Process for immobilization of a lipase

[0001]

Technical Field

[0002] The present invention relates to process for immobilizing a lipase on a support having a functional amino group, a process for producing a triglyceride composition using said immobilized lipase and to the use of the lipase in transesterification reactions.

Background Art

[0003] Lipases (E.C. 3.1.1.3), belonging to the group of enzymes, catalyse

specifically ester bonds in tri-, di-, and mono-acylglycerols to glycerol and fatty acids. They further catalyse other reactions such as

interesterifications, esterifications, acidolysis, alcoholysis and aminolysis. The high costs of lipases make enzymatic processes economically unattractive. Immobilization of the lipases is a way to increase the industrial susceptibility of lipases and allows recovery of the lipase protein. Lipases can be immobilized on different supports applying various ways of pretreatment of the support or the lipase.

[0004] Nevena et al.. ( NEVENA, Z. Immobilization of lipase from Candida rugosa on Sepabeads: the effect of lipase oxidation by periodates. Bioprocess BiosystEng. 2011 , no.34, p.803-810. ) describes the use of certain Sepabeads® having either amino functional groups or epoxy groups as suitable support for the immobilization of a non-specific lipase from

Candida rugosa. Sepabeads® having amino functional groups needed activation with glutaraldehyde or sodium-periodate to show improved activity.

[0005] Palomo et al.( PALOMO, Jose M, et al. Interfacial adsorption of lipases on very hydrophobic support (octadecyl-Sepabeads): immobilization, hyperactivation and stabilization of the open form of lipases. Journal of Molecular Catalysis B: Enzymatic. 2002, vol.19, no.20, p.279-286. ) tested the immobilization of various lipases on very hydrophobic support such as octadecyl-Sepabead® .

[0006] Enzyme activity is vulnerable to immobilizations reagents such as

glutaraldehyde or immobilization support. To secure enzyme stability and activity after immobilization of the enzyme, often non-lipase proteins are added such as hen egg album or bovin serum albumin. However, these animal proteins are known to cause allergic reactions.

[0007] There remains a need for a simplified immobilization method without

additional activation of the support and the right choice of support which will retain lipase activity and stability such as thermo stability to enable the production of commercially relevant triglyceride compositions.

Summary of invention

[0008] The objective of the present invention is to provide an immobilization

process wherein pre-activation of the support could be avoided and if indeed a hydrophobic support such as Sepabeads® having octadecyl groups (EC-OD) are able to perform transesterification reaction to obtain products of commercial importance. Another aim of the present invention was to provide an immobilization process avoiding treatment with non- lipase protein such as animal derived albumin to secure activity and stability of the lipase to produce triglyceride compositions.

[0009] According to the present invention, there is provided a process for

immobilizing a lipase on a support containing a functional amino group in the presence of a surface-active material. The term functional amino group refers to an amino group which is engaged in interacting with or binding to the lipase and optionally, the support.

[0010] A further aspect of the invention is a process for producing a triglyceride by enzymatic transesterification by using a lipase, which is immobilized on a support having a functional amino group.

[0011] Also provided by the invention in another aspect is the use of the

immobilized lipase for producing a triglyceride fat composition comprising at least 15% by weight OPO. [0012] The support having a functional amino group can be any support having an amino group such as amino-epoxy, or alkyl amino having a carbon chain of C1-C24, preferably C2-C 0. The support comprises a methacrylic polymer. Preferably the polymer forms a matrix.

[0013] A preferred support of the present invention contains a functional

alkylamino group such as ethyl amino or hexyl amino.

[0014] The mechanism of action between the support and the lipase is either by ionic interaction or chemical binding, wherein the ionic interaction is preferred.

[0015] The surfactant can be formed from sugars, (both mono-di-and

polysaccharides), polyols (e.g. sorbitan and sorbitol) or polyethylene glycols having molecular weight from 350 to 35000, such as PEG s 600, 1500, 4000. Very suitable non-ionic surfactants are polyoxyethylene sorbitan C8-C24 fatty acid esters, in particular those derived from lauric acid, such as Tween 20® or derived from oleic acid such as Tween 80® .

