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Alkaline pectic enzyme producing engineering strain and its construction and method for producing alkaline pectic enzyme with the same

A technology of pectinase and engineering bacteria, which is applied in the field of bioengineering, can solve the problems of lack of research reports on genetic engineering of alkaline pectin ester lyase, staying in the stage of strain screening and research on enzyme characteristics, and shortening the fermentation time. , less variety, the effect of simplifying the purification process

Inactive Publication Date: 2008-04-09
JIANGNAN UNIV
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since the early 1990s, research on alkaline pectinase has also been carried out in our country, but it generally stays in the stage of strain screening and enzyme characteristics research. There are almost no reports on the genetic engineering of alkaline pectin ester lyase in China.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of Recombinant Alkaline Pectinase Engineering Bacteria CGMCC No.2143

[0035] (1) Cloning of the gene pel: design and synthesize the primers required for PCR according to the pel-related gene sequence:

[0036] P1: 5′-GCTTACGTAGCTGATTTAGGCCACCAGACG-3′

[0037] P2: 5′-AACGCGGCCGCTTAATTTAATTTACCCGCACCCG-3′

[0038] Both ends of the primers were introduced into SnaB I and Not I sites, and the PCR reaction was completed using Bacillus sp WSHB04-02 DNA as a template: Add the following components to a 200 μL PCR reaction tube: Buffer 5 μL, 2.5 mmol / L DNTPs 4 μL, template DNA 1 μL, Taq DNA Polymerase 1 μL, water up to 50 μL; reaction conditions: denaturation at 95°C for 5 min, 35 cycles of 95°C for 50 s, 42°C for 90 s, and 72°C for 5 min. The obtained PCR product was confirmed by electrophoresis analysis and purified by PCR product purification kit After digestion with SnaB I and Not I, the 1.2kb fragment was recovered and connected to the vector pPIC9...

Embodiment 2

[0040] Example 2 Construction of Recombinant Alkaline Pectinase Engineering Bacteria CGMCC No.2143

[0041] Primers used instead:

[0042]P1': 5'-GCTTACGTAGCTGATTTAGGCCATCAAACG-3'

[0043] P2': 5'-AACGCGGCCGCTTAATTTAATTTACCAGCACCCG-3'

[0044] All the other operating methods are the same as in Example 1.

Embodiment 3

[0045] Embodiment 3 utilizes the technique that engineering bacterium CGMCC No.2143 produces alkaline pectinase is:

[0046] (1) Starting strain: CGMCC No.2143;

[0047] (2) Seed cultivation:

[0048] Seed medium composition: in g / 1000mL, peptone 10-20, yeast extract 5-10, glucose 10-20, sodium chloride 5-15;

[0049] Seed culture: 250mL Erlenmeyer flask, liquid volume 50mL, culture temperature 28°C-30°C, shaker speed 200r / min, culture 24h (stable period);

[0050] (3) Cell culture

[0051] Bacteria medium composition: in g / 1000mL, yeast nitrogen base medium 12-15, biotin 0.0002-0.0005, glycerol 7-10.

[0052] Bacterial culture: collect seeds by centrifugation, wash with normal saline twice, transfer to 100mL medium (add 1mL 100×biotin); culture temperature 28°C-30°C, shaker speed 200r / min, culture for 48h;

[0053] (4) Fermentation culture:

[0054] Fermentation medium composition: in g / 1000mL, yeast nitrogen base medium 12-15, biotin 0.0002-0.0005, methanol 10-50, (NH ...

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Abstract

An engineering bacterium of producing alkaline pectinase, a construction thereof and a method of producing alkaline pectinase by using the alkaline pertain to the technical field of bioengineering. The invention uses nonpathogenic bacillus sp WSHB04-02 genes group as a model and adopts amplification technology of PCR. And then the sequence pel containing 1.2kb coding alkaline pectinase genes section is obtained. At the same time from the point of the demand for industrial production, the invention makes use of a shuttle expression carrier to restructure yeast Pichiapastoris(pel), which means to produce engineering bacterium CGMCC No.2143 of alkaline pectinase (E.C.4.2.2.2). The invention also provides CGMCC No.2143 as the method of producing alkaline pectinase for fermented yeast strain. The genes engineering bacterium aims at exocytosis of protein and the species of extracellular protein is few, which simplifies the purification technique of producing alkaline pectinase by using microorganism and reduces the cost and shoetens the time for ferment. Besides, the invention improves the production efficiency and lays a foundation for industrialization of the ferment method of producing alkaline pectinase by using microorganism.

Description

technical field [0001] An engineering bacterium producing alkaline pectinase, its construction and a method for producing alkaline pectinase with the bacterium belong to the technical field of bioengineering. Background technique [0002] Pectinase refers to a type of enzyme that can decompose pectin, and alkaline pectinase (E.C.4.2.2.2) refers to a type of pectinase with relatively high activity in the alkaline range. In addition to the application of alkaline pectinase in the purification of plant viruses and pulp bleaching, it has become a vital biological enzyme preparation in the pretreatment of cotton fabrics. The use of alkaline pectinase will change the traditional alkali scouring process, not only reduce environmental pollution, save a lot of process water, but also improve the quality of cotton fabrics. [0003] In the past ten years, foreign researchers have been trying to use genetic engineering to construct high-yield strains of pectin ester lyase. Alkaline pec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/56C12R1/84
Inventor 陈坚诸葛斌堵国成
Owner JIANGNAN UNIV
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