Acetylglucosamine hydrolase mutant and its application
A technology of glucose hydrolase and acetylamino, which is applied in the field of acetylglucosamine hydrolase mutants, can solve the problems of low production efficiency of glucosamine, low activity of N-acetylglucosamine hydrolase, etc., and achieves efficient hydrolysis, high catalytic activity, The effect of improving production efficiency
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Embodiment 1
[0046] Example 1. Construction of N-acetylglucosamine hydrolase (GlcNAc) expression vector.
[0047] The embodiment of the present invention clones the deacetylation gene TK-dac (wild type) from the genomic DNA of Thermococcus kodakaraensis KOD1, the nucleotide sequence is shown in SEQ ID NO: 4, with pET28a (+) as the carrier, connected to pET-28a (+) A recombinant expression plasmid was constructed on the expression plasmid and named: pET28a(+)-TK-dac.
[0048] The expression carrier selected in the present invention is pET28a(+)-TK-dac. Design forward primer N169S-F: 5'-AGCTTCAACCGCTCTGACCTGA-3' (SEQ ID NO.5), and reverse primer N169-R: 5'-CGGCAGGCCAGCGAAAGAAAC-3' (SEQ ID NO.6), provided by Nanjing Kings Synthesized by Rui Technology Co., Ltd. Using pET28a(+)-TK-dac as a template, introduce mutation points into the primers for PCR amplification; use DpnI digestive enzyme to recognize and cleavage pET28a(+)-TK-dac; use PNK kinase to phosphorylate the PCR product, and at the...
Embodiment 2
[0054] Example 2. Expression and SDS-PAGE analysis of N-acetylglucosamine hydrolase.
[0055] The plasmids pET28a(+)-TK-dac and pET-28a(+)-Tk-169S in Example 1 were transformed into Escherichia coli JM109 (DE3) for expression, thereby obtaining the pET28a(+)-TK-dac strain and pET -28a(+)-Tk-169S strain. The specific conversion method is:
[0056] Take out 100 μ L of competent Escherichia coli JM109 (DE3), add respectively the plasmids pET28a(+)-TK-dac and pET-28a(+)-Tk-169S in Example 1, mix and place on ice for 30min; After being heat-shocked in a water bath at 42°C for 45s, move it to ice, place it for 2-3min, add 900μL sterile SOB medium, and cultivate it in a constant temperature shaker at 37°C at 200rpm for 1h; centrifuge the bacterial solution at 12,000rpm for 1min, Most of the supernatant was discarded, and the remaining about 200 μL was resuspended evenly and spread all over the LB solid plate containing Kan resistance, and cultured overnight at 37°C. After the colo...
Embodiment 3
[0061] Embodiment 3, the enzyme activity determination of N-acetylglucosamine hydrolase
[0062] Utilize alkali titration to measure the activity of N-acetylglucosamine hydrolase, the N-acetylglucosamine hydrolase expressed by pET-28a(+)-Tk-169S transformation strain is named mutant enzyme N169S, and its amino acid sequence is as SEQ ID NO .2; the N-acetylglucosamine hydrolase expressed by the pET28a(+)-TK-dac strain is named wild enzyme TK-DAC, and its amino acid sequence is shown in SEQID NO.1. It can be seen from Table 2 and Table 3 that the activity of the mutant enzyme N169S is about 7 times that of the wild enzyme TK-DAC. It can be seen that the wild enzyme TK-DAC shown in SEQ ID NO.1 is mutated by the 169th amino acid residue Compared with the wild enzyme TK-DAC, the enzyme activity is significantly improved, about 7 times higher.
[0063] Table 2: Conditions for determining the enzyme activity of N-acetylglucosamine hydrolase by alkaline titration
[0064] ...
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