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Alkaline pectase mutant with improved heat stability

A thermostability and pectinase technology, applied in the field of bioengineering, can solve the problems of commercial alkaline pectinase that cannot be ignored in research, and achieve the effect of enhanced thermostability

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

AI Technical Summary

Problems solved by technology

Although the expression level of PGL is very high, the enzymatic properties such as catalytic efficiency and heat resistance of the enzyme need to be further improved
The research on alkaline pectinase in my country mainly focuses on the improvement of strain selection, fermentation process and application process. There is still a lack of commercial alkaline pectinase with high potency, which paves the way for further industrialization. Research in this area Can not be ignored

Method used

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  • Alkaline pectase mutant with improved heat stability
  • Alkaline pectase mutant with improved heat stability
  • Alkaline pectase mutant with improved heat stability

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Experimental program
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Embodiment 1

[0024] The construction of embodiment 1 mutant expression plasmid and the acquisition of recombinant Bacillus subtilis

[0025] 1. Construct a mutant expression vector using the pET-20b(+)-pgl plasmid as a template

[0026] 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 serine S at position 229 has a greater impact on the thermal stability of alkaline pectinase, and a mutation experiment was designed to mutate the serine S at position 229 into the other...

Embodiment 2

[0039] Expression of embodiment 2 mutant PGL

[0040] Seed medium composition (g / L): yeast powder 5, tryptone 10, NaCl 10, glucose 20, pH 7.0.

[0041] 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 .

[0042] Inoculate the recombinant strain E.coli BL21(DE3) containing the mutant expression vector pET-20b(+)-pglS229K 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.

[0043] 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 to cultivate. 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, an...

Embodiment 3

[0044] Enzymatic properties of PGL before and after mutation in embodiment 3

[0045] 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(+) -pglS229K) was fermented, and the enzymatic properties such as heat resistance were analyzed after the enzyme in the fermentation broth was purified. The resulting alkaline pectinase mutant was named S229K.

[0046] 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; but after mutation, alkaline pectinase (S229K) After doing the same treatment, it still maintains a high residual enzyme activity. After incubation at 30°C for 24 hours, the residual enzyme activity after mutation increased by 42.29% compared with that before mutation; after incubation at 50°C for 2 hours, the residual enzyme act...

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Abstract

The invention discloses an alkaline pectase mutant with an improved heat stability, and belongs to the technical field of bioengineering. The 229th serine S site of an alkaline pectase mutates to lysine K. The heat stabilities of the mutant at 30DEG C, 50DEG C and 55DEG C are respectively obviously higher than the heat stabilities of the alkaline pectase before the mutation. The optimal reaction temperature of the mutant rises by 5DEG C, the half life of the mutant at 55DEG C is 2.10 times the half life of the alkaline pectase, and the Km, Vmax, kcat and kcat / Km are improved. The alkaline pectase mutant provided by the invention is more suitable for industrial production demands, and meets social production requirements.

Description

technical field [0001] The invention relates to an alkaline pectinase mutant with improved thermal stability, 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 o...

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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