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Alkaline pectase mutant with improved heat resistance

A pectinase and mutant technology, applied in the field of bioengineering, can solve problems such as high potency and research that cannot be ignored

Inactive Publication Date: 2014-08-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the expression level of PGL is very high, the enzymatic properties such as the catalytic efficiency and heat resistance of the enzyme need to be further improved, and the current molecular transformation is only trying to express heterologously in different hosts to improve the enzyme activity, heat resistance, and catalytic activity. Research on enzymatic properties such as efficiency is very little done in China at present
In the past ten years, the research direction of alkaline pectinase in foreign countries has focused on the separation and purification of enzymes, enzymatic characteristics and protein structure characteristics, etc., while the research on alkaline pectinase in my country mainly focuses on strain selection, fermentation and so on. In terms of improvement of technology and application technology, and my country still lacks commercial alkaline pectinase with high titer, which paves the way for further industrialization, research in this area cannot be ignored

Method used

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  • Alkaline pectase mutant with improved heat resistance
  • Alkaline pectase mutant with improved heat resistance
  • Alkaline pectase mutant with improved heat resistance

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Experimental program
Comparison scheme
Effect test

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 wild-type alkaline pectinase and 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 shown. By comparing the heat-resistant pectinase sequences: CAD56882 from Bacillus licheniformis, BAA96478 from Bacillus sp.strain P-4-N, and AAD35518 from Thermotoga maritima MSB82, the optimum temperatures are 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 72 mmol L -1 , KH 2 PO 4 17mmol L -1 .

[0044] Inoculate the recombinant strain E.coli BL21(DE3) containing the mutant expression vector pET-20b(+)-pglI325F from a glycerol tube into 100 μg mL -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 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 ind...

Embodiment 3

[0046] Enzymatic properties of PGL before and after mutation in embodiment 3

[0047] 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(+) -pglI325F) was fermented, and the enzymes in the fermentation broth were 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 p...

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Abstract

The invention discloses an alkaline pectase mutant with an improved heat resistance, and belongs to the technical field of bioengineering. The 325th isoleucine I site of alkaline pectase from Bacillus sp.WSHB04-02 mutates to phenylalanine F. The residual enzyme activity of the obtained mutated alkaline pectase undergoing heat insulation at 50DEG C for 2h is 4.27 times that of the alkaline pectase before the mutation; and the above situation shows that the heat resistance of the mutated alkaline pectase I325F at 50DEG C is obviously improved, and the I325F still has a stable structure at 50DEG C for a long time. The half life of the mutated alkaline pectase I325F at 55DEG C is 1.89 times the half life of the alkaline pectase before the mutation; and the Km, Vmax, kcat and kcat / Km are improved. The enzymatic properties of PGL provided by the invention are greatly improved, so the PGL is suitable for industrial production demands, and meets social production requirements.

Description

technical field [0001] The invention relates to an alkaline pectinase mutant with improved heat resistance, which belongs to the technical field of bioengineering. Background technique [0002] Alkaline pectinase (E.C.4.2.2.2, PGL for short), the full name of polygalacturonic acid lyase, has high activity under alkaline conditions, and can use trans-elimination to cut off the α-1,4 of polylacturonic acid -Glycosidic bonds, decomposing pectin into unsaturated oligogalacturonic acid. The enzyme widely exists in bacteria, yeast, fungi, plants and some parasitic nematodes. The homology of alkaline pectinase from different sources is quite different. Alkaline pectinase is widely used in various fields such as food, textile, papermaking, environment, and biotechnology, and plays a huge role in tea and coffee fermentation, textile and plant fiber processing, oil extraction, and pectin-containing industrial wastewater treatment. [0003] There are many accompanying impurities on ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21
CPCC12N9/88C12N15/70C12Y402/02002
Inventor 陈坚堵国成刘松汪明星
Owner JIANGNAN UNIV
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