[0016] The surfactant concentration in the aqueous solution should be sufficient to ensure effective loading of the support by the enzyme. Very good results were obtained by applying an aqueous solution with a surfactant concentration of at least 0.01 wt%, preferably 0.01-10, most preferably 0.1-5 wt.%.

[0017] An ideal amount of lipase in g to support in g is between 1-20 wt.% by weight, preferably 5-15% by weight.

[0018] The contact times applied can vary between wide ranges. Suitably,

however, contact times between 1 and 72 hours are applied.

[0019] The aqueous lipase solution has preferable a concentration between 1 to

20 g/l.

[0020] Although the lipase enzyme can be any prior art lipase, a preference is expressed for a lipase which is selected from 1) 1 ,3-specific lipases from Rhizomucor miehei ^' , Rhizopus oryzae and Thermomyces lanuginosus 2) lipases from Penicillium camembert/i specific for the hydrolysis of partial glycerides , such as Amano G, and 3) lipases specific for the hydrolysis of esters or triglycerides, preferably a lipase from Candida rugosa . In particular preferred is a 1 ,3- specific lipase from Rhizopus oryzae such as Lipase D from Amano.

[0021] Immobilization of the lipase can be performed in many different ways.

Suitably, the contact between support, lipase and/or surfactant is performed as a batch process, as a continuous process in a fixed bed, as a continuous process in a fluidized bed or in a continuously stirred tank, while the contacting is performed with a continuous motion of the lipase solution.

[0022] The immobilized lipase according to the invention can be applied in any enzymatic conversion process, such as hydrolysis of triglycerides, diglycerides or esters, but also the esterification or transesterification of fatty acids or diglycerides or triglycerides. These processes are also part of our invention, with the prerequisite that an immobilized lipase according to our invention be used in the process.

[0023] Preferred processes for making triglyceride is the production of

triglycerides compositions comprising symmetrical triglycerides of the general formula ABA, such as OPO or SOS, wherein O is oleic acid, P is palmitic acid and S is a saturated fatty acid selected from palmitic acid and stearic acid. A particular preferred triglyceride composition of the invention comprises at least 15% by weight OPO.

[0024] Triglyceride fats and oils are important commercial products and are used extensively in, for example, the food industry. Some triglycerides are nutritionally important and the triglyceride 1 ,3-dioleoyl-2-palmitoyl glyceride (OPO) is known to be an important component of human milk fat.

[0025]

[0026] Examples

[0027]

[0028] The following non-limiting examples illustrate the invention and do not limit its scope in any way. In the examples and throughout this specification, all percentages, parts and ratios are by weight unless indicated otherwise.

[0029]

[0030] Example 1 : Various Sepabeads® with aqueous Lipase D preparation ] Preparation of the lipase solutions: Seven lipase solutions were prepared according to Table 1. Sample N°7 was the control sample. All reagents were mixed at 150 rpm at room temperature between 3 to 24 hours and then centrifuged to receive the immobilized lipase as a pellet.

Table 1

Sample, N° Sepabeads® Amount of Lipase in g

(functional group) Sepabeads in g

0.12 in 70 ml

1 EC-HA 1.5

(Hexylamino)

2 EC-OD 0.12 in 70 ml

(Octadecyl) 1.5

3 EC-BU 1.5 0.12 in 70 ml

(Butyl)

4 EC-HFA 1.5 0.12 in 70 ml

(Amino-Epoxy)

5 EC-EA 1.5 0.12 in 70 ml

(Ethylamino) 6 EC-EP 1.5 0.12 in 70 ml

(Epoxy)

7 No support 0 0.18 in 75 ml

(equal to 0.12 in

70 ml)

Example 2 (comparative)

The acidolysis reaction was performed at 60 °C with all seven

preparations using the following acidolysis assay:

1 g Immobilized enzyme (use the pellet after centrifugation)

35 g Palm oil stearin fraction (Feedstock)

49 g Oleic acid

0.126 g H ₂ 0

Composition Feedstock to be found in Table 2.

Table 2

[0039] The carbon number was determined by GC according to AOCS Ce 5.86.

