Preparation method of glucaric acid and uronic acid dehydrogenase-NADH oxidase and application thereof in preparation

A technology of uronic acid dehydrogenase and glucaric acid, which is applied in the field of glucaric acid, can solve the problems of low enzyme reactivity, high cost, long conversion time, etc., and achieves simple and easy-to-control operation, low pollution, and improved catalytic efficiency. Effect

Active Publication Date: 2017-11-21
NANJING NORMAL UNIVERSITY
View PDF10 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problems of long conversion time, low ...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method of glucaric acid and uronic acid dehydrogenase-NADH oxidase and application thereof in preparation
  • Preparation method of glucaric acid and uronic acid dehydrogenase-NADH oxidase and application thereof in preparation
  • Preparation method of glucaric acid and uronic acid dehydrogenase-NADH oxidase and application thereof in preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The uronic acid dehydrogenase-NADH oxidase gene was synthesized by Shanghai Shenggong, as shown in the base sequence of SEQ ID NO.3, which was derived from the uronic acid dehydrogenation of Thermobispora bispora The enzyme (TbUDH) (WP_013131340) gene is linked to the Thermotoga maritima NADH oxidase (TmNOX) gene composition through Linker and obtained through codon optimization. TbUDH is the NAD screened from the whole genome sequencing of Thermotoga maritima. Dependent dehydrogenase gene, TmNOX is an oxidoreductase gene (Tm1433) screened from the whole genome sequencing of Thermotoga marina. The base sequence of SEQ ID NO.3 is used with the expression plasmid pET-28a(+) with NcoⅠ After double digestion with XhoⅠ, ligate with T4DNA ligase at 16°C overnight and transform the host E.coli JM109(DE3). When grown on LB medium at 37°C to OD=0.6-0.8, add final concentration of 0.5mM IPTG to induce culture 8-12h, centrifuge at 4℃ and 4000rpm to remove the supernatant, wash the ...

Embodiment 2

[0037] The uronic acid dehydrogenase-NADH oxidase gene was synthesized by Shanghai Shenggong, as shown in the base sequence of SEQ ID NO.4, which is derived from the uronic acid dehydrogenation of Thermobispora bispora The enzyme (TbUDH) (WP_013131340) gene is connected through Linker to Thermotiga maritima NADH oxidase (TmNOX) gene composition and obtained through codon optimization. TbUDH is selected from the whole genome sequencing of Thermotiga maritima NAD-dependent dehydrogenase gene, TmNOX is a hypothetical protein gene (Tm1432) screened from the whole genome sequencing of Thermotoga marina. The same method is used to obtain the cell extract, the amino acid sequence of the cell extract extract As shown in SEQ ID NO.2.

Embodiment 3

[0039] The primers SEQ ID NO. 5: 5'-CCGTTCCATGGACTACAGGATGTGC-3' and SEQ ID NO. 6: 5'-CCGCTCGAGCGGATATATCTTTCTTCCCTT-3' were designed according to the GH10 xylanase gene (TmXynB) of Thermotiga maritima. The GH67α-glucuronidase gene (TmAguA) sequence design primer of Thermotoga T. maritima SEQ ID NO. 7: 5'-GGAATTCCATATGAAAATATTACCTTCTGTGTTGAT-3' and SEQ ID NO. 8: 5'-CCCTCGAGTCTTTCTTCTATCTTTTTCTCCAG-3', selection The genomic DNA of T. maritima bacteria (provided by Jinling Women's College of Nanjing Normal University) was used as a template for PCR amplification to obtain the target gene, and then inserted into the expression plasmid, and the recombinant plasmid containing the xylanase and α-glucuronidase genes was transformed into the host After cultured in the medium at 25~37℃ to an OD600 value of 0.8-1.0, add IPTG or lactose to induce culture at ℃ for 6-10h, and then centrifuge the bacterial solution at 0-10℃ at 4000-6000rpm. Wash the precipitate 3 times with water, resuspend ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Diameteraaaaaaaaaa
Login to view more

Abstract

The invention discloses a preparation method of glucaric acid and uronic acid dehydrogenase-NADH oxidase and an application thereof in preparation. The preparation method includes: taking xylan extracted in wood fiber plants as a raw material; preparing glucuronic acid by adopting a high-temperature xylanase and alpha-glucuronidase hydrolyzing technology; preparing the glucaric acid by oxidizing the glucuronic acid generated through the double-functional uronic acid dehydrogenase-NADH oxidase. With the method, use amount of NAD can be effectively saved, the NAD is regenerated in situ by taking the NADN oxidase as uronic acid dehydrogenase to promote circulation of coenzyme, the uronic acid dehydrogenase continuously catalyzes the glucuronic acid to be converted into the glucaric acid, catalytic efficiency is improved, and conversion rate can be up to 100%; meanwhile, the preparation method has the advantages of high efficiency, rapidness, specificity and moderate reaction conditions, and chemical substances of acid, alkali and the like are not involved in the entire preparation process; the preparation method is low in energy consumption and small in pollution, and operation is simple and easy to control.

Description

Technical field [0001] The invention relates to glucaric acid in the fields of food, medicine and chemical industry, and clean production, in particular to an enzymatic preparation method of glucaric acid and a uronic acid dehydrogenase-NADH oxidase in the preparation and application thereof. Background technique [0002] Gluconic acid is a kind of glucose derivative, which can be used as a raw material to prepare a variety of polymers and biomass new energy. It has a wide range of applications in the chemical industry and is considered one of the "most valuable biorefining products". For example, it can be used as the basic monomer for the synthesis of polyamides, hydroxylated nylon (PHPAs) and polydimethylsiloxane (BDMS) polyamides, to synthesize non-toxic and biodegradable materials for household washing Agents, preservatives and concrete admixtures. Gluconic acid also has the effect of regulating hormones in the body, improving the immune function of the body, and reducing t...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12P7/58C12N9/02
CPCC12N9/0006C12N9/0036C12P7/58C12Y101/01203C12Y106/03001
Inventor 薛业敏李亚贤张宗慧
Owner NANJING NORMAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap