Study On Enrichment Of Polyunsaturated Fatty Acids With Thermomyced Lanuginosus Lipase

Many studies report that polyunsaturated fatty acids (PUFAs), such as DHA, EPA,DPA, has a positive effect on heart disease, cancer inhibition, mental development, and soon. Generally, PUFAs can be only synthesized by human bodies in a small part, and mostof them are absorbed through diet. In recent years, a lot of PUFAs-rich products appearedon the market, and are quickly sold out. Therefore, R&D of PUFAs-rich products are ofgreat commercial significance. Currently, there are two main preparation methods forpolyunsaturated fatty acids which are physical and chemical method and enzymaticcatalysis method. Compared with physical and chemical method, enzymatic catalysismethod has great advantages due to its mild reaction conditions, pollution-free, moresafety, and it has attracted more and more attention. In addition, in our laboratory,Thermomyced lanuginosus lipase (TLL, a kind of enzyme suits to enrich PUFAs from oils)can be highly overexpressed via multiple synergetic strategies, which effectively cut theproduction cost of the enzyme. Therefore, in this article, the enrichment of PUFAs wasconducted using TLL prepared in our laboratory, and the hydrolysis conditions werefurther optimized. The main contents and results are summarized below.1. Enzymatic properties of TLL were studied using pNP ester hydrolysis. Its optimalsubstrate was nitrophenol caprylate (pNC), the optimum temperature was30℃, and theoptimum pH is8.0. After incubation at30℃in different duration, the residual activitiesalways retained more than50%of its original one. K+, Ca2+, Mg2+, Mn2+, and CTAB(Cetyl trimethyl ammonium bromide) could strongly activate TLL, while Ni2+, Cu2+, Fe2+,Fe3+, Zn2+, TritonX-100, and Tween20showed significantly inhibitation, and SDS hadlittle effect. Moreover, TLL had good tolerance to hexane, whereas it was subtle tobutanol.2. The hydrolysis conditions were optimized when using Thermomyced lanuginosuslipase to enrich PUFAs from algal oil. First, the reaction conditions of the hydrolysis wereoptimized by single factor experiments. The obtained best conditions were: reaction time4h, enzyme loading12,000U/g-oil, reaction temperature40℃, additives0.3g/g-oil, oil-water ratio1:3, pH8.0and0.5M Tris-HCl. Then, based on the results of single factorexperiments, orthogonal experiments were employed to further optimize the hydrolysisconditions in consideration of interaction of parameters. Using KOH-CH3CH2OH solutionas the deacidification reagent, the highest content and recovery rate of DHA has beentested. The content of DHA increased to41.6%,2.08fold as that of crude oil, and therecovery rate attained81.6%. However, there was some DHA ethyl ester which in someway influences the quality of the prepared product.3. Considering the negative effect of DHA ethyl ester, molecular distillation wasexplored to deacidify the enzymatic hydrolyzed mixture. The distillation temperature andagitating rate were optimized via single-factorial experiments. The optimal conditionswere distillation temperature120℃and agitating rate150rpm. Under these conditions,the highest content of DHA reached37.8%,1.89fold as that of crude oil, where no DHAethyl ester was produced.