Immobilized sorbitol dehydrogenase as well as immobilization method and application of immobilized sorbitol dehydrogenase

A sorbitol dehydrogenase and volume technology, which is applied in the directions of immobilized enzymes, biochemical equipment and methods, oxidoreductases, etc., can solve the problems of groping blanks in production process conditions, and achieves the improvement of the number of reuses, the method is simple, the mechanical high intensity effect

Inactive Publication Date: 2017-02-22
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no suitable immobilized sorbitol dehydrogenase product for the production of D-fructose, D-tagatose or L-sorbo

Method used

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  • Immobilized sorbitol dehydrogenase as well as immobilization method and application of immobilized sorbitol dehydrogenase
  • Immobilized sorbitol dehydrogenase as well as immobilization method and application of immobilized sorbitol dehydrogenase
  • Immobilized sorbitol dehydrogenase as well as immobilization method and application of immobilized sorbitol dehydrogenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 Preparation of free sorbitol dehydrogenase

[0033] Referring to the method disclosed in the invention patent CN105154457A, prepare Escherichia coli engineering bacteria containing sorbitol dehydrogenase, induce expression for 15 h, centrifuge the bacterial liquid at 4 °C and 8 000 r / min for 15 min, discard the supernatant, and collect the The cells were washed twice with sodium phosphate buffer (pH 7.0) and suspended in the same buffer, and the cells were broken using a high-pressure homogenizer (-20°C, 8.0×107Pa). After centrifugation at 4 °C and 10 000 r / min for 30 min, the cell disruption solution was discarded, and the supernatant was the crude enzyme solution of sorbitol dehydrogenase.

Embodiment 2

[0034] Example 2 Computer Simulation Assisted Immobilized Enzyme Condition Optimization

[0035] Using the online tool swiss-model (https: / / swissmodel.expasy.org / ), and using PDB:1K2W as a template, carry out 3D structural homology modeling on the amino acid sequence of sorbitol dehydrogenase obtained in Example 1, and Its volume size is predicted to be 7 × 7 × 2.5 nm (Fig. 1). Submit the 3D homology model of sorbitol dehydrogenase to the online server PDB2PQR (http: / / nbcr-222.ucsd.edu / pdb2pqr_2.1.1 / ), analyze its surface potential under different pH environmental conditions, and predict the appropriate The pH range of the immobilization is 4~5.5 (Figure 2).

Embodiment 3

[0036] Example 3 Immobilization operation of sorbitol dehydrogenase

[0037] Prepare 2 mg / mL SBA15 mesoporous material (Nanjing Xianfeng Nano Material Technology Co., Ltd.) with sodium acetate buffer at pH 5.5. After ultrasonication for 15 min, stir magnetically for 30 min to obtain evenly dispersed mesoporous materials. To the material solution, add the free sorbitol dehydrogenase in Example 1, the final concentration is 1 mg / mL, stir or shake at a low speed for 0.25 h at room temperature, take out and filter, rinse with sodium acetate buffer, and place at 4°C Preserved, the protein immobilization capacity is 545 mg / g.

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Abstract

The invention discloses immobilized sorbitol dehydrogenase as well as an immobilization method and application of immobilized sorbitol dehydrogenase. A mesoporous material SBA15 with an adaptive pore diameter is used as a carrier and free sorbitol dehydrogenase is immobilized under a proper pH (Potential of Hydrogen) condition to obtain immobilized enzyme of the sorbitol dehydrogenase; and the prepared immobilized sorbitol dehydrogenase is used as a catalyst, and 20U-300U of NADH (Nicotinamide Adenine Dinucleotide Hydrogen) oxidase and 0.05mmol/L-0.5mmol/L of NAD<+> are added under the conditions that the concentration of a primer (sorbitol or D-galactitol or L-iditol) is 1.5g/L-100g/L and react for 3h-12h under the conditions that the pH is 8.0-9.0, the temperature is 20 DEG C-30 DEG C and the speed is 180rpm-280rpm, so as to obtain D-fructose (or D-tagatose or L-sorbose); after reaction is finished, a solution is filtered and the sorbitol dehydrogenase is recycled; and 75% of catalytic activity can still be kept after the sorbitol dehydrogenase is repeatedly used for 5 times. According to the immobilized sorbitol dehydrogenase disclosed by the invention, immobilization condition optimization is carried out through computer simulation, the immobilization efficiency is high and the protein supporting quantity reaches 545mg/g.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an immobilized sorbitol dehydrogenase, an immobilized method and application thereof. Background technique [0002] Sorbitol dehydrogenase (SDH, EC 1.1.1.14) is a class of oxidoreductases that can selectively oxidize the dihydroxy groups of polyols to ketone groups to generate corresponding ketoses. In our previous invention patent (CN105154457A), we disclosed a sorbitol dehydrogenase derived from Pseudomonas syringae and belonging to the short-chain dehydrogenase family, which can efficiently oxidize sorbitol, galactitol and L-ido Sugar alcohols are the corresponding ketose sugars (D-fructose, D-tagatose and L-sorbose). Compared with traditional chemical methods, oxidoreductases have significant advantages in terms of catalytic efficiency and stereoselectivity. However, the disadvantages of free enzymes such as poor stability, non-reusability, and unfavorable product se...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N11/00C12P19/02
CPCC12N9/0006C12N11/00C12P19/02C12Y101/01014Y02P20/50
Inventor 沙凤严明
Owner NANJING UNIV OF TECH
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