**3.3 Lipase transesterification**

#### **3.3.1 Materials and methods**

The experimental study was done with rapeseed oil of Romanian origin or soya oil and by using the lipase from the yeast *Candida rugosa* DSM 70761 obtained in aerobic bio processing, isolated from the cultivation medium and immobilized by adsorption on Merck Celite support (lipase activity of 4701 UEA / g support).

The transesterification was done in two variants: (a) in anhydrous medium without organic solvents adding; (b) in hexane (Biosolve).

The experimental working procedure was: the transesterification reaction was performed in Erlenmayer flasks of 100 mL, containing the tested vegetable oil in a concentration to determine a final triglycerides content of 0.08 mol / L and methanol (this last reagent in molar ratios between 3:1 and 8:1 with the triglycerides substrate). The immobilized enzyme was added in a chosen concentration after a period of 30 minutes at 370C. The reaction was done with continuous mixing of 250 rpm. Each 4 hours' sample from the liquid was analysed by thin layer chromatography and gas chromatography to determine the reaction advancement.


The apparatus was a gas chromatograph 6890N – AGILENT with FID detector and autosampler 7683B; column HP 5, L=30m; φ=0.32mm.

Reagents: N-hexane; the methylic esters of several fatty acids mostly presented in the vegetable oils (rapeseed oil or soya oil).

Working conditions:


The evaluation is done by the determination of the content in palmitic, oleic, arachidonic and erucic acids. The external standard method is applied.

Three experimental models were studied:

a. Batch enzymatic transesterification with methanol and without organic solvent, characterised by : vegetable oil concentration of 0.09 M; methanol concentration of 0.54 M (ratio of 8:1 methanol: triglycerides substrate); biocatalyst concentration of 5000 UEA / 100 mL reaction medium; reaction temperature of 370C; mixing of 250 rpm; reaction total duration of 24 hr.

