn-3
position in glycerophospholipids (including phosphatidylcholine, phosphatidylethanolamine
and phosphatidylserine) to 1,2-diacylglycerol and the corresponding
phosphate esters. Phospholipase C is to be used in refining vegetable oils intended
for human consumption.
1.1 Genetic modification
Phospholipase C is produced by pure culture fermentation of a genetically
modified strain of P. pastoris, which expresses the phospholipase C gene
derived from deoxyribonucleic acid (DNA) purified from a soil sample. The
phospholipase C gene was sequenced and shown to be devoid of DNA sequences
associated with haemolytic activity characteristic of certain microbial
phospholipases.
Pichia pastoris is a methylotrophic yeast, which is not known to be associated
with a disease of humans or animals. The phospholipase C production strain was
constructed by transformation of the P. pastoris host strain SMD1168 with a purified
DNA fragment containing multiple copies of the phospholipase C gene, the P.
pastoris HIS4 gene and non-coding DNA sequences necessary for expression of
both genes; and insertion of the DNA fragment into a predetermined location in the
P. pastoris genome. The P. pastoris HIS4 gene encodes histidinol dehydrogenase
and serves as a selectable marker to identify the transformed cells. The DNA
fragment used in transformation was inserted at the alcohol oxidase 1 (AOX1) locus
by homologous recombination.
1.2 Product characterization
Phospholipase C is produced by pure culture fed-batch fermentation of the
phospholipase C production strain. The fermentation medium consists of foodgrade
materials and contains glycerol as primary carbon source. After the cellular
mass has reached a desired density, methanol is added to induce the expression
of phospholipase C. The enzyme is secreted into the fermentation medium and is
subsequently recovered by purification and concentration. The purified enzyme
concentrate is formulated and standardized to a desired activity. Methanol is
removed during purification steps, and its residues in the final product are less than
9 mg/l. The phospholipase C enzyme preparation is a yellow or brown liquid, which
typically contains 7% total organic solids (TOS).
The phospholipase C enzyme preparation conforms to the General
Specifications and Considerations for Enzyme Preparations Used in Food
Processing (Annex 1, reference 184). It will be used in refining vegetable oils to
hydrolyse phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine,
present in the crude oil. The resulting esters, phosphorylcholine and
phosphorylethanolamine, as well as phospholipase C itself, will be removed from
the oil during subsequent purification steps, whereas 1,2-diacylglycerol, which is
also formed as a result of phospholipid hydrolysis, will remain in the oil.