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Engineering bacterium for producing alkaline pectinase and application thereof

A pectinase and alkaline technology, applied in the field of engineering bacteria for alkaline pectinase production, can solve the problems of high energy consumption, long fermentation period and the like

Active Publication Date: 2013-04-17
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, there have been reports of expressing alkaline pectinase with recombinant Pichia pastoris, but this technology has a long fermentation cycle and high energy consumption

Method used

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  • Engineering bacterium for producing alkaline pectinase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Acquisition and preservation of engineered bacteria

[0026] 1. The acquisition of engineering bacteria

[0027] 1. Extract the whole genome DNA of soil.

[0028] 2. Design degenerate primers based on the sequence of alkaline pectinase-related genes.

[0029] 3. Using whole genome DNA as a template, PCR amplification is performed with the degenerate primers designed in step 2 to obtain PCR amplification products.

[0030] 4. Sequencing the PCR amplified product of step 3 and discovering a new gene.

[0031] 5. Insert the new gene into the vector pET28b between the NcoI and XhoI restriction sites to obtain a recombinant plasmid.

[0032] 6. Transform the recombinant plasmid into Escherichia coli BL21(DE3) to obtain a recombinant strain (engineered strain), named BL21(DE3)PGL04.

[0033] 2. Preservation of engineered bacteria

[0034] BL21(DE3)PGL04 belongs to Escherichia coli. BL21(DE3)PGL04 has been deposited in the General Microbiology Center of the China Microbial Cultu...

Embodiment 2

[0035] Example 2: Production of alkaline pectinase using engineered bacteria

[0036] 1. Preparation of culture medium

[0037] Seed culture medium (pH7.0): Take 10g tryptone, 5g yeast extract and 5g sodium chloride, dissolve in water and dilute to 1L; sterilize at 121°C for 30min.

[0038] Fermentation medium (pH7.0): Take 10g tryptone, 15g yeast extract, 5g sodium chloride, 10g glycerol, 10mM potassium dihydrogen phosphate and 65mM dipotassium hydrogen phosphate, dissolve in water and dilute to 1L; kill at 121℃ Bacteria 30min.

[0039] 2. Application of engineering bacteria to produce alkaline pectinase

[0040] 1. Inoculate Escherichia coli BL21(DE3)PGL04 to the seed culture medium, and shake culture (100r / min) to OD at 35℃ 600nm = 3, which is the seed liquid.

[0041] 2. Inoculate the seed solution of step 1 into 40mL fermentation medium (using 250mL shake flask) to obtain OD 600nm The initial fermentation system with =0.1; the fermentation process is as follows (43 hours in total...

Embodiment 3

[0058] Example 3: Application of engineered bacteria to produce alkaline pectinase

[0059] 1. Preparation of culture medium

[0060] Seed culture medium (pH 7.2): Take 15g tryptone, 8g yeast extract and 10g sodium chloride, dissolve in water and dilute to 1L; sterilize at 121°C for 30min.

[0061] Fermentation medium (pH7.2): Take 15g tryptone, 23g yeast extract, 10g sodium chloride, 15g glycerol, 17mM potassium dihydrogen phosphate and 75mM dipotassium hydrogen phosphate, dissolve in water and dilute to 1L; kill at 121℃ Bacteria 30min.

[0062] 2. Application of engineering bacteria to produce alkaline pectinase

[0063] 1. Inoculate Escherichia coli BL21 (DE3) PGL04 to the seed medium, and shake culture (200r / min) at 37℃ to OD 600nm = 6, which is the seed liquid.

[0064] 2. Inoculate the seed solution of step 1 into 20mL fermentation medium (using 250mL shake flask) to obtain OD 600nm Fermentation initial system = 0.2; the fermentation process is as follows (53 hours in total): fi...

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Abstract

The invention discloses an engineering bacterium for producing alkaline pectinase and an application thereof. The engineering bacterium disclosed by the invention belongs to Escherichia coli, is named as BL21 (DE3) PGL04 and preserved with the number of CGMCC No.5697. The invention further discloses a method for producing the alkaline pectinase. The method comprises the following steps: (1) inoculating the Escherichia coli BL21 (DE3) PGL04 into a fermentation medium so as to obtain a fermentation initial system with OD600nm of 0.1-0.2; and (2) fermenting the fermentation initial system as follows: first, continuing to culture the fermentation initial system in a shaking way for 3-5 hours at 35-37 DEG C, then adding IPTG to the fermentation initial system until the final concentration is 50-100 microns, and finally continuing to culture the fermentation initial system in a shaking way for 40-48 hours at 25-30 DEG C so as to obtain the alkaline pectinase. The engineering bacterium disclosed by the invention can be used for producing the alkaline pectinase and further can be used for the hemp degumming industry.

Description

Technical field [0001] The invention relates to an engineered bacterium for the production of alkaline pectinase and its application. Background technique [0002] Pectin is an important component of plant cell walls, including protopectin, pectinic acid and pectinic acid. The presence of pectin substances can increase the firmness of plant cell walls and help maintain the specific shape and structure of plants, but it increases the difficulty of industrial operations for deep processing of plants. [0003] Pectinase refers to a class of enzymes that can decompose pectin. It is commonly found in higher plants and microorganisms in nature and is an important industrial enzyme preparation. [0004] Alkaline pectinase (EC: 4.2.2.2) is a type of enzyme that breaks the pectin backbone by trans elimination under alkaline conditions to produce unsaturated oligogalacturonic acid. It is used in the textile industry. The hemp degumming, cotton refining and other fields have a wide range of a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/26C12P19/14C12R1/19
CPCY02P20/52
Inventor 宋江宁李小曼王辉林马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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