A urease mutant with improved application performance
A mutant and urease technology, applied in the field of genetic engineering and enzyme engineering, can solve problems such as difficulty in using alcoholic beverages, instability, etc., and achieve the effect of improving affinity and degradation rate
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Embodiment 1
[0034] Embodiment 1: Preparation of recombinant bacteria containing urease gene
[0035] Use primers P1-F / P1-R to linearize plasmid pRSF-Duet-1 by PCR. The PCR reaction system is: 2×Phanta Max Master Mix 25μL, upstream primer (10μM) 2μL, downstream primer (10μM) 2 μL, template DNA 1 μL, ddH 2 O 20 μL (PCR reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd.) to obtain linearized pRSF-Duet-1.
[0036] Chemically synthesize the nucleotide sequence shown in SEQ ID NO.2, and connect it to the linearized plasmid pRSF-Duet-1 (using ClonExpress II One Step Cloning Kit, each 100 μg and 70 μg of the fragment and the linearized plasmid, at 37 °C ligation for 30 min) to obtain the recombinant plasmid P-1-WT, and transform the recombinant plasmid into Escherichia coli JM109 (for specific construction steps, refer to the instructions for use of JM109 competent cells of Beijing Suolaibao Technology Co., Ltd.). Utilize the positive transformant to extract the plasmid, trans...
Embodiment 2
[0046] Embodiment 2: the preparation of mutant M325V, M373A, M373T
[0047] By analyzing the docking structure of the urease protein and the substrate urethane molecule, we chose to mutate its 325th and 373rd methionines, and designed corresponding site-directed mutagenesis primers (Table 2). Using the recombinant plasmid P-1-WT as a template, the primers in Table 2 were used to amplify the recombinant plasmid P-1-WT.
[0048] Table 2 Urease Mutation Primer Sequence
[0049] Primer Primer sequence (5'-3') M373mut-A GCAGGCG NNK GGCAG
SEQ ID NO.11 M373mut-S CTGCC MNN CGCCTGC
SEQ ID NO.12 M325mut-A TGATATG NNK ATGGTCTGCCATC
SEQ ID NO.13 M325mut-S ACCAT MNN CATATCAAGATGCTCG
SEQ ID NO.14
[0050] PCR reaction system: 2×Phanta Max Master Mix 25 μL, upstream primer (10 μM) 2 μL, downstream primer (10 μM) 2 μL, template DNA 1 μL, ddH 2 O 20 μL.
[0051] The PCR amplification conditions were: pre-denaturation ...
Embodiment 3
[0053] Example 3: Expression and purification of urease mutants
[0054] The urease mutant strain was inoculated in liquid LB medium (containing 50 μg / mL kanamycin), cultivated at 37° C. for 12 hours at 220 rpm to obtain seed liquid, and inoculated the seed liquid into TB medium (containing 50 μg / mL kanamycin), 37 ℃, 220rpm cultivated to OD 600 =0.6-0.8, add 0.1mM IPTG and 6mM Ni 2 SO 4 , 30° C., 220 rpm induction culture for 15 hours to obtain a fermentation broth. The fermentation broth was centrifuged at 4°C and 8000r / min for 15min, and the bacterial sediment was collected. The bacterial cells were washed twice with 20mmol / L pH7.4 phosphate buffer solution, and 50mL of Binding Buffer (20mmol / L pH 7.4 phosphoric acid Salt buffer solution, 0.5mol / L NaCl) to resuspend the bacterial cells, ultrasonically break in an ice-water bath (crushing conditions: 130w, 2s / 4s, 20min), centrifuge at 4°C, 12000r / min for 20min, and collect the supernatant to obtain crude urease Enzyme so...
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