Immobilisation of lecitase® ultra for production of diacylglycerols by glycerolysis of soybean oil
Highlights
► Lecitase® ultra was immobilised as a catalyst for glycerolysis of soybean oil. ► The immobilised lecitase® ultra (IM-LU) had a high activity in production of DAG. ► IM-LU performed a good performance of reusability when being recycled 28 times. ► IM-LU possessed sn-1, 3-specificity to triacylglycerols in glycerolysis reaction.
Introduction
Diacylglycerols (DAG), in different degrees of purity, are used as additives or carriers in the food, medicinal and cosmetic industries (Fureby, Tian, Adlercreutz, & Mattiasson, 1997). Dietary DAG exhibits antiobesity activity and can prevent postprandial hypertriacylglycerolemia in experimental animals and humans, and mechanisms have been proposed (Murase et al., 2001, Reyes et al., 2008, Taguchi et al., 2000). The versatility of DAG oil is evident in numerous applications, for example, as a cooking oil, frying oil, salad oil, salad dressing and mayonnaise, shortenings and margarines, chocolates, and ice cream fats (Lo, Tan, Long, Yusoff, & Lai, 2008). Some researchers (Mori, Masui, Tanaka, & Yasukawa, 1999) have invented a W/O-emulsified fat composition that has good stability and spreadability and is suitable for use as a margarine. The W/O composition is made up of 40% to less than 95% (w/w) of DAG and 5% to less than 60% (w/w) of triacylglycerols (TAG).
Several chemoenzymatic and biotechnological methods are available for the preparation of DAG (Guanti et al., 2004, Villeneuve et al., 2000). The enzymatic synthesis of DAG has garnered considerable interest by reason of the milder conditions, higher selectivity and greener process as compared to chemical processes. In general, DAG can be enzymatically produced by direct esterification, glycerolysis, interesterification, partial hydrolysis, and/or the combination of partial hydrolysis and esterification (Blasi et al., 2007, Cheong et al., 2007, Yang et al., 2004).
Lecitase® ultra (E.C.3.1.1.32) is a phospholipase manufactured and marketed by Novozymes, Denmark. This commercial preparation is a protein-engineered carboxylic ester hydrolase from the fusion of lipase genes from Thermomyces lanuginose and phospholipase genes from Fusarium oxysporum (Fernandez-Lorente et al., 2008). Most attempts have been directed toward the application of phospholipase activity of this enzyme for degumming of vegetable oils and modifying of phospholipids (Kim et al., 2007, Sheelu et al., 2008, Yang et al., 2006). However, very little effort has been directed to exploring applications of its lipase activity in acylglycerol synthesis (Mishra, Kumaraguru, Sheelu, & Fadnavis, 2009).
One of the main hurdles for the production of DAG by the enzymatic method is the cost of enzyme. Our previous works (Wang et al., 2009, Wang et al., 2010) have reported the application of free lecitase® ultra for partial hydrolysis of TAG to produce DAG-enriched oil. This enzyme has also been described to suffer interfacial activation, similarly to lipases (Fernandez-Lorente et al., 2008). Furthermore, the commercial lecitase® ultra is a free enzyme, which is not easy to reuse in the reaction system. As a result, a technology of immobilisation of this enzyme should be developed in order to reduce the cost of using this enzyme.
The objective of this work was to immobilise lecitase® ultra onto macroporous resin, and to synthesise DAG by glycerolysis, using the immobilised lecitase® ultra (IM-LU) as a catalyst. The enzymatic productions of DAG, by IM-LU-catalysed glycerolysis of soybean oil in organic solvent and solvent-free systems were studied. The optimum enzyme immobilisation and glycerolysis reaction conditions were investigated. This new process for production of DAG is promising since the cost of the enzyme is dramatically reduced by reusing IM-LU.
Section snippets
Materials
Lecitase® ultra and Lipozyme TL IM were purchased from Novozymes (Copenhagen, Denmark). The standards of 1-monoolein, 1,3-diolein, 1,2-diolein and triolein were purchased from Sigma (St. Louis, MO). Soybean oil was purchased from Zhongliang Co., Ltd. (Dongguan, China). Glycerol was purchased from Tianjin Chemical Reagent Factory (Tianjin, China). Tributyrin was purchased from Tokyo Chemical Co., Ltd. (Tokyo, Japan). All other reagents and solvents used were of analytical or HPLC grade.
Preparation of IM-LU
The
Optimisation of the immobilisation procedure
After being pretreated according to the method of Gao, Tan, Nie, and Wang (2006), five types of macroporous resins (AB-8, X-5, DA-201, D3520 and D-101) and two anion resins (D202 and D318) were screened for immobilisation of lecitase® ultra in this study. The enzyme immobilised on macroporous resin DA-201 was found to exhibit the highest enzyme activity and retained activity as compared to others (data not shown). Therefore, macroporous resin DA-201 was selected as the support matrix for
Conclusions
Lecitase® ultra was successfully immobilised onto a kind of polar macroporous resin, DA-201, to obtain IM-LU with an enzyme activity of 1682 U/g, which was adopted for catalysing glycerolysis of soybean oil to produce DAG. The solvent-free system was more suitable for IM-LU-catalysed glycerolysis reaction than solvent systems. Under the optimised reaction conditions (glycerol/soybean oil mole ratio 10:1, initial water content 5 wt.%, and enzyme load 5 wt.%), a product with a 53.7 wt.% DAG content
Acknowledgements
The financial support from the National Natural Science Foundation of China under Grant 31000793, the Ministry of Science and Technology of People’s Republic of China under Grant 2010AA101505, the Science and Technology Council of Guangdong under Grants 2009A020700003 and 2009B080701063 is gratefully acknowledged.
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