An Alkaline Pectinase Mutant with Improved Heat Stability
A pectinase and mutant technology, applied in the field of bioengineering, can solve the problems of high titer and high titer that cannot be ignored in research
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Embodiment 1
[0026] The construction of embodiment 1 mutant expression plasmid and the acquisition of recombinant Bacillus subtilis
[0027] 1. Construct a mutant expression vector using the pET-20b(+)-pgl plasmid as a template
[0028] The nucleotide sequence of the gene encoding the wild-type alkaline pectinase and the signal peptide consisting of 21 amino acids is shown in SEQ ID NO.1, and the amino acid sequence of the wild-type mature alkaline pectinase is shown in SEQ ID NO.2. By comparing the heat-resistant pectinase sequences: CAD56882 of Bacilluslicheniformis, BAA96478 from Bacillussp.strainP-4-N, and AAD35518 from Thermotogamaritima MSB82, the optimum temperatures were 69°C, 70°C and 90°C, respectively. Combined with the three-dimensional structure of alkaline pectinase, it is speculated that the isoleucine I at position 325 has a greater impact on the thermal stability of alkaline pectinase, and a mutation experiment was designed to mutate the isoleucine I at position 325 into ...
Embodiment 2
[0041] Expression of embodiment 2 mutant PGL
[0042] Seed medium composition (g / L): yeast powder 5, tryptone 10, NaCl 10, glucose 20, pH 7.0.
[0043] Composition of fermentation medium: yeast powder 24g / L, tryptone 12g / L, glycerol 5g / L, K 2 HPO 4 72mmolL -1 , KH 2 PO 4 17mmolL -1 .
[0044] Inoculate the recombinant bacteria E.coliBL21(DE3) containing the mutant expression vector pET-20b(+)-pglI325F from a glycerol tube into 100μgmL -1 In the seed medium of ampicillin, the filling volume is 20mL / 250mL. The culture temperature was 37° C., and the culture was shaken on a shaker at 200 rpm for 10 h.
[0045] The seed solution cultivated for 10h was inoculated with 100μgmL with a 3% (V / V) inoculum -1 In the fermentation medium of ampicillin, the filling volume is 50mL / 500mL, at 37°C, 200rmin -1 to cultivate. Bacteria grow to a certain stage (OD 600 =0.6), adding a final concentration of 0.4mMIPTG for induction, while adjusting the temperature to 30°C, and inducing fe...
Embodiment 3
[0046] Enzymatic properties of PGL before and after mutation in embodiment 3
[0047] According to the method described in Example 2, E.coliBL21(DE3) containing the unmutated expression vector pET-20b(+)-pgl and the mutant strain E.coliBL21(DE3)(pET-20b(+)-pglI325F ) is fermented, and the enzymes in the fermentation broth are purified and analyzed for enzymatic properties such as heat resistance. The resulting alkaline pectinase mutant was named I325F.
[0048] Depend on figure 1 It can be seen that the enzyme activity loss of alkaline pectinase (WT) before mutation is obvious after incubation at 30°C for 24h, 50°C for 2h, and 55°C for 1h; After the same treatment, the residual enzyme activity was increased compared with that before the mutation, especially when incubated at 50°C for 2 hours. After incubation at 50°C for 2 hours, the residual enzyme activity after mutation was 4.27 times that before mutation; this shows that the heat resistance of the mutated alkaline pecti...
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