pqq-sgdh mutant, polynucleotide and glucose detection device
A technology of polynucleotides and mutants, applied in the field of soluble pyrroloquinoline quinone-dependent glucose dehydrogenase, which can solve problems such as poor substrate specificity, death, and elevated blood sugar levels
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Embodiment 1
[0099] Example 1: Cloning and expression of wild-type PQQ-sGDH in E. coli
[0100] The wild-type PQQ-sGDH coding gene of Acinetobacter calcoaceticus strain LMD79.41 was synthesized in vitro by Nanjing KingScript Biotechnology Co., Ltd., and then inserted into the plasmid PET30a (purchased from Novagen) to obtain the recombinant plasmid, then 10 μl of the obtained recombinant plasmid was introduced into the host cell Escherichia coli strain BL21, that is, the Escherichia coli transformed cell, and then cultured in 1 ml LB liquid medium at 37°C for 1 hour, the Bacteria were spread on agar plates and grown overnight at 37°C. Select bacterial spots from the agar plate for DNA sequencing, then inoculate the bacterial spots that have been successfully introduced into 50ml of LB medium and culture at 37°C until OD 600 When it was 1.0, 50 μl of IPTG was added to induce the expression of the wild-type PQQ-sGDH coding gene, and after continuing to cultivate for 3 hours, the bacteria we...
Embodiment 2
[0102] Example 2: Preparation of PQQ-sGDH mutants by PCR site-directed mutagenesis
[0103] Using the recombinant plasmid isolated in Example 1 as a starting template, PCR amplification was used to perform site-directed mutagenesis, so that A at position 194 was replaced by F.
[0104]The primers used to obtain the A194F mutation are shown in SEQ ID NO 3 and SEQ ID NO 4:
[0105] SEQ ID NO 3:
[0106] 5'-TGACCAAGGGCGTAACCAGCTTTTCTATTTGTTCTTGCCAAATCAAGCAC-3'
[0107] SEQ ID NO 4:
[0108] 5'-GTGCTTGATTTGGCAAGAACAAATAGAAAAGCTGGTTACGCCCTTGGTCA-3'.
[0109] Using the recombinant plasmid constructed in Example 1 as a starting template, PCR amplification was used to perform site-directed mutagenesis, so that Q at position 192 was replaced by A or S.
[0110] Primers used to obtain the Q192A mutation are shown in SEQ ID NO 5 and SEQ ID NO 6:
[0111] SEQ ID NO 5:
[0112] 5'-ATTGGTGACCAAGGGCGTAACGCGCTTGCTTATTTGTTCTTGCCAA-3'
[0113] SEQ ID NO 6:
[0114] 5'-TTGGCAAGAACAAATAAG...
Embodiment 3
[0123] Example 3: Purification of wild-type PQQ-sGDH or PQQ-sGDH mutants
[0124] First, the thalline collected in Example 1 (carrying the wild-type PQQ-sGDH coding gene), or the thalline collected in Example 2 (carrying the PQQ-sGDH mutant coding gene), was cultivated to OD at 37°C. 600 When it is 1.0, add IPTG50μl, induce wild-type PQQ-sGDH or PQQ-sGDH mutant coding gene expression, then collect E. Sodium dihydrogen phosphate, pH 7.0) was resuspended, and the wild-type PQQ-sGDH or PQQ-sGDH mutants present in the cells were released by high-pressure or ultrasonic disrupting methods, and then the supernatant was collected by centrifugation, so that the obtained supernatant The supernatant contains the crude enzyme solution of wild-type PQQ-sGDH or PQQ-sGDH mutant.
[0125] Next, load the obtained crude enzyme liquid sample onto the His-tag adsorption column equilibrated with phosphate buffer A in advance, first use phosphate buffer B (0.2M sodium chloride, 50mM imidazole, 20m...
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