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Method for cholate-metal ion composite modified whole cell and application of cholate-metal ion composite modified whole cell

A technology of metal ions and whole cells, applied in the direction of microorganism-based methods, biochemical equipment and methods, immobilized on or in inorganic carriers, can solve the problems of microorganisms reducing mass transfer efficiency and large size of microspheres, and achieve Effects of overcoming damage and death, fast material conversion, and overcoming mass transfer resistance

Pending Publication Date: 2020-01-03
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method mainly relies on sodium alginate, chitosan and Ca 2+ The microspheres formed by etc. realize the direct embedding of microorganisms, and the size of the microspheres is large (3.6mm), which reduces the mass transfer efficiency for the microorganisms embedded in the material
There is no interaction between the immobilized material and the microbial cell membrane, and the mass transfer efficiency of substances in and out of the cell membrane is not changed

Method used

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  • Method for cholate-metal ion composite modified whole cell and application of cholate-metal ion composite modified whole cell
  • Method for cholate-metal ion composite modified whole cell and application of cholate-metal ion composite modified whole cell
  • Method for cholate-metal ion composite modified whole cell and application of cholate-metal ion composite modified whole cell

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Experimental program
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Effect test

Embodiment 1

[0031] The cultivation and gene expression of embodiment 1 bacterial strain

[0032] Escherichia coli E. coli BL21(DE3) belongs to conventional bacteria and was purchased from Beijing Biolab Technology Co., Ltd.

[0033] Escherichia coli E. coli Trans1-T1 for transformational replication of plasmid, Escherichia coli E. coli BL21(DE3) was used for gene expression and strain fermentation, and the plasmid pET28a was purchased from Quanshijin Company. The cytochrome P450 monooxygenase was donated by Professor Liu Luo of Beijing University of Chemical Technology. After the whole gene sequence was synthesized by Nanjing Qinke Company, PCR primers were designed, amplified by PCR, and recovered by agarose gel electrophoresis. Nco I and Hind Cut the vector pET28a and P450 gene sequence with III cutting enzyme, connect with T4 DNA ligase, obtain the recombinant plasmid pET28a-P450, and transform it into Escherichia coli E. coli Trans1-T1 Competent is used to preserve the rec...

Embodiment 2

[0044] Example 2 Preparation of cholate-metal ion immobilized cell complex

[0045] Prepare the collected bacteria with Tris-Hcl (pH 7.2, 0.05mmol / L) buffer to make 1~10 OD 600 concentration of solution A.

[0046] Prepare deoxycholate into solution B with a concentration of 100-300mmol / L.

[0047] Prepare magnesium chloride into solution C with a concentration of 200-800mmol / L.

[0048] Mix solution A and solution C, add solution B, stir at room temperature for 15~60min, and wash twice with Tris-Hcl (pH 7.2, 0.05mmol / L) buffer solution to obtain cholate-metal ion complex-modified whole cells.

[0049] The thalline here is any one in embodiment 1.

[0050] Among them, the Escherichia coli modified with cholate-metal ion complex is recorded as complex (E. coli); see figure 1 ;

[0051] The Bacillus subtilis modified by the cholate-metal ion complex is denoted as complex (Bacillus subtilis);

[0052] Saccharomyces cerevisiae modified with the cholate-metal ion complex is ...

Embodiment 3

[0053] Example 3 Universal binding mechanism of cholate-metal ion complexes to different cells

[0054] With the escherichia coli in embodiment 1 E. coli BL21(DE3) is the immobilized target bacterium, and the Escherichia coli modified by the cholate-metal ion complex is prepared by Example 2 E. coli BL21(DE3), see figure 1 .

[0055] Use ethanol solutions with volume fractions of 30%, 50%, 80%, 90%, and 100% to wash the bacterial strains modified by the cholate-metal ion complex step by step, wash away excess materials, and obtain a single cholate - Whole cells modified with metal ion complexes ( figure 2 (a), the strain is Escherichia coli; figure 2 (b) the strain is Bacillus subtilis; figure 2 (c) The strain is Saccharomyces cerevisiae). The complex is still combined with the cell membrane after multiple washings, indicating that the material is stable after being embedded in the cell membrane, and there is a good combination between the complex and the cell mem...

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Abstract

The invention discloses a method for a cholate-metal ion composite modified whole cell and an application of the cholate-metal ion composite modified whole cell. The method comprises the following steps: preparing a metal ion solution of 100mmol / L to 600mmol / L, and subjecting the metal ion solution to stirring and mixing with a Tris-Hcl resuspended strain cell solution with a pH of 7.1 to 8.0 anda concentration of 0.1mol / L, so as to obtain a mixed solution A; preparing an aqueous surfactant solution of 50mmol / L to 300mmol / L, and dropwise adding the aqueous surfactant solution into the mixed solution A until the solution is turbid at a dropwise adding rate of 5 microliters per second to 20 microliters per second with stirring at room temperature, so as to obtain a mixed solution B; and subjecting the mixed solution B to centrifugation so as to obtain precipitates, and flushing the precipitates for 1 to 3 times with Tris-Hcl with a pH of 7.1 to 8.0 and a concentration of 0.05mol / L to wash away unreacted ingredients, thereby obtaining the cholate-metal ion composite modified whole cell. According to the method, the cholate-metal ion composite modified whole cell is prepared by adopting an aqueous-phase solution mixed method, the operation is simple, the conditions are moderate, the synthesis is rapid, the cost is low, the cell stability is high, the intracellular enzyme catalysisefficiency is high, and the cholate-metal ion composite modified whole cell has biological and physical catalytic activity.

Description

technical field [0001] The invention relates to the technical field of microbial catalysis, in particular to a method for compound modification of whole cells with cholate-metal ions and an application thereof. Background technique [0002] Biocatalysis is divided into microbial catalysis and enzyme catalysis. Microbial biocatalysis has the characteristics of green environmental protection and high efficiency. Compared with enzymes, microbial biocatalysis does not require the addition of expensive cofactors, and can achieve joint catalysis of multiple enzymes, which has incomparable advantages in enzyme catalysis. However, in a complex catalytic system, microorganisms face the disadvantages of high mass transfer resistance of the cell membrane, easy cell death, low catalytic efficiency, and difficult product recovery. The traditional field of immobilized technical materials (such as calcium carbonate microspheres, activated carbon particles, polyurethane particles, etc.) i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/02C12N11/14C12P17/06C12R1/19
CPCC12N11/02C12N11/14C12P17/06
Inventor 陈可泉陆秋豪曾金磊刘逸李春秋李辉欧阳平凯
Owner NANJING UNIV OF TECH
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