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A method for enzymatically preparing d-tagatose

An enzymatic preparation and tagatose technology, which is applied in the directions of botanical equipment and methods, biochemical equipment and methods, enzymes, etc., can solve the problems of increasing downstream separation supporting links, increasing production costs, and limiting conversion rate, and achieves simplification. The effect of post-separation and other operational processes, reduction of downstream operations, and reduction of production costs

Active Publication Date: 2019-07-16
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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  • A method for enzymatically preparing d-tagatose
  • A method for enzymatically preparing d-tagatose
  • A method for enzymatically preparing d-tagatose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

Embodiment 2

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

[0030] Use NcoI and BamHI to digest pET-28a (purchased from Novagen Merck China) and the target gene containing two restriction sites (artificially synthesized in Example 1), respectively, and recover the double-digested target fragment and Expression vector, the double-digested expression vector pET-28a and the target gene (the gene shown in SEQ ID NO: 1) were connected overnight with T4-DNA ligase (purchased from TaKaRa Company) to obtain the recombinant vector pET-28a -gdh; add 10 μL of the ligation product to 100 μL of Escherichia coli BL21 competent cells, place on ice for 30 min, and heat shock at 42°C for 90 s. Place on ice for 2 minutes. Add pre-warmed 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 the bacterial solution to an LB plate containing 30 μg / mL kanamycin, and culture ov...

Embodiment 3

[0031] Example 3: Cloning of NADH oxidase gene

[0032] Streptococcus thermophiles were purchased from China General Microorganism Culture Collection Center (CGMCC). MRS medium for Streptococcus thermophilus culture (g L-1): beef extract 10.0, yeast extract 5.0, casein peptone 10.0, glucose 5.0, sodium acetate 5.0, diamine citrate 2.0, Tween 80 1.0, phosphoric acid Dipotassium hydrogen dipotassium 2.0, magnesium sulfate 0.2, manganese sulfate 0.05, calcium carbonate 20.0, completely dissolved in 950mL deionized water, adjust pH to 6.8 with 5mmol·L-1 NaOH, constant volume 1L, 110℃, autoclaved for 15min .

[0033] Streptococcus thermophilus was inoculated in 5 mL of MRS liquid medium, cultured at 42°C until logarithmic growth phase, and the genome of Streptococcus thermophilus was extracted using DNAKit (TIANGEN, China). The primers used to construct the expression vectors are provided with enzyme cutting sites, and the primer sequences are as follows:

[0034] The upstream p...

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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 in particular relates to a D-tagatose production method 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 prevalence of diseases such as diabetes and obesity, the voice of using D-tagatose as a functional sweetener to replace sucrose has been increasing. D-tagatose is a rare monosaccharide that occurs naturally. It is found in the gums secreted by trees, sterilized milk, hot cocoa, cheese and cheese, etc., but it is difficult to obtain directly from nature on a large scale . [0003] At present, in the industry, the production of D-tagatose mainly adopts the chemical method using alkali...

Claims

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Application Information

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Patent Type & Authority Patents(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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