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Method for synthesizing NADPH through immobilized enzyme catalysis

A technology for immobilizing enzymes and kinases, applied in the directions of immobilizing enzymes, biochemical equipment and methods, enzymes, etc., can solve the problems of cumbersome synthesis steps, long production cycle and high production cost, achieve high recovery rate of enzyme activity and simplify production. Process, high stability effect

Active Publication Date: 2018-01-09
山东蓝康药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Therefore, the technical problem to be solved in the present invention is to overcome the low yield of NADPH synthesis in the prior art, the high production cost, and the problems of cumbersome synthesis steps and long production cycle; thereby providing a continuous catalytic preparation of NADPH with immobilized enzymes, And the production method with simple process, short period, low cost and high product yield

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  • Method for synthesizing NADPH through immobilized enzyme catalysis
  • Method for synthesizing NADPH through immobilized enzyme catalysis
  • Method for synthesizing NADPH through immobilized enzyme catalysis

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Embodiment 1

[0047] The invention provides a preparation method of immobilized NAD kinase, specifically comprising the following steps:

[0048] 1. Preparation of NAD kinase-producing bacteria

[0049] Artificially synthesized NAD kinase expression gene derived from Mycobacterium tuberculosis (Mycobacterium tuberculosis), the gene sequence is shown in SEQ ID NO.1, which is connected to the pET24a vector (Novagen Company, 69749-3) through NdeI and HindIII sites, and sequenced After being correct, it was loaded into Ecoli BL21 strain, and the high-expression strain was screened as the NAD kinase producer.

[0050] 2. Preparation of NAD kinase

[0051] (1) Melt the strain tube with NAD kinase producing bacteria at room temperature, then use an inoculation loop to dip a ring and draw a line on the seed medium (kanamycin sulfate 5mg / L) plate containing kanamycin sulfate, The composition of the seed medium is shown in Table 1, and it was activated overnight at 37°C.

[0052] Table 1 Seed Medi...

Embodiment 2

[0071] The present invention provides a preparation method of immobilized NAD kinase, which differs from the preparation method shown in Example 1 only in that:

[0072] 1. Add glutaraldehyde with a mass concentration of 1% to the enzyme carrier LX-1000HFA to activate for 1.5 hours, and filter to obtain the activated carrier.

[0073] 2. Add the activated LX-1000HFA to the enzyme solution prepared in Implementation 1 at a mass ratio of protein: carrier = 1:5 (or wet bacteria: carrier = 1:2), and then adjust the pH to 5.0. Stir slowly at 100 rpm at a temperature of 30°C, and fix for 25 hours.

Embodiment 3

[0075] The invention provides a kind of preparation method of immobilized glucose dehydrogenase, specifically comprises the following steps:

[0076] 1. Preparation of glucose dehydrogenase-producing bacteria

[0077]Artificially synthesize the glucose dehydrogenase expression gene derived from Bacillus subtilis (Bacillus subtilis), the gene sequence is as shown in SEQ ID NO.2, and it is connected to the pET24a vector (Novagen Company, 69749-3) through the NdeI and HindIII sites, After the sequence was correct, it was loaded into the Ecoli BL21 strain, and the high-expression strain was screened as the glucose dehydrogenase-producing strain.

[0078] 2. Preparation of glucose dehydrogenase

[0079] According to the preparation method provided in Example 1, glucose dehydrogenase enzyme solution was prepared by using glucose dehydrogenase-producing bacteria.

[0080] 3. Preparation of immobilized glucose dehydrogenase

[0081] According to the preparation method provided in E...

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Abstract

The invention discloses a method for synthesizing NADPH through immobilized enzyme catalysis. The method comprises the following steps: (1) in the presence of divalent metal ions, preparing a first reaction liquid by taking NAD and metaphosphate as substrates, adding immobilized NAD kinase into the first reaction liquid, and performing catalyzing synthesis of NADP; and (2) removing immobilized NADkinase in the first reaction liquid, continuously adding glucose for preparing a second reaction liquid, adding immobilized glucose dehydrogenase into the second reaction liquid, and performing catalyzing synthesis of NADPH. The method disclosed by the invention realizes that NADPH is synthesized through continuous reaction, production technology is simple, production period is short, productioncost is low, and product quality is more stable; besides, the immobilized enzyme catalysis is adopted for preparing NADPH, an immobilized enzyme can be continuously and repeatedly used, the productioncost is further reduced, introduction of protein impurities into a product is avoided, and NADPH can be beneficially produced with high purity and high yield when the immobilized enzyme is applied tothe method.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to a method for catalyzing the synthesis of NADPH by immobilized enzymes. Background technique [0002] Reduced coenzyme II (triphosphopyridine nucleotide, reduced nicotinamide adenine dinucleotide phosphate, referred to as: NADPH) is an extremely important nucleotide coenzyme, which is an oxidized coenzyme I (Nicotinamide Adenine Dinucleotide, nicotinamide adenine Dinucleotide (abbreviation: NAD) is a phosphorylated derivative of the 2'-position of the ribose ring system linked to adenine. As the most important electron donor and reductant for biosynthesis in cells, NADPH can provide hydrogen ions for reductive biosynthesis and participate in the synthesis of biomolecules such as amino acids, fatty acids, cholesterol, monooxygenases, and steroid hormones. In addition to participating in biosynthesis, NADPH also participates in the hydroxylation reaction in vivo and the biotransfo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/36C12N11/00
Inventor 秦正红
Owner 山东蓝康药业有限公司
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