A kind of β-galactosidase modified by site-directed mutagenesis and its construction method
A construction method, mutant technology, applied in the direction of microorganism-based methods, glycosylases, botanical equipment and methods, etc., can solve problems that have not been applied, and achieve the effect of improving enzyme activity
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Embodiment 1
[0022] Example 1 Preparation of β-gal mutants
[0023] (1) Construction of recombinant plasmid pET-28a(+)-β-gal: a recombinant vector encoding the gene of wild-type β-gal (shown in the amino acid sequence of SEQ ID No. 3).
[0024] First, according to the original amino acid sequence (GenBank: AF184246), the codon-optimized synthetic gene is carried out with the lactic acid bacteria expression host to obtain the optimized β-gal coding gene (nucleotide sequence as shown in SEQ ID No.4), and the above-mentioned β-gal The gal coding gene and the pET-28a(+) vector were double-digested with BamHI and Xhol enzymes, respectively, and then recovered separately, and the recovered coding gene fragment and the pET-28a(+) vector were ligated with T4 DNA ligase to obtain Recombinant plasmid vector pET-28a(+)-β-gal, the recombinant plasmid vector was transformed into cloning host E.coliTOP10. Sequence the obtained transformant to verify whether it is the correct gene clone (identical to th...
Embodiment 2
[0029] Embodiment 2 Contains the expression and enzymatic activity of the β-gal mutant genetically engineered bacteria of the present invention
[0030] (1) Expression and purification of genetically engineered bacteria:
[0031] The above-mentioned genetically engineered bacteria containing the β-gal mutation were expanded and expressed, the induction temperature was 10°C, the induction time was 13-14h, and the concentration of the inducer isopropyl-β-D-thiogalactoside (IPTG) was 0.1mM, centrifuge the induced bacterial liquid at 8000r / min at 4°C for 5min, collect the bacterial liquid, resuspend the bacterial cells with pH 7.5 phosphate buffer, and use an ultrasonic cell disruptor to disrupt the bacterial cells. Centrifuge at 8000r / min for 20min, and the obtained supernatant is the crude enzyme for the following purification.
[0032] Draw 1mL of Ni-IDA filler into the chromatography column, rinse with sterile water and equilibrate the Ni column with binding solution. Aspira...
example 3
[0035] Example 3 Enzyme immobilization between β-gal-A229C and PuriMag Si-SH through thiol-dithiol exchange reaction
[0036]The purchased thiol magnetic beads PuriMag Si-SH (~40μg protein / mg beads) and the β-gal before and after transformation were respectively in 0.1M NaBH pH 7.0 4 In the buffer solution system, at 30°C, shake and combine for 6h, then place the reaction test tube on the magnetic stand, let stand for 2min, remove the supernatant, add the same volume of blocking buffer, shake for 2h, let stand for 2min, remove the supernatant , then add 1mL washing buffer, resuspend the solution, let stand for 2min, remove the supernatant, repeat washing 3 times, finally add substrate FDG to the reaction system, let stand at room temperature for about 30min. Such as image 3 As shown, according to the above enzyme immobilization steps, β-gal-A229C and PuriMag Si-SH successfully completed the enzyme immobilization through the sulfhydryl-disulfhydryl exchange reaction, so after...
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