PGL (Polygalacturonate Lyase) mutant capable of improving secretion performance
A technology of secreting performance and pectinase, which is applied in the field of bioengineering, can solve the problems of commercial alkaline pectinase which cannot be ignored, and has high potency, and achieves the goal of enhancing heat resistance, enzymatic properties and enzymatic activity. Effect
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Embodiment 1
[0027] The construction of embodiment 1 mutant expression plasmid and the acquisition of recombinant Bacillus subtilis
[0028] 1. Construct a mutant expression vector using the pET-20b(+)-pgl plasmid as a template
[0029] The nucleotide sequence of the gene encoding wild-type alkaline pectinase and a signal peptide consisting of 21 amino acids is shown in SEQ ID NO.1, and the amino acid sequence of wild-type mature alkaline pectinase is shown in SEQ ID NO.2. Show. By analyzing the three-dimensional structure of alkaline pectinase, it is speculated that the isoleucine I at position 58 has a greater impact on the secretion and expression of alkaline pectinase, and a mutation experiment was designed to mutate the isoleucine I at position 58 into valine Acid V.
[0030] Using the pET-20b(+)-pgl plasmid as a template, the plasmid containing the mutant gene was amplified in vitro by PCR (polymerase chain reaction), and the isoleucine at position 58 was mutated into valine.
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Embodiment 2
[0046] Expression of embodiment 2 mutant PGL
[0047] Seed medium composition (g / L): yeast powder 5, tryptone 10, NaCl 10, glucose 20, pH 7.0.
[0048] Composition of fermentation medium: yeast powder 24g / L, tryptone 12g / L, glycerin 5g / L, K 2 HPO 4 72 mmol L -1 , KH 2 PO 4 17mmol L -1 .
[0049] Inoculate the recombinant strain E.coli BL21(DE3) (pET-20b(+)-pglI58V) containing the mutant expression vector pET-20b(+)-pglI58V in a 100 μg mL -1 In the seed medium of ampicillin, the filling volume is 20mL / 250mL. Culture temperature 37℃, 200rpm min -1 Incubate on a shaker for 10 h.
[0050] The seed solution cultivated for 10 h was inoculated with 3% (V / V) inoculum containing 100 μg mL -1 In the fermentation medium of ampicillin, the filling volume is 50mL / 500mL, at 37°C, 200r min -1 nourish. Bacteria grow to a certain stage (OD 600 =0.6), adding a final concentration of 0.4mM IPTG for induction, while adjusting the temperature to 30°C, and inducing fermentation for 48h...
Embodiment 3
[0051] Example 3 PGL extracellular enzyme activity before and after mutation
[0052] According to the method described in Example 2, E.coli BL21(DE3) containing the unmutated expression vector pET-20b(+)-pgl and the mutant strain E.coli BL21(DE3)(pET-20b(+) -pgl I58V) for fermentation.
[0053] figure 1 SDS-PAGE protein electrophoresis showed that the extracellular secretion expression of alkaline pectinase I58V was significantly increased after mutation, the extracellular enzyme activity of alkaline pectinase was 129.65U / mL before mutation, and the extracellular enzyme activity of alkaline pectinase increased after mutation It was 337.58U / mL, which was 2.60 times that before the mutation. Combined with Table 1, the specific enzyme activity of alkaline pectinase after mutation increased from 179.14U / mg to 295.63U / mg, which was 1.65 times that before mutation. It shows that after the mutation, the secretion ability of alkaline pectinase I58V is enhanced, and the enzyme acti...
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