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Inositol oxygenase mutant and application thereof

A technology of inositol oxygenase and mutants, applied in the field of bioengineering, can solve problems such as environmental pollution, use restrictions, and increase in glucaric acid production

Active Publication Date: 2016-11-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the preparation of glucaric acid is mainly through the method of chemical oxidation of glucose. The nitric acid oxidation method can oxidize glucose into glucaric acid and some small molecular substances by controlling the reaction process to achieve the purpose of the reaction. However, due to the production of a large amount of toxic gas or liquid, causing certain pollution to the environment, so the use of this method is subject to certain restrictions
2,2,6,6-tetramethylpiperidinyloxy free radical (TEMPO) oxidation method has mild reaction conditions and high selectivity, but it is difficult to control temperature, pH and other conditions during the reaction to achieve the best conditions, and the catalyst is very expensive
However, the production of glucaric acid still needs to be improved

Method used

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  • Inositol oxygenase mutant and application thereof
  • Inositol oxygenase mutant and application thereof
  • Inositol oxygenase mutant and application thereof

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Experimental program
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Effect test

Embodiment 1

[0037] The construction of embodiment 1 inositol oxygenase miox mutant strain

[0038] Using site-directed mutagenesis kit (TakaRa), design two pairs of primers (as shown in Table 1), and use pUC57-miox (see Liu, Y., et al., Production of glucaric acid from myo-inositol inengineeredPichia pastoris.Enzyme for the plasmid construction method) and Microbial Technology, 2016.91: p.8-16.) as a template for PCR, the 59-position lysine inside the inositol oxygenase molecule is mutated to valine; or the 171-position arginine is mutated to serine , respectively named K59V and R171S. After the product was recovered, the transformation was verified, and the correct transformants were named pUC57-mioxK59V and pUC57-mioxR171S, respectively. Using the correctly sequenced strain pUC57-mioxK59V as a template, the arginine at position 171 inside the inositol oxygenase molecule was mutated to serine, and the correct transformant obtained was named pUC57-mioxK59V / R171S.

Embodiment 2

[0039] Example 2 Construction of recombinant Pichia pastoris pPIC9KGAP-Udh-mioxK59V, pPIC9KGAP-Udh-mioxR171S, pPIC9KGAP-Udh-mioxK59V / R171S

[0040] Using pGAPZB (commercialized plasmid, purchased by Invitrogen) as a template, using GAP-F\R as a primer pair to amplify the promoter, using the Pseudomonas putida genome as a template, using udh-F\R as a primer pair to amplify udh Gene (Gene ID: 1045739). At the same time, the pUC57-mioxK59V, pUC57-mioxR171S, and pUC57-mioxK59V / R171S plasmids were used as templates, and miox-F / R were used as primers to amplify the inositol oxygenase mutant gene respectively, and the fusion PCR method was used to combine the three mutant genes with The udh gene was fused, and restriction sites SacI and NotI were introduced into the upstream and downstream of the fusion fragment respectively. After double enzyme digestion and ligation transformation, after identification, the recombinant plasmid was linearized and electroporated into the expression ...

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Abstract

The invention discloses an inositol oxygenase mutant and application thereof, and belongs to the technical field of bioengineering. The method comprises the steps that an inositol oxygenase mutant with improved enzyme activity is obtained through site-specific mutagenesis and co-expressed with aldehyde dehydrogenase (udh) in yeast, and the glucaric acid synthetic route is enhanced. Through comparisons, the yield of recombinant Pichia pastoris GS115-udh-mioxK59V / R171S is the highest, and glucaric acid 1.20g / L is produced in a YPD-MI culture medium during flask shaking fermentation culture. A metabolic engineering strategy is adopted, microbiological strains are improved to synthesize the target product glucaric acid, and a certain foundation is laid for efficient production of glucaric acid through a biological method.

Description

technical field [0001] The invention relates to an inositol oxygenase mutant and application thereof, belonging to the technical field of bioengineering. Background technique [0002] Glucaric acid (Glucaric acid, Saccharic acid, GA), also known as glucuronic acid, is a sugar acid formed by oxidizing the aldehyde group and C-6 hydroxyl group of glucose to carboxylic acid, mainly produced by oxidation of glucose. Glucaric acid and its lactones are found in some plants and mammals, especially in some cruciferous vegetables (such as cauliflower, alfalfa sprouts, etc.) and fruits (cherry, grapefruit, citrus, etc.). As an important reaction intermediate, glucaric acid has important application value in the production of polymer materials, food additives and clinical medicine, and has been selected as one of the "most valuable biorefinery products" by the U.S. Department of Energy one. [0003] At present, the preparation of glucaric acid is mainly through the method of chemical...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19C12P7/58C12R1/865C12R1/73C12R1/84
CPCC12N9/0006C12N9/0069C12N15/815C12N2800/102C12N2800/60C12P7/58C12Y101/01203C12Y113/99001
Inventor 康振陈坚堵国成巩旭
Owner JIANGNAN UNIV
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