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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 produced by alkaline pectinase, can solve the problems of long fermentation cycle and high energy consumption

Active Publication Date: 2012-07-18
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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, acquisition and preservation of engineering bacteria

[0026] 1. Acquisition of engineering bacteria

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

[0028] 2. According to the gene sequence related to alkaline pectinase, degenerate primers were designed.

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

[0030] 4. Sequencing the PCR amplification product in step 3, and discovering a new gene.

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

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

[0033] 2. Preservation of engineering bacteria

[0034] BL21(DE3)PGL04 belongs to Escherichia coli. BL21(DE3)PGL04 was deposited in the General Microorganism Ce...

Embodiment 2

[0035] Embodiment 2, application engineering bacterium produces alkaline pectinase

[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 (pH 7.0): Take 10g tryptone, 15g yeast extract, 5g sodium chloride, 10g glycerin, 10mM potassium dihydrogen phosphate and 65mM dipotassium hydrogen phosphate, dissolve in water and dilute to 1L; extinguish at 121°C Bacteria 30min.

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

[0040] 1. Inoculate Escherichia coli BL21(DE3)PGL04 into the seed medium, and shake culture at 35°C (100r / min) to OD 600nm =3, namely the seed solution.

[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 of =0.1; the fermentation process is as follows...

Embodiment 3

[0058] Embodiment 3, application engineering bacterium produces alkaline pectinase

[0059] 1. Preparation of culture medium

[0060] Seed culture medium (pH7.2): 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 (pH 7.2): Take 15g tryptone, 23g yeast extract, 10g sodium chloride, 15g glycerin, 17mM potassium dihydrogen phosphate and 75mM dipotassium hydrogen phosphate, dissolve in water and dilute to 1L; extinguish at 121°C Bacteria 30min.

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

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

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

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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 engineering bacterium used for the production of alkaline pectinase and its application. Background technique [0002] Pectin is an important component of plant cell walls, including protopectin, pectic acid and pectin ester acid. The presence of pectin substances can increase the firmness of plant cell walls, which is conducive to maintaining the specific shape and structure of plants, but 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 kind of enzyme that breaks the main chain of pectin by trans-elimination under alkaline conditions to produce unsaturated oligogalacturonic acid. It is used in the textile industry It has a wide range of applications in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/26C12P19/14C12R1/19
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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