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Preparation method of glucaric acid

A technology of glucaric acid and glucuronic acid is applied in the preparation of glucaric acid, the field of "one-pot-two-step" enzyme-catalyzed preparation of glucaric acid, which can solve the problem of low utilization rate of raw materials, degradation of xylan, and increasing process Complexity and other problems, to achieve the effect of high utilization rate of raw materials, prevention of product inhibition, and green-friendly yield

Active Publication Date: 2020-04-07
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But this method uses xylan as the starting material, and xylanase can not fully degrade xylan into a single product, so only a part of the raw material is converted into intermediate glucuronic acid, and the utilization rate of the raw material is low; The step of preparing the intermediate glucuronic acid from polysaccharides needs to be carried out at a higher temperature (60-90°C), and the prepared intermediate glucuronic acid needs to be separated, which undoubtedly increases the complexity of the process

Method used

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  • Preparation method of glucaric acid
  • Preparation method of glucaric acid
  • Preparation method of glucaric acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 prepares glucaric acid by inositol (1.8g / L)

[0048] In this example, the inositol oxidase is from Cryptococcus neoformans, and the NCBI accession number is AAN85573.1. The gene miox was synthesized by BGI and optimized for codons. In this example, the aldolate dehydrogenase is from Agrobacterium tumefaciens, and the NCBI accession number is DAA06454.1. Agrobacterium tumefaciens was purchased from China General Microorganism Culture Collection Center (CGMCC). The gene udh was obtained from genomic DNA by PCR using corresponding primers. The above genes were respectively cloned into pET21a vectors by enzyme-linking or simple cloning methods to obtain the corresponding recombinant expression vectors pET21a-MIOX and pET21a-UDH. The above plasmids were respectively transferred into Escherichiacoli BL21 (DE3), and the protein expression and purification were carried out. The protein expression results were as follows: figure 2 shown.

[0049] (1) Inositol ...

Embodiment 2

[0056] Embodiment 2 utilizes NAD + Regeneration of glucaric acid from inositol (1.8g / L)

[0057] Utilize NAD + Regeneration of the catalytic pathway for the production of glucaric acid from inositol as Figure 4 shown.

[0058] The preparation method of inositol oxidase and aldolase dehydrogenase is the same as that in Example 1. In this example, the NADH oxidase is from Streptococcus mutans, and the NCBI accession number is BAA08707.1. The gene nox was synthesized by BGI and optimized for codons. The gene was cloned into the pET29a vector by restriction linking or Simple cloning to obtain the corresponding recombinant expression vector pET29a-NOX. The above plasmids were respectively transformed into E.coli BL21(DE3), and the protein expression and purification were carried out. The protein expression results were as follows: figure 2 shown.

[0059] (1) Inositol oxidase activation

[0060]A mixture containing 50 mM MOPS buffer (pH 6.5), 5 g / L inositol oxidase, 2.0 m...

Embodiment 3

[0066] Embodiment 3 utilizes NAD + Regeneration of glucaric acid from inositol (9g / L)

[0067] The preparation method of inositol oxidase and aldolase dehydrogenase is the same as that in Example 1. The preparation method of NADH oxidase is the same as that in Example 2. The initial concentration of inositol was 9g / L.

[0068] (1) Inositol oxidase activation

[0069] A mixture containing 50 mM MOPS buffer (pH 6.5), 10 g / L inositol oxidase, 2.0 mM ammonium ferrous sulfate and 5.0 mM ascorbic acid was incubated in an ice-water bath for 60 min to activate inositol oxidase.

[0070] (2) Generation of intermediate glucuronic acid

[0071] A 0.2 mL reaction system containing 200 mM MOPS buffer (pH 7.5), 9 g / L inositol and 2.1 g / L activated inositol oxidase was reacted at 30° C. and 1000 rpm. Use high performance liquid chromatography (HPLC) to detect until the output of intermediate glucuronic acid no longer changes. HPLC detection condition is the same as embodiment 1.

[00...

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Abstract

The invention discloses a method for preparing glucaric acid from inositol by adopting a ''one pot-two step'' enzyme catalysis means, and comprises the following steps: catalyzing inositol by utilizing inositol oxidase so as to produce an intermediate glucuronic acid; and then, converting the intermediate glucuronic acid into the glucaric acid by utilizing aldehyde dehydrogenase. Compared with existing gluconic acid production methods, the method disclosed by the invention has the advantages of being simple in production process, green, friendly, high in yield, conducive to large-scale preparation and the like.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for preparing glucaric acid, and more specifically to a method for preparing glucaric acid by a "one-pot-two-step" enzyme-catalyzed method. Background technique [0002] Glucaric acid (GA for short) is a six-carbon monosaccharide derivative that is widely found in vegetables, fruits and other plants, as well as in a few mammals. It has important biological functions and can be used to lower cholesterol and treat Cancer, its calcium salt can be used as a food additive. At the same time, glucaric acid is also a high value-added organic acid, which has a wide range of applications in the field of fine chemicals, such as the basic unit of polymer synthesis to synthesize polyamides, hydroxylated nylon (PHPAs) and polydimethyl Siloxane (BDMS) polyamides, synthetic biodegradable polymers, slow-release fertilizers, various films, etc., can also be used as raw materials t...

Claims

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Application Information

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IPC IPC(8): C12P7/58
CPCC12P7/58
Inventor 游淳李元刘珊
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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