Immobilization method of Thermus lipase

A lipase and lipase gene technology, applied in the field of lipase immobilization, can solve the problems of easy inactivation, difficult recovery, and limited application, and achieve easy magnetic separation and recovery, good superparamagnetism, and good operational stability Effect

CN102994491AInactive Publication Date: 2013-03-27ZHEJIANG UNIV
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2013-03-27
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention discloses an immobilization method of Thermus lipase, comprising the following steps of: mixing an aminated nanometre magnetic carrier Fe3O4 / SiO2 with a buffering solution having a pH of 8.0; adding 25% by mass glutaraldehyde aqueous solution, and magnetically stirring for 4 hours at a room temperature; then adding the Thermus lipase (Thermus thermophilus WL) buffering solution dissolved by using a Tris-HCl buffering solution, and magnetically stirring for 3.5 hours at a room temperature; magnetically separating, removing a supernatant a, and washing a precipitate a by the buffering solution; and carrying out freeze-drying to obtain the immobilized Thermus lipase. The immobilized Thermus lipase obtained by the method disclosed by the invention has a heat-resistant range from 40 to 100 DEG C and a pH value application range from 4.0 to 9.0 and is strong in tolerance for metal ions, enzyme inhibitors and decontaminants, great in superparamagnetism and good in operation stability, and magnetic separation and recovery are easy to perform.
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Description

(1) Technical field

[0001] The invention relates to a lipase immobilization method, in particular to a Thermus lipase immobilization method. (2) Background technology

[0002] Lipase (lipase, EC3.1.1.3) is a special class of acyl hydrolase, which can catalyze ester hydrolysis, ester synthesis, transesterification, lactone synthesis, polypeptide synthesis, polymer synthesis and three-dimensional synthesis at the oil-water interface. Chemical reactions such as isomer resolution. So far, various lipases from animals, plants and microorganisms have been isolated and purified. It is an enzyme catalyst widely studied and widely used in detergents, oils and food processing, organic synthesis, surface cleaning, medicine, cosmetics , spices, leather and paper industry, biodiesel preparation, oil modification, etc.

[0003] Thermus lipase is a type of thermophilic lipase isolated from thermophilic microorganisms (>55°C) in high-temperature environments such as deep-sea hydrotherm...

Examples

Embodiment 1

[0039] Example 1. Preparation of Thermus lipase (Thermus thermophilus WL)

[0040] Thermus lipase (Thermus thermophilus WL) is derived from the thermophilic bacteria (Thermus thermophilus WL) isolated from deep-sea hydrothermal vents. The Thermus lipase (Thermus thermophilus WL) gene is 486 bp in length and contains a reading frame encoding 161 amino acids (Genbank sequence registration number: AAS81965.1).

[0041] When using Escherichia coli E.Coli BL21(DE3) as the host cell, the Escherichia coli [Escherichia coli BL21(DE3)] containing the Thermus lipase gene was obtained, which was preserved in China on June 15, 2012 The General Microbiology Center of the Microbial Culture Collection Management Committee (CGMCC for short), the preservation number is CGMCC No: 1.12252.

[0042] The preparation method of described Thermus lipase is:

[0043] (1) Preparation of free Thermus lipase (Thermus thermophilus WL): Pick a single colony of Escherichia coli containing the Thermus lipa...

Embodiment 2

[0046] Example 2. Carrier preparation and Thermus lipase immobilization

[0047] (1) High-performance magnetic nanospheres (ie Fe 3 o 4 Nanoparticles) Preparation

[0048] Fe 3 o 4 Nanoparticles are prepared by an improved chemical co-precipitation method, and the reaction formula is

[0049] Fe 2+ +2Fe 3+ +8OH-→Fe 3 o 4 +4H 2 o

[0050] Specific process: Weigh 4.054g FeCl respectively 3 ·6H 2 O and 1.988g FeCl 2 4H 2 O (considering Fe 2+ Oxidation in the preparation and subsequent process, select Fe 3+ / Fe 2+ The amount ratio of the substance is 3 / 2) placed in a 50mL beaker, add 25mL double-distilled water to dissolve and then add 90mL double-distilled water to mix to form a 115mL mixed solution, add the mixed solution to a 500mL three-necked bottle, feed nitrogen, and After stirring mechanically (1000rpm) in a water bath at 30°C for 5 minutes, add 25% NH with a mass concentration dropwise with a syringe (0.7 mm in diameter) 3 ·H 2 O10mL and 5mL of PEG (molec...

Embodiment 3

[0058] Example 3. Determination of immobilized Thermus lipase enzyme activity

[0059] The assay of lipase activity is measured with p-nitrophenol laurate (C12, p-nitrophenyl laurate) as substrate, see (Sigurgisladóttir S, Konraosdóttir M, Jónsson A, Kristjánsson J, Matthiasson E.Lipase activity of thermophilic bacteria from Icelandic hot springs.Biotechnol Lett.1993,15:361-366.), the specific operation is as follows:

[0060] 1) Preparation of C12 solution: Dissolve a certain amount of C12 in absolute ethanol to prepare a 25mM C12 ethanol solution, and store it in the dark (it is best to use it immediately).

[0061] 2) Preparation of diluted enzyme solution sample: the immobilized Thermus lipase obtained by the method in Example 2 was diluted with 50 mM Tris-HCl buffer solution (pH 8.0) to an 80 mg / L enzyme solution. Prepare calcium chloride buffer solution with 50mM Tris-HCl buffer solution (pH8.0) to make calcium chloride buffer solution with a final concentration of 40mM...