Glucose dehydrogenase dna molecule, carrier and bacterial strain and application

A technology of glucose dehydrogenase and DNA molecules, which is used in the construction of mutants and engineering bacteria, the production of DNA molecules, proteins or polypeptide sequences of glucose dehydrogenase. stability issues

Active Publication Date: 2021-06-08
安琪酶制剂(宜昌)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The problem of the prior art that the present invention solves is: the specific enzyme activity of the genetically engineered bacterium of glucose dehydrogenase in the prior art is low, and expression is unstable, is difficult for the defective of realizing industrialization

Method used

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  • Glucose dehydrogenase dna molecule, carrier and bacterial strain and application
  • Glucose dehydrogenase dna molecule, carrier and bacterial strain and application
  • Glucose dehydrogenase dna molecule, carrier and bacterial strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Embodiment 1 provides a kind of route of constructing the mutant gene of the present invention, as follows:

[0102] (1) Screening of mutant genes

[0103] Using the keyword "glucose dehydrogenase" to search on NCBI, after comparative analysis, the glucose dehydrogenase (GDH) gene sequence in Bacillus subtilis subsp. Subtilis str.AG1839 was selected as the research template, in which Bacillus subtilis The sequence of the glucose dehydrogenase of subsp. Subtilis str.AG1839 is SEQ ID NO.3,

[0104]Wherein, the sequence of SEQ ID NO.3 is as follows:

[0105] atgtatccgg atttaaaagg aaaagtcgtc gctattacag gagctgcttc agggctcgga 60

[0106] aaggcgatgg ccattcgctt cggcaaggag caggcaaaag tggttatcaa ctattatagt 120

[0107] aataaacaag atccgaacga ggtaaaagaa gaggtcatca aggcgggcgg tgaagctgtt 180

[0108] gtcgtccaag gagatgtcac gaaagaggaa gatgtaaaaa atatcgtgca aacggcaatt 240

[0109] aaggagttcg gcacactcga tattatgatt aataatgccg gtcttgaaaa tcctgtgcca 300

[0110] tctcacgaaa tgccgctcaa ...

Embodiment 2

[0182] Example 2 provides a comparison experiment of the specific enzyme activity of the glucose dehydrogenase produced by the strain prepared in Example 1 of the present invention and the glucose dehydrogenase produced by the original strain.

[0183] Among them, the specific enzyme activity is the number of enzyme activity units per mg per protein, that is, the enzyme activity units are divided by the mass of the enzyme protein.

[0184] The target fragment gene of known sequence and the gene fragment of glucose dehydrogenase and the 149-170 double-mutated mutant were respectively connected to the large fragment of plasmid pBAD, and 10 μL of the ligation reaction was added to 100 μL of competent bacteria. Gently mix in the EP tube, ice bath for 30min, heat shock at 42°C for 90s, take it out and quickly ice bath for 2min to cool the bacteria. Add 1 mL of LB liquid medium without antibiotics (containing 10 μL of arabinose inducer), shake at 200 rpm for 1 h at 37 °C. Take 100 ...

Embodiment 3

[0188] Embodiment 3 provides the application of the glucose dehydrogenase prepared in Embodiment 1 in the field of biosynthesis.

[0189]

[0190] Formula (Ⅱ)

[0191] Add 2.4kg substrate (S)-6-chloro-5-hydroxy-3-oxohexanoic acid tert-butyl ester (67% concentration), 25kg sodium phosphate buffer, 2kg glucose solid, 2kg Glucose dehydrogenase solution (2500U / ml), 9kg ketoreductase (80U / ml) and 500mg coenzyme NADP+, magnetic stirring, reaction temperature 30°C, pH=7.0. Among them, the gas phase detection conditions are: chromatographic column type, HP-5, injection volume 1 μL; injector temperature, 270°C; column oven temperature, programmed temperature rise of 100°C for 3 minutes, then 10°C / min to 220°C; FID Detector temperature, 270°C. The gas phase detection conditions were used to detect the completeness of the reaction.

[0192] Then add 300g of activated carbon to the reaction system and stir for 30min, then filter, the filtrate is extracted twice with 40L and 20L of e...

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Abstract

The invention relates to the field of genetic engineering, in particular to DNA molecules, protein or polypeptide sequences, vectors, bacterial strains and applications for producing glucose dehydrogenase. The base sequence of the glucose dehydrogenase DNA molecule is shown in SEQ ID NO.1; or it is selected from genes encoding the following protein (a) or (b): (a) the amino acid shown in SEQ ID NO.2 (b) a polypeptide or protein derived from (a) that has undergone substitution, deletion or addition of one or several amino acids in the amino acid sequence defined in (a) and has glucose dehydrogenase activity. The invention connects the mutant gene with the plasmid pBAD to be more stable, and under the induction of arabinose, the fermentation enzyme activity is higher, which can reach more than 2400U / mL, has the characteristics of high expression level, simple fermentation control, etc., and is suitable for large-scale production .

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a mutant for producing glucose dehydrogenase and a method for constructing engineering bacteria, in particular to a DNA molecule, protein or polypeptide sequence, carrier, bacterial strain and application for producing glucose dehydrogenase. Background technique [0002] (1) As one of the three major (three highs: hyperglycemia, hyperlipidemia, and hypertension) killers that endanger human health in modern society, hyperlipidemia is an important object of medical diagnosis and treatment. Hyperlipidemia often causes atherosclerosis and then leads to coronary heart disease, hypertension and cerebrovascular disease. Statins are the drugs of choice for lowering blood lipids. Statins currently on the market or under development include rosuvastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, rosuvastatin, and pitavastatin. The global output value of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/04C12N15/70C12N1/21C12R1/19
CPCC12N9/0006C12N15/70C12N2800/101C12N2830/002C12Y101/9901
Inventor 余华顺俞学锋李知洪姚鹃戴秋红龚大春王健李建华邹林汉吴尧李明
Owner 安琪酶制剂(宜昌)有限公司
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