Engineered ribokinase as well as coding gene, expression vector, engineering bacterium and application thereof
A ribokinase, encoding gene technology, applied in the field of biopharmaceuticals and biotransformation, can solve the problems of high price of 5-phosphate ribose, no further research, etc., and achieve the effect of good catalytic activity and improved yield
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Embodiment 1
[0026] Embodiment 1: the construction of gene cloning and expression vector
[0027] The amino acid sequence of the wild-type ribokinase derived from Escherichia coli can be retrieved from NCBI, as shown in Sequence 1, and then the corresponding nucleic acid was synthesized by common techniques in the art and cloned into the expression vector pet28a. Transform the recombinant expression plasmid into competent cells of E.coil BL21(DE3), the transformation condition is 42°C, heat shock for 90 seconds, the transformation solution is spread on the LB plate containing chloramphenicol, and cultured upside down at 37°C overnight, that is Recombinant transformants were obtained.
Embodiment 2
[0028] Example 2: Expression of ribokinase and preparation of enzyme solution
[0029] Transfer 5 μL of the frozen RK bacterial solution into 5 mL (containing 50 μL / ml kanamycin sulfate) LB medium, and activate overnight.
[0030] The activated bacterial liquid was put into 500mL LB medium, cultured at 37°C until the OD600 was about 0.7, and pre-cooled to 20°C.
[0031] Add the IPTG inducer with a final concentration of 0.2 mM, and then put it into a shaker at 20° C. for 20 h.
[0032] The cultured bacterial liquid was centrifuged and washed three times with pH 8.0, 50mM Tris-His buffer (containing 100mM NaCl)
[0033] The washed bacteria were resuspended with pH 8.0, 50 mM Tris-His buffer (containing 100 mM NaCl), and ultrasonically lysed. The lysed broken bacteria solution was taken out and centrifuged, and the supernatant was taken for subsequent reactions.
Embodiment 3
[0034] Example 3: Construction of ribokinase mutant library
[0035] According to the crystal structure of ribokinase, the key amino acids (14, 16, 42, 46, 143, 265, 273) around the active center were selected and mutated into alanine (A), a typical representative of small-medium-large volume, respectively. Leucine (L), Phenylalanine (F). The specific method is as follows:
[0036] The PCR system is: 5 μL of 10xBuffer, 5 μL of 2mM dNTPs, 1 μL of plasmid DNA template (50ng / μL), 2 μL of upstream and downstream primers (10 μM), 0.5 μL of high-fidelity enzyme, 1 μL of DMSO, and 34 μL of ddH2O. The codons of the PCR primers at the mutation positions are as follows:
[0037] Mutant Primer sequence N14A CTGGGTAGCGTTGCGGCAGATCATGTTC N14L CTGGGTAGCGTTCTGTGCAGATCATGTTC N14F CTGGGTAGCGTTTTTGCAGATCATGTTC D16A GTTAATGCAGCGCATGTTCTG D16L GTTAATGCACTGCATGTTCTG D16F GTTAATGCATTTCATGTTCTG G42A GTTATTCCGGGTGCGAAAGGCGCAAATC G42L...
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