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

Inactive Publication Date: 2014-08-06
JIANGNAN UNIV
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Problems solved by technology

[0005] 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, there is still a lack of commercial alkaline pectinase with high potency, which paves the way for further industrialization. Research in this area cannot be ignored

Method used

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  • PGL (Polygalacturonate Lyase) mutant capable of improving secretion performance
  • PGL (Polygalacturonate Lyase) mutant capable of improving secretion performance
  • PGL (Polygalacturonate Lyase) mutant capable of improving secretion performance

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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.

[0...

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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Abstract

The invention discloses a PGL (Polygalacturonate Lyase) mutant capable of improving secretion performance and belongs to the technical field of bioengineering. A 58-th isoleucine I point of PGL deriving from Bacillus sp.WSHB04-02 is mutated into valine V. The extracellular enzyme activity of the mutated PGL is improved to 337.58 U / mL, and is 2.60 times that of the PGL which is not mutated, and the expression quantity of extracellular secretion is obviously increased. In addition, the optimum reaction temperature of the mutant is improved by 5 DEG C, and the optimum enzyme activity can be better shown under the condition of high temperature; Km is obviously improved, the capacity of the mutant in combination with a substrate is improved to be 4.5 times than that of the mutant which is not mutated, and kcat, t1 / 2,kcat / Km and the like are improved. The enzymatic property of the PGL provided by the invention is greatly improved, and requirements for industrial production and social production are met.

Description

technical field [0001] The invention relates to an alkaline pectinase mutant with improved secretion performance, which belongs to the technical field of bioengineering. Background technique [0002] Alkaline pectinase (E.C.4.2.2.2, referred to as PGL), the full name of polygalacturonate lyase, the enzyme widely exists in bacteria, yeast, fungi, plants and some parasitic nematodes. 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. Alkaline pectinase has high activity under alkaline conditions, and can use trans-elimination to cut the α-1,4-glycosidic bond of polylacturonic acid, and decompose pectin into unsaturated oligogalacturonic acid . [0003] There are many accompanying impurities on the outermost surface of the primary cell wall of cotton...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/10C12N15/70C12R1/19C12R1/125
CPCC12N9/88C12Y402/02002
Inventor 陈坚堵国成刘松汪明星
Owner JIANGNAN UNIV
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