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Preparation of nano gel immobilized multienzyme system and application thereof in synthesizing 1,3-propylene glycol

A technology of nanogel and multi-enzyme system, applied in the direction of immobilized on or in the inorganic carrier, multi-enzyme system, fermentation, etc., can solve problems such as conformational changes and molecular movement restrictions, enzyme activity reduction, and damage to the microenvironment. Achieve the effect of reducing the influence of diffusion, inhibiting agglomeration, and simple reaction

Inactive Publication Date: 2010-12-15
SOUTHEAST UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

In the sol-gel nano-immobilized enzyme process, acid, alkali or salt are generally used as catalysts in the sol-gel process, and the enzyme is easily inactivated under conditions of high, low pH or high ionic strength; in the sol-gel process In the process, a large number of OH and COOH on the surface of the enzyme molecule interact strongly with the polymer fragments containing M-O(H)-M and M-OH at the initial stage of gel network formation (Xu Songwei, 2004), resulting in a change in the conformation of the enzyme molecule, causing Reduced enzyme activity
In addition, the ethanol produced by the hydrolysis of the precursor in the sol-gel process destroys the microenvironment suitable for the survival of the enzyme, and the gel restricts the enzyme to some microenvironments that are not suitable for the maintenance of enzyme activity. The conformational changes and molecular movements of the enzyme are compared with those in the solution. restricted

Method used

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  • Preparation of nano gel immobilized multienzyme system and application thereof in synthesizing 1,3-propylene glycol
  • Preparation of nano gel immobilized multienzyme system and application thereof in synthesizing 1,3-propylene glycol
  • Preparation of nano gel immobilized multienzyme system and application thereof in synthesizing 1,3-propylene glycol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment one different alcohol dispersants to TiO 2 load factor effect

[0033] Bacteria: Klebsiella pneumoniae (K.pneumoniae)

[0034] Take 1000mL fermentation broth to collect the bacteria by centrifugation, wash and weigh, add 10 times the mass of 0.9% normal saline, mix well, ultrasonically crush in an ice bath environment, and centrifuge to obtain crude enzyme liquid, which is precipitated with ammonium sulfate with a saturation of 50% , after desalting by dialysis, a multi-enzyme system solution with a concentration of 20.0 g / L was prepared with Tris-HCl buffer solution.

[0035] Weigh 50mg TiO respectively 2 (Particle size 16nm, specific surface area 280m 2 / g) Nanocarriers were put into No. 1-7 test tubes, No. 1 test tube had no dispersant, and 5mL Tris-HCl buffer solution was used as a blank, and ultrasonically dispersed for 30min. Add 5mL of different alcohol dispersant solutions to No. 2-7 test tubes, and ultrasonically disperse for 30min. Then add 5mL...

Embodiment 2

[0045] Example 2 Effects of Different pH Loading Processes on the Loading Rate of Glycerol Dehydrogenase Loaded on Nanogel Carriers

[0046] The preparation of the multi-enzyme system solution was the same as in Example 1.

[0047] With glycerol as dispersant, weigh the same amount of TiO 2 (Particle size 16nm, specific surface area 280m 2 / g) 12 parts of powder, add different pH Tris-HCl buffer solutions (pH=5.0~10.0), ultrasonically disperse for 30 minutes, add a certain amount of enzyme solution, shake and adsorb in a shaker at 37°C for 1 hour, take it out and centrifuge at 8000rpm After 10 minutes, the lower precipitate was washed with Tris-HCl buffer solution until the supernatant was free of protein, and the washing liquid and supernatant were combined for analysis of the enzyme loading rate under different pH conditions. The average value of two parallel samples was taken, and the experimental results are shown in Table 2.

[0048] Table 2 Effects of different pH loa...

Embodiment 3

[0051] The thermostability of embodiment three nano-gel immobilized multi-enzyme system

[0052] Bacteria: Klebsiella pneumoniae (K.pneumoniae)

[0053] Glycerol dehydrogenase GDH enzyme activity assay: 5mL GDH reaction solution (containing 30mmol / L (NH 4 ) 2 SO 4 , 0.2mol / L glycerol, 2mmol / L NAD + , 1μmol / L (NH 4 ) 2 Fe(SO 4 ) 2 , prepared with potassium carbonate buffer solution with pH = 11) at 37°C, add the enzyme used in the experiment to start the reaction, react at a constant temperature in a shaker for 40 minutes, transfer to a centrifuge tube and centrifuge at 8000r / min for 10 minutes, and the supernatant in Absorbance A was measured at 340 nm. One enzyme activity unit (U) is the amount of enzyme required to consume 1 mol of substrate in one minute under the conditions.

[0054] The preparation of the multi-enzyme system solution was the same as in Example 1.

[0055] Weigh a certain amount of TiO 2 For powder, use glycerol as dispersant, ultrasonically dis...

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Abstract

The invention relates to preparation of a nano gel immobilized multienzyme system. The preparation method comprises the following steps: constructing a nano gel-multienzyme assembling system by using a nano gel dispersion-adsorption immobilized enzyme technology, carrying out self-coupling, and catalyzing by multiple enzymes to synthesize the 1,3-propylene glycol. The nano gel suspension, which can be evenly dispersed in the water solution, is prepared to directly absorb the immobilized multienzyme system. The method can effectively inhibit the aggregation and enhance the dispersibility; the invention fully displays the excellent properties of nano gel carrier, such as high specific surface and the like, provides a location for simultaneously coupling multiple biological enzymes (multienzyme immobilization), and reduces the influence of the carrier on the dispersion of a substrate and products; and compared with the microbe fermentation method, the invention has the advantages of low cost, simple reaction, no cell pollution and the like, and the nano gel immobilized multienzyme system can not be easily degraded by the microbes. The invention provides a new effective catalytic method for synthesizing nano gel immobilized enzymes.

Description

technical field [0001] The present invention relates to the preparation of a nano-gel immobilized multi-enzyme system and its application in the synthesis of 1,3-propanediol (1,3-PD). By preparing a nano-gel suspension uniformly dispersed in an aqueous solution, using Dispersion-adsorption immobilization technology, construction of nanogel-multi-enzyme assembly system, multi-enzyme coupling catalytic synthesis of 1,3-PD. This method can effectively inhibit agglomeration, improve dispersibility, form a uniform and stable nanogel suspension, and exert the excellent properties of nanocarriers such as high specific surface area. Diffusion effects of small carriers on substrates and products. The use of nanogel immobilized multi-enzyme system to catalyze the synthesis of 1,3-PD has the advantages of low cost, simple reaction, no cell pollution, and not easy to be degraded by microorganisms compared with microbial fermentation methods. The invention belongs to the technical field ...

Claims

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Application Information

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IPC IPC(8): C12N11/18C12N11/14C12P7/18
Inventor 吴敏何琴倪恨美张玲卜长飞
Owner SOUTHEAST UNIV
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