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Sorbitol dehydrogenase gene from pseudomonas syringae and application of sorbitol dehydrogenase gene

A technology of sorbitol dehydrogenase and Pseudomonas, which is applied in the field of genetic engineering and enzyme engineering, can solve the problems of low activity, unsuitable for industrial application, and difficulty in distinguishing xylitol, etc., and achieve high conversion rate and important industrial The effect of applying value

Active Publication Date: 2015-12-16
苏州科宁多元醇有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

from sheep liver, Bacillus subtilis Due to the broad substrate specificity of SDH isolated in , it is difficult to distinguish xylitol (similar in structure to sorbitol) in the sample; Schneider et al. Pseudomonas sp. SDH with low activity to xylitol (only 2% of sorbitol) was isolated, but it was not suitable for industrial application due to low yield; then, IkukoMasuda et al. Pseudomonas sp. KS-E1806 cloned an SDH gene and constructed genetically engineered bacteria for efficient production of sorbitol dehydrogenase, and applied for patent protection in Europe, the United States, and Japan (Patent No.: EP1262551(A3), US2003022336(A1), JP2002355046 (A))

Method used

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  • Sorbitol dehydrogenase gene from pseudomonas syringae and application of sorbitol dehydrogenase gene
  • Sorbitol dehydrogenase gene from pseudomonas syringae and application of sorbitol dehydrogenase gene

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Embodiment 1

[0031] Cloning of embodiment 1 sorbitol dehydrogenase gene

[0032] Pseudomonas syringae was purchased from the China General Microorganism Culture Collection Center (CGMCC), medium LB (g L -1 ): Yeast extract 5g, peptone 10g, NaCl 10g, add distilled water to 1L.

[0033] Pseudomonas syringae was inoculated in 5mL LB liquid medium, cultured at 30°C to logarithmic growth phase, and the genome of Pseudomonas syringae 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] Upstream primers (SDH-sense containing EcoR I) is:

[0035] 5'-CG GAATTC AAACGACTTGAAGGTAAAAGCG-3'

[0036]Downstream primers (SDH-anti containing xho I) is:

[0037] 5'-CCG CTCGAG TCAGTTCATCCAGTTGCCACCA-3'

[0038] All primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd. PCR conditions of the gene: denaturation at 94°C for 7 minutes, cycled 30 times ...

Embodiment 2

[0039] Example 2 Recombinant expression vector pET-28a- sdh build

[0040] use xho Ⅰ and EcoR Ⅰ Respectively digest pET-28a (purchased from Novagen Merck China) and the amplified target gene containing two restriction sites (obtained by PCR amplification in Example 1), and recover the double-digested target fragment and express Carrier, the expression vector pET-28a that has been double digested 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- sdh ; 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 preheated 0.45mL SOC medium (2% (W / V) peptone, 0.5% (W / V) yeast extract powder, 0.05% (W / V) NaCl, 2.5mMKCl, 10mMMgCl 2 , 20 mM glucose. ). 220rpm, 37°C, 1h. Add 200 μL of bacterial liquid to the LB plate containing 30 μg / mL kan...

Embodiment 3

[0041] Induced expression of embodiment 3 sorbitol dehydrogenase gene in Escherichia coli BL21

[0042] pick recombinant bacteria E. coli BL21 (containing pET-28a- sdh ) and control bacteria E. coli BL21 (containing pET-28a) was added to LB liquid medium containing 30 μg / mL kanamycin, and cultured overnight at 37°C with shaking. Then they were inoculated into fresh LB liquid medium containing 30 μg / mL kanamycin according to 2% inoculation amount, and cultured at 37°C until OD 600 At about 0.6, add IPTG to a final concentration of 0.2mmol·L -1 , 16°C, 220rpm, after induction of expression for 24h, centrifuge (4°C, 5000rpm, 15min), resuspend the bacteria sludge with 100mM Tris-HCl buffer (pH9.0), and sonicate the cells (power 300W, ultrasonic 3s, intermittent 5s, total 5min), centrifuge (4°C, 12000rpm, 15min). SDS-PAGE analysis showed that the recombinant bacteria E. coli BL21 (containing pET-28a- sdh ) expresses a protein with a molecular weight of about 27kDa (see ...

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Abstract

The invention discloses a sorbitol dehydrogenase gene from pseudomonas syringae and application of the sorbitol dehydrogenase gene. The gene nucleic acid sequenc is shown as SEQ ID NO:1, a recombinant vector is built, cloning and expression are conducted in escherichia coli, and the sorbitol dehydrogenase amino acid sequence of the gene coding is shown as SEQ ID NO:2. The sorbitol dehydrogenase can effectively catalyze conversion of various important polyhydric alcohols and ketose corresponding to the polyhydric alcohols, the conversion rate to substrate of the sorbitol dehydrogenase is larger than 99%, compared with a traditional production method depending on isomerase, the later-period separation and decoloration costs are greatly lowered, and the sorbitol dehydrogenase gene has important industrial application value.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme engineering, in particular to a sorbitol dehydrogenase gene derived from Pseudomonas syringae and application thereof. Background technique [0002] Sorbitol dehydrogenase (Sorbitoldehydrogenase, referred to as SDH, EC1.1.1.14) widely exists in microorganisms, plants and animals. It was first discovered in cat liver by Breusch, F.L., etc. (1942). key enzymes. When unutilized glucose enters the polyol pathway, aldose reductase first reduces it to sorbitol and simultaneously oxidizes NADPH to NADP + . In turn, SDH utilizes NAD + The hydrogen acceptor oxidizes the C2 hydroxyl group of sorbitol to produce fructose, which is phosphorylated by hexokinase to form 6-phosphate-fructose and returns to the glycolytic pathway. At present, researchers have investigated dozens of sorbitol dehydrogenases from different sources. It was only by Philippsen, A., Pauly, T.A, etc. who successively us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/04C12N1/21C12N1/19C12N1/15C12P7/18
Inventor 严明沙凤魏淼李艳章志林
Owner 苏州科宁多元醇有限公司
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