Method for preparing whole cells of gluconobacter oxydans by using glycerol as carbon source

A technology for oxidizing glucose and acid bacteria, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, oxidoreductases, etc., to achieve the effects of mild reaction conditions, reduced preparation costs, and convenient operation

Inactive Publication Date: 2018-12-28
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The search found that there is no report on the method of preparing whole cells of Gluconobacter oxidans using glycerol as a carbon source

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  • Method for preparing whole cells of gluconobacter oxydans by using glycerol as carbon source
  • Method for preparing whole cells of gluconobacter oxydans by using glycerol as carbon source

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Knockout of genes related to glycerol metabolites

[0034] (1) Knockout of the membrane-bound alcohol dehydrogenase large subunit GOX1068

[0035] The membrane-bound alcohol dehydrogenase large subunit GOX1068 has a sequence length of 2274 bases, and its nucleotide sequence is shown in SEQID NO.1.

[0036] The bacterial strain constructed in the present invention is genetically modified on the basis of the original bacteria of G.oxydans 621H.

[0037] Genomic DNA of strain G.oxydans 621H was prepared by a conventional method, and the genomic DNA of G.oxydans 621H could be extracted by referring to the small-scale preparation method of the bacterial genome in the "Guidelines for Experiments of Molecular Biology" published by Science Press; Using the G.oxydans 621H genome as a template, the upstream and downstream homology arms of the GOX1068 coding gene were amplified by PCR using primers "GOX1068up.f and GOX1068up.r" and "GOX1068down.f and GOX1068down.r". T...

Embodiment 2

[0056] Embodiment 2: the preparation of four kinds of bacterium whole-cell catalysts

[0057] (1) Streak the strains of Gluconobacter oxydans G.oxydans 621H and G.oxydansΔGOX1068ΔGOX0854 on the sorbitol complex medium plate containing 1.5-1.8% agar by mass volume ratio and 50 μg / mL cefoxitin, respectively, G. oxydans 621H was shaken at 30±1°C for 24±1 hours, G.oxydansΔGOX1068ΔGOX0854 was shaken at 30±1°C for 36±1 hours;

[0058] (2) First-class seeds: under sterile conditions, use a sterile toothpick to pick out the single colony on the plate of step (1) respectively, and then inoculate them into 5 mL of sorbitol complex culture containing 50 μg / mL cefoxitin medium, G.oxydans 621H was shaken at 30±1°C for 24±1 hours, and G.oxydansΔGOX1068ΔGOX0854 was shaken at 30±1°C for 36±1 hours;

[0059](3) Secondary seeds: Under aseptic conditions, the bacterial solution obtained in step (2) was inoculated into 50 mL of sorbitol complex medium with a volume ratio of 2-4% inoculum, and G....

Embodiment 3

[0066] Embodiment 3: the comparison of the catalytic effect of four kinds of biocatalysts that embodiment 2 obtains

[0067] Obtain four kinds of whole cell bacterium liquids of two kinds of medium sources as biocatalyst with embodiment 2, make the cell final concentration (OD 600nm ) is 13, under strict aerobic conditions at 30°C and pH 7.0 to 7.5, the conversion concentration of xylose is about 7.8g / L; 150 rpm water bath shaker shakes; catalyzes 6-hour sampling to detect xylose consumption and xylose The formation of sugar acid: centrifuge the obtained conversion solution at 13,000±500 rpm for 5 minutes to remove the added biocatalyst, absorb the supernatant and perform high performance liquid chromatography analysis to determine the concentration of xylose.

[0068] The results show that the xylose consumption process is shown in the attached figure 1 , reacted for 6 hours, G.oxydans621H, G.oxydansΔGOX1068ΔGOX0854 whole cells prepared with sorbitol as carbon source and G....

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Abstract

The invention discloses a method for preparing whole cells of gluconobacter oxydans by using glycerol as a carbon source. The method includes performing genetic engineer transformation on wild-type gluconobacter oxydans to make the wild-type gluconobacter oxydans grow with the glycerol as the carbon source, to make a recombinant gluconobacter oxydans DeltaGOX1068DeltaGOX0854, and then inoculatingthe obtained recombinant gluconobacter oxydans in a glycerol inorganic salt medium, to obtain the whole cells of gluconobacter oxydans after shaking culture through a shaking table at 30+ / -1 DEG C. Experiments prove that the process of preparing the whole cells in the method is simple and convenient, a prepared whole cell biocatalyst has catalytic efficiency equivalent to that of a gluconobacter oxydans 621H whole cell biocatalyst prepared in a sorbitol complex medium, and the biocatalyst has a low preparation cost and has good application values.

Description

technical field [0001] The invention relates to a method for preparing whole cells of Gluconobacter oxydans, in particular to a method for preparing whole cells of Gluconobacter oxydans by using glycerol as a carbon source. Background technique [0002] Gluconobacter oxidans is a Gram-negative, strictly aerobic acidophilic bacterium belonging to the genus Gluconobacter in the family Acetobacteriaceae (Gupta A et al., J. Mol. Microbiol. Biotechnol., 2001, 3:445-456) . Gluconobacter oxidans is able to incompletely oxidize a large number of alcohols and carbohydrates, and many compounds are oxidized in the periplasmic space of Gluconobacter oxidans in a chemo-, site-, stereo-selective manner to the corresponding Ketones, aldehydes and organic acids (Keliang G et al., Appl. Microbiol. Biotechnol., 2006, 70: 135-139; Deppenmeier U et al., J. Mol. Microbiol. Biotechnol., 2002, 16: 69-80 ). In addition, Gluconobacter oxydans has the characteristics of high oxidation rate and low...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/58C12R1/01
CPCC12N9/0006C12P7/58
Inventor 高超严金鑫徐静马翠卿许平
Owner SHANDONG UNIV
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