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Sorbose dehydrogenase and sorbosone dehydrogenase, and applications thereof

The technology of sorbose dehydrogenase and sorbone dehydrogenase is applied in the field of microbial biology and can solve the problems of long cycle, increased process control, nutrient composition of fermentation medium and energy waste, etc.

Inactive Publication Date: 2013-07-03
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The two-step fermentation method involves two fermentations, three kinds of bacteria participate, and the cycle is long. Especially the second step of mixed bacteria fermentation increases the difficulty of process control, causes waste of nutrients and energy in the fermentation medium, increases environmental pollution, and improves production. cost

Method used

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  • Sorbose dehydrogenase and sorbosone dehydrogenase, and applications thereof
  • Sorbose dehydrogenase and sorbosone dehydrogenase, and applications thereof
  • Sorbose dehydrogenase and sorbosone dehydrogenase, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Extraction of Gluconobacter oxydans CGMCC No.1.637 Genomic DNA

[0031] (1) Cultivate Gluconobacter oxidans CGMCC No.1.637 (purchased from the Institute of Microbiology, Chinese Academy of Sciences) until the OD value is about 2.0, and collect 5 mL of bacterial liquid by centrifugation at 4°C and 8000 rpm for 5 min.

[0032] (2) Add 450 μL TE, 50 μL 10% SDS, 5 μL proteinase K, and vortex at 1800 rpm to fully resuspend.

[0033] (3) Cleavage at 80°C for 10 minutes, let cool to room temperature, add 5 μL RNaseA, mix by inverting 10 times, and bathe in 37°C water for 2 hours.

[0034] (4) Add 30 μL of 3M KAC, shake at 1800 rpm for 30 seconds, and place at -20°C for 20 minutes.

[0035] (5) Centrifuge at 12000 rpm at 4°C for 20 min, and pipette the supernatant into another centrifuge tube.

[0036] (6) Add 60 μL of 5M NaCl and 300 μL of isopropanol, invert and mix 20 times, and place at -20°C for 20 minutes.

[0037] (7) Centrifuge at 12000 rpm and 4°C for 20 m...

Embodiment 3D

[0046] Embodiment 3DCIP and active electrophoresis detection activity

[0047] The pMD18T-SNDHSDH / DH5α cells were collected by centrifugation and lysed by ultrasound. After centrifugation, the supernatant was taken for activity detection by DCIP method and Native electrophoresis. Sorbose and xylose were used as substrates respectively, and the results showed SDH and SNDH activities (Table 2) .

[0048] Table 2 DCIP and activity electrophoresis detection activity results

[0049]

[0050] Note: "+" means no activity, "-" means no activity.

Embodiment 4

[0051] Example 4 Construction of pBBR1MCS2-SNDHSDH-1.637 plasmid

[0052] The successfully constructed pMD18T-SNDHSDH plasmid and pBBR1MCS2 (GenBank accession number U23751) plasmid vector were digested with Xba I / SalI respectively, the target fragment was recovered by the agarose gel recovery kit, ligated at 16°C (10 μL system) overnight, and transformed into DH5a , to screen for positive recombinants. Identification by bacterial liquid PCR ( Figure 6 ) and sequencing identification showed that the pBBR1MCS2-SNDHSDH plasmid was constructed successfully.

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PUM

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Abstract

The invention provides sorbose dehydrogenase and sorbosone dehydrogenase for gluconobacter oxydans CGMCC No. 1. 637, amino acid sequences of the sorbose dehydrogenase and sorbosone dehydrogenase are shown as SEQ ID No. 2 and SEQ ID No. 4. The invention also provides genes or gene clusters for coding the above proteins. SNDH-SDH gene clusters with self-controlling sequences are connected to broad host range plasmids pBBRiMCS2, and are introduced to the gluconobacter oxydans 621H through conjugative transfer, so that transformation from the sorbose to keto-gulonic acid can be realized.

Description

technical field [0001] The invention belongs to the technical field of microbial biology, and in particular relates to the sorbose dehydrogenase and sorbone dehydrogenase genes and encoded proteins of Gluconobacter oxydans 1.637. Background technique [0002] Gluconobacter oxydans (Gluconobacter oxydans) belongs to the Acetobacteraceae Gluconobacter genus, which is closely related to human production and life, and has broad application prospects in food, beverage, pharmaceutical, chemical and other industries. [0003] At present, VC production generally adopts a two-step fermentation method. The first step is to convert the substrate sorbitol into sorbose by Gluconobacter oxydans, and the second step is to convert sorbose into ketogulonic acid, which is completed by mixed fermentation of large and small bacteria. The two-step fermentation method involves two fermentations, three kinds of bacteria participate, and the cycle is long. Especially the second step of mixed bacter...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/63C12N1/21C12P7/60C12R1/01C12R1/19
Inventor 张惟材熊向华汪建华葛欣陈微微侯伟
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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