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Method for preparing D-tagatose by enzyme process

A technology for enzymatic preparation of tagatose, applied in botany equipment and methods, biochemical equipment and methods, enzymes, etc., can solve the problems of increasing downstream separation supporting links, increasing production costs, and conversion rate limitations, etc., to achieve simplification The effect of operating procedures such as post-separation, reducing downstream operations, and reducing production costs

Active Publication Date: 2016-07-06
江苏中酶生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biggest bottleneck problem of this technology is: the conversion rate is limited by the chemical balance, and the theoretical conversion rate of D-galactose is only about 40%. It needs to increase the downstream separation and other supporting links, which increases the input and increases the production cost.

Method used

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  • Method for preparing D-tagatose by enzyme process
  • Method for preparing D-tagatose by enzyme process
  • Method for preparing D-tagatose by enzyme process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Source and acquisition of galactitol dehydrogenase gene

[0028] This example provides a polynucleotide molecule encoding a polypeptide with galactitol dehydrogenase activity. The nucleotide molecule is artificially synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and has a nucleotide sequence of SEQ ID NO: 1 , It encodes the 239 amino acid polypeptide of SEQ ID NO: 2.

Embodiment 2

[0029] Example 2: Construction of recombinant expression vector pET-28a-gdh

[0030] PET-28a (purchased from Novagen Merck China) and the target gene containing two restriction sites (obtained by artificial synthesis in Example 1) were digested with NcoⅠ and BamHI, respectively, and the target fragments that had been double digested and Expression vector, the double digested expression vector pET-28a and the target gene (the gene shown in SEQ ID NO: 1) are ligated overnight with T4-DNA ligase (purchased from TaKaRa) to obtain the recombinant vector pET-28a-gdh ; Add 10 μL of the ligation product to 100 μL of E. coli BL21 competent cells, place on ice for 30 minutes, and heat shock at 42°C for 90 seconds. Place on ice for 2 minutes. Add preheated 0.45 mL SOC medium (2% (W / V) peptone, 0.5% (W / V) yeast extract, 0.05% (W / V) NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose). 220rpm, 37°C, 1h. Add 200 μL of bacterial solution to an LB plate containing 30 μg / mL of kanamycin, and incubate...

Embodiment 3

[0031] Example 3: Cloning of NADH oxidase gene

[0032] Streptococcusthermophiles were purchased from China Common Microorganism Collection and Management Center (CGMCC). MRS medium for culture of Streptococcus thermophilus (g·L-1): beef extract 10.0, yeast extract 5.0, casein peptone 10.0, glucose 5.0, sodium acetate 5.0, citrate diamine 2.0, Tween 801.0, hydrogen phosphate Dipotassium 2.0, magnesium sulfate 0.2, manganese sulfate 0.05, calcium carbonate 20.0, completely dissolved in 950mL deionized water, adjusted to pH 6.8 with 5mmol·L-1 NaOH, constant volume 1L, 110°C, autoclaved for 15min.

[0033] The Streptococcus thermophilus was inoculated into 5mLMRS liquid medium, cultivated to logarithmic growth phase at 42°C, and the genome of Streptococcus thermophilus was extracted using DNAKit (TIANGEN, China). The primers used to construct the expression vector are equipped with restriction sites, and the primer sequence is as follows:

[0034] The upstream primer (NOX-sense contai...

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Abstract

The invention discloses a method for preparing D-tagatose by an enzyme process. The method comprises the following steps: 1) separately establishing engineering bacteria containing galactitol dehydrogenase gene and engineering bacteria containing NADH oxidase gene or establishing coexpression engineering bacteria containing galactitol dehydrogenase gene and NADH oxidase gene; 2) fermenting the established engineering bacteria to obtain a whole cell containing galactitol dehydrogenase and NADH oxidase; and 3) with the whole cell containing galactitol dehydrogenase and NADH oxidase or the free enzyme of galactitol dehydrogenase and NADH oxidase as a catalyst, catalyzing the galactitol by taking NAD+ as a hydrogen acceptor in a weakly alkaline condition to obtain D-tagatose. Compared with traditional production method depending on L-arabinose isomerase, in the invention, the substrate conversion rate is high (>99.0%) and the product is easy to separate; and moreover, the co-product of a coenzyme circulation system adopted in the method is water which does not influence the separation and purification of the principal product.

Description

Technical field [0001] The invention belongs to the field of biochemical industry, and specifically relates to a method for producing D-tagatose based on double enzyme coupling of galactitol dehydrogenase and NADH oxidase. Background technique [0002] D-tagatose is a six-carbon sugar, the ketose form of galactose, and the epimer of fructose. Its sweetness is 92 of that of sucrose, but its calories are only 38% of that of sucrose. In recent years, with the improvement of living standards and the generalization of diseases such as diabetes and obesity, the call for D-tagatose as a functional sweetener to replace sucrose has been increasing. D-tagatose is a rare monosaccharide naturally occurring. It can be found in the gums secreted by trees, sterilized milk, hot cocoa, cheese and cheese, but it is difficult to obtain directly from nature on a large scale. . [0003] At present, the production of D-tagatose mainly adopts chemical methods using alkali metal salts as catalysts or bi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/02C12N15/70C12N15/53
CPCC12N9/0006C12N9/0036C12N15/70C12N2800/101C12P19/02C12Y101/01016C12Y106/99003
Inventor 严明
Owner 江苏中酶生物科技有限公司
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