Novel bacillus arabinose isomerase gene cloning and expression method and use
A technology of arabinose and bacillus, applied in the field of bioengineering, can solve problems such as unapproved use, and achieve the effect of increasing production
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Embodiment 1
[0032] Example 1: PCR amplification of Bacillussp.SYBChb4L-arabinose isomerase gene L-AI, protein sequence functional region prediction
[0033] The bacterial genomic DNA extraction kit (TaKaRa Company) was used to extract the total genomic DNA of Bacillussp.SYBChb4 bacteria according to its operation, its quality and purity were identified by agarose gel electrophoresis, and its concentration was determined by an ultraviolet spectrophotometer. Design upstream and downstream primers according to the whole gene sequencing: h1 (SEQ ID NO: 3, 5'-ATGTTAACAAGTCAGAAG-3') and h2 (SEQ ID NO: 4, 5'-TCACAGTAGAATGCATTTC-3'), using the PCR method, using the above-mentioned Bacillussp.SYBChb4 genome as Template, with the above-mentioned h1 and h2 as specific primers, amplify the full-length coding frame sequence (1446bp) of the Bacillussp.SYBChb4L-arabinose isomerase gene (such as figure 1 shown). The PCR reaction conditions are: using genomic DNA as a template, in a 50 μL reaction syste...
Embodiment 3
[0039] Embodiment 3: the fermentation experiment of recombinant bacterial strain
[0040] The recombinant Escherichia coli constructed as in Example 2 was cultured in shake flasks. Pick a single colony of recombinant Escherichia coli on the Amp resistant plate and inoculate it into LB liquid medium containing ampicillin antibiotic. See Example 2 for the recipe of the culture medium. Cultivate at 37°C and 200rpm for about 12 hours. When the thalline grows to the logarithmic phase, insert it into 50ml of fermentation medium (diluted 10 times by lactose refining wastewater) according to the inoculum size of 2%. Cultivate for about 24 hours at 200 rpm.
Embodiment 4
[0041] Embodiment 4: Quantitative detection of tagatose
[0042] D-tagatose and D-galactose in each eluted sample can be detected qualitatively and quantitatively by high performance liquid chromatography (HPLC). HPLC conditions: chromatographic column: Sugarpark 16.5mm×30mm; mobile phase: pure water; detector: Waters2410 differential refractive index detector; column temperature: 85°C; flow rate: 0.4mL / min;
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