Sorbose dehydrogenase and sorbosone dehydrogenase, and applications thereof

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

[0031] (1) Cultivate Gluconobacter oxydans CGMCC No.1.637 (purchased from the Institute of Microbiology, Chinese Academy of Sciences) to an OD value of about 2.0, and centrifuge at 4°C and 8000 rpm for 5 minutes to collect 5 mL of bacterial cells.

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

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

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

[0035] (5) Centrifuge at 12000 rpm and 4°C for 20 minutes, and transfer the supernatant to another centrifuge tube.

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

[0037] (7) Centrifuge at 12000 rpm and 4°C for 20 min, and discard the supernatant.

[0038] (8) Add 100 μL of 70% pre-cooled ethanol to rinse the DNA pellet, centrifuge at 12000 rpm and 4°C for 10 min, and discard the supernatant.

[0039] (9) Repeat washing once and discard the supernatant. Dry at room temperature.

[0040] (10) Add 30 μL of TE to dissolve the genomic DNA pellet, and freeze at -20°C for use.

[0041] Take 2μL sample for 1% agarose gel electrophoresis detection, the sample concentration is about 50ng/μL, the electrophoresis graph shows that there is no obvious RNA band, which meets the requirements of PCR amplification template ( figure 1 ).

[0042] Example 2 Amplification of Gluconobacter oxydans 1.637 sorbose dehydrogenase and sorbitol dehydrogenase genes

[0043] Using the genomic DNA of CGMCC No.1.637 extracted in Example 1 as a template, using the primers shown in Table 1, PCR amplification of SDH, SNDH genes and SNDH-SDH gene clusters were performed by 1% agarose electrophoresis analysis ( figure 2 , image 3 ). Cut the gum to recover the target fragment, connect the pMD18T vector, transform DH5α, pick the blue spot colony for culture, and identify the positive recombinant by PCR ( Figure 4 , Figure 5 ). The positive bacterial strains were identified by PCR and sent to BGI for gene sequencing. After sequencing, the correct clones were extracted and the plasmids were stored and named pMD18T-SDH, pMD 18T-SNDH, and pMD 18T-SNDHSDH.

[0044] Table 1 PCR primers

[0045] SDH-F:

[0046] Example 3 DCIP and activity electrophoresis to detect activity

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

[0048] Table 2 DCIP and activity electrophoresis test results

[0049]

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

[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, and the target fragment was recovered by the agarose gel recovery kit, and ligated at 16°C (10μL system) overnight to transform DH5a , Screen positive recombinants. Identification by bacterial liquid PCR ( Image 6 ) And sequencing identification showed that the plasmid pBBR1MCS2-SNDHSDH was successfully constructed.