[0040] Table 3 provides the results of the various acidolysis reactions of

feedstock after 24 h.

Table 3 HA OD BU HFA EA EP Control

Carbon

number

C48 59.9 60.1 60.0 57.4 60.3 60.1 60.3

C50 25.4 25.3 25.3 26.8 25.1 25.3 25.2

C52 9.2 9.2 9.2 10.2 9.1 9.2 9.1

C54 2.1 2.1 2.2 2.5 2.1 2.1 2.1

C56 0.2 0.1 0.2 0.2 0.2 0.2 0.2

[0041] After 24 h nearly no product OPO or OOP (C52) was produced for all lipase preparations

[0042] Example 3

[0043] 70 ml of the lipase preparation of Example 1 was mixed with 2.4 g hen egg albumin, 0.65 g Twee n 20® and 1.5 g of the respective supports. The acidolysis reaction was performed according to example 2.

[0044] Table 4 shows the results after acidolysis (24 hours) by using various sepabeads with aqueous lipase D solution in the presence of hen egg albumin and TWEEN 20.

Table 4

HA OD BU HFA EA EP

Carbon

number

C48 7.2 9.5 61.2 6.9 7.2 61.4

C50 29.3 31.3 24.8 29.0 25.0 24.7

C52 41.5 41.5 8.8 42.8 42.3 8.8

C54 20.4 16.9 2.0 20.4 20.7 1.9 C56 0.5 0.4 0.0 0.5 0.4 0.0

[0045] Example 4

[0046] 70 ml of the lipase preparation of Example 1 was mixed with 250 mg PEG 1500, 0.65 g Tween 20® and 1.5 g of the respective supports. The acidolysis reaction was performed according to example 2. As comparison immobilization on polypropylene (Accurel) under same reaction conditions was performed.

[0047] Table 5 shows the results after acidolysis (24 hours) by using various sepabeads with aqueous lipase D solution in the presence of PEG 1500 and Tween 20® .

Table 5

[0048] Example 5

[0049] Multiple usage of Lipase D immobilized on support EC-HA.

[0050] 70 ml of the lipase preparation of Example 1 was mixed with 30 mg PEG 600, 0.65 g Tween 20® and 1.5 g of support EC-HA. The acidolysis reaction was performed according to example 2. After 3.5 hours the acidolysis reaction was stopped and the immobilized lipase separated from the reaction mixture by filtration. The immobilized lipase is collected and used for the second run of the acidolysis assay . These runs were repeated eight times. At each run a sample (- 2 ml) at time 3.5 hours were taken for carbon number analysis.

[0051] Table 6 shows the results after acidolysis by reusing the immobilized

lipase D on EC-HA support in subsequent 8 runs

[0052]

Table 6

[0053] Example 6

[0054] Lipase D solution (0.9 g/77 ml) was mixed with various Tween in amounts provided in Table 7 and stirred for 15 min. To each of the preparations 1 ,5 g of Sepabead EC-HA was added and the mixture was stirred for 24 hours. Then immobilized enzyme was filtered off and tested in the acidolysis reaction as described in example 2. Table 7 shows the results of 5 different Tween's after acidolysis after 3.5 h.

Table 7

Carbonnumber Tween 20 Tween 40 Tween 60 Tween 80 Tween 85

Amount in g 0.650 0.676 0.693 0.694 0.974

C46 1.5 2.14 2.79 1.34 2.4

C48 16.6 41.19 54.37 18.02 46.5 C50 34.2 31.4 27.49 33.26 30.5

C52 36.9 18.73 11.85 36.11 15.8

C54 10.6 5.78 3.48 10.77 4.5

C56 0.3 0.53 0 0.38 0.3

[0055] Example 7

[0056] Example 6 was repeated with Tween 80® with the difference that the premixing of the lipase solution with Tween 80® was skipped. Lipase solution, Tween 80® and support material were put together and the mixture was stirred for 24 hours. Then immobilized lipase was filtered off and tested in the acidolysis reaction as described in example 2. Table 8 shows the results after acidolysis after 3.5 h.

[0057]

Table 8

[0058] The results demonstrate that premixing of Tween 80® with lipase solution is not required.