Immobilization method for cells containing nitrilase

A nitrilase and cell technology, applied in the field of immobilization of nitrilase-containing cells, can solve the problems of low reaction batch, low substrate solubility, high toxicity of bisnitrile substrates, etc., and achieves good reusability and operation. Simple, low-cost effects

Inactive Publication Date: 2015-09-16
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the problem of low reaction batches caused by the high toxicity of the bisnitrile substrate and the low solubility of the substrate in the process of producing 1-cyanocyclohexylacetic acid catalyzed by nitrilase, and provides a kind of cell containing nitrilase immobilization method

Method used

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  • Immobilization method for cells containing nitrilase
  • Immobilization method for cells containing nitrilase
  • Immobilization method for cells containing nitrilase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of recombinant Escherichia coli E.coli BL21(DE3) / pET28b(+)-F168V and preparation and performance determination of wet cells

[0035] The recombinant Escherichia coli E.coli BL21(DE3) / pET28b(+)-F168V constructed in this laboratory is used as the production strain, and the method is as follows:

[0036] (1) Construction of recombinant bacteria: the Acidovorax facilis ZJB09122 nitrilase mutant gene F168V (nucleotide sequence shown in SEQ ID NO.1, has been published in Xin-Hong Zhang et al / Process Biochemistry49 (2014) 2141-2148 ) was ligated with PGEM-T vector and then introduced into E.coli JM109, F168V / PGEM-T and plasmid pET28b(+)-Nit were double digested, ligated overnight with ligase, and the ligation product pET28b(+)- F168V was introduced into host E.coli BL21(DE3) to obtain recombinant Escherichia coli E.coli BL21(DE3) / pET28b(+)-F168V.

[0037] The PGEM-T carrier connection conditions are as follows: 10 μL of the connection system is added to...

Embodiment 2

[0050] (1) Take the wet thallus obtained by the method in Example 1, mix them according to the wet weight of the thallus: phosphate buffer (pH=7.0, 100mM)=1:12 (m / v), then take 8.3g of wet thallus and add Prepare 100mL bacterial suspension in 100ml phosphate buffered saline.

[0051] (2) Add 0.4g diatomite to the 100mL bacterial suspension in step (1), mix well on the magnetic stirrer, then mix the polyethyleneimine aqueous solution with 3ml concentration of 5% (v / v) and the magnetic stirrer fully Mix under stirring for 0.5 h.

[0052] (3) Add 1 ml of 25% (v / v) glutaraldehyde aqueous solution to the solution in step (2), discard the supernatant after cross-linking at 25° C. for 0.5 h, and vacuum filter to obtain immobilized cells.

[0053] (4) Take 0.625 g of the immobilized cells prepared in step (3) in 10 ml of pH 7.0 phosphate buffer, add 200 mM substrate 1-cyanocyclohexyl acetonitrile, react at 40°C for 10 min, take samples, and detect the product 1-cyano by HPLC Accordi...

Embodiment 3

[0058] (1) Take the wet thalline prepared by the method of Example 1, mix according to the wet weight of the thallus: phosphate buffer (pH=7.0, 100mM)=1:10 (m / v) to obtain a bacterial suspension, that is, take 10g of wet thalline The bacteria were added to 100mL phosphate buffer to make 100mL bacterial suspension.

[0059] (2) in step (1) 100mL bacterium suspension, add the diatomite of 0.8g, mix on the magnetic stirrer, be the polyethyleneimine aqueous solution mixing of 5% (v / v) with 5ml concentration again, in magnetic force Mix for 1 h under vigorous agitation with a stirrer.

[0060] (3) Add 1 ml of 25% (v / v) glutaraldehyde aqueous solution to the solution in step (2), discard the supernatant after cross-linking at 15° C. for 0.5 h, and vacuum filter to obtain immobilized cells.

[0061] (4) Immobilized cell enzyme activity, enzyme activity recovery rate, half-life and continuous operation batch yield test method at 500 mM substrate concentration are the same as in Examp...

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Abstract

The invention discloses an immobilization method for cells containing nitrilase. The immobilization method comprises the steps that wet vectors obtained by a recombinant genetic engineering strain containing nitrilase genes through fermental cultivation is added into a buffer solution or distilled water to prepare bacterial suspension; a carrier is added into the bacterial suspension, and even stirring is conducted; then polyethyleneimine is added, and stirring for flocculation is conducted for 0.5-2 h; then glutaraldehyde is added to conduct crosslinking, and after stirring and crosslinking are conducted at 10-25 DEG C by 0.5-2 h, liquid supernatant is discarded; after vacuum filtration, cells containing nitrilase are obtained. The immobilization method for cells containing nitrilase has the advantages that the cost is low, operation is easy, and reusability is good; prepared immobilization recombinant escherichia coli whole cells producing nitrilase are used for catalyzing 1-cyanocyclohexane acetonitrile to produce 1-cyanocyclohexane acetic acid; after conversion is continuously conducted by 30 times, the conversion yield is still more than 80%; and a good industrialized application prospect is achieved.

Description

(1) Technical field [0001] The invention relates to a method for immobilizing cells, in particular to a method for immobilizing cells containing nitrilase. (2) Background technology [0002] Nitrilase has the characteristics of high catalytic efficiency and good regioselectivity. It can quickly and efficiently hydrolyze the cyano group on the methylene group in 1-cyanocyclohexyl acetonitrile into a carboxyl group and retain the cyano group on the cyclohexyl group. It can be used in biological Catalyst for the synthesis of 1-cyanocyclohexylacetic acid, an intermediate of the antiepileptic drug gabapentin. Compared with the disadvantages of free cell reaction process, such as easy damage, low reuse rate, and difficult product recovery, after the whole cell of nitrilase is fixed into granules through physical and chemical methods, the tolerance of cells to toxic nitrile substrates can be enhanced. It is beneficial to the continuous multi-batch use of the catalyst and the separ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N11/04
Inventor 郑裕国邹树平黄季维薛亚平
Owner ZHEJIANG UNIV OF TECH
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