Alkaline pectate lyase producing gene engineering bacteria and construction and use thereof

A technology of pectate lyase and genetically engineered bacteria, applied in the field of genetic engineering, can solve the problems of no research report on Aspergillus alkaline pectate lyase, poor recombinant expression effect, low enzyme yield, etc., and achieve enzyme production The effect of high protein content, less protein types, and short fermentation time

Inactive Publication Date: 2011-09-21
YANCHENG TEACHERS UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the presence of other carbon sources such as glucose or sucrose, the expression of pectinase will be inhibited. Although solid fermentation and promoter region mutation technology are effective methods to solve the problem of carbon sources such as glucose or sucrose inhibiting the natural expression of fungal pectinase, But the yield of the enzyme is still low
In order to improve the expression efficiency of the enzyme, the predecessors used other Aspergillus and yeast as heterologous expression hosts for recombi

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Alkaline pectinase acid lyase engineering bacteria Escherichia coli pel build

[0022] (1) Artificial total synthesis of Aspergillus nidulans pel gene, the nucleotide sequence of which is shown as SEQ ID NO.1), the sequencing indicates that the synthesis is correct, and the pel The gene sequence was cloned into the vector pET-20b(+), and the recombinant plasmid was obtained pel / pET-20b(+).

[0023] (2) Construction of genetically engineered bacteria: recombinant plasmids will be obtained pel / pET-20b(+) electroporation Escherichia coli BL 21 (DE3), resulting in recombinant Escherichia coli pel / pET-20b(+) / Escherichia coli BL 21 (DE3), named Escherichia coli pel.

Embodiment 2

[0024] Example 2 Production of Alkaline Pectate Lyase by Fermentation

[0025] Genetically engineered bacteria Escherichia colipel Inoculate in seed culture medium, 250 mL Erlenmeyer flask, 50 mL filling volume, culture temperature 37°C, shaker speed 200 r / min, after 8 hours of culture, transfer to fermentation medium for staged fermentation, the pre-fermentation conditions are : The cultured seeds were inoculated into the fermentation medium, the culture conditions were 37°C, 200 r / min, and cultured to OD 600 0.8, time 4 h, no inducers to induce and promote enzyme production, such as calcium chloride, glycine, triton-100, IPTG, were added to the fermentation medium at this stage. The post-fermentation conditions were: post-culture temperature 37°C, shaker speed 200 r / min, culture 2 h, 20 mM calcium chloride, 0.1% glycine, 0.1% triton-100, 1 mM IPTG were added to the fermentation medium. After the fermentation, the activity of alkaline pectinase acid lyase was 400 U / mL.

...

Embodiment 3

[0029] Embodiment 3 fermentation produces alkaline pectate lyase

[0030] Genetically engineered bacteria Escherichia colipel Inoculate in seed culture medium, 500 mL Erlenmeyer flask, 100 mL liquid volume, culture temperature 37°C, shaker speed 200 r / min, after 8 hours of culture, transfer to fermentation medium for staged fermentation, the initial fermentation conditions are : The cultured seeds were inoculated into the fermentation medium, the culture conditions were 37°C, 200 r / min, and cultured to OD 600 0.8 for 4 h, no lactose was added to the fermentation medium at this stage. The post-fermentation conditions were as follows: post-culture temperature 37°C, shaker speed 200 r / min, culture for 4 h, adding 5 g of lactose to the fermentation medium, and the enzyme activity of alkaline pectinase acid lyase was 385 U / mL after fermentation .

[0031] The seed medium is: in g / 1000 mL, peptone 10, yeast extract 5, sodium chloride 10, penicillin 0.05, pH 7.0.

[0032] The ferm...

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Abstract

The invention discloses alkaline pectate lyase producing gene engineering bacteria and construction and use thereof and belongs to the field of genetic engineering. In the invention, the Escherichia coli pel engineering bacteria capable of secreting and expressing pectinase Pel in vitro with high efficiency are constructed from an industrial prospective by using a totally synthetic Aspergillus nidulans pel gene. The invention also provides a method for producing pectinase by using the engineering bacteria. When the engineering bacteria are used, the fermentation time is short, and at 37 DEG C, the total fermentation lasts for 6 hours, wherein the early fermentation lasts for 4 hours and post fermentation lasts for 6 hours; the enzyme active yield is as high as 400U/mL; in addition, the produced pectinase can be secreted in vitro directly, hybrid proteins are reduced, and the purification is easy. Therefore, the engineering bacteria have better industrial application prospect.

Description

technical field [0001] The invention relates to a genetic engineering bacterium producing alkaline pectate lyase and its construction and application, in particular to a genetic engineering bacterium for efficiently secreting and expressing alkaline pectate lyase, which belongs to the field of genetic engineering. Background technique [0002] Pectinase (pectinases) refers to a class of enzymes that decompose pectin substances. Microorganisms such as bacteria, fungi, and actinomycetes can produce pectinases. The pectinase enzyme system contains all the enzymes that degrade the main chain and side chain of pectin, and contains about 40 different enzymes; the pectinase that acts on the main chain is mainly pectate lyase (Pectate lyase, Pel, EC 4.2.2.2), pectin acid hydrolase (polygalacturonase, PG, EC 3.2.1.15), pectin ester lyase (pectin lyase, Pec, EC 4.2.2.10). [0003] Alkaline pectate lyase (EC.4.2.2.2) is a pectinase that acts on the non-methylated pectin region. It has...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/60C12N15/63C12N9/88C02F3/34C12R1/19
Inventor 赵庆新沈敏吴生才葛月潭成秀梅
Owner YANCHENG TEACHERS UNIV
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